Treatment of myeloperoxidase-positive ANCA-associated vasculitis with H2S-releasing compounds

Abstract

Treatment of Myeloperoxidase-Positive ANCA-Associated Vasculitis with H2S-Releasing Compounds. The present invention relates to hydrogen sulfide (H2S) emitter compounds and compositions for use in the treatment of myeloperoxidase (MPO)-induced neutrophil-mediated tissue damage, particularly positive antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), including MPO-induced granulomatosis with polyangiitis (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg-Strauss syndrome), microscopic polyangiitis (MPA), and various forms of the disease in various organs such as the kidney and lung.

Description

[Technical Field]

[0001] The present invention relates to hydrogen sulfide (HS) emitter compounds and compositions for use in the treatment of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV), including MPO granulomatosis with polyangiitis (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg-Strauss syndrome), microscopic polyangiitis (MPA), and renal-confined vasculitis (renal-confined microscopic polyangiitis or renal-confined necrotizing crescentic glomerulonephritis, NCGN). [Background technology]

[0002] Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a group of rare autoimmune diseases characterized by severe systemic vasculitis and the presence of autoantibodies against circulating autoantigens, the most important of which are circulating antiproteinase 3 (PR3) or antimyeloperoxidase (MPO) autoantibodies. AAV subtypes include granulomatosis with polyangiitis (formerly known as Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome), microscopic polyangiitis, and renal-limited vasculitis, all of which may be associated with PR3 or myeloperoxidase (MPO) [Jennette, JC and PH Nachman, ANCA Glomerulonephritis and Vasculitis. Clin J Am Soc Nephrol, 2017. 12(10): pp. 1680-1691; Kallenberg, CG, Pathophysiology of ANCA-associated small vessel vasculitis. Curr Rheumatol Rep, 2010. 12(6): pp. 399-405].

[0003] Granulomatosis with polyangiitis (GPA, Wegener's granulomatosis) is characterized by granulomas forming in the respiratory tract, focal glomerulonephritis, and necrotizing systemic vasculitis.

[0004] In rare variants, the necrotizing vasculitis manifestations of microscopic polyangiitis (MPA) can be detected without immune deposition.

[0005] Churg-Strauss syndrome (also known as eosinophilic granulomatosis with polyangiitis, EGPA) is a vasculitis associated with eosinophilic granulocytes, characterized by granuloma formation, and often accompanied by asthmatic symptoms.

[0006] In AAV, the kidneys are recognized as the most commonly affected vital organ, and the severity of renal manifestations indicates the outcome. Therefore, renal-limited vasculitis (also known as microscopic polyangiitis limited to the kidney or renal-limited necrotizing crescentic glomerulonephritis, NCGN) is a particularly important variant of the disease from a public health perspective [Galesic K, Ljubanovic D, Horvatic I. Treatment of renal manifestations of ANCA-associated vasculitis. J Nephropathol. 2013 Jan;2(1):6-19. doi: 10.5812 / nephropathol.8971. Epub 2013 Jan 1. PMID: 24475421; PMCID: PMC3886180.]

[0007] In patients with renal AAV, mortality is 2.7 times higher than in the general population. In the first year, the main causes of death are infection (48%) and active vasculitis (19%), while in later stages, cardiovascular disease (26%), malignant transformation (22%), and infection (20%) [88, 89] [Tan, JA, et al., Mortality in ANCA-associated vasculitis: a meta-analysis of observational studies. Ann Rheum Dis, 2017. 76(9): pp. 1566-1574; Flossmann, 0., et al., Long-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis, 2011. 70(3): pp. 488-94].

[0008] The autoimmune nature of the disease suggests that the majority of AAV patients are found to be positive for at least one of the ANCA autoantigens. AAV disease is grouped based on the type of autoantigen, e.g., PR3-positive, MPO-positive, and ANCA-negative vasculitis [Kitching, AR, et al., ANCA-associated vasculitis. Nat Rev Dis Primers, 2020. 6(1): p. 71.].

[0009] MPO, or myeloperoxidase, is one of the most abundant autoantigens in AAV. According to Galesic, K. et al., the prevalence of MPO in Wegener's granulomatosis is 20%, in microscopic polyangiitis 50%, in Churg-Strauss syndrome 60%, while in renal-limited microscopic polyangiitis it is 70%, while the prevalence of ANCA-negative variants of the same diseases is 5%, 10%, 30%, and 10%, respectively, and the prevalence of PR3 also varies greatly [Galesic, K. et al., 2013, see below].

[0010] The role of hydrogen sulfide in autoimmune inflammatory diseases is a topic of considerable debate. Among its wide range of biological functions, various protective functions in inflammatory processes have been proposed for this small endogenous mediator [Whiteman, M. and PG Winyard, Hydrogen sulfide and inflammation: the good, the bad, the ugly, and the promising. Expert Rev Clin Pharmacol, 2011. 4(1): pp. 13-32.]. Interactions with heme proteins may represent an important mechanism in these processes [Palinkas, Z., et al., Interactions of hydrogen sulfide with myeloperoxidase. Br J Pharmacol, 2015. 172(6): pp. 1516-32.].

[0011] However, H2S can easily become toxic, depending on the conditions and concentration. H2S toxicity is caused by the reaction of H2S with ferric iron in oxidized cytochrome oxidase, which inhibits cellular respiration and leads to cellular hypoxia [Gall Tamas et al. Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin / heme-mediated vascular damage in atherosclerosis, Redox Biology, Volume 57, 2022, 102504, ISSN 2213-2317.]

[0012] The interaction of H2S with the MPO enzyme has been investigated, and a reversible inhibitory effect of H2S on the peroxidase and chlorination activities of MPO was observed. Nevertheless, kinetic experiments have shown that the interaction between H2S and MPO is quite complex, and H2S also functions as a substrate for MPO [Palinkas, Z., et al., Interactions of hydrogen sulfide with myeloperoxidase. Br J Pharmacol, 2015. 172(6): pp. 1516-32.] [Garai, D., et al., Mechanisms of myeloperoxidase-catalyzed oxidation of H2S by H2O2 or O2 to produce potent protein Cys-polysulfide-inducing species. Free Radic Biol Med, 2017. 113: pp. 551-563.]

[0013] Therefore, this dual nature of H2S places a heavy burden on investigators to reach an accurate conclusion as to whether H2S is beneficial in a particular disease, and complex experiments with unpredictable outcomes or results are required.

[0014] In fact, on the other hand, the present inventors have also found that sulfide and sulfide donors can effectively inhibit the formation of MPO oxidized products in the supernatant of PMA-activated neutrophils [Garai, D., et al. Measurements for Sulfide-Mediated Inhibition of Myeloperoxidase Activity. In: Belhowski, J. (eds) Vascular Effects of Hydrogen Sulfide. Methods in Molecular Biology, 2019, vol. 2007. Humana, New York, NY. https: / / doi.org / 10.1007 / 978-1-4939-9528-8_14], and MPO is an autoantigen in ANCA vasculitis, and its activity is irrelevant from the perspective of AAV pathogenesis. Indeed, currently, persistent MPO positivity, an increase in MPO levels, or even a change from MPO-negative to MPO-positive serotype are at most only weakly predictive of future disease recurrence and should not be used to guide treatment decisions according to the KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases [KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases Kidney International, (2021) 100(45) Supplement, pages S1 to S276].

[0015] Prior art results suggest that H2S may have an effect on the pathophysiological processes of neutrophils due to its important physiological effects in inflammation, but the discovery of a complex network of effects indicates that the overall effect is uncertain and unpredictable.

[0016] Currently, there are no data regarding the role of MPO activity as a predictor in the treatment of autoimmune diseases such as AAV.

[0017] Furthermore, there is clear evidence that neutrophil function was not affected by hydrogen sulfide donors when activated with PMA, LPS, or Escherichia coli.

[0018] For example, treatment with the hydrogen sulfide donor GYY4137 did not inhibit PMA- or LPS-induced oxidative burst. In fact, pretreatment of neutrophils with 200 μM GYY4137 prior to PMA activation increased ROS production compared to untreated samples. Treatment with GYY4137 also inhibited apoptosis and promoted cell viability in LPS-induced neutrophils. Sulfide was demonstrated to modestly reduce ROS formation in LPS-induced neutrophils, but these effects were measured only after 17–18 hours of incubation [Petrushanko, LY, et al. Influence of the Donor of Hydrogen Sulfide GYY4137 on the Activation of Human Neutrophils by E. coli Lipopolysaccharides. Mol Biol 53, 79–86 (2019)].

[0019] Furthermore, another research group has shown that various sulfide-releasing compounds, such as sodium sulfide, diallyl disulfide, diallyl trisulfide, and cysteine, also inhibited neutrophil apoptosis and promoted the oxidative burst upon PMA and bacterial activation [Farahat S. et al. Effect of Hydrogen Sulfide on Essential Functions of Polymorphonuclear Leukocytes. Toxins. 2023; 15(3): 198.]

[0020] Rinaldi, L. et al. further demonstrated that in the presence of NaH2S (1.83 mM), neutrophils killed bacteria more efficiently than in the absence of sulfide, and that the sulfide donor also reduced neutrophil apoptosis [Rinaldi, L., Gobbi, G., Pambianco, M. et al. Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3. Lab Invest 86, 391-397 (2006). https: / / doi.org / 10.1038 / labinvest.3700391].

[0021] At first glance, none of these publications mention AAV, an autoimmune disease. There is no suggestion in the prior art that sulfide may inhibit priming, ANCA-induced degranulation, and oxidative burst. Rather, the prior art would suggest that hydrogen sulfide may even enhance neutrophil activation by preventing neutrophil apoptosis, an undesirable process in autoimmune diseases.

[0022] Realistically, prior art treatment options for AAV disease are limited.

[0023] Initially, patients typically receive immunosuppressive treatment with cyclophosphamide or rituximab, which may be augmented with other treatment options, such as glucocorticoids or methotrexate, depending on the patient's condition. Recommendation 9.3.1.1 of the KDIGO 2021 Clinical Practice Guidelines for the Management of Glomerular Diseases [KDIGO 2021, see below] suggests the use of a combination of glucocorticoids and cyclophosphamide or rituximab as initial treatment for de novo AAV(1B).

[0024] To maintain remission, patients may be treated with azathioprine or methotrexate, but only a small percentage of patients achieve remission without pharmaceutical treatment [Chen, M. and CG Kallenberg, ANCA-associated vasculitides—advances in pathogenesis and treatment. Nat Rev Rheumatol, 2010. 6(11): pp. 653-64; Ozaki, S., ANCA-associated vasculitis: diagnostic and therapeutic strategy. Allergol Int, 2007. 56(2): pp. 87-96].

[0025] Unfortunately, the combination of immunosuppressants and steroid treatment predisposes patients to infections. Additional hormonal disorders and concomitant conditions such as malignancies of the bladder, hematopoietic tissue, or skin may occur, and septic shock may develop as a long-term side effect [Haubitz, M. Acute and Long-term Toxicity of Cyclophosphamide. 2007].

[0026] New suggested treatments over the past 15 years are being investigated.

[0027] Tumor necrosis factor alpha (TNFα) appears to be an important mediator of AAV disease, particularly GPA, and monoclonal antibodies against TNFα have been proposed for treatment, but safety concerns may arise. According to a review by Unizony S. and Stone, JH, results suggest that monoclonal anti-TNFα inhibitors offer no role beyond conventional immunosuppression in newly diagnosed patients, while firm conclusions cannot be drawn in refractory disease [Unizony S. and Stone, JH Experimental therapies for vasculitis CHAPTER 42 Oxford Textbook of Vasculitis, Third edition, Oxford University Press 2014 Eds. Ball, Gene V., Fessler, Barri J., and Bridges S. Louis].

[0028] Later, it was recognized as US8865684B2 (Singapore University), and in the now-expired US20130331360A1, Contains GYY4137 A series of delayed types H 2 S-emitter compounds and their use as vasodilators has been disclosed, thus suggesting its use in cardiovascular disease, but not suggesting or implying its use in vasculitis.

[0029] Vasoconstriction is not part of the pathophysiology of antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis [Duvuru Geetha and J Ashley Jefferson. ANCA-Associated Vasculitis: Core Curriculum 2020, Am J Kidney Dis, 2020 Jan;75(1):124-137]. Therefore, Treatment of ANCA-associated vasculitis involves vasodilatory action and vasodilators does not include any therapeutic approach related to[van Daalen et al. Developments in the Histopathological Classification of ANCA-Associated Glomerulonephritis. Clin J Am Soc Nephrol 2020 Aug 7;15(8):1103-1111.] and [Rovin et al., Executive summary of the KDIGO 2021 Guideline for the Management of Glomerular Diseases Kidney Int. 2021 Oct;100(4):753-779.].

[0030] Accumulating evidence of ANCA-mediated neutrophil activation suggests neutrophil-directed therapies. Potential therapeutic strategies for AAV include removal of the causative autoantibody (plasma exchange), modulation of the neutrophil FcγR signaling pathway, inhibition of neutrophil extracellular trap formation, and anti-cytokine therapy [Unizony S. and Stone, JH Experimental therapies for vasculitis CHAPTER 42 Oxford Textbook of Vasculitis, Third edition, Oxford University Press 2014 Eds. Ball, Gene V., Fessler, Barri J., and Bridges S. Louis].

[0031] Neutrophil priming The cell surface expression of MPO, which acts as an autoantigen for ANCA, This process is known to involve the translocation of ATP, which ultimately induces the respiratory burst [Jennette, JC and RJ Falk, Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat Rev Rheumatol, 2014. 10(8): p. 463-73.] MPO activity does not play a role in this process.

[0032] Book The inventors surprisingly found that sulfide donors can also inhibit the translocation of the ANCA antigen MPO, thereby inhibiting neutrophil priming. .

[0033] Furthermore, the present inventors Sulfide donors can effectively inhibit neutrophil degranulation during ANCA activation and can effectively inhibit neutrophil activation by IgG isolated from ANCA patients. To my surprise, I found that...

[0034] on the other hand, Neutrophil phagocytosis and bacterial killing are not inhibited in the presence of sulfide Sulfide and sulfide donor compounds had no effect on PMA-induced activation of neutrophilic granulocytes - a surprising fact indicating that sulfide is not detrimental in this respect.

[0035] The prior art is silent regarding the use of sulfide donor molecules in the treatment of ANCA vasculitis, particularly when MPO functions as an autoantigen. Rather, based on prior art observations, the fact that sulfide may inhibit priming and ANCA-induced degranulation and oxidative burst is not clearly inferred from currently available data in the literature. Summary of the Invention [Problem to be solved by the invention]

[0036] In a systematic series of experiments, the inventors investigated how sulfides could interfere with these processes, H 2 We unexpectedly found that S donor compounds are useful in mitigating MPO-related pathological processes in AAV, while surprisingly not affecting the activity of normal neutrophils required to maintain immune homeostasis. Notably, increased serum levels of H2S demonstrated activity in MPO-related disease processes, whereas neutrophil phagocytosis and bacterial killing were not inhibited in the presence of sulfide, and sulfide and sulfide-donor compounds had no effect on PMA-induced activation of neutrophilic granulocytes (a bacterial infection model) or NADPH oxidase (NOX) activity. Oral administration of the compounds is particularly preferred. Sulfide-donor compounds provided protection from renal injury in a mouse model of MPO-ANCA vasculitis.

[0037] Therefore, the present inventors have surprisingly found that a novel type of AAV treatment is possible in patients with MPO-positive serotypes by increasing H2S serum levels, i.e., by H2S donors. Slow-acting H2S-releasing compounds are preferred. [Means for solving the problem]

[0038] 1. The present invention relates to an H2S donor compound for use in the treatment of antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) in a mammalian patient with AAV of the myeloperoxidase-ANCA serotype (MPO-ANCA vasculitis), particularly where the AAV is myeloperoxidase (MPO)-positive AAV and the subject has increased levels of ANCA relative to MPO.

[0039] In a preferred embodiment, MPO levels are measured in said patient and AAV is diagnosed clinically.

[0040] Preferably, MPO levels are measured by IIF and / or ELISA. Preferably, MPO levels are above a pre-defined threshold level.

[0041] 2. Preferably, said H2S donor compound is a slow H2S releaser compound.

