Chemically modified microorganism, pharmaceutical composition and nutraceutical composition

Description

1 / 43 “CHEMICALLY MODIFIED MICROORGANISM, PHARMACEUTICAL COMPOSITION AND NUTRACEUTICAL COMPOSITION”

[001] The invention is in the field of drug delivery, in particular the intestinal delivery of antioxidant drugs intended to combat oxidative stress.

[002] More specifically, the invention relates to the use, as a medicine or food supplement, in particular to combat oxidative stress and, more particularly, to treat inflammatory diseases, of a chemically modified microorganism containing a Superoxide Dismutase (SOD) mimic. The invention also relates to a pharmaceutical composition, as well as a nutraceutical composition, food composition or food supplement containing such a microorganism.

[003] Superoxide dismutases (SODs) are metalloenzymes involved in cellular antioxidant defenses. In the body, SODs are responsible for maintaining superoxide, a reactive oxygen species (ROS), at strictly controlled levels and contribute to preventing oxidative stress situations that are known to be implicated in various diseases. At their active site, all SODs contain a metal cation that cycles between two redox states to successively reduce superoxide to H2O2 and oxidize it to O2. In humans, three different types of eukaryotic SODs have been described, depending on the metal contained in their active site: two copper / zinc SODs, SOD1 found in the cytosol and mitochondrial intermembrane space and SOD3 found in the extracellular environment, within the extracellular matrix and on the cell surface, and manganese SOD (SOD2), also called MnSOD, located in the mitochondrial matrix (Weisiger and Fridovich, 1973, Journal of Biological Chemistry).248: 4793-4796).

[004] Inflammatory Bowel Diseases (IBDs) are disorders Petition 870250088303, dated 09 / 29 / 2025, page 74 / 132 2 / 43 chronic gastrointestinal disorders (IBDs) are characterized by chronic inflammation of part of the gastrointestinal tract. These include Crohn's disease and ulcerative colitis, which differ at least in the location of the inflammation. Both can cause diarrhea, rectal bleeding, anemia, weight loss, and abdominal pain. IBDs constitute a global health problem, as their incidence is particularly high and continues to increase in developed countries, reaching a cumulative lifetime incidence of 1% in Europe. The number of IBD cases worldwide is estimated at 6.8 million people. To date, there is no curative therapy to treat IBDs, and patients primarily receive medications aimed at controlling inflammation in order to alleviate symptoms and prevent flare-ups. The main treatments include corticosteroids and immunomodulators, which comprise small molecules and monoclonal antibodies that target specific inflammatory pathways or cytokines (Paramsothy et al.)., 2018, Mucosal Immunol. 11: 1558-1570). However, these expensive medications are not always effective and can induce loss of response and adverse effects. There is, therefore, an urgent need for new, safe and effective therapeutic alternatives to combat IBDs.

[005] Inflammatory bowel diseases have been described as being accompanied by an overexpression of intestinal MnSOD in an enzymatically inactive form and by an underexpression of intestinal cytoplasmic Cu / Zn SOD (Kruidenier et al., 2003, J. Pathol. 201: 7-16). These deficiencies in the SOD antioxidant system may cause, or at least exacerbate, the oxidative stress observed in IBDs, which is known to be closely related to chronic inflammation (Krzystek-Korpacka et al., 2020, Diagnostics. 10: 601). SOD-based antioxidant treatment has therefore emerged as a promising therapy for IBDs. MnSODs, in particular, have been shown to efficiently reduce lipid peroxidation and Petition 870250088303, dated 09 / 29 / 2025, page 75 / 132 3 / 43 neutrophil recruitment and attenuation of inflammation in murine models of colitis induced by both DSS and TNBS (Naito et al., 2005, CDTIA. 4: 511-515). Document WO 2020 / 050460 describes a composition for the treatment of IBDs, comprising a mutant strain of the bacterium Bacillus amyloliquefaciens with a high production rate of the SOD enzyme and / or the purified SOD enzyme from such strain after incubation of the cells with a manganese salt. However, the use of these purified enzymes as therapeutics is limited by their short half-life, the immunogenicity triggered, and their low cellular penetration.

[006] To overcome these deficiencies, low molecular weight synthetic SOD mimetics, also called SOD mimetics, which mimic the activity of SOD, i.e., capable of catalyzing superoxide dismutation, have been examined as therapeutic candidates for the treatment of IBDs. A wide variety of synthetic inorganic antioxidant complexes that mimic SOD have been reported, including iron, copper, zinc, and manganese complexes. These synthetic inorganic complexes generally have a molecular weight of less than 10 kDa and often less than 5 kDa. Manganese complexes, in particular, appeared to be more favorable compared to Cu, Fe, and Ni complexes, since manganese, if released, is better tolerated by cells and does not catalyze the Fenton or Haber-Weiss reactions that lead to the formation of the extremely reactive and toxic HO* radical.

[007] Many manganese-based SOD mimetics have been described in the literature, including, for example, complexes with ligands such as salen derivatives, cyclic polyamine, 1,2-ethanediamine-centered ligands, porphyrins, phthalocyanines, etc. (Vincent et al., 2021, Journal of Inorganic Biochemistry 219: 111431).

[008] As an example, a manganese complex that mimics the Petition 870250088303, dated 09 / 29 / 2025, page 76 / 132 4 / 43 SOD, called Mn1, with formula (V): It has been reported to exert intracellular anti-inflammatory activity in vivo in a murine model of DBNS-induced colitis, demonstrated by analysis of weight variation and macroscopic scores, when administered orally in carbonate buffer (Mathieu et al., 2017, Inorg. Chem., 56: 2545-2555). This activity was, however, limited, likely due to the instability of this inorganic complex in the biological environment. Furthermore, although the use of a basic buffer as a vehicle for the metal complex improves its stability in acidic environments, such a vehicle is not desirable for administration in humans.

[009] The publication by Karlsson et al., 2015, Drug Discovery Today, 20: 411-421, reveals the intracellular ingestion of a SOD mimic, Mn Salen, in MnSOD-deficient Cryptococcus neoformans.

[0010] The present invention aims to propose a method for the efficient treatment of inflammatory diseases, particularly IBDs. More specifically, the invention falls within the context of the use of synthetic SOD mimics to combat oxidative stress, particularly in the treatment of inflammatory diseases, and aims to propose a solution to increase the bioavailability and efficacy of these active ingredients at their site of action, particularly in the intestine, compared to the solutions proposed by the prior art for the administration of these compounds.

[0011] The inventors have now discovered that these objectives Petition 870250088303, dated 09 / 29 / 2025, page 77 / 132 5 / 43 can be achieved by using microorganisms as vectors for the targeted administration of SOD mimetics in the intestine. They discovered, in particular, that a synthetic inorganic complex can be accumulated in microorganism cells and that, when these cells are administered orally to an individual and pass into the stomach, they protect the inorganic complex they contain from contact with the highly acidic gastric juices and thus prevent decomplexation that would otherwise have been induced by this contact. A high proportion of the inorganic complex administered to the individual is therefore advantageously intact when the cells reach their site of action in the intestine.

[0012] Thus, protected during the progression of the microorganism cells in the gastrointestinal tract, the inorganic complex remains intact and retains all its catalytic capacity for superoxide dismutation when it reaches its site of action in the intestine.

[0013] A first object of the invention is, therefore, a chemically modified microorganism, that is to say, a microorganism modified by the introduction of a chemical compound, said microorganism being selected from bacteria and yeasts, and containing a synthetic inorganic complex that is a superoxide dismutase mimic, for its use as a medicine or food supplement.

[0014] This chemically modified microorganism, in which the SOD mimic is vectored, is particularly easy to prepare, by simple passive penetration of the inorganic complex through the cell wall and its accumulation inside the cell.

[0015] When administered orally to an individual, the cell wall advantageously protects the inorganic complex from acidic gastric conditions and prevents its dissociation and degradation, so that it is advantageously delivered to the intestine with all its bioactivity, in particular its Petition 870250088303, dated 09 / 29 / 2025, page 78 / 132 6 / 43 ability to correct any intestinal hyperpermeability induced by colitis. This bioactivity was confirmed, in particular, by an assay performed in a murine model of acute colitis.

[0016] In the context of an intended therapeutic application of the chemically modified microorganism of the invention, involving its administration to a subject, both the microorganism and the inorganic complex are advantageously chosen so as not to be harmful to said subject.

[0017] Therefore, the inorganic complex of the invention is preferably pharmaceutically and / or physiologically acceptable, meaning that it does not produce any adverse, allergic or other undesirable reaction when administered to a subject, in particular to a mammal and more particularly to a human being.

[0018] The microorganism is advantageously chosen so as to be non-pathogenic in relation to the individual to whom it is intended to be administered, in particular non-pathogenic to mammals and, more particularly, to humans. The microorganism is preferentially commensal in relation to that individual.

