Chemically modified microorganism containing superoxide dismutase simulated inorganic complex and application of chemically modified microorganism in treatment of inflammatory diseases

Abstract

The present invention relates to a chemically modified microorganism selected from bacteria and saccharomycetes, said chemically modified microorganism comprising a superoxide dismutase mimetic inorganic complex, in particular a human manganese superoxide dismutase mimetic. Such microorganisms are particularly suitable for the treatment of inflammatory diseases by oral administration, in particular for inflammatory bowel diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery, specifically relating to the intestinal delivery of antioxidant drugs for combating oxidative stress.

[0002] More specifically, the present invention relates to the use of chemically modified microorganisms containing superoxide dismutase (SOD) mimics as pharmaceuticals or dietary supplements for combating oxidative stress, and more specifically for treating inflammatory diseases. The invention also relates to pharmaceutical compositions comprising such microorganisms, as well as nutritional supplement compositions, food compositions, or dietary supplements. Background Technology

[0003] Superoxide dismutase (SOD) is a metalloenzyme involved in cellular antioxidant defense. In the body, SOD is responsible for maintaining superoxide, a reactive oxygen species (ROS), at strictly controlled levels and helps prevent oxidative stress states known to be associated with a variety of diseases. All SODs contain a metal cation at their active site, which cycles between two redox states, successively reducing superoxide to H₂O₂ and then oxidizing H₂O₂ to O₂. Based on the metal contained in their active sites, three different eukaryotic SODs in the human body are described: two copper / zinc SODs, namely SOD1, which exists in the cytosol and mitochondrial intermembrane space, and SOD3, which exists in the extracellular matrix and cell surface in the extracellular environment; and manganese SOD (SOD2), also known as MnSOD, which exists in the mitochondrial matrix (Weisiger and Fridovich, 1973, *Journal of Biochemistry* 248:4793-4796).

[0004] Inflammatory bowel disease (IBD) is a chronic disease characterized by chronic inflammation of parts of the gastrointestinal tract. This category includes Crohn's disease and ulcerative colitis, which differ at least in the location of inflammation. Both diseases can cause diarrhea, bloody stools, anemia, weight loss, and abdominal pain. Due to its particularly high and continuously rising prevalence in developed countries, IBD has become a global health problem, with a lifetime cumulative prevalence of 1% in Europe. The estimated number of IBD cases worldwide is 6.8 million. Currently, there is no cure for IBD; patients mostly receive medication to control inflammation, thereby alleviating symptoms and preventing acute exacerbations. The main treatments include corticosteroids and immunomodulators, the latter including small molecules and monoclonal antibodies targeting specific inflammatory pathways or cytokines (Paramsothy et al., 2018, *Mucosal Immunology* 11: 1558-1570). However, these expensive drugs are not always effective and can cause loss of drug response and adverse reactions. Therefore, there is an urgent need to develop new, safe, and effective alternative treatments to combat IBD.

[0005] Studies have shown that inflammatory bowel disease (IBD) is often accompanied by overexpression of the inactive form of MnSOD in the gut, while cytoplasmic copper / zinc SOD is underexpressed (Kruidenier et al., 2003, *Journal of Pathology* 201: 7-16). These deficiencies in the SOD antioxidant system may lead to or at least exacerbate oxidative stress observed in IBD, and oxidative stress is known to be closely associated with chronic inflammation (Krzystek-Korpacka et al., 2020, *Diagnostics* 10: 601). Therefore, SOD-based antioxidant therapy has become a promising treatment for IBD. Studies have shown that MnSOD can effectively reduce lipid peroxidation and neutrophil infiltration, and simultaneously alleviate the inflammatory response in a mouse colitis model induced by dextran sulfate sodium (DSS) and trinitrobenzenesulfonic acid (TNBS) (Naito et al., 2005, CDTIA 4: 511-515). WO 2020 / 050460 discloses a composition for treating IBD comprising a mutant strain of Bacillus amyloliquefaciens, with high yields of its SOD enzyme and / or SOD enzyme purified from this strain after incubation with manganese salts. However, the application of these purified enzymes as therapeutic agents is limited by their short half-life, triggered immunogenicity, and low cell permeability. Summary of the Invention

[0006] To overcome these shortcomings, synthetic low-molecular-weight SOD mimics were tested as candidate drugs for IBD management. These synthetic low-molecular-weight SOD mimics, also known as SOD analogs, can mimic SOD activity, i.e., the ability to catalyze superoxide dismutase (SOD) reactions. Several synthetic SOD-mimicking antioxidant inorganic complexes have been reported, including iron, copper, zinc, and manganese complexes. The molecular weights of these synthetic inorganic complexes are typically less than 10 kDa, and often less than 5 kDa. If manganese is released, cells are more tolerant to it, and manganese does not catalyze the Fenton or Haber-Weiss reactions, which form highly reactive and toxic H2O. • Because of its free radicals, manganese complexes are significantly more advantageous than copper, iron, and nickel complexes.

[0007] The literature introduces many manganese-based SOD mimics, including complexes containing ligands such as salon derivatives, cyclic polyamines, 1,2-ethylenediamine central ligands, porphyrins, and phthalocyanines (Vincent et al., 2021, Journal of Inorganic Biochemistry 219:111431).

[0008] For example, it has been reported that analysis of weight changes and macroscopic scores has demonstrated that in a DBNS-induced colitis mouse model, when administered orally with carbonate buffer, structural formula (V): (V) A manganese complex mimicking SOD (called Mn1) exhibits intracellular anti-inflammatory activity in vivo (Mathieu et al., 2017, Inorganic Chemistry, 56: 2545-2555). However, this activity is relatively limited, which may be attributed to the instability of this inorganic complex in the biological environment. Furthermore, although using alkaline buffers as solvents for metal complexes can improve their stability in acidic environments, such solvents are not suitable for human administration.

[0009] A 2015 paper by Karlsson et al. published in Drug Discovery Today 20:411-421 disclosed the intracellular uptake of the SOD mimic Salen-manganese complex in MnSOD-deficient Cryptococcus neoformans.

[0010] This invention aims to provide a highly effective method for treating inflammatory diseases, particularly IBD. More specifically, this invention relates to the application of synthetic SOD mimics in combating oxidative stress (especially in the treatment of inflammatory diseases), and aims to provide a solution that improves the bioavailability and efficacy of such active ingredients at the site of action (particularly the intestine) compared to existing drug delivery methods for such compounds.

[0011] The inventors have now discovered that the aforementioned objective can be achieved by using microorganisms as carriers for targeted delivery of SOD mimics within the gut. Specifically, the inventors have discovered a synthetic inorganic complex that accumulates within microbial cells, protecting the inorganic complex contained within these cells from contact with highly acidic gastric juices when these cells are orally administered and pass through the subject's stomach, thereby preventing such contact from triggering dissociation in other ways. Therefore, when the cells reach their site of action in the gut, the majority of the inorganic complex administered to the subject retains its integrity.

[0012] Therefore, during the process of microbial cells traveling in the gastrointestinal tract, the protected inorganic complexes remain intact and fully retain their superoxide dismutase catalytic activity when they reach the intestinal site of action.

[0013] Therefore, the primary objective of this invention is to provide a chemically modified microorganism, namely a microorganism modified by introducing chemical compounds selected from bacteria and yeast, and containing synthetic inorganic complexes as superoxide dismutase mimics, which can be used as a medicine or dietary supplement.

[0014] The preparation of this chemically modified microorganism is particularly simple, requiring only that the inorganic complex passively penetrates the cell wall and accumulates within the cell, wherein the SOD mimic is carrier-based.

[0015] When subjects orally ingest this chemically modified microorganism, its cell wall effectively protects the inorganic complex from the acidic conditions of the stomach, preventing its dissociation and degradation, thus allowing it to be delivered to the intestine with intact biological activity, particularly its ability to fully correct intestinal hyperpermeability caused by colitis. This biological activity has been specifically demonstrated through assays in a mouse model of acute colitis.

[0016] For the intended therapeutic application of the chemically modified microorganisms of the present invention, it is desirable to prefer microorganisms and inorganic complexes that are harmless to the subject, the application involving the administration of the microorganisms to the subject.

[0017] Therefore, the inorganic complexes of the present invention preferably have pharmaceutical and / or physiological acceptability, i.e., when administered to subjects, they do not produce adverse, allergic or other adverse reactions, specifically mammals, and more specifically humans.

[0018] The microorganisms are preferably non-pathogenic to the intended recipient, specifically non-pathogenic to mammals, and more specifically non-pathogenic to humans. The microorganisms are preferably symbiotic with the recipient.