[0042] Preferably, the compound is To inhibit neutrophil granulocyte activation by anti-MPO antibodies (MPO-ANCA-associated vasculitis) in mammalian subjects with AAV To inhibit neutrophil degranulation in mammalian subjects with AAV to inhibit neutrophil priming (by translocation of the ANCA antigen MPO) in mammalian subjects with AAV, and / or To prevent kidney damage / protect the kidneys in mammalian subjects with AAV Used.

[0043] In certain embodiments, the H2S donor compounds are useful in treating MPO-positive AAV at an early stage or preventing the onset of clinical symptoms, preferably by inhibiting neutrophil priming in mammalian subjects with AAV.

[0044] In certain embodiments, H2S donor compounds are useful in the treatment of renal-confined vasculitis and / or any kidney-associated form of AAV.

[0045] Preferably, the H2S donor compound does not inhibit neutrophil NOX activity, phagocytosis, and bacterial killing. Preferably, the H2S donor compound does not inhibit pathogen-induced neutrophil activity.

[0046] Preferably, the H2S donor compound is a low molecular weight compound.

[0047] 3. Preferably, the AAV is selected from granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA) (Churg-Strauss syndrome), renal-confined vasculitis (AAV), and pulmonary AAV.

[0048] In a further preferred embodiment, the AAV is a renal-confined vasculitis, preferably renal-confined microscopic polyangiitis or renal-confined necrotizing crescentic glomerulonephritis, NCGN.

[0049] In a more preferred embodiment, the AAV is expressed in the lung.

[0050] In a further particularly preferred embodiment, the AAV is expressed in the kidney.

[0051] In a more preferred embodiment, the AAV is expressed in at least the lung and kidney.

[0052] In a highly preferred embodiment, the compound is for use in microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA) (Churg-Strauss syndrome), or renal-confined vasculitis, preferably renal-confined microscopic polyangiitis or renal-confined necrotizing crescentic glomerulonephritis, NCGN, very preferably EGPA and / or renal-confined vasculitis.

[0053] 4. Preferably, the compound increases the level of H2S in the serum of a mammalian subject after administration of the compound to the subject when compared to a normal H2S serum level (or the H2S serum level when the compound is not administered (or before administration)), preferably increasing the H2S serum level in the subject between 1 hour and 24 hours after administration of the compound.

[0054] The normal level may be a predetermined threshold level or range measured and calculated in a control subject not administered the compound, or a control serum level of H2S measured in a control subject not administered (or prior to administration) the compound.

[0055] Preferably, said increased H2S serum level is significantly higher than normal serum levels as measured by any suitable statistical method.

[0056] Preferably, H2S serum level is meant to refer to the level of bioavailable sulfide (H2S) throughout this literature as measured by the protocol of Ditroi et al. [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: pp. 146-158.] Preferably, the increase in the subject's H2S serum level is at least 1.5-fold, highly preferably at least 2-fold, and even more preferably at least 3-fold higher than the normal H2S serum level as measured by the method of Ditroi et al. at a particular time point.

[0057] Preferably, the compounds of the present invention are H2S emitters, preferably slow H2S emitters, that significantly increase the level of bioavailable sulfide (H2S) in the serum of a mammalian subject after administration of the compound to the subject at time points between 1 hour and 24 hours after administration of the compound, when compared to normal H2S serum levels as a control.

[0058] Preferably, the H2S donor compounds of the present invention, especially the slow H2S releaser compounds, are low molecular weight compounds.

[0059] 5. In a particularly preferred embodiment, said compound is administered orally to said mammalian patient.

[0060] Particularly preferably, the H2S donor compound is degraded and the H2S is released in the gastrointestinal tract, preferably in the stomach, or in conditions that model the gastrointestinal tract or stomach.

[0061] 6. In a general embodiment, the compound has the general formula (Y): MLQ, (Y) (In the formula, Q is an H2S releasing moiety that upon metabolism, when present in the body of a mammalian patient, releases H2S into the bloodstream of said mammalian patient; Preferably Q contains a dithiol group (-SS-), or Q is a phosphinodithionate group (=P(S)S - ), L is an organic linking moiety; M is a moiety covalently attached to the remainder of the molecule by a hydrolyzable bond, such that upon hydrolysis and metabolism to release H2S, M is converted into a compound that is tolerable to, and preferably beneficial to, a mammalian patient; Either L or M may be absent, or both may be present. It has.

[0062] In certain embodiments, the compound is degraded to release H2S under conditions that model the gastrointestinal system of a mammalian subject, preferably under gastric model conditions.

[0063] 7. In a preferred embodiment, the compound has the general formula (X) [ka] (In the formula, Q is an H2S releasing moiety that upon metabolism, when present in the body of a mammalian patient, releases H2S into the bloodstream of said mammalian patient; In a preferred embodiment Q comprises a 5- to 6-membered heterocycle containing a dithiol group (—SS—); or Q is a phosphinodithionate group (=P(S)S - ) L is a linking moiety, In a preferred embodiment, L is C1-C8 alkylene (preferably methylene), -, -O-, S, -NH-, aryl, C1-C4 alkylaryl, or a 5- to 6-membered heterocycle, which is optionally linked to Q via a C1-C4 alkylene; At least two, preferably three or four of R1, R2, R3, R4, and R5 are H; R1, R2, R3, R4, and R5 are independently selected from the following: ·H, halogen, pseudohalogen, -CN, -OH, -SH, -NO2, -NH2, -NHCH3, -COOH, CONH2, Substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl) , C1 to C8 carboxyl (preferably C2 to C8 alkylcarboxyl, C3 to C8 alkenylcarboxyl or C3 to C8 alkynylcarboxyl), C2 to C8 carboxylic acid ester (preferably C2 to C8 alkyl ester, C3 to C8 alkenyl ester or C3 to C8 alkynyl ester), when the substituents are present, the substituents are selected from halide, pseudohalide, -OH, -SH, -OMe, -NO2, -NH2 and -NHMe, -OCOR 16 , -COOR 17 , -OR18 , -CONHR 19 (R 16 , R 17 , R 18 , and R 19 represents H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C 1 to C8 carboxyl (preferably C2 to C8 alkylcarboxyl, C3 to C8 alkenylcarboxyl or C3 to C8 alkynylcarboxyl), C2 to C8 carboxylic acid ester (preferably C2 to C8 alkyl ester, C3 to C8 alkenyl ester or C3 to C8 alkynyl ester), the substituents selected from the above, and when the substituents are present, they are selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, -NH2 and -NHMe; Preferably, R1 and R3 are -OCOR 16 , -COOR 17 , -OR 18 are independently selected from R 16 , R 17 , and R 18 is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkylester, C3-C8 alkenylester or C3-C8 alkynylester), the aforementioned substituents, wherein the aforementioned substituents, if present, are selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, -NH2, -NHMe; More preferably R2, R3, and R5 are H; R 15 is selected from C1-C8 alkylene, C1-C8 alkyl ether, C1-C8 carboxylate, preferably -(CH2) n -(CO)O-, where n is 0, 1, 2 or 3, preferably 0 or 1; or a pharmaceutically acceptable salt and / or solvate and / or complex thereof.

[0064] 8. In a preferred embodiment, the compound has the general formula I [ka] (In the formula, R7 is selected from the following: 3- to 10-membered, preferably 5- to 10-membered, more preferably 5- or 6-membered heterocycles -NH-R 23 (R 23 is selected from C1-4 alkyl and C1-4 alkylcarbonyl, R8 (preferably R8 is L) is selected from: -NH- -CH2-, -O-, S or NH, preferably CH2, O or NH, or R 15 means nothing (GYY, Lawesson's reagent, JK donor), A is selected from the following: -S - (A is -S - R6 is absent), -S- and -S - (If A is -S-, R6 may be selected from H, a 5- to 10-membered, preferably 6-membered, optionally substituted heterocycle, C1-4 alkyl, or C6-C10 aryl, or A is -S-, and R6 and R7 together form a moiety having the formula [ka] R1 to R5 and R8 are independently as defined above, and the substituents on the two rings may be the same or different; -O - -O- (if A is -O-, R6 is H, a 5- to 10-membered, preferably 6-membered heterocycle, C1-4 alkyl, or C6-C10 aryl, or and / or a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings, optionally having at least one 1- to 8-membered open-chain moiety, and optionally containing 1 to 4, preferably 1 to 3, heteroatoms; and / or R6 is an organic moiety that, upon hydrolysis, is converted into a compound that is tolerable to, and preferably beneficial to, a mammalian patient; R1, R2, R3, R4, and R5 are as defined above, or preferably, R1, R2, R3, R4, and R5 are independently selected from H, halogen, —CN, —OH, —SH, —NO2, —NH2, —NHCH3, —COOH, CONH2, and at least two of R1, R2, R3, R4, and R5 are H; Preferably, R3 is -OCH3, or R1, R2, R3, R4, and R5 are each H; or a pharmaceutically acceptable salt and / or solvate and / or complex thereof, In certain embodiments, dichloromethane complexes and / or morpholinium salts It has.

[0065] 9. In certain embodiments, the compound has the general formula I.2 [ka] (In the formula, R9 is H, and the H may be dissociated, and -S-R9 is -S - and A is selected from absent and -O-; If A is -O-, R6 is a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings, optionally having at least one 1- to 8-membered open-chain moiety, and optionally containing 1 to 4, preferably 1 to 3, heteroatoms; or If A is absent, R6 is a morpholino group linked via a 5- to 10-membered heterocycle, preferably a 6-membered heterocycle, preferably an O- and / or N-containing heterocycle, especially an N-containing heterocycle, R1, R2, R3, R4, and R5 are selected from H, C1-4 alkyl, C1-4 alkoxy, halide, and at least two of R1, R2, R3, R4, and R5 are H; preferably, R3 is -Ome, or R1, R2, R3, R4, and R5 are each H; or a pharmaceutically acceptable salt and / or solvate and / or complex thereof It has.

[0066] 10. In a particularly further embodiment, the compound has the general formula 1.3 [ka] (R 10 is a morpholino group linked via a 5- to 10-membered, preferably 6-membered, heterocycle, preferably an O- and / or N-containing heterocycle, especially an N-containing heterocycle; R1, R2, R3, R4, and R5, and R9 are as defined above. and Particularly preferred are compounds of the general formula 1.3.1 [ka] (In the formula, R 11 is C1-4 alkyl) and Preferably, the compound for use according to claim 10 is GYY4137.

[0067] 11. In a further specific embodiment, the compound has the general formula 1.4 [ka] wherein R1, R2, R3, R4, and R5 are as defined above, and preferably each is H; R6 and R9 are defined above. It has.

[0068] 12. In an alternative embodiment, the compound has the general formula II [ka] (wherein R1, R2, R3, R4, and R5 are as defined above (claim 8), At least two, preferably three or four of R1, R2, R3, R4, and R5 are H; Preferably, R1, R2, R3, R4, and R5 are independently selected from the group consisting of: H, halogen, pseudohalogen, -CN, -OH, -SH, -NO2, -NH2, -NHCH3, -COOH, CONH2, preferably OH, and -OCOR 16 , -COOR 17 , -OR 18 (R 16 , R 17 , R 18 , and R 19is H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C 8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents selected from the above, which, when present, are selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, ONO2, -NH2, -NHMe, Preferably, R1 is -OCOR 16 , -COOR 17 , -OR 18 Selected from R 16 , R 17 , and R 18 is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkylester, C3-C8 alkenylester or C3-C8 alkynylester), the aforementioned substituents, wherein the aforementioned substituents, if present, are selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, ONO2, -NH2, -NHMe; More preferably, R2, R3, R4, and R5 are H and R1 is as defined above, or Even more preferably, R1 is -OH, -OCOR 16Selected from R 16 is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 carboxylic acid ester (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the aforementioned substituents, when present, being selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, ONO2, -NH2, -NHMe; R 14 is a group having the formula III.2 [ka] In formula III.2, R1, R2, R4, and R5 are each independently H, halogen, pseudohalogen, -CN, -OH, -SH, -NO2, -NH2, -NHCH3, -COOH, CONH2, substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, Preferably, independently selected from H or OCH3, R 13 is selected from H and OCH3, preferably [ka] (selected from (preferably as part M when dependent on claim 7 or 8), In a preferred embodiment, the compound is ADT-OH. [ka]

[0069] 13. Preferably, the compound is [ka] is selected from.

[0070] 14. In a further alternative embodiment, the HS donor compound is a naturally occurring HS donor compound selected from allicin, alliin, diallyl trisulfide, diallyl disulfide, diallyl tetrasulfide, ajoene (e.g., E-ajoene or Z-ajoene), anthionine, ovothiol, S-allylmercaptocysteine ​​(SAMC), 3H-1,2-dithiole-3-thione, and alpha lipoic acid.

[0071] 15. In a further alternative embodiment, the H2S donor compound is a substituted or unsubstituted 5-membered heterocycle containing a dithiol group (-SS-), preferably a 1,2 dithiolane group or a 1,2 dithiolene group, preferably a group having formula 5: [ka] (In the formula, R 13 is H or C1-8 alkyl) Includes:

[0072] 16. In a preferred embodiment of the H2S donor compound for use according to any one of paragraphs 1 to 15, the composition is administered daily, preferably once, twice, or three times daily, for a period of at least 1 month, 2 months, 3 months, 6 months, 1 year, or more.

[0073] Pharmaceutical compositions (drugs) 17. The invention also relates to a pharmaceutical composition comprising an H2S donor compound for use according to any one of paragraphs 1 to 15, preferably any one of paragraphs 5 to 15, wherein the pharmaceutical composition is formulated for systemic administration.

[0074] 18. The pharmaceutical composition preferably comprises an H2S donor compound for use according to claim 18, formulated for oral administration, preferably in capsule and / or tablet form; Protected from light, moisture, and decay that forms H2S during storage.

[0075] 19. A pharmaceutical composition preferably comprises an H2S donor compound for use as described in claim 18, said composition being formulated for intravenous administration.

[0076] 20. The present invention also relates to a pharmaceutical composition comprising an H2S donor compound for use according to claim 20, The composition is formulated in powder form, and the powder is dissolved in an appropriate solution (e.g., Ringer's solution, Ringer's lactate solution, buffer solution, etc.) and intravenous injection or infusion, intraperitoneal injection or infusion, or It can be reconstituted as an injection.

[0077] 21. The pharmaceutical composition preferably comprises an H2S donor compound for use according to claim 18, said composition being a liquid formulation, e.g. Eye drops Nasal drops It is formulated as

[0078] 22. The present invention also relates to a pharmaceutical composition comprising an H2S donor compound for use according to any one of paragraphs 1 to 15, preferably according to any one of paragraphs 5 to 15, The composition is for topical administration and Preferably, it is formulated in the form of an ointment, gel, or emulsion, The topical dosage form includes excipients that protect the H2S donor compound from moisture and / or degradation to form H2S during storage, and optionally from light.

[0079] 23. The composition of any one of paragraphs 17 to 22 is formulated, including packaged and stored as necessary to provide an adequate shelf life.

[0080] 23. A pharmaceutical composition or formulation according to any one of paragraphs 17 to 22, wherein the composition comprises a light-protective package.

[0081] 24. In a preferred embodiment, the AAV patient is diagnosed as MPO-ANCA positive prior to treatment.

[0082] A patient with AAV is a patient who is considered to exhibit AAV symptoms according to any of the guidelines described herein (in certain embodiments, the 2012 revised International Chapel Hill Consensus Conference nomenclature of vasculitides, [Jennette JC, 2012, infra], or other diagnostic or assessment tools, such as the Birmingham Vasculitis Activity Score (BVAS) and Vasculitis Damage Index (VDI) or as referred to in [Ball, Gene V., Fessler, Barri J. and Bridges S. Louis, Eds., Oxford Textbook of Vasculitis, Third edition, Oxford University Press 2014], or known in the art or known at a given time). In this regard, levels of diagnosis suggesting that a patient may have or develop AAV include: To begin treatment, to perform further diagnostic steps (e.g., biopsy), and / or To detect ANCA Advising (or justifying) is sufficient.

[0083] In this regard, methods are described or cited in the section "Diagnosis of MPO-ANCA associated vasculitis patients" and are incorporated herein by reference where specifically discussed or appropriate.

[0084] A level of "advising" includes cases where symptoms from at least two organs are indicative of AAV, or when an effective protocol advises initiation of treatment or further investigation.

[0085] In certain embodiments, the mammalian patient has a high ANCA titer and multiple affected organ systems (as defined by Houben E et al., 2016, infra).

