Use of bacterioruberins and their glycosylated derivatives to prevent and treat diseases involving deregulation of protein aggregation, such as neurodegenerative diseases

Abstract

Use of bacterioruberins and their glycosylated derivatives for preventing and treating diseases involving deregulation of protein aggregation, such as neurodegenerative diseases The invention relates to a composition comprising at least one bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it for use in a method of treating or preventing a disease involving deregulation in protein aggregation, such as for example a degenerative disease, advantageously a neurodegenerative disease, fibrosis, advantageously pulmonary fibrosis, or diabetes.The invention also relates to a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in a method of treating or preventing a disease presenting a deregulation in the aggregation of proteins, and a method of treating or preventing a degenerative disease. Figure for the abstract: NONE.

Description

Title of the invention: Use of bacterioruberins and their glycosylated derivatives for preventing and treating diseases involving deregulation of protein aggregation, such as neurodegenerative diseases Field of invention

[0001] The present invention relates to a composition comprising at least one bacterio-ruberin and / or at least one glycosylated bacterioruberin for the treatment or prevention of a disease involving a deregulation of protein aggregation, such as degenerative diseases, advantageously neurodegenerative, in particular a disease chosen from Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy or amyotrophic lateral sclerosis (ALS) as well as ocular neurodegenerative diseases chosen from macular degeneration, retinitis pigmentosa and retinopathy. State of the art

[0002] Degenerative diseases, and in particular neurodegenerative diseases such as Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy or amyotrophic lateral sclerosis as well as ocular neurodegenerative diseases, are chronic, disabling pathologies with slow progression. They generally cause a deterioration in the functioning of nerve cells, in particular neurons, which can lead to cell death or neurodegeneration. The disorders induced by neurodegenerative diseases are varied and can be cognitive-behavioral, sensory and motor (Dugger et al. 2017).

[0003] It is difficult to gauge the overall impact of neurodegenerative diseases on the global human population; the World Health Organization (WHO) estimates that up to one billion human beings could be affected if we consider all the manifestations of these conditions, the boundaries of which are sometimes blurred; these figures are likely to increase in view of the increasing aging of the population in developed and developing countries.

[0004] As research progresses, many similarities are emerging linking these diseases to each other, particularly at the cellular level through the aggregation of atypical or unfolded proteins and induced neuronal death. The discovery of these similarities offers hope for therapeutic advances that could simultaneously improve many diseases, particularly by acting on the mechanisms of intracellular protein aggregation in neurons.

[0005] Carotenoids are highly conjugated linear isoprenoid compounds responsible for the majority of the yellow, orange, and red pigmentation observed in organisms on Earth (Armstrong, 1997). Carotenoid biosynthesis occurs in all living things, except animals in which carotenoids are introduced through the diet (Britton, 1995). Although approximately 1,000 different carotenoids have been identified in nature and they exhibit highly varied structural features, all known carotenoids share a lipophilic linear conjugated backbone, obtained by passing through highly conserved biosynthetic pathways (Britton, 2004). Carotenoids are synthesized from the linear condensation of isoprene units, derived from primary metabolism (Armstrong, 1994).Covalent modifications at each end of the chain give rise to the observed structural diversity of known carotenoids (Armstrong, 1997). Desaturation of the chains generates the chromophore, characteristic of carotenoids, which results in a region of easily excitable delocalized electrons; these properties underlie two fundamental characteristics common to all carotenoids, namely their photochemical properties and their antioxidant action (Britton, 1995).

[0006] The term carotenoid groups together molecules from the carotene and xanthophyll families.

[0007] Among the carotenoids with the greatest antioxidant potential, bacterioruberins, tetrahydroxylated carotenes with 50 carbon atoms, should be mentioned. Bacterioruberins and their derivatives are found in extremophile bacteria, including halophilic archaea and some psychrophilic actinobacteria; in these microorganisms, they play an important role in the protection of DNA and membranes against solar irradiation as well as the thermal and osmotic environmental stresses that these organisms are constantly confronted with (Mandelli et al. 2012). In particular, these carotenes are found in the psychrophilic actinobacterium Arthrobacter (Micrococcus) agilis. This bacterium is also capable of synthesizing glycosylated forms of bacterioruberins, i.e., whose terminal hydroxyl groups are substituted with sugars (Fong et al. 2001).

[0008] It is known that several carotenoids can help prevent, slow down or treat neurodegenerative diseases. Thus, patent application WO2014 / 155189 discloses the use of several xanthophylls, including lutein and zeaxanthin for the treatment and prevention of PD and PALS.

[0009] Application WO2008 / 038119 discloses the treatment of PD by a composition containing: (a) a coenzyme Q10 complex and at least one cyclodextrin; and (b) at least one carotenoid, in particular a carotene chosen from α-carotene, [3-carotene and lycopene.

[0010] It is also known that glycosylated carotenoids can be useful in the treatment and prevention of neurodegenerative diseases: for example, a neuroprotective action has been associated with crocin, a glycosylated carotenoid responsible for the yellow color of saffron (Farkhondeh et al. 2018).

[0011] Despite the usefulness of carotenoids already used in the prevention of neurodegenerative diseases, there remains a clear need for new remedies capable of more effectively countering the emergence of these pathologies. Aims of the invention

[0012] The invention aims to solve the technical problem of providing a compound or composition having chaperone activity, i.e. having the capacity to combat the denaturation and aggregation of proteins, thus protecting cellular proteins.

