EP4380684A1 - Protein or its compositions for use in treatment of the microbiota - Google Patents
Protein or its compositions for use in treatment of the microbiotaInfo
- Publication number
- EP4380684A1 EP4380684A1 EP22741379.6A EP22741379A EP4380684A1 EP 4380684 A1 EP4380684 A1 EP 4380684A1 EP 22741379 A EP22741379 A EP 22741379A EP 4380684 A1 EP4380684 A1 EP 4380684A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- protein
- diseases
- microbiota
- family
- intestinal
- 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
Links
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Classifications
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- 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
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
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- A61P3/00—Drugs for disorders of the metabolism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- 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
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- 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
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- 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
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- 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
- the measured values for the general population with a variety of ages (12 individuals aged 25-70 years) and conditions were in the range of values over 1, similarly to what was observed for a younger population (11 individuals aged 18-36 years); conversely for individuals affected by diseases correlating with changes in the microbiota the value of the F/B ratio was below 1 and ranged between 0.12 and 0.25, in particular for Crohn's disease (6 individuals), ulcerative colitis (5 individuals), multiple sclerosis (5 individuals), obese individuals with metabolic syndrome (28 individuals); surprisingly, for individuals with Down's syndrome the average F/B ratio value was instead over 1, that is similar to healthy individuals and indeed particularly high. Moreover, in the microbiota of Down's syndrome individuals there is a surprising absence of certain risk species such as particular bacteria (including Akkermansia and Dorea), which are instead found at high levels in the intestines of patients with neurodegenerative diseases.
- particular bacteria including Akkermansia and Dorea
- 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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Abstract
The present invention relates to 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 diseases linked to disorders of the immune system, 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, in humans or animals; a composition comprising 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 diseases linked to disorders of the immune system, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal microbiota; a diagnostic kit for assessing the presence of S100B protein in the intestinal microbiota; an assay regimen for a protein belonging to the S100 family, in particular S100B protein; a method for identifying whether a protein of the S100 family or another protein or peptide is useful as a harmoniser and/or stabiliser and/or modulator and/or regulator of the intestinal microbiota.
Description
“Protein oritscompositionsforusein treatmentofthemicrobiota”
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Field oftheInvention
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 beneficially harmonising and/or stabilising and/or modulatingand/orregulatingtheintestinalmicrobiota;a diagnostickit fordetectingthepresenceofS100B protein in theintestinalmicrobiota; an assay procedure for measuring the concentration of a protein belongingtotheS100family,in particularS100B protein;amethodfor identifying whether a protein belonging to the S100 family or another protein or peptide is usefulas a harmoniser and/or stabiliser and/or modulatorand/orregulatoroftheintestinalmicrobiota.
Background totheInvention
Theintestinalmicrobiotaisthecommunity 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. The several hundred species that populate it often cannot be cultured but can be studied using DNA (mfDNA, metagenome) and constitute a dynamic equilibrium. In recent years 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). In addition, there has even been defined 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.
For these and other reasons, products capable of modifying the microbiota, such as probiotics and prebiotics, have been developed over time.
The 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.
There has therefore been felt a particular need to provide new products or compositions for beneficially harmonising and/or stabilising and/or modulating and/or regulating intestinal and/or oral and/or pharyngeal and/or pulmonary and/or cutaneous and/or genital microbiota, in humans or animals, with the aim of using them in the treatment of 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.
As is well known, 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.
The name S100 derives from the fact that these proteins, and particularly S100B protein, are 100% soluble in ammonium sulphate at neutral pH.
Most 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 Ca2+ 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.
There are many studies on the role of S100B protein in the central nervous system in the literature, and there are also patents that describe infant supplements containing S100B protein for infant brain development and growth.
Although 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.
In particular, in the intestine it was considered to be an indirect and incidental finding due for example to the release of cells originating from nerves or histiocytes.
The present inventors have instead realised that 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)].
This research has been developed through a bioinformatic approach and an ongoing study involving confirmations through in vitro and in vivo studies in patients and animals.
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.
