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

Abstract

The present invention relates to hydrogen sulfide (H2S) releaser compounds and compositions for use in the treatment of myeloperoxidase (MPO) induced neutrophil mediated tissue damage, in particular positive anti-neutrophil cytoplasmic autoantibody (ANCA) associated vasculitis (AAV), comprising myeloperoxidase granulomatous polyvasculitis (Wegner's granulomatous disease), eosinophilic granulomatous polyvasculitis (EGPA; ), polyvasculitis under microscope (MPA), and various forms of diseases in various organs, such as kidney and lung.

Description

Technical Field

[0001] The present invention relates to hydrogen sulfide (H2S)-releasing agent compounds and compositions for the treatment of antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), including myeloperoxidase granulomatosis with polyangiitis (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA; formerly known as Churg-Strauss syndrome), microscopic polyangiitis (MPA), and localized renal vasculitis (localized renal microscopic polyangiitis or localized renal necrotizing crescentic glomerulonephritis, NCGN). Background Art

[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 self-antigens, the most important of which are circulating anti-proteinase 3 (PR3) or anti-myeloperoxidase (MPO) autoantibodies. AAV subtypes include granulomatosis with polyangiitis (formerly Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA; formerly Chargar-Strauss syndrome), microscopic polyangiitis, and localized renal vasculitis, all of which are associated with PR3 or myeloperoxidase MPO [Jennette, JC, and PH Nachman, ANCA Glomerulonephritis and Vasculitis. Clin J Am Soc Nephrol, 2017. 12(10): p. 1680-1691; Kallenberg, CG, Pathophysiology of ANCA-associated small vessel vasculitis. Curr Rheumatol Rep, 2010. 12(6): p. 399-405.].

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

[0004] In a rare variant, microscopic polyangiitis (MPA), symptoms of necrotizing vasculitis may be detected in the absence of immune deposits.

[0005] Chargar-Strauss syndrome (also known as eosinophilic granulomatosis with polyangiitis, EGPA) is an eosinophil-associated vasculitis characterized by the formation of granulomas and often accompanied by asthma symptoms.

[0006] In AAV, the kidneys are often found to be the most affected of the vital organs, and the severity of renal symptoms indicates the outcome. Therefore, localized renal vasculitis (or microscopic polyangiitis limited to the kidney or localized renal necrotizing crescentic glomerulonephritis, NCGN) is a disease variant of particular public health importance. [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] The mortality rate in patients with renal AAV is 2.7 times that of the general population. The main causes of death in the first year after onset are infection (48%) and active vasculitis (19%), while in the later years, cardiovascular disease (26%), malignant transformation (22%), and infection (20%) are the main causes. [88,89] [Tan, JA, et al., Mortality in ANCA-associated vasculitis: a meta-analysis of observational studies. Ann Rheum Dis, 2017. 76(9): p. 1566-1574. Flossmann, O., et al., Long-term patient survival in ANCA-associated vasculitis. Ann Rheum Dis, 2011. 70(3): p. 488-94.]

[0008] The autoimmune nature of the disease means that the vast 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, for example, there are PR3-positive vasculitis, MPO-positive vasculitis, and ANCA-negative vasculitis [Kitching, AR, et al., ANCA-associated vasculitis. Nat Rev Dis Primers, 2020. 6(1): p.71.].

[0009] MPO (i.e., myeloperoxidase) is one of the most abundant autoantigens in AAV. According to a study by Galesic, K. et al., the prevalence of MPO in Wegener's granulomatosis is 20%, in microscopic polyangiitis is 50%, in Chargar-Strauss syndrome is 60%, and in limited renal microscopic polyangiitis 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, below].

[0010] The role of hydrogen sulfide in autoimmune inflammatory diseases is highly controversial. Among the wide range of biological functions of hydrogen sulfide, this small endogenous mediator has multiple protective functions in inflammatory processes [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): p.13-32.]. Interactions with heme proteins may represent a key mechanism in these processes (Palinkas, Z., et al., Interactions of hydrogensulfide with myeloperoxidase. Br J Pharmacol, 2015. 172(6): p.1516-32.].

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

[0012] The interaction between H2S and the MPO enzyme has been studied, and it has been found that H2S has a reversible inhibitory effect on the peroxidase and chlorination activities of MPO. However, kinetic experiments have shown that the interaction between H2S and MPO is very complex. Nevertheless, H2S can also be used as a substrate for MPO. [Palinkas, Z., et al., Interactions of hydrogen sulfide with myeloperoxidase. Br J Pharmacol, 2015. 172(6): p. 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: p. 551-563.]

[0013] Therefore, this Janus-faced feature of H2S places a huge burden on researchers, making it difficult for them to draw correct conclusions, i.e., whether H2S is beneficial for specific diseases, and requiring complex experiments, but the effects or results of the experiments are unpredictable.

[0014] In fact, although the present inventors also found that sulfide and sulfide donors can effectively inhibit the formation of MPO oxidants in the supernatant of PMA-activated neutrophils [Garai, D., et al. Measurements for Sulfide-Mediated Inhibition of Myeloperoxidase Activity. In: Beltowski, 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], MPO is an autoantigen in ANCA vasculitis and its activity is irrelevant from the perspective of AAV etiology. Indeed, at present, the persistence of MPO positivity, rising MPO levels, or even a change from an MPO-negative to an MPO-positive serotype is at best modestly predictive of future disease relapse and should not be used to guide treatment decisions according to the KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases Kidney International, (2021) 100(45) Supplement, pages S1 to S276.

[0015] Existing technical results indicate that although H2S may affect the pathophysiological processes of neutrophils due to its significant physiological effects in inflammation, the discovery of a complex effect network shows that the overall effect is uncertain and unpredictable.

[0016] There are currently no data on the role of MPO activity in predicting treatment of autoimmune diseases such as AAV.

[0017] Furthermore, there is clear evidence that neutrophil function is not affected by hydrogen sulfide donors in the presence of PMA, LPS, or E. coli activation.

[0018] For example, treatment with the hydrogen sulfide donor GYY4137 did not inhibit the oxidative burst induced by PMA or LPS. In fact, pretreatment of neutrophils with 200 μM GYY4137 before PMA activation increased ROS production compared to untreated samples. Treatment with GYY4137 also inhibited LPS-induced apoptosis of neutrophils and improved cell viability. It was demonstrated that sulfide moderately reduced the formation of ROS in LPS-induced neutrophils, but these effects were measured after 17-18 hours of incubation. [Petrushanko, IY, 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] In addition, another research group showed that various sulfide-releasing compounds (such as sodium sulfide, dipropylene trisulfide, dipropylene disulfide, and cysteine) can also inhibit neutrophil apoptosis and promote oxidative burst after 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. even demonstrated that in the presence of NaH2S (1.83 mM), neutrophils were more efficient at killing bacteria than in the absence of sulfide, and that sulfide donors 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] Notably, none of these publications mention AAV as an autoimmune disease. There is no suggestion in the prior art that sulfide can inhibit priming, ANCA-induced degranulation, and the oxidative burst. Instead, the prior art seems to suggest that hydrogen sulfide can even increase neutrophil activation by preventing neutrophil apoptosis—an undesirable process in autoimmune diseases.

[0022] Indeed, current therapeutic options for AAV diseases are limited.

[0023] Initially, patients typically receive immunosuppressive therapy with cyclophosphamide or rituximab, and depending on the patient's condition, immunosuppressive therapy can be enhanced with other treatment options (e.g., glucocorticoids or methotrexate). Recommendation 9.3.1.1 of the KDIGO 2021 Clinical Practice Guideline for the Management of Glomerular Diseases (hereinafter referred to as KDIGO 2021) recommends that glucocorticoids combined with cyclophosphamide or rituximab be used as initial treatment for newly diagnosed AAV (1B).

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

[0025] Unfortunately, the combination of immunosuppressant and steroid therapy can make patients susceptible to infections. Other complications may also develop, such as hormonal imbalances, malignancies of the bladder, hematopoietic tissue, or skin, and even septic shock due to long-term side effects [Haubitz, M. Acute and Long-term Toxicity of Cyclophosphamide. 2007].

[0026] Over the past 15 years, new proposed therapies have been considered.

[0027] Because tumor necrosis factor α (TNFα) appears to be an important mediator in AAV disease, particularly GPA, monoclonal antibodies targeting TNFα have been proposed for treatment. However, safety concerns may arise. According to a review by Unizony S. and Stone, JH, results indicate that monoclonal anti-TNF-α inhibitors have no effect in newly diagnosed patients in addition to traditional immunosuppression, and no definitive conclusions can be drawn for 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] US20130331360A1 (grant number US8865684B2 (National University of Singapore), now expired) discloses a series of slow H2S releaser compounds, including GYY4137, and their use as vasodilators , and thus their use in cardiovascular diseases has been proposed, however, their use in vasculitis has not been suggested or implied.

[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]. ANCA-associated vasculitis Treatment does not include vasodilator effects and vasodilators Any related treatments[van Daalen et al. Developments in the Histopathological Classification of ANCA-Associated Glomerulonephritis. Clin J Am Soc Nephrol 2020Aug 7;15(8):1103-1111.] and [Rovin et al., Executive summary of the KDIGO 2021 Guideline for the Management of Glomerular Diseases Kidney Int.2021Oct;100(4):753-779.].

[0030] As evidence of ANCA-mediated neutrophil activation continues to accumulate, neutrophil-directed therapies have also been taught. Therapeutic strategies for AAV may include removing harmful autoantibodies (plasma exchange), modulating the neutrophil FcγR signaling pathway, inhibiting 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] Known neutrophil priming includes MPO translocates to the cell surface to serve as an autoantigen for ANCA , which ultimately induces the respiratory burst [Jennette, JC and RJ Falk, Pathogenesis of antineutrophilcytoplasmic autoantibody-mediated disease. Nat Rev Rheumatol, 2014. 10(8): p. 463-73.]. MPO activity does not play a role in this process.

[0032] The present inventors unexpectedly discovered that sulfide donors can also inhibit the translocation of ANCA antigen MPO, thereby inhibiting the neutral Granulocyte activation .

[0033] Furthermore, the present inventors unexpectedly discovered that Sulfide donors can effectively inhibit neutrophil shedding during ANCA activation. granulocytes and inhibit the activation of neutrophils by IgG isolated from ANCA patients.

[0034] at the same time, The phagocytic and bactericidal activities of neutrophils were not inhibited in the presence of sulfide, and sulfide and sulfide-donating compounds had no effect on PMA-induced neutrophil activation—a surprising fact that suggests that sulfide is harmless in this regard.

[0035] The prior art does not mention the use of sulfide-donating molecules in the treatment of ANCA vasculitis, particularly where MPO acts as an autoantigen. In contrast, the fact that sulfide can inhibit priming and ANCA-induced degranulation and oxidative burst is not obvious from the data available in the literature, according to the prior art.

[0036] In a systematic series of experiments, the inventors investigated how sulfide interferes with these processes and Unexpectedly We found that H2S-donating compounds can help alleviate MPO-related pathological processes in AAV, while surprisingly, not affecting maintenance Normal neutrophil activity is necessary to maintain immune homeostasis Specifically, although elevated H2S serum levels are active in MPO-associated disease processes, neutrophil phagocytosis and bactericidal activity were not inhibited in the presence of sulfide, and sulfide and sulfide-donating compounds had no effect on PMA-induced neutrophil activation (a bacterial infection model) or NADPH oxidase (NOX) activity. Oral administration of the compound is particularly preferred. In a mouse model of MPO-ANCA vasculitis, sulfide-donating compounds provided protection from renal damage.

[0037] Thus, the present inventors have surprisingly discovered that novel AAV treatments can be performed on patients with MPO-positive serotypes by increasing H2S serum levels (ie, via H2S donors). Slow H2S releaser compounds are preferred. Summary of the Invention

[0038] 1. The present invention relates to an H2S donor compound for use in treating anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) in a mammalian patient suffering from AAV of the myeloperoxidase-ANCA serotype (MPO-ANCA vasculitis). Specifically, the AAV is myeloperoxidase (MPO)-positive AAV, wherein the subject has elevated ANCA levels against MPO.

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

[0040] Preferably, the MPO level is measured by IIF and / or ELISA.Preferably, the MPO level is above a predetermined threshold level.

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

[0042] Preferably, the compound is used

[0043] - Inhibition of neutrophil granulocyte activation by anti-MPO antibodies in mammalian subjects with AAV (MPO-AAV associated vasculitis)

[0044] - inhibiting neutrophil degranulation in a mammalian subject suffering from AAV,

[0045] - inhibiting neutrophil priming (via translocation of the ANCA antigen MPO) in a mammalian subject with AAV, and / or

[0046] -Preventing kidney damage in a mammalian subject suffering from AAV / protecting the kidneys of a mammalian subject suffering from AAV.

[0047] In specific embodiments, H2S donor compounds are useful in treating or preventing the manifestation of clinical symptoms of early-stage MPO-positive AAV, preferably by inhibiting neutrophil priming in mammalian subjects with AAV.

[0048] In specific embodiments, H2S donor compounds are useful in treating localized renal vasculitis and / or any kidney-associated form of AAV.

[0049] Preferably, the H2S donor compound does not inhibit NOX activity, phagocytosis and bactericidal activity of neutrophils. Preferably, the H2S donor compound does not inhibit pathogen-induced neutrophil activity.

[0050] Preferably, the H2S donor compound is a low molecular weight compound.

[0051] 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 limited vasculitis (AAV), and pulmonary AAV.

[0052] In a further preferred embodiment, AAV is a localized renal vasculitis, preferably a localized renal microscopic polyangiitis or a localized renal necrotizing crescentic glomerulonephritis (NCGN).

[0053] In a further preferred embodiment, the AAV is expressed in the lung.

[0054] In a further particularly preferred embodiment, AAV is expressed in the kidney.

[0055] In a further preferred embodiment, AAV is expressed in at least the lungs and kidneys.

[0056] In a highly preferred embodiment, the compound is used in microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA) (Chargé-Strauss syndrome), or localized renal vasculitis, preferably localized renal microscopic polyangiitis or localized renal necrotizing crescentic glomerulonephritis (NCGN), with EGPA and / or localized renal vasculitis being highly preferred.

[0057] 4. Preferably, after administration of the compound to a mammalian subject, the compound increases the level of H2S in the serum of the subject, preferably, the subject has an elevated H2S serum level after a time point of 1 hour to 24 hours after administration of the compound, if compared to a normal H2S serum level (or a H2S serum level without (or before) administration of the compound).