[0019] Food-grade bacteria and yeasts are particularly preferred in the context of the invention. By food-grade microorganisms, it is understood here to mean microorganisms: - listed in the QPS (“Qualified Presumption of Safety”) list of the European Food Safety Authority (EFSA), last updated (“QPS list 2022”), available on the Knowledge Junction at Zenodo (https: / / doi.org / 10.5281 / zenodo.1146566); EFSA has written guidelines for the safety assessment and characterization of microorganisms (including probiotics) to be used as feed additives or as production organisms (i.e., administration). Petition 870250088303, dated 09 / 29 / 2025, page 79 / 132 7 / 43 (EFSA Panel on Additives and Products or Substances Used in Animal Feed); - and / or listed in the U.S. Food and Drug Administration's "Generally Recognized as Safe (GRAS)" document, dated September 6, 2019, accessed January 30, 2021.

[0020] The microorganism used according to the invention may be deficient in the enzyme SOD, or it may produce such an enzyme.

[0021] By superoxide dismutase (SOD) mimetic / mimic, it is conventionally referred to here as a low molecular weight synthetic compound (in particular, less than 10 kDa and possibly less than 5 kDa) capable of catalyzing the dismutation of superoxide in an aqueous medium, i.e., having a similar effect in protecting against oxidative damage as SOD. More particularly, SOD mimetics are capable of performing the catalytic redox cycle to oxidize O2- to O2 and reduce O2 to H2O2. This implies that the redox potential of the redox pair involved is between 0.18 V / NHE and 0.89 V / NHE (at pH 7).

[0022] It is within the competence of the specialist in the area to identify inorganic complexes that mimic SOD.

[0023] To date, a huge diversity of such complexes, in particular manganese complexes, has been reported in the state of the art. They involve ligands such as salen derivatives, cyclic polyamine, nitrogen-centered tri- or dipodal ligands, 1,2-ethanediamine-centered ligands, deferrioxamine derivatives, polyaminocarboxylate or polycarboxylate ligands, peptides, porphyrins, phthalocyanines, texaphyrins, corroles or biliverdin and their derivatives, etc. Examples of SOD mimics, which can be used according to the invention, are cited, in particular, in the publication by Vincent et al., 2021, J. Inorg. Biochem. 219, 111431.

[0024] Otherwise, to determine if a given Petition 870250088303, dated 09 / 29 / 2025, page 80 / 132 8 / 43 inorganic complex is a SOD mimic, that is, it has an intrinsic SOD activity, the person skilled in the art can apply the so-called indirect McCord and Fridovich assay, which is commonly used to identify SOD mimics by a superoxide dismutation test, and which is described in the publication by McCord and Fridovich, 1969, J. Biol. 244 (22), 6049-6065. Schematically, this assay comprises the determination of the catalytic rate (kcat) for superoxide dismutation in a HEPES buffer (e.g., at 50 mM, pH 7.4), the inorganic complex to be tested being in competition with a redox marker such as ferricitochrome c, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)2H-tetrazolium-5-carboxanilide (XTT) or nitro tetrazolium blue (NBT), e.g. at 100 μM, to react with superoxide that is continuously produced by means of a xanthine (200 μM) / xanthine oxidase enzymatic system. The reduction of the redox marker is monitored by UV-visible spectroscopy, e.g. at 550 nm for ferricitochrome c, for different amounts of the inorganic complex. The reduction rates in the absence and presence of the complex are determined for each amount of inorganic complex added.If s1 is the slope of the reduction curve before the addition of the inorganic complex and s2 is the slope after the addition of the same, for each inorganic complex the percentage of inhibition is given by I (%) = (s1 - s2) / s1*100. The IC50 value, which is the concentration of the inorganic complex required to induce a 50% inhibition of the redox marker reduction, is obtained for I = 50%; (s1 s2) / s2 can also be plotted against the concentration of the inorganic complex, providing a linear correlation. The IC50 is then obtained for (s1 s2) / s2 = 1. The kcat of the inorganic complex is then obtained from the IC50 and the known value of the kcat of the redox marker (kredox) (e.g., for XTT, kxTT = 2.9x104M'1.s'1 at pH 7.4), according to the equation: kkat = kredox x [redox] / IC50, where [redox] is the concentration of the redox marker in the medium of Petition 870250088303, dated 09 / 29 / 2025, page 81 / 132 9 / 43 test, for example 100 μM.

[0025] An inorganic complex can be qualified as a SOD mimic when the dismutation kinetics of superoxide catalyzed by this complex, as measured in the assay above, i.e., kcat, is faster than the autodismutation of superoxide (autodismutation, without any catalyst) at the same pH, as tabulated in the publication by Bielski et al., 1985, J. Chem. Ref Data, 14(4), 1041-1100, for example, faster than 6.105M-1.s-1 at pH 7.

[0026] Controls for the reliability of this assay, in particular to verify that the tested inorganic complex does not inhibit superoxide production by inhibiting the xanthine oxidase reaction, can be performed as described in Durot et al., 2005, Eur. J. Inorg. Chem., 17, 3513-3523.

[0027] In specific embodiments of the invention, the inorganic complex is a mimic of human manganese superoxide dismutase.

[0028] More generally, the metal in the inorganic complex is preferentially manganese (II) or manganese (III), which have the advantage, particularly compared with other metals such as iron, copper or nickel, of not increasing oxidative stress when released into the body.

[0029] Any inorganic SOD mimic complex, in particular any manganese-based SOD mimic described in the prior art, falls within the scope of the invention, in particular those having ligands such as salen derivatives, cyclic polyamine, nitrogen-centered tri- or dipodal ligands, 1,2-ethanediamine-centered ligands, deferrioxamine derivatives, polyaminocarboxylate or polycarboxylate ligands, peptides, porphyrins, phthalocyanines, texafirin, corroles or biliverdin and their derivatives, mentioned in the above-referenced publication by Vincent et al., 2021. Petition 870250088303, dated 09 / 29 / 2025, page 82 / 132 10 / 43

[0030] By way of example, the microorganism of the invention may contain: - Mn(II) dichloro[(4aR,13aR,17aR,21aR)- 1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7nitrilo-7Hdibenzo[b,h][1,4,7,10]tetraazacycloheptadecinekN5,kN13,kN18,kN21,kN22] (Imisopasem manganese), a specific manganese(II) pentaazamacrocil (cyclic polyamine); - such as Mn(III) porphyrins: Mn(III) meso-tetrakis(N-ethylpyridinium-2yl)porphyrin (MnTE-2-PyP5+); Mn(III) meso-tetrakis(N-(2'-n-butoxyethyl)pyridinium-2yl)porphyrin (MnTnBuOE-2-PyP5+); Mn(III) 5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP); - Mn(III) 2,2'-[1,2-ethanediylbis(nitrilome-tylidino)]bis[6-methoxy-phenol); - etc.

[0031] In specific embodiments of the invention, the inorganic complex is inspired by the active site of MnSOD and is built upon a ligand with a 1,2-N,N'-diaminoethane core structure functionalized with Lewis bases and in which the diamino structure is stiffened with a C-based ring in order to limit the release of Mn. The inorganic complex may, in particular, have the general formula (I): in which: n, mep, identical or different, are integers between 1 and 3, each of which n, mep preferably equals 1. Petition 870250088303, dated 09 / 29 / 2025, p. 83 / 132 11 / 43 R1 represents a hydrogen atom or a linear, branched and / or cyclic alkyl group, preferably having from 1 to 12 carbon atoms, in particular from 2 to 6 carbon atoms, optionally substituted by at least one, i.e., one or more, aromatic cyclo(s) or polycyclo(s), preferably having from 6 to 14 carbon atoms, such as anthracene, or R1 represents a peptide comprising from 1 to 20, in particular from 1 to 9, amino acid residues, R2, R2', R3, R3' all represent a hydrogen atom, or R2' and R3' represent a hydrogen atom and R2 and R3 form, together with the carbon atoms to which they are attached, an aliphatic ring or heterocycle, in particular a 5, 6, 7 or 8 membered ring or heterocycle, optionally substituted by a Lewis base, a phenol group, an imidazole ring, a pyridine ring and / or by one or more identical or different alkyl groups, each preferably having from 1 to 12 carbon atoms, in particular from 1 to 6 carbon atoms, each of said alkyl groups being optionally substituted by a Lewis base, a phenol group, an imidazole ring and / or a pyridine ring, or R2, R2', R3, R3' form, together with the carbon atoms to which they are attached, an aromatic ring or heterocycle, in particular a 5, 6, 7 or 8 membered ring or heterocycle members, optionally replaced by a Lewis base, a phenol group, an imidazole ring,a pyridine ring and / or one or more identical or different alkyl groups, each preferably having from 1 to 12 carbon atoms, in particular from 1 to 6 carbon atoms, each of said alkyl groups being optionally replaced by a Lewis base, a phenol group, an imidazole ring and / or a pyridine ring, Ar1 and Ar2, which may be identical or different, each represent a 5- or 6-membered aromatic heterocycle, preferably comprising one or two heteroatoms, in particular one or two atoms Petition 870250088303, dated 09 / 29 / 2025, p. 84 / 132 12 / 43 of nitrogen, such as a pyrrole ring, an imidazole, a pyridine or a pyrimidine, said aromatic heterocycle being optionally substituted by one or more identical or different substituents, each of which is selected from a bromine atom, a chloride atom, a methyl group, a methoxy group and a nitro group, R4 represents a phenyl group, optionally substituted by one or more identical or different substituents, each of which is selected from a bromine atom, a chloride atom, a methyl group, a methoxy group and a nitro group, or R4 represents a group of formula -R5-CO-, wherein the carbonyl group is bonded to the oxygen atom and R5 is bonded to the -(CH2)p- group, and R5 represents a linear, branched and / or cyclic alkyl, preferably having from 1 to 3 carbon atoms, optionally substituted by a Lewis base.