[0086] 25. The present invention also relates to a method of treatment in which said composition or formulation is administered to a mammalian patient with an MPO-ANCA serotype as defined herein in order to prevent or treat MPO-associated AAV in said patient.

[0087] In certain embodiments, the present invention relates to a method of treatment for treating a condition defined in any one of paragraphs 1 to 24.

[0088] In a particular embodiment, oral administration is applied.

[0089] In certain embodiments, diagnostic tests are performed as disclosed herein to identify patient groups to be treated.

[0090] definition A "subject," as used herein, is an individual of an animal species, preferably a vertebrate, more preferably a mammal or avian species, especially a mammalian species; highly preferably, the individual is a primate, hominid, or human. A "patient" is a subject receiving or intended to receive medical or veterinary care, observation, supervision, diagnosis, or treatment.

[0091] "Treatment" of a subject refers to any process, act, therapy, etc. in which a subject or patient receives assistance, particularly medical or veterinary assistance, for the purpose of directly or indirectly improving the subject's or patient's condition. Improving the subject's condition can include restoring or maintaining normal organ or tissue function, preferably at least partially restoring or maintaining health (treatment by a medical or veterinarian). Treatment typically refers to the administration of an effective amount of a compound or composition described herein. Preferably, administration of the compound is oral and leads to adequate serum levels of H2S in the patient. Particularly preferred is treatment by a medical or veterinarian in an early stage of disease, e.g., the neutrophil priming stage, in MPO-ANCA-positive patients, particularly AAV patients.

[0092] Broadly construed, treatment includes prevention (or prophylaxis), i.e., treatment by a physician or veterinarian at an early stage of disease, including prevention of the onset of disease symptoms or prevention of signs of disease, in MPO-ANCA-positive patients. In a more specific sense, prophylaxis is not covered.

[0093] "Disease activity" of ANCA-associated vasculitis refers to signs or symptoms resulting from active disease in any organ system.

[0094] "Remission" is defined as the absence of signs of vasculitis. Remission may also be understood as the abatement of clinical symptoms of the disease while MPO positivity persists.

[0095] "Relapse" is defined as the occurrence of increased disease activity after a period of partial or complete remission.

[0096] A "pharmaceutical composition" of the present invention is a composition of matter comprising at least one H2S donor compound of the present invention for use in MPO-ANCA-positive patients, said composition comprising an active agent and at least one additional substance. Preferably, the compound of the present invention is present in an effective amount, preferably an amount that results in effective H2S serum levels in the mammalian subject. The composition may also comprise additional biologically active substances useful, for example, in combination therapy. Additionally, the composition may comprise biologically acceptable carriers, formulating agents, excipients, etc., which may be known in the art.

[0097] The term "effective amount" refers to the amount of compound needed to exert the effect of the active agent in the composition. A "therapeutically effective amount" is sufficient to relieve an AAV patient or prevent one or more of the symptoms or characteristic parameters of a condition, e.g., a disorder or disease (or prevent the appearance or manifestation of an AAV symptom or progression of a disease state).

[0098] An "HS donor compound" is a compound that decomposes in response to an environmental stimulus (e.g., water, light, a nucleophile such as a thiol, enzymatic action, or other stimulus) to release HS. Preferably, administration of an effective amount of a compound of the present invention results in an effective serum level of HS in a mammalian subject. Preferably, an "HS donor compound" has a "sulfide releaser moiety," as used herein to refer to the sulfur-containing portion of the compound that is the portion of the molecule responsible for the release of HS in the patient's body, preferably resulting in elevated serum levels of HS in the patient. In particular, an HS donor compound is a compound defined by Powell, Chadwick R. et al. [in: Powell, Chadwick R., Kearsley M. Dillon, and John B. Matson. "A review of hydrogen sulfide (HS) donors: Chemistry and potential therapeutic applications." Biochemical pharmacology 149 (2018): 110-123]. In certain embodiments, the H2S is released under biological conditions, e.g., in a subject or in conditions that provide a model of biological conditions, including an in vitro model.

[0099] A "slow H2S releaser" compound is an H2S donor compound that releases H2S under appropriate reference conditions, e.g., under the same or appropriate corresponding conditions, more slowly, preferably over a period of 10 times longer, more preferably over a period of 100 times longer, than from a reference sulfide compound, preferably from Na2S. Examples of measuring the duration of hydrogen sulfide release from Na2S in a cellular environment are shown in Figure 2 / A by Vitvitsky et al. [Vitvitsky, Victor, et al. "The mitochondrial NADH pool is involved in hydrogen sulfide signaling and stimulation of aerobic glycolysis." Journal of Biological Chemistry 296 (2021)] or [Lee ZW et al. The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects in vitro and in vivo. PLoS One. 2011;6(6):e21077. doi: 10.1371 / journal.pone.0021077]. Other methods of measurement are known in the art.

[0100] The terms "sulfide" and "HS" are used interchangeably, particularly bioavailable sulfide and HS, and include all protonated isomers (especially HS, HS) unless otherwise indicated. - , S2 - ) is included.

[0101] As defined herein, a "low molecular weight compound" refers to a compound having a molar weight or molecular weight (used interchangeably herein) of less than 2000 Da or less than 1500 Da, particularly less than 1000 Da, and preferably less than 900 Da. Preferably, the low molecular weight compound is an inorganic compound, a metallo-organic compound, or an organic compound. In certain embodiments, the compound is different from polymer molecules of biological origin (particularly biological molecules isolated from nature), such as peptides or nucleic acids; in certain embodiments, it is a synthetic or semi-synthetic compound. The low molecular weight compound may be a small molecule or salt, such as a Li salt, for example, an organic Li salt or LiCl. In a preferred embodiment, the low molecular weight compound is understood as a compound that can penetrate the cell membrane and enter cells without using cell surface receptors, and in particular, its molecular weight is less than 1000 Da, preferably less than 900 Da.

[0102] A "moiety" is used herein to mean a part of a molecule that can in principle be derived from another moiety by removing a hydrogen atom or group or any part thereof.

[0103] A "sulfide releaser moiety," as used herein, is a sulfur-containing portion of a compound of the present invention that is the part of the molecule responsible for releasing H2S in the patient's body, preferably resulting in elevated H2S levels in the patient's serum.

[0104] As used herein, the term "alkyl", alone or in combination, refers to a straight-chain or branched-chain (where appropriate) saturated hydrocarbon group, preferably containing 1 to 15, 1 to 10, or 1 to 8 carbon atom(s), or particularly 1 to 6, or 1 to 4, 1 to 3, or 1 to 2 carbon atom(s) [i.e., "C 1~15 "," "C 1~10 "," "C 1~8 "," "C 1~6 ", or especially "C 1~4 "," "C 1~3 " or "C 1~2"Alkyl group], for example, particularly preferably means a methyl, ethyl, propyl or isopropyl group.

[0105] As used herein, the term "alkoxy" refers to an alkyl-O- group, in which the alkyl group is as previously described. The bond to the rest of the molecule or complex, i.e., the parent moiety, is through the oxygen (or, in the case of a carbon atom, an ether oxygen).

[0106] The term "alkoxyalkyl" means an alkyl group substituted with an alkoxy group, i.e., an alkyl-O- group as defined above. The bond to the alkyl moiety is through the oxygen, i.e., the oxygen is an ether oxygen.

[0107] As used herein, the terms "carbonyl," "alkyl-carbonyl," "alkenyl-carbonyl," and "alkynyl-carbonyl" refer to moieties having a carbonyl group optionally substituted with an alkyl group, an alkenyl group, and an alkynyl group, respectively. In the broader sense, the group can be linked by an alkyl, an alkenyl, or an alkynyl, or through the carbonyl group. In preferred embodiments, i.e., in the narrower sense, the group is attached to the parent moiety through the carbon of the carbonyl group. In preferred embodiments, "alkyl-carbonyl," "alkenyl-carbonyl," and "alkynyl-carbonyl" are alkanoyl, alkenoyl, and alkynoyl, respectively.

[0108] This definition of broad and narrow applies to any group similar to a functional group used herein, even if not specifically defined.

[0109] As used herein, the terms "carboxyl," "alkyl-carboxyl," "alkenyl-carboxyl," and "alkynyl-carboxyl" are defined to mean moieties having a carboxyl group optionally substituted with an alkyl, alkenyl, and alkynyl group, respectively, where the bond to the parent moiety is through the carboxyl group. These groups can be linked by the alkyl, alkenyl, or alkynyl, or through the carboxyl group (the latter being an ester).

[0110] "Alkenyl," as used herein, alone or in combination, means a straight or branched chain unsaturated hydrocarbon group containing at least one carbon-carbon double bond, said hydrocarbon group preferably having from 2 to 20, preferably from 2 to 15, from 2 to 10, or from 2 to 8 carbon atoms, or from 2 to 6, from 2 to 4, from 2 to 3, or from 2 carbon atoms [i.e., "C 2~20 "," "C 2~15 "," "C 2~10 "," "C 2~8 "," "C 2~6 " or "C 2~4 "," "C 2~3 " or "C2" alkyl group].

[0111] "Alkynyl," as used herein, is defined mutatis mutandis in an analogous manner to alkenyl.

[0112] As used herein, a "heterocyclic" ring is a cyclic moiety having, in addition to carbon atom(s), at least one non-carbon atom as a member(s) of the ring(s). A heterocyclic ring may contain multiple rings, for example, an aromatic heterocyclic ring, fused to another ring of the aromatic heterocyclic ring, which may or may not be aromatic; i.e., if not aromatic, it may form a cyclic substituent of the aromatic heterocyclic ring. In a preferred embodiment, when a heteroaryl contains multiple, particularly two, fused rings, both rings are aromatic. Preferably, the ring(s) of the heterocyclic moiety are 5- to 6-membered ring(s).

[0113] The term "heterocycloalkyl" refers to a "heterocyclic" ring having a straight or branched (where appropriate) saturated hydrocarbon group, optionally with alkyl substituents on the heterocycle and optionally on the heteroaryl, and / or derivable from a cycloalkyl group as defined above, in which at least one of the ring carbon atoms has been replaced with a heteroatom, such as, but not limited to, nitrogen or oxygen.

[0114] As used herein, an "aromatic" moiety may be described as a planar cyclic moiety (ring), in which the single bonds between the ring-forming atoms (called σ-bonds) are formed from the overlap of hybridized atomic sp2 orbitals in the line between the carbon nuclei, and the delocalized π-bond system is formed from the overlap of atomic p orbitals of each ring-forming atom above and below the plane of the ring, and the number of π-electrons provided by the ring-forming atoms, participating according to molecular orbital theory, must be equal to 4n+2 (Hückel's rule), where n is 1, 2, 3, etc., preferably 1 or 2, with n=1 for a single ring containing six π-electrons. The ring-forming atoms typically donate one or two π-electrons to the delocalized π-electron system.

[0115] The term "heteroaryl" is defined herein as a group or molecule containing an aromatic heterocycle, preferably a moiety having at least one heteroatom as a "member" incorporated within an aromatic ring. Examples of heteroatoms include nitrogen, oxygen, and sulfur, preferably nitrogen and oxygen. In some embodiments, a heteroaryl may contain an aromatic heterocycle fused to another ring of the aromatic heterocycle, which may or may not be aromatic; i.e., if not aromatic, it may form a cyclic substituent of the aromatic heterocycle. In preferred embodiments, when a heteroaryl contains multiple, particularly two, fused rings, both rings are aromatic. The members of a heteroaryl are related to the carbon atom(s) or heteroatom(s) that form the ring.

[0116] The term "aryl" as used herein refers to a group containing any carbon-based aromatic ring, preferably a monocyclic or bicyclic group, with bicyclic groups preferably containing two fused rings. In a preferred embodiment, the aryl group consists solely of carbon as ring atoms, i.e., "members." In a broad sense, the term aryl also optionally includes "heteroaryl." Optionally, the term "aryl" is limited to non-heteroaryl, which also includes the term aryl, defining groups containing aromatic groups that do not contain heteroatoms. Aryl groups may be substituted or unsubstituted (i.e., optionally substituted). When an aryl group is substituted, it may be substituted with any substituent, examples of which include C 1~4 Alkyl, C 2~4 Alkenyl, C 1~3 Alkyloxy, C 1~3 Alkanoyl, C 1~3 Alkylamines, C 1~3 Alkylamides, halogens, etc.

[0117] The term "aralkyl," as used herein, refers to an arylalkyl group that is linked to the parent molecule via an alkyl group and that may be further optionally substituted with one or more, preferably 1 to 3 or 1 to 2, alkyl substituents. Thus, the aryl group may be substituted with alkyl substituents, and preferably each substituent is C 1~4 It is not larger than alkyl.

[0118] "Aryl" or "heteroaryl" may include monocyclic rings, fused rings, or polycyclic rings in which a single ring is joined by a single bond, preferably monocyclic or bicyclic rings.

[0119] As used herein, the term "fused ring" means that a ring is fused with at least one other ring to form a compound group containing two or more rings, and a single bond between two member atoms of the ring is common to the two rings, i.e., shared by the two rings, along with the two members. An example of a fused ring is a polycyclic aryl. A polycyclic aryl is herein understood as a group containing multiple rings of a carbon-based group, in which at least one ring is an aryl, and optionally also contains a cycloalkyl and / or heterocycloalkyl.

[0120] A "substituted" moiety includes substituents selected from groups and moieties defined herein; however, the substituents are preferably small, i.e., short, i.e., consisting of no more, preferably fewer, atoms than the moiety it replaces. In the present invention, "optionally substituted," i.e., "unsubstituted or substituted," means that the moiety may be substituted with any substituent.

[0121] In the general formulae herein, H atoms are typically not shown, but one of ordinary skill in the art will understand the formulae and be able to recognize the complete structure.

[0122] Terms expressing options, such as optional embodiments or variations, are used throughout, and among these, "particularly" refers to embodiments or variations of particular interest, and "preferred" refers to those that are preferred for some reason.

[0123] The singular forms "a," "an," and "the," or at least "a," "an," include plural referents unless the context clearly dictates otherwise.

[0124] The terms "comprises" or "comprising" or "including" herein should be construed as having a non-exhaustive meaning, allowing for the addition or involvement of further elements, e.g., features or method steps or members or components, to anything that includes the listed elements. "Comprising" may be substituted for "including" where implementation of a given language variant requires it, or may be limited to "consisting essentially of" when no other elements other than those listed are essential to enable the invention to be practiced, or to "consisting of" where no other elements must be present.