[0058] Normal levels include predetermined threshold levels or ranges measured and calculated in control subjects not administered the compound, or control serum levels of H2S measured in control subjects not administered the compound (prior to administration of the compound).

[0059] Preferably, the elevated H2S serum level is significantly higher than the normal serum level if measured by any relevant statistical method.

[0060] Preferably, the HS serum level is intended to refer to the bioavailable sulfide (HS) level measured according to the protocol of Ditroi et al. throughout the document. [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect HS measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: p. 146-158.] Preferably, the elevated HS serum level is at least 1.5 times, more preferably at least 2 times, even more preferably at least 3 times higher than the normal HS serum level measured by the method of Ditroi et al. at a specific time point.

[0061] Preferably, the compounds of the present invention are HS releasers, preferably slow HS releasers, which, after administration of the compound to a mammalian subject, significantly increase the level of bioavailable sulfide (HS) in the serum of the mammalian subject after a time point of 1 hour to 24 hours after administration of the compound, as compared to normal HS serum levels as a control.

[0062] Preferably, the H2S donor compounds, in particular the slow H2S releaser compounds of the present invention are low molecular weight compounds.

[0063] 5. In a particularly preferred embodiment, the compound is administered orally to the mammalian patient.

[0064] Particularly preferably, the H2S donor compound decomposes and the H2S is released in the gastrointestinal tract, preferably in the stomach, or under conditions simulating the gastrointestinal tract or stomach.

[0065] 6. In a general embodiment, the compound has the general formula (Y)

[0066] MLQ, (Y)

[0067] in

[0068] Q is the portion of H2S released which, once present in the body of a mammalian patient, releases H2S into the bloodstream of said mammalian patient after metabolism,

[0069] Preferably

[0070] Q contains a dithiolene group (-SS-), or

[0071] Q contains a dithiophosphonate group (=P(S)S - )

[0072] L is an organic linking part,

[0073] M is a moiety covalently bonded to the remainder of the molecule via a hydrolyzable bond, wherein upon hydrolysis and upon metabolism and release of H2S, M is converted into a compound tolerable to said mammalian patient, preferably a compound beneficial to said mammalian patient,

[0074] L or M may be missing, or both may be missing.

[0075] In embodiments, the compound decomposes and releases H2S under conditions that simulate the gastrointestinal system of a mammalian subject, preferably under gastric model conditions.

[0076] 7. In a preferred embodiment, the compound has the general formula (X)

[0077]

[0078] in

[0079] Q is the portion of H2S released which, once present in the body of a mammalian patient, releases H2S into the bloodstream of said mammalian patient after metabolism,

[0080] In a preferred embodiment

[0081] Q includes a 5- to 6-membered heterocyclic ring including a dithiolene group (-SS-), or

[0082] Q includes a dithiophosphonate group (=P(S)S - )

[0083] L is the connecting part,

[0084] In a preferred embodiment, L is C1-C8 alkylene (preferably methylene), -, -O-, S, -NH-, aryl, C1-C4 alkylaryl, 5 to 6 membered heterocycle, L is optionally linked to Q via C1-C4 alkylene,

[0085] wherein at least two, preferably three or four of R1, R2, R3, R4 and R5 are H,

[0086] wherein R1, R2, R3, R4 and R5 are independently selected from

[0087] -H, halogen, pseudohalogen, -CN, -OH, -SH, -NO2, -NH2, -NHCH3, -COOH, CONH2,

[0088] -substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide, 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents, if any, being selected from halides, pseudohalides, -OH, -SH, -OMe,

[0089] -NO2, -NH2, -NHMe,

[0090] --OCOR 16 、-COOR 17 、-OR18 、-CONHR 19 , where R 16 、R 17 、R 18 and R 19 selected from H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide, 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), wherein the substituent, the substitution, if any, is selected from halide, pseudohalide, -OH, -SH, -Ome,

[0091] -NO2, -NH2, -NHMe,

[0092] Preferably, R1 and R3 are independently selected from OCOR 16 、-COOR 17 、-OR 18 , where 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), and the substituent, if any, is selected from halide, pseudohalide, -OH, -SH, -Ome,

[0093] -NO2, -NH2, -NHMe,

[0094] More preferably

[0095] Wherein R2, R3 and R5 are H,

[0096] R 15 Selected from C1-C8 alkylene, C1-C8 alkyl ether, C1-C8 carboxylate, preferably -(CH2) n -(CO)O-, wherein n is 0, 1, 2 or 3, preferably 0 or 1,

[0097] or a pharmaceutically acceptable salt and / or solvate and / or complex thereof.

[0098] 8. In a preferred embodiment, the compound has the general formula I

[0099]

[0100] in

[0101] R7 is selected from

[0102] -3- to 10-membered heterocyclic ring, preferably 5- to 10-membered heterocyclic ring, more preferably 5- to 6-membered heterocyclic ring

[0103] -NH-R 23 , where R 23 is selected from C1-4 alkyl, C1-4 alkylcarbonyl,

[0104] R8 (preferably wherein R8 is L) is selected from

[0105] --NH-

[0106] --CH2-, -O-, S or NH, preferably CH2, O or NH,

[0107] - or R 15 means nothing (GYY, Lawesson reagent, JK donor),

[0108] A is selected from

[0109] --S - , where if A is -S - , then R6 does not exist,

[0110] --S- and -S - , where if A is -S-, then

[0111] -R6 can be selected from H, 5 to 10 membered optionally substituted heterocycle, preferably 6 membered optionally substituted heterocycle, C1-4 alkyl or C6-C10 aryl, or

[0112] -A is -S-, and R6 and R7 together form a moiety having formula (I.1)

[0113]

[0114] wherein R1 to R5 and R8 are independently as defined above, wherein the substituents of the two rings may be the same or different,

[0115] --O -

[0116] --O-, where if A is -O-, then

[0117] R6 can be selected from H, 5 to 10 membered heterocyclic ring, preferably 6 membered heterocyclic ring, C1-4 alkyl group or C6-C10 aryl group, or

[0118] a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings and optionally at least one 1- to 8-membered open-chain moiety, optionally containing 1 to 4, preferably 1 to 3, heteroatoms, and / or

[0119] R6 is an organic moiety which, upon hydrolysis, is converted into a compound tolerable to the mammalian patient, preferably a compound beneficial to said mammalian patient,

[0120] 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, wherein at least two of R1, R2, R3, R4 and R5 are H;

[0121] Preferably, R3 is -OCH3 or R1, R2, R3, R4 and R5 are each H,

[0122] or a pharmaceutically acceptable salt and / or solvate and / or complex thereof,

[0123] In specific embodiments, the dichloromethane complex and / or the morpholinium salt.

[0124] 9. In a specific embodiment, the compound has the general formula I.2

[0125]

[0126] in

[0127] R9 is H, wherein said H is dissociable, such that -S-R9 becomes -S - ,

[0128] A is selected from the group consisting of absent and -O-, and

[0129] Where A is -O-, then R6 is

[0130] a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings and optionally at least one 1- to 8-membered open-chain moiety, optionally containing 1 to 4, preferably 1 to 3, heteroatoms, or

[0131] If A does not exist, then R6 is

[0132] 5- to 10-membered heterocycle, preferably 6-membered heterocycle, preferably a heterocycle containing O and / or N, in particular a morpholinyl group attached via N,

[0133] R1, R2, R3, R4 and R5 are selected from H, C1-4 alkyl, C1-4 alkoxy, halide, wherein at least two of R1, R2, R3, R4 and R5 are H; preferably R3 is -Ome, or each of R1, R2, R3, R4 and R5 is H,

[0134] or a pharmaceutically acceptable salt and / or solvate and / or complex thereof;

[0135] 10. In further specific embodiments, the compound has the general formula 1.3

[0136]

[0137] where R 10 is a 5- to 10-membered heterocycle, preferably a 6-membered heterocycle, preferably a heterocycle containing O and / or N, in particular a morpholinyl group attached via N,

[0138] R1, R2, R3, R4, R5 and R9 are as defined above.

[0139] Particularly preferably, the compound has the general formula 1.3.1

[0140]

[0141] where R 11 is a C1-4 alkyl group,

[0142] Preferably, the compound for use according to claim 9 is GYY4137.

[0143] 11. Further, the compound has the general formula 1.4

[0144]

[0145] wherein R1, R2, R3, R3 and R5 are as defined above, preferably each of them is H,

[0146] R6 and R9 are as defined above.

[0147] 12. In an alternative embodiment, the compound has the general formula II (preferably, as moiety M if dependent on claim 6 or 7),

[0148]

[0149] wherein R1, R2, R3, R4 and R5 are as defined above (in claim 7)

[0150] wherein at least two, preferably three or four of R1, R2, R3, R4 and R5 are H,

[0151] Wherein, preferably R1, R2, R3, R3 and R5 are independently selected from the group consisting of the following (preferably R1 is selected from the group consisting of the following

[0152] -H, halogen, pseudohalogen, -CN, -OH, -SH, -NO2, -NH2, -NHCH3, -COOH, CONH2, preferably OH, and

[0153] -OCOR 16 、-COOR 17 、-OR 18 , where R 16 、R 17 、R 18 and R 19 is selected from H and the following substituted or unsubstituted groups:

[0154] C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide, C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 alkynylcarbonyl), C1-C8 carboxyl R1 is preferably selected from the group consisting of: -OCOR 16 、-COOR 17 、-OR 18 , where 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), and the substituent, if any, is selected from halide, pseudohalide, -OH, -SH, -Ome, -NO2, ONO2,

[0155] -NH2, -NHMe,

[0156] More preferably, R2, R3, R4 and R5 are H, and R1 is as defined above, or

[0157] Even more preferably, R1 is selected from -OH, -OCOR 16 , where R 16 is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents, if any, being selected from halides, pseudohalides, -OH, -SH, -Ome, -NO2, ONO2, -NH2, -NHMe,

[0158] R 14 is a group having formula III.2

[0159]

[0160] Wherein, in formula III.2:

[0161] R1, R2, R4 and R5 are independently selected from 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,

[0162] Preferably H or OCH3,

[0163] R 13 Selected from H and OCH3, preferably

[0164]

[0165] Wherein in a preferred embodiment, the compound is ADT-OH

[0166]

[0167] 13. Preferably, the compound is selected from

[0168]

[0169] 14. In a further alternative embodiment, the H2S donor compound is a natural H2S donor compound selected from the following: allicin, alliin, diallil-trisulfide, diallildisulfide, diallil tetrasulfide, ajoene (e.g., E-ajoene or Z-ajoene), anthionine, ovothiol, S-Allylmercaptocysteine ​​(SAMC), 3H-1,2-dithiole-3-thione, α-lipoic acid.

[0170] 15. In a further alternative embodiment, wherein the H2S donor compound comprises a substituted or unsubstituted 5-membered heterocyclic ring comprising a dithiole group (-SS-, preferably a 1,2-dithiolane group or a 1,2-dithiolene group), preferably a group having Formula 5:

[0171]

[0172] where R 13 is H or C1-8 alkyl, preferably wherein

[0173] 16. In a preferred embodiment, the H2S donor compound for use according to any one of paragraphs 1 to 15, wherein the composition is administered daily, preferably once, twice or three times a day, for at least one month, two months, three months, six months, one year or longer.

[0174] Pharmaceutical composition (drug)

[0175] 17. The present invention also relates to a pharmaceutical composition comprising the H2S donor compound for use according to any one of paragraphs 1 to 15, preferably according to any one of paragraphs 5 to 15,

[0176] The compositions are formulated for systemic administration.

[0177] 18. The pharmaceutical composition according to claim 17, preferably comprising a H2S donor compound for said use,

[0178] The composition is formulated for oral administration, preferably

[0179] in the form of capsules and / or tablets; wherein

[0180] protecting the composition from light, moisture, and decay that could result in the formation of H2S during storage;

[0181] 19. The pharmaceutical composition according to claim 17, preferably comprising a H2S donor compound for said use, said composition being formulated for intravenous administration.

[0182] 20. The present invention also relates to a pharmaceutical composition comprising a H2S donor compound for said use according to claim 19,

[0183] The composition is formulated in the form of a powder which can be

[0184] intravenous injection or infusion,

[0185] Intraperitoneal injection or infusion or

[0186] During infusion,

[0187] Reconstitute with an appropriate solution (eg, Ringer's solution, Ringer's lactate solution, buffered solution, etc.).

[0188] 21. A pharmaceutical composition according to claim 17, preferably comprising a H2S donor compound for said use, said composition being formulated as drops, e.g.

[0189] - Eye drops,

[0190] -Nose drops.

[0191] 22. The present invention also relates to a pharmaceutical composition comprising the H2S donor compound for use according to any one of paragraphs 1 to 15, preferably according to any one of paragraphs 5 to 15,

[0192] The composition is formulated for topical administration.

[0193] Preferably in the form of an ointment, gel or lotion,

[0194] The topical administration form comprises an excipient to protect the H2S donor compound from moisture and / or decay, preferably from light, to form H2S during storage.

[0195] 23. Formulating the composition of paragraphs 17 to 22, including packaging and storage as required to achieve an appropriate shelf life.

[0196] The pharmaceutical composition or formulation of any of paragraphs 17 to 22, comprising light-proof packaging.

[0197] 24. In a preferred embodiment, prior to treatment, the AAV patient is diagnosed as MPO-ANCA positive.

[0198] An AAV patient is one who is considered to be exhibiting symptoms of AAV 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 any other diagnostic or rating tool, such as the Birmingham Vasculitis Activity Score (BVAS) and the 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 who is known in the art or known at a given time to be exhibiting symptoms of AAV). In this regard, it is sufficient that the diagnostic level indicates that the patient may have or may develop AAV, provided that such level suggests (or warrants)

[0199] Start treatment,

[0200] Further diagnostic steps (e.g., biopsy) and / or

[0201] Detection of anti-antibodies (ANCA).

[0202] In this regard, the methods described or referenced in the section "Diagnosis of MPO-ANCA associated vasculitis patients" are specifically contemplated or, if appropriate, incorporated herein by reference.

[0203] The "recommendation" level includes situations where symptoms implicate AAV in at least two organs or where current protocols recommend initiation of treatment or further investigation.

[0204] In certain embodiments, the mammalian patient has elevated ANCA titers and multiple affected organ systems (as defined in Houben E et al. 2016, infra).