[0019] This invention specifically prefers food-grade bacteria and yeasts. The food-grade microorganisms described in this invention refer to: - Microorganisms listed in the European Food Safety Authority (EFSA) "Qualified for Safety (QPS)" list, with the most recent update ("2022 QPS List"), which can be found in the Zenodo platform's Knowledge Junction section (https: / / doi.org / 10.5281 / zenodo.1146566) - EFSA has developed guidelines for the safety assessment and characterization of microorganisms (including probiotics) used as feed additives or in the production (i.e., delivery) of organisms (EFSA Animal Feed Additives and Products or Substances Group). - and / or microorganisms listed in the U.S. Food and Drug Administration’s “Generally Recognized As Safe (GRAS)” document issued on September 6, 2019, with a search date of January 30, 2021.

[0020] The microorganisms used in this invention may lack SOD enzymes or may produce them.

[0021] In a conventional manner, the superoxide dismutase (SOD) mimicry in this invention refers to a synthetic low-molecular-weight compound capable of catalyzing the dismutation of superoxide in an aqueous medium, particularly a compound with a molecular weight less than 10 kDa and possibly less than 5 kDa, i.e., a compound possessing antioxidant damage protection similar to SOD. More specifically, the SOD mimicry can catalyze redox cycles, converting O2... -Oxidized to O2, while simultaneously turning O2 - It is reduced to H₂O₂. This indicates that the redox potential of the redox couple involved is between -0.18V / NHE and 0.89V / NHE (pH 7).

[0022] Identifying inorganic complexes that act as SOD analogs falls within the skill scope of those skilled in the art.

[0023] To date, numerous such complexes, particularly manganese complexes, have been reported in the art. These complexes involve ligands such as salon derivatives, cyclic polyamines, tridentate or bidentate nitrogen-centered ligands, 1,2-ethylenediamine-centered ligands, deferoxamine derivatives, polyaminocarboxylic acid or polycarboxylic acid ligands, polypeptides, porphyrins, phthalocyanines, telsafuran, carboxylic acid, or biliverdin and their derivatives. Vincent et al., in their 2021 article published in the *Journal of Inorganic Biochemistry* 219, 111431, specifically cited examples of SOD mimics usable according to the present invention.

[0024] In other respects, to determine whether a given inorganic complex is a SOD mimic, i.e., whether a given inorganic complex possesses intrinsic SOD activity, those skilled in the art can employ the so-called McCord-Fridovich indirect assay, a method commonly used to identify SOD mimics via superoxide dismutation reaction assays, as described by McCord and Fridovich in their 1969 article published in the *Journal of Biochemistry* 244(22), 6049-6065. According to the diagram, this assay involves determining the catalytic rate (kJ) of superoxide in HEPES buffer (e.g., 50 mM, pH 7.4). cat The inorganic complexes to be tested competed with redox markers such as ferrocytochrome c, 2,3-bis(2-methoxy-4-nitro-5-sulfonic acid phenyl)-2H-tetrazole-5-carboxyaniline (XTT), or nitrotetrazole blue (NBT) at a concentration of 100 μM, thereby reacting with superoxide continuously generated by the enzymatic xanthine (200 μM) / xanthine oxidase system. The reduction of the redox markers was monitored by UV-Vis spectroscopy for different amounts of the inorganic complexes; for example, for ferrocytochrome c, monitoring was performed at a wavelength of 550 nm. The reduction rate was simultaneously determined with and without the added inorganic complex for each amount. 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 inorganic complex, then the inhibition percentage of each inorganic complex is given by I (%) = (s1 - s2) / s1 × 100. IC can be calculated when I = 50%. 50The value of is the concentration of the inorganic complex required to induce 50% inhibition of the redox marker reduction; furthermore, (s1 - s2) / s2 can be linearly fitted with the inorganic complex concentration, and the two show a linear correlation. Then, when (s1 - s2) / s2 = 1, the IC can be calculated. 50 Then, according to the following equation, from IC 50 and redox markers (k redox ) of k cat Known values ​​(e.g., for XTT, k at pH 7.4) XTT = 2.9×10 4 M -1 .s -1 The k of the inorganic coordination compound was calculated. cat :k cat = k redox × [redox] / IC 50 , Wherein, [redox] represents the concentration of the redox marker in the measurement medium, for example, 100 μM.

[0025] When the kinetic rate (i.e., k) of the superoxide dismutation reaction catalyzed by a certain inorganic complex was measured in the above determination... cat When the rate of spontaneous disproportionation (self-disproportionation, without catalyst) of superoxide is greater than that of superoxide under the same pH conditions, the inorganic complex can be identified as a SOD mimic, as shown in the literature data published by Bielski et al. in the Journal of Physical and Chemical Reference Data 14(4), 1041-1100 in 1985, for example, the rate is greater than 6.10 at pH 7. 5 M -1 .s -1 .

[0026] To ensure the reliability of this assay, and in particular to confirm that the inorganic complex being tested does not inhibit superoxide formation by inhibiting xanthine oxidase reaction, a control experiment can be performed as described in the article by Durot et al. published in the European Journal of Inorganic Chemistry, 2005, 17, 3513-3523.

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

[0028] More generally, the metal in the inorganic complex is preferably manganese (II) or manganese (III), and specifically, these manganese ions have the advantage of not increasing oxidative stress when released in an organism compared to other metals such as iron, copper or nickel.

[0029] Any SOD analog inorganic complex described in the prior art, especially any manganese-based SOD analog, falls within the scope of this invention. In particular, complexes having ligands such as salon derivatives, cyclic polyamines, tridentate or bidentate nitrogen-centered ligands, 1,2-ethylenediamine-centered ligands, deferoxamine derivatives, polyaminocarboxylic acid or polycarboxylic acid ligands, polypeptides, porphyrins, phthalocyanines, telsafuran, carboxylic acid, or biliverdin and their derivatives, as described in the aforementioned document by Vincent et al., 2021.

[0030] As an example, the microorganisms of the present invention may include: - 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-eicoserohydro-11,7-nitroso-7H-dibenzo[b,h][1,4,7,10]tetraazacycloheptadecene-kN5,kN13,kN18,kN21,kN22]manganese(II) (imipammonium), a specific pentaazacycloheptadecene (cyclic polyamine); - as a manganese(III) porphyrin: manganese(III) meso-tetra(N-ethylpyridin-2-yl)porphyrin (MnTE-2-PyP) 5 + Manganese(III) meso-tetra(N-(2'-butoxyethyl)pyridin-2-yl)porphyrin (MnTnBuOE-2-PyP) 5+ Manganese(III) 5,10,15,20-tetra(4-benzoic acid)porphyrin (MnTBAP); -manganese(III) 2,2'-[1,2-ethylenedimethylbis(nitrosomethylimine)]bis[6-methoxyphenol); -etc.

[0031] In a specific embodiment of the present invention, the inorganic complex is inspired by the active site of MnSOD and is based on 1,2- N , N’ The inorganic complex is constructed with a central skeleton of diaminoethane and ligands functionalized with Lewis bases. The diamino structure is rigidly fixed via a carbonyl ring, thereby limiting manganese release. Specifically, the inorganic complex may have the general formula (I): (I) in: n, m, and p may be the same or different, and are integers between 1 and 3. Ideally, n, m, and p should each be equal to 1. R1 represents a hydrogen atom or a straight-chain, branched, and / or cyclic alkyl group, wherein the hydrogen atom or straight-chain, branched, and / or cyclic alkyl group preferably has 1-12 carbon atoms, particularly 2-6 carbon atoms, wherein the carbon atoms may be substituted by at least one (i.e., one or more) aromatic rings or polycyclic rings, wherein the aromatic rings or polycyclic rings preferably have 6-14 carbon atoms, such as anthracene, or R1 represents a peptide comprising 1-20 (particularly 1-9) amino acid residues. R2, R2', R3, and R3' all represent hydrogen atoms. R2' and R3' may represent hydrogen atoms, and R2 and R3, together with the carbon atoms attached to them, constitute an aliphatic ring or heterocycle, particularly a 5-membered, 6-membered, 7-membered, or 8-membered ring or heterocycle. The aliphatic ring or heterocycle may be substituted with a Lewis base, a phenolic group, an imidazole ring, a pyridine ring, and / or one or more identical or different alkyl groups. Each alkyl group preferably has 1-12 carbon atoms, particularly 1-6 carbon atoms, and each alkyl group may be substituted with a Lewis base, a phenolic group, an imidazole ring, and / or a pyridine ring. R2, R2', R3, R3', and the carbon atoms attached to them together form an aromatic ring or heterocycle, particularly a 5-membered, 6-membered, 7-membered, or 8-membered ring or heterocycle. The aromatic ring or heterocycle may be substituted with a Lewis base, a phenolic group, an imidazole ring, a pyridine ring, and / or one or more identical or different alkyl groups. Each alkyl group preferably has 1-12 carbon atoms, particularly 1-6 carbon atoms. Each alkyl group may be substituted with a Lewis base, a phenolic group, an imidazole ring, and / or a pyridine ring. Ar1 and Ar2 can be the same or different, each Ar1 and Ar2 representing a 5-membered or 6-membered aromatic heterocycle, wherein the aromatic heterocycle preferably includes one or two heteroatoms, particularly one or two nitrogen atoms, such as pyrrole, imidazole, pyridine, or pyrimidine rings, and the aromatic heterocycle may be substituted by one or more identical or different substituents, each substituent being selected from bromine, chlorine, methyl, methoxy, and nitro groups. R4 represents a phenyl group, which may be substituted with one or more identical or different substituents, each substituent being selected from bromine, chlorine, methyl, methoxy, and nitro groups. Alternatively, R4 represents a group with the formula -R5-CO-, where the carbonyl group is attached to an oxygen atom, and R5 is attached to -(CH2). p - Groups are linked together, R5 represents a straight-chain, branched and / or cyclic alkyl group, wherein the straight-chain, branched and / or cyclic alkyl group preferably has 1-3 carbon atoms, and the carbon atoms may be substituted by Lewis bases.

