Chemically modified microorganisms containing superoxide dismutase-mimicking inorganic complexes, and their use in the treatment of inflammatory diseases.

Abstract

This invention relates to chemically modified microorganisms selected from bacteria and yeasts, comprising superoxide dismutase-mimicking inorganic complexes, particularly mimics of human manganese superoxide dismutase. These microorganisms are particularly useful for the treatment of inflammatory diseases, especially inflammatory bowel disease, by oral administration.

Description

[Technical Field]

[0001] This invention belongs to the field of drug delivery, particularly the intestinal delivery of antioxidant drugs aimed at combating oxidative stress.

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

[0003] Superoxide dismutase (SOD) is a metalloenzyme involved in the antioxidant defense of cells. In living organisms, SOD plays a role in maintaining superoxide, a reactive oxygen species (ROS), at a strictly controlled concentration, and contributes to the prevention of oxidative stress, which is known to be involved in various diseases. All SODs contain a metal cation in their active site, and this metal cation cycles between two states, redox and superoxide, continuously reducing superoxide to H2O2 and then oxidizing it back to O2. In humans, three different eukaryotic SODs have been described depending on the metal contained in the active site. These are two types of copper / zinc SOD: SOD1, found in the cytosol and mitochondrial intermembrane space; and SOD3, found in the extracellular environment of the extracellular matrix and cell surface; and manganese SOD (SOD2; also called MnSOD), which is localized in the mitochondrial matrix (Non-Patent Literature 1).

[0004] Inflammatory bowel disease (IBD) is a chronic disease characterized by chronic inflammation of a part of the gastrointestinal tract. IBD includes Crohn's disease and ulcerative colitis, which differ at least in the location of inflammation. All can cause diarrhea, rectal bleeding, anemia, weight loss, and abdominal pain. IBD is particularly prevalent and continues to increase in developed countries, and with a lifetime cumulative incidence of 1% in Europe, it constitutes a global health problem. The number of IBD cases worldwide is estimated at 6.8 million. Currently, there is no curative treatment for IBD, and patients receive medications primarily aimed at controlling inflammation to alleviate symptoms and prevent flare-ups. Main treatments include corticosteroids and immunomodulatory drugs, including small molecule and monoclonal antibodies targeting specific inflammatory pathways or cytokines (Non-Patent Literature 2). However, these expensive drugs are not always effective and can induce abscesses and adverse events. Therefore, there is an urgent need for novel, safe, and effective alternative treatments to combat IBD.

[0005] Inflammatory bowel disease (IBD) is described as being accompanied by overexpression of an enzymatically inactive form of intestinal MnSOD and low expression of intestinal cytoplasmic Cu / Zn SOD (Non-Patent Literature 3). These deficiencies in the SOD antioxidant system may cause, or at least exacerbate, oxidative stress seen in IBD, which is known to be closely associated with chronic inflammation (Non-Patent Literature 4). Therefore, SOD-based antioxidant therapies have been found to be promising treatments for IBD. In particular, MnSOD has been shown to efficiently reduce lipid peroxidation and neutrophil recruitment, and to alleviate inflammation in both DSS-induced colitis and TNBS-induced colitis mouse models (Non-Patent Literature 5). Patent Literature 1 discloses a Bacillus amyloliquefaciens mutant strain with a high rate of SOD enzyme production, and / or a composition for the treatment of IBD comprising SOD enzyme purified from such strains after incubation of cells of such strains with a manganese salt. However, the therapeutic use of these purified enzymes is limited due to their short half-lives, immunogenicity, and low cell permeability.

[0006] To overcome these drawbacks, synthetic low molecular weight SOD mimics (also called SOD mimetics), which mimic SOD activity, i.e., the activity to catalyze the disproportionation of superoxide, have been tested as therapeutic candidates for IBD management. A variety of synthetic SOD mimetic antioxidant inorganic complexes containing iron, copper, zinc, and manganese complexes have been reported. These synthetic inorganic complexes generally have a molecular weight of less than 10 kDa, often less than 5 kDa. Manganese complexes in particular are considered to be more favorable compared to Cu, Fe, and Ni complexes, because manganese is readily tolerated by cells even if released, and does not catalyze the Fenton reaction or the Haber-Weiss reaction that leads to the formation of highly reactive and toxic HO· radicals.

[0007] Many manganese-based SOD mimics have been reported in the literature, such as complexes having ligands such as salen derivatives, cyclic polyamines, ligands centered on 1,2-ethylenediamine, porphyrins, phthalocyanines, etc. (Non-Patent Document 6).

[0008] As an example, a SOD mimicking manganese complex called Mn1 of formula (V):

[0009] TIFF2026515321000001.tif58170

[0010] has been reported. This complex has been reported to exhibit intracellular anti-inflammatory activity in vivo as shown by analysis of body weight fluctuations and macroscopic scores when orally administered in carbonate buffer in a DBNS-induced colitis mouse model (Non-Patent Document 7). However, this activity is limited, and the cause is likely the instability of this inorganic complex in the biological environment. Furthermore, although using a basic buffer as a vehicle for the metal complex improves the stability of the metal complex in an acidic environment, such a vehicle is not desirable for human administration.

[0011] Non-Patent Document 8 discloses the intracellular uptake of Mn-salen, a SOD mimic, in Cryptococcus neoformans lacking MnSOD.

Prior Art Documents

Patent Documents

[0012]

Patent Document 1

Non-Patent Documents

[0013]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

[0014] The present invention aims to propose a method for efficiently treating inflammatory diseases, particularly IBD. More specifically, the present invention belongs to the context of the use of synthetic SOD mimetic compounds to counteract oxidative stress, particularly for the treatment of inflammatory diseases, and aims to propose a solution that enhances the bioavailability and efficacy of these active ingredients, particularly at the site of action, in the intestines, compared to solutions proposed in the prior art for administering such compounds. [Means for solving the problem]

[0015] The inventors have newly discovered that these objectives can be achieved by using microorganisms as vectors for targeted delivery of SOD mimetic bodies in the intestines. In particular, the inventors found that synthetic inorganic complexes can accumulate within microbial cells, and that when these cells are orally administered to a target and pass through the stomach, the inorganic complexes contained within the cells are protected from contact with the highly acidic gastric juice, thereby preventing the decomposition that would otherwise be induced by such contact. Therefore, even when the cells reach the site of action in the intestines, a large proportion of the inorganic complexes administered to the target are favorably preserved in their complete state.

[0016] Thus, by being protected as microbial cells migrate through the digestive tract, the inorganic complex maintains its integrity even when it reaches the site of action in the intestine, and retains its maximum superoxide disproportionation catalytic activity.

[0017] Therefore, the first object of the present invention is a chemically modified microorganism, i.e., a microorganism modified by the introduction of a chemical compound, wherein the microorganism is selected from bacteria and yeast and comprises a synthetic inorganic complex that is a superoxide dismutase mimetic, for use as a drug or nutritional supplement. [Brief explanation of the drawing]