[0205] 25. The present invention also relates to a method of treatment, wherein the composition or formulation is administered to a mammalian patient with an MPO-ANCA serotype as defined herein to prevent or treat MPO-associated AAV in the patient.

[0206] In a specific embodiment, the invention relates to a therapeutic method for treating a condition as defined in any one of paragraphs 1 to 24.

[0207] In specific embodiments, oral administration is employed.

[0208] In specific embodiments, diagnostic tests are performed as disclosed herein to identify groups of patients to be treated.

[0209] definition

[0210] As used herein, a "subject" is an individual of an animal species, preferably a vertebrate, more preferably a mammalian or avian species, particularly a mammalian species, and highly preferably the individual is a primate, ape, or human. A "patient" is a subject who is or is intended to be receiving medical or veterinary care, observation, supervision, diagnosis, or treatment.

[0211] "Treatment" of a subject refers to any process, action, therapy, etc. in which assistance is provided to the subject or patient, in particular medical assistance or veterinary assistance for the purpose of directly or indirectly improving the condition of the subject or patient. Improving the condition of the subject may include restoring or maintaining the normal function of an organ or tissue, preferably at least partially restoring or maintaining health (medical treatment or veterinary treatment). Treatment typically refers to the administration of an effective amount of a compound or composition as described herein. Preferably, the administration of the compound is oral and results in an appropriate serum level of HS in the patient. Particularly preferred treatments include medical or veterinary treatments in the early stages of the disease, for example, at the stage of neutrophil priming in MPO-ANCA-positive patients, particularly AAV patients.

[0212] In a broad sense, treatment includes medical or veterinary treatment at an early stage of the disease, including prevention (or prophylaxis), i.e., preventing the onset of disease symptoms or preventing the manifestation of the disease in an MPO-ANCA positive patient. In a more limited sense, prevention is not covered.

[0213] "Disease activity" in ANCA-associated vasculitis refers to signs or symptoms due to active disease in any organ system.

[0214] "Remission" is defined as the disappearance of vasculitis manifestations. Remission can be understood as the alleviation of clinical symptoms of the disease, but MPO positivity persists.

[0215] Relapse is defined as an increase in disease activity following a period of partial or complete remission.

[0216] A "pharmaceutical composition" of the present invention is a composition of matter comprising at least one HS donor compound of the present invention for use in MPO-ANCA-positive patients, the composition comprising an active agent and at least one other substance. Preferably, the compound of the present invention is present in an effective amount, preferably an amount that elicits effective serum levels of HS in a mammalian subject. The composition may also contain other biologically active substances, for example, for use in combination therapy. Furthermore, the composition may contain biologically acceptable carriers, formulations, excipients, and the like as known in the art.

[0217] The term "effective amount" defines the amount of compound in a composition required to exert the effect of the active agent. A "therapeutically effective amount" is sufficient to alleviate or prevent (or prevent the appearance or manifestation of AAV symptoms, or worsening of a disease state) one or more symptoms or characteristic parameters of a condition (e.g., a disorder or disease) in an AAV patient.

[0218] "H2S donor compound" is a compound that degrades in response to environmental stimuli (e.g., water, light, nucleophiles such as thiols, enzymes, or other stimuli) to release H2S. Preferably, once an effective amount of the compound of the present invention is administered, an effective serum level of H2S in a mammalian subject will be caused. Preferably, the "H2S donor compound" has a "sulfide releaser moiety" used herein as the sulfur-containing moiety of the compound, which is a molecular moiety responsible for the release of H2S in the patient's body, preferably causing an increase in H2S levels in the patient's serum. Specifically, the H2S donor compound is a compound as defined by Powell, Chadwick R. et al., [Powell, Chadwick R., Kearsley M.Dillon, and John B.Matson." A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications." Biochemical pharmacology 149 (2018): 110-123]. In particular aspects, H2S is released under biological conditions, eg, in a subject or under conditions that provide a model of a biological condition, including an in vitro model.

[0219] A "slow H2S releaser" compound is an H2S donor compound from which H2S is released more slowly, preferably for a time that is 10 times longer, more preferably 100 times longer, than from a reference sulfide compound (preferably from Na2S) under appropriate reference conditions, e.g. under the same or appropriately comparable conditions. Examples of measuring the time period over which hydrogen sulfide is released 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 aerobicglycolysis." Journal of Biological Chemistry 296 (2021)] or [Lee ZW et al. The slow-releasing hydrogen sulfide donor, GYY4137, exhibits novel anti-cancer effects invitro and in vivo. PLoS One. 2011; 6 (6): e21077. doi: 10.1371 / journal.pone.0021077]. Other measurement methods are known in the art.

[0220] The terms "sulfide" and "H2S", particularly bioavailable sulfide and H2S, are used interchangeably and include all protonated isomers (particularly H2S, HS - 、S2 - ), unless otherwise stated.

[0221] As defined herein, a "low molecular weight compound" is a compound having a molar mass or molecular weight (used interchangeably herein) of less than 2000 Da or 1500 Da, in particular less than 1000 Da, preferably 900 Da. Preferably, the low molecular weight compound is an inorganic compound, an organometallic compound or an organic compound. In an embodiment, the compound is different from a polymeric molecule of biological origin (in particular a biomolecule isolated from nature), such as a peptide or a nucleic acid; in a specific embodiment, it is a synthetic or semi-synthetic compound. The low molecular weight compound may be a small molecule or a salt, such as a lithium salt, for example an organic lithium salt or LiCl. In a preferred embodiment, a low molecular weight compound is understood to be a compound that can penetrate into the cell through the cell membrane without the use of cell surface receptors, in particular wherein its molecular weight is less than 1000 Da, preferably 900 Da.

[0222] As used herein, "moiety" refers to a portion of a molecule that can in principle be obtained by removing another moiety (even a hydrogen atom or radical or any part thereof).

[0223] As used herein, a "sulfide-releasing moiety" is a sulfur-containing moiety of a compound of the invention that is the portion of the molecule responsible for releasing H2S in a patient, preferably causing an increase in H2S levels in the patient's serum.

[0224] The term "alkyl" as used herein, alone or in combination, refers to a saturated straight-chain or branched (if appropriate) hydrocarbon group preferably containing 1 to 15, 1 to 10 or 1 to 8 carbon atoms, or in particular 1 to 6, or 1 to 4, 1 to 3, or 1 to 2 carbon atoms [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"], such as methyl, ethyl, propyl or isopropyl being particularly preferred.

[0225] As used herein, the term "alkoxy" refers to an alkyl-O- group wherein the alkyl group is as previously described and the bond to the remainder of the molecule or complex (i.e., the parent moiety) is through the oxygen (or ether oxygen if to a carbon atom).

[0226] The term "alkoxyalkyl" refers to an alkyl group substituted with an alkoxy group, ie, an alkyl-O- group as previously described. The bond to the alkyl portion is through the oxygen (ie, it is an ether oxygen).

[0227] 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, alkenyl, and alkynyl group, respectively. Broadly speaking, the group can be attached through an alkyl, alkenyl, alkynyl, or via a carbonyl group. In preferred embodiments (i.e., in a more narrow 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.

[0228] This broad and narrow definition applies to any similar group having a functional group as used herein, even if not separately defined.

[0229] As used herein, the terms "carboxyl," "alkyl-carboxyl," "alkenyl-carboxyl," and "alkynyl-carboxyl" refer to moieties having a carboxyl group optionally substituted with alkyl, alkenyl, and alkynyl groups, respectively, wherein the bond to the parent moiety is through the carboxyl group. The group may be attached through the alkyl, alkenyl, or alkynyl group or via the carboxyl group (in the latter case, an ester).

[0230] "Alkenyl" as used herein, alone or in combination, refers to an unsaturated straight or branched chain hydrocarbon group containing at least one carbon-carbon double bond, preferably containing 2 to 20, preferably 2 to 15, 2 to 10 or 2 to 8 carbon atoms, or 2 to 6, 2 to 4, 2 to 3 or 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].

[0231] As used herein, the definition of "alkynyl" is analogous to that of alkenyl, mutatis mutandis.

[0232] As used herein, "heterocycle" refers to a cyclic moiety having at least one non-carbon atom as a ring member in addition to carbon atoms. A heterocycle may comprise multiple rings; for example, it may comprise an aromatic heterocycle and another ring fused to the aromatic heterocycle, which may or may not be aromatic; that is, if it is not aromatic, it may form a cyclic substituent of the aromatic heterocycle. In a preferred embodiment, if the heteroaryl group comprises multiple, particularly two, fused rings, both rings are aromatic. Preferably, the rings of the heterocyclic moiety are 5- to 6-membered.

[0233] The term "heterocycloalkyl" refers to a "heterocyclic" ring having a saturated linear or branched (if applicable) hydrocarbon group and which may have alkyl substituents on the heterocyclic ring, optionally heteroaryl and / or may be derived from a cycloalkyl as defined above, wherein at least one carbon atom of the ring is replaced by a heteroatom (such as, but not limited to, nitrogen or oxygen).

[0234] As used herein, an "aromatic" moiety can be described as a planar cyclic moiety (ring) in which single bonds (called σ bonds) between the ring atoms are formed by overlap of aligned hybrid atomic sp2-orbitals between carbon nuclei, and in which a delocalized π bond system is formed by overlap of atomic p orbitals of each of the ring atoms above and below the plane of the ring, wherein according to molecular orbital theory, the number of π electrons donated by the ring atoms must be equal to 4n+2 (Hückel's rule), where n=1, 2, 3, etc., preferably 1 or 2, and for a single ring with six π electrons, n=1. The ring atoms typically donate one or two π electrons to the delocalized π electron system.

[0235] The term "heteroaryl" is defined herein as a group or molecule containing an aromatic heterocyclic ring, preferably containing at least one heteroatom incorporated into an aromatic ring as a "member". Examples of heteroatoms include nitrogen, oxygen, and sulfur, preferably nitrogen and oxygen. In embodiments, a heteroaryl group may comprise an aromatic heterocyclic ring and another ring fused to the aromatic heterocyclic ring, which ring may or may not be aromatic; that is, if it is not aromatic, it may form a cyclic substituent of the aromatic heterocyclic ring. In preferred embodiments, if the heteroaryl group comprises multiple, particularly two, fused rings, both rings are aromatic. The members of a heteroaryl group, with respect to the ring atoms, may be carbon atoms or heteroatoms.

[0236] The term "aryl" as used herein is a group containing any carbon-based aromatic ring, preferably a monocyclic or bicyclic group, wherein the bicyclic group preferably comprises two fused rings. In a preferred embodiment, the aryl group is composed only of carbon as a ring atom (i.e., "member"). In a broader sense, the term aryl group also optionally includes "heteroaryl". Optionally, the term "aryl" is limited to the non-heteroaryl group also included in the term aryl, and defines a group comprising an aromatic group that does not contain heteroatoms. The aryl group can be substituted or unsubstituted (i.e., optionally substituted). If the aryl group is substituted, it can be substituted by any substituent, and the example of the substituent includes C 1-4 Alkyl, C 2-4 Alkenyl, C 1-3 Alkoxy, C 1-3 Alkanoyl, C 1-3 Alkylamine, C 1-3 Alkylamide, halogen, etc.

[0237] As used herein, the term "aralkyl" refers to an arylalkyl group linked to a parent molecular weight through an alkyl group, which may be further optionally substituted with one or more, preferably one to three or one to two, alkyl substituents. Thus, an aryl group may be substituted with an alkyl substituent, preferably with each substituent being no more than C 1-4 alkyl.

[0238] "Aryl" or "heteroaryl" may include a single ring, a fused ring, or multiple rings in which the individual rings are linked by single bonds, preferably a single ring or a bicyclic ring.

[0239] As used herein, the term "fused ring" refers to a group of a compound comprising two or more rings fused to at least one other ring, wherein a single bond between two member atoms of the ring is common to the two members, i.e., shared by the two rings. An example of a fused ring is a polycyclic aryl. Polycyclic aryl is understood herein to be a group comprising multiple rings of a carbonyl group, wherein at least one ring is an aryl group, and which optionally may also comprise a cycloalkyl group and / or a heterocycloalkyl group.

[0240] "Substituted" moieties include substituents selected from the groups and moieties defined herein; however, the substituents are preferably smaller, i.e., shorter, i.e., composed of no more, preferably less, atoms than the moiety they replace. In the present invention, "optionally substituted," i.e., "unsubstituted or substituted," means that it can be substituted with any substituent.

[0241] H atoms are typically not shown in the general formulae of the specification, however one skilled in the art would be able to understand the formulae and recognize the complete structure.

[0242] Throughout the specification, terms expressing optionality, such as optional embodiments or variations, where "particularly" refers to embodiments or variations of particular interest and "preferred" refers to embodiments or variations that are preferred for some reason, are used.

[0243] The singular forms "a," "an," and "the," or "at least one," include plural referents unless the context clearly dictates otherwise.

[0244] The terms "comprises" or "comprising" or "including" should be interpreted herein as having a non-exhaustive meaning and allowing for the addition or reference of further elements, e.g., features or method steps or members or components, to whatever includes the listed elements. If the practice of a given language variant so requires, "comprises" may be replaced with "comprising," or may be limited to "consisting essentially of" if elements other than the listed elements are not essential to the practice of the invention, or may be limited to "consisting of" in the absence of any other elements.

[0245] abbreviation

[0246] AAV: ANCA-associated vasculitis

[0247] ANCA: antineutrophil cytoplasmic antibodies

[0248] EGPA: Eosinophilic granulomatosis with polyangiitis (or Chargar-Strauss syndrome).

[0249] GPA: Granulomatosis with polyangiitis (or Wegener's granulomatosis),

[0250] IgG immunoglobulin

[0251] MPA Microscopic Polyangiitis

[0252] MPO: myeloperoxidase

[0253] NCGN: Necrotizing crescentic glomerulonephritis,

[0254] NOX: NADPH oxidase

[0255] PAS (Periodic Acid-Schiff Stain): Periodic acid-Schiff stain

[0256] PBS (Phosphate buffered saline): Phosphate buffered saline

[0257] PMA (Phorbol 12-myristate 13-acetate)

[0258] PR3: Proteinase-3

[0259] SOD (Superoxide Dismutase): Superoxide Dismutase BRIEF DESCRIPTION OF THE DRAWINGS

[0260] Figure 1 Effects of sulfide on neutrophil activation

[0261] (A) Effect of sodium sulfide on H2O2 production by PMA-activated neutrophils.