[0013] Thus, the invention also aims to solve the technical problem of providing a compound or composition protecting at least one intracellular or extracellular protein from both oxidative stress and denaturation.

[0014] The invention aims to solve the technical problem of providing a compound or composition useful in the treatment and prevention of a disease exhibiting deregulation in protein aggregation, such as, for example, degenerative diseases. Description of the invention

[0015] Surprisingly, the Applicant has discovered that a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising them, has a chaperone activity, thus making them useful in the treatment and prevention of diseases involving deregulation in the aggregation of proteins, such as for example a degenerative disease, advantageously a neurodegenerative disease, fibrosis, advantageously pulmonary fibrosis, or diabetes. Among the neurodegenerative diseases, mention may be made in particular of neurodegenerative diseases characterized by the accumulation of protein aggregates in neurons, such as PALS and PD. In the experimental part, it is shown that bacterioruberins, and even more so glycosylated bacterioruberins, make it possible to stabilize proteins and slow down their inactivation / denaturation.The chaperone effect of these molecules can play an important role in the treatment of these pathologies, by protecting neurons.

[0016] The present invention also relates to a composition comprising a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in the treatment or prevention of a disease involving a deregulation in the aggregation of proteins, such as for example a degenerative disease, advantageously a neurodegenerative disease, fibrosis, advantageously pulmonary fibrosis, or diabetes.

[0017] The invention relates in particular to a composition comprising a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in the treatment or prevention of a disease involving deregulation in the aggregation of proteins by reducing the formation of toxic protein aggregates, and in particular in neurons.

[0018] The invention relates in particular to a composition comprising a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in the treatment or prevention of a disease involving deregulation in the aggregation of proteins by reducing the denaturation of proteins.

[0019] Thus the present invention relates to a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in the treatment or prevention of a degenerative disease, advantageously of a neurodegenerative disease.

[0020] The present invention also relates to a composition comprising a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in the treatment or prevention of a degenerative disease, advantageously of a neurodegenerative disease.

[0021] The present invention also relates to a method for treating or preventing a degenerative disease, advantageously a neurodegenerative disease in which a composition comprising at least one bacterioruberin and / or at least one glycosylated bacterioruberin is administered to a subject in need thereof.

[0022] Typically, the neurodegenerative disease is chosen from Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy or amyotrophic lateral sclerosis (ALS) as well as ocular neurodegenerative diseases chosen from macular degeneration, retinitis pigmentosa and retinopathy, advantageously for the treatment of Alzheimer's disease (ALS), Parkinson's disease (PD).

[0023] The compositions according to the invention may also be useful for treating other pathologies presenting a deregulation in the aggregation of proteins, such as for example, fibrosis, advantageously pulmonary fibrosis, or diabetes.

[0024] Bacterioruberin (CAS No. 32719-43-0), also known as “a-Bacterioruberin”.

[0025] “-Bacterioruberin” has the following structure:

[0026] [Chem.l] OH

[0027] α-Bacterioruberin comprises 4 terminal hydroxyl groups, each of which may be substituted by ether bond with a sugar group, or even one or more covalently bonded sugars. By "glycosylated form of bacterioruberin" or "glycosylated bacterioruberin" is meant a bacterioruberin of which at least one hydroxyl group is substituted with one or more, for example two or three, sugar residues by means of an ether bond between the backbone of bacterioruberin and the sugar.

[0028] An “isolated glycosylated bacterioruberin” according to the invention is obtained by synthesis by biotechnology, by chemical synthesis, typically followed by purification, or, alternatively by purification of a glycosylated bacterioruberin naturally contained in a natural bacterium.

[0029] For example, a “glycosylated bacterioruberin” according to the invention has the following structure:

[0030] [Chem.2] GOLD GOLD

[0031] in which R is independently selected from a hydrogen atom, one or more, for example two or even three, sugar residues and where R at least one occurrence represents one or more, for example two or even three sugar residues.

[0032] In a preferred embodiment, the sugar is a hexose or a deoxyhexose. selected from the group consisting of allose, altrose, glucose, mannose, gulose, idose, galactose, fucose, fructose, fucose.

[0033] In a preferred embodiment, a composition according to the invention comprises at least one glycosylated bacterioruberin chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, decaglycosylated bacterioruberins, undecaglycosylated bacterioruberins, and do-decaglycosylated bacterioruberins. Advantageously, it is at least one glycosylated bacterioruberin chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, and tetraglycosylated bacterioruberins.

[0034] Advantageously, said composition comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins and tetraglycosylated bacterioruberins; and preferably a mixture of monoglycosylated bacterioruberins and diglycosylated bacterioruberins. In a preferred embodiment, the composition according to the invention is essentially free of non-glycosylated forms of bacterioruberin.

[0035] Advantageously, the total extract of carotenoids containing the glycosylated bacterioruberins according to the invention is a bacterial extract, preferably of Actinobacterium, even more advantageously of the Micrococcoccaceae family. Advantageously, these are the species Micrococcus roseus and Arthobacter agilis.

[0036] Glycosylated bacterioruberins can be obtained by extraction and purification, for example by chromatography, of total extracts of carotenoids of actinobacterium of the genera Micrococcus or Arthrobacter, advantageously the species A. agilis and / or M. roseus. The species A. agilis is also known under the name Micrococcus agilis. Thus the extracts and strains described in the publications Strand et al. 1997, Fong et al. 2001 and in the patent application WO 2014 / 167247 can be used as a source of glycosylated bacterioruberins. Preferably, the strains of A. agilis strains used as sources of glycosylated bacterioruberins within the meaning of the invention are strain MB813 (described in Fong et al. 2001) and / or SB5 (described in patent application WO 2014 / 167247). Methods for obtaining total carotenoid extracts from these bacterial species are known to those skilled in the art and are, for example, described in Strand et al. 1997, Fong et al.2001 as well as patent application WO 2014 / 167247. However, these methods do not allow the isolation of the different glycosylated bacterioruberins.