[0125] Abbreviation AAV:ANCA-associated vasculitis ANCA: anti-neutrophil cytoplasmic antibody EGPA: Eosinophilic granulomatosis with polyangiitis (or Churg-Strauss syndrome). GPA: Granulomatosis with Polyangiitis (or Wegener's Granulomatosis), IgG: immunoglobulin MPA: Microscopic polyangiitis MPO: myeloperoxidase NCGN: necrotizing crescentic glomerulonephritis NOX:NADPH oxidase PAS: Periodic acid-Schiff staining PBS: phosphate buffered saline PMA: phorbol 12-myristate 13-acetate PR3: proteinase-3 SOD: Superoxide dismutase [Brief explanation of the drawings]

[0126] [Figure 1] Effect of sulfide on neutrophil activation. (A) Effect of sodium sulfide on HO production by PMA-activated neutrophils. Samples were treated with different concentrations of NaH2S (0–100 μM) for 5 min before activation with PMA. The value obtained for samples not treated with sulfide was considered 100%. HO is produced by the cellular NOX2 enzyme complex. HO in the cell supernatant was measured using the FOX-1 assay. HO production was unaffected by the addition of Na2S up to 50 μM. (B) Effect of sodium sulfide on the oxidative burst of PMA-activated neutrophils. Cells were treated with different concentrations of sodium sulfide (0–50 μM) and incubated for 20 min in the presence of WST-1, 5 μg / ml catalase, and 100 ng / ml PMA. WST-1 was reduced by superoxide produced by NOX2 in the cell supernatant, which was measured spectrophotometrically at 560 nm. (C) Effect of GYY4137 sulfide donor on the oxidative burst of PMA-activated neutrophils. Samples were prepared using WST-1 reagent, treated with different concentrations of GYY4137 (0–100 μM), and induced with PMA for 20 minutes. Reduced WST-1 was measured at 560 nm from the cell supernatant. (D) The effect of GYY4137 sulfide donor on the oxidative burst of ANCA-activated neutrophils was measured using the WST-1 assay. Cells were prepared using WST-1 assay conditions and treated with GYY4137 either before treatment with 2 ng / ml TNF-α (black columns) or 15 minutes after the start of TNF-α treatment (patterned columns). 15 minutes after the addition of TNF-α, 300 μg / ml of normal IgG or ANCA IgG was added and incubated for 60 minutes. Reduced WST-1 was measured from cell supernatants by photometric measurement at 560 nm. [Figure 2] Figure 1 shows the effect of GYY4137 on neutrophil degranulation. Neutrophilic granulocytes were incubated in HBSS buffer in the presence of cytochalasin B for 10 minutes and then treated with different concentrations of GYY4137 (0-100 μM) for 5 minutes. The cells were then activated with PMA or ANCA, and β-glucuronidase activity or protein levels were examined in the cell supernatant. β-glucuronidase activity was determined by measuring the μg of phenolphthalein liberated over 19 hours, which was measured spectrophotometrically at 520 nm. (A) Effect of GYY4137 on β-glucuronidase activity. Cells were treated with 3% Triton X under β-glucuronidase assay conditions in the absence or presence of GYY4137. The cell supernatant was incubated for 19 hours in the presence of different concentrations of GYY4137 (10-100 μM) and phenolphthalein-glucuronide. (B) Effect of GYY4137 on neutrophil degranulation in PMA-activated cells. Cells were prepared using the β-glucuronidase assay conditions and treated with GYY4137 as described above. 100 ng / ml PMA was added to the samples and incubated for 30 minutes. Phenolphthalein liberated by β-glucuronidase in the supernatant was measured spectrophotometrically at 520 nm. (C) Cells were prepared using the β-glucuronidase assay conditions, treated with GYY4137, and activated with PMA. SDS-PAGE and silver staining were performed using 40 μl of each supernatant. (D) Cells were prepared using the β-glucuronidase assay conditions and treated with GYY4137 sulfide donor (0–100 μM) as described above. Cells were treated with TNF-alpha, and then 300 μg / ml ANCA IgG was added to the samples and incubated for 30 minutes. Phenolphthalein in the supernatant released by β-glucuronidase was measured spectrophotometrically at 520 nm. In the figure, 0% inhibition represents the value from the donor sample that was not treated with sulfide. [Figure 3]This figure shows the effect of sulfide on MPO translocation to the cell surface. In the presence of 10 ng / ml TNF-α, neutrophils translocate MPO to the cell surface. In this experiment, neutrophils were incubated on gelatin (0.2%)-treated glass coverslips in 24-well plates for 30 minutes to allow them to adhere. GYY4137 (25 μM or 50 μM) or DPBS (Dulbecco's phosphate buffered saline) was added 5 minutes later (after TNF-α) or simultaneously with TNF-α treatment. The order of the treatments indicated in the figure indicates the order of treatments. Cells were incubated for 30 minutes after the addition of TNF-α and then fixed with 4% PFA. Prior to permeabilization, surface MPO (second column) was labeled with a goat anti-MPO antibody followed by an Alexafluor 488 rabbit anti-goat secondary antibody. After permeabilization with 1% Triton X, granular MPO (column 3) was labeled with goat anti-MPO antibody followed by Alexafluor 568 rabbit anti-goat secondary antibody, and nuclei (column 1) were labeled with Hoechst 33258. After labeling, cells were mounted on glass slides with Prolong™ Glass Antifade Mountant and observed under STED microscope. At the scale shown in (A), more neutrophils can be seen, while at the scale shown in (B), single-cell granular MPO within the cell (column 3) and cell surface MPO located to one side (polar configuration) can be observed at different magnifications (scales). [Figure 4]Figure 1 shows the effect of sulfide on neutrophil phagocytosis and bacterial killing. (A) Neutrophils (4 × 104 cells / well) were incubated with fluorescently labeled E. coli K-12 bioparticles in the presence or absence of different concentrations of GYY4137 (10–100 μM) on an EZ-slide chamber slide for 2 hours. After incubation, the wells were washed and then observed under a fluorescence microscope. A negative control was prepared using cytochalasin D, a phagocytosis inhibitor. (B) Plate assay to determine the effect of sulfide on neutrophil phagocytosis. Neutrophils (3 × 104 cells / well) were prepared and treated with GYY4137 and bioparticles as described above. Fluorescence was detected using a TECAN Spark 10M at excitation and emission wavelengths of 485 / 525 nm. GYY4137 was added 1 hour before (black squares) or simultaneously with the addition of bioparticles (gray circles with black borders). Correlation analysis showed that the presence of GY4137 did not affect phagocytosis at the applied concentrations. (C) Opsonized Staphylococcus aureus was incubated with isolated neutrophilic granulocytes in the presence or absence of 100 μM hydrogen sulfide for 20 minutes. Colony-forming unit assays were performed, and bacterial killing was calculated based on the original, extracellular, and intracellular bacterial concentrations. Treatment with hydrogen sulfide did not significantly affect bacterial killing compared to untreated samples. [Figure 5]Histopathological findings in kidney tissue from Rag2 knockout mice after passive transfer of splenocytes from MPO-immunized MPO-deficient mice. (A, B) Black arrows indicate cellular crescents, where proliferating mural cells, monocytes, and other inflammatory cells fill Bowman's capsule. Gray dotted arrows indicate crushed glomeruli (PAS, X553). (C) Black arrows indicate fibrocellular crescents. The figure also shows large cells with distinct, large nuclei and surrounding fibrotic tissue (PAS, X645). (D) As inflammation progresses, more fibrotic lesions can be detected in the crescentic areas (black arrows). The development of fibrotic tissue leads to the disappearance of cells previously present in Bowman's capsule (see gray dotted arrows in 5 / A). Cells are replaced by fibrin and collagen (PAS, X700). (E, F) When severe inflammation persists, dissolution of Bowman's capsule can be detected (black arrows). In this scenario, a series of cells mobilized to the injury site and infiltrated the inflammatory area (see 5 / A white straight arrow) (PAS, X482). (G, H) Black arrows indicate glomerular sclerosis. This type of glomerular injury cannot be repaired, and function cannot be restored (PAS, X260). [Figure 6] Figure 1 shows the effect of GYY4137 sulfide donor treatment in an ANCA mouse model. (A) The number of affected glomeruli was counted in six different slices of each kidney sample. Affected glomeruli showed cellular, fibrocellular, and fibrous crescents, as well as segmental or diffuse sclerotic lesions. Black columns represent untreated samples, and gray dotted columns represent GYY4137-treated pairs. Each pair received splenocytes from one Rag2-deficient mouse (immunized with BSA or MPO). (B) Columns and numbers indicate the percentage of affected glomeruli in sulfide donor-treated samples compared to their untreated counterparts. The gray line represents 0% baseline (the number of affected glomeruli was the same in treated and untreated mice), and the last column shows the average percentage across all samples. [Figure 7]This figure shows the intraperitoneal treatment of healthy (Rag2- / - immunodeficient) mice with GYY4137 and per os treatment with ATB. ATB (60 μmol / kg) has previously been tested in the ANCA mouse model, where it was administered orally (per os) and proved ineffective. In this experiment, it was administered to healthy mice. GYY4137 (150 μmol / kg) was administered intraperitoneally to animals of the same breed. H2S levels were measured from the animal serum as described in the Examples. Briefly, blood samples were collected from the canthus of the mouse's eye after isoflurane anesthesia and incubated at room temperature for 30 minutes. The clotted blood samples were centrifuged, and the serum was then collected in a new tube. 25 μl of serum sample was mixed with 66 μl of premixed reagent-buffer solution, followed by immediate vigorously vortexing. After exactly 10 minutes at 20°C, the reaction was quenched with TCA and vigorously vortexed. The precipitated protein was removed, and the supernatant was transferred to an autosampler vial and cooled. Calibration samples for quantification were prepared in the same manner using a standardized NaHS solution and further diluted after derivatization. HPLC measurements were performed on a C18(2) column using a gradient elution profile with 0.1% TFA in water and 0.1% TFA in acetonitrile. The fluorescence detector excitation wavelength was set at 390 nm, and the emission wavelength was set at 475 nm. Preparation was performed as previously described with a modified chromatographic setup [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: pp. 146-158.]. Intraperitoneally administered GYY4137 resulted in significantly higher serum levels than orally administered ATB346. [Figure 8]Figure 1 shows a comparison of donor mix administration and GYY4137 administration. A sulfide donor mix (HS donor mix) containing GYY4137 (150 μmol / kg, 5 mg / ml stock), N-acetylcysteine ​​(40 μmol / kg, 0.64 mg / ml stock), and pyridoxal 5'-phosphate (20 μmol / kg, 0.49 mg / ml stock) was prepared. (A) The HS donor mix was administered IP and per os to healthy mice. Serum concentrations were measured before sulfide treatment (0 h) and at 1 and 24 h after administration. Surprisingly, oral administration resulted in significantly higher serum levels. Serum concentrations were highest at 1 h (post-administration), but the difference, or ratio, between oral and IP administration was highest at 24 h. (B) GYY4137 at a dose of 150 μmol / kg, GYY4137 at a dose of 750 μmol / kg, and an HS donor mix containing GYY4137 at a dose of 150 μmol / kg were orally administered to healthy mice, and HS serum levels were measured. Higher doses of GYY4137 resulted in higher serum concentrations compared with lower dose forms (pure and mixed). Intraperitoneally administered GYY4137 significantly increased HS serum levels (see Figure 7), whereas IP-administered HS donor mix did not (Figure 8A). [Figure 9] Figure 1 shows the measurement of serum sulfide concentrations in mice treated with GYY4137 or GKK-895. Mice were treated intraperitoneally with 150 μmol / kg GYY4137 or 110 μmol / kg GKK-895. Blood samples were collected from the canthus of the eye at the indicated time points after isoflurane anesthesia. Hydrogen sulfide concentrations were measured from the collected blood serum samples using a monobromobimane assay. Treatment with both GYY4137 and GKK-895 resulted in a significant increase in circulating sulfide concentrations, with GKK-895 producing higher measured sulfide values ​​2 and 4 hours after injection. [Figure 10]Figure 1 shows a comparison of GYY4137 and GKK-895 treatment in mouse kidney samples. Kidney samples from an ANCA vasculitis mouse model were prepared for histological analysis and evaluated by spectrophotometry. The percentage of affected glomeruli in treated samples was calculated compared to their control pairs. Samples treated with 150 μmol / kg GYY4137 (shown in dark gray) and that also showed a reduction in kidney pathology are shown in this figure, with an average reduction in affected glomeruli of 56.8%. Samples treated with 90 μmol / kg GKK-895 (shown in light gray) showed an average reduction in affected glomeruli of 57.65% compared to their control pairs. [Figure 11] This figure shows the pathogenesis of ANCA-associated vasculitis. (A) Proinflammatory cytokines and chemokines (e.g., TNF-alpha) are released due to local or systemic processes, leading to increased expression of endothelial adhesion molecules and the initiation of neutrophil priming. (B) Neutrophil priming results in increased expression of adhesion molecules on neutrophils, inducing the translocation of ANCA antigens to the cell surface. (C) The F(ab)2 region of ANCA recognizes the ANCA antigen on the cell surface, activating neutrophils and allowing them to bind to the vascular wall and initiate transmigration. (D) ANCA-mediated neutrophil activation induces ROS production and degranulation, leading to vasculitis. DETAILED DESCRIPTION OF THE INVENTION

[0127] Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) has a particularly complex etiological background. The pathogenic role of ANCA has been supported by several studies, including in vitro and in vivo studies, as well as clinical trials and both in vitro and in vivo experiments. Several factors, such as neutrophils, complement, and effector T cells, are also involved in AAV pathogenesis [Chen, M., Kallenberg, C. ANCA-associated vasculitides—advances in pathogenesis and treatment. Nat Rev Rheumatol 6, 653-664 (2010)]. https: / / doi.org / 10.1038 / nrrheum.2010.158]. The patient group was of the MPO-positive serotype, and prior art evidence suggests that MPO as an autoantigen contributes to the development of MPO-positive AAV.

[0128] In a systematic series of experiments, we investigated how sulfide might interfere with these processes and unexpectedly found that H2S-emitting compounds are useful in the treatment of MPO-positive serotypes of AAV.

[0129] Neutrophil phagocytosis and bacterial killing were not inhibited in the presence of sulfide, and sulfide and sulfide-donor compounds had no effect on PMA-induced activation of neutrophil granulocytes, but were able to efficiently inhibit neutrophil degranulation upon ANCA activation, as well as neutrophil activation by IgG isolated from ANCA patients.

[0130] Sulfide donors can inhibit TNF-alpha-induced ANCA antigen MPO translocation and thereby neutrophil priming, for which active MPO has been reported to be a mediator.

[0131] Finally, sulfide donor compounds protected against renal injury in a mouse model of MPO-ANCA vasculitis.

[0132] The inventors also provide evidence that increasing the H2S dose and serum H2S levels results in increasing efficacy, indicating that the beneficial effects are due to H2S release. Slow-acting H2S donors are preferred. These H2S donors that provide high serum levels upon administration can be easily screened in test animals.

[0133] Thus, in an unexpected manner, the H2S donor compounds of the present invention act in multiple ways against this autoimmune disease, while sparing normal neutrophil function, which is so important to a healthy immune system.

[0134] The role of hydrogen sulfide is a topic of considerable debate in autoimmune inflammatory diseases, and H2S can be toxic depending on the condition and concentration. H2S has been shown to be protective against many different processes that contribute to atherogenesis in various models, although these models appear to have limitations [Gall Tamas et al. Overview on hydrogen sulfide-mediated suppression of vascular calcification and hemoglobin / heme-mediated vascular damage in atherosclerosis, Redox Biology, Volume 57, 2022, 102504, ISSN 2213-2317.]

[0135] This dual nature of H2S places a heavy burden on investigators to reach precise conclusions about whether H2S is beneficial or not in specific diseases, and complex experiments are required with unpredictable outcomes or results.

[0136] The factors and biological processes associated with AAV are discussed in more detail below; for an overview, see FIG. 11.

[0137] Inflammatory cytokines induce the translocation of ANCA antigens, such as MPO and PR3, to the cell surface, which primes neutrophil cells for activation based on antibody recognition.

[0138] Thus, neutrophil priming involves the translocation of MPO to the cell surface, which serves as an antigen for ANCA, ultimately inducing a respiratory burst [Jennette, JC and RJ Falk, Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat Rev Rheumatol, 2014. 10(8): pp. 463-73.] In this pathogenic process, ANCA-induced neutrophils infiltrate the walls of small and medium-sized blood vessels, generating large amounts of cytotoxic reactive oxygen species (ROS) in the extracellular space, in this case the interstitium of the blood vessel wall. In these processes, MPO may play a central role, as ANCA-induced release of toxic oxygen radicals and harmful granule enzymes from cytokine-primed neutrophils may mediate vascular inflammation [Falk, RJ, et al., Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA, 1990. 87(11): p. 4115-9.]

[0139] Some background on neutrophil granulocytes: These cells play a key role in innate immunity, providing the first line of defense against pathogenic microorganisms. During an infection, they migrate to the site of infection, where they kill pathogens through phagocytosis and antimicrobial proteins [Jaillon, S., et al. Neutrophils in innate and adaptive immunity. In Seminars in immunopathology. 2013, Springer.; Laskay, T., G. van Zandbergen, and W. Solbach, Neutrophil granulocytes as host cells and transport vehicles for intracellular pathogens: apoptosis as infection-promoting factor. Immunobiology, 2008. 213(3-4): pp. 183-91.; Cassatella, M.A., Neutrophil-derived proteins: selling cytokines by the pound. Advances in immunology, 1999. 73: pp. 369-509.] Activated neutrophils produce a repertoire of proinflammatory molecules such as CXC and CC chemokines, interleukins, interferons, colony-stimulating factors, and tumor necrosis factor-α (TNF-α) [Cassatella, MA, 1999, see below].

[0140] We also investigated the effect of sulfide on the autoimmune inflammatory process of ANCA vasculitis, which is mediated by neutrophilic granulocytes. We first demonstrated that sulfide inhibited the ANCA antibody-induced oxidative burst mechanism in a dose-dependent manner, regardless of whether sulfide was added before or after priming with TNF-α.

[0141] It has been known in the art that autoantibody activation also induces the release of granule enzymes in the extracellular space [Flint, J., MD Morgan, and CO Savage, Pathogenesis of ANCA-associated vasculitis. Rheum Dis Clin North Am, 2010. 36(3): pp. 463-77.] Sulfide also interferes with degranulation during ANCA activation, and observations have shown that treatment with the sulfide donor GYY4137 inhibited the release of β-glucuronidase in a dose-dependent manner. These effects were not observed in PMA-activated neutrophil samples, suggesting that sulfide acts by inhibiting cellular autoimmune activation.