[0262] Prior to activation with PMA, samples were treated with various concentrations of NaH2S (0-100 μM) for 5 minutes. The values ​​obtained for samples not treated with sulfide were considered 100%. H2O2 is produced by the cellular NOX2 enzyme complex. H2O2 was measured in cell supernatants using the FOX-1 assay. Addition of up to 50 μM Na2S did not affect H2O2 generation.

[0263] (B) Effect of sodium sulfide on the oxidative burst of PMA-activated neutrophils. Cells were treated with various concentrations of sodium sulfide (0-50 μM) and incubated for 20 minutes in the presence of WST-1, 5 μg / ml catalase, and 100 ng / ml PMA. WST-1 reduced superoxide generated by NOX2 in the cell supernatant and was measured spectrophotometrically at 560 nm.

[0264] (C) Effect of GYY4137 sodium sulfide donor on the oxidative burst in PMA-activated neutrophils. Samples were prepared with WST-1 reagent, treated with various concentrations of GYY4137 (0-100 μM), and induced with PMA for 20 minutes. Reduced WST-1 was measured in cell supernatants at 560 nm.

[0265] (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 before treatment with 2 ng / ml TNF-α (black bars) or 15 minutes after the start of TNF-α treatment (patterned bars). 15 minutes after the addition of TNF-α, 300 μg / ml healthy IgG or ANCA IgG was added and incubated for 60 minutes. Reduced WST-1 was measured photometrically in cell supernatants at 560 nm.

[0266] Figure 2 Effect of GYY4137 on neutrophil degranulation. Neutrophils were incubated in HBSS buffer in the presence of cytochalasin B for 10 minutes and then treated with various concentrations of GYY4137 (0-100 μM) for 5 minutes. Cells were then activated with PMA or ANCA, and β-glucuronidase activity or protein levels were assessed in the cell supernatants. β-glucuronidase activity was determined spectrophotometrically at 520 nm by measuring μg of phenolphthalein released over 19 hours. (A) Effect of GYY4137 on β-glucuronidase activity. Cells were treated with 3% Triton X under β-glucuronidase assay conditions in the absence of GYY4137. Cell supernatants were incubated in the presence of various concentrations of GYY4137 (10-100 μM) and phenolphthalein-glucuronide for 19 hours. (B) Effect of GYY4137 on neutrophil degranulation in PMA-activated cells. Cells were prepared using β-glucuronidase assay conditions and treated with GYY4137 as described above. 100 ng / ml PMA was added to the sample and incubated for 30 minutes. Phenolphthalein released by β-glucuronidase in the supernatant was measured spectrophotometrically at 520 nm. (C) Cells were prepared using β-glucuronidase assay conditions and treated with GYY4137 and activated by PMA. 40 μl of each supernatant was used for SDS-PAGE and silver staining. (D) Cells were prepared using β-glucuronidase assay conditions and treated with GYY4137 sulfide donor (0-100 μM) as described above. Cells were treated with TNF-α and then 300 μg / ml ANCA IgG was added to the sample and incubated for 30 minutes. Phenolphthalein released by β-glucuronidase in the supernatant was measured spectrophotometrically at 520 nm. In the figures, the 0% inhibition mark value is from the sample not treated with the sulfide donor.

[0267] Figure 3 Effect of sulfide on the translocation of MPO on the cell surface. Neutrophils translocate MPO to the cell surface in the presence of 10 ng / ml TNF-α. In this experiment, neutrophils were incubated on gelatin (0.2%) treated glass coverslips on 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 for 5 minutes or added simultaneously with TNF-α treatment. The order of the inscriptions shows the order of treatment for the figure. After adding TNF-α, the cells were incubated for 30 minutes and then fixed with 4% PFA. Surface MPO (second column) was labeled with goat anti-MPO antibody before permeabilization and then labeled with Alexafluor 488 rabbit anti-goat secondary antibody. After permeabilization with 1% Triton X, granular MPO (third column) was labeled with goat anti-MPO antibody, followed by rabbit anti-goat Alexa fluor 568 secondary antibody, and cell nuclei (first column) were labeled with Hoechst 33258. After labeling, cells were mounted on glass slides using Prolong™ Glass Antifade Mountant and observed using STED microscopy. (A) Scale bar shows more neutrophils, while (B) single cells show intracellular granular MPO (third column) and cell surface MPO (polar arrangement) located on one side, due to different magnifications (scale bar).

[0268] Figure 4 Effects of sulfide on neutrophil phagocytosis and bactericidal activity. (A) In the presence or absence of different concentrations of GYY4137 (10-100 μM), 4*10 4 Neutrophils were incubated with fluorescently labeled E. coli K-12 BioParticles for 2 hours. After incubation, the wells were washed and observed using a fluorescence microscope. A negative control was provided using cytochalasin D, a phagocytic inhibitor. (B) Plate assay to determine the effect of sulfide on neutrophil phagocytosis. Neutrophils were prepared at a concentration of 3 x 10 4cells / well and treated with GYY4137 and bioparticles as described above. Fluorescence values ​​were detected using TECAN Spark 10M at excitation and emission wavelengths of 485 / 525 nm. GYY4137 was added 1 hour before the addition of bioparticles (black squares) or at the same time as the addition of bioparticles (grey circles with black borders). Based on correlation analysis, the presence of GY4137 did not affect phagocytosis at the applied concentration. (C) Opsonized S. aureus was incubated with isolated neutrophils in the presence or absence of 100 μM hydrogen sulfide and incubated for 20 minutes. Colony forming unit determination was performed and the bactericidal rate was calculated based on the original, extracellular and intracellular bacterial concentrations. Treatment with hydrogen sulfide had no significant effect on the bactericidal effect compared to untreated samples.

[0269] Figure 5 Histopathological findings in the kidneys of Rag2-knockout mice following passive transfer of splenocytes from MPO-immunized, MPO-deficient mice. (A, B) Black arrows indicate cellular crescents in the Bowman capsule, populated by proliferating parietal cells, monocytes, and other inflammatory cells. Dashed gray arrows indicate flattened glomeruli (PAS, X553). (C) Black arrows show fibroblastic crescents. Also shown are large cells with prominently enlarged nuclei and surrounding fibrotic tissue (PAS, X645). (D) As inflammation progresses, more fibrotic lesions (black arrows) can be detected in the crescent regions. The appearance of fibrotic tissue leads to the disappearance of cells previously present in the Bowman capsule (see dashed gray arrow in 5 / A). These cells are replaced by fibrin and collagen (PAS, X700). (E, F) If severe inflammation persists, dissolution of the Bowman capsule can be detected (black arrows). In this case, a range of cells are recruited to the site of injury and infiltrate the inflamed area (see straight white arrow in 5 / A) (PAS, X482). (G, H) Black arrows indicate glomerulosclerosis. This type of glomerular damage cannot be repaired and function cannot be restored (PAS, X260).

[0270] Figure 6AEffect of GYY4137 sulfide donor treatment in the NCA mouse model. (A) The number of affected glomeruli was counted in 6 different sections of each kidney sample. The affected glomeruli showed symptoms of cells, fibrocytes and fibrotic crescents, as well as segmental or diffuse sclerotic lesions. The black columns represent untreated samples and the gray dashed columns represent GYY4137-treated pairs. Each pair of mice received spleen cells from 1 Rag 2-deficient mouse (immunized with BSA or MPO). (B) The bar graph and numbers represent the percentage of affected glomeruli in sulfide donor-treated samples compared to untreated sample pairs. The gray line represents the 0% baseline (the number of affected glomeruli in treated and untreated mice is the same), and the last column represents the average percentage for all samples.

[0271] Figure 7 Healthy (Rag2- / - immunodeficient) mice were treated orally with GYY4137 and intraperitoneally with ATB.

[0272] ATB (60 μmol / kg) was previously tested in the ANCA mouse model, administered orally (po) and proved to be inefficient. In this experiment, it was administered to healthy mice.

[0273] GYY4137 (150 μmol / kg) has been administered intraperitoneally to the same species of animals.

[0274] H2S levels were measured from animal serum according to the method described in the example. Briefly, after isoflurane anesthesia, blood samples were collected from the corners of the mice's eyes and incubated at room temperature for 30 minutes. The coagulated blood samples were centrifuged and the serum was collected into a new test tube. 25 μl of serum sample was mixed with 66 μl of premixed reagent buffer solution and immediately vortexed vigorously. After reacting at 20.0°C for exactly 10 minutes, the reaction was quenched with TCA and vortexed vigorously. The precipitated protein was removed and the supernatant was transferred to an autosampler bottle and cooled. Quantitative calibration samples were prepared in the same manner using standardized NaHS solutions and further diluted after derivatization. HPLC measurements were performed on a C18(2) column using a gradient elution profile of water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The excitation wavelength of the fluorescence detector was set at 390 nm and the emission wavelength was 475 nm. A modified chromatographic apparatus was prepared as previously described [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: p. 146-158.]

[0275] Intraperitoneal administration of GYY4137 has resulted in significantly higher serum levels compared to oral administration of ATB346.

[0276] FIG8 Administration of donor mixture compared to GYY4137

[0277] A sulfide donor mixture (H2S donor mixture) was prepared 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).

[0278] (A) HS donor mixture was administered to healthy mice by intraperitoneal injection and oral administration. Serum concentrations were measured before sulfide treatment (0 hour) and 1 hour and 24 hours after administration. Surprisingly, oral administration resulted in significantly higher serum levels. Although serum concentrations reached a maximum at 1 hour after administration, the difference (i.e., ratio) between oral and intraperitoneal administration serum concentrations reached a maximum at 24 hours.

[0279] (B) GYY4137 at a dose of 150 μmol / kg, GYY4137 at a dose of 750 μmol / kg, and an H2S donor mixture containing GYY4137 at a dose of 150 μmol / kg were orally administered to healthy mice, and H2S serum levels were measured. Higher doses of GYY4137 resulted in higher serum concentrations compared to lower dose forms (pure and mixed).

[0280] Intraperitoneal administration of GYY4137 significantly increased H2S serum levels (see Figure 7 ), whereas intraperitoneal administration of a H2S donor mixture did not increase H2S serum levels (see Figure 8A ).

[0281] Figure 9 Serum sulfide concentrations were measured in mice treated with GYY4137 or GKK-895. Mice were treated intraperitoneally with 150 μmol / kg GYY4137 or 110 μmol / kg GKK-895. At the indicated time points, blood samples were collected from the canthus of the eye after isoflurane anesthesia. Hydrogen sulfide concentrations in the collected serum samples were measured using the monobromobis(methane) assay. Concomitant treatment with GYY4137 and GKK-895 resulted in a significant increase in circulating sulfide concentrations, with GKK-895 resulting in higher measured sulfide values ​​at 2 and 4 hours post-injection.

[0282] Figure 10 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 spectroscopically. The percentage of affected glomeruli in treated samples was calculated compared to control samples. Samples treated with 150 μmol / kg GYY4137 (marked in dark grey) also showed reduced renal symptoms, with the figure depicting an average reduction of 56.8% in affected glomeruli. Samples treated with 90 μmol / kg GKK-895 (marked in light grey) showed an average reduction of 57.65% in affected glomeruli compared to control pairs.

[0283] Figure 11 Pathogenesis of ANCA-associated vasculitis. (A) Inflammatory cytokines and chemokines (eg, TNF-α) are released as a result of local or systemic processes, which leads to increased expression of endothelial adhesion molecules and initiates neutrophil priming. (B) Neutrophil priming results in increased expression of adhesion molecules in neutrophils and induces translocation of ANCA antigens to the cell surface. (C) The F(ab)2 region of ANCA recognizes ANCA antigens on the cell surface, activating neutrophils, which bind to the vessel wall and initiate migration. (D) ANCA-mediated neutrophil activation induces ROS production and degranulation, leading to vasculitis. DETAILED DESCRIPTION

[0284] Antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) has a particularly complex etiology. The pathogenic role of ANCA is supported by numerous studies, including in vitro and in vivo studies, clinical trials, and in vitro and in vivo experiments. Multiple factors, such as neutrophils, complement, and effector T cells, also contribute to the pathogenesis of AAV. [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]. A subgroup of patients has an MPO-positive serotype, and existing technical evidence suggests that MPO acts as an autoantigen and contributes to the development of MPO-positive AAV.

[0285] In a systematic series of experiments, the present inventors have investigated how sulfide interferes with these processes and unexpectedly discovered that H2S-releasing compounds can be used to treat MPO-positive AAV serotypes.

[0286] Although neutrophil phagocytosis and bactericidal activity were not inhibited in the presence of sulfide, and sulfide and sulfide-donating compounds had no effect on PMA-induced neutrophil activation, they could effectively inhibit neutrophil degranulation during ANCA activation and effectively inhibit neutrophil activation by IgG isolated from ANCA patients.

[0287] Sulfide donors can also inhibit TNF-α-induced translocation of the ANCA antigen MPO, for which active MPO has been reported to be a mediator, thereby suppressing neutrophil priming.

[0288] Finally, sulfide-donating compounds protected against renal damage in a mouse model of MPO-ANCA vasculitis.

[0289] The inventors also provide evidence that increased HS doses and HS levels in serum lead to enhanced effects, indicating that the beneficial effects are due to released HS. Slow HS donors are preferred. HS donors that provide increased serum levels after administration can be easily screened by testing animals.

[0290] Thus, in an unexpected manner, the H2S donor compounds of the present invention combat this autoimmune disease at multiple points, without compromising normal neutrophil function, which is crucial for a healthy immune system.

[0291] The role of hydrogen sulfide in autoimmune inflammatory diseases is highly controversial, and depending on the conditions and concentration, H2S can be prone to toxicity. Although H2S has been found to have protective effects against many different processes leading to atherosclerosis in various models, these models appear to have limitations. [Gáll Tamás 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.]

[0292] This two-sided nature of H2S places a huge burden on researchers, making it difficult for them to draw correct conclusions whether H2S is beneficial for specific diseases and requiring complex experiments where the effects or results are unpredictable.

[0293] The factors and biological processes associated with AAV are discussed in more detail below. Figure 11 .

[0294] Inflammatory cytokines induce the translocation of ANCA antigens (such as MPO and PR3) to the cell surface, thereby preparing neutrophils for antibody-based activation.