[0032] In this invention, a Lewis base refers to a group capable of donating a pair of electrons to an electron acceptor such as manganese(II) to form a non-permanent coordinate covalent bond. Examples of Lewis bases that may be included in the inorganic compounds of this invention include those with the structural formulas -OH, -COOH, -SH, -NH2, and -NHR.a -NR a (R b ) groups, wherein R a and R b They may be the same or different, and each represents a C1-C3 alkyl, alkenyl, or phenyl group.

[0033] All stereoisomers, diastereomers, and enantiomers of inorganic complexes having the above general formula (I), as well as isomers having all the structural formulas below, are within the scope of this invention. These isomers can be used alone or in any mixture thereof. Specifically, they are respectively used with C a Atoms and C b The atomically bonded nitrogen atoms may be in a cis configuration. They are preferably in a trans configuration. Any salt of an inorganic complex having structural formula (I) or any of the following structural formulas is within the scope of this invention.

[0034] Inorganic complexes having structural formula (I) may meet one or more of the following characteristics, which may exist in any combination.

[0035] For example, R1 can be n-propyl.

[0036] In a specific embodiment of the present invention, R1 represents a hydrogen atom when at least R2 and R3 do not represent hydrogen atoms.

[0037] When Ar1 or Ar2 represents a substituted 6-membered aromatic heterocycle (such as a pyridine or pyrimidine ring), it is relative to -(CH2). m -or-(CH2) n - The nitrogen atom at the position of the carbon atom connected to it, wherein the substituent is preferably located in the ortho position, more preferably in the para position (i.e., relative to the nitrogen atom coordinated with manganese (II)).

[0038] When Ar1 or Ar2 represents an imidazole ring, at least one substituent is bonded to a nitrogen atom, except for the nitrogen atom coordinated to manganese(II).

[0039] When R4 represents a phenyl group, this group is preferably attached to a carbon atom (-(CH2)) relative to the carbon atom bonded to the oxygen atom. p - A certain position on.

[0040] The inorganic complexes of the present invention may have the general formula (II): (II) The definitions of m, n, p, R1, R2, R2', R3, R3', Ar1, and Ar2 are as described above.

[0041] Specifically, the inorganic complex may have the general formula (IIa): (IIa) The definitions of m, n, R1, R2, R2', R3, R3', Ar1, and Ar2 are as described above.

[0042] Preferably, in the above formulas (II) and (IIa), m equals 1 and / or n equals 1.

[0043] In a specific embodiment of the present invention, the inorganic complex has the general formula (III): (III) The definitions of m, n, p, R1, R2, R2', R3, R3', and R4 are as described above.

[0044] Specifically, the inorganic complex may have the general formula (IIIa): (IIIa) The definitions of p, R1, R2, R2', R3, R3', and R4 are as described above.

[0045] Preferably, in equations (III) and (IIIa) above, p equals 1.

[0046] In a specific embodiment of the present invention, the inorganic complex has a coordination layer, which is composed of two imidazole rings and a phenoxy ion.

[0047] The inorganic complex preferably has the general formula (IV): (IV) The definitions of R1, R2, R2', R3, and R3' are as described above.

[0048] The specific inorganic complex having structural formula (IV) is manganese(II)-[ having the above structural formula (V). N -(hydroxybenzyl)- N , N’ [-bis[2-(N-methylimidazolyl)methyl]-ethane-1,2-diamine], referred to in this invention as Mn1. Specifically, Mn1 exhibits distinct anti-superoxide anion activity when removed from the cellular environment, and possesses antioxidant and anti-inflammatory effects on intestinal epithelial cells and macrophages. As mentioned above, Mn1 can also alleviate DNBS-induced colitis in mouse models, particularly based on changes in body weight. The kJ of Mn1 obtained by the McCord-Fridovich assay described above... cat It is 7.10 6 M -1 .s -1 .

[0049] In other respects, the inorganic complexes of the present invention may have one of the structural formulas (VI), (VII) and (VIII): (VI) (VII) (VIII).

[0050] For the purposes of this invention, inorganic complexes with a further rigidified 1,2-diaminoethane group (here, a 6-membered cyclohexyl group is used as an example, i.e., the complexes of the above structural formulas (VII) and (VIII)) are particularly preferred, and the inorganic complexes are significantly more inert in the biological environment than Mn1.

[0051] The microorganisms themselves are selected from bacteria and yeasts, preferably from such microorganisms that lack pro-inflammatory activity. In specific embodiments of the invention, the microorganisms have anti-inflammatory effects. For the purposes of this invention, probiotics with naturally beneficial properties (especially anti-inflammatory properties) can be used. The World Health Organization (WHO, 2001) defines probiotics as "live microorganisms that, when ingested in adequate amounts, have a beneficial effect on the health of the host." This definition has recently been reviewed and confirmed by an expert panel (Hill et al., Expert Consensus Document: International Society for the Study of Probiotics and Prebiotics (ISAPP) Consensus Statement on the Scope and Correct Use of the Terminology "Probiotics". Nature Reviews Gastroenterology & Hepatology 2014, 11, 506-514; Salminen et al., International Society for the Study of Probiotics and Prebiotics (ISAPP) Consensus Statement on the Definition and Scope of Postbiotics. Nature Reviews Gastroenterology & Hepatology 2021, 18, 649-667).

[0052] Furthermore, for the purposes of this invention, microorganisms whose cell walls have good permeability to inorganic complexes are preferred.

[0053] The microorganisms of this invention can be yeasts, such as Saccharomyces boulardii (Saccharomyces boulardii). Saccharomyces boulardii ) or Yersinia lipophila ( Yarrowia lipolytica ) bacterial strains.

[0054] In an alternative embodiment of the invention, the microorganism is selected from bacteria, which has the advantages of being easy to handle and growing rapidly.

[0055] Since high manganese content is beneficial for promoting the persistent presence of manganese in cells in the form of ligand coordination, for the purposes of this invention, naturally manganese-rich bacteria, i.e. bacteria with a manganese content of about 1 mM or higher, are particularly preferred.

[0056] In a specific embodiment of the present invention, the microorganism is lactic acid bacteria, particularly Lactobacillus (Lactobacillus genus). Lactobacillus) or Lactococcus spp. Lactococcus These bacteria possess the advantages of being able to survive over a wide pH range (especially in the acidic medium of the gastrointestinal tract), having good permeability to inorganic complexes, and having a high manganese content. These advantages promote enhanced ligand-metal coordination, maximizing the number of inorganic complexes assembled within the cell.

[0057] The bacteria can be selected from the genus Lactobacillus ( Lactobacillus Lactococcus spp. Lactococcus Bifidobacterium spp. Bifidobacterium ) or Bacillus spp. ( Bacillus Specifically, Lactobacillus plantarum is preferred. Lactobacillus plantarum ) and Lactococcus lactis ( Lactococcus lactis ) bacterial strains.

[0058] The bacteria can also be Escherichia coli species that are non-pathogenic to mammals (especially humans).