[0018] [Figure 1] Figure 1 is a bar graph showing the Mn concentration in cell lysates measured by ICP-MS for either the empty bacterium MD007 ("HEPES") or the bacterium MD007 into which SOD mimetic compounds (Mn1, Mn1C) or MnCl2 have been introduced. [Figure 2] Figure 2 is a graph showing the time course of body weight measurements in mice that received DNBS injection (day 0) and were orally administered daily with empty MD007 bacteria or MD007 bacteria introduced with an SOD mimetic (Mn1C) or MnCl2 (days -1 to 2). Body weight is expressed as a percentage of the initial body weight of the mouse before the start of the assay. [Figure 3] Figure 3 is a graph showing the quantification of FITC-dextran as a marker of intestinal barrier permeability in mice that received DNBS injection and oral administration of empty MD007 bacteria or MD007 bacteria introduced with SOD mimetic (Mn1C) or MnCl2. The data represent the mean ± SD from three independent assays (approximately 24 mice). The p-value was calculated using a non-parametric Mann-Whitney test (one-sided test) because the Gaussian distribution of the data could not be verified by the Anderson-Darling normality test. The mean rank of each column was compared to that of the DNBS control, and each comparison was performed independently. **: p<0.01 compared to the DNBS control; ns means no significant difference. [Figure 4] Figure 4 is a bar graph showing the count of viable cells in freeze-thaw aliquots of either empty MD007 bacteria ("HEPES") or MD007 bacteria introduced with SOD mimetic organisms (Mn1, Mn1C). [Figure 5] Figure 5 is a bar graph showing the Mn content in cell lysates (average of 3 experiments) measured by ICP-MS for empty MD007 bacteria ("HEPES") or MD007 bacteria introduced with SOD mimetic compounds (Mn1, Mn1C). [Figure 6]Figure 6 shows the area under the curve of body weight measured from day 0 to day 3 (D0 to D3) in mice that received DNBS injection (day 0) and were orally administered daily with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). [Figure 7] Figure 7 is a graph showing the macroscopic scores determined on the day of euthanasia for mice that received DNBS injection and were orally administered daily for four days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with the SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using eight mice per assay / condition). [Figure 8] Figure 8 is a graph showing the concentration of lipocalin 2 (LCN-2) measured in the colon of mice that received DNBS injection and were orally administered daily for 4 days either Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). [Figure 9] Figure 9 is a graph showing the concentration of lipocalin 2 (LCN-2) measured in the serum of mice that received DNBS injection and were orally administered daily for 4 days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). [Figure 10] Figure 10 shows the mRNA levels of SOD2 normalized by the mRNA levels of GADPH and TBP, measured in colon samples of mice that received DNBS injection and were orally administered daily for 4 days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with the SOD mimetic Mn1C (average for 16 mice under each condition). [Figure 11]Figure 11 is a graph showing the time course of body weight measurements in mice that received DNBS injection (day 0) or not ("vehicle"), and were orally administered Hepes ("DNBS vehicle") or empty MG1363 bacteria ("DNBS MG1363") daily. Body weight is expressed as a percentage of the initial body weight of the mice before the start of the assay. [Figure 12] Figure 12 shows the area under the curve in Figure 11 (from day 0 to day 5: D0 to D5). [Figure 13] Figure 13 is a graph showing the macroscopic scores determined on the day of euthanasia (day 5: "D5" or day 6: "D6") for mice that received DNBS injections and were orally administered either Hepes ("DNBS vehicle") or empty MG1363 bacteria ("DNBS MG1363") daily for 5 days. [Figure 14] Figure 14 is a bar graph showing the body weight on day 5 (D5) of mice that received daily oral administration of either DNBS injection (day 0) or no injection ("vehicle"), Hepes ("DNBS vehicle"), empty MG1363 bacteria ("DNBS MG1363"), or MG1363 bacteria with Mn1 introduced. Body weight is expressed as a percentage of the initial body weight of the mouse before the start of the assay (D0). [Modes for carrying out the invention]

[0019] Chemically modified microorganisms in which SOD mimetic molecules are vectorized are particularly easy to prepare because the inorganic complex simply and passively passes through the cell wall and accumulates inside the cell.

[0020] When administered orally to subjects, the cell wall favorably protects the inorganic complex from the acidic conditions of the stomach, preventing its dissociation and degradation. As a result, the inorganic complex is favorably delivered to the intestines while retaining its full biological activity, particularly its full ability to correct intestinal hyperpermeability induced by colitis. This biological activity has been confirmed, in particular, by assays performed in a mouse model of acute colitis.

[0021] In the context of the therapeutic application of chemically modified microorganisms as intended by the present invention, when the microorganisms are administered to a target, both the microorganisms and the inorganic complexes are advantageously selected so as not to be harmful to the target.

[0022] Therefore, the inorganic complexes of the present invention are preferably pharmaceutically and / or physiologically acceptable. This means that they do not cause adverse reactions, allergic reactions, or other undesirable reactions when administered to subjects, particularly mammals, more specifically humans.

[0023] Microorganisms are advantageously selected to be nonpathogenic to the target to which they are intended, particularly to mammals, and more specifically to humans. The microorganisms are preferably commensal to the target.

[0024] In the context of the present invention, food-grade bacteria and yeasts are particularly preferred. Food-grade microorganisms, as used herein, refer to the following microorganisms: • Microorganisms listed in the latest version of the European Food Safety Authority's (EFSA) QPS ("Qualified Presumption of Safety") list ("2022 QPS List"), available at Zenodo's Knowledge Junction (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 production (i.e., delivery) microorganisms (EFSA Panel on Additives and Products or Substances used in Animal Feed); • Microorganisms listed in the U.S. Food and Drug Administration's "Generally Recognized as Safe (GRAS)" document (September 6, 2019; retrieved January 30, 2021).

[0025] The microorganisms used in this invention may be deficient in SOD enzymes, or they may produce such enzymes.

[0026] In this specification, a superoxide dismutase (SOD) mimetic is, as is customary, a synthetic low molecular weight compound (particularly one with a molecular weight of less than 10 kDa, and in some cases less than 5 kDa) capable of catalyzing the disproportionation of superoxide in an aqueous medium. That is, it has a protective effect against oxidative damage, similar to SOD. More specifically, SOD mimetic compounds undergo a catalytic redox cycle, and O2 - Oxidizes to O2, and O2 - It can be reduced to H2O2. This means that the redox potential of the redox pair involved is in the range of -0.18V / NHE to 0.89V / NHE (pH 7).

[0027] Identifying inorganic complexes that mimic SOD is within the scope of the skills of those skilled in the art.

[0028] To date, a wide variety of such complexes, particularly manganese complexes, have been reported in the prior art. These include ligands such as salen derivatives, cyclic polyamines, tripod or bipodial nitrogen ligands, 1,2-ethanediamine ligands, desferrioxamine derivatives, polyaminocarboxylate or polycarboxylate ligands, peptides, porphyrins, phthalocyanines, texaphyrin, corol, or biliverdin and its derivatives. Examples of SOD mimetic compounds that may be used according to the present invention are described in particular in Vincent et al., 2021, J. Inorg. Biochem. 219, 111431.

[0029] In addition, in order to determine whether a certain inorganic complex is a SOD mimetic, that is, whether it has intrinsic SOD activity, one of ordinary skill in the art may apply the so-called indirect assay of McCord and Fridovich. This assay is generally used to identify SOD mimetics by testing superoxide disproportionation and is described in the literature of McCord and Fridovich, 1969, J. Biol. Chem. 244(22), 6049-6065. Briefly, this assay involves measuring the catalytic rate (k cat ) for superoxide disproportionation in a HEPES buffer (e.g., 50 mM, pH 7.4), and regarding the reaction with superoxide continuously generated by the enzymatic xanthine (200 μM) / xanthine oxidase system, the inorganic complex to be tested is made to compete with a redox marker such as (e.g., 100 μM) ferricytochrome c, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT), or nitroblue tetrazolium (NBT). The reduction of the redox marker is measured by UV-visible spectrophotometry, for example, at 550 nm for ferricytochrome c, for different amounts of the inorganic complex. For each amount of the inorganic complex added, both the reduction rate when the complex is absent and the reduction rate when it is present are determined. If the slope of the reduction curve before adding the inorganic complex is s1 and the slope after adding is s2, the inhibition rate for each inorganic complex is given by I(%) = (s1 - s2) / s1 × 100. The value of IC 50 , which is the concentration of the inorganic complex required to inhibit the reduction of the redox marker by 50%, is obtained at I = 50%. It is also possible to plot (s1 - s2) / s against the inorganic complex concentration, and a linear correlation is obtained. And IC 50 is obtained when (s1 - s2) / s = 1. Then, the k cat of the inorganic complex is calculated according to the following formula from the value of IC 50 and the known k cat of the redox marker (k redox )(for example, for XTT, k XTT = 2.9×10 4 M -1 ·s-1 ) k cat =k redox ×[redox] / IC 50 In the formula, [redox] is the concentration of the redox marker in the assay medium, for example, 100 μM.

[0030] The inorganic complex can be considered a SOD mimetic because of the kinetics of superoxide disproportionation catalyzed by this complex, as measured in the above assay, i.e., k cat However, this is the case when the rate is faster than the self-disproportionation of superoxide at the same pH (disproportionation without the use of a catalyst). The rate of self-disproportionation is shown in a table in Bielski et al., 1985, J. Chem. Phys. Ref Data, 14(4), 1041-1100, for example, at pH 7, it is 6.10 5 M -1 ·s -1 It's faster than that.

[0031] A control for the reliability of this assay, in particular to confirm that the tested inorganic complex does not inhibit superoxide production by inhibiting the xanthine oxidase reaction, may be performed as described in Durot et al., 2005, Eur. J. Inorg. Chem., 17, 3513-3523.

[0032] In certain embodiments of the present invention, the inorganic complex is a mimic of human manganese superoxide dismutase.

[0033] More generally, the metal of the inorganic complex is preferably manganese(II) or manganese(III), which have the advantage of not increasing oxidative stress when released in the body, especially compared to other metals such as iron, copper, or nickel.