Several further scientific studies on patients with Crohn's disease had included healthy individuals for comparison, and in particular a recent paper further suggested both the hypothesis of a role for the microbiota and the presence of S100B protein and a role for it in the intestinal mucosa, with experimental data already published (Di Liddo, R. et al., 2020), with both the present inventors as co-authors.
The extensive scientific literature and these two studies strongly support a possible role for S100B protein and the intestinal microbiota in the pathogenesis of the disease, but above all the bioinformatic study has suggested a prospect for a physiological function and an interaction between S100B and some proteins in the intestinal microbiota proteome.
Furthermore, the inventors think that 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.
It has been also noted that S100B hyperproducers (Down’s syndrome), characterised by an accelerated ageing process for several tissues, surprisingly show a youthful and healthy microbiota.
In particular, the inventors have surprisingly found that 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.
Summary of the Invention
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.
It is a further object of the present invention to provide a composition comprising 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 diseases linked to disorders of the immune system, such as allergies, rheumatoid arthritis or autoimmune diseases, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal microbiota.
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.
It is also an object of the present invention to provide a dosage regimen comprising the administration of a composition comprising S100B 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.
It is also an object of the present invention to provide a method for identifying whether a protein of the S100 family or another protein
or peptide is useful as a harmoniser and/or stabiliser and/or modulator and/or regulator of the intestinal microbiota. The compositions and/or formulations of the invention may be prepared according to methods in the known art.
Detailed description of the invention
It is an object of the present invention to provide 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 diseases linked to disorders of the immune system, 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, in humans or animals.
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.
Preferably, 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.
Preferably, 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. Alternatively, 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.
Other proteins of the same family are also commercially available for research or technological purposes.
By “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.
By “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.
By “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.
By “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.
It is an object of the present invention to provide a composition comprising 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 diseases linked to disorders of the immune system, such as allergies, rheumatoid arthritis or autoimmune diseases, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal microbiota.
By “composition” according to the present invention is typically meant a pharmaceutical-type composition, a dietary supplement or a
medical device.
It is therefore an object of the present invention to provide a 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.
Preferably the composition according to the present invention comprises S100B protein and/or its epitope and/or its functional and/or structural domain.
The terms "harmonisation", "stabilisation", "modulation" and "regulation" are to be understood as defined above.
In a preferred aspect, 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.
In fact, the inventors of this patent application found that proteins of the S100 family can act as an enhancer of the activity of prebiotics and/or probiotics.
In a preferred aspect of the invention, 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.
In a preferred aspect of the invention, 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.
Preferably 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:
1) Place a tea infusion (typically 1-5 L) that is slightly sweetened (typically between 2 and 10%, preferably 5%, e.g. sucrose or glucose or fructose,) in 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, add the fruit or plant species containing S100B protein, fresh or dried, cover with gauze to allow the contents to breathe in a clean environment.
2) Allow to ferment for 5-22 days, preferably 10 days, at room temperature, preferably 25°C.
3) Filter and place in bottles and store at 4°C.
Analysis of S100B performed on both the fermented mother and the beverage showed an improvement in the microflora and the presence of S100B.
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.
An assay regime for a protein belonging to the S100 family, specifically S100B protein, is also an object of the present invention.
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.
It is also an object of the present invention to provide a method for identifying whether a protein of the S100 family or another protein or peptide is useful as a harmoniser and/or stabiliser and/or modulator and/or regulator of the intestinal microbiota, including: the comparison of protein levels in faeces before and after treatment with this protein, e.g. by ELISA, Western Blot or other methods, depending on the respective biodiversity indices, such as OTU, Shannon or other parameters; extrapolation of the trend line, where x = concentration of S100B protein preferably in ng/ml and y = biodiversity index, preferably according to Shannon, when it shows a trend (regression equation) of the type y = kx+n, preferably with a gradient of between 5 and 45 degrees, preferably between 10 and 20 degrees, for treatments between 10 and 45 days, where the constant k indicates the rate of change of the biodiversity phenomenon per unit of protein concentration expressed in ng/ml and n is a constant, according to the more general semi-elasticity formula of the type y where k and n can respectively take a value of preferably 0.15, typically between 0.05 and 3.02, and preferably 2.59, typically between 0.09 and 26.41.