[0032] A Lewis base, in this context, refers to a group that can donate an electron pair to an electron acceptor, such as manganese (II), in order to form a non-permanent coordinate covalent bond. Examples of Lewis bases that can be included in the inorganic compound of the invention are the groups with formulas -OH, -COOH, -SH, -NH2, -NHRa, NRa(Rb), wherein Ra and Rb, identical or different, each represent a C1-C3 alkyl or alkenyl group or a phenyl group.

[0033] All stereoisomers, diastereomers and enantiomers of the inorganic complex of general formula (I) above, as well as of all formulas below, fall within the scope of the invention, individually or in any of their mixtures. In particular, the nitrogen atoms bonded respectively to the Ca and Cb atoms may be in a cis configuration. They are preferably in a trans configuration.

[0034] Any salt of the inorganic complex of formula (I), or any of the formulas below, is within the scope of the invention. Petition 870250088303, dated 09 / 29 / 2025, p. 85 / 132 13 / 43

[0035] The inorganic complex of formula (I) may respond to one or more of the characteristics below, in any of their combinations.

[0036] Ri can be, for example, an n-propyl group.

[0037] In particular embodiments of the invention, when at least R2 and R3 do not represent a hydrogen atom, then Ri represents a hydrogen atom.

[0038] When Ar1, respectively Ar2, represents a substituted six-membered aromatic heterocycle, such as a pyridine or pyrimidine ring, the substituent(s) is / are preferably in the ortho position, and more preferably in the para position, with respect to the nitrogen atom located in the α position with respect to the carbon atom bonded to -(CH2)m-, respectively -(CH2)n- (i.e., with respect to the nitrogen atom coordinated with manganese(II)).

[0039] When Ar1, respectively Ar2, represents an imidazole ring, at least one of the substituents is attached to a nitrogen atom other than the one coordinated with manganese(II).

[0040] When R4 represents a phenyl group, this group is preferentially attached to an α-(CH2)p- carbon atom in the α position relative to the carbon atom bonded to the oxygen atom.

[0041] The inorganic complex of the invention may have the general formula (II): where m, n, p, R1, R2, R2', R3, R3', Ar1 and Ar2 are as defined Petition 870250088303, dated 09 / 29 / 2025, p. 86 / 132 14 / 43 above.

[0042] It may have in particular the general formula (Ila): where m, n, Ri, R2, R2', R3, R3', Ar1 and Ar2 are as defined above.

[0043] Preferably, in formulas (II) and (IIa) above, m equals 1 and / or n equals 1.

[0044] In particular embodiments of the invention, the inorganic complex has the general formula (III): (III) where m, n, p, R1, R2, R2', R3, R3' and R4 are as defined above.

[0045] It may have in particular the general formula (IIIa): where p, R1, R2, R2', R3, R3' and R4 are as defined above. Petition 870250088303, dated 09 / 29 / 2025, p. 87 / 132 15 / 43

[0046] Preferably, in formulas (III) and (IlIa) above, p is equal to 1.

[0047] In particular embodiments of the invention, the inorganic complex has a coordination sphere consisting of two imidazole rings and a phenolate ion.

[0048] The inorganic complex then preferentially has the general formula (IV): where Ri, R2, R2', R3 and R3' are as defined above.

[0049] A particular inorganic complex of formula (IV) is Mn(II)-(M(hydroxybenzyl)-M,M-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine)), herein called Mn1, of formula (V) above. In particular, Mn1 has the advantages of showing clear anti-superoxide activity outside the cellular context, as well as antioxidant and anti-inflammatory effects in intestinal epithelial cells and macrophages. As indicated above, Mn1 also improves DNBS-induced colitis in a murine model, in particular according to weight variation. The kcat of Mn1, as obtained by the McCord and Fridovich assay mentioned above, is 7.106M'1.s'1.

[0050] The inorganic complex of the invention may otherwise, for example, have one of the formulas (VI), (VII) and (VIII): Petition 870250088303, dated 09 / 29 / 2025, p. 88 / 132 16 / 43

[0051] Inorganic complexes with higher rigidity of the 1,2-diaminoethane moiety, exemplified herein with a 6-membered cyclohexyl moiety (complexes of formulas (VII) and (VIII) above), being advantageously more inert than Mn1 in the biological environment, are particularly preferred in the context of the invention.

[0052] The microorganism itself, chosen from bacteria and yeasts, is preferably selected from microorganisms devoid of pro-inflammatory effects. In specific embodiments of the invention, the microorganism possesses an anti-inflammatory effect. Probiotics with native beneficial properties, in particular anti-inflammatory properties, can be used in the context of the invention. Probiotics have been defined by the World Health Organization (WHO, 2001) as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. This definition has also been re-examined and validated more recently by a group of experts (Hill et al., Expert consensus).

[0053] document: The International Scientific Association for Petition 870250088303, dated 09 / 29 / 2025, page 89 / 132 17 / 43 Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic.. Nat. Rev. Gastroenterol. Hepatol. 2014, 11, 506-514; Salminen et al. The International Scientific Association of Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of postbiotics.. Nat. Rev. Gastroenterol. Hepatol. 2021, 18, 649-667).

[0054] In addition, microorganisms whose cell wall has good permeability to inorganic complexes are favored in the context of the invention.

[0055] The microorganism of the invention may be a yeast, for example of the species Saccharomyces boulardii or Yarrowia lipolytica.

[0056] In other embodiments of the invention, the microorganism is chosen from bacteria, which have the advantage of being easy to handle and growing quickly.

[0057] Bacteria naturally rich in manganese, i.e., with a manganese content of approximately 1 mM or more, are particularly preferred in the context of the invention, since a high manganese content advantageously promotes the persistence, within the cells, of manganese in its ligand-coordinated form.

[0058] In specific embodiments of the invention, the microorganism is a lactic acid bacterium, in particular of the genera Lactobacillus or Lactococcus. Such bacteria combine, in particular, the advantages of being able to survive in a wide pH range, particularly in the acidic environment of the gastrointestinal tract, of good penetration of inorganic complexes into it, and of a high manganese content, which forces ligand-metal coordination and maximizes the amount of inorganic complex gathered in the cell.

[0059] Bacteria may be selected from the genera Lactobacillus, Lactococcus, Bifidobacterium or Bacillus, with the species Lactobacillus plantarum and Lactococcus lactis being particularly preferred. Petition 870250088303, dated 09 / 29 / 2025, pp. 90 / 132 18 / 43

[0060] The bacteria may also be bacteria of the species Escherichia coli, which are not pathogenic to mammals, particularly humans.

[0061] In preferred embodiments of the invention, the microorganism is modified so as to improve the targeted and efficient release of the encapsulated inorganic complex in a suitable zone to obtain therapeutic efficacy, more particularly at the site of inflammation in the intestine. Preferably, this is achieved by weakening the cell wall of the microorganism before administering it to the individual, so that when the chemically modified microorganism of the invention is administered to the individual, after passing through the stomach and reaching the intestine, during its transit through it, and as its wall has been previously weakened by the prior weakening treatment, it advantageously lyses and releases the SOD-mimicking inorganic complex in the intestine.

[0062] Therefore, in specific embodiments of the invention, the cell wall of the microorganism has been genetically, chemically, and / or enzymatically weakened. As a consequence, this cell wall is more easily lysed than that of the original, non-weakened microorganism. In general, cells whose cell wall has been weakened grow more slowly and precipitate more rapidly than their non-weakened counterparts. Microorganisms whose cells have been weakened can, for example, be identified using a membrane permeability determination kit, such as the Baclight® kit, to measure the ability of a reagent such as propidium iodide to penetrate the cells. Microorganisms whose cell wall has been weakened exhibit greater accumulation of this reagent than original microorganisms with non-weakened cell walls.