[0034] SOD-mimicking inorganic complexes described in the prior art, particularly manganese-based SOD mimics, are all included within the scope of the present invention, and in particular, those having ligands such as salen derivatives, cyclic polyamines, tripod or bipodial nitrogen ligands, 1,2-ethane-diamine ligands, desferrioxamine derivatives, polyaminocarboxylate or polycarboxylate ligands, peptides, porphyrins, phthalocyanines, texaphyllines, corols, or biliverdin and its derivatives, as described in the literature of Vincent et al., 2021 referenced above.

[0035] For example, the microorganisms of the present invention may include the following: · 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-11,7-nitrilo-7Hdibenzo[b,h][1,4,7,10]tetraazacycloheptadesine-kN5,kN13,kN18,kN21,kN22](imisopasemmanganese) (a specific example of a manganese(II) pentaazamacrocycle (cyclic polyamine)); · As Mn(III) porphyrin: Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl) porphyrin (MnTE-2-PyP 5+ );Mn(III) mesotetrakis[N-(2'-n-butoxyethyl)pyridinium-2-yl]porphyrin (MnTnBuOE-2-PyP 5+ );Mn(III)5,10,15,20-tetrakis(4-benzoic acid)porphyrin (MnTBAP); ·Mn(III)2,2'-[1,2-ethanediylbis(nitrilomethyridine)]bis[6-methoxyphenol]; ·others.

[0036] In certain embodiments of the present invention, the inorganic complex is inspired by the active site of MnSOD and is constructed based on a ligand having a Lewis base-functionalized 1,2-N,N'-diaminoethane central skeleton, and the diamino structure is rigidified with a carbon ring to restrict the release of Mn. The inorganic complex may in particular have general formula (I).

[0037] TIFF2026515321000002.tif71170

[0038] During the ceremony, n, m, and p may be the same or different, and are integers between 1 and 3, with each of n, m, and p preferably equal to 1; R1 represents a hydrogen atom, or a linear, branched, and / or cyclic alkyl group, wherein the alkyl group preferably has 1 to 12 carbon atoms, particularly 2 to 6 carbon atoms, and may be substituted by at least one, i.e., one or more aromatic rings or polycyclic rings, wherein the polycyclic ring preferably has 6 to 14 carbon atoms, such as anthracene; Alternatively, R1 represents a peptide containing 1 to 20 amino acid residues, particularly 1 to 9 amino acid residues. R2, R2', R3, and R3' all represent hydrogen atoms. Alternatively, R2' and R3' represent hydrogen atoms, and R2 and R3, together with the carbon atoms to which they are bonded, form an aliphatic ring or heterocycle, particularly a 5-membered, 6-membered, 7-membered, or 8-membered ring or heterocycle, and the aliphatic ring or heterocycle may be substituted with a Lewis base, a phenol group, an imidazole ring, a pyridine ring, and / or one or more identical or different alkyl groups, each alkyl group preferably having 1 to 12 carbon atoms, particularly 1 to 6 carbon atoms, and each alkyl group may also be substituted with a Lewis base, a phenol group, an imidazole ring, and / or a pyridine ring. Alternatively, R2, R2', R3, and R3' together with the carbon atoms to which they are bonded form an aromatic ring or heterocycle, particularly a 5-membered, 6-membered, 7-membered, or 8-membered ring or heterocycle, and the aromatic ring or heterocycle may be substituted with a Lewis base, a phenol group, an imidazole ring, a pyridine ring, and / or one or more identical or different alkyl groups, each of which preferably has 1 to 12 carbon atoms, particularly 1 to 6 carbon atoms, and each of which may also be substituted with a Lewis base, a phenol group, an imidazole ring, and / or a pyridine ring. Ar1 and Ar2 may be the same or different, each representing a 5-membered or 6-membered aromatic heterocycle, preferably containing one or two heteroatoms, particularly one or two nitrogen atoms, such as a pyrrole, imidazole, pyridine, or pyrimidine ring, and the aromatic heterocycle may be substituted with one or more identical or different substituents, each substituent selected from a bromine atom, a chlorine atom, a methyl group, a methoxy group, and a nitro group. R4 represents a phenyl group which may be optionally substituted with one or more identical or different substituents, each of which substituents is selected from a bromine atom, a chlorine atom, a methyl group, a methoxy group, and a nitro group. Alternatively, R4 represents the group in formula -R5-CO-, where the carbonyl group is bonded to the oxygen atom, and R5 is -(CH2) p -Bonded to a base, R5 represents a linear, branched, and / or cyclic alkyl group which may be substituted with a Lewis base, preferably having 1 to 3 carbon atoms.

[0039] In this specification, a Lewis base refers to a group that can donate an electron pair 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 the present invention include -OH, -COOH, -SH, -NH2, and -NHR. a , -NR a (R b The basis for each of the equations is listed, and here R a and R bThese may be the same or different, and each represents a C1-C3 alkyl or alkenyl group, or a phenyl group.

[0040] The inorganic complex of the above general formula (I), and all inorganic complexes of the following formulas, whether their stereoisomers, diastereomers, or enantiomers, whether alone or as mixtures thereof, are included within the scope of the present invention. In particular, C a and C b The nitrogen atoms bonded to each atom may be in a cis configuration. Preferably, they are in a trans configuration.

[0041] Any salt of the inorganic complex of formula (I), or any salt of any inorganic complex of any of the following formulas, is included within the scope of the present invention.

[0042] The inorganic complex of formula (I) may satisfy one or more of the following characteristics in any combination.

[0043] For example, R1 may be an n-propyl group.

[0044] In certain embodiments of the present invention, when at least R2 and R3 do not represent hydrogen atoms, R1 represents a hydrogen atom.

[0045] When Ar1 or Ar2 represents a six-membered aromatic heterocycle such as a substituted pyridine or pyrimidine ring, the substituent is -(CH2) m -, or -(CH2) n With respect to the nitrogen atom located at the α-position relative to the carbon atom bonded to the carbon atom (i.e., relative to the nitrogen atom coordinated to manganese(II)), it is preferably at the ortho position, more preferably at the para position.

[0046] When Ar1 or Ar2 represents an imidazole ring, at least one of the substituents is bonded to the nitrogen atom that is not coordinated to manganese(II).

[0047] When R4 represents a phenyl group, this group has a carbon atom at the α-position relative to the carbon atom bonded to the oxygen atom, which is -(CH2). p It is preferable that it is bonded to -

[0048] The inorganic complex of the present invention may have general formula (II).

[0049] TIFF2026515321000003.tif73170

[0050] In the formula, m, n, p, R1, R2, R2', R3, R3', Ar1, and Ar2 are as defined above.

[0051] In particular, inorganic complexes may have the general formula (IIa).

[0052] TIFF2026515321000004.tif71170

[0053] In the formula, m, n, R1, R2, R2', R3, R3', Ar1, and Ar2 are as defined above.

[0054] Preferably, in formulas (II) and (IIa) above, m is equal to 1 and / or n is equal to 1.

[0055] In certain embodiments of the present invention, the inorganic complex has general formula (III).

[0056] TIFF2026515321000005.tif71170

[0057] In the formula, m, n, p, R1, R2, R2', R3, R3', and R4 are as defined above.

[0058] In particular, inorganic complexes may have the general formula (IIIa).

[0059] TIFF2026515321000006.tif71170

[0060] In the formula, p, R1, R2, R2', R3, R3', and R4 are as defined above.

[0061] Preferably, in formulas (III) and (IIIa) above, p is equal to 1.

[0062] In certain embodiments of the present invention, the inorganic complex has a coordination sphere consisting of two imidazole rings and one phenolate ion.

[0063] In this case, it is preferable that the inorganic complex has general formula (IV).

[0064] TIFF2026515321000007.tif71170

[0065] In the formula, R1, R2, R2', R3, and R3' are as defined above.

[0066] One specific inorganic complex of formula (IV) is Mn(II)-(N-(hydroxybenzyl)-N,N'-bis[2-(N-methyl-imidazolyl)methyl]-ethane-1,2-diamine)) of formula (V) above, which is referred to as Mn1 in this specification. In particular, Mn1 has the advantage of exhibiting clear anti-superoxide activity even under extracellular conditions, as well as antioxidant and anti-inflammatory effects on intestinal epithelial cells and macrophages. Furthermore, as described above, Mn1 improves DNBS-induced colitis in a mouse model, particularly in evaluations based on weight change. cat The value obtained by the McCord and Fridovich methods described above is 7.10. 6 M -1 ·s -1 That is the case.

[0067] The inorganic complex of the present invention may also have any of the formulas (VI), (VII), and (VIII), for example.

[0068] TIFF2026515321000008.tif179170

[0069] Inorganic complexes in which the 1,2-diaminoethane moiety is further rigidified (for example, with a 6-membered cyclohexyl moiety) (complexes of formulas (VII) and (VIII) above) are more favorably inert than Mn1 in the biological environment and are particularly preferred in the context of the present invention.