The question of the beneficial harmonisation of microbiota as a result of the presence of S100B protein does not in fact exclusively relate to the presence or absence of a particular microbial species, but rather to the relative qualitative and quantitative relationship between the various hundreds of species present, the direction in which this biodiversity changes, and how it remains stable over time. Because of this, treatment proceeds in parallel with evaluation of both the protein and the microbiota, for example after introduction of the protein in purified and/or recombinant form and/or through the in vivo introduction of the DNA and/or RNA gene sequence of S100B and/or through gene vectors containing the genetic information and capable of having it expressed in vivo in host cells and/or through plants and/or plant extracts containing it. 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.
This is based on the assessment of modulation of the S100B protein (e.g. by the ELISA method, or Western blot) and in parallel by measuring the biodiversity of the microbiota through mass sequencing (NGS) or through the quantification of specific indicators by Real Time PCR (RT-PCR).
Preferably, the protein used according to the invention is S100B.
The following examples illustrate, but do not limit, the present invention.
Example 1
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. Differences in the composition of the microbiota were observed following treatment with S100B, showing an improvement in the biodiversity of the intestinal microbiota, and in particular a change in the number and representation of species, as summarised by the increase in biodiversity indices which rose from 1.8- 2.3 to values between 2.6-3.3 from the third week of treatment for the Shannon index and from 0.32-0.42 to 0.48-0.58 for the Equitability index, calculated according to standard procedures known to those
skilled in the art.
This experiment shows that the administration of S100B does not lead to a situation of intestinal dysbiosis associated with the loss of species that are of value for the physiological intestinal balance, or associated with the prevalence and overrepresentation of undesirable species, but rather administration of the protein favoured variability in the number of species and their relative composition in the intestinal microbiota, supporting beneficial harmonisation, modulation and stabilisation.
Example 2
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.
Surprisingly, the values of the biodiversity indices for the microbiota as observed in the different samples analysed are directly proportional to the values of SIOOB. In other words, as S100B increases, there is also a proportional increase in biodiversity. To put it simply, stool samples with an S100B value of more than 2 picograms per millilitre (16 observations) show a higher biodiversity value (Shannon = 2.750±0.8) than those with lower values (20 observations), between 1.9- 0.0 picograms per millilitre and lower biodiversity (Shannon = 1.950±1.3), and do so with high statistical significance (p = 0.0001). In short, 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.
Example 3
In the same manner of application and relative procedures used and reported in Examples 1 and 2, similar results are observed when analysing the faeces of mice treated with drugs that may interfere with
S100B. In particular, faeces were collected from 20 mice, 10 of which were treated and 10 untreated according to standard protocols known to those skilled in the art, by administering a substance known to interfere with S100B production (Arun die Acid). Significant differences in biodiversity values were observed when the faeces of treated mice were examined in comparison with untreated controls, in particular a change in biodiversity indices as reported by Shannon values of 2.354±0.8 and 1.350±0.9 respectively.
This example shows that the action of the protein may also be accomplished by acting on substances that modulate its production or functional activity, ultimately influencing the composition of the microbiota.
Example 4
In order to assess the effect of an increase in S100B in humans, a “natural experiment” was used, seeking data on individuals who genetically overexpress S100B and assessing the presence of dysbiosis and/or beneficial harmonisation and regulation of the intestinal microbiota. The microbiota sequences obtained from the faeces of 17 Down’s syndrome patients who constitutionally produce more S100B protein were analysed, as the gene is present in the minimal region characteristic of the chromosomal aberration characteristic of this chromosomal syndrome (Trisomy 21), making the gene present in 3 copies instead of 2, as in diploid individuals not affected by this genetic disorder. These 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.