[0037] Surprisingly, the Applicant has developed a method for efficiently isolating the glycosylated forms of bacterioruberin from an extract of carotenoids from A. agilis. The present invention describes a method for purifying and isolate bacterioruberin and its glycosylated forms.

[0038] Thus, the present invention also relates to isolated bacterioruberin and / or an isolated glycosylated bacterioruberin, as well as mixtures thereof, in particular for the uses and applications described in the present invention.

[0039] In other words, the invention covers a separated and purified glycosylated bacterioruberin, in particular from an extract of extremophile bacteria, and preferably of Arthrobacter agilis, for its use in a method of treatment or prevention of a degenerative disease, advantageously of a neurodegenerative disease.

[0040] In a preferred embodiment, a total extract of carotenoids containing the glycosylated bacterioruberins according to the invention corresponds to the carotenoids contained in the raw material MIRORUBERINE marketed by the company GREENTECH and corresponding to the INCI designation Micrococcus lysate. Alternatively, the glycosylated bacterioruberins according to the invention can be obtained by biotechnology, or by chemical synthesis, for example by means of controlled glycosylation of the native forms of bacterioruberins, typically of α-bacterioruberins, for example starting from α-bacterioruberin. This glycosylation can be obtained chemically or by biotechnology, preferably by biotechnology using suitable glycosyltransferases.For example, the raw material HA-LORUBIN marketed by the company HALOTEK GMBH can be used as a source of α-bacterioruberin in the synthesis of glycosylated bacterioruberins within the meaning of the invention.

[0041] Advantageously, a composition according to the invention comprises α-bacterioruberin. α-bacterioruberin can be obtained by extraction from the aforementioned actinobacteria, which also synthesize glycosylated forms of bacterioruberin. Alternatively, α-bacterioruberin can be extracted from cultures of one or more halophilic archaea, such as, for example, the species Halobacterium salinarum, Halorubrum sodomense, Haloarcula valismortis, Salinibacter ruber. Thus, the raw material HALORUBINE marketed by the company HALOTEK and corresponding to the INCI designation Halobacterium salinarum carotenoides can be used in the compositions according to the invention.

[0042] In an alternative embodiment, a composition according to the invention comprises at least one bacterioruberin and one glycosylated bacterioruberin. In other words, this composition comprises a mixture of glycosylated forms of bacterioruberin, and non-glycosylated forms, and advantageously a mixture of α-bacterioruberin, monoglycosylated bacterioruberins, diglycosylated bacterioruberins. Advantageously, the ratio between non-glycosylated forms and glycosylated forms is between 2 / 1 and 1 / 2.

[0043] In one embodiment, a composition according to the invention comprises one or several glycosylated bacterioruberins and substantially does not include a non-glycosylated form of bacterioruberin. By "substantially does not include a non-glycosylated form of bacterioruberin" or "substantially free of non-glycosylated forms of bacterioruberin" is meant that the non-glycosylated form of bacterioruberin is sought to be avoided and eliminated, but may be present in trace amounts. Preferably such trace amounts are not detectable by analysis.

[0044] Advantageously, a composition according to the invention comprises a mixture of monoglycosylated bacterioruberins, diglycosylated bacterioruberins, tetraglycosylated bacterioruberins; and preferably a mixture of glycosylated bacterioruberins essentially consisting of monoglycosylated bacterioruberins and diglycosylated bacterioruberins, and said mixture preferably comprising 20 to 80% by mass of monoglycosylated bacterioruberins and 20 to 80% by mass of diglycosylated bacterioruberins relative to the total mass of the mixture of glycosylated bacterioruberins.

[0045] The pharmaceutical compositions comprising at least one bacterioruberin and / or one glycosylated bacterioruberin according to the invention are generally in dosed form. Thus, the composition comprising at least one bacterioruberin and / or one glycosylated bacterioruberin may be in the form of a tablet, dragee, capsule, suppository, injectable or oral solution, or even drop and it is suitable for administration by oral, oromucosal, rectal, vaginal, intramuscular, parenteral or ophthalmic route.

[0046] Among the pharmaceutical compositions according to the invention, mention will be made more particularly of those which are suitable for oral, oromucosal, parenteral (intravenous, intramuscular or subcutaneous), per or transcutaneous, intravaginal, rectal, nasal, perlingual, buccal, ocular or respiratory administration.

[0047] The pharmaceutical compositions according to the invention for parenteral injections include in particular sterile aqueous and non-aqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for the reconstitution of injectable solutions or dispersions.

[0048] The pharmaceutical compositions according to the invention, for solid oral administrations, include in particular simple or coated tablets, sublingual tablets, sachets, capsules, granules, and for oral, nasal, buccal or ocular liquid administrations, include in particular emulsions, solutions, suspensions, drops, syrups and aerosols.

[0049] Pharmaceutical compositions for rectal or vaginal administration are preferably suppositories or ovules, and those for per or transcutaneous administration include in particular powders, aerosols, creams, ointments, gels and patches.

[0050] The pharmaceutical compositions cited above illustrate the invention but do not limit it in any way.