[0142] Neutrophil priming by inflammatory cytokines is an important part of this type of activation, so we performed experiments with TNF-α to examine the effect of sulfide on this process. The data showed that sulfide effectively inhibited MPO translocation to the cell surface and may interfere with the signaling pathways that regulate the priming mechanism (Figure 3).

[0143] We used the ANCA mouse model published by Xiao and colleagues [Xiao H, Heeringa P, Hu P, Liu Z, Zhao M, Aratani Y, Maeda N, Falk RJ, Jennette JC. Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest. 2002 Oct;110(7):955-63] to investigate the potential protective effect of sulfide in MPO-ANCA-associated vasculitis. The original publication showed that the most prominent symptoms appeared in the kidney. The severity of symptoms in animals was highly variable, with diverse symptoms and varying numbers of affected glomeruli. Crescent formation and fibrosis in the kidney were also observed in the treatment groups. Surprisingly, treatment with GYY4137 reduced the number of affected glomeruli by an average of 64.9%.

[0144] This is probably the best animal model, although other models are known to those skilled in the art (Salama AD, Little MA. Animal models of antineutrophil cytoplasm antibody-associated vasculitis. Curr Opin Rheumatol. 2012 Jan;24(1):1-7.).

[0145] Furthermore, ATB346 and GYY4137 have been tested in an ANCA mouse model and healthy (Rag2 knockout) mice. IP-administered GYY4137 was shown to be active in an ANCA vasculitis mouse model and to increase serum H2S levels, whereas orally administered ATB346 did not increase serum H2S levels in healthy mice and was not shown to be beneficial in reducing renal injury in the ANCA mouse model under the conditions applied.

[0146] Therefore, a high H 2 S serum levels correlated with improved renal status in model animals.

[0147] In further experiments, a sulfide donor mix (HS donor mix) containing GYY4137 preparation (150 μmol / kg), N-acetylcysteine ​​(40 μmol / kg), and pyridoxal 5'-phosphate (20 μmol / kg) was administered IP or intravenously to healthy mice (Figure 8 / B).

[0148] Intraperitoneally administered GYY4137 significantly increased H2S serum levels (see Figure 7), whereas IP administered H2S donor mix did not - a finding closely correlated with the fact that IP administered GYY4137, but not IP administered donor mix, improved renal status in an ANCA vasculitis animal model (Figure 8 / A).

[0149] The conclusion can be drawn that increased H2S serum levels are a prerequisite for real therapeutic effect in ANCA vasculitis.

[0150] Additionally, the inventors have surprisingly found that oral administration is preferable to IP administration.

[0151] The present data indicate that sulfide donor molecules offer a novel treatment option for MPO-mediated ANCA vasculitis. The results indicate that sulfide can reduce oxidative damage during the active phase and may also promote the induction of remission through the inhibition of autoimmune activation of neutrophilic granulocytes.

[0152] These findings were also substantiated by experiments using a novel hydrogen sulfide donor, GKK-895. Treatment with low concentrations of GKK-895 increased blood sulfide concentrations more than did high doses of GYY4137. Increased blood sulfide levels correlated with beneficial effects in an ANCA mouse model, further demonstrating a strong correlation between increased blood sulfide levels upon treatment with the donor molecule and reduced renal symptoms of ANCA vasculitis.

[0153] Diagnosis of patients with MPO-ANCA-associated vasculitis Analysis has shown that AAV diagnosis is accompanied by careful attention and can be successful despite several subtypes of the disease and various symptoms. The 2012 Revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides defines the AAV categories microscopic polyangiitis (MPA), granulomatosis with polyangiitis (Wegener's syndrome) (GPA), and eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome) (EGPA) and their characteristic features [Jennette JC. Overview of the 2012 revised International Chapel Hill Consensus Conference nomenclature of vasculitides. Clin Exp Nephrol. 2013 Oct;17(5):603-606.; Jennette JC, et al. 2012 revised International Chapel Hill Consensus Conference Nomenclature of Vasculitides. Arthritis Rheum. 2013 Jan;65(1):1-11. doi: 10.1002 / art.37715. PMID: 23045170.].

[0154] In the present invention, the detection of MPO positivity and signs of small-vessel vasculitis provides a clear definition of the diagnostic spectrum. A 2016 study by Houben E et al. suggested that high ANCA titers and multiple affected organ systems help distinguish between AAV and other systemic diseases, and that a diagnostic scoring system incorporating these factors should be considered [Houben E, et al. Diagnosing ANCA-associated vasculitis in ANCA-positive patients: A retrospective analysis on the role of clinical symptoms and the ANCA titer. Medicine (Baltimore). 2016 Oct;95(40):e5096].

[0155] However, MPO and PR3 ANCA may be positive in a variety of diseases that mimic AAV. High ANCA titers and multiple affected organ systems may help distinguish between AAV and other systemic diseases in anti-PR3 and anti-MPO positive patients. A diagnostic scoring system incorporating these factors should be considered.

[0156] AAV, which includes GPA or Wegener's granulomatosis, microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA or Churg-Strauss syndrome), and other related autoimmune diseases, manifests in the airways and kidneys, among other organs, and is characterized by necroinflammation of small blood vessels (i.e., arterioles, capillaries, and venules) and the appearance of minimal immune-type (i.e., minimal or absent immune complex deposition in the vessel walls) necroinflammation in the microcirculation, and in some patients, the presence of antibodies against neutrophil and macrophage granule components: i.e., myeloperoxidase (MPO-ANCA) and / or proteinase-3 (PR3-ANCA). [Unizony S. and Stone, JH Experimental therapies for vasculitis CHAPTER 42 Oxford Textbook of Vasculitis, Third edition, Oxford University Press 2014 Eds. Ball, Gene V., Fessler, Barri J. and Bridges S. Louis]

[0157] Clinical signs associated with NCGN include microscopic hematuria with dysmorphic red blood cells and red blood cell casts, and proteinuria.

[0158] Patients with systemic vasculitis may present with extrarenal manifestations affecting one or several organ systems, with or without renal involvement. The systems usually involved are the upper and lower respiratory tract, skin, eyes, and nervous system. [KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases Kidney International, (2021) 100(45) Supplement, pages S1 to S276]

[0159] Systemic symptoms include fever, fatigue, weight loss, and muscle or joint pain. Systemic vasculitis is a multisystem disorder. For example, the following individual organs may be affected (typical symptoms are listed): eyes (e.g., redness, blurred vision, or vision loss), ears (tinnitus or hearing loss), sinuses (pain, runny nose, or nosebleeds), skin (rash, ulcers, or soreness, typically deep and slow to heal), lungs (cough and chest pain, shortness of breath, difficulty breathing, or hemoptysis), kidneys (proteinuria, foamy urine, or hematuria), and nervous system (numbness and / or tingling). Additional nonspecific symptoms include fever, polymyalgia, polyarthralgia, headache, and fatigue, which overlap with other diseases such as infections or malignancies. Symptoms may also overlap with those of other inflammatory diseases.

[0160] Symptoms may manifest in many ways, including nonspecific and more specific symptoms. In primary care, blood tests may reveal leukocytosis, thrombocytosis, elevated erythrocyte sedimentation rate and C-reactive protein levels, normochromic-normocytic anemia, and elevated serum creatinine, the latter an indicator of renal damage. Urinalysis and urinary sediment may reveal hematuria and proteinuria. Elevated serum creatinine indicates that renal damage has already occurred. Chest x-ray may show infiltrates, nodules, or cavities in the lung parenchyma and is recommended in patients with transpulmonary symptoms [Berden A et al. Diagnosis and management of ANCA-associated vasculitis, Clinical Review, BMJ (2012) 344 e26 doi: 10.1136 / bmj.e26].

[0161] In the absence of generally reliable serological markers, accurate clinical tools may be applied to assess disease activity. For this purpose, reliable disease assessment tools have been developed. The Birmingham Vasculitis Activity Score (BVAS) and Vasculitis Damage Index (VDI) have been adopted and used by most research groups involved in clinical trials in vasculitis. These are internationally recognized assessment tools and have been compared with clinical trials. The Birmingham Vasculitis Activity Score (BVAS) has also been specifically applied to Wegener's granulomatosis [Flossmann O et al. Development of comprehensive disease assessment in systemic vasculitis. Ann Rheum Dis. 2007 Mar;66(3):283-92.]

[0162] In AAV, kidney biopsy is also an important option for both the primary diagnosis and recurrent disease. Biopsy remains the gold standard. Kidney biopsy should always be considered in patients in whom the kidney is diseased or suspected to be the affected organ.

[0163] Nevertheless, in cases with positive MPO- or PR3-ANCA serology, low suspicion for secondary vasculitis, and a clinical picture compatible with small-vessel vasculitis, treatment can be initiated, and biopsies can be performed even after treatment initiation.

[0164] However, with regard to disease recurrence, ANCA positivity or an increase in ANCA levels is considered to be only weakly predictive of future disease recurrence and should not be used to guide treatment decisions.

[0165] However, in the present invention, the patient group to be treated is MPO-ANCA positive patients. Therefore, the MPO serotype should be diagnosed in patients presenting with AAV symptoms.

[0166] Methods used to detect MPO and determine MPO-ANCA specificity The test of choice for treating MPO in patients is indirect immunofluorescence (IIF).

[0167] The preferred method for demonstrating ANCA by IIF was established in the International Consensus Statement on Testing and Reporting of ANCA [Savige, J. et al. International consensus statement on testing and reporting of antineutrophil cytoplasmic antibodies (ANCA). American Journal of Clinical Pathology, 111, 507-13.]. Post-preparation methods include evaluation using incident-light fluorescence microscopy. Methods are available from the European Vasculitis Society (https: / / vasculitis.org / ) [Damoiseaux, J. and Tervaert, JWC Autoantibodies in vasculitis CHAPTER 6 Oxford Textbook of Vasculitis, Third edition, Oxford University Press 2014 Eds. Ball, Gene V., Fessler, Barri J., and Bridges S. Louis].

[0168] The MPO specificity of ANCA autoantibodies may also be determined by enzyme-linked immunosorbent assay (ELISA). Direct non-competitive ELISA, in which MPO is bound to a microtiter plate, is the preferred choice for detecting antigen-specific antibodies. ELISA is a quantitative assay that should use a reference standard. Such a standard is available for MPO-ANCA (IUIS-CDC reference preparation), allowing results to be obtained in international units. Several types of direct ELISA are available, as known to those skilled in the art. Chemiluminescence-type assays are also available (Damoiseaux, J. and Tervaert, JWC 2014, infra and documents cited therein; see the chapter "Methods used to determine ANCA specificity").

[0169] A second-generation test for detecting ANCA is the so-called capture ELISA, in which the first coating of the solid phase consists not of antigen but of monoclonal antibodies specific for individual antigens, to which the antigen preparation is added. It has been suggested that this capture of antigens by immobilized antibodies provides superior recognition by MPO.

[0170] The later developed anchor ELISA is a so-called third-generation assay for detecting MPO. In these assays, purified antigen is conjugated to a linker peptide, which is used to bind the antigen to a solid phase. Similar to capture ELISAs, third-generation, or anchor, ELISAs preserve the three-dimensional structure of the antigen, ultimately resulting in superior sensitivity (Damoiseaux, J. and Tervaert, JWC 2014, infra and cited therein; see the chapter "Methods used to determine ANCA specificity").

[0171] ANCA tests are commercially available, but currently require trained personnel to use.

[0172] According to the Oxford Textbook of Vasculitis [Oxford Textbook of Vasculitis Third Edition, Oxford University Press 2014 Eds. Ball, Gene V., Fessler, Barri J. and Bridges S. Louis, page 66, Autoantibodies in vasculitis CHAPTER 6, Conclusions], there is an international consensus that ANCA, including MPO-ANCA, should be detected by a combination of IIF and antigen-specific assays.

[0173] A recent 2022 review [Walker Brandon S. et al. Performance of MPO-ANCA and PR3-ANCA immunoassays for the stratification of specific ANCA-associated vasculitis: A systematic review and meta-analysis Autoimmunity Reviews 21 (2022) 103100] performed a meta-analysis to identify diagnostic accuracy studies using PR3-ANCA or MPO-ANCA to evaluate granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA).

[0174] The present invention should be used in the MPO serotype of AAV patients, and thus MPO-ANCA-positive patients will be treated. Thus, in a preferred variant, MPO-ANCA is detected in AAV patients in the present invention. In a specific embodiment, MPO-ANCA is detected in a subject, and the subject is treated with a compound for use according to the present invention.

[0175] In a preferred embodiment, the AAV clinical subtype is GPA, MPA, or EGPA.

[0176] In a preferred embodiment, IIF and an antigen-specific assay, such as an ELISA, are performed to detect MPO-ANCA. In particular, the ELISA may be a first-generation, second-generation, or third-generation ELISA as described above.

[0177] In one embodiment, disease progression is monitored in said AAV patients with MPO-ANCA serotype. In this regard, an increase in MPO levels measured at two different time points is an indication of relapse, and treatment should be adjusted. In this regard, in a preferred embodiment, a combination of IIF and ELISA is performed, or ELISA, preferably second- or third-generation ELISA, is performed.

[0178] Compounds useful in the present invention GYY4137 An example of a sulfide donor compound used by the present inventors is GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate), which is often used as its dichloromethane complex (Formula 1) [Rose, P. et al. GYY4137, a Novel Water-Soluble, HS-Releasing Molecule, Methods in Enzymology, Volume 554, 2015 Elsevier Inc ISSN 0076-6879]. [ka]

[0179] The chemical synthesis of GYY4137 and its release of H2S in vitro and in vivo have been described by Li L et al. [Li L, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogen sulfide. Circulation. 2008 May 6;117(18):2351-60.]

[0180] The authors also describe that GYY4137 slowly releases H2S both in aqueous solution in vitro and in anesthetized rats in vivo after intravenous or intraperitoneal administration. [ka]

[0181] Typically, GYY4137 is used in the art by intraperitoneal administration.

[0182] We prepared and tested both intraperitoneal (ip) and oral (per os) formulations of GYY in mice (see Examples) and unexpectedly found that the oral formulation resulted in higher serum levels, possibly making it preferable and more effective for treating MPO-ANCA vasculitis than intraperitoneal application.

[0183] GYY4137 derivatives may also be useful in the present invention.

[0184] Such derivatives have been described, for example, by Huang et al. [Huang, CW et al., A novel slow-releasing hydrogen sulfide donor, FW1256, exerts anti-inflammatory effects in mouse macrophages and in vivo., Pharmacological Research 113 (2016) 533-546].

[0185] Examples of such compounds are dithiophosphordiamines, which are fast-acting H2S releasers, and benzo[d][1,3,2]oxazaphospholes, which are intermediate and / or delayed releasers (delayed medium releasers).

[0186] A further option is a delayed emitter. [ka] It is a derivative of GYY4137.

[0187] Aspirin derivatives Anetholetrithione (ADT-OH, Formula 4) is known as an H2S-releasing compound. [ka]

[0188] Anethole trithione is useful because it is an HS donor itself but can also bind to other compounds, such as nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin or other NSAIDs (Song et al., Hydrogen sulfide donors in research and drug development, Issue Med. Chem. Commun., 2014, 5, 557; IDS). ADT-OH is released from these compounds in vivo and acts as an HS donor, while other molecular counterparts, such as aspirin derivatives, also have their own effects.

[0189] However, some other types of NSAIDs, such as non-selective cyclooxygenase (COX-1 and COX-2) inhibitors, such as diclofenac and naproxen, may have long-term adverse gastrointestinal or renal effects on renal conditions and are therefore less advantageous in the present invention (AAV disease itself has adverse effects on the kidneys) [Li, L. et al. Anti-inflammatory and gastrointestinal effects of a novel diclofenac derivative. Free Radicals Biol. Med., 2007, 42, 706.] [ka]

[0190] ADT-OH, ACS14, and ACS21 can be prepared and administered orally as described by Sparatore Anna et al. [Sparatore A, et al. Pharmacological profile of a novel H2S-releasing aspirin Free Radical Biology & Medicine 46 (2009) 586-592].