[0070] The microorganisms themselves are selected from bacteria and yeasts, preferably from among those that do not exhibit pro-inflammatory effects. In certain embodiments of the present invention, the microorganisms have anti-inflammatory properties. Probiotics having unique beneficial properties, particularly anti-inflammatory properties, may be used in the context of the present invention. Probiotics are defined by the World Health Organization (WHO, 2001) as "live microorganisms that, when administered in appropriate amounts, provide health benefits to the host." This definition has more recently been reviewed and validated by a group of experts (Hill et al., Expert consensus document: The International Scientific Association for 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).

[0071] Furthermore, microorganisms having cell walls that exhibit good permeability to inorganic complexes are preferred in the context of this invention.

[0072] The microorganism of the present invention may be a yeast, for example, the yeast species Saccharomyces boulardii or Yarrowia lipolytica.

[0073] In another embodiment of the present invention, the microorganism is selected from bacteria, which have the advantages of being easy to handle and growing quickly.

[0074] Bacteria that naturally contain a large amount of manganese, i.e., bacteria with a manganese content of about 1 mM or more, are particularly preferred in the context of the present invention. This is because a high manganese content advantageously increases the persistence of manganese in the form coordinated by ligands within the cell.

[0075] In certain embodiments of the present invention, the microorganism is a lactic acid bacterium, particularly a lactic acid bacterium of the genus Lactobacillus or Lactococcus. Such bacteria have the advantages of being able to survive over a wide pH range, especially in the acidic media of the digestive tract, allowing inorganic complexes to penetrate well into the bacteria, and having a high manganese content, thereby promoting ligand-metal coordination and maximizing the amount of inorganic complexes constructed within the cell.

[0076] The bacteria may be selected from the genera Lactobacillus, Lactococcus, Bifidobacterium, or Bacillus, with Lactobacillus plantarum and Lactococcus lactis being particularly preferred.

[0077] The bacteria may also be nonpathogenic to mammals, particularly humans, such as the Escherichia coli species.

[0078] In a preferred embodiment of the present invention, the microorganisms are modified to improve the targeted and efficient release of the encapsulated inorganic complex at a site suitable for obtaining therapeutic efficacy, more specifically at the site of intestinal inflammation. This is preferably achieved by weakening the cell wall of the microorganism before administration to the subject. That is, when the chemically modified microorganisms of the present invention are administered to a subject, they pass through the stomach and reach the intestines. Because the cell walls of the chemically modified microorganisms are weakened in advance by pretreatment, they are favorably lysed during their migration through the intestinal tract and release the SOD-mimicking inorganic complex in the intestines.

[0079] Therefore, in certain embodiments of the present invention, the cell walls of microorganisms are weakened genetically, chemically, and / or enzymatically. As a result, these cell walls are more easily lysed than the cell walls of the original, unweakened microorganisms. Generally, cells with weakened cell walls grow more slowly and settle more quickly than cells with unweakened cell walls. Microorganisms with weakened cell walls can be identified using membrane permeability testing kits (e.g., BacLight® kits) that measure the ability of reagents such as propidium iodide to penetrate into the cell. Microorganisms with weakened cell walls accumulate this reagent more readily than the original, unweakened microorganisms.

[0080] Any method known to those skilled in the art for weakening the cell walls of microorganisms without killing them is included within the scope of the present invention. The step of weakening the cell walls of microorganisms may be performed before, simultaneously with, or after the step of contacting the microorganisms with the inorganic complex in order to promote the penetration of the inorganic complex into the microorganisms. Preferably, the weakening step is performed before the contact step. In this case, the rate of penetration of the inorganic complex into the microbial cells is advantageously increased.

[0081] For example, microorganisms may be cultured for several hours, preferably 6 to 18 hours, for example, about 12 hours, in the presence of a cell wall-weakening agent such as glycine or threonine.

[0082] In certain embodiments of the present invention, the microorganisms are nutritionally dependent on a certain substance, preferably an amino acid, particularly a D-type amino acid, and are cultured for at least 1 hour, preferably at least 3 hours, in a medium that does not contain the substance / amino acid. Such amino acid deficiency induces weakening of the microbial cell wall. The amino acid may be, for example, alanine, phenylalanine, or thymidine.

[0083] For example, the Lactobacillus plantarum strain MD007 was deposited on March 23, 2023, with deposit number CNCM I-5940 (identification reference code LBH791-MD007) in the French National Collection of Microorganism Cultures (CNCM) (25 Rue du Docteur Roux, Paris, France) at the Pasteur Institute, and can be used in the context of this invention. This strain was obtained by making the Lactobacillus plantarum strain ATCC BAA-793 / NCIMB 8826 nutritionally resistant to alanine, as described in the literature by Palumbo et al. (2004; FEMS Microbiology Letters, 233: 131-138). Preferably, this strain is used after 1 to 5 hours of alanine deficiency.

[0084] Alternatively, Lactococcus lactis MG1363 strain (containing SOD enzyme) may be used in the context of the present invention.

[0085] According to the present invention, chemically modified microorganisms can be used as drugs for treating diseases or as nutritional supplements.

[0086] As used herein, the term “treatment” means obtaining a desired pharmacological and physiological effect, which may be preventive or curative. Therefore, as used herein, “treatment” includes preventing or partially preventing the onset of a disease or its symptoms or condition in a subject not yet diagnosed with the disease; and / or partially or completely curing a disease, its symptoms or condition, or adverse effects resulting from such disease.

[0087] The chemically modified microorganisms of the present invention can be used to counteract oxidative stress. Here, "counteract" means to prevent or reduce oxidative stress.

[0088] The chemically modified microorganisms of the present invention can be used to treat a wide range of pathophysiological processes involving oxidative stress, such as diabetes, inflammatory and neurodegenerative diseases, post-ischemia reperfusion injury, cancer, skin diseases such as psoriasis associated with oxidative stress, or any other disease associated with oxidative stress.

[0089] The chemically modified microorganisms of the present invention can be used in the treatment of inflammatory diseases, more specifically inflammatory bowel diseases such as Crohn's disease or ulcerative colitis.

[0090] The subjects of treatment with chemically modified microorganisms according to the present invention, who are suffering from or susceptible to the disease, are preferably mammals, particularly humans. For example, the subjects may be dogs or cats.

[0091] In a preferred embodiment of the present invention, the chemically modified microorganism of the present invention is administered orally to the subject.

[0092] Alternatively, it can be administered topically, particularly in the context of treating skin diseases.

[0093] In the context of drugs, the chemically modified microorganisms of the present invention are preferably administered to a subject in a therapeutically effective amount, that is, an amount suitable for delivering a therapeutically effective amount of SOD-mimicking inorganic complex to the subject.

[0094] The term "therapeutic effective dose" refers to the amount of a compound sufficient to treat a disease when administered to a subject for the treatment of that disease. The therapeutic effective dose of a compound depends on several factors, including the disease and its severity, the age and weight of the subject, the specific compound used, the route of administration, and the form of administration. Therefore, the amount of the chemically modified microorganism of the present invention administered to the subject is determined by the clinician for each case. In particular, the amount of the microorganism should be sufficiently small so as not to induce toxicity, such as manganese poisoning if the metal is manganese.

[0095] For example, a dose equivalent to 0.10-0.20 mg / kg / day of manganese, or for instance, 0.14 mg / kg / day, can be applied.

[0096] In the context of nutritional supplements, it is preferable that the chemically modified microorganisms of the present invention be administered to a subject in an appropriate amount to deliver an appropriate amount of the SOD-mimicking inorganic complex to the subject in order to obtain the desired effect, such as beneficial effects on health, comfort, and / or well-being.

[0097] For example, the chemically modified microorganisms of the present invention may be administered to the subject once or twice a day. In particular, during disease flare-up, the subject may be administered twice a day, for example, in the morning and evening, and during remission, it may be administered once a day.

[0098] The present invention may also be expressed as a method for therapeutically treating subjects suffering from diseases, particularly inflammatory diseases, more specifically inflammatory bowel diseases such as Crohn's disease or ulcerative colitis, the method comprising administering a therapeutically effective amount of the chemically modified microorganisms of the present invention to a subject requiring such treatment. The method may have any of the above features or combinations of features related to the use of the chemically modified microorganisms of the present invention as a drug. The chemically modified microorganisms may be administered to subjects as a component of a drug, or as a component of a food composition, nutraceutical composition, or dietary supplement.