[0051] Among the inert, non-toxic, human-acceptable or pharmaceutically acceptable excipients or vehicles, mention may be made, by way of indication and without limitation, of diluents, solvents, preservatives, wetting agents, emulsifiers, dispersing agents, binders, swelling agents, disintegrating agents, retardants, lubricants, absorbents, suspending agents, colorants, flavorings, etc.

[0052] The useful dosage varies according to the age and weight of the patient, the route of administration, the pharmaceutical composition used, the nature and severity of the condition. For example, the composition according to the invention can be administered once a month, week or day and it can contain from 1 mg to 1 g of glycosylated bacterioruberins and / or non-glycosylated bacterioruberins or any of their mixtures.

[0053] The glycosylated bacterioruberins according to the invention are suitable for use in food supplements and nutraceuticals. The methods for formulating the food supplements are known to those skilled in the art. Advantageously, the food supplements are in the form of a tablet or capsule. Each dose may contain, for example, from 1 mg to 1 g of glycosylated bacterioruberins and / or non-glycosylated bacterioruberins and any of their mixtures.

[0054] In a tablet, microcrystalline cellulose is for example used as a bulking agent. It is used from 10 to 30% by weight relative to the total weight of the food supplement, more advantageously around 20% by weight.

[0055] Dicalcium phosphate and tricalcium phosphate are used as compressing agents to prepare tablets. Dicalcium phosphate is used from 10 to 30% by weight relative to the total weight of the food supplement, more preferably around 15% by weight. Tricalcium phosphate is used in an amount ranging from 2.5 to 7.5% by weight relative to the total weight of the food supplement, and more preferably around 5% by weight.

[0056] Hydrated silica, magnesium stearate and colloidal silica can advantageously be used as fluidizers in the food supplement in the form of tablets or capsules. They are introduced in an amount of around 2% by weight, 1% by weight and 0.6% by weight relative to the total weight of the food supplement, respectively.

[0057] Other adjuvants, such as flavorings (natural or chemical flavorings, fruit or other) or pigments are advantageously incorporated into the preparation of the food supplement.

[0058] When the food supplement is in the form of a soft capsule or gelatin capsule, the shell of these soft capsules or gelatin capsules may contain in particular animal gelatin such as fish gelatin, glycerin, or a material of plant origin such as a cellulose or starch derivative, or a plant protein. In a preferred embodiment, one or more glycosylated bacterioruberins according to the invention incorporated in the capsules may be solubilized in a fatty substance, advantageously caprylic and / or capric triglyceride, and preferably stabilized by tocopherol. Thus, a food grade of the raw material MIRO-RUBERIN marketed by the company GREENTECH and corresponding to the INCI designations caprylic / capric triglyceride & tocopherol & Micrococcus lysate may be used in the food supplements according to the invention.

[0059] The manner in which the invention can be implemented and the advantages which result therefrom will become more apparent from the following examples of implementation, given for informational and non-limiting purposes, with the support of the appended figures.

[0060] [fig. 1] [fig.l] shows the composition of a carotenoid extract from the SB5 isolate of the species A. agilis.: BR= a-Bacterioruberin, BR-MonoG: monoglycosylated form, BR-DiG: di-glycosylated form, BR-DiG2: Other form of BR-DiG, BR-TetraG: tetraglycosylated form.

[0061] [fig.2] [fig.2] shows the percentage of heat protection of an extract total carotenoids from A. agilis (Snow bacteria extract -> SBE), bacterioruberin (BR), monoglycosylated bacterioruberins (BR-MonoG) and diglycosylated bacterioruberins (BR-DiG).

[0062] [fig.3] [fig.3] shows the percentage of protection against oxidative stress of a total carotenoid extract of A. agilis (Snow bacteria extract -> SBE), bacterioruberin (BR), monoglycosylated bacterioruberins (BR-MonoG) and diglycosylated bacterioruberins (BR-DiG).

[0063] [fig.4], [fig.5] Figures 4 and 5 respectively represent the results on a neurite network ([fig.4]) and Tau hyperphosphorylation ([fig.5]) of glutamate-injured cortical neurons and the protection conferred by the neurotrophin BDNF and A. agilis carotenoids (SBE). Results are expressed as a percentage of the control condition as mean + / - standard error (n = 4-6). Statistical treatment: One-way ANOVA followed by Fisher's Least Significant Difference (LSD) test. * = p < 0.05 was considered significant. Examples of achievements

[0064] Example I - Purification of glycosylated bacterioruberins from a carotenoid extract of the bacterium A. agilis I -1 Aim of the study

[0065] The aim of this study is to isolate and quantify the molecules contained in an extract of total carotenoids from the bacterium A. agilis. 1-2 Materials and methods

[0066] 1-2.1 Extract

[0067] The total carotenoid extract of the bacterium A. agilis contained in the GREENTECH raw material called “Miroruberine” and corresponding to the INCI designation Micrococcus lysate was used in this study, derived from the strain SB5. This extract called “SBE” can be obtained using the method described in the publication of the patent application WO2014167247.

[0068] 1-2.2 Column and thin layer chromatography

[0069] The SBE extract was taken up in tetrahydrofuran (THF) until completely solubilized. A step of separation of the glycosylated Bacterioruberins by silica gel chromatography was carried out in a glass column after dilution of the SBE in THF.