[0063] Any method for weakening the cell wall of a microorganism, without destroying it, known to a person skilled in the art, falls under Petition 870250088303, dated 09 / 29 / 2025, pp. 91 / 132 19 / 43 within the scope of the invention. The step(s) of weakening the microorganism's cell wall may be performed before, during, or after the contact step of the inorganic complex with the microorganism, in order to promote its penetration into the microorganism. Preferably, they are performed before this contact step. The penetration rate of the inorganic complex into the microorganism's cells is then advantageously increased.

[0064] As an example, the microorganism can be cultivated in the presence of a cell wall weakening agent, such as glycine or threonine, for a few hours, preferably for 6 to 18 hours, for example, for approximately 12 hours.

[0065] In specific embodiments of the invention, the microorganism is auxotrophic for a particular substance, preferably for an amino acid, in particular of the D series, and has been cultivated in a culture medium devoid of said substance / amino acid for at least 1 hour, preferably at least 3 hours. Such amino acid deprivation induces a weakening of the microorganism's cell wall. The amino acid may be, for example, alanine, phenylalanine or thymidine.

[0066] For example, the Lactobacillus plantarum strain MD007, deposited on March 23, 2023, in the French National Collection of Microorganism Cultures (CNCM) at the Pasteur Institute (25 Rue du Docteur Roux, Paris, France) under CNCM accession number I-5940 (identification reference LBH791-MD007), can be used in the context of the invention. This strain was obtained by making the Lactobacillus plantarum ATCC BAA-793 / NCIMB 8826 strain auxotrophic to alanine, as described in the publication by Palumbo et al., 2004, FEMS Microbiology Letters, 233: 131-138. Preferably, this strain is used after a 1 to 5 hour alanine deprivation.

[0067] Otherwise, the Lactococcus lactis MG1363 strain (which has a SOD enzyme) can be used in the context of the invention. Petition 870250088303, dated 09 / 29 / 2025, pp. 92 / 132 20 / 43

[0068] According to the invention, the chemically modified microorganism is used as a medicine, to treat a disease or as a food supplement.

[0069] The term “treat,” as used in this document, means to obtain a desired pharmacological and physiological effect, which may be prophylactic or curative. The term “treat,” as used in this document, therefore includes: preventing, or partially preventing, the occurrence of a disease, symptom, or condition in an individual who has not yet been diagnosed with the disease; and / or partially or completely curing a disease, symptom, or condition, or an adverse effect attributed to the disease.

[0070] The chemically modified microorganism of the invention can be used to combat oxidative stress. By "combat," it is understood here to prevent or reduce oxidative stress.

[0071] The chemically modified microorganism of the invention can be used to treat a wide range of pathophysiological processes in which oxidative stress is involved, including diabetes, inflammatory and neurodegenerative diseases, reperfusion injury after ischemia, cancer, skin diseases associated with oxidative stress, such as psoriasis, or any other disease related to oxidative stress.

[0072] The chemically modified microorganism of the invention can be used, in particular, to treat inflammatory diseases, and more particularly inflammatory bowel diseases, such as Crohn's disease or ulcerative colitis.

[0073] The subject treated with the chemically modified microorganism of the invention, which suffers from the disease or is susceptible to contracting it, is preferably a mammal, in particular a human being. It could also be, for example, a dog or a cat.

[0074] In preferred embodiments of the invention, the Petition 870250088303, dated 09 / 29 / 2025, pp. 93 / 132 21 / 43 The chemically modified microorganism of the invention is administered to the subject orally.

[0075] Otherwise, it can be administered topically, especially in the context of treating skin diseases.

[0076] In the context of a medicament, the chemically modified microorganism of the invention is preferably administered to the subject in a therapeutically effective amount, that is, an amount that is adequate to deliver a therapeutically effective amount of the SOD mimic inorganic complex to the subject.

[0077] By “therapeutically effective amount” is meant here the amount of a compound which, when administered to a subject to treat a disease, is sufficient to accomplish such treatment of the disease. The therapeutically effective amount of a compound depends on several factors, such as the disease and its severity, the age, weight, etc., of the individual to be treated, the specific compound used, the route and form of administration, etc. The amount of the chemically modified microorganism of the invention to be administered to the individual will therefore be determined by the physician for each individual case. It must, in particular, be low enough not to induce toxicity, such as manganism, when the metal is manganese.

[0078] As an example, a dosage equivalent to 0.10 to 0.20, for example 0.14 mg / kg / day of manganese, may be applied.

[0079] In the context of a dietary supplement, the chemically modified microorganism of the invention is preferably administered to the subject in an amount suitable for administering to the subject an amount of the SOD mimic inorganic complex that is adequate to obtain a desired effect, such as a beneficial effect on health, comfort and / or well-being.

[0080] The chemically modified microorganism of the invention Petition 870250088303, dated 09 / 29 / 2025, pp. 94 / 132 22 / 43 can, for example, be administered to the subject once or twice a day. In particular, it can be administered to the subject twice a day, for example, in the morning and evening, during disease flare-ups, and once a day during remission phases.

[0081] The invention can also be expressed in terms of a method for therapeutically treating an individual suffering from a disease, in particular an inflammatory disease, more particularly an inflammatory bowel disease such as Crohn's disease or ulcerative colitis, said method comprising administering to the individual in need a therapeutically effective amount of a chemically modified microorganism of the invention. This method may exhibit any of the features or combination of features described above in relation to the use as a medicine of a chemically modified microorganism according to the invention. The chemically modified microorganism may be administered to the individual as a component of a medicine or as a component of a food composition, a nutraceutical composition or a food supplement.

[0082] The invention also relates to the use of a chemically modified microorganism, according to the invention, for the manufacture of a medicament, in particular a medicament for the treatment of an inflammatory disease, more particularly an inflammatory bowel disease, such as Crohn's disease or ulcerative colitis. This use may meet any of the features or combination of features described above in relation to the use as a medicament of a chemically modified microorganism, according to the invention.

[0083] The invention also relates to the use of a chemically modified microorganism, according to the invention, for the manufacture of a food composition, a nutraceutical composition or a supplement. Petition 870250088303, dated 09 / 29 / 2025, pp. 95 / 132 23 / 43 food, in particular to combat oxidative stress. This use may meet any of the characteristics or combination of characteristics described above in relation to the use as a medicine or food supplement of a chemically modified microorganism, according to the invention.

[0084] The invention also relates to a pharmaceutical composition containing a chemically modified microorganism, as defined above, in a pharmaceutically suitable vehicle. In this pharmaceutical composition, the active ingredient is the SOD-mimicking inorganic complex encapsulated in the microorganism cells.

[0085] A “pharmaceutically suitable vehicle” here means a vehicle that is useful in the preparation of a pharmaceutical composition or formulation and that is generally safe, non-toxic and neither biologically nor otherwise undesirable to the subject to be treated, in particular mammals and more particularly humans.

[0086] The vehicle of the composition of the invention may be solid, semi-solid or liquid. It may be a diluent, an adjuvant or any other conventional vehicle per se for the constitution of pharmaceutical compositions.

[0087] The chemically modified microorganism can be contained in any form, particularly in lyophilized form.

[0088] The pharmaceutical composition of the invention can be formulated in any galenic form, in particular in a form that is suitable for administration in mammals and, in particular, in humans. It is preferably formulated in a form suitable for oral administration, such as powder, capsules, solution or oral suspension, or in a form suitable for topical administration, especially on the skin and / or mucous membranes of the individual, such as a cream, etc.

[0089] The pharmaceutical composition of the invention may comprise one or more conventional excipients / additives on their own, for example Petition 870250088303, dated 09 / 29 / 2025, pp. 96 / 132 24 / 43 preservatives, sweeteners, flavorings, suspending agents, dispersants, lubricants, stabilizers, buffers or any mixture thereof.

[0090] It may also contain one or more other active agents, which may or may not act synergistically with the inorganic complex of the invention, for example, other anti-inflammatory agent(s) and / or analgesic agent(s).

[0091] The invention also relates to the therapeutic use of a pharmaceutical composition as defined above, in particular for treating an inflammatory disease, more particularly an inflammatory bowel disease such as Crohn's disease or ulcerative colitis in a subject, in particular a mammal and more particularly a human being.

[0092] The invention also relates to a nutraceutical composition, food composition or food supplement containing a chemically modified microorganism, as defined above, in a physiologically suitable vehicle. In this composition / supplement, the active ingredient is the SOD-mimicking inorganic complex encapsulated in the microorganism cells.

[0093] A “physiologically acceptable vehicle” here means a vehicle that is physiologically tolerable and does not produce an allergic or similar undesirable reaction when administered to a subject, in particular a mammal and more particularly a human.

[0094] The vehicle of the composition / supplement may be solid, semi-solid or liquid. It may be a diluent, an adjuvant or any other conventional vehicle per se for the constitution of nutraceutical compositions, food compositions or food supplements.