[0191] Compound formulations were prepared using a 2 ml / kg mixture containing 0.5% w / v carboxymethylcellulose and DMSO (9 / 1, v / v) and the active agent, which was provided at the following doses: ACS14 (50 mg / kg), aspirin (23 mg / kg), ACS21 (45 mg / kg), salicylic acid (18 mg / kg), and ADTOH (29 mg / kg). Administration was once daily for 7 consecutive days. The doses of ACS14, ACS21, salicylic acid, and ADTOH used in these experiments were equimolar to the dose of aspirin. [Sparatore, A. et al., 2009, see below]

[0192] Examples of natural H2S donors Natural H2S donors may also be applied in the present invention.

[0193] Examples of such compounds are provided below and can be purchased from a variety of exemplary suppliers. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0194] Further exemplary H2S-donors that do not contain -SS- groups are, for example: [ka] [ka] [ka]

[0195] Pharmaceutical Preparations and Administration of the Invention It is within the skill of one in the art to prepare compositions or pharmaceutical preparations for use from the compounds of the present invention.

[0196] For long term storage, it is advantageous if the composition is stored in a dry form, for example, crystallized or lyophilized.

[0197] Suitable vehicles are known in the art, and a typical vehicle used is, for example, carboxymethyl-cellulose.

[0198] Furthermore, some compounds may be sensitive to hydrolysis, suggesting the use of buffers, especially in injectable formulations, but also to avoid the formation of acidic or basic pH even in dry formulations.

[0199] Injectable formulations may be reconstituted before use and therefore only soluble components should be used.

[0200] Low solubility can be a problem that limits drug loading and needs to be resolved.

[0201] Typical excipients may include the following categories and examples:

[0202] Disintegrants such as cross-linked polymers, polyvinylpyrrolidone (crospovidone), cross-linked sodium carboxymethylcellulose (croscarmellose sodium), especially the latter.

[0203] For example, binders including: Saccharides, such as disaccharides (lactose, saccharose); polysaccharides (e.g., cellulose, starch, etc.), modified polysaccharides such as microcrystalline cellulose, cellulose ethers, or derivatized saccharides such as carboxymethylcellulose, may be useful; Sugar alcohols, such as xylitol, sorbitol or mannitol; especially mannitol (E421) Protein-type binders such as gelatin; (especially lightweight gelatin capsules).

[0204] Lubricants such as magnesium stearate or other stearic acid derivatives (or talc or silica, etc.) may be applied.

[0205] Polymers that act as stabilizers, surfactants, thickeners and solubility enhancers, such as povidone (polyvinylpyrrolidone, PVP) or other synthetic polymers such as polyethylene glycol (PEG), preferably povidone.

[0206] In the present invention, the compounds used are typically rather hydrophobic and highly photosensitive compounds. Therefore, when formulating them into pharmaceutical compositions, these issues should be taken into consideration. It is advantageous if the composition is protected from light.

[0207] A preferred formulation is a capsule, such as a light gelatin capsule or a hard capsule, in which the active agent is protected from light. In some embodiments, the packaging should protect the azidated compound from light. Opaque capsules are preferred for oral administration.

[0208] An example is Lonza Capsugel, a TiO2-free light-protective capsule (see Lonza Press Release "Lonza Expands its Capsugel® Capsule Offering to Include Titanium Dioxide-Free White Hard Gelatin Capsules" May 9, 2022, Basel, Switzerland).

[0209] Methods for encapsulating or incorporating increasing loads into polymer matrices, including nanoparticles and microparticles, and related compounds have been disclosed as nanoemulsion formulations, etc., and are known in the art.

[0210] Hereinafter, the present invention will be further characterized by non-limiting examples.

[0211] Materials and Methods Preparation of sodium sulfide and GYY4137 sulfide donor stock solutions Sodium sulfide stock solution was freshly prepared, sterile filtered (0.2 μm pore size), and stored on ice in a capped plastic tube until use. Larger crystals of sodium sulfide were rinsed and then dissolved in ultrapure degassed water. The stock solution was appropriately diluted, and the concentration was measured at 240 nm. Contaminants produced by sulfide oxidation were checked by adding 400 μM DTNB and measuring the absorbance at 560 nm. If the difference between the calculated concentrations from the two measured absorbances was less than 5%, the stock solution was deemed suitable for use. GYY4137 (Sigma-Aldrich, product number SML0100, PubChem substance ID: 329825158) was dissolved in DMSO or DTNB at the appropriate concentration and sterile filtered through a 0.2 μm pore size [Palinkas, Z., et al., Interactions of hydrogen sulfide with myeloperoxidase. Br J Pharmacol, 2015. 172(6): p. 1516-32.].

[0212] GYY4137 (P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid, compound and morpholine (1:1), morpholin-4-ium 4-methoxyphenyl(morpholino)phosphinodithioate), e.g., in the form of a dichloromethane complex, can be purchased for experimental purposes, e.g., from Merck (Sigma-Aldrich), catalog number SML0100.

[0213] Human samples All human samples were prepared from peripheral venous blood obtained with informed consent from healthy donors or patients with active ANCA disease. Sample handling was performed in accordance with national regulations (ethical decision: BPR-021 / 00084-2 / 2014; study number: 84 / 2014, project number: 4678-20161,2).

[0214] Isolation of immunoglobulins from human venous blood Immunoglobulins were isolated using protein G agarose resin according to the manufacturer's protocol. Blood serum samples were diluted 1:1 with binding buffer, loaded onto the agarose column, mixed, and incubated at room temperature for 2 hours. The column was washed with 15 ml of binding buffer, and the IgG fraction was then recovered by acidic elution using 100 mM glycine solution (pH 2.5). A 0.5 ml flow-through fraction was collected, and the pH was neutralized with 1 M phosphate buffer (pH 7.5). The fractions containing the highest protein concentration were pooled and analyzed by Ca elution. 2+ and Mg 2+ The solution was dialyzed against 500 ml of HBSS containing no HBSS twice. The protein concentration was determined using Bradford reagent, and the solution was sterile filtered (pore size 0.2 μm).

[0215] Isolation and activation of neutrophilic granulocytes Peripheral venous blood was obtained from healthy adult donors with informed consent. Erythrocytes were sedimented with a 1% dextran solution (1% dextran dissolved in DPBS), and the pale yellow supernatant was pipetted into a plastic tube and centrifuged at 500 g for 5 minutes. The supernatant was decanted, and the cells were then resuspended in 10 ml of DPBS and centrifuged at 500 g for 5 minutes. Polymorphonuclear cells were resuspended in 5 ml of DPBS, layered on top of 5 ml of Hystopaque 1077 solution, and centrifuged continuously at 800 g for 30 minutes. After gradient centrifugation using Hystopaque, the polymorphonuclear cells were sedimented to the bottom of the tube along with the remaining red blood cells. Erythrocytes were lysed with 0.2% NaCl solution, and isotonicity was then restored by adding an equal volume of 1.6% NaCl solution. The cells were washed with DPBS, resuspended in HBSS solution, and kept on ice until use. Viability and cell concentration were measured using trypan blue solution [Boyum, A., Isolation of lymphocytes, granulocytes, and macrophages. Scand J Immunol, 1976. Suppl 5: pp. 9-15.]. Polymorphonuclear cell suspensions were used within 2 hours of preparation. Neutrophilic granulocytes were activated with 100 ng / ml 12-phorbol 13-myristate acetate (PMA) and incubated for 20 minutes in the appropriate assay buffer. An equal volume (1 μl) of DMSO was added to all control samples. For autoimmune cell activation, 300 μg / ml IgG isolated from ANCA patients or healthy donors was used. Cells were first pretreated with different concentrations of GYY4137 for 5 minutes, and then 2 ng / ml TNF-α was added to the samples and incubated for 15 minutes. After priming with TNF-α, samples were treated with purified IgG and incubated for 60 min unless otherwise specified.

[0216] Effect of hydrogen sulfide on NOX2 activity in neutrophilic granulocytes FOX reagent (100 μM xylenol orange, 250 μM ammonium iron(II) sulfate, 100 mM sorbitol, and 25 mM sulfuric acid diluted in 30 ml of ion-exchanged water) was prepared according to the protocol published by Wolff [Jiang, ZY, et al. Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange. Comparison with the TBA assay and an iodometric method. Lipids, 1991. 26(10): pp. 853–856.] and stored on ice until use. Neutrophil granulocyte samples were prepared in L-tyrosine assay buffer as discussed above, using PMA as an activator and adding 1 mM Na azide to inhibit MPO activity and prevent hydrogen peroxide decomposition. The supernatant was diluted 50-fold with 50 mM phosphate buffer, and 50 μl of FOX assay reagent was added to 140 μl of diluted sample and mixed thoroughly. For the standard curve, 140 μl of a dilution series of hydrogen peroxide (0.5-4 μM) in DPBS was mixed with 50 μl of FOX reagent. The samples and standards were incubated at room temperature for 40 minutes, and the optical density was measured at a wavelength of 560 nm.

[0217] Determining the effect of sulfide on neutrophil granulocyte activation The oxidative burst of neutrophils was measured based on NOX2 activity upon activation with PMA or IgG from healthy donors or MPO-ANCA patients. WST-1 reagent reacts with superoxide produced in the extracellular space [Ngamwongsatit, P., et al., WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxic Bacillus species using CHO cell line. Journal of Microbiological Methods, 2008. 73(3): pp. 211-215.; Tan, AS, and MV Berridge, Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1, to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents. J Immunol Methods, 2000. 238(1-2): pp. 59-68.] Neutrophil activation was performed in the presence of 300 μM WST-1 and 20 μg / ml catalase, and in the presence or absence of different concentrations of sulfide (5–50 μM) or GYY4137 sulfide donor (20–100 μM). Cells were incubated in the presence of sulfide for 5 min before or after the addition of TNF-α. PMA-activated samples were incubated for 20 min, and ANCA-activated samples for 90 min. All samples were placed on ice for 5 min to slow the reaction. All samples were centrifuged at 2000 g for 5 min, and the supernatants were then pipetted into a 96-well plate and the absorbance was measured at 450 nm. The concentration of superoxide produced was determined using WST-1 (37 × 10 3 M -1 cm -1) and taking into account that 2 moles of superoxide are required to reduce 1 mole of WST-1.

[0218] Effect of sulfide on neutrophil priming by TNF-α A 24-well sterile plate containing sterile glass cover slips was treated with 0.1% gelatin for 1 hour at 37°C. The plate was washed three times with 500 μl of DPBS and 2.5 × 10 5 Neutrophils were incubated for 30 minutes and then treated with the appropriate concentration of GYY4137 for 5 minutes before or simultaneously with the addition of 10 ng / ml TNF-α. Neutrophils were primed for 15 minutes at 37°C in the presence or absence of GYY4137. The supernatant was discarded, and cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature. Nonspecific antigens were then blocked with 5% goat serum overnight at 4°C. Cells were washed three times with 500 μl of DPBS, and surface MPO was then labeled with rabbit anti-MPO antibody (1:2000) for 1 hour at room temperature. Wells were washed three times and then labeled with goat anti-rabbit Alexa Flour 488 (1:500) secondary antibody in the dark for 1 hour at room temperature. Cells were then permeabilized with 0.1% Triton X for 15 minutes in the dark to label intracellular MPO. The cells were then washed three times as described above and then labeled with rabbit anti-MPO antibody. After the washing step, intracellular MPO was labeled with goat anti-rabbit Alexa Fluor 568 (1:500) secondary antibody for 1 hour in the dark. For nuclear staining, 0.5 ng / ml Hoechst 33258 was used for 15 minutes at room temperature. The cells were washed, and then the coverslips were mounted on glass slides using Prolong™ Glass Antifade Mountant and incubated for 15 minutes. Microscopic analysis was performed using a STED microscope system.

[0219] Measurement of neutrophil degranulation in the presence of sulfide or sulfide donors 2×10 6Neutrophil granulocytes at a final concentration of 10 cells / ml were incubated with 5 μM cytochalasin B in HBSS at 37°C for 10 minutes. Different concentrations of sulfide or GYY4137 were added to the cells and incubated for 5 minutes. For PMA, activated sodium sulfide (0–50 μM) was used, and after 5 minutes, cells were activated with PMA. For autoimmune activation with IgG, neutrophils were treated with GYY4137 sulfide donor (0–80 μM). Samples were then primed with 4 ng / ml TNF-α for 5 minutes and then incubated with ANCA or healthy IgG (300 μg / ml) for 30 minutes. For positive controls, 1% Triton X was added to the samples, incubated for 20 minutes, and then vortexed. All samples were placed on ice for 5 minutes and centrifuged at 2000 g for 5 minutes. 100 μl of the supernatant was then pipetted into a 96-well plate with 100 μl of 1 mM phenolphthalein-glucuronide dissolved in 100 mM Na acetate solution and incubated for 19 hours. The enzymatic reaction was stopped with 100 μl of 400 mM glycine buffer containing 200 mM NaCl, resulting in the production of pink phenolphthalein, which was measured spectrophotometrically at 540 nm. One unit of enzyme activity was the amount liberated from 10 μg of phenolphthalein in 19 hours. For the standard curve, 1 mg of phenolphthalein was dissolved in 200 μl of 96% Drum-Grate ethanol, vortexed, and then 800 μl of ion-exchanged water was added to make a 1 mg / ml solution. Dilutions were performed as quickly as possible. The figures show 100 μl of the phenolphthalein diluent, 100 μl of HBSS, and 100 μl of glycine buffer. Protein levels in neutrophil supernatants from PMA-activated samples were analyzed by SDS-PAGE and silver staining.

[0220] Effect of sulfide on neutrophil phagocytosis Phagocytosis experiments were performed using the Vybrant™ Phagocytosis Assay Kit according to the manufacturer's instructions. Briefly, fluorescently labeled E. coli K-12 bioparticles and trypan blue stock solutions were prepared. 4Neutrophils per well were incubated in a black 96-well plate for 1 hour in the presence or absence of GYY4137 (20-100 μM) to allow cell adhesion. After the incubation period, the supernatant was removed, and 100 μl of the prepared fluorescent bioparticle suspension was added. The cells were then incubated at 37°C for 2 hours in the presence or absence of GYY4137 (20-100 μM). In two different experimental conditions, the cells were treated with GYY4137 for 1 hour or 5 minutes before adding the fluorescent bioparticle suspension. After 2 hours, the bioparticle solution was removed, and 100 μl of trypan blue solution was immediately added and incubated at room temperature for 1 minute. The trypan blue was removed, and 100 μl of HBSS was added to each well. Fluorescence was measured at excitation and emission wavelengths of 480 and 520 nm, respectively. Samples for microscopy were prepared in 2 × 10 wells on EZ-Slide chamber slides. 5 The cells were prepared in the same manner with a cell number of 1000 / well.

[0221] Effect of sulfide on bacterial killing by neutrophils. S. aureus was cultured on Columbia sheep blood agar plates, incubated overnight at 37°C, and then stored at 4°C. For experiments, single colonies were transferred from the plates to 15 ml of nutrient broth, incubated overnight at 37°C, then centrifuged at 1000 g for 5 minutes and washed twice with PBS. Concentrations were determined by a turbidity curve (A550 of 0.2, approximately 1 x 10 8 For opsonization, bacteria were cultured at 1 × 10 in PBS containing 10% human blood serum. 7 The tubes were subjected to end-over-end rotation at 6 rpm at 37°C for 20 minutes.

[0222] The effect of hydrogen sulfide on bacterial killing was measured using a modified colony-forming unit (CFU) assay [Hampton MB, et al. A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J Leukoc Biol. 1994 Feb;55(2):147-52. doi: 10.1002 / jlb.55.2.147. Erratum: Hampton MB, et al. J Leukoc Biol 1994 Jul;56(1):104. PMID: 8301210.] For experimental reactions, 500 μL of isolated human neutrophils (1 × 107 / mL, prewarmed to 37°C for 10 min) were added to 500 μL of freshly opsonized bacteria (1 × 108 / mL) and 50 μL of prewarmed serum (10%), prepared with or without 100 μM NaHS. For control reactions, neutrophils were replaced with 500 μL of Hank's buffer. Reactions were incubated at 37°C with end-over-end rotation (6 rpm). After 20 min of incubation, 50 μL of sample was pipetted into 950 μL of ice-cold PBS to quench neutrophil activity. Samples were centrifuged at 100 g for 5 min at 4°C (to sediment neutrophils rather than bacteria), and the neutrophil pellet was washed two more times with 1 mL of ice-cold PBS. The supernatant was collected at each step to measure bacteria not internalized by neutrophils. The pellet was resuspended in 2.5 mL of water, adjusted to pH 11.00 with NaOH for 5 minutes, and then vortexed thoroughly. The control, supernatant, and each sample containing intracellular bacteria were then diluted in water (pH 11) to yield approximately 50 colonies per half-plate when plated on Columbia sheep blood agar, and the plates were incubated overnight at 37°C. Colonies were counted, and the number of intracellular colonies was normalized for the control and supernatant samples to compare killing in untreated and hydrogen sulfide-treated samples.