Using the Firmicutes/Bacteroides (F/B) ratio as an index for assessing the composition and health status of the microbiota, it was observed that, in addition to the reduction in biodiversity, there is a considerable reduction in F/B ratio in individuals with chronic and degenerative diseases in comparison with samples of the non-diseased population of different ages and the group of individuals with Down’s
syndrome, who instead show a surprisingly favourable F/B ratio corresponding to that of healthy individuals.
In particular, the measured values for the general population with a variety of ages (12 individuals aged 25-70 years) and conditions were in the range of values over 1, similarly to what was observed for a younger population (11 individuals aged 18-36 years); conversely for individuals affected by diseases correlating with changes in the microbiota the value of the F/B ratio was below 1 and ranged between 0.12 and 0.25, in particular for Crohn's disease (6 individuals), ulcerative colitis (5 individuals), multiple sclerosis (5 individuals), obese individuals with metabolic syndrome (28 individuals); surprisingly, for individuals with Down's syndrome the average F/B ratio value was instead over 1, that is similar to healthy individuals and indeed particularly high. Moreover, in the microbiota of Down's syndrome individuals there is a surprising absence of certain risk species such as particular bacteria (including Akkermansia and Dorea), which are instead found at high levels in the intestines of patients with neurodegenerative diseases.
Altogether these observations point to a protective action on the microbiota in individuals who genetically possess an additional copy of the gene coding for S100B protein and who may overexpress it. Although the chromosomal alteration caused by chromosome 21 trisomy leads to a number of changes in the tissues, from the point of view of the intestinal microbiota these individuals nevertheless do not manifest dysbiosis and are in fact more protected, showing high biodiversity typical of the microbiota of healthy individuals and beneficial regulation of the intestinal microbiota. Data on the microbiota sequences and clinical characteristics of the individuals analysed can be obtained from known sources.
Example 5
In silico analysis of functional and/or structural domains in other living beings including bacteria, fungi, animals, plants, with homologues of S100B and thus functional or structural portions similar to S100B (SlOOB-like). In some of these species, subsequent in vitro sampling and analysis were also carried out to identify proteins or their
SlOOB-like portions, performing the analyses by enzyme -linked immunosorbent assay (ELISA) according to standard protocols known to those skilled in the art. In short, sequences of the structural and/or functional domains of S100B were identified, selected and used to search for the presence of similar regions among proteins encoded by the genomes of all living species available in international databases (NCBI and others), using bioinformatics methods known to those skilled in the art.
Through this research numerous living species which therefore contain the genetic information required to be able to produce proteins with similar functions to S100B and which can therefore display such properties in the leaves, fruits or parts of those particular plant species at various stages of their natural development were selected. For some of these species, readily available parts were selected, e.g. leaves, seeds, flowers, and protein extraction and the search for homologues of S100B were also performed by means of enzyme-linked immunosorbent assay (ELISA) according to procedures known to those skilled in the art. For some of these species the presence of SlOOB-like components was confirmed not only in silico, but also in vitro. Among others, one example is species belonging to the genus Salvia, in whose leaves the presence of a SlOOB-like component at values even above 50 ng/ml was found following the ELISA test.
Surprisingly, it is precisely this family of plants, their extracts and derivatives, that have been used for their medicinal properties since antiquity, and indeed the common Salvia officinalis contains principles already known to act on the gastro-intestinal system, while the very etymology of the word Salvia derives from the Latin word salus, meaning “health”.
From the many species selected after searching for relevant profiles in the amino acid sequence of S100B protein and analysis by alignment with all the sequences available from the various microbial, animal and plant species in the databases, the results were filtered and ranked in order to choose those that were most similar and met the requirements of homology with the structural and functional domains of S100B. These included Spinacia oleracea, Artocarpus heterophyllus,
Adansonia digitata, Carpinus fangiana, Durio zibethinus, Herrania umbratical and others such as Salvia splendens, Helianthus annuus, Dendrobium catenatum, Musa acuminata and Olea europaea, and some edible fungi including Laccaria bicolor and Fistulina hepatica, and some marine animal species, including Limulus polyphemus, a species already known for its biotechnological applications.