[0059] In a preferred embodiment of the invention, the modified microorganisms improve the targeted and efficient release of encapsulated inorganic complexes into suitable areas, thereby achieving better therapeutic effects, particularly at sites of inflammation in the intestine. Preferably, before administering the microorganisms to the subject, their cell walls are weakened, so that after administration of the chemically modified microorganisms of the present invention to the subject, after the chemically modified microorganisms pass through the stomach and reach the intestine, during their intestinal transit, and when the cell walls of the microorganisms have been previously weakened and become brittle, the microorganisms will efficiently lyse and release SOD-mimicking inorganic complexes into the intestine.

[0060] Therefore, in specific embodiments of the present invention, the cell walls of the microorganisms are weakened through genetic, chemical, and / or enzymatic methods. Consequently, these cell walls are more prone to lysis compared to the unweakened original microorganisms. Generally, cells with weakened cell walls grow slower and settle faster than the unweakened control group. Microorganisms with weakened cell walls can be identified using membrane permeability testing kits such as the Baclight® kit to determine the ability of reagents such as propidium iodide to penetrate cells. Microorganisms with weakened cell walls accumulate more reagents than the unweakened original microorganisms.

[0061] Any method of weakening the microbial cell wall known to those skilled in the art without causing microbial death is within the scope of this invention. The microbial cell wall weakening step can be performed before, during, or after the step of contacting the inorganic complex with the microorganism to promote its penetration. Preferably, the weakening step is performed before this contact step. This will subsequently increase the permeability of the inorganic complex in the microbial cell.

[0062] For example, microorganisms can be cultured for several hours, preferably 6-18 hours, such as about 12 hours, in the presence of cell wall weakening agents such as glycine or threonine.

[0063] In a specific embodiment of the invention, the microorganism is an auxotroph of a given substance, preferably an amino acid, particularly a D-type amino acid, and the microorganism has been cultured in a medium lacking the given substance / amino acid for at least 1 hour, preferably at least 3 hours. This amino acid deprivation induces weakening of the microbial cell wall. The amino acid may be alanine, phenylalanine, or thymidine.

[0064] For example, in the context of this invention, *Lactobacillus plantarum* (…) can be used. Lactobacillus plantarum The strain MD007 was deposited on March 23, 2023, at the French National Center for the Preservation of Microbial Cultures (CNCM), part of the Pasteur Institute (address: 25 Rue du Roux, Paris, France), with accession number CNCM I-5940 (identification reference number LBH791-MD007). This strain was obtained by converting *Lactobacillus plantarum* strain ATCC BAA-793 / NCIMB 8826 into an alanine auxotroph, as described by Palumbo et al. in their 2004 article published in *FEMS Microbiology Letters* 233:131-138. Preferably, this strain is used 1-5 hours after alanine deprivation treatment.

[0065] In other respects, for the purposes of this invention, the Lactococcus lactis strain MG1363 (which has SOD enzyme) can be used.

[0066] According to the present invention, the chemically modified microorganisms can be used as drugs, for treating diseases, or as dietary supplements.

[0067] The term "treatment" as used in this invention refers to achieving the desired pharmacological and physiological effects, which may be preventative or therapeutic. Therefore, the term "treatment" as used in this invention includes: preventing or partially preventing the occurrence of a disease, symptoms, or condition in a subject who has not yet been diagnosed with the disease; and / or partially or completely curing a disease, symptoms, or condition, or adverse reactions caused by the disease.

[0068] The chemically modified microorganisms of this invention can be used to combat oxidative stress. The term "combine" in this invention refers to the prevention or mitigation of oxidative stress.

[0069] The chemically modified microorganisms of the present invention can be used to treat a variety of pathophysiological processes involving oxidative stress, including diabetes, inflammatory and neurodegenerative diseases, ischemia-reperfusion injury, cancer, psoriasis and other skin diseases or any other diseases related to oxidative stress.

[0070] The chemically modified microorganisms of the present invention can be specifically used to treat inflammatory diseases, and more specifically to treat inflammatory bowel diseases such as Crohn's disease or ulcerative colitis.

[0071] Subjects who are currently ill or susceptible to illness and have been treated with the chemically modified microorganisms of this invention are preferably mammals, particularly humans. For example, subjects may also be dogs or cats.

[0072] In a preferred embodiment of the invention, the subject orally ingests the chemically modified microorganism.

[0073] In other respects, the aforementioned chemically modified microorganisms can be applied topically, particularly in the treatment of skin diseases.

[0074] In terms of the medicine, it is preferable to administer the chemically modified microorganism of the present invention to the subject in a therapeutically effective amount, which is the dose of the SOD-mimicking inorganic complex suitable for delivering a therapeutically effective amount to the subject.

[0075] In this invention, "therapeutic effective dose" refers to the dose of a compound administered to a subject that is sufficient to treat the disease. The therapeutic effective dose of a compound depends on various factors, such as the disease and its severity, the age and weight of the subject, the specific compound used, the route of administration, and the dosage form. Therefore, the dose of the chemically modified microorganisms of this invention administered to the subject will be determined by a licensed physician on a case-by-case basis. Specifically, the dose should be sufficiently low to avoid inducing toxicity, such as manganese poisoning when the metal is manganese.

[0076] For example, a dose equivalent to 0.10-0.20 mg (e.g., 0.14 mg) of manganese per kilogram of body weight per day can be administered.

[0077] In the case of dietary supplements, the chemically modified microorganisms of the present invention are preferably administered to subjects at a dose suitable for delivering to subjects a dose of SOD-mimicking inorganic complexes that achieves the desired effects, such as promoting health, improving comfort and / or improving overall condition.

[0078] For example, the chemically modified microorganisms of this invention can be administered to the subject 1-2 times daily. Specifically, during the flare-up of the disease, the microorganisms can be administered to the subject twice daily, for example, once in the morning and once in the evening, and during the remission period, they can be administered once daily.

[0079] This invention can also be described as a therapeutic treatment method for a subject suffering from an inflammatory disease, more specifically, a subject suffering from inflammatory bowel disease such as Crohn's disease or ulcerative colitis. The method includes administering a therapeutically effective amount of the chemically modified microorganism of this invention to the subject in need. This method may have any of the features or combinations thereof described above related to the use of the chemically modified microorganism as a medicine according to this invention. The chemically modified microorganism may be administered to the subject as a pharmaceutical component, a food composition component, a nutritional pharmaceutical composition component, or a dietary supplement component.

[0080] This invention also relates to the use of chemically modified microorganisms in pharmaceutical production, specifically as a treatment for inflammatory diseases, and more specifically as a treatment for inflammatory bowel diseases such as Crohn's disease or ulcerative colitis. This use may conform to any of the features or combinations thereof described above relating to the use of chemically modified microorganisms as pharmaceuticals according to this invention.

[0081] This invention also relates to the use of the chemically modified microorganisms according to the invention in the production of food compositions, nutritional pharmaceutical compositions, or dietary supplements, and more specifically to the use of the chemically modified microorganisms according to the invention in resisting oxidative stress. This use may conform to any of the features or combinations thereof associated with the use of the chemically modified microorganisms according to the invention as pharmaceuticals or dietary supplements.

[0082] This invention also relates to a pharmaceutical composition comprising, in a pharmaceutically suitable solvent, the chemically modified microorganisms as defined above. In this pharmaceutical composition, the active ingredient is an SOD-mimicking inorganic complex encapsulated within microbial cells.

[0083] In this invention, "pharmaceutical-suitable solvent" refers to a solvent that can be used to prepare a pharmaceutical composition or dosage form. The solvent is generally safe and non-toxic, and has no adverse effects on the subjects to be treated (specifically mammals, more specifically humans) in biological and other aspects.

[0084] The solvent for the compositions of the present invention may be a solid, a semi-solid, or a liquid. The solvent may be a diluent, an adjuvant, or any other conventional solvent used to constitute a pharmaceutical composition.

[0085] The chemically modified microorganisms can be contained in any form, especially in a lyophilized form.

[0086] The pharmaceutical compositions of the present invention can be formulated into any pharmaceutical dosage form, particularly dosage forms suitable for administration to mammals, and especially dosage forms suitable for administration to humans. The pharmaceutical compositions are preferably formulated into dosage forms suitable for oral administration, such as powders, capsules, oral solutions, or suspensions, or into dosage forms suitable for topical administration, particularly dosage forms applied to the skin and / or mucous membranes of a subject, such as creams.

[0087] The pharmaceutical compositions of the present invention may include one or more conventional excipients / additives that can be used alone, such as any one of preservatives, sweeteners, flavoring agents, suspending agents, dispersants, lubricants, stabilizers, buffers, or mixtures thereof. The pharmaceutical compositions may also contain one or more other active agents that may or may not synergize with the inorganic complexes of the present invention, such as other anti-inflammatory agents and / or analgesics.