[0070] 1. Suspension of silica gel in a DCM / methanol mixture (10 / 1) before being cast into the column

[0071] 2. After sedimentation of the silica gel, 1 cm of sand is added before carrying out 3 washes with the DCM / methanol mixture

[0072] 3. 0.5 ml of SBE diluted in THF is placed on the sand and left there for 5 minutes

[0073] 4. 50 ml of DCM / methanol mixture (10 / 1) are added gradually allowing to collect fractions 1, 2, 3 and 4 separately

[0074] 5. 40 ml of DCM / methanol mixture (8 / 2) are added gradually allowing the separate collection of fractions 5 and 6

[0075] 6. 40 ml of DCM / methanol mixture (5 / 5) are added gradually allowing the collection of fraction 7

[0076] 7. 40 ml of DCM / methanol mixture (3 / 7) are added gradually to allow the collection of fraction 8

[0077] 8. All fractions are then compared by TLC (DCM / methanol (10 / 1)) with the SBE and quantified by absorption (using the maximum absorption of each fraction).

[0078] 1-2.3 Separation of fractions by HPLC

[0079] Equipment: Nexera XR, binary pump (Shimadzu)

[0080] Column: Cl8; Intersustainable Swift 5pm 4.6 x 150 mm Producer: GL Sciences

[0081] Mobile phases:

[0082] A: 20% H2O in MeOH

[0083] B: 20% EtOAc in MeOH

[0084] Flow: 1.5 ml / min

[0085] Injection volume: 50 pL

[0086] [Tables 1] AB 1 min 100 0 20 min 0 100 1-3 Results and Discussion

[0087] In this purification, the first step of separation by column chromatography made it possible to collect the various fractions whose purity was confirmed by TLC and by HPLC-DAD, by comparing it to the absorbance spectrum of the native extract; the quantification of the different forms was carried out by UV absorption at 500nm.

[0088] Each of the molecules in each of the fractions collected by chromatography was identified by Maldi-TOF-TOF spectroscopy using an AUTOFLEX device (Brucker). The method used is "CHCA and DHB Matrix without TFA in reflector acquisition". The distribution between the different forms was calculated by combining the results obtained with the quantifications carried out on these fractions by HPLC-DAD ([fig.l]). Fraction 1 corresponds to beta-carotene, a secondary product of the synthesis of bacterioruberin, but this molecule represents only 0.79% of the extract. Fraction 4 presents the same profile of Halobacter salinarium extract (Halorubin) whose major molecule is bacterioruberin (BR). This molecule represents approximately half of the dry extract ([fig.l]).

[0089] Two diglycosylated forms, migrating separately (BR-DiGl and BR-DiG2) were identified. The diglycosylated forms represent >22% of the extract.

[0090] The monoglycosylated form BR-MonoG represents >26% of the extract. The tetra-glycosylated form (BR-TetraG) represents only 0.01% of the extract ([fig.l]).

[0091] Example II - Protection and stabilization of proteins by an extract of carotenoids from A. agilis as well as the isolated bacterioruberins and glycosylated forms composing it II-1 Aim of the study

[0092] The aim of this study is to compare the protein protection capacities of the different components of a carotenoid extract of the actinobacterium A. agilis separated by chromatography and HPLC. More specifically, we tested all the major fractions to evaluate their capacity to protect the alkaline phosphatase enzyme:

[0093] - Against denaturation via their effect of protecting proteins from denaturation (AP-Heat test) (chaperone effect)

[0094] - Against oxidation (APox test) (Protective effect against oxidative stress). II-2 Materials and methods

[0095] II-2.1 Samples to be tested

[0096] [Tables2] SBE SBE = Snow bacteria extract; Total extract of carotenoids from A. agilis; this extract the raw material corresponding to the INCI designation î Micrococcus lysate (GREENTECH); the extraction method described in patent application WO2014-A-16727 BR a-bacterioruberin extracted from the GREENTECH raw material corresponding to the INCI designation Micrococcus lysate (GREENTECH) isolated and purified according to example I BR-MonoG monoglycosylated bacterioruberin extracted from the GREENTECH raw material corresponding to the INCI designation Micrococcus lysate (GREENTECH) isolated and purified according to example I BR-DiG diglycosylated bacterioruberin extracted from the GREENTECH raw material corresponding to the INCI designation Micrococcus lysate (GREENTECH) isolated and purified according to example I

[0097] Four doses were tested for SBE, BR, BR-MonoG and BR-DiG: 20 pM, 10 pM, 5 pM, 2.5 pM, 1.25 pM

[0098] II-2.2 APox and AP-Heat test

[0099] APox test and protocol:

[0100] This test is described in patent application FR3002544-A1- and measures the ability of a substance to protect the alkaline phosphatase enzyme from oxidative stress.

[0101] Materials required:

[0102] - Bovine alkaline phosphatase (PA) (Sigma P0114)

[0103] - PA liquid substrate (Sigma P7998)

[0104] - 30% hydrogen peroxide

[0105] - FeO4S solution (30mg in 1ml of H2O)

[0106] - 96-well flat-bottom plate

[0107] - 405nm plate reader

[0108] In each well, the following are deposited:

[0109] - 10 pl of PA diluted to 10-5 in MgSO4 10 2M.

[0110] - 4 pl of molecule to be tested, solvent (negative control) or H2O (positive control) +6 pL of H2O

[0111] - 30 µl of hydrogen peroxide solution (from a stock solution composed of 940pl H2O + 40pl H2O2 30% + 20 pl FeO4S 10 2) or 30pl of H2O (positive control)

[0112] Incubate for 15 min at 37°C.

[0113] Add 50 μl of liquid substrate.

[0114] Reading of DO at 405nm for 20 min at 37°C.