[0295] Thus, neutrophil priming involves the translocation of MPO to the cell surface to serve as an antigen for ANCA, which ultimately induces a respiratory burst [Jennette, JC and RJ Falk, Pathogenesis of antineutrophil cytoplasmic autoantibody-mediated disease. Nat Rev Rheumatol, 2014. 10(8): p. 463-73.]. In this pathogenic process, ANCA-induced neutrophils infiltrate the walls of small and medium-sized blood vessels and produce a large amount of cytotoxic reactive oxygen species (ROS) that enter the extracellular space, in this case into the interstitial space of the blood vessel wall. In these processes, MPO may play a central role, as the toxic oxygen free radicals and harmful granzymes released by neutrophils primed by ANCA-induced cytokines may mediate vascular inflammation. [Falk, RJ, et al., Anti-neutrophilcytoplasmic autoantibodiesinduce neutrophilsto degranulate and produce oxygenradicals in vitro. Proc Natl Acad Sci US A, 1990.87(11):p.4115-9. ].

[0296] To understand some background about neutrophils, these cells play an important role in innate immunity and provide the first line of defense against pathogenic microorganisms. In the event of an infection, they migrate to the site of infection where they kill the pathogen through phagocytosis and the production of 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): p. 183-91.. Cassatella, MA, Neutrophil-derived proteins: selling cytokines by the pound. Advances in immunology, 1999. 73: p. 369-509.] Activated neutrophils produce a range of inflammatory molecules, such as CXC and CC chemokines, interleukins, interferons, colony-stimulating factors, and tumor necrosis factor-α (TNF-α) [Cassatella, MA, 1999, infra].

[0297] The present inventors also investigated the effects of sulfide on the autoimmune inflammatory process of neutrophil-mediated ANCA vasculitis. First, they demonstrated that sulfide inhibited the oxidative burst mechanism induced by ANCA antibodies in a dose-dependent manner, regardless of whether sulfide was added before or after TNF-α initiation.

[0298] It is known in the art that activation of autoantibodies can also induce the release of granzymes in the extracellular space [Flint, J., M.D., Organ and CO. S. Savage, Pathogenesis of ANCA-associated vasculitis. Rheum Dis Clin North Am, 2010. 36(3): p. 463-77.]. Sulfide also interferes with degranulation during ANCA activation, and observations show that treatment with the GYY4137 sulfide donor inhibits 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 autoimmune activation of cells.

[0299] Since priming of neutrophils by inflammatory cytokines is an important part of this activation, the inventors conducted experiments using TNF-α to investigate the effects of sulfide on this process. The data showed that sulfide effectively inhibited the translocation of MPO to the cell surface, suggesting that sulfide may interfere with the signaling pathways that regulate the priming mechanism (Figure 3).

[0300] The present inventors 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 effects of sulfide in MPO-ANCA-associated vasculitis. The original publication showed that the most prominent symptoms were manifested in the kidneys. The severity of symptoms in the animals showed great variability, with a variety of symptoms and different numbers of affected glomeruli. Although renal crescent formation and fibrosis were also found in the treated groups, surprisingly, treatment with GYY4137 reduced the number of affected glomeruli by an average of 64.9%.

[0301] While this is considered the best animal model, 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.]

[0302] Furthermore, ATB346 and GYY4137 have been tested in both ANCA mouse models and healthy (Rag2 knockout) mice. While intraperitoneal administration of GYY4137 has been shown to be active in the ANCA vasculitis mouse model and to increase H2S serum levels, oral administration of ATB346 neither increased H2S serum levels in healthy mice nor was shown to help reduce renal damage in the ANCA mouse model under the conditions applied.

[0303] Therefore, higher H2S serum levels were associated with improved renal conditions in model animals.

[0304] In further experiments, a sulfide donor mixture (H2S donor mixture) containing GYY4137 formulation (150 μmol / kg), N-acetylcysteine ​​(40 μmol / kg) and pyridoxal 5'-phosphate (20 μmol / kg) was administered IP or peros to healthy mice (Figure 8 / B).

[0305] Intraperitoneal administration of GYY4137 significantly increased H2S serum levels (see Figure 7 This finding correlates well with the fact that IP-administered GYY4137, but intraperitoneally administered H2S donor mixture, improved renal pathology in an animal model of ANCA vasculitis (Figure 8 / A).

[0306] It can be concluded that increased H2S serum levels are a condition of actual medical benefit in ANCA vasculitis.

[0307] Furthermore, the present inventors have unexpectedly discovered that oral administration is superior to intraperitoneal administration.

[0308] The present data suggest that sulfide-donating molecules offer a novel therapeutic option for MPO-mediated ANCA vasculitis. Our results suggest that sulfide can reduce oxidative damage during active phases and may also promote remission induction by inhibiting autoimmune activation of neutrophils.

[0309] Experiments with the novel hydrogen sulfide donor GKK-895 also confirmed this finding. Treatment with lower concentrations of GKK-895 increased blood sulfide concentrations more than treatment with higher doses of GYY4137. Elevated blood sulfide levels were associated with beneficial effects in an ANCA mouse model, further demonstrating a strong correlation between elevated blood sulfide levels and reduced renal symptoms of ANCA vasculitis following treatment with the donor molecule.

[0310] Diagnosis of patients with MPO-ANCA-associated vasculitis

[0311] The analysis showed that despite the presence of several AAV subtypes and a wide range of disease symptoms, a successful diagnosis can be made with caution. The 2012 revision of the International Chapel Hill Consensus Conference Nomenclature of Vasculitides defines the categories of AAV: microscopic polyangiitis (MPA), granulomatosis with polyangiitis (Wegener's granulomatosis) (GPA), and eosinophilic granulomatosis with polyangiitis (Czag-Strauss) (EGPA), as well as the characteristics of these diseases. [Jennette JC.Overview of the 2012revisedInternational Chapel Hill Consensus Conference nomenclature ofvasculitides.Clin Exp Nephrol.2013Oct;17(5):603-606.;Jennette JC, et al. 2012revisedInternational Chapel Hill Consensus Conference Nomenclature ofVasculitides.Arthritis Rheum.2013Jan;2(1):6-19.doi:10.5812 / nephropathol.8971.PMID:23045170.]

[0312] The present invention provides a clear definition of the diagnostic range for MPO positive detection and signs of small vessel vasculitis. Houben E et al. pointed out in their 2016 study that higher ANCA titers and multiple affected organ systems help distinguish AAV from other systemic illnesses, and a diagnostic scoring system that combines 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 titre. Medicine (Baltimore). 2016 Oct; 95(40): e5096]

[0313] However, MPO and PR3 ANCA may be positive in a variety of AAV-like diseases. Higher ANCA titers and multiple affected organ systems may help distinguish AAV from other systemic diseases in patients with anti-PR3 and anti-MPO positivity. Diagnostic scoring systems that incorporate these factors should be considered.

[0314] AAV, which includes GPA or Wegener's granulomatosis, microscopic polyangiitis (MPA), eosinophilic granulomatosis with polyangiitis (EGPA or Tschag-Strauss syndrome), and other related autoimmune diseases, presents with symptoms in organs such as the respiratory tract and kidneys and is characterized by necrotizing inflammation of small vessels (ie, arterioles, capillaries, and venules) and pauciimmune (ie, little or no immune complex deposition in the vessel wall) necrotizing inflammation of the microcirculation, and in some patients, the presence of antibodies to components of neutrophil and macrophage granules: ie, myeloperoxidase (MPO-ANCA) and / or proteinase 3 (PR3-ANCA).

[0315] [Unizony S. and Stone, JHExperimental therapies for vasculitis CHAPTER42Oxford Textbook of Vasculitis,Third edition,Oxford University Press2014Eds.Ball,Gene V.,Fessler,Barri J.and Bridges S.Louis]

[0316] Clinical manifestations associated with NCGN include microscopic hematuria with dysmorphic red blood cells and red blood cell casts, and proteinuria.

[0317] Patients with systemic vasculitis may present with extrarenal manifestations affecting one or more organ systems, with or without renal involvement. Commonly affected systems include the upper and lower respiratory tract, skin, eyes, and nervous system.

[0318] [KDIGO 2021 Clinical Practice Guideline for the Management ofGlomerular Diseases Kidney International,(2021)100(45)Supplement,pages S1 toS276]

[0319] Systemic symptoms include fever, fatigue, weight loss, and muscle or joint pain. Systemic vasculitis is a multisystem disorder. For example, the following organs may be affected (list typical symptoms): eyes (e.g., redness, blurred vision, or blindness), ears (tinnitus or hearing loss), sinuses (pain, runny nose, or nosebleeds), skin (rash, ulcers, or sores that are often deep and slow to heal), lungs (cough and chest pain, difficulty breathing, wheezing, or hemoptysis), kidneys (protein-containing urine, foamy urine, or blood in the urine), nervous system (numbness and / or tingling). Other nonspecific symptoms include fever, polymyalgia, polyarthralgia, headache, malaise, etc., which overlap with other diseases such as infection or malignancy. Symptoms may also overlap with those of other inflammatory diseases.

[0320] Symptoms may manifest in a variety of ways, including nonspecific symptoms and more specific symptoms. In primary care, blood tests may show leukocytosis, thrombocytosis, elevated erythrocyte sedimentation rate and C-reactive protein values, normochromic normocytic anemia, and elevated serum creatinine, which indicates renal damage. Urinalysis and urine 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 within the lung parenchyma and is reasonable in patients with pulmonary symptoms. [Berden A et al. Diagnosis and management of ANCA associated vasculitis, Clinical Review, BMJ (2012) 344e26 doi:10.1136 / bmj.e26]

[0321] Due to the lack of universally reliable serological markers, accurate clinical tools can be used to assess disease activity. To this end, we have created reliable disease assessment tools. The Birmingham Vasculitis Activity Score (BVAS) and the Vasculitis Damage Index (VDI) have been adopted and used by most research groups involved in vasculitis clinical trials. These are internationally recognized assessment tools and are also very effective for comparative clinical trials. The Birmingham Vasculitis Activity Score (BVAS) has also been specifically adapted for Wegener's granulomatosis. [Flossmann O et al., Development of comprehensive disease assessment in systemic vasculitis. Ann Rheum Dis. 2007 Mar; 66(3): 283-92.]

[0322] In AAV, renal biopsy is an important option, both at initial diagnosis and for recurrent disease. Biopsy remains the gold standard. A renal biopsy should be considered in patients with renal involvement or involvement of an organ considered to be involved.

[0323] Nonetheless, if MPO or PR3-ANCA serology is positive and the clinical presentation is consistent with small-vessel vasculitis with a low likelihood of secondary vasculitis, treatment can be initiated, and a biopsy can even be performed after initiation of treatment.

[0324] However, regarding disease recurrence, ANCA positivity or elevated ANCA levels are considered only modestly predictive of future disease recurrence and should not be used to guide treatment decisions.

[0325] The patient group to be treated in the present invention is MPO-ANCA positive patients. Therefore, for patients with AAV symptoms, the MPO serotype must be diagnosed.

[0326] Methods for detecting MPO and determining MPO-ANCA specificity

[0327] One option for treating MPO in patients is indirect immunofluorescence (IIF).

[0328] The International Consensus Statement on Testing and Reporting of Antineutrophil Cytoplasmic Antibodies (ANCA) defines IIF as the method of choice for demonstrating 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.] After preparation, the method involves evaluation using a fluorescence microscope with incident light illumination. This method is recommended by the European Vasculitis Society ( https: / / vasculitis.org / ) is available. 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]

[0329] The MPO specificity of ANCA autoantibodies can 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 test in which a reference standard is used. This standard is suitable for MPO-ANCA (IUIS-CDC reference preparation) and allows the results to be obtained in international units. Several types of direct ELISA are available, as known to those skilled in the art. Chemiluminescent assays are also available. [Damoiseaux, J. and Tervaert, JWC 2014, infra, and documents cited therein; see the section "Methods used to determine ANCA specificity"]

[0330] The second generation of ANCA detection tests are so-called capture ELISAs, in which the first coating of the solid phase does not consist of the antigen, but of monoclonal antibodies specific for the corresponding antigen, to which the antigen preparation is added. This has been shown to provide better recognition for MPO by capturing the antigen by the immobilized antibody.

[0331] The later developed anchored ELISA is the so-called third generation of MPO assays. In these assays, purified antigen is coupled to a linker peptide, which is used to bind the antigen to a solid phase. The third generation (i.e., anchored ELISA) is similar to the capture ELISA, allowing the three-dimensional structure of the antigen to be maintained, ultimately achieving better sensitivity. [Damoiseaux, J. and Tervaert, JWC 2014, infra, and documents cited therein; see the section "Methods used to determine ANCA specificity"].

[0332] ANCA tests are commercially available, however, their use currently requires specialized personnel.

[0333] According to the 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 international consensus that ANCA should be detected by a combination of IIF and antigen-specific assays (including MPO-ANCA).

[0334] 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] conducted a meta-analysis to identify studies using PR3-ANCA or MPO-ANCA for the diagnostic accuracy of granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis (EGPA).

[0335] The present invention will be used for AAV patients with the MPO serotype, and thus for the treatment of MPO-ANCA positive patients. Thus, in a preferred variant, MPO-ANCA is detected in AAV patients of 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.

[0336] In preferred embodiments, the AAV clinical subtype is GPA, MPA, or EGPA.

[0337] In a preferred embodiment, IIF and an antigen-specific assay (eg, ELISA) are performed to detect MPO-ANCA. In particular, the ELISA is a first generation, second generation, or third generation ELISA as described above.

[0338] In embodiments, AAV patients with the MPO-ANCA serotype are monitored for disease progression. In this regard, elevated MPO levels measured at two different time points indicate relapse and the need for adjustment of treatment. In this regard, in preferred embodiments, a combination of IIF and ELISA is performed, or an ELISA, preferably a second or third generation ELISA, is performed.

[0339] Compounds useful in the present invention

[0340] GYY4137

[0341] An example of a sulfide donor compound used by the present inventors is GYY4137 (morpholin-4-ium 4-methoxyphenyl(morpholino)phosphonodithioate). It is typically used as a dichloromethane complex (Formula 1). Rose, P. et al. (GYY4137, a Novel Water-Soluble, H2S-Releasing Molecule, Methods in Enzymology, Volume 554, 2015 Elsevier Inc. ISSN 0076-6879).

[0342]

[0343] Li L et al. describe the chemical synthesis of GYY4137 and its in vitro and in vivo release of H2S. Li L, et al. Characterization of a novel, water-soluble hydrogen sulfide-releasing molecule (GYY4137): new insights into the biology of hydrogensulfide. Circulation. 2008 May 6; 117(18): 2351-60.