[0088] The present invention also relates to the therapeutic use of the pharmaceutical compositions defined above, specifically for the treatment of inflammatory diseases, and more specifically for the treatment of inflammatory bowel diseases, such as Crohn's disease or ulcerative colitis, in subjects (specifically mammals, more specifically humans).

[0089] This invention also relates to a nutritional pharmaceutical composition, food composition, or dietary supplement comprising chemically modified microorganisms as defined above in a pharmaceutically suitable solvent. In this composition / supplement, the active ingredient is an SOD-mimicking inorganic complex encapsulated within microbial cells.

[0090] In this invention, "physiologically acceptable solvent" refers to a solvent that is physiologically tolerable and will not cause allergies or similar adverse reactions when administered to a subject (specifically, a mammal, more specifically, a human).

[0091] The solvent for the composition / supplement may be solid, semi-solid, or liquid. The solvent may be a diluent, adjuvant, or any other conventional solvent used to constitute a nutritional pharmaceutical composition, food composition, or dietary supplement.

[0092] The chemically modified microorganisms can be contained in any form, especially in a lyophilized form.

[0093] The nutritional pharmaceutical compositions, food compositions, or dietary supplements of the present invention are preferably in dosage forms suitable for oral administration.

[0094] The nutritional pharmaceutical composition, food composition, or dietary supplement may include one or more probiotics, prebiotics, vitamins, polyphenols, minerals, materials suitable for oral administration (liquid or gel type, such as solvents, diluents, non-toxic solubilizers that do not have a harmful effect on the composition components) and / or any other ingredients or excipients, such as proteins, amino acids, carbohydrates, lipids, oligosaccharides, other micronutrients, metal salts, or cations.

[0095] The present invention also relates to the use of the nutritional pharmaceutical compositions, food compositions or dietary supplements as defined above in combating oxidative stress and / or treating inflammation in subjects (particularly mammals, and more particularly humans).

[0096] According to the present invention, a method for producing chemically modified microorganisms includes contacting microbial cells with a composition containing the superoxide dismutase-mimicking inorganic complex, preferably for at least one hour to several hours, thereby allowing the inorganic complex to passively penetrate into and accumulate within the microbial cells. This contact step is preferably performed under the following conditions: - The contact duration is 1-12 hours, preferably 1-5 hours, for example, about 2 hours; - and / or at 37°C; -and / or under oscillating conditions; - and / or in a liquid composition, preferably at a pH between 7 and 8, for example, a 0.1 M solution of 4-[2-(hydroxyethyl)-1-piperazin-1-yl]ethanesulfonic acid (HEPES); - and / or when the optical density of the cell suspension at a wavelength of 600 nm is between 0.6 and 0.8; The concentration of the inorganic complex in the liquid composition is preferably 0.1-5 mM, for example, substantially equal to 0.4 mM. This concentration range is high enough to allow for the penetration of a large amount of the inorganic complex into the microbial cells, while also being low enough to avoid the accumulation of large amounts of other forms of metals in the microbial cells, such as avoiding the accumulation of manganese in the form of MnCl2 when the metal is manganese, while also ensuring non-toxicity to the cells.

[0097] As described above, the method may include a microbial cell wall weakening step before, during, or after (preferably before) the step of contacting microbial cells with a composition containing the superoxide dismutase-mimicking inorganic complex.

[0098] This step can be performed using any method known to those skilled in the art, such as culturing the microorganisms in a medium supplemented with cell wall weakening agents such as glycine (concentration between 10-20 g / L) for 1-12 hours. The culture medium can be any suitable medium for culturing the microorganisms. Specifically, the culture medium includes all nutrients and other elements necessary for the survival of the microorganisms, which may optionally include all nutrients and other elements required for microbial growth.

[0099] In a specific embodiment of the invention where the microorganism is an auxotroph of a given amino acid such as alanine, the method includes a cell wall depletion step of culturing the microorganism in a culture medium lacking the stated amino acid. The culture medium includes all nutrients and other elements necessary for microbial survival and optional growth, such as de Mann-Rogoza-Sharp (MRS) medium, but lacking the stated amino acid. This depletion step can be performed for 1-5 hours, for example, about 3 hours. Preferably, this step is initiated when the optical density of the culture at 600 nm has reached 0.6 and before the microbial cells are contacted with a composition containing a superoxide dismutase-mimicking inorganic complex.

[0100] In all cases, the culturing step is preferably carried out at 37°C.

[0101] An example of the method for preparing chemically modified auxotrophic microorganisms according to the present invention includes the following sequential steps: - Preferably, the microorganisms, especially bacteria, are cultured at 37°C in a medium such as MRS supplemented with auxotrophic substances for the microorganisms until an optical density value between 0.6 and 0.8 at a wavelength of 600 nm is obtained; - Separate the cells from the culture medium, for example by centrifugation, and optionally wash the cells, for example using MRS medium, preferably three times; - Culture cells in MRS medium lacking the substance for 1-5 hours, for example 3 hours (“substance deprivation”). - Separate the cells from the culture medium, for example by centrifugation, and optionally wash the cells with 0.1 M HEPES solution, preferably three times; - Resuspend cells in a liquid solution containing an inorganic complex of SOD (e.g., 0.1 M HEPES solution), preferably at a concentration between 0.1 and 5 mM, such as 0.4 mM; - Incubate the suspension at 37°C for 1-5 hours, for example 2-3 hours, preferably under shaking conditions; - Separate the cells from the liquid culture medium, for example by centrifugation, and optionally wash the cells with 0.1 M HEPES solution; Finally, the cells are recovered, for example, in a 0.1 M HEPES solution containing 16% glycerol.

[0102] The resulting chemically modified cells, containing a large amount of SOD-mimicking inorganic complexes, can then be administered to subjects in need for prophylactic or therapeutic treatment of diseases associated with oxidative stress, particularly inflammatory bowel disease. Administration should preferably be rapid after obtaining the chemically modified cells, more specifically, within hours of obtaining them.

[0103] In other respects, the cells may be used after freeze-drying; or frozen at -80°C in a buffer such as HEPES buffer supplemented with 16% glycerol by volume, and temporarily thawed before administration to the subject to be treated. Attached Figure Description