[0099] The present invention also relates to the use of chemically modified microorganisms according to the present invention for the manufacture of drugs, and more particularly to the manufacture of drugs for the treatment of inflammatory diseases, more specifically inflammatory bowel diseases such as Crohn's disease or ulcerative colitis. This use may satisfy any of the features or combinations of features described above in relation to the use of chemically modified microorganisms according to the present invention as drugs.

[0100] The present invention also relates to the use of the chemically modified microorganisms according to the present invention for the production of food compositions, nutraceutical compositions, or dietary supplements, particularly for the production of those for combating oxidative stress. This use may satisfy any of the features or combinations of features described above in relation to the use of the chemically modified microorganisms according to the present invention as drugs or dietary supplements.

[0101] The present invention also relates to a pharmaceutical composition comprising a chemically modified microorganism as defined above in a pharmaceutically appropriate vehicle. In this pharmaceutical composition, the active ingredient is an SOD-mimicking inorganic complex encapsulated within the microbial cell.

[0102] In this specification, the term "pharmaceutically appropriate vehicle" means a vehicle that is useful for the preparation of a pharmaceutical composition or formulation, is generally safe, non-toxic, and does not have any properties that are biologically or otherwise undesirable to the target of treatment, particularly mammals, and more specifically to humans.

[0103] The vehicle of the composition of the present invention may be a solid, a semi-solid, or a liquid. The vehicle may be a diluent, an adjuvant, or another vehicle known in itself in the composition of a pharmaceutical composition.

[0104] Chemically modified microorganisms can be included in the vehicle in any form, particularly in freeze-dried form.

[0105] The pharmaceutical composition of the present invention may be formulated into any dosage form (galenical form), and may be formulated in a form particularly suitable for administration to mammals, and especially to humans. The pharmaceutical composition is preferably formulated in a form suitable for oral administration, such as a powder, capsule, oral solution, or suspension, or in a form particularly suitable for topical administration to the skin and / or mucous membranes of the target, such as a cream.

[0106] The pharmaceutical composition of the present invention may contain one or more excipients / additives known on their own, such as preservatives, sweeteners, fragrances, suspending agents, dispersants, lubricants, stabilizers, buffers, or mixtures thereof.

[0107] Furthermore, the pharmaceutical composition may contain one or more other active ingredients, whether or not they act synergistically with the inorganic complex of the present invention, and examples of such ingredients include other anti-inflammatory agents and / or analgesics.

[0108] The present invention also relates to therapeutic uses of the pharmaceutical compositions as defined above, and more particularly to uses for treating inflammatory diseases, more specifically inflammatory bowel diseases such as Crohn's disease or ulcerative colitis, in mammals, and more specifically in humans.

[0109] The present invention also relates to a nutraceutical composition, food composition, or dietary supplement comprising a chemically modified microorganism as defined above in a physiologically suitable vehicle. In this composition or supplement, the active ingredient is an SOD-mimicking inorganic complex encapsulated within the microbial cell.

[0110] In this specification, "physiologically acceptable vehicle" means a vehicle that, when administered to a subject, particularly a mammal, more specifically a human, is physiologically acceptable and does not produce an allergic reaction or similar undesirable reaction.

[0111] The vehicle of the composition / supplement may be solid, semi-solid, or liquid. The vehicle may be a diluent, an adjuvant, or any other vehicle known on its own for the composition of the nutraceutical composition, food composition, or dietary supplement.

[0112] The chemically modified microorganisms may be included in any form, and in particular in a freeze-dried form.

[0113] The nutraceutical composition, food composition, or nutritional supplement of the present invention is preferably in a form suitable for oral administration.

[0114] The composition or food may contain one or more probiotics, prebiotics, vitamins, polyphenols, minerals, materials suitable for oral administration (liquid or gel type, e.g., solvents, diluents, or non-toxic solubilizers that do not interact with the components of the composition in a harmful manner), and / or any other components or excipients, e.g., proteins, amino acids, carbohydrates, lipids, oligosaccharides, other micronutrients, metal salts, or cations.

[0115] The present invention also relates to the use of the above-defined nutraceutical compositions, food compositions, or dietary supplements for combating oxidative stress and / or treating inflammation in subjects, particularly in mammals, and more specifically in humans.

[0116] The method for producing chemically modified microorganisms according to the present invention comprises contacting the cells of the microorganism with a composition containing the superoxide dismutase mimic inorganic complex, preferably for at least one to several hours, thereby enabling the inorganic complex to penetrate into the microbial cells by a passive mechanism and accumulate inside.

[0117] This contact step is preferably: 1 to 12 hours, preferably 1 to 5 hours, for example, between approximately 2 hours; • and / or at 37°C; · and / or under shaking; • and / or in a liquid composition, preferably in a liquid composition with a pH of 7 to 8, for example, in 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 600 nm is 0.6 to 0.8; It will continue.

[0118] The concentration of the inorganic complex contained in the liquid composition is preferably 0.1 to 5 mM, for example, substantially equal to 0.4 mM. Such a concentration range is advantageously high enough to allow a large amount of the inorganic complex to penetrate into the microbial cells, while being low enough to avoid the accumulation of other forms of the metal (for example, manganese in the form of MnCl2 if the metal is manganese) in the microbial cells and to ensure that no toxicity to the cells occurs.

[0119] As described above, the method may include a step of weakening the cell wall of a microorganism before, during, or after (preferably before) the step of contacting the microbial cells with a composition containing a superoxide dismutase-mimicking inorganic complex.

[0120] Such a process can be carried out by any method known to those skilled in the art, for example, by culturing microorganisms for 1 to 12 hours in a culture medium to which a cell wall-weakening agent, such as glycine, has been added, for example, at a concentration of 10 to 20 g / L. The culture medium may be any medium suitable for culturing microorganisms. In particular, the culture medium contains all the nutrients and other elements necessary for the survival and, optionally, growth of the microorganisms.

[0121] In certain embodiments of the present invention, if the microorganism is nutritionally dependent on a specific amino acid such as alanine, the method includes a cell wall weakening step of culturing the microorganism in a medium that does not contain that amino acid. The medium contains all the nutrients and other elements necessary for at least the survival and optionally growth of the microorganism, and is, for example, De Man, Rogosa and Sharpe (MRS) medium, but does not contain the aforementioned amino acid. Such a deficiency step can be carried out for 1 to 5 hours, for example, about 3 hours. The deficiency step is preferably started when the optical density of the culture at 600 nm reaches 0.6, before the step of contacting the microbial cells with a composition containing a superoxide dismutase-mimicking inorganic complex.

[0122] In all cases, the culture process is preferably carried out at 37°C.

[0123] An example of a method for preparing chemically modified nutrient-requiring microorganisms according to the present invention includes the following sequence of steps: • Culturing microorganisms, especially bacteria, preferably at 37°C in a culture medium such as MRS supplemented with substances that the microorganisms require for nutrition, until the optical density at 600 nm reaches 0.6 to 0.8; • Separate the cells from the culture medium by centrifugation or the like, and wash the cells preferably three times with MRS medium or the like, if desired. • Culturing cells in MRS medium free of the aforementioned substance for 1 to 5 hours, for example, 3 hours ("substance deficiency"); • Separate the cells from the culture medium by centrifugation or the like, and wash the cells preferably three times with a 0.1 M HEPES solution or the like, if desired. • Resuspend the cells in a solution such as a 0.1 M HEPES solution containing a SOD-mimicking inorganic complex, preferably at a concentration of 0.1 to 5 mM, for example, 0.4 mM; Incubate the suspension at 37°C for 1 to 5 hours, for example, 2 to 3 hours, preferably with shaking; • Separate the cells from the liquid medium by centrifugation or similar means, and wash with a 0.1 M HEPES solution or similar solution if desired. Finally, collect the cells in a 16% glycerol-0.1M HEPES solution or similar.

[0124] Chemically modified cells containing large amounts of SOD-mimicking inorganic complexes obtained in this manner may be administered to subjects in need to prevent or treat diseases associated with oxidative stress, particularly inflammatory bowel disease. This administration is preferably carried out promptly after obtaining the chemically modified cells, more specifically within a few hours of obtaining them.