[0115] AP-Heat test and protocol:

[0116] This test is a modification of the APox test, to measure the potential for protecting proteins not against oxidative stress but against denaturation (thermal stress). Indeed, under the effect of heat, proteins denature and enzymes lose their activity. By adjusting the temperature and incubation time, it is possible to determine the conditions necessary to inhibit 90% of the activity of alkaline phosphatase (55°C for 1 hour).

[0117] Materials required:

[0118] - Bovine alkaline phosphatase (PA) (Sigma P0114) - Liquid alkaline phosphatase substrate (Sigma P7998) - Heating block - 96-well flat-bottom plate - 405nm plate reader

[0119] In each well, we deposit:

[0120] - 100L of PA diluted to 105 in 10 2M MgSO4 - 4pL of molecule to be tested or solvent alone + 6pL of H2O

[0121] Incubate for 15 min with shaking at 37°C.

[0122] Incubate for 1 hour at 37°C or 55°C on a heating block.

[0123] Add 50pL of liquid substrate.

[0124] Reading of DO at 405nm for 20min at 37°C.

[0125] The protective power of a molecule called X against denaturation (PPX) is calculated by taking the ratio of alkaline phosphatase (AP) activity at 55°C (stressed condition) to its activity at 37°C (basal condition) in the presence of the molecule. This ratio is then normalized using the same ratio but obtained in the presence of the solvent alone.

[0126] Schematically, the calculation is as follows:

[0127] PPX = (APA

[0128] With:

[0129] APAX55: PA activity in the presence of molecule X at 55°C

[0130] APAX37: PA activity in the presence of molecule X at 37°C

[0131] APAS55: PA activity in the presence of solvent at 55°C

[0132] APAS37: PA activity in the presence of solvent at 37°C

[0133] And considering that:

[0134] - APAX37 corresponds to 100% enzyme protection - APAX37 x APAs55 / APAs37 corresponds to zero protection.

[0135] The activity of the enzyme for each condition is calculated by taking the average of the optical density values ​​measured at 405nm for the replicates, values ​​from which the average value of the so-called “blank” wells (reagents alone) is subtracted, i.e.:

[0136] APA= [(DO replicate 1 - DO blank) + (DO replicate 2 - DO blank) + (DO replicate 3 - DO blank)] / 3

[0137] These calculations can only be applied with DO values ​​located in the linear part of the curve, generally between 0.15 and 1.5.

[0138] All enzyme activity measurements were performed on an EnSight - Perkin Elmer 96-well plate reader. II-3 Results and discussion

[0139] The results of the so-called "AP-heat" test are shown in [fig.2]. The glycosylated forms of bacterioruberin (BR-MonoG, BR-DiG) as well as the SBE extract, which corresponds to a mixture of a-bacterioruberin (BR) and glycosylated forms of this carotenoid, in particular the diglycosylated form (BR-DiG), protect the protein more effectively than BR itself. This effect is found consistently at all concentrations tested.

[0140] The results of the APOX test are shown in [fig.3]. These results show that the glycosylated forms have a higher protective potential than the non-glycosylated BR and that this effect is in perfect agreement with the chaperone effect. In this case again the protection of the protein from oxidation is particularly pronounced for the diglycosylated form of bacterioruberin (BR-DiG) as well as the total SBE extract. This effect is found consistently at all concentrations tested.

[0141] These results indicate that bacterioruberin (BR) but especially the glycosylated forms of bacterioruberin (in particular BR-MonoG, BR-DiG), as well as the SBE extract, which corresponds to a mixture of a-bacterioruberin (BR) and glycosylated forms of this carotenoid, and in particular the diglycosylated form (BR-DiG), make it possible to protect intracellular proteins from both oxidative stress and denaturation, which is generally followed by the formation of aggregates in cells. Thus, bacterioruberin (BR) but especially the glycosylated forms of bacterioruberin (in particular BR-MonoG, BR-DiG), as well as the SBE extract, which corresponds to a mixture of a-bacterioruberin (BR) and glycosylated forms of this carotenoid, and in particular the diglycosylated form (BR-DiG), are therefore suitable for use in the development of treatments aimed at reducing the formation of aggregates. toxic proteins, for example in neurons, or in a disease involving dysregulation in protein aggregation.

[0142] Example III - Neuroprotective effect on neuron model III-1 Aim of the study

[0143] The aim of the study was to evaluate the neuroprotective effect of a carotenoid extract from the bacterium A. agilis rich in glycosylated bacterioruberins called "SBE" on glutamate excitotoxicity in a cellular model mimicking Alzheimer's disease (ALS).

[0144] The glutamatergic system, and in particular NMDA receptors (glu-tamatergic receptors) plays a major role in learning and memory processes. Synaptic plasticity can be regulated by NMDA receptor signaling.

[0145] Overactivation of NMDA receptors is a common pathological feature in many neurodegenerative diseases, including those leading to cognitive impairment such as Alzheimer's disease. In this light, early pharmacological treatment with substances reducing glutamate overstimulation is an avenue to treat patients diagnosed with cognitive decline. Tau protein is a microtubule-associated protein involved in microtubule stability and axonal transport. Pathological hyperphosphorylation of Tau triggers the formation of neurofibrillary tangles and actively participates, in association with beta-amyloid protein oligomers, in the neurodegenerative process of ALS. Moreover, glutamate excitotoxicity and Tau phosphorylation are closely related phenomena.