[0104] The features and advantages of the present invention will be explained through the following embodiments and in conjunction with Figure 1-14 To be more clear, these examples are intended to be illustrative and not limiting of the invention, wherein: - Figure 1 The bar chart shows the manganese concentration in cell lysates of MD007 bacteria without SOD, MD007 bacteria loaded with SOD mimics (Mn1, Mn1C) or MD007 bacteria loaded with MnCl2 as determined by ICP-MS. - Figure 2 The graph shows the changes in body weight over time in mice treated with DNBS injection (day 0) followed by daily oral administration of empty MD007 bacteria, MD007 bacteria loaded with SOD mimic (Mn1C), or MD007 bacteria loaded with MnCl2 (days 1 to 2); body weight is expressed as a percentage of the mice's initial body weight before measurement. - Figure 3 This paper presents quantitative curves of FITC-glucan, a marker of intestinal barrier permeability in mice treated with DNBS injection followed by oral administration of either empty MD007 bacteria, MD007 bacteria loaded with SOD mimicry (Mn1C), or MD007 bacteria loaded with MnCl2. Data represent the mean ± standard deviation of three independent measurements (approximately 24 mice). Since the Anderson-Darling normality test did not validate a Gaussian distribution, the nonparametric Mann-Whitney test (one-tailed test) was used to calculate p-values. The mean rank of each column was compared to the DNBS control group; each comparison was performed independently. **: p < 0.01 compared to the DNBS control group; ns indicates no statistical significance. - Figure 4The bar chart shows the count of live cells in thawed aliquots of MD007 bacteria after cryopreservation, either empty (“HEPES”) or loaded with SOD mimics (Mn1, Mn1C). - Figure 5 The bar chart shows the manganese content in cell lysates of MD007 bacteria without SOD or loaded with SOD mimics (Mn1, Mn1C) as determined by ICP-MS (average of 3 experiments). - Figure 6 The area under the curve (AUC) of body weight monitoring in mice treated with DNBS injection (day 0) followed by daily oral administration of Hepes (“Hepes”), empty MD007 bacteria (“MD007 control”), or MD007 bacteria loaded with SOD mimics Mn1 or Mn1C, from day 0 to day 3 (D0–D3) is shown (data collected from 3 independent assays, 8 mice per assay for each condition). - Figure 7 The macroscopic score charts of mice treated with DNBS injection followed by oral administration of Hepes (“Hepes”), empty MD007 bacteria (“MD007 control group”) or MD007 bacteria loaded with SOD mimics Mn1 or Mn1C for four consecutive days on the day of euthanasia are shown (data collected from three independent assays, with eight mice per assay for each condition). - Figure 8 The graph shows the concentration of lipid carrier protein 2 (LCN-2) in the colon of mice treated with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control group”), or MD007 bacteria loaded with SOD mimics Mn1 or Mn1C for four consecutive days after DNBS injection (data collected from three independent assays, with eight mice per assay for each condition). - Figure 9 The graph shows the concentration of lipid carrier protein 2 (LCN-2) in the serum of mice treated with Hepes (“Hepes”), empty MD007 bacteria (“MD007 control group”), or MD007 bacteria loaded with SOD mimics Mn1 or Mn1C for four consecutive days after DNBS injection (data collected from three independent assays, with eight mice per assay for each condition). - Figure 10The results show the normalized SOD2 mRNA levels (mean values ​​from 16 mice per condition) in mouse colon samples treated with Hepes (“Hepes”), empty MD007 bacteria (“MD007 Hepes”), or MD007 bacteria loaded with SOD mimic Mn1C, administered orally for four consecutive days after DNBS injection. - Figure 11 The graph shows the changes in body weight over time in mice treated with DNBS injection (day 0) or without DNBS injection (“solvent”) or daily oral administration of Hepes (“DNBS solvent”) or empty vector MG 1363 bacteria (“DNBS MG1363”) – expressed as a percentage of the mice’s initial body weight before determination. - Figure 12 Showing Figure 11 The area under the curve (between day 0 and day 5: D0-D5); - Figure 13 The macroscopic score chart shows the results of mice euthanized on the day of death (day 5: "D5" or day 6: "D6") after being treated with Hepes ("DNBS solvent") or empty vector MG1363 bacteria ("DNBS MG 1363") orally for 5 consecutive days following DNBS injection. - Figure 14 The bar chart shows the body weight of mice on day 5 (D5) after DNBS injection (day 0) or without DNBS injection (“solvent”) or daily oral administration of Hepes (“DNBS solvent”) or Mn1-loaded MG1363 bacteria – expressed as a percentage of the mice’s initial body weight before determination (D0). Detailed Implementation

[0105] Example 1 - Synthetic SOD analogues inorganic complexes The ligands used to prepare the inorganic complexes are EnPI2C and EnPI2CP, with structural formulas (Ixa) and (Ixb), respectively: (Ixa) (Ixb) The SOD mimic Mn(II) complex was prepared by mixing the two ligands with MnCl2 in 0.1 M HEPES buffer (pH 7.5) at a molar ratio of 1:1.3 and allowing it to stand at ambient temperature for 2 hours to allow the ligands to fully coordinate with manganese(II).

[0106] The following SOD analog metal complexes were obtained: Mn1C (structure formula (VII) above) was prepared by EnPI2C, and Mn1CP (structure formula (VIII) above) was prepared by EnPI2CP.

[0107] Example 2 - Bacterial SOD mimicry The experiment used *Lactobacillus plantarum* strain MD007, which was deposited on March 23, 2023, at the French National Center for the Preservation of Microbial Cultures (CNCM), part of the Pasteur Institute (address: 25 Rue du Roux, Paris, France), with accession number CNCM I-5940 (identification reference number LBH791-MD007). This strain was obtained by converting *Lactobacillus plantarum* strain ATCC BAA-793 / NCIMB 8826 into an alanine auxotroph, as described by Palumbo et al. in their 2004 article published in *FEMS Microbiology Letters* 233:131-138.

[0108] Overnight MD007 cell culture in MRS medium supplemented with alanine (MRS-alanine) was diluted to OD with MRS-alanine. 600nm = 0.2, then let stand to allow cells to grow until OD is reached. 600nm = 0.6. The culture was then centrifuged, the precipitate was washed twice, and resuspended in alanine-free MRS.

[0109] Bacteria were subjected to alanine deprivation for 3 hours, which weakened them and accelerated their lysis in the mouse intestine.

[0110] After alanine deprivation treatment, the culture was centrifuged again, the precipitate was washed twice, and resuspended in 0.1 M HEPES. The culture was then divided into four aliquots, and SOD mimics Mn1, Mn1C, Mn1CP, and MnCl2 were added to each aliquot until a final concentration of 0.4 mM was achieved. The bacterial suspension was then incubated at 37°C with shaking for 2 hours.

[0111] Finally, the culture was centrifuged and washed with 0.1 M HEPES, then resuspended in 0.1 M HEPES containing 16% glycerol (HEPES-gly), and stored in 2 mL aliquots at -80°C to avoid repeated freeze-thaw cycles.

[0112] Example 3 - Quantitative analysis of manganese by ICP-MS For each inorganic complex and the control MnCl2, take one sample of bacterial suspension (OD). 600nm=1) The sample was divided into equal parts, and manganese was quantified by inductively coupled plasma mass spectrometry (ICP-MS). A negative control without bacterial loading (HEPES) was also analyzed.

[0113] Select 55 Mn is used as an isotope to avoid interference from isotopes.

[0114] ICP-MS experiments were performed using an Agilent 7700 series ICP-MS system and an ASX-500 series autosampler, with the following parameters: RF power: 1550 W; sampling depth: 10 mm; helium flow rate: 5 mL / min; measurements were repeated 5 times, with 100 scans per scan, and an integration time / mass of 1 s.

[0115] The bacterial suspension was diluted with 2% HNO3 to lyse the bacteria and release manganese from all coordination sites. To achieve complete bacterial lysis, the 2% HNO3 solution was allowed to stand at ambient temperature for 1 hour, followed by filtration. A calibration curve was established using commercially available multi-element standards, and the result was measured by OD0.05 of the bacterial suspension. 600nm The total metal content is standardized.

[0116] The results obtained for HEPES, MnCl2, Mn1, and Mn1C are as follows: Figure 1 As shown. The results indicate that the bacteria loaded with SOD mimics according to the present invention have a high manganese content.

[0117] Example 4 - Induction of DNBS colitis in mice and administration of bacteria In vivo assays were performed on pathogen-free male C57BL / 6 mice at the animal facility of the French National Institute for Agriculture, Food and Environment (INRAE, Jouy-en-Rossa).

[0118] The timeline for colitis induction and bacterial loading inoculation in mice via gavage is as follows. This assay lasted for 5 days. For the first 4 days, mice were administered the bacterial loading via gavage daily. On the second day, after intraperitoneal anesthesia, mice were rectally injected with dinitrobenzenesulfonic acid (DNBS) (2.75 mg per mouse, dissolved in 20 μL PBS-ethanol (70 / 30 v / v)) through a 4 cm plastic tube inserted into the colon to induce colitis. On the fifth and final day, mice were euthanized using cervical dislocation. Mice were weighed daily.

[0119] The study groups were as follows: a colitis control group (DNBS + HEPES-gly), an empty bacterial control group (DNBS + MD007), and two bacterial-loaded groups: "DNBS + MD007_MnCl2" and "DNBS + MD007_Mn1C". Each group consisted of 8 mice. This assay was independently replicated 3 times, meaning a total of 24 mice were used for each condition.

[0120] The bacterial load determined in mice was achieved by ensuring that all mice received the same total amount of manganese at a fixed dose of 150 nmol. This corresponds to a bacterial count of 5 × 10⁻⁶. 8 -2×10 9 Changes between CFUs. The control group was given the same dose of MD007 as the control group (DNBS + MB007), a dose equal to the maximum viable bacterial load in the bacterial load group. Finally, the colitis control group received 200 μL of HEPES-gly.

[0121] Example 5 - Severity assessment of colitis 5.1 / mouse weight The evolution of mouse body weight during the measurement period is as follows: Figure 2 As shown.

[0122] As shown in the figure, mice fed with MD007 internalized the SOD mimic Mn1C experienced limited weight loss and accelerated weight recovery. In fact, the weight loss (reaching its maximum on day 2) was only 13%, and the mice had recovered to approximately 90% of their initial weight by day 3.

[0123] In addition, mice treated with the untreated MD007 strain experienced a slightly smaller decrease in body weight than untreated mice, indicating that the strain has very low probiotic activity.

[0124] 5.2 / euthanasia of mice After euthanizing the mouse, open the abdominal cavity, remove the colon, open it longitudinally, and rinse away the feces.

[0125] Then, the colon was longitudinally separated into multiple pieces for use in subsequent experiments.