[0095] The chemically modified microorganism can be contained in any form, particularly in lyophilized form.

[0096] The nutraceutical composition, food composition or Petition 870250088303, dated 09 / 29 / 2025, pp. 97 / 132 25 / 43 The dietary supplement of the invention is preferably in a form suitable for oral administration.

[0097] It may contain one or more probiotics, prebiotics, vitamins, polyphenols, minerals, materials suitable for oral administration (of the liquid or gel type, such as a solvent, a diluent, a non-toxic solubilizing agent that does not interact with the components of the composition in a harmful way) and / or any other ingredient or excipient, such as proteins, amino acids, carbohydrates, lipids, oligosaccharides, other micronutrients, salts or metallic cations, etc.

[0098] The invention also relates to the use of a nutraceutical composition, food composition or dietary supplement as defined above to combat oxidative stress and / or to treat inflammation in a subject, in particular a mammal and more particularly a human.

[0099] A method for producing a chemically modified microorganism according to the invention comprises placing the microorganism cells in contact with a composition containing the inorganic superoxide dismutase mimic complex, preferably for at least 1 hour to several hours, in order to allow the inorganic complex to penetrate, by a passive mechanism, into the microorganism cells and accumulate therein.

[00100] This contact step is preferably carried out: - for a period between 1 and 12 hours, preferably between 1 and 2 hours, for example, about 2 hours; - and / or at 37 °C; - and / or under agitation; - and / or in a liquid composition, preferably with a pH between 7 and 8, such as a 0.1 M 4-[2-(hydroxyethyl)-1-piperazin-1-yl]ethanesulfonic acid (HEPES) solution; Petition 870250088303, dated 09 / 29 / 2025, pp. 98 / 132 26 / 43 - and / or when the optical density of the cell suspension at 600 nm is between 0.6 and 0.8.

[00101] The concentration of the inorganic complex in the liquid composition is therefore preferably between 0.1 and 5 mM, for example, substantially equal to 0.4 mM. This concentration range is advantageously high enough to achieve the penetration of a large amount of the inorganic complex into the microorganism cells, and low enough to avoid the accumulation of a large amount of other forms of the metal, for example, when the metal is manganese, manganese in the form of MnCl2, while ensuring the absence of toxicity to the cells.

[00102] As explained above, the method may comprise, before, during or after the contact step of the microorganism cells with a composition containing said inorganic superoxide dismutase mimic complex, preferably before this step, a step of weakening the cell wall of the microorganism.

[00103] This step can be carried out by any method known to those skilled in the art, for example, by cultivating the microorganism in a culture medium supplemented with a cell wall weakening agent, such as glycine, for example, at a concentration between 10 and 20 g / L, for example, for a period of 1 to 12 hours. The culture medium can be any medium suitable for the cultivation of the microorganism. It includes, in particular, all the nutrients and other elements necessary for the survival and, optionally, the growth of the microorganism.

[00104] In particular embodiments of the invention, wherein the microorganism is auxotrophic for a given amino acid, such as alanine, the method comprises a step of weakening the cell wall by cultivating said microorganism in a culture medium devoid of said amino acid. The culture medium includes at least all the nutrients Petition 870250088303, dated 09 / 29 / 2025, pp. 99 / 132 27 / 43 and other elements necessary for the survival and, optionally, the growth of the microorganism, such as a De Man, Rogosa and Sharpe (MRS) medium, and is devoid of the aforementioned amino acid. This deprivation step can be carried out for a period between 1 and 5 hours, for example, about 3 hours. It is preferably initiated when the optical density at 600 nm of the culture reaches 0.6 and before the contact step of the microorganism cells with a composition containing the inorganic superoxide dismutase mimic complex.

[00105] In all cases, the cultivation stage is preferably carried out at 37 °C.

[00106] An example of a method for preparing a chemically modified auxotrophic microorganism according to the invention comprises successive steps of: - Cultivate the microorganism, particularly bacteria, preferably at 37 °C, in a culture medium such as MRS, supplemented with the substance for which the microorganism is auxotrophic, until an optical density at 600 nm between 0.6 and 0.8 is obtained; - Separate the cells from the culture medium, for example by centrifugation, and optionally wash the cells, for example with MRS medium, preferably three times; - Cultivate the cells in MRS medium devoid of the aforementioned substance for 1 to 5 hours, for example for 3 hours ("substance deprivation"); - Separate the cells from the culture medium, for example by centrifugation, and optionally wash the cells, for example with a 0.1M HEPES solution, preferably three times; - Resuspend the cells in a liquid solution, for example a 0.1M HEPES solution containing the inorganic mimic complex. Petition 870250088303, dated 09 / 29 / 2025, pp. 100 / 132 28 / 43 of SOD, preferably at a concentration between 0.1 and 5 mM, for example 0.4 mM; - Incubate the suspension at 37 °C for 1 to 5 hours, for example for 2 to 3 hours, preferably with shaking; - Separate the cells from the liquid medium, for example by centrifugation, and optionally wash the cells, for example with a 0.1M HEPES solution; - and finally recover the cells, for example in a 0.1M 16% glycerol-HEPES solution.

[00107] The chemically modified cells thus obtained, containing a high quantity of the inorganic SOD-mimicking complex, can then be administered to an individual in need of them for the preventive or curative treatment of a disease related to oxidative stress, in particular an inflammatory bowel disease. This administration should preferably be carried out soon after obtaining the chemically modified cells, more particularly a few hours after obtaining them.

[00108] Otherwise, the cells can be lyophilized and used as such; or they can be frozen, for example at -80 °C, in a buffer, such as a HEPES buffer supplemented with glycerol, for example at 16% v / v, and thawed extemporaneously before being administered to the subject to be treated.

[00109] The features and advantages of the invention will become clearer in light of the following implementation examples, provided for illustrative purposes only and in no way limiting the invention, with the support of Figures 1 to 14, in which: Figure 1 shows a bar graph representing the concentration of Mn measured by ICP-MS in bacterial cell lysates. Petition 870250088303, dated 09 / 29 / 2025, pp. 101 / 132 29 / 43 Empty MD007 (“HEPES”) or loaded with SOD mimics (Mn1, Mn1C) or with MnCte; Figure 2 shows a graph representing the weight monitored over time of mice subjected to a DNBS injection (on Day 0) and treated daily orally with empty MD007 bacteria or bacteria loaded with a SOD mimic (Mn1C) or with MnCl2 (from Day -1 to Day 2); weights are expressed as a percentage of the mice's initial weight before the start of the assay; Figure 3 shows a graph representing the quantification of FITC-dextran as a marker of intestinal barrier permeability in mice subjected to DNBS injection and orally treated with empty MD007 bacteria or those loaded with a SOD mimic (Mn1C) or with MnCl2. Data represent the mean ± SD for three independent assays (approximately 24 mice). P-values ​​were calculated using the non-parametric Mann-Whitney test (one-tailed test) because the Gaussian distribution of the data was not validated by the Anderson-Darling normality test. The mean ranks of each column were compared to those of the DNBS control; each comparison is independent. **: p < 0.01 versus DNBS control, and ns means not significant; - Figure 4 shows a bar graph representing the enumeration of viable cells in frozen-thawed aliquots of empty MD007 bacteria (“HEPES”) or loaded with SOD mimetics (Mn1, Mn1C); - Figure 5 shows a bar graph representing the Mn content measured by ICP-MS in cell lysates of empty MD007 bacteria (“HEPES”) or loaded with SOD mimetics (Mn1, Mn1C) (average of 3 experiments); Figure 6 shows a graph representing the area under the weight monitoring curve between day 0 and day 3 (D0-D3) of mice subjected to a DNBS injection (on day 0) and treated daily via Petition 870250088303, dated 09 / 29 / 2025, pp. 102 / 132 30 / 43 oral with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control”) or loaded with Mn1 or Mn1C SOD mimetics (data collected from 3 independent assays, with 8 mice / assay / condition); - Figure 7 shows a graph representing the macroscopic scores, determined on the day of euthanasia, of mice subjected to DNBS injection and treated daily for 4 days orally with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control”) or loaded with SOD Mn1 or Mn1C mimetics (data collected from 3 independent assays, with 8 mice / assay / condition); - Figure 8 shows a graph representing the concentration of lipocalin 2 (LCN-2) measured in the colon of mice subjected to DNBS injection and treated daily for 4 days orally with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control”) or bacteria loaded with Mn1 or Mn1C SOD mimetics (data collected from 3 independent assays, with 8 mice / assay / condition); - Figure 9 shows a graph representing the concentration of lipocalin 2 (LCN-2) measured in the serum of mice subjected to DNBS injection and treated daily for 4 days orally with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control”) or bacteria loaded with SOD Mn1 or Mn1C mimetics (data collected from 3 independent assays, with 8 mice / assay / condition); - Figure 10 shows the mRNA levels of SOD2, normalized with those of GADPH and TBP, measured in colon samples from mice subjected to DNBS injection and treated daily for 4 days orally with Hepes (“Hepes”), empty MD007 bacteria (“MD007 Hepes”) or loaded with the SOD mimic Mn1C (average in 16 mice per condition); Figure 11 shows a graph representing weight. Petition 870250088303, dated 09 / 29 / 2025, pp. 103 / 132 31 / 43 monitored over time of mice subjected to an injection of DNBS (on Day 0) or not (“Vehicle”) and treated daily orally with Hepes (“DNBS Vehicle”) or with empty MG1363 bacteria (“DNBS MG1363”) weights are expressed as a percentage of the mice's initial weight before the start of the assay; Figure 12 shows the area under the curve of the curves in the figure (between day 0 and day 5: D0-D5); - Figure 13 shows a graph representing the macroscopic scores, determined on the day of euthanasia (day 5: “D5” or day 6: “D6”), of mice subjected to DNBS injection and treated daily for 5 days orally with Hepes (“DNBS Vehicle”) or with empty MG1363 bacteria (“DNBS MG1363”); Figure 14 shows a bar graph representing the weight on day 5 (D5) of mice subjected to DNBS injection (on day 0) or not (“Vehicle”) and treated daily orally with Hepes (“DNBS Vehicle”), with empty MG1363 bacteria (“DNBS MG1363”) or with MG1363 bacteria loaded with Mn1 - weights are expressed as a percentage of the mice's initial weight before the start of the assay (D0). Example 1 - Synthesis of inorganic complexes mimetic of SOD