[0344] The authors also described that GYY4137 slowly released H2S in aqueous solution in vitro and in anesthetized rats after intravenous or intraperitoneal administration.

[0345]

[0346] Typically, GYY4137 is used in the art by intraperitoneal administration.

[0347] The present inventors have prepared intraperitoneal (ip) and oral (per os) formulations of GYY and tested them in mice (see Examples) and surprisingly found that the oral formulation resulted in higher serum levels. There is reason to believe that the oral formulation is more preferred and effective for treating MPO-ANCA vasculitis than intraperitoneal administration.

[0348] Derivatives of GYY4137 may also be used in the present invention.

[0349] For example, such derivatives are described 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].

[0350] Examples of such compounds are dithiophosphor diamines, which are fast H2S releasers, and benzo[d][1,3,2]oxazaphospholes, which are medium and / or slow releasers (slow-medium releasers).

[0351] Another option is the derivatives of GYY4137, which are slow-release formulations and are

[0352]

[0353] Aspirin derivatives

[0354] Anethole trithione (ADT-OH, Formula 4) is a known H2S-releasing compound.

[0355]

[0356] While anethole trithione itself is an H2S donor, it is also useful because it can be attached to other compounds, such as non-steroidal 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]. These compounds release ADT-OH in the body, which in turn acts as an H2S donor, while other counterparts of this molecule (here, aspirin derivatives) also have their own effects.

[0357] However, several 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 (where AAV disease itself has anti-nephrotic effects). [Li, L. et al. Anti-inflammatory and gastrointestinal effects of a novel diclofenac derivative. Free Radicals Biol. Med., 2007, 42, 706.]

[0358]

[0359] ADT-OH, ACS14, and ACS21 can be prepared and orally administered according to the method 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].

[0360] Compound formulations were prepared using 2 ml / kg of a 9 / 1, v / v mixture containing 0.5% w / v carboxymethylcellulose and DMSO, along with the active agents. The formulations were prepared 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, infra].

[0361] Examples of natural H2S donors

[0362] Natural H2S donors can also be used in the present invention.

[0363] Examples of such compounds are given below and are commercially available from various exemplary suppliers.

[0364] a. Allicin:

[0365] ChemFaces Catalog No. CFN90201

[0366]

[0367] b. Propylene trisulfide:

[0368] Merck (Sigma Aldrich) Catalog Number: SMB00289

[0369] c. Propylene disulfide:

[0370] Merck (Sigma-Aldrich) Catalog Number: SMB00289

[0371] d. Propylene tetrasulfide:

[0372] Abcam catalog number: ab143603

[0373] eE-Allicene:

[0374] MedChemExpress Catalog Number: HY-106784

[0375] fZ-Allicene:

[0376] Merck (Sigma-Aldrich) Catalog Number: A0228

[0377] g. Lenthionine:

[0378] [Morita, Katsura et al. Isolation, Structure, and Synthesis of Lenthionine and Its Analogs., Chemical and Pharmaceutical Bulletin, 1967, Volume 15, Issue 7, Pages 988-993]

[0379] hS-Allylmercaptocysteine ​​(SAMC):

[0380] MedChemExpress. Catalog number: HY-145532

[0381] i. Alpha Lipoic Acid:

[0382] Merck (Supelco catalog number: PHR2561

[0383] j.3H-1,2-dithiolene-3-thione:

[0384] Abcam, catalog number: ab141925

[0385] Other exemplary HS donors that do not contain a -SS- group are as follows:

[0386]

[0387] k. Ovothiol:

[0388] [Mirzahosseini, Arash et al., A cost-effective synthesis of enantiopureovothiol A from L-histidine, its natural precursor” Volume 2014, Issue 6, pp.1-9]

[0389] lS-propyl-L-cysteine-S-oxide:

[0390]

[0391] m.LKT LABS, product number P6855

[0392] n. Alliin ((±)-L-alliin):

[0393] Merck (Sigma Aldrich) catalog number 74264

[0394] Pharmaceutical preparations and administration of the present invention

[0395] It is within the skill of those skilled in the art to prepare compositions or pharmaceutical formulations using the compounds of the present invention.

[0396] For long term storage it is advantageous if the composition is stored in dry form, for example crystallized or lyophilized.

[0397] Suitable carriers are known in the art. A typical carrier used is, for example, carboxymethyl cellulose.

[0398] Furthermore, several compounds may be sensitive to hydrolysis, which suggests the use of buffers, especially in injectable formulations; however, even in dry formulations, it may be advisable to avoid acidic or basic pH.

[0399] Injectable preparations can be reconstituted before use so that only the soluble components are used.

[0400] Poor solubility leading to limited drug loading is a problem that must be addressed.

[0401] Typical excipients may include the following categories and examples:

[0402] Disintegrants such as cross-linked polymers, polyvinyl pyrrolidone (crospovidone), cross-linked sodium carboxymethylcellulose (croscarmellose sodium), especially the latter.

[0403] Adhesives, including for example

[0404] - Sugars, such as disaccharides (lactose, sucrose); polysaccharides (such as cellulose, starch, etc.), modified polysaccharides (such as microcrystalline cellulose, cellulose ethers, etc.) or derivatized sugars (such as carboxymethyl cellulose, etc.) can all be used;

[0405] - sugar alcohols, such as xylitol, sorbitol or mannitol; in particular mannitol (E421)

[0406] - Protein-based binders, such as gelatin; (especially in lightweight gelatin capsules).

[0407] Lubricants such as magnesium stearate or other stearic acid derivatives (or talc or silica etc.) may be used.

[0408] Polymers with stabilizers, surfactant thickeners, solubility enhancers, such as povidone (polyvinyl pyrrolidone, PVP) or other synthetic polymers such as polyethylene glycol (PEG), preferably povidone.

[0409] In the present invention, the compounds used are generally quite hydrophobic, light-sensitive compounds. Therefore, these issues should be taken into account when formulating them into pharmaceutical compositions. It is advantageous if the composition is protected from light.

[0410] The 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 embodiments, the packaging should protect the azide compound from light. For oral administration, opaque capsules are preferred.

[0411] Examples are Lonza Capsugel light-protected capsules that do not contain TiO2 (see [Lonza Press Release "Lonza Expandsits Capsule Offering to Include Titanium Dioxide-FreeWhite Hard Gelatin Capsules" May 9, 2022, Basel, Switzerland]).

[0412] Methods for encapsulation or incorporation into polymer matrices (including nano- and microparticles) to increase loading are also known, and related compounds are disclosed as nanoemulsified formulations, and analogs are known in the art.

[0413] The present invention is further characterized by the following non-limiting examples.

[0414] Materials and methods

[0415] Preparation of sodium sulfide and GYY4137 sulfide donor stock solution

[0416] Freshly prepared sodium sulfide stock solution, sterile filtered (pore size 0.2 μm) and stored in a capped plastic tube on ice until use. Relatively large sodium sulfide crystals were rinsed and then dissolved in ultrapure degassed water. The stock solution was appropriately diluted and the concentration was determined at 240 nm. Contamination with sulfide oxidation products was checked by adding 400 μM DTNB and measuring the absorbance at 560 nm. The stock solution was suitable for use when the difference in concentration calculated from the two measured absorbances was less than 5%. GYY4137 (Sigma-Aldrich, product number SML0100, PubChem substance ID: 329825158) was dissolved in DMSO or DTNB at the appropriate concentration and sterile filtered with 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.].

[0417] GYY4137 (P-(4-methoxyphenyl)-P-4-morpholino-dithioic acid, compound with morpholine (1:1), morpholin-4-ium 4-methoxyphenyl(morpholino)phosphonodithioate), for example, in the form of a dichloromethane complex, can be purchased for experimental purposes, for example, from Merck (Sigma-Aldrich), catalog number SML0100.

[0418] human samples

[0419] All human samples were prepared from peripheral venous blood obtained from healthy donors or patients with active ANCA disease with informed consent. Sample processing was carried out in accordance with national regulations [Ethical Decision: BPR-021 / 00084-2 / 2014; Research No.: 84 / 2014, Project No.: 4678–2016 1, 2].

[0420] Isolation of immunoglobulins from human venous blood

[0421] Immunoglobulins were separated using protein G agarose resin according to the manufacturer's protocol. Serum samples were diluted with binding buffer (1:1), 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 then acid eluted with 100 mM glycine solution (pH 2.5) to recover the IgG fraction. 0.5 ml of the flow-through fraction was collected and the pH was neutralized with 1 M phosphate buffer (pH 7.5). The fractions with the highest protein concentration were pooled and separated in a Ca-free column. 2+ and Mg 2+The protein concentration was measured using Bradford reagent and the solution was sterile filtered (pore size 0.2 μm).

[0422] Neutrophil isolation and activation

[0423] Peripheral venous blood was obtained from healthy adult donors with informed consent. The red blood cells were precipitated in 1% dextran solution (1% dextran dissolved in DPBS), the pale yellow supernatant was pipetted into a plastic tube, and centrifuged at 500g for 5 minutes. The supernatant was discarded, and the cells were resuspended in 10ml DPBS and centrifuged at 500g for 5 minutes. The polymorphonuclear cells were suspended in 5ml DPBS and layered on 5ml Hystopaque 1077 solution and centrifuged at 800g for 30 minutes without stopping. After gradient centrifugation using Hystopaque, the polymorphonuclear cells were precipitated at the bottom of the tube together with the remaining red blood cells. The red blood cells were lysed with 0.2% NaCl solution, and then an equal volume of 1.6% NaCl solution was added to restore the isotonic state. The cells were washed with DPBS, resuspended in HBSS solution and placed 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: p. 9-15.]. Polymorphonuclear cell suspensions were used within 2 hours of preparation. Neutrophils were activated with 100 ng / ml 12-phorbol-13-myristate (PMA) and incubated for 20 minutes in appropriate assay buffer. An equal volume (1 μl) of DMSO was added to all control samples. To activate cellular autoimmunity, 300 μg / ml IgG isolated from ANCA patients or healthy donors was used. First, cells were pretreated with different concentrations of GYY4137 for 5 minutes, and then 2 ng / ml TNF-α was added to the sample and incubated for 15 minutes. After priming with TNF-α, samples were treated with purified IgG and incubated for 60 minutes, unless otherwise specified.

[0424] Effects of hydrogen sulfide on NOX2 activity in neutrophils

[0425] Fox reagent was prepared according to the protocol published by Wolff et al. (100 μM xylenol orange, 250 μM ammonium ferric sulfate (II), 100 mM sorbitol, and 25 mM sulfuric acid diluted in 30 ml of ion exchange water) [Jiang, ZY, et al. Lipidhydroperoxide measurement by oxidation of Fe2+ in the presence of xylenolorange. Comparison with the TBA assay and an iodometric method. Lipids, 1991. 26 (10): p. 853-856.] and stored on ice until use. Neutrophil samples were prepared in L-tyrosine assay buffer using PMA as an activator as described above, and 1 mM sodium azide was added to inhibit MPO activity and prevent hydrogen peroxide degradation. 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. To obtain a 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.

[0426] Determination of the Effect of Sulfide on Neutrophil Activation

[0427] Neutrophil oxidative burst is measured based on NOX2 activity after PMA or IgG activation in healthy donors or MPO-ANCA patients. WST-1 reagent is used, which reacts with superoxide produced into the extracellular space [Ngamwongsatit, P., et al., WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cellline. Journal of Microbiological Methods, 2008. 73(3): p. 211-215.; Tan, A.S. and M.V. 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): p. 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 varying concentrations of sulfide (5-50 μM) or GYY4137 sulfide donor (20-100 μM). Cells were incubated in the presence of sulfide for 5 minutes before or after the addition of TNF-α. PMA-activated samples were incubated for 20 minutes, ANCA-activated samples were incubated for 90 minutes, and all samples were placed on ice for 5 minutes to slow the reaction. All samples were centrifuged at 2000 g for 5 minutes, and the supernatant was then pipetted into a 96-well plate and the absorbance measured at 450 nm. The superoxide concentration generated was calculated using the molar extinction coefficient of WST-1 (37x10 3 M -1 cm -1 ) was calculated because 2 mol of superoxide are required to reduce 1 mol of WST-1.

[0428] Effects of sulfide on TNF-α-mediated neutrophil priming

[0429] A 24-well sterile plate containing a glass sterile cover slip was treated with 0.1% gelatin at 37°C for 1 hour. The plate was washed three times with 500 μl DPBS and 2.5 x 10 5 Neutrophils were incubated for 30 minutes and then treated with GYY4137 at an appropriate concentration 5 minutes before or simultaneously with the addition of 10 ng / ml TNF-α. Neutrophils were activated for 15 minutes at 37°C in the presence or absence of GYY4137. The supernatant was discarded and the cells were fixed with 3.7% paraformaldehyde for 15 minutes at room temperature, followed by blocking of nonspecific antigens with 5% goat serum overnight at 4°C. The cells were washed 3 times with 500 μl DPBS and then labeled with rabbit anti-MPO antibody (1:2000) for surface MPO for 1 hour at room temperature. The wells were washed 3 times and then labeled with goat anti-rabbit AlexaFlour 488 (1:500) secondary antibody for 1 hour in the dark at room temperature. The cells were then permeabilized with 0.1% Triton X for 15 minutes in the dark to label intracellular MPO. The cells were then washed 3 times and then labeled with rabbit anti-MPO antibody as described above. After washing, intracellular MPO was labeled with goat anti-rabbit Alexa Fluor 568 secondary antibody (1:500) for 1 hour in the dark. For nuclear staining, 0.5 ng / ml Hoechst 33258 was used for 15 minutes at room temperature. Cells were washed, and coverslips were mounted on glass slides with Prolong™ Glass Antifade Mountant and incubated for 15 minutes. Microscopic analysis was performed using a STED microscope system.