[0126] 5.3 / Intestinal permeability measurement Three hours prior to euthanasia, mice were administered a fluorescently labeled FITC-glucan (12 mg per mouse, dissolved in 200 μL PBS) via gavage. Immediately before euthanasia, blood was collected via submandibular vein puncture under heparin anticoagulation. The collected whole blood was then centrifuged to separate plasma. Fluorescence intensity measurements were used to determine the FITC-glucan concentration in the plasma sample. For this purpose, fluorescence readings were performed using a fluorescence spectrophotometer (excitation wavelength = 488 nm, emission wavelength reading = 520 nm) on 80 μL plasma samples in microplates.

[0127] The results are as follows Figure 3 As shown.

[0128] It is known that DNBS injection induces damage to intestinal tight junctions, enhances barrier permeability, and promotes the entry of FITC-glucan into the bloodstream via the gastrointestinal tract. Quantitative analysis showed that, compared with untreated mice, the plasma samples of mice treated with MD007 + Mn1C had significantly lower levels of FITC-glucan. This indicates that Mn1C delivered by the MD007 vector has a significant effect in alleviating DNBS-induced intestinal permeability damage.

[0129] Example 6 - Supplementary experiments on Lactobacillus plantarum strain MD007 6.1 / Viability of loaded bacteria and quantitative analysis of manganese within bacteria The following three independent experiments were conducted. The results shown are the average of these three experiments.

[0130] At 37°C, Lactobacillus plantarum strain MD007 was grown in MRS containing D-alanine (200 μg / mL).

[0131] Inoculate fresh culture medium with overnight culture to allow OD 600nm Reaching 0.2, cells grow to OD 600nm After reaching a concentration of 0.7, the bacteria were washed three times with MRS medium without D-alanine. The washed bacteria were then incubated for 3 hours at 37°C in D-alanine-free medium, followed by SOD mimic loading. After alanine starvation, the bacteria were washed three times with Hepes (0.1 M, pH 7.4) to achieve a concentration of approximately 10. 9 1 bacteria / 200 μL.

[0132] After removing the supernatant from the culture medium, incubate the bacteria at 37°C with Hepes (0.1 M, pH 7.4) alone, or incubate in the same buffer with the tested compound (Mn1 or Mn1C) at the desired concentration for 2 hours or 24 hours. Mn1C and Mn1 solutions should be prepared fresh to prevent degradation and manganese oxidation. After centrifugation and washing with Hepes (0.1 M, pH 7.4), resuspend the bacteria in 0.1 M Hepes containing 16% glycerol to achieve a concentration of approximately 10. 9 100 bacteria / 200 μL. This volume corresponds to the maximum routine daily dose administered to mice via gavage. Store the bacteria in 2 mL aliquots at -80°C, avoiding repeated freeze-thaw cycles. Freeze the mouse gavage samples at -80°C and bring them to room temperature before administration to the mice.

[0133] To assess bacterial viability, overnight cultures and cryopreserved tubes were controlled using colony-forming units (CFU) testing. The formulation was serially diluted 1 / 10 with PBS, and known dilutions were inoculated onto the corresponding media. The plates were incubated at 37°C for 48 hours. Colony counting was then performed visually. The results are shown below. Figure 4 As shown in the figure. The results indicate that bacteria loaded with Mn1 or Mn1C do not affect cell viability.

[0134] Manganese in bacterial lysates was quantified by ICP-MS. For ICP-MS analysis, the bacterial pellet recovered by centrifugation was acidified with 2% HNO3 to lyse cells and release Mn from all coordination sites. Before analysis, samples were finely filtered through a 0.2 μm filter to remove bacterial debris. A calibration curve was established using commercially available manganese standards. Calibration was typically performed using a concentration range of 0–100 ppb. Results are shown below. Figure 5 As shown. The results indicate that the bacteria loaded with SOD mimics according to the present invention have a high manganese content.

[0135] 6.2 / Mouse treatment Male C57BL / 6 mice (6 weeks old) were housed under specific pathogen-free (SPF) conditions. Mice were housed under standard conditions for at least one week prior to the experiment to allow them to acclimatize. All animal studies were conducted at accredited research institutions and were approved by both the French government and the local ethics committee (authorization number: 16744-201807061805486_v2).

[0136] Mice were given empty / loaded bacterial gavage one day prior to DNBS-induced inflammation (D-1) to ensure bacterial colonization in the colon. On the day of DNBS-induced inflammation (D0), mice were anesthetized by intraperitoneal injection of ketamine (75 mg / kg, Imalgene, Boehringer Ingelheim Animal Health)-xerazine (9 mg / kg, Rompun, KVP). DNBS (3 mg / mouse, dissolved in 30% ethanol / PBS) was administered rectally via catheter, inserted approximately 3.5 cm into the colon. Bacteria were administered to mice daily via gavage (approximately 10 mg / kg). 9 The mice were given either bacteria (200 μL / bacteria), bacteria-loaded protein (CMB), or Hepes for 4 or 5 days. The amount of CMB administered to the mice was determined to ensure that all mice received the same total amount of manganese, at a fixed dose of 150 nmol per dose, equivalent to 1 × 10⁻⁶. 9 -2×10 9 The dosage of bacteria was determined. The amount of bacteria administered to the DNBS MD007 control group was equivalent to the maximum amount of bacteria administered to the CMB treatment group. Mice were monitored and closely watched throughout the experiment. On the last day (D3 or D4), mice were euthanized by cervical dislocation. The study groups were named as follows: colitis group receiving no bacteria but instead Hepes buffer (“Hepes”), colitis group receiving empty bacterial vector (“MD007 control”), and two colitis groups treated with CMB: “MD007 Mn1” and “MD007 Mn1C”. Each group consisted of 8 mice. This assay was independently replicated 3 to 4 times, i.e., 24 or 32 mice were used for each condition.

[0137] The body weight of all mice was monitored over time. The area under the body weight monitoring curve for days 0-3 (D0-D3) is shown in the figure. Figure 6 As shown in the figure, mice fed with MD007 internalized SOD mimics Mn1 and Mn1C showed limited weight loss and even some weight gain.

[0138] 6.3 / Assess the severity of colitis in treated mice After bacterial loading, the peritoneum of each euthanized mouse was opened, the colon was removed and opened longitudinally, and feces were flushed away. A macroscopic score was then immediately assessed. This score evaluates colonic status by considering the following indicators: colonic wall thickening, presence of ulcers, congestion, adhesions to other intra-abdominal tissues, and consistency of colonic contents (indicators of intestinal transit abnormalities). Results are as follows: Figure 7 As shown, treatment with bacteria loaded with Mn1 or Mn1C significantly improved macroscopic scores.

[0139] The colon was then separated longitudinally into two pieces. The right side was then separated transversely into four parts for subsequent analysis.

[0140] The concentrations of lipid carrier protein 2 (LCN-2) in mouse colon and plasma were measured. For plasma LCN-2 concentration determination, centrifuged blood samples (2000 g, 20°C for 10 minutes) were used. A commercially available sandwich enzyme-linked immunosorbent assay kit (Mouse Lipcarrier Protein-2 / NGAL DuoSet ELISA; R&D Systems Europe) was used according to the manufacturer's instructions. For colon LCN-2 concentration determination, colonic supernatant was used, and the kit was the same as for plasma. Colonic tissue blocks were weighed and placed in 0.5% HTAB (cetyltrimethylammonium bromide) buffer, and mechanically dissociated at 10000 rpm for three 20-second cycles using a Precellys® (Bertin) device equipped with a mixture of 1.4 mm and 2.8 mm ceramic beads. The samples were then briefly centrifuged, and the supernatant was collected. The results are shown below. Figure 8 LCN-2 concentration in colon and Figure 9 Serum LCN-2 concentration. The results showed that treatment of mice with bacteria loaded with Mn1 or Mn1C significantly reduced the concentration of lipid transporter 2.

[0141] The following quantitative analysis was performed on SOD2 gene transcription in different parts of the colon. Total RNA was extracted from colonic homogenate using the Qiagen RNeasy kit according to the manufacturer's instructions, and β-mercaptoethanol was used as a reducing agent to achieve irreversible denaturation of RNases. Reverse transcription was then performed using oligo(dT)12-18 primers and SuperScript® II reverse transcriptase (Invitrogen®) to generate complementary DNA. SOD2 gene expression was quantitatively analyzed by quantitative PCR using a Takyon® rox Sybr mastermix DTTP Blue (Eurogentec) on a StepOne® real-time PCR system. The following primer pairs were used: 5'-ATTAACGCGCAGATCATGCA-3' (forward) (SEQ ID No: 1) and 5'-TGTCCCCCACCATTGAACTT-3' (reverse) (SEQ ID No: 2) The annealing temperature is set to 60℃.