[00110] The ligands used in the preparation of the inorganic complexes were EnPI2C and EnPI2CP, with respective formulas (Ixa) and (Ixb): (Ixa) Petition 870250088303, dated 09 / 29 / 2025, pp. 104 / 132 32 / 43 \ \----( (Ixb)

[00111] Mn(II) SOD-mimetic complexes were prepared by mixing these respective ligands and MnCl2 in a molar ratio of 1:1.3 in 0.1 M HEPES (pH 7.5) and left for 2 h at room temperature to provide complete complexation of the ligands with manganese(II).

[00112] The following SOD-mimetic metal complexes were obtained: Mn1C (formula (VII) above) from EnPl2C, Mn1CP (formula (VIII) above) from EnPl2CP. Example 2 - Loading bacteria with SOD mimetics

[00113] The MD007 strain of L. plantarum, deposited on March 23, 2023, in the French National Collection of Microorganism Cultures (CNCM) of the Pasteur Institute (25 Rue du Docteur Roux, Paris, France) under accession number CNCM I-5940 (identification reference LBH791-MD007), was used for the experiments. This strain was obtained by transforming the alanine-auxotrophic Lactobacillus plantarum ATCC BAA-793 / NCIMB 8826 strain, as described in the publication by Palumbo et al., 2004, FEMS Microbiology Letters, 233: 131-138.

[00114] The overnight culture of MD007 cells in alanine-supplemented MRS medium (MRS-alanine) was diluted to OD600 nm = 0.2 in MRS-alanine and the cells were allowed to grow until ODgqq nm = 0.6 was reached. The culture was then centrifuged and the pellet was washed twice and resuspended in alanine-free MRS.

[00115] The bacteria were deprived of alanine for 3 hours Petition 870250088303, dated 09 / 29 / 2025, pp. 105 / 132 33 / 43 to weaken them in order to accelerate their lysis in the intestines of mice.

[00116] After alanine deprivation, the culture was centrifuged again and the pellet was washed twice and resuspended in 0.1 M HEPES. At this point, the culture was divided into 4 parts and the SOD mimics Mn1, Mn1C and Mn1CP, and MnCl2 were added, respectively, to a final concentration of 0.4 mM. The bacterial suspensions were incubated for 2 hours at 37 °C, under agitation.

[00117] Finally, after centrifugation and washing of the cultures with 0.1 M HEPES, the bacteria were resuspended in 0.1 M HEPES containing 16% glycerol (HEPES-gly) and stored at -80 °C in 2 mL aliquots to avoid freeze-thaw cycles. Example 3 - Quantification of Mn by ICP-MS analysis

[00118] For each inorganic complex, and for the control MnCl2, one of the aliquots of the bacterial suspension (at OD600nm =1), was used for Mn quantification by ICP-MS (Inductively Coupled Plasma Mass Spectrometry). A negative control with discharged bacteria (HEPES) was also analyzed.

[00119] 55Mn was selected as the isotope to avoid isobaric interference.

[00120] ICP-MS experiments were performed on a 7700 series ICP-MS system with an Agilent ASX500 series autosampler, with the following parameters: RF power: 1550 W; sampling depth: 10 mm; helium flow rate: 5 mL / min, measurement replicated five times with 100 scans per replicate and an integration / mass time of 1 s.

[00121] The bacterial suspensions were diluted in 2% HNO3 to lyse the bacteria and release Mn from all coordination sites. Petition 870250088303, dated 09 / 29 / 2025, pp. 106 / 132 34 / 43 To achieve complete bacterial lysis, 2% HNO3 solutions were left for 1 h at room temperature and then filtered. A calibration curve was established using a commercial multielement standard, and the total amount of metal was normalized by the OD600nm of the bacterial suspension.

[00122] The results obtained are shown in figure 1, for HEPES, MnCl2, Mn1 and Mn1C. They show a high manganese content in the bacteria loaded with SOD mimics according to the invention. Example 4 - Induction of DNBS colitis in mice and administration of bacteria.

[00123] In vivo assays were conducted in pathogen-free male C57BL / 6 mice at the animal facilities of the National Institute for Agricultural and Environmental Research (INRAE, Jouy-en-Josas).

[00124] The schedule for inducing colitis and oral administration of the bacteria-laden mice was as follows. The assay lasted 5 days. The bacteria-laden mice were administered daily intragastrically to the mice for the first 4 days. On the second day, the mice were anesthetized intraperitoneally and colitis was induced by intrarectal injection of dinitrobenzene sulfonic acid (DNBS) (2.75 mg per mouse in 20 µl of PBS-ethanol (70 / 30 v / v)) through a plastic tube inserted 4 cm into the colon. On the fifth and final day, the mice were euthanized by cervical dislocation. The mice were weighed daily.

[00125] The groups studied are as follows: control group with colitis (DNBS + HEPES-gly), control group with empty bacteria (DNBS + MD007) and 2 groups with loaded bacteria: “DNBS + “MD007_MnCl2” and “DNBS + MD007_Mn1C”. Each group consisted of 8 mice. The assay was repeated three times independently, which Petition 870250088303, dated 09 / 29 / 2025, pp. 107 / 132 35 / 43 means that, in total, 24 mice were used for each condition.

[00126] The amount of loaded bacteria administered to the mice was determined so that all received the same amount of total manganese, fixed at 150 nmol per dose. This corresponds to a number of bacteria ranging between 5 x 10⁸ and 2 x 10⁹ CFU. The amount of MD007 administered to the control group “DNBS + MB007” was chosen as equal to the maximum amount of viable loaded bacteria administered to the mice in the loaded bacteria groups. Finally, the control group with colitis received 200 pL of HEPES-gly. Example 5 - Assessment of colitis severity 5.1 / Weight of the mice

[00127] The evolution of the weight of the mice throughout the trial is shown in Figure 2.

[00128] As can be seen in this figure, mice fed Mn1C, an SOD mimic internalized by MD007, were able to limit weight loss and accelerate weight recovery. In fact, weight reduction (maximum on the second day) reached only 13%, and the mice recovered about 90% of their initial weight on the third day.

[00129] Furthermore, mice treated with empty MD007 lost slightly less weight than untreated mice, suggesting a small probiotic activity of the strain. 5.2 / Dissection of mice after euthanasia

[00130] The abdominal cavity of the sacrificed mice was opened and the colon was removed, opened longitudinally and washed of feces.

[00131] The colon was then separated longitudinally into pieces for subsequent experiments. 5.3 / Intestinal permeability assay

[00132] Three hours before the euthanasia of the mice, Petition 870250088303, dated 09 / 29 / 2025, pp. 108 / 132 36 / 43 of these mice received a fluorescent marker via gavage: fluorescein isothiocyanate (FITC)-dextran (12 mg per mouse in 200 μl of PBS). Immediately before euthanasia, blood was collected in the presence of heparin via submandibular vein puncture. The collected whole blood was then centrifuged to recover the plasma. Measuring the fluorescence intensity allowed the determination of the FITC-dextran concentration in the plasma samples. For this purpose, a fluorescence reading was performed with a spectrofluorometer (excitation wavelength = 488 nm, emission reading = 520 nm) in a microplate, using 80 μl of plasma sample.