[0146] In this example, BDNF (Brain-Derived Neurotrophic Factor) was used as a positive control, as this neurotrophin is known to promote the survival and differentiation of neurons in vivo and in vitro. III-2 Materials and methods

[0147] III-2-1. Primary culture of cortical neurons

[0148] All experiments were carried out in accordance with the regulations in force in the European Union (Directive 2010 / 63 / EU).

[0149] Murine cortical neurons were cultured as described by Callizot et al., 2013. Cells were mechanically dissociated by three forced passages through the tip of a 10 ml pipette. Cells were then centrifuged at 515 xg for 10 minutes at 4 °C. The supernatant was removed and the pellet was taken up in a defined culture medium consisting of Neurobasal medium with a 2% solution of B27 supplement, 2 mmol / liter of L-glutamine, 2% PS solution and 10 ng / mL of BDNF. Viable cells were counted in a Neubauer cytometer, using the trypan blue exclusion assay. Cells were seeded at a density of 25,000 per well in a 96-well plate pre-coated with poly-L-lysine and cultured at 37 °C in a CO2 (5%) incubator. The medium was changed every 2 days. Experiments were subsequently performed on 96-well plates (n = 6 culture wells per condition). Of the 96 wells in each plate, only 60 were used. The wells in the first and last rows and columns were not used to avoid any edge effects and were filled with sterile water.

[0150] III-2-2 Test compounds and glutamate intoxication

[0151] The following compounds were tested in this example:

[0152] [Tables3] Test compound Concentration Control (vehicle) - Glutamate (20 pM, 20 min) / vehicle SBE 1 pM BDNF homodimer 1.85 nM = 50 ng / mL

[0153] The test compounds were solubilized in DMSO and the concentration was adjusted to ensure a DMSO concentration in the culture medium of 0.1%. On day 13 of culture, the compounds were pre-incubated with primary cortical neurons for 1 hour, before glutamate application. Subsequently, on the same day, the cortical neurons were exposed to glutamate for 20 min. Glutamate was added to a final concentration of 20 pM (diluted in the control medium) in the presence of SBE or BDNF (used as a positive control). After 20 minutes, the glutamate was removed and fresh culture medium with the test compounds was added for an additional 48 hours.

[0154] III-2-3 Evaluation of the effects of compounds by immunolabeling

[0155] 48 hours after glutamate intoxication, the cell culture supernatant was removed using multichannel automated pipettes. Cells were then washed with phosphate-buffered saline (PBS). Cortical neurons were fixed with a cold solution of ethanol (95%) and acetic acid (5%) for 5 min at -20 °C. They were washed again twice in PBS and then permeabilized. Nonspecific sites were blocked with a PBS solution containing 0.1% saponin and 1% FCS, for 15 min at room temperature. Cells were incubated for 2 hours with respectively:

[0156] a) a mouse monoclonal antibody anti-MAP-2 (microtubule-associated-protein 2) at a dilution of 1 / 400 in PBS, with 1% fetal calf serum and 0.1% saponin. This antibody binds specifically to neurons and neurites, allowing the study of the neurite network.

[0157] b) a mouse monoclonal antibody AT100 anti-phosphorylated Tau on Thr212 / Ser214 at a dilution of 1 / 400 in PBS containing 1% fetal calf serum and 0.1% saponin. This antibody allows the study of hyperphosphorylation of the Tau protein.

[0158] These antibodies were revealed with the secondary antibodies Alexa Fluor 488 IgG goat anti-mouse, Alexa Fluor 568 IgG goat anti-chicken anti-mouse, Alexa Fluor 568 IgG goat anti-rabbit. These secondary antibodies were incubated with the neuron preparations at a dilution of 1 / 400 in PBS containing 1% FCS, 0.1% saponin, for 1 hour at room temperature.

[0159] For each condition, 30 images per well were automatically taken using ImageXpress (Molecular Devices) at 20x magnification. All images were generated using the same acquisition parameters. From images, analyses were automatically performed by Custom Module Editor® (Molecular Devices). The following parameters were examined:

[0160] - Total neurite network (MAP-2 positive neurite length) - Tau protein hyperphosphorylation (Tau / MAP-2 overlap, pm2 of overlap)

[0161] III-2-4 Statistical processing of data

[0162] Data are expressed as a percentage of the control. All values ​​show the mean + / - standard error of the mean of 4-6 wells per condition. Graphs and statistical analyses across conditions (ANOVA followed by Fisher's LSD test [all groups versus glutamate group]) were performed using GraphPad Prism software version 8.1.2. * P < 0.05 was considered significant. III-3 Results and discussion

[0163] Neurite network integrity: Glutamate intoxication induced a significant reduction (60%) in neurite network density ([fig.4]). As expected, BDNF exerted a significant protective effect on neurite network integrity (total neurite network = 85%,). SBE application significantly improved neurite network integrity. The total neurite network length reached 83%, a significant result comparable to that obtained with neurotrophin.

[0164] Hyperphosphorylation of the Tau protein (AT100}: Glutamate intoxication induced a significant increase in the AT 100 area corresponding to hyperphosphorylation of the Tau protein and accumulation of the protein in the cytoplasm of neurons (+193% of the negative control (100% = value 0); [fig.5]). As expected, treatment with the neurotrophin BDNF leads to a significant reduction and significant increase in Tau hyperphosphorylation (+121%). Like BDNF, SBE application significantly reduced Tau phosphorylation (+137%).

[0165] In synthesis, the SBE extract exerts a neuroprotective effect on the neurite network and this effect is accompanied by a significant reduction in the hyperphosphorylation of the Tau protein in the cytoplasm of neurons.