[0125] Alternatively, the cells may be freeze-dried and used as is. Or, the cells may be frozen at -80°C in a buffer such as HEPES buffer supplemented with glycerol at, for example, 16% (v / v), and thawed immediately before administration to the target of treatment. [Examples]

[0126] The features and advantages of the present invention will be further clarified by the following embodiments with reference to Figures 1 to 14. These embodiments are for illustrative purposes only and do not limit the present invention in any way. Figure 1 is a bar graph showing the Mn concentration in cell lysates measured by ICP-MS of either empty bacterial MD007 ("HEPES") or bacterial MD007 into which SOD mimetic compounds (Mn1, Mn1C) or MnCl2 have been introduced. Figure 2 is a graph showing the time course of body weight measurements in mice that received DNBS injection (day 0) and were orally administered daily with empty MD007 bacteria or MD007 bacteria introduced with an SOD mimetic (Mn1C) or MnCl2 (days -1 to 2). Body weight is expressed as a percentage of the initial body weight of the mouse before the start of the assay. Figure 3 is a graph showing the quantification of FITC-dextran as a marker of intestinal barrier permeability in mice that received DNBS injection and oral administration of empty MD007 bacteria or MD007 bacteria introduced with SOD mimetic (Mn1C) or MnCl2. The data represent the mean ± SD from three independent assays (approximately 24 mice). The p-value was calculated using a non-parametric Mann-Whitney test (one-sided test) because the Gaussian distribution of the data could not be verified by the Anderson-Darling normality test. The mean rank of each column was compared to that of the DNBS control, and each comparison was performed independently. **: p<0.01 compared to the DNBS control; ns means no significant difference. Figure 4 is a bar graph showing the count of viable cells in freeze-thaw aliquots of either empty MD007 bacteria ("HEPES") or MD007 bacteria introduced with SOD mimetic organisms (Mn1, Mn1C). Figure 5 is a bar graph showing the Mn content in cell lysates (average of 3 experiments) measured by ICP-MS for empty MD007 bacteria ("HEPES") or MD007 bacteria introduced with SOD mimetic compounds (Mn1, Mn1C). Figure 6 shows the area under the curve of body weight measured from day 0 to day 3 (D0 to D3) in mice that received DNBS injection (day 0) and were orally administered daily with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). Figure 7 is a graph showing the macroscopic scores determined on the day of euthanasia for mice that received DNBS injection and were orally administered daily for four days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). Figure 8 is a graph showing the concentration of lipocalin 2 (LCN-2) measured in the colon of mice that received DNBS injection and were orally administered daily for 4 days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). Figure 9 is a graph showing the concentration of lipocalin 2 (LCN-2) measured in the serum of mice that received DNBS injection and were orally administered daily for 4 days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with SOD mimetic Mn1 or Mn1C (data were collected from three independent assays, using 8 mice per assay / condition). Figure 10 shows the mRNA levels of SOD2 normalized by the mRNA levels of GADPH and TBP, measured in colon samples of mice that received DNBS injection and were orally administered daily for 4 days with Hepes ("Hepes"), empty MD007 bacteria ("MD007 control"), or MD007 bacteria introduced with the SOD mimetic Mn1C (average for 16 mice in each condition). Figure 11 is a graph showing the time course of body weight measurements in mice that received DNBS injection (day 0) or not ("vehicle"), and were orally administered Hepes ("DNBS vehicle") or empty MG1363 bacteria ("DNBS MG1363") daily. Body weight is expressed as a percentage of the initial body weight of the mouse before the start of the assay. Figure 12 shows the area under the curve in Figure 11 (from day 0 to day 5: D0 to D5). Figure 13 is a graph showing the macroscopic scores determined on the day of euthanasia (day 5: "D5" or day 6: "D6") of mice that received DNBS injections and were orally administered Hepes ("DNBS vehicle") or empty MG1363 bacteria ("DNBS MG1363") daily for 5 days. Figure 14 is a bar graph showing the body weight on day 5 (D5) of mice that received daily oral administration of either DNBS injection (day 0) or no injection ("vehicle"), Hepes ("DNBS vehicle"), empty MG1363 bacteria ("DNBS MG1363"), or MG1363 bacteria with Mn1 introduced. Body weight is expressed as a percentage of the initial body weight of the mouse before the start of the assay (D0).

[0127] Example 1 - Synthesis of SOD-mimicking inorganic complexes The ligands used in the preparation of the inorganic complexes were EnPI2C and EnPI2CP, respectively, as shown in formulas (Ixa) and (Ixb).

[0128] TIFF2026515321000009.tif103170

[0129] The SOD mimetic Mn(II) complex was prepared by mixing each ligand with MnCl2 in a molar ratio of 1:1.3 in 0.1 M HEPES (pH 7.5) and holding at room temperature for 2 hours to achieve complete complex formation with the ligand manganese(II).

[0130] The following SOD-mimicking metal complexes were obtained: Mn1C from EnPl2C (formula (VII) above), and Mn1CP from EnPl2CP (formula (VIII) above).

[0131] Example 2 - Introduction of SOD mimetic into bacteria The L. plantarum MD007 strain, deposited on March 23, 2023, as deposit number CNCM I-5940 (identification reference code LBH791-MD007) in the French National Collection of Microorganism Cultures (CNCM) (25 Rue du Docteur Roux, Paris, France) of the Pasteur Institute, was used in the experiment. This strain was obtained by making the Lactobacillus plantarum ATCC BAA-793 / NCIMB 8826 strain nutrient-dependent for alanine, as described in the literature by Palumbo et al. (2004; FEMS Microbiology Letters, 233: 131-138).

[0132] MD007 cells cultured overnight in MRS medium supplemented with alanine (MRS-alanine) were subjected to OD in MRS-alanine. 600nm = Dilute to 0.2, then OD the cells. 600nm The culture was allowed to grow until it reached 0.6. The culture was then centrifuged, the pellet was washed twice, and resuspended in alanine-free MRS.

[0133] To accelerate bacterial lysis in the mouse intestines, bacteria were weakened by depriving them of alanine for 3 hours.

[0134] After alanine deficiency, the culture medium was centrifuged again, the pellet was washed twice, and resuspended in 0.1 M HEPES. At this point, the culture was divided into four parts, and SOD mimetic compounds Mn1, Mn1C, and Mn1CP, as well as MnCl2, were added to each to a final concentration of 0.4 mM. The bacterial suspension was incubated at 37°C for 2 hours with stirring.

[0135] Finally, the culture medium was centrifuged, washed with 0.1 M HEPES, and the bacteria were resuspended in 0.1 M HEPES (HEPES-gly) containing 16% glycerol. To avoid freeze-thaw cycles, the cultures were stored at -80°C in 2 mL aliquots.

[0136] Example 3 - Mn determination by ICP-MS analysis For each inorganic complex and the control MnCl2, the bacterial suspension (OD) was used. 600nm Mn quantification was performed by ICP-MS (Inductively Coupled Plasma Mass Spectrometry) using one of the aliquots from =1). A negative control using unintroduced bacteria (HEPES) was also analyzed.

[0137] To avoid isobaric interference, as an isotope 55 I selected Mn.

[0138] ICP-MS experiments were performed using an Agilent 7700 series ICP-MS system and ASX-500 series autosampler under the following conditions: RF power 1550W; sampling depth 10mm; helium flow rate 5mL / min; measurements repeated 5 times with 100 sweeps per replicate; integration time / mass 1s.

[0139] The bacterial suspension was diluted with 2% HNO3 to lyse the bacteria and release Mn from all coordination sites. To achieve complete bacterial lysis, the 2% HNO3 solution was allowed to stand at room temperature for 1 hour, and then filtered. A calibration curve was created using commercially available multi-element standards, and the total metal content was measured using the OD of the bacterial suspension. 600nm It was normalized using this method.

[0140] The results obtained for HEPES, MnCl2, Mn1, and Mn1C are shown in Figure 1. These results indicate that bacteria into which the SOD mimetic according to the present invention has a high manganese content.

[0141] Example 4 - Induction of DNBS colitis and administration of bacteria in mice In vivo assays were performed on pathogen-free male C57BL / 6 mice at the animal facility of the National Institute of Agricultural and Environmental Research (INRAE, Jouy-en-Josas).

[0142] The timeline for inducing colitis and forcing oral administration of the introduced bacteria to mice is as follows. The assay was continued for 5 days. The introduced bacteria were administered intragastricly daily for the first 4 days. On day 2, mice were anesthetized intraperitoneally, and colitis was induced by rectal injection of dinitrobenzenesulfonic 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 final day, day 5, the mice were euthanized by cervical dislocation. The body weight of the mice was measured daily.

[0143] The groups examined were as follows: a control colitis group (DNBS+HEPES-gly), a control group using empty bacteria (DNBS+MD007), and two groups using introduced bacteria: "DNBS+MD007_MnCl2" and "DNBS+MD007_Mn1C". Each group consisted of 8 mice. The assay was repeated three times independently. In other words, a total of 24 mice were used for each condition.