[00133] The results are shown in Figure 3.

[00134] It is known that DNBS injection causes impairment of intestinal tight junctions, resulting in increased barrier permeability, which favors the passage of FITC-dextran from the gastrointestinal tract into the bloodstream. Significantly lower levels of FITC-dextran were quantified in plasma samples from mice treated with MD007 + Mn1C compared to untreated mice. This demonstrates the efficacy of MD007-vectored Mn1C in significantly attenuating DNBS-induced intestinal permeability damage. Example 6 - Additional experiments with the L. plantarum MD007 strain 6.1 / Viability of loaded bacteria and quantification of Mn in bacteria

[00135] Three independent experiments are performed as described below. The results shown are the average of these three experiments.

[00136] The Lactobacillus plantarum MD007 strain is cultivated in MRS containing D-alanine (200 μg / mL) at 37 °C. Petition 870250088303, dated 09 / 29 / 2025, pp. 109 / 132 37 / 43

[00137] An overnight culture is used to inoculate the fresh medium to obtain an OD600nm of 0.2, and the cells are cultured to an OD600nm of 0.7 and washed three times with MRS medium without D-alanine. The washed bacteria are incubated in medium without D-alanine for 3 hours at 37°C before the addition of SOD mimic. After alanine deprivation, the bacteria are washed three times with Hepes (0.1 M, pH 7.4) to achieve a concentration of approximately 109 bacteria per 200 µL.

[00138] After removing the supernatant from the culture medium, the bacteria are incubated with either Hepes (0.1 M, pH 7.4) or with the tested compounds (Mn1 or Mn1C) at the desired concentration in the same buffer for two or twenty-four hours at 37 °C. Solutions of Mn1C and Mn1 are prepared extemporaneously to avoid manganese degradation and oxidation reactions. After centrifugation and washing of the cultures with Hepes (0.1 M, pH 7.4), the bacteria are resuspended in 0.1 M Hepes containing 16% glycerol to achieve a concentration of approximately 10⁹ bacteria per 200 µL. This volume corresponds to the maximum and usual volume administered by gavage to mice daily. The bacteria are stored at -80 °C in 2 mL aliquots to avoid freeze-thaw cycles. Samples intended for gavage in mice are frozen at -80 °C and warmed to room temperature before being administered to the mice.

[00139] To assess bacterial viability, overnight cultures and frozen flasks are controlled by the CFU (colony-forming unit) test, diluting the preparation in PBS by serial dilution of 1 / 10 and seeding a known volume of the dilutions onto the corresponding medium. The plates are incubated at 37 °C for 48 h. The colonies are then counted by visual observation. The results obtained are shown in Figure 4. It can be observed that the bacterial load with Mn1 or Mn1C does not affect viability. Petition 870250088303, dated 09 / 29 / 2025, pp. 110 / 132 38 / 43 cell phone.

[00140] Manganese quantification in bacterial lysates is performed by ICP-MS. For ICP-MS analysis, bacterial pellets recovered by centrifugation are acidified in 2% HNO3 to lyse cells and release Mn from all coordination sites. Samples are carefully filtered through 0.2 µm filters to remove bacterial residues before analysis. A calibration curve is established using a commercial Mn standard. A concentration range from zero to 100 ppb is generally used for calibration. The results are shown in Figure 5. They show a high manganese content in the bacteria loaded with SOD mimics according to the invention. 6.2 / Treatment of mice

[00141] Male C57BL / 6 mice (6 weeks old) were maintained under specific pathogen-free conditions (SPF). The mice were housed under standard conditions for at least 1 week prior to the experiments for acclimation. All animal studies were conducted in accredited research facilities and approved by local ethics committees, as well as the French government (authorization no.: 16744201807061805486_v2).

[00142] Gavage of these mice with empty or loaded bacteria was initiated the day before DNBS inflammation induction (D-1) to ensure that bacteria were already present in the colon. On D0, mice were anesthetized with an intraperitoneal injection of a mixture of ketamine (75 mg / kg, Imalgene, Boehringer Ingelheim Animal Health) and xylazine (9 mg / kg, Rompun, KVP). DNBS (3 mg / mouse in 30% ethanol / PBS) was administered by intrarectal injection using a catheter approximately 3.5 cm into the colon. Bacteria (approximately 10⁹ bacteria / 200 pL), loaded bacteria (MBC), or Hepes were administered daily intragastrically to Petition 870250088303, dated 09 / 29 / 2025, pp. 111 / 132 39 / 43 mice were treated for 4 or 5 days. The amount of CMB administered to the mice was determined so that all mice received the same amount of total manganese, fixed at 150 nmol per dose, which corresponds to a dose ranging from 1x10⁹ to 2x10⁹ bacteria. The amount of bacteria administered to the DNBS MD007 control group was determined to correspond to the maximum number of bacteria administered to the CMB-treated groups. The mice were supervised throughout the experiment with special attention. On the last day (D3 or D4), the mice were sacrificed by cervical dislocation. The study groups are named as follows: a bacteria-free colitis group that received Hepes buffer (“Hepes”), a colitis group that received empty bacteria (“MD007 control”), and 2 CMB-treated colitis groups: “MD007 Mn1” and “MD007 Mn1C”. Each group consisted of 8 mice.The trial was repeated three or four times independently, meaning that, in total, 24 or 32 mice were used for each condition.

[00143] The weight of all mice was monitored over time. The area under the weight monitoring curve between day 0 and day 3 (D0-D3) is shown in Figure 6. As can be seen in this Figure, mice fed internalized SOD by MD007 mimic Mn1 and Mn1C, which allowed limiting the weight loss of the mice and even increasing their weight. 6.3 / Evaluation of colitis severity in treated mice

[00144] After treatment with the loaded bacteria, the abdominal cavity of each euthanized mouse was opened and the colon was removed, opened longitudinally, and washed to remove feces. The macroscopic score was then immediately evaluated. This score assesses the state of the colon taking into account: thickening of the colonic wall, presence of ulcers, hyperemia, adhesion to other intra-tissues. Petition 870250088303, dated 09 / 29 / 2025, pages 112 / 132 40 / 43 abdominal measurements and consistency of colonic contents (indicator of intestinal transit abnormalities). The results obtained are shown in Figure 7. Treatment with Mn1- or Mn1C-laden bacteria significantly improved the macroscopic score.

[00145] The colons were then separated longitudinally into two parts. The right parts were separated laterally into four parts intended for further analysis.

[00146] Lipocalin 2 (LCN-2) concentration was measured in the colon and plasma of mice. For the determination of plasma LCN-2 concentration, centrifuged blood samples (2000 g for 10 min at 20 °C) were used. A commercially available sandwich enzyme immunoassay (Mouse Lipocalin-2 / NGAL DuoSet ELISA; R&D Systems Europe) was used according to the manufacturer's instructions. For the determination of LCN-2 concentrations in the colon, colon supernatants were used with the same kit used for plasma. For this purpose, colon pieces were weighed and mechanically dissociated in 0.5% HTAB buffer (hexadecyltrimethylammonium bromide) using a Precellys® device (Bertin) (3 cycles of 20 s at 10,000 rpm) in the presence of a mixture of 1.4 and 2.8 mm ceramic beads. The samples were then briefly centrifuged and the supernatants were collected.The results obtained are shown in Figure 8 for the concentration of LCN-2 in the colon and in Figure 9 for the concentration of LCN-2 in the serum, respectively. They show that treatment of mice with bacteria carrying Mn1 or Mn1C resulted in a significant decrease in the concentration of lipocalin 2.

[00147] SOD2 gene transcription was quantified in colonic fragments as follows. Total RNA was extracted from colon homogenates using a Qiagen RNeasy kit, according to the instructions of Petition 870250088303, dated 09 / 29 / 2025, pages 113 / 132 41 / 43 manufacturer. β-mercaptoethanol was used as a reducing agent for the irreversible denaturation of RNase enzymes. Reverse transcription was then performed to produce complementary DNA with the oligo(dT)12-18 primer and the SuperScript® II reverse transcriptase enzyme (Invitrogen®). SOD2 gene expression was quantified by quantitative PCR on a StepOne® Real-time PCR system using Takyon® rox Sybr mastermix DTTP blue (Eurogentec). The following primer pair was used: 5'-ATTAACGCGCAGATCATGCA-3' (forward) (SEQ ID No: 1) and 5'-TGTCCCCCACCATTGAACTT-3' (reverse) (SEQ ID No: 2) and the annealing temperature was set to 60 °C.