[0223] mouse ANCA vasculitis animal model Breeding pairs of Black 6 recombinase activating gene 2-deficient (Rag2- / -) mice and MPO-deficient (Mpo- / -) mice were purchased from Jackson Laboratories and maintained by the Department of Experimental Pharmacology of the National Institute of Oncology (ethical decision number: PE / EA / 00419-4 / 2022). Mice aged 10 to 14 weeks were used in all experiments, and different groups were used as shown in the following table (Table 1). [Table 1]

[0224] Animal experiments were performed according to the protocol published by Falk [Xiao, H., et al., Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest, 2002. 110(7): pp. 955-63]. MPO knockout mice were immunized with murine MPO according to the following protocol. On day 1, MPO KO mice were injected with a total of 200 μl of murine MPO (mMPO) or 20 μg of BSA containing complete Freud's adjuvant. 30–40 μl was injected into the hind footpad, and 120–160 μl was injected subcutaneously into the abdominal region. On day 14, mice were injected IP with 20 μg of mMPO / BSA in incomplete Freud's adjuvant, and on day 28, they were boosted IP with 20 μg of mMPO / BSA in DPBS buffer. Ten days after the boost, blood was collected from the canthus of the eye under isoflurane anesthesia, and antibody titers were determined by anti-MPO ELISA. If the titer was low, mice were boosted again with 20 μg of mMPO or BSA, and titers were determined again 7 days later. If the titer was appropriate, mice were terminated, and splenocytes were isolated. Spleens were removed, gently homogenized in RPMI 1640, filtered through a 70 μM sterile filter, and washed twice with chilled RPMI 1640. Residual red blood cells were lysed using red blood cell lysis buffer according to the manufacturer's instructions. Cells were resuspended in 600 μl of RPMI 1640. Cells were counted using trypan blue solution, and the total cell number was 3 × 10 7 From 6 x 10 7The antibody titers varied between groups. Splenocytes from one mouse were intravenously injected into two Rag2- / - immunodeficient mice. Pairs were treated with 100 μg / kg GYY4137 or DPBS every other day for 5 weeks. After 4 weeks, antibody titers were confirmed using an anti-MPO ELISA. After 5 weeks, mice were euthanized with isoflurane, and lung and kidney tissue samples were fixed in 10% paraformaldehyde and prepared for light microscopic analysis. Lung samples were stained with hematoxylin and eosin (H&E), kidney samples were stained with H&E and periodic acid-Schiff stain, and selected samples were stained with Masson's trichrome.

[0225] In an alternative experiment, pairs were treated with 90 μmol / kg GKK895 or DPBS three times a week for three weeks. GKK895 is a derivative of GYY4137. After three weeks, mice were euthanized with isoflurane, and kidney tissue samples were fixed in 10% paraformaldehyde and prepared for light microscopy. Kidney samples were stained with H&E and periodic acid-Schiff stain.

[0226] To compare GYY4137 and GKK-895 treatment in mouse kidneys, kidney samples from the ANCA vasculitis mouse model were prepared for histological analysis and evaluated by spectrophotometric analysis. The percentage of affected glomeruli in treated samples was calculated compared to their control pairs. Samples treated with 150 μmol / kg GYY4137 (shown in dark gray) and showing a reduction in renal pathology are shown in this figure, with an average reduction in affected glomeruli of 64.9%. Samples treated with 90 μmol / kg GKK-895 (shown in light gray) showed an average reduction in affected glomeruli of 56.9% compared to their control pairs (Figure 10). The figure shows only GYY-treated samples in which treatment was effective.

[0227] Healthy (Rag2- / - immunodeficient) mice and measurement of H2S serum levels Healthy Rag2- / - immune-deficient mice (see above) were used for IP and per os administration experiments and were maintained and treated as described above, mutatis mutandis.

[0228] Blood samples were collected from the canthus of the mouse under isoflurane anesthesia and incubated for 30 minutes at room temperature. The clotted blood samples were centrifuged at 2000 g for 10 minutes, and the serum was then collected into a new tube. 25 μl of serum sample was mixed with 66 μl of premixed reagent-buffer solution (65 μl of 200 mM HEPES, pH 8.2 + 1 μl of 100 mM MBB in ACN) followed by immediate vigorously vortexing. After exactly 10 minutes at 20°C, the reaction was quenched by adding 5 μl of 50% TCA (w / v) and vigorously vortexing. Precipitated proteins were removed by centrifugation at 3000 g for 5 minutes, and the supernatant was transferred to an autosampler vial and maintained at 4°C. Calibration samples for quantification were prepared in the same manner using standardized NaHS solution and further diluted after derivatization. For HPLC measurements, 3 μl was injected onto a Phenomenex Luna C18(2) 250 × 2 mm 3 μm column using a gradient elution profile of 0.1% TFA in water and 0.1% TFA in acetonitrile. The fluorescence detector excitation wavelength was set at 390 nm, and the emission wavelength was set at 475 nm. Preparation was performed as previously described with a modified chromatographic setup [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: pp. 146-158.]

[0229] Measurement of serum sulfide concentrations in mice treated with GYY4137 or GKK-895 Measurement of serum sulfide concentrations in mice treated with GYY4137 or GKK-895. Mice were treated intraperitoneally with 150 μmol / kg GYY4137 or 110 μmol / kg GKK-895. Blood samples were collected from the canthus of the eye at the indicated time points under isoflurane anesthesia. All blood samples were incubated at room temperature for 30 minutes and then centrifuged at 2000 g for 10 minutes. Hydrogen sulfide concentrations were measured from the collected blood serum samples using a monobromobimane assay [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: pp. 146-158.] Both GYY4137 and GKK-895 treatments resulted in significant increases in circulating sulfide concentrations, with GKK-895 producing higher measured sulfide values ​​at 2 and 4 hours after injection.

[0230] It can be concluded that serum levels of H2S are an important factor for the efficacy of these compounds.

[0231] Effect of sulfide on reactive oxygen species production by 12-phorbol 13-myristate acetate (PMA)-activated neutrophils First, we investigated the effect of sulfide on neutrophil activation by 12-phorbol 13-myristate acetate (PMA).

[0232] FOX reagent (100 μM xylenol orange, 250 μM ammonium iron(II) sulfate, 100 mM sorbitol, and 25 mM sulfuric acid diluted in 30 ml of ion-exchanged water) was prepared and stored on ice until use. Neutrophil granulocyte samples were prepared in Hank's balanced salt solution (phenol red-free) using PMA as an activator and 1 mM sodium azide to inhibit MPO activity and prevent hydrogen peroxide decomposition. The supernatant was diluted 50-fold with 50 mM phosphate buffer, and 50 μl of FOX assay reagent was added to 140 μl of the diluted sample and mixed thoroughly. For the standard curve, 140 μl of a dilution series of hydrogen peroxide (0.5–4 μM) in DPBS was mixed with 50 μl of FOX reagent. Samples and standards were incubated for 40 min at room temperature, and the optical density was measured at a wavelength of 560 nm (see Methods and [Wolff, SP, Ferrous Ion Oxidation in Presence of Ferric Ion Indicator Xylenol Orange for Measurement of Hydroperoxides. Oxygen Radicals in Biological Systems, Pt C, 1994. 233: pp. 182-189]). HO production by PMA-activated neutrophils, measured by the FOX assay, was unaffected by the applied sulfide concentration range (5-50 μM) (Fig. 1A).

[0233] Effect of sulfide on the oxidative burst of PMA- or ANCA-activated neutrophils. In the case of MPO-ANCA activation of neutrophils, the FOX assay cannot be used to assess superoxide formation because it requires the addition of sodium azide, which interferes with the antigenic function of cell-surface MPO. Therefore, we applied a different method. Superoxide production was measured using the WST-1 reagent [Tan, AS and MV Berridge, "Superoxide produced by activated neutrophils efficiently reduces the tetrazolium salt, WST-1, to produce a soluble formazan: a simple colorimetric assay for measuring respiratory burst activation and for screening anti-inflammatory agents." J Immunol Methods, 2000. 238(1-2): pp. 59-68]. The reduction of WST-1 to a reporter yellow product in PMA-stimulated neutrophil samples was not inhibited by sodium sulfide (Figure 1B) or the sulfide donor GYY4137 (Figure 1C). Treated with IgG isolated from ANCA patients The cells are produced significantly higher concentrations of superoxide than samples treated with IgG isolated from healthy donors (p=<0.05).

[0234] Surprisingly, the sulfide donor GYY4137 inhibited superoxide production in a dose-dependent manner, with IC 50 The IC value was 46.7 μM (Fig. 1D). This effect was enhanced by treatment with a sulfide donor after priming, which resulted in a slightly higher IC 50The NOX2 activity was significantly higher when GYY4137 was added 5 min before priming with TNF-α (Fig. 1D, black columns) than when the sample had a value of 62.6 μM (Fig. 1D, patterned columns). These results indicate that sulfide may also interfere with the neutrophil priming process. These observations demonstrate that sulfide does not affect NOX2 activity, given that treatment with sodium sulfide or GYY4137 does not affect PMA activation. However, GYY4137 may efficiently inhibit neutrophil activation by IgG isolated from ANCA patients.

[0235] Effect of sulfide on neutrophil degranulation To investigate the effect of sulfide on neutrophil degranulation, we used a β-glucuronidase assay based on the release of phenolphthalein from phenolphthalein-glucuronide [Falk, RJ, et al., Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA, 1990. 87(11): pp. 4115-4119]. The interference of sulfide with the assay conditions was measured in neutrophil samples treated with 1% Triton X. Triton X was used to disrupt the cell membrane and obtain maximum β-glucuronidase activity in the supernatant. The results show that the average unit of enzyme activity, calculated based on the amount of phenolphthalein (μg) released at 19 h, was 10.88 (±1.88) units. The data also show that the addition of different concentrations of GYY4137 did not affect enzyme activity or interfere with the assay conditions (Figure 2 / A). β-glucuronidase activity in the supernatants of PMA-treated samples was comparable, and again, the presence of sulfide did not affect enzyme activity (Figure 2 / B). We further confirmed using SDS-PAGE followed by silver staining that the protein levels in the supernatants correlated well with the measured enzyme activity. No decrease in protein concentration was detected in GYY4137-treated samples, again suggesting that sulfide or GYY4137 did not interfere with the assay (Figure 2 / C). In the case of ANCA activation, the degree of degranulation was similar to that in Triton X- or PMA-treated samples, with a mean enzyme activity of 9.77 (±1.36) units. However, unexpectedly, in this case, the presence of GYY4137 effectively inhibited phenolphthalein release in a dose-dependent manner, resulting in an IC 50 The value was 6.47 μM (Figure 2D). Thus, surprisingly, the sulfide donor GYY4137 could effectively inhibit neutrophil degranulation upon ANCA activation, but had no effect upon PMA activation.

[0236] Effect of sulfide on neutrophil priming with TNF-α Proinflammatory cytokines such as TNF-α induce the translocation of ANCA antigens, such as MPO and PR3, to the cell surface, priming neutrophil cells for antibody-recognition-based activation. We investigated the interference of sulfide with TNF-α-induced neutrophil priming by differential immunofluorescence labeling of surface MPO using a green fluorescent tag (Figure 3, second column) and granular MPO using a red fluorescent tag (Figure 3, third column). In the black-and-white figures, fluorescence is shown in gray but can be identified via the location in the figures provided herein. In TNF-α-treated samples, MPO translocated to the membrane (Figure 3, second row), whereas no MPO could be detected on the surface of untreated control samples (Figure 3, first row). Neutrophils treated with 25 μM or 50 μM GYY4137 5 min before or together with TNF-α showed reduced MPO signaling on the membrane and abundant presence in azurophilic granules (Figure 3, rows 3–5). These results suggest that GYY4137 can inhibit TNF-α-induced translocation of the ANCA antigen MPO, thereby inhibiting neutrophil priming.

[0237] Effect of sulfide on phagocytosis Control neutrophils phagocytosed E. coli bioparticles (Fig. 4 / A1-2), and this process was inhibited by cytochalasin D, which inhibits phagocytosis by interfering with the formation of new actin fibers [Parod, RJ and JD Brain, Immune opsonin-independent phagocytosis by pulmonary macrophages. J Immunol, 1986. 136(6): pp. 2041-2047] (Fig. 4 / A15-16). Addition of GYY4137 5 min before or together with the bioparticles did not affect phagocytosis (Fig. 4 / A3-14). A plate assay was also used to quantitatively investigate the effect of sulfide on phagocytosis. Treatment with GYY4137 did not decrease the reported relative fluorescence signal at any given concentration (20-100 μM), confirming that sulfide did not inhibit phagocytosis (Fig. 4 / B, black squares). No inhibition was observed even when a sulfide donor was given simultaneously with the bioparticles (Figure 4B, gray circle). Thus, neutrophil phagocytosis is not inhibited in the presence of sulfide. Hydrogen sulfide also had no effect on bacterial killing by neutrophil granulocytes (Figure 4C).

[0238] The sulfide donor GYY4137 protects against renal injury in a mouse model of MPO-ANCA vasculitis Several studies have described different ANCA mouse models (animal models of ANCA-associated vasculitis). The method we chose to establish in our laboratory involves adoptive transfer of activated B cells from mice immunized with MPO or, for control, with BSA, and non-immunized mice. While this method does not strictly result in an autoimmune reaction, it robustly produces symptoms similar to those observed in patients with ANCA-associated vasculitis [Xiao, H., et al., Antineutrophil cytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest, 2002. 110(7): pp. 955-63.]. Using this model, we investigated the effect of the hydrogen sulfide donor molecule GYY4137 on the development and severity of AAV-associated symptoms.

[0239] GN is usually focal and segmental, but can be generalized or diffuse. Glomerular inflammation is present, accompanied by necrotic lesions. The necrotic lesions are invariably accompanied by crescents. The crescents are formed by proliferating parietal epithelial cells and monocytes; lymphocytes and granulocytes are occasionally present. Crescents contain only cells, not collagen, and are called cellular crescents (Figure 5A, B). The next step in the process is when fibroblasts migrate into Bowman's space, synthesize collagen, which gradually replaces the crescents; when cellular components are mixed with collagen, the lesion is called fibrocellular crescent (Figure 5C); and in advanced stages, when epithelial cells are absent and only fibrous tissue is present, the lesion is called fibrous crescent (Figure 5D). These three stages indicate active or chronic lesions and, therefore, response to treatment. Extensive destruction of Bowman's capsule is often present (Figure 5 / E); in these cases, granulomas and multinucleated cells are usually found around the glomerulus (Figure 5 / F), but these granulomas do not represent Wegener's granulomatosis. Glomerulosclerosis is scarring (hardening) of the glomerulus (Figure 5 / G, H). It is caused by activation of glomerular cells to produce scar material. This may be produced by the glomerular cells themselves or may be stimulated by molecules called growth factors that are delivered to the glomerulus by circulating blood that penetrates the glomerular filter.

[0240] Rag2-deficient mice receiving splenocytes from control mice or BSA-immunized MPO-deficient mice did not develop inflammation in their kidneys. Rag2-deficient mice receiving splenocytes from MPO-immunized MPO-deficient mice developed the above-mentioned symptoms with great variability (Figure 6A). Kidney samples from two different mouse pairs (MPO16, MPO18) showed elevated levels of inflammation in GYY4137-treated samples compared with untreated samples. One mouse from each pair (MPO5, MPO7) died 24 hours after intravenous injection of splenocytes, and therefore we excluded both pairs from the experiment. Semiquantitative comparative analysis of mouse pairs that developed symptoms upon adoptive splenocyte transfer showed a reduction in renal vasculitis symptoms as a result of GYY4137 treatment. The number of affected glomeruli in treated mice was 64.9% lower compared to their untreated counterparts (excluding MPO3, MPO16 and MPO18) (Fig. 6 / B).