[0144] The amount of introduced bacteria administered to the mice was fixed at 150 nmol per dose, so that the total amount of manganese administered was the same for all mice. This is 5 × 10 8 ~2×10 9 This corresponds to the bacterial count of CFU. The amount of MD007 administered to the control "DNBS+MB007" group was set to the same amount as the maximum amount of surviving introduced bacteria administered to mice from the introduced bacterial group. Finally, 200 μL of HEPES-gly was administered to the control colitis group.

[0145] Example 5 - Evaluation of Colitis Severity 5.1 / Mouse weight Figure 2 shows the changes in mouse body weight throughout the entire assay period.

[0146] As is clear from this figure, mice that ingested the SOD mimetic Mn1C, which was internalized in MD007, showed suppressed weight loss and accelerated weight recovery. In fact, weight loss (maximum on day 2) was limited to 13%, and the mice recovered to approximately 90% of their initial weight by day 3.

[0147] Furthermore, mice treated with empty MD007 showed only slightly less weight loss compared to untreated mice, suggesting that this strain possesses slight probiotic activity.

[0148] 5.2 / Dissection of mice after euthanasia The abdominal cavity of the euthanized mice was opened, the colon was removed, and a longitudinal incision was made to wash away the feces.

[0149] Subsequently, the colon was divided into multiple sections lengthwise for further experiments.

[0150] 5.3 / Intestinal Permeability Assay Three hours before euthanasia, mice were orally administered fluorescein isothiocyanate (FITC)-dextran (12 mg per mouse in 200 μL of PBS), a fluorescent marker. Immediately before euthanasia, blood was collected by submandibular vein puncture in the presence of heparin. The collected whole blood was then centrifuged to obtain plasma. The FITC-dextran concentration in the plasma sample was measured by fluorescence intensity. Fluorescence readings were performed using a fluorescence spectrophotometer with 80 μL of plasma sample in a microplate (excitation wavelength = 488 nm, emission measurement = 520 nm).

[0151] The results are shown in Figure 3.

[0152] DNBS infusion is known to cause damage to intestinal tight junctions, increasing barrier permeability and thereby promoting the transfer of FITC-dextran from the gastrointestinal tract to the bloodstream. Compared to untreated mice, plasma samples from mice treated with MD007+Mn1C showed significantly lower quantified FITC-dextran concentrations. This indicates that Mn1C vectorized with MD007 has a significant effect in mitigating DNBS-induced damage to intestinal permeability.

[0153] Example 6 - Additional experiments using L. plantarum MD007 strain 6.1 / Viability of introduced bacteria and quantitative determination of Mn in bacteria Three independent experiments were conducted as described below. The results shown are the average of these three experiments.

[0154] Lactobacillus plantarum strain MD007 was grown in MRS containing D-alanine (200 μg / mL) at 37°C.

[0155] The culture that has been incubated overnight is inoculated into fresh culture medium, OD 600nmAfter making it 0.2, OD 600nm The cells are grown until the pH is 0.7, and then washed three times with MRS medium that does not contain D-alanine. After washing, the bacteria are incubated in D-alanine-free medium at 37°C for 3 hours, and then the SOD mimetic is introduced. After alanine starvation, the bacteria are washed three times with Hepes (0.1M, pH 7.4), and the bacterial count per 200 μL is approximately 10 9 Adjust the concentration to a level where it is sufficient.

[0156] After removing the supernatant, the bacteria are incubated with Hepes (0.1M, pH 7.4) alone, or with the desired concentration of the test compound (Mn1 or Mn1C) in the same buffer solution, at 37°C for 2 hours or 24 hours. The Mn1C and Mn1 solutions are prepared each time to prevent degradation and manganese oxidation. The culture is centrifuged and washed with Hepes (0.1M, pH 7.4), and the bacteria are resuspended in 0.1M Hepes containing 16% glycerol, with approximately 10 cells per 200 μL. 9 Adjust to a concentration that results in a single dose. This volume corresponds to the maximum and typical volume for daily forced oral administration to mice. Store the bacteria in 2 mL aliquots at -80°C to avoid freeze-thaw cycles. Freeze the sample for forced oral administration to mice at -80°C and allow it to return to room temperature before administration to the mice.

[0157] To evaluate bacterial viability, a CFU (colony-forming unit) test was performed on cultures incubated overnight and frozen vials. In this test, the prepared sample was diluted in PBS in 1 / 10 serial dilutions, and the corresponding known volume was plated onto the corresponding culture medium. The plate was incubated at 37°C for 48 hours. Then, the colonies were visually counted. The results are shown in Figure 4. It can be seen that introducing Mn1 or Mn1C into the bacteria did not affect cell viability.

[0158] Quantitative determination of manganese in bacterial lysates is performed by ICP-MS. For ICP-MS analysis, the bacterial pellet recovered by centrifugation is acidified with 2% HNO3 to lyse the cells and release Mn from all coordination sites. Before analysis, the sample is carefully filtered through a 0.2 μm filter to remove bacterial residue. A calibration curve is created using a commercially available Mn standard solution. A concentration range of 0 to 100 ppb is typically used for calibration. The results are shown in Figure 5. These results indicate that bacteria introduced with the SOD mimetic according to the present invention have high manganese levels.

[0159] 6.2 / Mice Treatment Male C57BL / 6 mice (6 weeks old) were reared under specific pathogen-free (SPF) conditions. For acclimatization, the mice were reared under standard conditions for at least one week prior to the experiment. All animal experiments were conducted in an accredited research facility and approved by the French government (license number: 16744-201807061805486_v2) as well as the local ethics committee.

[0160] Forced oral administration of empty or introduced bacteria to these mice was started the day before inflammation induction by DNBS (D-1) to ensure that bacteria were present in the colon beforehand. On D0, the mice were anesthetized by 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 rectally via catheter at a location approximately 3.5 cm into the colon. Bacteria (approximately 10 cells) 9 Mice were administered intragastricly to 4 or 5 consecutive days using either 1 cell / 200 μL of introduced bacteria (CMB) or Hepes. The amount of CMB administered to the mice was fixed at 150 nmol per dose, ensuring that the total amount of manganese administered was the same for all mice. This amount corresponds to 1 × 10⁶ bacterial cells. 9 ~2×10 9This corresponds to one individual. The amount of bacteria administered to the DNBS MD007 control group was set to correspond to the maximum number of bacteria administered to the CMB-treated group. Special care was taken in the management of the mice throughout the experimental period. On the final day (D3 or D4), the mice were euthanized by cervical dislocation. The groups examined were named as follows: a colitis group administered with Hepes buffer instead of bacteria ("Hepes"), a colitis group administered with empty bacteria ("MD007 control"), and two colitis groups treated with CMB ("MD007 Mn1" and "MD007 Mn1C"). Each group consisted of 8 mice. The assay was repeated independently 3 or 4 times. That is, a total of 24 or 32 mice were used for each condition.

[0161] The body weight of all mice was monitored over time. Figure 6 shows the area under the curve for body weight monitoring from day 0 to day 3 (D0 to D3). As shown in this figure, mice that ingested SOD mimetic Mn1 and Mn1C, which are internalized in MD007, were able to suppress body weight loss and even increase body weight.

[0162] 6.3 / Evaluation of Colitis Severity in Treated Mice After treatment with the introduced bacteria, the abdominal cavity of each euthanized mouse was opened, the colon was removed, and the colon was incised longitudinally to flush out the feces. Immediately thereafter, a macroscopic score was evaluated. This score assesses the condition of the colon, considering thickening of the colonic wall, presence of ulcers, congestion, adhesion to other intraperitoneal tissues, and the characteristics of the colonic contents (indicators of transport abnormalities). The results are shown in Figure 7. Treatment with bacteria introduced with Mn1 or Mn1C significantly improved the macroscopic score.

[0163] Subsequently, the colon was divided longitudinally into two sections. The right-hand section was then divided transversely into four sections for further analysis.

[0164] Lipocalin-2 (LCN-2) concentrations were measured in the colon and plasma of mice. For plasma LCN-2 concentration, centrifuged blood samples (2000g for 10 minutes, 20°C) were used. A commercially available sandwich enzyme immunosorbent assay (Mouse Lipocalin-2 / NGAL DuoSet ELISA; R&D Systems Europe) was used according to the instructions for use. For colon LCN-2 concentration, the colon supernatant was used, employing the same kit as for plasma. To this end, colon samples were weighed and mechanically dissociated using a Precellys® (Bertin) instrument in 0.5% HTAB (hexadecyltrimethylammonium bromide) buffer in the presence of a mixture of 1.4 and 2.8 mm ceramic beads (10,000 rpm for 20 seconds x 3 cycles). The samples were then briefly centrifuged, and the supernatant was collected. The results are shown in Figure 8 for colon LCN-2 concentration and in Figure 9 for serum LCN-2 concentration. These studies demonstrate that treatment of mice with bacteria introducing Mn1 or Mn1C results in a significant decrease in lipocalin 2 levels.