Example 6
Among the microorganisms found in the microbiota of patients with IBD and which have been associated with inflammatory damage are the Clostridia.
It was found that the value for this species is low in normal samples in comparison with those treated with S100B inhibitors.
In a test performed on 26 mice in the presence and absence of S100B inhibitors, and in particular Arundic Acid and Pentamidine, it was observed that the biodiversity of the microbiota was reduced in the presence of S100B inhibitors. The Shannon index measured in a group of 5 untreated mice fell from 2.6 to 1.85 in the homologous group of 7 mice treated with Pentamidine and Arundic Acid. Furthermore, the same effect was also found in mice subjected to severe experimental inflammatory conditions, such as those used according to the EAE protocol for the induction of experimental autoimmune encephalomyelitis by the prior administration of antigens and adjuvants. The Shannon index in a group of 8 EAE animals rose to 3.24, but was reduced to an average value of 2.90 in a homologous group of 6 mice treated with Pentamidine and Arundic Acid. Inhibition of the natural presence of S100B through the use of chemical inhibitors resulted in a loss of biodiversity, both under physiological conditions and during the inflammatory process.
Example 7
Among the microorganisms found in the microbiota of patients with IBD and which have been associated with inflammatory damage are the Clostridia.
Administration of S100B protein diluted in water (150 microlitres of a 1 mg/ml solution per os for over 14 days) in Mus musculus doubled the amount of S100B present in faeces (from 0.95 to 1.94 ng/ml).
Analysis of the microbiota from faeces taken during week 3 of treatment showed a significant reduction in the presence of sequences (reads) of Clostridium within the composition of the microbiota, and in particular at least a 7-fold reduction in average values in controls given only the water vehicle (7214 reads) in comparison with those treated (1234 reads).
This observation shows how the administration of S100B per os reduces the presence of risk species in the intestinal lumen in intestinal inflammatory processes and in particular IBD, leading to a beneficial effect on IBD pathology. The treatment to synchronise and harmonise the biodiversity of the microbiota also reduced the presence of other undesirable species involved in the pathogenesis of IBD, including Listeria and Bacteroides, in the animals treated, indicating a broader protective, preventive and curative action for IBD through interference with the microbiota.
Example 8
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. In particular, in a trial with two Knock Out mice compared to five identical control mice of the same strain, but genetically unmodified and reared under identical conditions, analysis of the intestinal microbiota showed a decrease in the Shannon index from 2.4 to 1.9, as well as a reduction in the average value of identifiable microbial species with an OTU value that decreased from 380 to 202, indicating a change in biodiversity.
Example 9
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. 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. To this end, S100B was administered to three volunteer individuals and the effects on the microbiota were assessed after a period of time. For administration, 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.
The volunteers took no drugs either before or during the treatment, as well as no supplements or probiotics or prebiotics, and maintained their usual diet and lifestyle.
Analysis of the microbiota before and after treatment and comparison of the data showed a clear overall improvement in Equitability, that is to say the tendency for equal distribution of the
various species in a harmonious, balanced and beneficial manner, with a median value below 0.40 in the pre-treatment samples rising to over 0.45 following the administration of S100B. In addition, the Firmicutes/ Bacteroides ratio in each individual tended to correct itself towards optimal values between 0.8 and 1, increasing if lower and decreasing if higher, as in one case where the treatment raised a rather low F/B of 0.5 to F/B = 0.73, or reduced an excessively high F/B of 1.3 to 0.8, or caused no change either up or down when the F/B value was already close to optimal values, close to F/B=l. Furthermore, the disappearance of potentially pathogenic species associated with the risk of developing IBD, Crohn's Disease and colon cancer, such as Clostridium perfringens, Citrobacter freundii and Fusobacterium nucleatum, was observed in each of the individuals, and if present these were no longer identifiable after treatment with S100B. Together these observations show the efficacy of the protein in beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal microbiota even when the source is of plant origin, and in particular from preparations obtained from Durio zibethinus.
Example 10
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.
Example 11
An intestinal-renal axis has also been described among the various axes through which the microbiota acts on different organs. In fact, 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).