[0430] Measuring neutrophil degranulation in the presence of sulfide or sulfide donors

[0431] The final concentration was 2x10 6Neutrophils were incubated with 5 μM cytochalasin B at 37°C in HBSS for 10 minutes. Different concentrations of sulfide or GYY4137 were added to the cells and incubated for 5 minutes. In the case of PMA activation, sodium sulfide (0-50 μM) was used and the cells were activated with PMA after 5 minutes. For autoimmune activation using IgG, neutrophils were treated with GYY4137 sulfide donor (0-80 μM), and the 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, centrifuged at 2000g for 5 minutes, and then 100μl of supernatant was pipetted into a 96-well plate containing 100μl of 1mM phenolphthalein-glucuronide dissolved in 100mM sodium acetate solution and incubated for 19 hours. The enzymatic reaction was stopped using 100μl of 400mM glycine buffer and 200mM NaCl to produce pink phenolphthalein, which was measured spectrophotometrically at 540nm. One unit of enzyme activity is the amount of 10μg of phenolphthalein released within 19 hours. For the standard curve, 1mg of phenolphthalein was dissolved in 200μl of 96% drum grate ethanol, vortexed, and then 800μl of ion-exchanged water was added to a concentration of 1mg / ml. Use as soon as possible after dilution. Abb 100μl of phenolphthalein diluent, 100μl of HBSS, and 100μl of glycine buffer. Protein levels in neutrophil supernatants of PMA-activated samples were analyzed by SDS-PAGE and silver staining.

[0432] Effects of sulfide on neutrophil phagocytosis

[0433] Phagocytosis assay was performed using Vybrant TM The phagocytosis assay was performed according to the manufacturer's instructions. Briefly, fluorescently labeled E. coli K-12 BioParticles and trypan blue stock solutions were prepared. 4*10 4Neutrophils in cells / 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 incubation for a period of time, the supernatant was removed and 100 μl of the prepared fluorescent BioParticles suspension was added and incubated at 37 ° C for 2 hours in the presence or absence of GYY4137 (20-100 μM). Under two different experimental conditions, the cells were treated with GYY4137 for 1 hour or 5 minutes before adding the fluorescent BioParticles 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. 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. Similarly, 2x10 5 Prepare samples for microscopic analysis using the number of cells per well.

[0434] Effects of sulfide on the bactericidal effect of neutrophils

[0435] Staphylococcus aureus was cultured on Columbia sheep blood agar plates, incubated overnight at 37°C, and then stored at 4°C. In the experiment, a single colony on the plate was transferred to 15 ml of nutrient broth, incubated overnight at 37°C, then centrifuged at 1000g for 5 minutes and washed twice in PBS. The concentration was measured using a turbidity curve (A550 of 0.2 is approximately 1x10 8 For opsonization, bacteria were plated at 1 x 10 7 The cells were suspended in PBS containing 10% human serum (collected from at least 5 healthy donors) at a concentration of 1 mL / ml. The tubes were inverted at 6 rpm for 20 minutes at 37°C.

[0436] A modified colony forming unit (CFU) assay was used to measure the bactericidal effect of hydrogen sulfide [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 the experimental reactions, 500 μL of isolated human neutrophils (1×107 / mL, preheated to 37°C for 10 minutes) were prepared in the presence or absence of 100 μM NaHS and then added to 500 μL of freshly opsonized bacteria (1×108 / mL) and 50 μL of preheated serum (10%). For the control reaction, neutrophils were replaced with 500 μL Hank buffer. The reaction was incubated with rotation up and down (6 rpm) at 37 ° C. After incubation for 20 minutes, 50 μL samples were pipetted into 950 μL ice-cold PBS to terminate neutrophil activity. The sample was centrifuged at 100 g for 5 minutes at 4 ° C (to precipitate neutrophils but not precipitate bacteria), and then the neutrophil pellet was washed twice more with 1 mL ice-cold PBS. Supernatant was collected in each step to measure bacteria not absorbed by neutrophils. The precipitate was resuspended in 2.5 mL water, the pH value was adjusted to 11.00 with NaOH, stirred for 5 minutes, and then thoroughly vortexed. Then each sample (including control, supernatant and intracellular bacteria) was diluted in water (pH 11) so that when plated on Columbia sheep blood agar, 50 colonies were produced per half plate and incubated overnight at 37 ° C. The number of colonies was counted and the number of intracellular colonies was corrected with the control and supernatant samples to compare the bactericidal effects of untreated and hydrogen sulfide-treated samples.

[0437] mice

[0438] Animal Models of ANCA Vasculitis

[0439] Black 6 recombinase activator gene 2-deficient (Rag2– / –) mice and MPO-deficient (Mpo– / –) mice were purchased from Jackson Laboratories and maintained by the Laboratory of Experimental Pharmacology at the National Institute of Oncology (Ethics Decision: PE / EA / 00419-4 / 2022). All experiments used mice aged 10–14 weeks and used the different groups shown in the table below (Table 1).

[0440] Control anti-BSA anti-MPO

[0441] spleen cells spleen cells

[0442] PBS 2 4 10 150 μmol GYY4137 / kg 2 4 10 90 μmol / kg GKK-895 2 - 6

[0443] Animal experiments were performed according to the protocol published by Falk [Xiao, H., et al., Antineutrophilcytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest, 2002. 110(7): p. 955-63]. MPO knockout mice were immunized with mouse MPO based on the following protocol. On day 1, 20 μg of mouse MPO (mMPO) or BSA was injected with complete Freund's adjuvant in a total of 200 μl into MPO KO mice. 30-40 μl was injected into the hind paw pad and 120-160 μl was injected subcutaneously into the abdominal area. On day 14, 20 μg of MPO / BSA and incomplete Freund's adjuvant were injected intraperitoneally, and on day 28, the mice were boosted with an intraperitoneal injection of 20 μg of mMPO / BSA dissolved in DPBS buffer. After 10 days, blood was drawn from the corner of the eye under isoflurane anesthesia, and the antibody titer was checked by anti-MPO ELISA. If the titer was low, the mice were boosted again with 20 μg MPO or BSA, and the titer was checked again after 7 days. If the titer was appropriate, the mice were sacrificed and splenocytes were isolated. The spleen was removed, gently homogenized in RPMI 1640, filtered with a 70 μM sterile filter, and washed twice with cold RPMI 1640. The remaining red blood cells were lysed with red blood cell lysis buffer according to the manufacturer's instructions. The cells were suspended in 600 μl RPMI 1640. The cells were counted using trypan blue solution, and the total cell count was 3x10 7 Up to 6x10 7Splenocytes obtained from one mouse were injected intravenously into two Rag2- / - immunodeficient mice. Paired mice were treated with 100 μg / kg GYY4137 or DPBS every other day for 5 weeks. Antibody titers were assessed using an anti-MPO ELISA after 4 weeks. After 5 weeks, mice were euthanized with isoflurane, and lung and kidney tissue samples were fixed with 10% paraformaldehyde and prepared for light microscopy analysis. Lung samples were stained with hematoxylin and eosin (H&E), and kidney samples were stained with H&E, periodic acid-Schiff, and selected samples were stained with Masson's trichrome.

[0444] In another experiment, the same pairs received either 90 μmol / kg of GKK895 or DPBS three times weekly for three weeks. GKK895 is a derivative of GYY4137. After five weeks, the mice were euthanized with isoflurane, and kidney tissue samples were fixed with 10% paraformaldehyde and prepared for light microscopy. Kidney samples were stained with H&E and periodic acid-Schiff stain.

[0445] To compare GYY4137 and GKK-895 treatment in mouse kidney samples, kidney samples from an ANCA vasculitis mouse model were prepared for histological analysis and evaluated spectroscopically. The percentage of affected glomeruli in treated samples was calculated compared to control samples. Samples treated with 150 μmol / kg GYY4137 (marked in dark grey) also showed reduced renal symptoms, with the figure depicting an average reduction of 64.9% in affected glomeruli. Samples treated with 90 μmol / kg GKK-895 (marked in light grey) showed an average reduction of 56.9% in affected glomeruli compared to control pairs ( Figure 10 ). The figure shows only the samples treated with GYY, and the treatment effect is good.

[0446] Healthy (Rag2- / - immunodeficient) mice and measurement of H2S serum levels

[0447] Healthy mice Rag2- / - immunodeficient mice (see above) were used for IP and per os administration experiments and were housed and treated as described above, with necessary modifications.

[0448] Blood samples were collected from the canthus of the mouse eye under isoflurane anesthesia and incubated at room temperature for 30 minutes. The coagulated blood samples were centrifuged at 2000g for 10 minutes, and then the serum was collected in a new test tube. 25 μl serum samples were mixed with 66 μl premixed reagent buffer solution (65 μl 200mM HEPES pH 8.2+1 μl 100mM MBB acetonitrile solution) and then vortexed vigorously immediately. After reacting for just 10 minutes at 20.0°C, the TCA quenching reaction was vortexed vigorously by adding 5 μl 50% TCA (w / v). The protein was removed by centrifugation at 3000g for 5 minutes and the supernatant was transferred to an automatic sampling bottle and kept at 4°C. Quantitative calibration samples 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 x 2 mm 3 μm column using a gradient elution profile of water containing 0.1% TFA and acetonitrile containing 0.1% TFA. The fluorescence detector was set at an excitation wavelength of 390 nm and an emission wavelength of 475 nm. A modified chromatographic apparatus was prepared as previously described [Ditroi, T., et al., Comprehensive analysis of how experimental parameters affect H2S measurements by the monobromobimane method. Free Radic Biol Med, 2019. 136: p. 146-158.].

[0449] Measurement of serum sulfide concentrations in mice treated with GYY4137 or GKK-895

[0450] Serum sulfide concentrations were measured in mice treated with GYY4137 or GKK-895. Mice were treated with intraperitoneal injections of 150 μmol / kg GYY4137 or 110 μmol / kg GKK-895. At the designated time points, blood samples were collected from the canthus under isoflurane anesthesia. All blood samples were incubated at room temperature for 30 minutes and then centrifuged at 2000 g for 10 minutes. The concentration of hydrogen sulfide in the collected serum samples was measured using the 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: p.146-158.]. Simultaneous treatment with GYY4137 and GKK-895 resulted in a significant increase in circulating sulfide concentrations, with GKK-895 resulting in higher measured sulfide values ​​at 2 hours and 4 hours after injection.

[0451] It can be concluded that serum levels of H2S are an important factor in the effects of these compounds.

[0452] Effect of sulfide on reactive oxygen species production in 12-phorbol 13-myristate (PMA)-activated neutrophils ring

[0453] First, we investigated the effects of sulfide on 12-phorbol 13-myristate (PMA)-activated neutrophils.

[0454] Prepare 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) and store on ice until use. Neutrophil samples were prepared in Hank's balanced salt solution (without phenol red) using PMA as an activator, and 1 mM sodium azide was added to inhibit MPO activity and prevent degradation of hydrogen peroxide. 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. To obtain a 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 (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: p. 182-189]). H2O2 production measured by the FOX assay of PMA-activated neutrophils was not affected in the applied sulfide concentration range (5-50 μM) (Figure 1 / A).

[0455] Effects of sulfide on the oxidative burst of neutrophils activated by PMA or ANCA

[0456] In the case of MPO-ANCA activated neutrophils, the Fox assay cannot be used to assess superoxide formation because it involves the addition of sodium azide, which may interfere with the antigenic function of cell surface MPO, so we used a different approach. 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): p. 59-68]. In PMA-stimulated neutrophil samples, sodium sulfide (Figure 1 / B) or the sulfide donor GYY4137 (Figure 1 / C) did not inhibit the reduction of WST-1 to a yellow reporter product. Cells treated with IgG isolated from ANCA patients produced significantly higher concentrations of superoxide than samples treated with IgG isolated from healthy donors (p = <0.05).

[0457] Surprisingly, the sulfide donor GYY4137 inhibited superoxide production in a dose-dependent manner, IC 50 This effect was more pronounced when GYY4137 was added 5 minutes before priming with TNF-α (Figure 1 / D, black bars), compared to samples treated with sulfide donors after priming, with an IC 50 The value was slightly higher, at 62.6 μM (Figure 1 / D, patterned bar). These results suggest that sulfide may also interfere with the priming process of neutrophils. These observations confirm that sulfide has no effect on NOX2 activity, as treatment with sodium sulfide or GYY4137 had no effect on PMA activation. However, GYY4137 can effectively inhibit neutrophil activation by IgG isolated from ANCA patients.

[0458] Effects of sulfide on neutrophil degranulation

[0459] To investigate the effect of sulfide on neutrophil degranulation, we used a β-glucuronidase assay based on the release of phenolphthalein from phenolphthalein-glucuronic acid [Falk, RJ, et al., Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro. Proc Natl Acad Sci USA, 1990. 110(7): p. 955-63]. The interference of sulfide with the assay conditions was measured on 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 showed that the average units of enzyme activity calculated based on the amount of phenolphthalein released (μg) over 19 hours were 10.88 (± 1.88) units. The data also showed that the addition of different concentrations of GYY4137 did not affect the enzyme activity or interfere with the assay conditions (Figure 2 / A). PMA-treated samples had similar β-glucuronidase activity in the supernatant, and the presence of sulfide had no effect on the enzyme activity (Figure 2 / B). We further confirmed by SDS-PAGE and silver staining that the protein levels in the supernatant correlated well with the measured enzyme activity. No decrease in protein concentration was detected in GYY4137-treated samples, again indicating that sulfide or GYY4137 did not interfere with the assay (Figure 2 / C). In the case of ANCA activation, the extent of degranulation was similar to that of Triton X- or PMA-treated samples, with an average enzyme activity of 9.77 (±1.36) units. However, unexpectedly, in this case, the presence of GYY4137 effectively inhibited the release of phenolphthalein in a dose-dependent manner, with an IC 50 The value was 6.47 μM (Figure 2 / D). Therefore, it is surprising that the sulfide donor GYY4137 can effectively inhibit neutrophil degranulation upon ANCA activation, but has no effect upon PMA activation.

[0460] Sulfide on TNF- α Effects of induced neutrophil priming

[0461] Inflammatory cytokines (such as TNF-α) induce ANCA antigens (such as MPO and PR3) to translocate to the cell surface, thereby preparing neutrophils for activation based on antibody recognition. The present inventors studied the interference of sulfide on TNF-α-induced neutrophil activation by differential immunofluorescence labeling of surface MPO using green (Figure 3-column 2) fluorescent labels and granular MPO using red (Figure 3-column 3) fluorescent labels. In the black and white figures, fluorescence is shown in gray, but can be identified by the position of the figures given herein. Samples treated with TNF-α translocate MPO to the membrane (Figure 3, row 2), while surface MPO is not detected in untreated control samples (Figure 3, row 1). Neutrophils treated with 25 μM or 50 μM GYY4137 5 minutes before or while adding TNF-α showed that the MPO signal on the membrane was weakened, while the signal in the azuridine blue granules was more (Figure 3, rows 3-5). These results indicate that GYY4137 can inhibit the expression of ANCA antigen MPO induced by TNF-α. translocation, thereby inhibiting neutrophil priming.