[0165] The transcription of the SOD2 gene in the colon was quantified as follows: Total RNA was extracted from colon homogenate using the Qiagen RNeasy kit according to the instructions for use. β-mercaptoethanol was used as a reducing agent for irreversible denaturation of RNases enzymes. Subsequently, reverse transcription was performed using oligo(dT)12-18 primers and SuperScript® II reverse transcriptase (Invitrogen®) to generate complementary DNA. SOD2 gene expression was quantified by quantitative PCR using 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 was set to 60°C.

[0166] The levels of housekeeping GAPDH and TATA-box binding protein (TBP) genes, which are known not to be affected in inflammatory states, were also quantified by RT-PCR, and SOD2 levels were normalized based on these levels. The results obtained for bacteria with Mn1C are shown in Figure 10. It can be seen that mice treated with bacteria with Mn1C have significantly lower SOD2 mRNA levels.

[0167] Example 7 - Lactococcus lactis MG1363 strain The Lactococcus lactis MG1363 strain, described in Sanders et al., 1995, J. Bacteriol 177: 5254-5260, was used in this experiment. This strain possesses the SOD enzyme.

[0168] 7.1 / Introduction to bacteria This strain is grown in M17 glucose at 30°C.

[0169] The culture that has been incubated overnight is inoculated onto fresh culture medium, and OD 600nm After making it 0.2, OD 600nm The cells were allowed to grow until the pH was 0.7. The bacteria were washed three times with Hepes (0.1M, pH 7.4), and the bacterial count was approximately 10 per 200 μL. 9 This was defined as the concentration at which a single unit is formed.

[0170] The introduction of Mn1 into bacteria was performed using the protocol described in Example 6.

[0171] 7.2 / Assessment of bacterial neutrality in DNBS-induced colitis The male C57BL / 6 mouse (6 weeks old) described in Example 6 was used in this experiment.

[0172] On the first day of the assay (D0), mice were anesthetized by intraperitoneal administration (IP) of ketamine and xylazine 0.06%. DNBS solution (3 mg / mouse) in 30 / 70 ethanol / PBS (v:v) was administered by rectal injection (IR) using a catheter to a location approximately 3.5 cm into the colon. Bacterial suspension (10 cells) 9Mice were administered intragastricly (200 μL / unit) or Hepes (100 μL) daily for 5 days. Special care was taken in the management of the mice throughout the experiment. On the final day (D5) or the following day (D6), the mice were euthanized by cervical dislocation.

[0173] The body weight of all mice was measured over time. Figure 11 shows the change in mouse body weight throughout the entire assay period, from day 0 to day 5. Figure 12 shows the area under the curve. As is clear from these figures, administration of empty MG1363 to DNBS-induced colitis mice did not have a significant effect on mouse body weight.

[0174] The macroscopic scores of the mice were evaluated on day 5 or day 6, as described in Example 6. The results are shown in Figure 13. It can be seen that the MG1363 bacterium does not affect DNBS-induced colitis.

[0175] 7.3 / Evaluation of Mn1-inducible MG1363 bacteria in DNBS-induced colitis On the first day of the assay (D0), mice were anesthetized by intraperitoneal administration of ketamine and xylazine 0.06%, and DNBS (3 mg / mouse) and DNBS solution in 30% ethanol / PBS (Sigma) were administered rectally via catheter to a location approximately 3.5 cm into the colon. One group of non-colitis controls received only Hepes (referred to as the "vehicle"). Bacteria (bacterial cells 10 9 Mice were administered intragastricly either (individual cells / 200 μL), introduced bacteria (CMB), or Hepes daily for 5 days. The amount of CMB administered to the mice was fixed at 150 nmol per dose, ensuring that the total amount of manganese administered was the same for all mice. Special care was taken in managing the mice throughout the experiment.

[0176] The body weight of all mice was measured over time. The body weight of the mice on day 5 is shown in Figure 14. These results indicate that administering MG1363 bacteria with Mn1 introduced to mice with DNBS-induced colitis increased the mice's body weight, which signifies an improvement in the mice's health. This improvement was not observed with empty bacteria.

[0177] 8 / Statistical Analysis (Examples 6 and 7) All statistical analyses were performed using GraphPad Prism software (GraphPad Software). Results are shown in dot plots along with the mean ± SEM. p-values ​​were calculated using a nonparametric Mann-Whitney test (one-sided test). Outliers detected by Prism software were excluded. The mean rank of each column was compared to that of the DNBS Hepes control. Each comparison was performed independently. ****: p<0.0001, ***: p<0.001, **: p<0.01, and *: p<0.05 (compared to the DNBS Hepes control or vehicle), where ns means no significant difference. A p-value less than 0.05 was considered statistically significant.

Claims

1. Chemically modified microorganisms selected from bacteria and yeasts for use as drugs or nutritional supplements, comprising a synthetic superoxide dismutase-mimicking inorganic complex.

2. The inorganic complex is a mimetic of human manganese superoxide dismutase, according to claim 1, a chemically modified microorganism for use.

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

4. The aforementioned inorganic complex has the general formula (I): It has, in the formula, n, m, and p may be the same or different, and are integers from 1 to 3. R １ represents a hydrogen atom, or a linear, branched, and / or cyclic alkyl group which may be substituted with at least one aromatic ring or polycyclic ring. Alternatively, R １ This represents a peptide containing 1 to 20 amino acid residues. R ２ , R ２ ', R ３ , R ３ ' represents a hydrogen atom in all cases. Alternatively, R ２ ’ and R ３ ’ represent a hydrogen atom, and R ２ and R ３ together with the carbon atom to which they are attached form an aliphatic ring or a heterocyclic ring, and the aliphatic ring or heterocyclic ring may be substituted by a Lewis base, a phenol group, an imidazole ring, a pyridine ring, and / or one or several identical or different alkyl groups, and each of the alkyl groups may be substituted by a Lewis base, a phenol group, an imidazole ring, and / or a pyridine ring. Alternatively, R ２ , R ２ ', R ３ , R ３ ' together with the carbon atoms to which they are bonded form an aromatic ring or heterocycle, and the aromatic ring or heterocycle may be substituted with a Lewis base, a phenol group, an imidazole ring, a pyridine ring, and / or one or more identical or different alkyl groups, and each of the alkyl groups may be substituted with a Lewis base, a phenol group, an imidazole ring, and / or a pyridine ring, Ar １ and Ar ２ These may be the same or different, each representing a five-membered or six-membered aromatic heterocycle, and may be substituted with one or more identical or different substituents, each of which is selected from a bromine atom, a chlorine atom, a methyl group, a methoxy group, and a nitro group. R ４ represents a phenyl group which may be optionally substituted with one or more identical or different substituents, each of which substituents is selected from a bromine atom, a chlorine atom, a methyl group, a methoxy group, and a nitro group. Alternatively, R ４ is formula -R ５ It represents the -CO- group, and in the formula, the carbonyl group is bonded to the oxygen atom, R ５ ha- (CH ２ ) It is bonded to the p-group, R ５ represents a linear, branched, and / or cyclic alkyl group which may be substituted with a Lewis base. A chemically modified microorganism for use according to any one of claims 1 to 3.

5. The aforementioned inorganic complex has the general formula (IV): It has, in the formula, R １ , R ２ , R ２ ', R ３ , and R ３ A chemically modified microorganism for use according to claim 4, wherein ' is as defined in claim 4.

6. The inorganic complex is given by formula (VII): A chemically modified microorganism for use according to claim 5, having the characteristics described.

7. A chemically modified microorganism for use according to any one of claims 1 to 6, which is a lactic acid bacterium, particularly a lactic acid bacterium of the genus Lactobacillus or Lactococcus.

8. A chemically modified microorganism for use according to any one of claims 1 to 7, wherein the cell wall is genetically, chemically, and / or enzymatically weakened.

9. A chemically modified microorganism for use according to claim 8, which is nutritionally required for amino acids and has been cultured for at least one hour in a medium that does not contain the amino acids.

10. A chemically modified microorganism for use according to any one of claims 1 to 9, for combating oxidative stress.

11. A chemically modified microorganism for use according to any one of claims 1 to 10, for the treatment of inflammatory diseases.

12. Chemically modified microorganisms for use according to claim 11, for the treatment of inflammatory bowel disease.

13. The chemically modified microorganism for use according to any one of claims 1 to 12, wherein the chemically modified microorganism is administered orally to a subject.

14. A chemically modified microorganism for use according to any one of claims 1 to 13, for treating a subject that is a mammal, preferably a human.

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

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

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