[00148] SOD2 levels were normalized with those of the GAPDH and TATA box-binding protein (TBP) genes, which are not affected under inflammatory conditions, also quantified by RT-PCR. The results obtained for Mn1C-laden bacteria are shown in Figure 10. It is observed that the SOD2 mRNA level is much lower in mice treated with Mn1C-laden bacteria. Example 7 - Lactococcus lactis strain MG1363

[00149] Lactococcus lactis MG1363, described in Sanders et al., 1995, J. Bacteriol 177: 5254-5260, is used in this experiment. This strain possesses a SOD enzyme. 7.1 / Bacterial Loading

[00150] The strain is grown in M17 glucose at 30 °C.

[00151] An overnight culture is used to inoculate fresh medium in order to obtain an OD600nm of 0.2, and the cells are cultured to an OD600nm of 0.7. The bacteria are washed three times with Hepes (0.1 M, pH 7.4) to achieve a concentration of approximately 109 bacteria per 200 pL.

[00152] The bacteria are loaded with Mn1 using the protocol described in Example 6. Petition 870250088303, dated 09 / 29 / 2025, pages 114 / 132 42 / 43 7.2 / Evaluation of bacterial neutrality in relation to DNBS-induced colitis

[00153] Male C57BL / 6 mice (6 weeks old), as described in Example 6, were used in this experiment.

[00154] On the first day of the trial (D0), mice were anesthetized with 0.06% ketamine and xylazine by intraperitoneal (IP) injection. A DNBS solution (3 mg / mouse) in 30 / 70 ethanol / PBS v:v was administered by intrarectal (IR) injection using a catheter approximately 3.5 cm into the colon. Bacterial suspensions (10⁹ bacteria / 200 pL) or Hepes (100 pL) were administered daily intragastrically to mice for 5 days. Mice were closely monitored throughout the experiment. On the last day (D5) or the following day (D6), mice were sacrificed by cervical dislocation.

[00155] The weight of all mice was monitored over time. The evolution of the mice's weight throughout the assay, from day 0 to day 5, is shown in Figure 11. The areas under the curves are shown in Figure 12. As can be seen in these Figures, administration of empty MG1363 to mice with DNBS-induced colitis does not significantly affect the weight of the mice.

[00156] The macroscopic score of the mice was evaluated as described in Example 6, on day 5 or day 6. The results are shown in Figure 13. It is observed that the bacterium MG1363 has no effect on DNBS-induced colitis. 7.3 / Evaluation of Mn1-laden MG1363 bacteria in relation to DNBS-induced colitis

[00157] On the first day of the trial (D0), mice were anesthetized with 0.06% ketamine and xylazine by intraperitoneal injection and DNBS (3 mg / mouse) and a 30% DNBS solution (Sigma) Petition 870250088303, dated 09 / 29 / 2025, pp. 115 / 132 43 / 43 ethanol / PBS were administered by intrarectal injection using a catheter approximately 3.5 cm into the colon. A control group without colitis received only Hepes and is called the “Vehicle” group. Bacteria (10⁹ bacteria / 200 pL), loaded bacteria (CMB), or Hepes were administered daily intragastrically to mice for 5 days. The amount of CMB given to the mice was determined so that all mice received the same amount of total manganese, fixed at 150 nmol per dose. The mice were supervised throughout the experiment with close attention.

[00158] The weight of all mice was monitored over time. The weights of the mice on day 5 are shown in Figure 14. These results demonstrate that administration of Mn1-laden MG1363 bacteria to mice with DNBS-induced colitis increases the weight of the mice, indicating an improvement in mouse health. This improvement is not obtained with empty bacteria. 8 / Statistical analysis (Example 6 and Example 7)

[00159] The statistical analysis was completed using the software GraphPad Prism (GraphPad Software). Results are presented as scatter plots with means ± SEM. P-values ​​were calculated using the non-parametric Mann-Whitney test (one-tailed test). Outliers found using Prism software were discarded. The mean rankings of each column were compared to those of the DNBS Hepes control; each comparison is independent. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01 and *: p < 0.05 versus control or DNBS Hepes vehicle, and ns means not significant. A p-value less than 0.05 was considered significant. Petition 870250088303, dated 09 / 29 / 2025, pp. 116 / 132

Claims

1 / 5 Claims 1. CHEMICALLY MODIFIED MICROORGANISM, characterized by being selected from bacteria and yeasts, containing a synthetic inorganic complex that mimics superoxide dismutase, for use as a medicine or food supplement.

2. CHEMICALLY MODIFIED MICROORGANISM for use, according to claim 1, characterized in that the inorganic complex is a mimic of human manganese superoxide dismutase.

3. CHEMICALLY MODIFIED MICROORGANISM for its use, according to any one of claims 1 to 2, characterized in that the metal of said inorganic complex is manganese (II) or manganese (III).

4. CHEMICALLY MODIFIED MICROORGANISM for use, according to any one of claims 1 to 3, characterized by the inorganic complex having the general formula (I): wherein n, mep, identical or different, are integers between 1 and 3, R1 represents a hydrogen atom or a linear, branched and / or cyclic alkyl, optionally substituted by at least one aromatic ring or polycycle, or Ri represents a peptide comprising from 1 to 20 residues Petition 870250088303, dated 09 / 29 / 2025, p. 117 / 132 2 / 5 of amino acids, R2, R2', R3, R3' all represent a hydrogen atom, or R2' and R3' represent a hydrogen atom and R2 and R3 form, together with the carbon atoms to which they are attached, an aliphatic ring or heterocycle, optionally substituted by a Lewis base, a phenol group, an imidazole ring, a pyridine ring and / or by one or more identical or different alkyl groups, each of the alkyl groups optionally substituted by a Lewis base,A phenol group, an imidazole ring and / or a pyridine ring, or R2, R2', R3, R3' form, together with the carbon atoms to which they are attached, an aromatic ring or heterocycle, optionally substituted by a Lewis base, a phenol group, an imidazole ring, a pyridine ring and / or by one or more identical or different alkyl groups, each of the alkyl groups optionally substituted by a Lewis base, a phenol group, an imidazole ring and / or a pyridine ring, Ar1 and Ar2, which may be identical or different, each representing a 5- or 6-membered aromatic heterocycle, optionally substituted by one or more identical or different substituents, each of which is selected from a bromine atom, a chloride atom, a methyl group, a methoxy group and a nitro group, R4 representing a phenyl group, optionally substituted by one or more identical or different substituents, each of which is selected from an atom of bromine,a chloride atom, a methyl group, a methoxy group and a nitro group, or R4 represents a group of formula -R5-CO-, in which the carbonyl group is bonded to the oxygen atom and R5 is bonded to the -(CH2)p- group, and R5 represents a linear, branched and / or cyclic alkyl, optionally substituted by a Lewis base. Petition 870250088303, dated 29 / 09 / 2025, p. 118 / 132 3 / 5, 5. CHEMICALLY MODIFIED MICROORGANISM for its use, according to claim 4, characterized by the inorganic complex having the general formula (IV): (IV) wherein Ri, R2, R2', R3 and R3' are as defined in claim 4.

6. CHEMICALLY MODIFIED MICROORGANISM for its use, according to claim 5, characterized in that said inorganic complex has the formula (VII): L- -I (VII).

7. CHEMICALLY MODIFIED MICROORGANISM for its use, according to any one of claims 1 to 6, characterized by being a lactic acid bacterium, in particular of the genera Lactobacillus or Lactococcus. Petition 870250088303, dated 09 / 29 / 2025, pp. 119 / 132 4 / 5 8. CHEMICALLY MODIFIED MICROORGANISM for use, according to any one of claims 1 to 7, characterized by the cell wall of which has been genetically, chemically and / or enzymatically weakened.

9. A CHEMICALLY MODIFIED MICROORGANISM for its use, according to claim 8, characterized by being auxotrophic for an amino acid and having been cultivated in a culture medium devoid of said amino acid for at least 1 hour.

10. CHEMICALLY MODIFIED MICROORGANISM for use according to any one of claims 1 to 9, characterized by being effective against oxidative stress.

11. A CHEMICALLY MODIFIED MICROORGANISM for use according to any one of claims 1 to 10, characterized by being for treating an inflammatory disease.

12. CHEMICALLY MODIFIED MICROORGANISM for its use, according to claim 11, characterized by being for treating an inflammatory bowel disease.

13. CHEMICALLY MODIFIED MICROORGANISM for its use, according to any one of claims 1 to 12, characterized in that the chemically modified microorganism is administered orally to a subject.

14. CHEMICALLY MODIFIED MICROORGANISM for use according to any one of claims 1 to 13, characterized in that it is for treating a subject that is a mammal, preferably a human.

15. PHARMACEUTICAL COMPOSITION, characterized by containing a chemically modified microorganism, as defined in any one of claims 1 to 9, in a pharmaceutically suitable vehicle.

16. NUTRACEUTICAL COMPOSITION, food composition or dietary supplement, characterized by containing a chemically modified microorganism, as defined in any of claims 1 to 9, in a physiologically acceptable vehicle. Petition 870250088303, dated 09 / 29 / 2025, pp. 120 / 132 5 / 5

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