[0166] Thus, a bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, can be used in a method of treatment or prevention of a neurodegenerative disease.

[0167] Bibliography

[0168] Armstrong GA (1994) Eubacteria show their true colors - genetics of carotenoid pigment biosynthesis from microbes to plants. J Bacteriol. 176:4795-4802.

[0169] Armstrong, GA (1997). Genetics of eubacterial carotenoid biosynthesis: A colorful case. In: Ornston, LN., editor. Annu Rev Microbiol. USA: Annual Reviews Inc.; . p. 629-659.

[0170] Britton G. (1995) Structure and properties of carotenoids in relation to function. FASEBJ. 9:1551-1558.

[0171] Britton, GL-JSPH. (2004) Carotenoids handbook. Basel; Boston: Birkhauser Verlag.

[0172] Callizot N, Combes M, Steinschneider R, Poindron P. (2013). Operational dissection of [3-amyloid cytopathic effects on cultured neurons. JNeurosci Res. 91: 706-16.

[0173] Dugger BN, Dickson DW (2017) Pathology of Neurodegenerative Diseases Cold Spring Harb Perspect Biol 9(7): a028035.

[0174] Farkhondeh T, Samarghandian S, Shaterzadeh Yazdi H, Samini F (2018) The protective effects of crocin in the management of neurodegenerative diseases: a review. Am J Neurodegener Dis 7:1-10.

[0175] Fong N, Burgess M, Barrow K, Glenn D (2001) Carotenoid accumulation in the psy-chrotrophic bacterium Arthrobacter agilis in response to thermal and sait stress Appl Microbiol BiotechnolSb, 750-756.

[0176] Mandelli F, Miranda VS, Rodrigues E, Mercadante AZ.. (2012) Identification of carotenoids with high antioxidant capacity produced by extremophile microorganisms. world J Microbiol Biotechnol

[0177] 28:1781-1790.

[0178] Strand A, Shivaji, S, Liaaen-Jensen (1997) Bacterial carotenoids 55. C50-carotenoids 25: revised structures of carotenoids associated with membranes in psychrotrophic Mi-crococcus roseus. Bioch. Syst. & Eco. 25(6) : 547-552

Claims

Claims

1. Composition comprising at least one bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it for use in a method of treatment or prevention of a disease involving deregulation in the aggregation of proteins, such as for example a degenerative disease, advantageously a neurodegenerative disease, fibrosis, advantageously pulmonary fibrosis, or diabetes.

2. Composition for its use according to claim 1, characterized in that the disease is a neurodegenerative disease chosen from Alzheimer's disease (ALS), Parkinson's disease (PD), Huntington's disease, posterior cortical atrophy, amyotrophic lateral sclerosis (ALS) as well as ocular neurodegenerative diseases chosen from macular degeneration, retinitis pigmentosa and retinopathy.

3. Composition for its use according to claim 1 or 2, characterized in that the neurodegenerative disease is Alzheimer's disease (ALS) or Parkinson's disease (PD).

4. Composition for its use according to any one of the preceding claims, characterized in that the at least one glycosylated bacterioruberin is chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins, pentaglycosylated bacterioruberins, hexaglycosylated bacterioruberins, heptaglycosylated bacterioruberins, octaglycosylated bacterioruberins, nonaglycosylated bacterioruberins, decaglycosylated bacterioruberins, undecaglycosylated bacterioruberins, and dodecaglycosylated bacterioruberins.

5. Composition for its use according to any one of the preceding claims, characterized in that the at least one glycosylated bacterioruberin is chosen from monoglycosylated bacterioruberins, diglycosylated bacterioruberins, triglycosylated bacterioruberins, tetraglycosylated bacterioruberins.

6. Composition for its use according to any one of the preceding claims, characterized in that it comprises at least one extract comprising at least one bacterioruberin and / or one bacterio- glycosylated ruberin.

7. Composition for its use according to any one of the preceding claims, characterized in that it comprises at least one bacterioruberin and one glycosylated bacterioruberin, advantageously a mixture of α-bacterioruberin, monoglycosylated bacterioruberin and diglycosylated bacterioruberin.

8. Composition for its use according to any one of the preceding claims, characterized in that the ratio between non-glycosylated forms and glycosylated forms of bacterioruberin is between 2 / 1 and 1 / 2.

9. Composition for use according to one of the preceding claims, characterized in that it is essentially free of non-glycosylated forms of bacterioruberin.

10. Composition for its use according to any one of the preceding claims, characterized in that it is in the form of a tablet, dragee, capsule, suppository, injectable or oral solution, or even drop and it is suitable for administration by oromucosal, oral, rectal, vaginal, intramuscular, parenteral or ophthalmic route.

11. Composition for use according to any one of the preceding claims, characterized in that it is in the form of a food supplement.

12. Composition for use according to any one of the preceding claims, characterized in that it contains from 1 mg to 1 g of glycosylated bacterioruberins and / or non-glycosylated bacterioruberins or mixtures thereof.

13. Bacterioruberin, preferably in glycosylated form, optionally in admixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for use in a method of treatment or prevention of a disease exhibiting deregulation in protein aggregation.

14. Bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use according to claim 14, characterized in that the disease is a degenerative disease, advantageously a neurodegenerative disease.

15. Composition comprising at least one bacterioruberin, preferably in glycosylated form, optionally in a mixture with different forms of glycosylated bacterioruberins, or an extract comprising it, for its use in a method of treating or preventing a degenerative disease, and advantageously a neurodegenerative disease.

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