[0241] H 2 S serum levels depend on sulfide donor molecule, dose, and administration Normal (healthy) mice were treated with various sulfide donor preparations both intraperitoneally and orally: GYY4137 was used at 150 and 750 μM / kg body weight. A donor mixture (HS donor mix) containing GYY4137 (150 μmol / kg, 5 mg / ml strain), N-acetylcysteine ​​(40 μmol / kg, 0.64 mg / ml strain), and pyridoxal 5'-phosphate (20 μmol / kg, 0.49 mg / ml strain) was prepared. ATB346 (otenaproxen, currently in Phase 2B efficacy trials; Antibe Therapeutics) was administered both orally and intraperitoneally at a dose of 60 μmol / kg. ATB346 (briefly ATB) is a novel hydrogen sulfide-releasing derivative of naproxen with apparently reduced toxicity and inhibits COX activity. ATB346 is both an anti-inflammatory agent and a novel nonsteroidal anti-inflammatory drug ("NSAID") that induces apoptosis in human melanoma cells.

[0242] ATB was tested in an ANCA mouse model and administered orally (per os) and proved ineffective. In further experiments, ATB (60 μmol / kg) was administered orally to healthy mice, serum levels were measured, and GYY4137 (150 μmol / kg) was administered intraperitoneally in parallel, and serum levels were compared. Surprisingly, IP-administered GYY4137 proved active in the ANCA vasculitis mouse model (see the examples above and Figures 7 and 8), significantly increasing serum H2S levels, whereas orally administered ATB346 did not, indicating a correlation between elevated serum levels and beneficial effects on kidneys damaged in AAV.

[0243] H2S levels were measured from the serum of the animals.

[0244] In further experiments, a sulfide donor mix (HS donor mix) containing GYY4137 (150 μmol / kg, 5 mg / ml strain), N-acetylcysteine ​​(40 μmol / kg, 0.64 mg / ml strain), and pyridoxal 5'-phosphate (20 μmol / kg, 0.49 mg / ml strain) was prepared. The components were dissolved in PBS.

[0245] The HS donor mix was administered IP and per os to healthy mice. Serum concentrations were measured at time 0, resulting in high concentrations of sulfide in serum samples. Per os administration of the donor mix resulted in a significant increase in serum sulfide compared to IP treatment (Figure 8B). IP treatment, despite containing 150 μmol / kg GYY4137 (along with N-acetylcysteine ​​and pyridoxal 5'-phosphate), did not increase sulfide concentrations in animal serum (Figure 8A).

[0246] Thus, intraperitoneally administered GYY4137 significantly increased H2S serum levels (see Figure 7), whereas IP administered H2S donor mix did not (Figure 8A) - a finding closely correlated with the fact that IP administered GYY4137 improved renal status in the ANCA vasculitis animal model, but no improvement was observed in the IP administered donor mix cases.

[0247] When the novel hydrogen sulfide donor GKK-895 was administered IP to healthy mice, serum concentrations were significantly elevated 2 and 4 hours after treatment compared with control and GYY4137-treated samples (Figure 9). This also demonstrated that GKK-895 at very low concentrations reduced renal symptoms of ANCA-associated vasculitis in an ANCA mouse model to the same extent as high concentrations of GYY4137 (Figure 10).

[0248] These findings provide further evidence that elevated blood hydrogen sulfide concentrations correlate with protective effects in the ANCA mouse model.

[0249] Taken together, the results lead to the conclusion that, indeed, any H2S donor compound, i.e., any compound that provides H2S to a subject, i.e., any compound that increases serum levels of H2S, is likely to be useful in the treatment of MPO-ANCA vasculitis.

Claims

1. ANTI-NEUTROPHIL CYTOPLASMIC AUTOANTIBODY (ANCA)-ASSOCIATED VASCULITIS (AAV) IN MAMMALIAN PATIENTS WITH MYELOPEROXIDASE-ANCA (MPO-ANCA) SEROTYPE AAV ２ The above-mentioned H is an S donor compound, preferably wherein the AAV is a myeloperoxidase (MPO) autoantigen-positive AAV, and the subject has an increased level of MPO-specific ANCA. ２ S donor compounds.

2. 2. The method of claim 1, wherein the AAV is a myeloperoxidase (MPO) autoantigen-positive AAV and the subject has an increased level of MPO-specific ANCA. ２ S donor compounds.

3. The H ２ S donor compounds are delayed H ２ an S-emitter compound, said compound being To prevent kidney damage and / or protect the kidneys in mammalian subjects with AAV, To inhibit neutrophil granulocyte activation by anti-MPO antibodies in mammalian subjects with AAV, - to inhibit neutrophil degranulation in mammalian subjects with AAV, and / or To inhibit neutrophil priming in mammalian subjects with AAV Used, 3. The method of claim 1 or 2, wherein the method is used to prevent kidney damage and / or to protect the kidneys in mammalian subjects with AAV. ２ S donor compounds.

4. 4. The method of claim 1, wherein the AAV is selected from microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA) (Churg-Strauss syndrome), and renal-confined vasculitis. ２ S donor compounds.

5. The compound is normal H ２ S serum level (or H before (or before) the compound is administered) ２ the bioavailable H in the serum of a mammalian subject after administering the compound to said subject, as compared to the H serum level. ２ and increasing H S levels in said subject, preferably at a time point between 1 hour and 24 hours after administration of said compound. ２ S serum levels increased, Preferably, the H ２ 5. The use of claim 1, wherein the increase in serum levels of H is at least twice the normal serum level. ２ S donor compounds.

6. The compound orally administered to said mammalian subject; or administered intraperitoneally to said mammalian subject; or administered intravenously to said mammalian patient; Preferably administered orally to said mammalian patient, H for use according to any one of claims 1 to 5 ２ S donor compounds.

7. The compound is represented by the general formula (Y) M-L-Q, (Y) (In the formula, Q is a compound that, in the body of a mammalian patient, induces H ２ H that releases S ２ S-releasing part, Preferably, Q contains a dithiol group (-S-S-), or Q is a phosphinodithionate group (=P(S)S － ), L is an organic linking moiety; M is a moiety covalently attached to the remainder of the molecule by a hydrolyzable bond, such that upon hydrolysis, M is converted into a compound that is tolerable to, and preferably beneficial to, a mammalian subject; Either L or M may be absent, or both may be present).

7. The method of claim 1, wherein the H ２ S donor compounds.

8. The compound is represented by the general formula (X) 【Chemistry 1】 (In the formula, Q, when administered to a mammalian patient, induces H ２ H that releases S ２ S-releasing part, Preferably, Q comprises a 5- to 6-membered heterocycle containing a dithiol group (—S—S—), or Q is a phosphinodithionate group (=P(S)S － ), L is a linking moiety, In a preferred embodiment, L is C1-C8 alkylene, (preferably methylene), -, -O-, S, -NH-, aryl, C1-C4 alkylaryl, or a 5- to 6-membered heterocycle, which is optionally linked to Q via a C1-C4 alkylene; R １ , R ２ , R ３ , R ４ , and R ５ at least two, preferably three or four of are H; R １ , R ２ , R ３ , R ４ , and R ５ are independently selected from H, halogen, pseudohalogen, -CN, -OH, -SH, -NO ２ , -NH ２ , -NHCH ３ ,-COOH,CONH ２ , - substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkylester, C3-C8 alkenylester or C3-C8 alkynylester), the above substituents (when the above substituents are present, halides, pseudohalides, -OH, -SH, -OMe, -NO ２ , -NH ２ , —NHMe), ・-OCOR １６ , -COOR １７ , -OR １８ , -CONHR １９ (R １６ , R １７ , R １８ , and R １９ represents H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkyl, and the substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO, —C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl, or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester, or C3-C8 alkynyl ester), and the substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO ２ , -NH ２ , —NHMe), Preferably, R １ and R ３ Is -OCOR １６ , -COOR １７ , -OR １８ and R １６ , R １７ , and R １８ is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkylester, C3-C8 alkenylester or C3-C8 alkynylester), and the aforementioned substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO ２ , -NH ２ , -NHMe; More preferably R ２ , R ３ , and R ５ is H, R １５ is selected from C1-C8 alkylene, C1-C8 alkyl ether, C1-C8 carboxylate, preferably —(CH ２ ) ｎ -(CO)O-, where n is 0, 1, 2 or 3, preferably 0 or 1; or a pharmaceutically acceptable salt and / or solvate and / or complex thereof. ２ S donor compounds.

9. The compound has the general formula I 【Chemistry 2】 (In the formula, R ７ is selected from the following: a 3- to 10-membered, preferably 5- to 10-membered, more preferably 5- or 6-membered heterocycle, ・NH-R ２３ (R ２３ is selected from C1-4 alkyl, C1-4 alkylcarbonyl), R ８ (Preferably, R ８ is L) is selected from the following: ・-NH- ・-CH ２ -, -O-, S or NH, preferably CH ２ , O or NH, ・or R １５ means nothing, A is selected from the following: ・-S － (A is -S － If R ６ is non-existent), -S- and -S － (When A is -S-, ・R ６ may be selected from H, a 5- to 10-membered, preferably 6-membered, optionally substituted heterocycle, C1-4 alkyl, or C6-C10 aryl; or ・A is -S- and R ６ and R ７ together with the other moieties of the formula to form a compound having formula I.1 【Transformation 3】 R １ From R ５ and R ８ are independently as defined above, and the substituents on the two rings may be the same or different), ・-O － -O- (when A is -O-, R ６ is H, a 5- to 10-membered, preferably 6-membered heterocycle, C1-4 alkyl, or C6-C10 aryl, or and / or a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings, optionally having at least one 1- to 8-membered open-chain moiety, and optionally containing 1 to 4, preferably 1 to 3, heteroatoms; and / or R ６ is an organic moiety that, upon hydrolysis, is converted into a compound that is tolerable to, and preferably beneficial to, a mammalian patient. R １ , R ２ , R ３ , R ４ , and R ５ is as defined above or preferably R １ , R ２ , R ３ , R ４ , and R ５ is H, halogen, -CN, -OH, -SH, -NO ２ , -NH ２ , -NHCH ３ ,-COOH,CONH ２ and R １ , R ２ , R ３ , R ４ , and R ５ at least two of are H; Preferably, R ３ Ha-OCH ３ or R １ , R ２ , R ３ , R ４ , and R ５ are H, respectively), or a pharmaceutically acceptable salt and / or solvate and / or complex thereof, In certain embodiments, dichloromethane complexes and / or morpholinium salts H for use according to any one of claims 1 to 8, having ２ S donor compounds.

10. The compound has the general formula I.2 【Chemistry 4】 (In the formula, R ９ is H, and the H may be dissociated, -S-R ９ Ha-S － and A is selected from absent and —O—; When A is -O-, R ６ teeth a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings, optionally having at least one 1- to 8-membered open-chain moiety, and optionally containing 1 to 4, preferably 1 to 3, heteroatoms; or If A is absent, then R ６ teeth a morpholino group linked via a 5- to 10-membered, preferably 6-membered, heterocycle, preferably an O- and / or N-containing heterocycle, in particular an N-containing heterocycle, R １ , R ２ , R ３ , R ４ , and R ５ is selected from H, C1-4 alkyl, C1-4 alkoxy, and halide; R １ , R ２ , R ３ , R ４ , and R ５ at least two of R ３ is -OMe, or R １ , R ２ , R ３ , R ４ , and R ５ are H, respectively), or a pharmaceutically acceptable salt and / or solvate and / or complex thereof H for use according to any one of claims 1 to 8, having ２ S donor compounds.

11. The compound has the general formula 1.3 【Transformation 5】 (R １０ is a morpholino group linked via a 5- to 10-membered, preferably 6-membered, heterocycle, preferably an O- and / or N-containing heterocycle, in particular an N-containing heterocycle; R １ , R ２ , R ３ , R.R. ４ , and R ５ , and R ９ is defined above) H for use according to claim 10, having ２ S donor compounds.

12. Preferably, the compound has the general formula 1.3.1 【Transformation 6】 (In the formula, R １１ is C1-4 alkyl) and Preferably, the compound for use according to claim 10 is GYY4137. ２ S donor compounds.

13. The compound has the general formula II 【Transformation 7】 (In the formula, R １ , R ２ , R ３ , R ４ , and R ５ is defined above (claim 8), R １ , R ２ , R ３ , R ４ , and R ５ at least two, preferably three or four of are H; Preferably, R １ , R ２ , R ３ , R ４ , and R ５ are independently selected from the group consisting of: １ is selected from the group consisting of: H, halogen, pseudohalogen, -CN, -OH, -SH, -NO ２ , -NH ２ , -NHCH ３ ,-COOH,CONH ２ , preferably OH, and ・-OCOR １６ , -COOR １７ , -OR １８ (R １６ , R １７ , R １８ , and R １９ represents H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamido, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkyl-heteroaryl, C1-C5 amido, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkyl, and the substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO, —C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl, or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester, or C3-C8 alkynyl ester), and the substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO ２ , ONO ２ , -NH ２ , —NHMe), Preferably, R １ Is -OCOR １６ , -COOR １７ , -OR １８ and R １６ , R １７ , and R １８ is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylic acid ester (preferably C2-C8 alkylester, C3-C8 alkenylester or C3-C8 alkynylester), and the aforementioned substituents, when present, are selected from halide, pseudohalide, —OH, —SH, —OMe, —NO ２ , -NH ２ , -NHMe; More preferably, R ２ , R ３ , R ４ , and R ５ is H and R １ is as defined above, or Even more preferably, R １ is -OH, -OCOR １６ and R １６ is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 carboxylic acid ester (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the aforementioned substituents, which, when present, are halide, pseudohalide, —OH, —SH, —OMe, —NO ２ , ONO ２ , -NH ２ , -NHMe; R １４ is a group having the formula III.2 【Transformation 8】 Formula III. 2nd year, R １ , R ２ , R ４ , and R ５ is H, halogen, pseudohalogen, -CN, -OH, -SH, -NO ２ , -NH ２ , -NHCH ３ ,-COOH,CONH ２ , substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, Preferably, H or OCH ３ are independently selected from R １３ is H and OCH ３ is selected from, preferably 【Chemistry 9】 (selected from (preferably as moiety M when dependent on claim 7 or 8) ２ S donor compounds.

14. 14. The method of claim 13, wherein the compound is selected from the following: ２ S donor compounds 【Chemistry 10】

15. The compound is a natural H-protein selected from allicin, alliin, diallyl trisulfide, diallyl disulfide, diallyl tetrasulfide, ajoene (e.g., E-ajoene or Z-ajoene), lenthionine, ovothiol, S-allylmercaptocysteine ​​(SAMC), 3H-1,2-dithiole-3-thione, and alpha lipoic acid. ２ 7. The H for use according to any one of claims 1 to 6, which is an S donor compound. ２ S donor compounds.

16. The compound is a substituted or unsubstituted 5-membered heterocycle containing a dithiol group (—S—S—), preferably a 1,2 dithiolane or 1,2 dithiolene group, preferably a group having formula 5: 【Chemistry 11】 (In the formula, R １３ is H or C1-8 alkyl H for use according to any one of claims 1 to 6 or claim 15, comprising ２ S donor compounds.

17. 17. The method of claim 1, wherein the composition is administered daily, preferably once, twice, or three times daily, for a period of at least one month, two months, three months, six months, one year, or more. ２ S donor compounds.

18. H for use according to any one of claims 1 to 16, preferably according to any one of claims 6 to 16 ２ A pharmaceutical composition comprising an S-donor compound, the pharmaceutical composition being formulated for systemic administration.

19. formulated for oral administration, preferably in capsule and / or tablet form; Preferably, during storage, ２ Protected from light, moisture, and decay forming S; 19. The method of claim 18, wherein the composition is preferably formulated in a light-protected package. ２ A pharmaceutical composition comprising an S-donor compound.

20. provided that said use is different from oral administration, - For intravenous or intraperitoneal administration It is formulated as:

19. The method of claim 18, wherein the method is formulated as an injection or infusion. ２ A pharmaceutical composition comprising an S-donor compound.

21. With the proviso that said use is preferably different from the oral administration according to any one of claims 7 to 16, For topical administration, Preferably, it is formulated in the form of an ointment, gel, or emulsion, The topical dosage form is ２ from moisture and / or decay forming S, and from light if necessary, H ２ containing an excipient that protects the S donor compound; 17. The method of claim 1, wherein the composition is formulated in a light-protected package. ２ A pharmaceutical composition comprising an S-donor compound.

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