Since it is now well established that any change in the composition and function of the microbiota represents a risk factor and a therapeutic target in the management of these and other chronic- degenerative diseases, some treatments have been directed precisely towards correcting the microbiota and acting on certain receptors including TLRs, which are involved in innate immunity and in maintaining the balance between species present in the microbiota and intestinal mucosa. Surprisingly, in silico analysis of the interactions of S100B domains (PF01023 and PF12202) with other proteins and metaproteomes of the microbiome has revealed associations at precisely this level, including through interaction with inflammation cytokines, including the immunoglobulin domain (PF13895), and interleukins (PF00340), as well as with natural peptides with antimicrobial action such as melittin (PF01372). Furthermore, change in the microbiota with respect to S100B concentration has demonstrated clear functional modulations in hydrolase and catalase activity (p<1-30), including the action of urease, which is capable of generating uraemic toxins, such as p-cresol, which is in fact produced by intestinal bacteria following the catabolism of thyroxine and phenylalanine, which is associated with kidney disease. Administration of S100B in both mice and humans shows modulation and harmonisation of biodiversity in both OTUs and precisely in these species targeted by current probiotic or prebiotic therapies, including changes of around 20% in OTUs and 60% for some of the main species of the microbiota implicated in these diseases, including Prevotella and Lactobacilli. The presence of S100B protein in the gut lumen also modifies these risk factors for kidney disease, favourably and beneficially modulating the biodiversity of the microbiota and also reducing the risk conditions for the gut-kidney axis.
Example 12
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. 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. Furthermore, in another similar construct, 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. This was in order to be able to obtain not only the purifiable recombinant protein, but also an organism capable of producing it in its natural, readily available form. 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. In conclusion, 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). In this way, both 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.
Example 13
Another fruit found to be positive for S100B is Giaca, from a plant belonging to the Rosidae species, Artocarpus heterophy llus.
Surprisingly, this plant has also been associated with health benefits and a beneficial effect on the microbiota. 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.
Example 14
To test for the presence of S100B in plant species, fresh fragments of plant, fruit, leaves, flowers and seeds were sampled, the total protein fraction was extracted, and the presence of S100B was verified using antibodies, essentially by ELISA. The protein extract was obtained using lysis solutions containing both detergents and also protease inhibitors, according to known methods. For example, for Salvia (Example 5), use was made of fragments of leaf and flower; for Durio zibethinus (Example 9), fragments of various parts of the fresh fruit, seed, leaf, peel, but also freeze-dried and sun-dried preparations, or preparations obtained by the sublimation technique following dehydration after freezing; for the Giaca fruit (Example 13) of the plant Artocarpus heterophyllus, use was made of dried and powdered or sublimated products. It emerged that the materials best containing antibody-recognisable and therefore biologically functional S100B protein domains are fresh products that are either adequately cooled and vacuum-preserved or obtained after sublimation. Furthermore, not all parts of the plant contained 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. In 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.
In preparations obtained by drying another plant belonging, like Durio, to the Rosidae clade and the Malvaceae family, namely Adansoma digitata or Baobab, concentrations of around 65 ng/ml were observed.
In the Giaca fruit, also belonging to the Rosidae clade, the protein was also observed in amounts exceeding 4 ng/ml in preparations obtained by freeze-drying as well as by sublimation.
It is therefore important that not just any part of the plant is used, but specifically that part that contains S100B protein, and in particular epitopes and/or functional or structural domains thereof that are adequately conserved, and that are neither degraded nor altered by production, extraction or storage processes.
REFERENCES
Di Liddo et al. S100B as a new faecal biomarker of inflammatory bowel diseases. Eur Rev Med Pharmacol Sci. 2020;24(l):323.
Orsini, et al. In Silico Evaluation of Putative S100B Interacting Proteins in Healthy and IBD Intestinal microbiota. Cells. 2020; 9(7):1697.
Claims
1. 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 diseases linked to disorders of the immune system 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, in humans or animals.
2. Protein according to claim 1, preferably selected from the group consisting of 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 for use in the treatment according to claim 1.