[0142] SOD2 levels were normalized using the housekeeping genes GAPDH and TATA box-binding protein (TBP), both of which are known to be unaffected under inflammatory conditions, and were also quantified by RT-PCR. For Mn1C-loaded bacteria, the results are as follows: Figure 10As shown in the figure, observations revealed that mice treated with Mn1C-loaded bacteria had significantly lower levels of SOD2 mRNA.

[0143] Example 7 - Lactococcus lactis MG1363 strain According to the article published by Sanders et al. in the Journal of Bacteriology in 1995 (177:5254-5260), this experiment used Lactococcus lactis strain MG1363. This strain possesses SOD enzyme.

[0144] 7.1 / Bacterial load The strain was grown in M17 glucose at 30°C.

[0145] Inoculate fresh culture medium with overnight culture to allow OD 600nm Reaching 0.2, cells grow to OD 600nm The concentration was 0.7. The bacteria were washed three times with Hepes (0.1 M, pH 7.4) to achieve a concentration of approximately 10 μL per 200 μL. 9 One bacterium.

[0146] Using the scheme described in Example 6, bacteria were loaded with Mn1.

[0147] 7.2 / Assessing the role of bacterial neutralization in DNBS-induced colitis This experiment used male C57BL / 6 mice (6 weeks old) as described in Example 6.

[0148] On day 1 (D0), mice were anesthetized by intraperitoneal injection (IP) of 0.06% ketamine-xerazine. DNBS solution (3 mg / mouse) was dissolved in 30 / 70 ethanol / PBS via rectal injection (IP) through a catheter inserted approximately 3.5 cm into the colon. Bacterial suspension (10 mg / mL) was administered to mice daily via gavage. 9 Mice were given either 100 μL of bacteria (200 μL) or Hepes (100 μL) for 5 days. Throughout the experiment, mice were monitored and closely observed. On the last day (D5) or the following day (D6), mice were euthanized by cervical dislocation.

[0149] Monitor the change in body weight of all mice over time. The evolution of mouse body weight during the period from day 0 to day 5 is shown below. Figure 11 As shown. The area under the curve is as follows. Figure 12 As shown in the figure, administration of empty MG1363 to mice with DNBS-induced colitis had no significant effect on mouse body weight.

[0150] As described in Example 6, the macroscopic scores of the mice were assessed on day 5 or day 6. The results were as follows: Figure 13As shown in the figure, observations indicate that MG1363 bacteria have no effect on DNBS-induced colitis.

[0151] 7.3 / Evaluation of the role of Mn1-loaded MG1363 bacteria in DNBS-induced colitis On day 1 (D0), mice were anesthetized via intraperitoneal injection of 0.06% ketamine-xeracilin. DNBS (3 mg / mouse) and a DNBS solution (Sigma) dissolved in 30% ethanol / PBS were then administered transrectally via catheter inserted approximately 3.5 cm into the colon. One group, serving as a non-colitis control, received only Hepes treatment and was referred to as the "solvent." Mice were administered bacteria (approximately 10 mg / mouse) via gavage daily. 9 The mice were administered either bacteria (200 μL / bacteria), bacterial-loaded manganese (CMB), or Hepes for 5 days. The amount of CMB administered to the mice was determined to ensure that all mice received the same total amount of manganese, at a fixed dose of 150 nmol per dose. The mice were monitored and closely observed throughout the experiment.

[0152] Monitor the change in body weight of all mice over time. The body weight of the mice on day 5 is as follows: Figure 14 As shown in the figure. These results indicate that administration of Mn1-loaded MG1363 bacteria to mice with DNBS-induced colitis increased the mice's body weight, suggesting an improvement in their health. No improvement was observed when empty bacterial vectors were used.

[0153] 8 / Statistical analysis (Examples 6 and 7) Statistical analysis was performed using GraphPad Prism software. Results are presented as scatter plots with mean ± SEM. The nonparametric Mann-Whitney test (one-tailed test) was used to calculate p-values. Outliers identified using Prism software were removed. The mean rank of each column was compared to the DNBS Hepes control group; each comparison was performed independently. ****: p < 0.0001, ***: p < 0.001, **: p < 0.01, *: p < 0.05. ns indicates no statistical significance compared to the DNBS Hepes control group or solvent. A p-value less than 0.05 was considered statistically significant.

Claims

1. A chemically modified microorganism selected from the group consisting of bacteria and yeasts, said chemically modified microorganism comprising a synthetic superoxide dismutase mimetic inorganic complex, for use as a medicament or a dietary supplement.

2. The chemically modified microorganism having the use of claim 1, wherein, The inorganic complex is a human manganese superoxide dismutase mimetic.

3. The chemically modified microorganism having the use of claim 1 or 2, wherein, The metal of the inorganic complex is manganese (II) or manganese (III).

4. The chemically modified microorganism having the use of any one of claims 1-3, wherein, The inorganic complex has the general formula (I): （I） wherein, n, m and p, which are the same or different, are integers between 1 and 3, R1 represents a hydrogen atom or a linear, branched and / or cyclic alkyl group, which can be optionally substituted with at least one aromatic or fused ring, or R1 represents a peptide comprising 1-20 amino acid residues, R2, R2', R3, R3' each represent a hydrogen atom, or R2' and R3' represent a hydrogen atom, R2 and R3, together with the carbon atom to which they are attached, form an aliphatic or heterocyclic ring, which can be optionally substituted with a Lewis base, a phenolic group, an imidazole ring, a pyridine ring and / or one or more alkyl groups, which can be the same or different, each of said alkyl groups being optionally substituted with a Lewis base, a phenolic group, an imidazole ring and / or a pyridine ring, or R2, R2', R3, R3', together with the carbon atom to which they are attached, form an aromatic or heterocyclic ring, which can be optionally substituted with a Lewis base, a phenolic group, an imidazole ring, a pyridine ring and / or one or more alkyl groups, which can be the same or different, each of said alkyl groups being optionally substituted with a Lewis base, a phenolic group, an imidazole ring and / or a pyridine ring, Ar1 and Ar2, which can be the same or different, each represent a 5- or 6-membered aromatic heterocyclic ring, which can be optionally substituted with one or more substituents, which can be the same or different, each substituent being selected from the group consisting of a bromine atom, a chlorine atom, a methyl group, a methoxy group and a nitro group, R4 represents a phenyl group, which can be optionally substituted with one or more substituents, which can be the same or different, each substituent being selected from the group consisting of a bromine atom, a chlorine 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 attached to the oxygen atom and R5 represents a linear, branched and / or cyclic alkyl group, which can be optionally substituted by a Lewis base. p - group, R5 represents a linear, branched and / or cyclic alkyl group, which can be optionally substituted by a Lewis base.

5. The chemically modified microorganism having the use of claim 4, wherein, The inorganic complex has the general formula (IV): (IV) wherein R1, R2, R2', R3 and R3' are as defined in claim 4.

6. The chemically modified microorganism having the use of claim 5, wherein, The inorganic complex has the general formula (VII): (VII)。 7. The chemically modified microorganism for use according to any one of claims 1-6, which is a lactic acid bacterium of the genus Lactobacillus or of the genus Streptococcus.

8. The chemically modified microorganism for use according to any one of claims 1-7, whose cell wall has been treated by genetic, chemical and / or enzymatic means to weaken it.

9. The chemically modified microorganism for use according to claim 8, which exhibits auxotrophy for a certain amino acid and has been cultured in a medium lacking said amino acid for at least 1 hour.

10. The chemically modified microorganism for use according to any one of claims 1-9, which is used to counteract oxidative stress.

11. The chemically modified microorganism for use according to any one of claims 1-10, which is used to treat an inflammatory disease.

12. The chemically modified microorganism having the use according to claim 11 for use in the treatment of inflammatory bowel disease.

13. The chemically modified microorganism having the uses of any one of claims 1-12, wherein, The chemically modified microorganism is administered to the subject by oral means.

14. The chemically modified microorganism having the use according to any one of claims 1-13 for use in the treatment of a mammalian subject, preferably for use in the treatment of a human.

15. A pharmaceutical composition comprising a chemically modified microorganism as defined in any one of claims 1-9 and a pharmaceutically suitable vehicle.

16. A nutraceutical composition, a food composition or a dietary supplement comprising a chemically modified microorganism as defined in any one of claims 1-9 and a physiologically acceptable vehicle.

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