3. Protein according to claim 1 or 2, in which the protein is S100B protein and/or its epitope and/or afunctional and/or structural domain thereof for use in the treatment according to claim 1.
4. S100B protein according to claims 1, 2 or 3, for use in the treatment of disorders of the intestinal microbiota.
5. S100B protein according to claim 4, where the disorder of the intestinal microbiota is associated with Crohn's disease or IBD.
6. Protein for use in the treatment according to any of claims 1 — 5, in which S100B protein may be either the pure protein or the protein obtained from extracts or through recombinant methods or through genetic engineering techniques, preferably gene therapy techniques or gene vectors enabling its expression in vivo, for example in the intestinal mucosa, and/or through in vivo introduction of the DNA and/or RNA
gene sequence of S100B, or S100B protein obtained from leaves of plants or from the fruits of specific plants or from fungi or marine animal/plant organisms as revealed by bioinformatic analysis, selected 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.
7. Composition comprising 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 diseases linked to disorders of the immune system, by beneficially harmonising and/or stabilising and/or modulating and/or regulating the intestinal microbiota.
8. Composition comprising a protein belonging to the S100 family, in particular S100B and/or its epitope and/or a functional and/or structural domain thereof, where the composition is of the type of a pharmaceutical, a dietary supplement, a medical device.
9. Composition according to claim 7, comprising S100B protein and/or its epitope and/or a functional and/or structural domain thereof.
10. Composition according to either of claims 7 or 9, comprising a protein of the S100 family and at least one physiologically acceptable excipient, and in addition a prebiotic and/or probiotic.
11. Composition according to claim 10, in which the at least one pharmaceutically acceptable excipient is selected from the group comprising: diluents, binders, colouring agents, sweeteners, disintegrating agents, controlled release compounds, waxes, oils, paraffins, talc, titanium dioxide, cellulose for use in the treatment according to claim 7.
12. Composition according to claims 7, 9 - 11, in a form selected from the group comprising preferably 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-quenching beverages, energy drinks, fruit juices, or milk and milk derivatives, including those fermented such as for example yoghurt or the like, or other ferments or fermented products for use in the treatment according to claim 7.
13. Composition according to claim 12, in which the ferment or fermented product comprises a protein of the S100 family, in particular S100B protein, for use in the treatment according to claim 7.
14. Device according to claim 8, comprising a protein belonging to the S100 family, in particular S100B, and the most common physiologically acceptable excipients.
15. Composition according to claims 7, 9 - 13, comprising a protein belonging to the S100 family, in particular S100B protein, said composition comprising in particular:
S100B protein in an amount by weight of 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, for use in the treatment according to claim 7.
16. 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.
17. Dosing regimen comprising the administration of a composition including S100B in tablet form 1-2 times daily at least 5-8 hours apart, after meals, for a period of at least 10-30 days.
18. Method for identifying whether a protein of the S100 family or another protein or peptide is useful as a harmoniser and/or stabiliser and/or modulator and/or regulator of the intestinal microbiota, comprising: the comparison of protein levels in faeces before and after treatment with said protein, for example by ELISA, Western Blot or other methods, depending on the respective biodiversity indices, such as OTU, Shannon or other parameters; extrapolation of the trend line, where x = concentration of S100B protein preferably in ng/ml and y = biodiversity index, preferably according to Shannon, when it shows a trend (regression equation) of the type y = kx+n with a gradient of between 5 and 45 degrees, preferably between 10 and 20 degrees, for treatments of between 10 and 45 days, where constant k indicates the rate of change of the biodiversity phenomenon per unit of protein concentration expressed in ng/ml and n is a constant, according to the more general semi-elasticity formula of the type y = ene(kx), where k and n can respectively take a value of preferably 0.15, typically between 0.05 and 3.02, and preferably 2.59, typically between 0.09 and 26.41.
19. Protein according to claim 2 for use in the treatment according to claim 1 by harmonising and/or stabilising and/or modulating and/or regulating a human or animal microbiota, in which the protein is S100B protein.
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