[0462] Effects of sulfide on phagocytosis

[0463] Control neutrophils phagocytosed E. coli bioparticles (Figure 4 / A1-2), and this process was inhibited by cytochalasin D, which inhibits phagocytosis by interfering with the formation of new actin filaments [Parod, R.J. and J.D. Brain, Immune opsonin-independent phagocytosis by pulmonary macrophages. J Immunol, 1986. 136(6): p.2041-7] (Figure 4 / A15-16). Adding GYY4137 5 minutes before or with the bioparticles had no effect on phagocytosis (Figure 4 / A3-14). The effect of sulfide on phagocytosis was also quantitatively investigated using a plate assay. Treatment with GYY4137 did not reduce the relative fluorescence signal reported at any given concentration (20-100 μM), confirming that sulfide had no inhibitory effect on phagocytosis (Figure 4 / B, black squares). No inhibition was observed when a sulfide donor was administered simultaneously with the bioparticles (Figure 4 / B, gray circles). Thus, neutrophil phagocytosis is not inhibited in the presence of sulfide. Hydrogen sulfide also did not affect neutrophil bacterial killing (Figure 4 / C).

[0464] The sulfide donor GYY4137 protects against renal damage in a mouse model of MPO-ANCA vasculitis

[0465] Several studies have described different ANCA mouse models (animal models of ANCA-associated vasculitis). The method we chose in our laboratory involves transplantation of activated B cells into mice immunized with MPO or into mice immunized with BSA and unimmunized controls. Although this approach does not result in a strict autoimmune reaction, it provides favorable evidence of symptoms similar to those observed in patients with ANCA-associated vasculitis [Xiao, H., et al., Antineutrophilcytoplasmic autoantibodies specific for myeloperoxidase cause glomerulonephritis and vasculitis in mice. J Clin Invest, 2002. 110(7): p.955-63.]. Using this model, we investigated the effect of the hydrogen sulfide donor molecule GYY4137 on the onset and severity of AAV-associated symptoms.

[0466] There is glomerular inflammation with necrotizing lesions, where GN is usually focal and segmental but can also be generalized and diffuse. Necrotic lesions are always accompanied by crescents. Crescents are formed by proliferating parietal epithelial cells and monocytes; sometimes lymphocytes and granulocytes are also present. Crescents that contain only cells and no collagen are called: cellular crescents ( Figure 5 / A, B). The next step in the process is the migration of fibroblasts into Bowman's space, where they synthesize collagen, gradually replacing the crescents; when the cellular components are mixed with the collagen, the lesion is called a fibroblastic crescent ( Figure 5 / C); in advanced stages, there is no epithelial cells, only fibrous tissue, and the lesion is called a fibrous crescent ( Figure 5 / D). These three stages indicate active or chronic disease and therefore response to treatment. Bowman's capsule is often extensively destroyed ( Figure 5 / E); in these cases, granulomas are often found ( Figure 5 / F) and multinucleated cells around the glomeruli. These granulomas do not represent Wegener's granulomatosis. Glomerulosclerosis is scarring (sclerosis) of the glomeruli ( Figure 5 Glomerulosclerosis is caused by the activation of glomerular cells, which produce scarring substances. This is likely due to stimulation by molecules called growth factors, which may be produced by the glomerular cells themselves or brought to the glomerulus by circulating blood entering the glomerular filter.

[0467] Rag2-deficient mice that received splenocytes from control or BSA-immunized MPO-deficient mice did not develop inflammation in the kidneys. Rag2-deficient mice that received splenocytes from MPO-deficient mice immunized with MPO developed the aforementioned symptoms, but with significant variability (Figure 6 / A). Kidney samples from two different mouse pairs (MPO16 and MPO18) showed elevated levels of inflammation in GYY4137-treated samples compared to untreated samples. One individual from two different mouse pairs (MPO5 and MPO7) died within 24 hours of intravenous splenocyte injection, and these two pairs were therefore excluded from the experiment. Semiquantitative comparative analysis of mouse pairs that developed symptoms after splenocyte transplantation revealed a reduction in renal vasculitis symptoms after GYY4137 treatment. Compared to untreated mouse pairs (excluding MPO3, MPO16, and MPO18), treated mice showed a 64.9% reduction in the number of affected glomeruli (Figure 6 / B).

[0468] H2S serum levels depend on the sulfide donor molecule, dose, and administration

[0469] Normal (healthy) mice were treated intraperitoneally and orally with various sulfide donor preparations:

[0470] - GYY4137 was used at 150 and 750 μM / kg body weight,

[0471] - Donor mix A (H2S donor mix) was prepared, 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).

[0472] ATB346 (otenaproxesul, currently in a Phase 2B efficacy trial; Antibe Therapeutics) was administered orally and intraperitoneally at a dose of 60 μmol / kg. ATB346 (abbreviated as ATB) is a novel naproxen derivative that releases hydrogen sulfide with significantly reduced toxicity, which inhibits COX activity. ATB346 is also an anti-inflammatory drug and a novel nonsteroidal anti-inflammatory drug (NSAID) that induces apoptosis in human melanoma cells.

[0473] ATB was tested in an ANCA mouse model, administered orally (by mouth) and proved to be inefficient. In further experiments, ATB (60 μmol / kg) was administered orally to healthy mice to measure serum levels, while GYY4137 (150 μmol / kg) was injected intraperitoneally and serum levels were compared. Surprisingly, although intraperitoneal administration of GYY4137 (which has been shown to be active in an ANCA vasculitis mouse model (see the examples above and Figure 7 and 8)) and significantly increased H2S serum levels, but oral administration of ATB346 did not increase H2S serum levels, suggesting that elevated serum levels are associated with beneficial effects on the damaged kidneys in AAV.

[0474] H2S levels were measured in the serum of the animals.

[0475] In further experiments, a sulfide donor mixture (H2S donor mixture) was prepared 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). These components were dissolved in PBS.

[0476] The H2S donor mixture was administered to healthy mice both IP and per os. Serum concentrations were measured at 0 hours, revealing elevated sulfide concentrations in serum samples. Per os application of the donor mixture resulted in a significant increase in serum sulfide compared to IP treatment (Figure 8 / B). Despite containing 150 μmol / kg GYY4137 (along with N-acetylcysteine ​​and pyridoxal 5'-phosphate), IP treatment did not increase sulfide concentrations in the animals' serum (Figure 8 / A).

[0477] Thus, intraperitoneal administration of GYY4137 significantly increased H2S serum levels (see Figure 7 ), whereas IP administration of the H2S donor mixture did not ( Figure 8A )—a finding that correlates well with the fact that IP-administered GYY4137 improved renal pathology in an animal model of ANCA vasculitis, whereas IP-administered donor mixtures did not.

[0478] When the novel hydrogen sulfide donor GKK-895 was intraperitoneally administered to healthy mice, serum concentrations were significantly increased 2 and 4 hours after treatment compared with the control group and GYY4137-treated samples ( Figure 9 The study also showed that in an ANCA mouse model, GKK-895 at a much lower concentration alleviated the renal symptoms of ANCA-associated vasculitis to the same extent as the higher concentration of GYY4137 ( Figure 10 ).

[0479] These findings further demonstrate that elevated blood hydrogen sulfide concentrations are associated with protection in the ANCA mouse model.

[0480] Taken together, the results lead to the conclusion that virtually any H2S donor compound, ie, any compound that provides H2S to a subject, ie, a compound that increases serum levels of H2S, may be useful in treating MPO-ANCA vasculitis.

Claims

1. An H2S donor compound for use in the treatment of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) in a mammalian patient suffering from AAV of the myeloperoxidase-ANCA (MPO-ANCA) serotype, preferably a myeloperoxidase (MPO) autoantigen-positive AAV, wherein the subject has elevated MPO-specific ANCA levels.

2. The H2S donor compound for use according to claim 1, wherein The AAV is a myeloperoxidase (MPO) autoantigen-positive AAV, wherein the subject has elevated MPO-specific ANCA levels.

3. The H2S donor compound for use according to any one of claims 1 or 2, wherein The H2S donor compound is a slow H2S releaser compound, and the compound is used preventing kidney damage in said mammalian subject suffering from AAV and / or protecting the kidneys of said mammalian subject suffering from AAV, - inhibiting activation of neutrophils by anti-MPO antibodies in a mammalian subject suffering from AAV, - inhibiting neutrophil degranulation in said mammalian subject suffering from AAV, and / or - inhibiting neutrophil priming in said mammalian subject suffering from AAV, In particular for preventing kidney damage in said mammalian subject suffering from AAV and / or protecting the kidneys of said mammalian subject suffering from AAV.

4. The H2S donor compound for use according to any one of claims 1 to 3, wherein The AAV is selected from the group consisting of microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) (Wegener's granulomatosis), eosinophilic granulomatosis with polyangiitis (EGPA) (Chargé-Strauss syndrome), and localized renal vasculitis.

5. The H2S donor compound for use according to any one of claims 1 to 3, wherein After administration of the compound to a mammalian subject, the compound increases the level of bioavailable HS in the serum of the subject, preferably, the subject has an elevated HS serum level after a time point of 1 hour to 24 hours after administration of the compound, if compared to a normal HS serum level (or a HS serum level without (or before) administration of the compound), Preferably, the elevated H2S serum level is at least twice the normal serum level.

6. The H2S donor compound for use according to any one of claims 1 to 4, wherein The compound is - orally administered to said mammalian patient, or - intraperitoneally administering to said mammalian patient, or - intravenously administering to said mammalian patient, Administration to said mammalian patient is preferably oral.

7. The H2S donor compound for use according to any one of claims 1 to 5, wherein The compound has the general formula (Y) MLQ, (Y) in Q is the H2S releasing moiety that releases H2S within the mammalian patient into the bloodstream of said mammalian patient, Among them, preferably Q contains a dithiolene group (-SS-), or Q contains a dithiophosphonate group (=P(S)S - ) L is an organic linking part, M is a moiety covalently bonded to the remainder of the molecule via a hydrolyzable bond, wherein upon hydrolysis, M is converted into a compound that is tolerable to said mammalian patient, preferably a compound that is beneficial to said mammalian patient, Either L or M may be missing, or both may be present.

8. The H2S donor compound for use according to any one of claims 1 to 6, wherein The compound has the general formula (X) in Q is the H2S releasing fraction that, upon administration to said mammalian patient, releases H2S into the bloodstream of said mammalian patient, Among them, preferably Q includes a 5- to 6-membered heterocyclic ring including a dithiolene group (-SS-), or Q includes a dithiophosphonate group (=P(S)S - ) L is the connecting part, 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, L is optionally linked to Q via a C1-C4 alkylene group, wherein at least two, preferably three or four of R1, R2, R3, R4 and R5 are H, and R1, R2, R3, R4 and R5 are independently selected from -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 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide, 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents, if any, being selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, -NH2, -NHMe, --OCOR 16 、-COOR 17 、-OR 18 、-CONHR 19 , where R 16 、R 17 、R 18 and R 19 selected from H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents, if any, being selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, -NH2, -NHMe, Preferably, R1 and R3 are independently selected from -OCOR 16 、-COOR 17 、-OR 18 , where 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), and the substituents, if any, are selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, -NH2, -NHMe, More preferably Wherein R2, R3 and R5 are H, R 15 Selected from C1-C8 alkylene, C1-C8 alkyl ether, C1-C8 carboxylate, preferably -(CH2) n -(CO)O-, wherein n is 0, 1, 2 or 3, preferably 0 or 1, or a pharmaceutically acceptable salt and / or solvate and / or complex thereof 9. The H2S donor compound for use according to any one of claims 1 to 7, wherein The compound has the general formula I in R7 is selected from - a 3- to 10-membered heterocycle, preferably a 5- to 10-membered heterocycle, more preferably a 5- to 6-membered heterocycle, --NH-R 23 , where R 23 is selected from C1-4 alkyl, C1-4 alkylcarbonyl, R8 (preferably wherein R8 is L) is selected from --NH- --CH2-, -O-, S or NH, preferably CH2, O or NH, - or R 15 The meaning is non-existent. A is selected from --S - , where if A is -S - , then R6 does not exist, --S- and -S - , where if A is -S-, then -R6 can be selected from H, 5 to 10 membered optionally substituted heterocycle, preferably 6 membered optionally substituted heterocycle, C1-4 alkyl or C6-C10 aryl, or -A is -S-, and R6 and R7 together with the other parts of the formula form a compound having formula I.1 wherein R1 to R5 and R8 are independently as defined above, wherein the substituents of the two rings may be the same or different, --O - --O-, where if A is -O-, then R6 can be selected from H, 5 to 10 membered heterocyclic ring, preferably 6 membered heterocyclic ring, C1-4 alkyl group or C6-C10 aryl group, or a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings and optionally at least one 1- to 8-membered open-chain moiety, optionally containing 1 to 4, preferably 1 to 3, heteroatoms, and / or R6 is an organic moiety which, upon hydrolysis, is converted into a compound tolerable to said mammalian patient, preferably a compound beneficial to said 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, wherein at least two of R1, R2, R3, R4 and R5 are H; Preferably, R3 is -OCH3 or each of R1, R2, R3, R4 and R5 is H, or a pharmaceutically acceptable salt and / or solvate and / or complex thereof, In specific embodiments, the dichloromethane complex and / or the morpholinium salt.

10. The H2S donor compound for use according to any one of claims 1 to 7, wherein The compound has the general formula I.2 in R9 is H, wherein said H is dissociable, such that -S-R9 becomes -S - , A is selected from the group consisting of absent and -O-, and Where A is -O-, then R6 is a 10- to 20-membered organic moiety having one or two 5- to 6-membered heterocyclic rings and optionally at least one 1- to 8-membered open-chain moiety, optionally containing 1 to 4, preferably 1 to 3, heteroatoms, or If A does not exist, then R6 is 5- to 10-membered heterocycle, preferably 6-membered heterocycle, preferably a heterocycle containing O and / or N, in particular a morpholinyl group attached via N, R1, R2, R3, R4 and R5 are selected from H, C1-4 alkyl, C1-4 alkoxy, halide, wherein at least two of R1, R2, R3, R4 and R5 are H; preferably R3 is -OMe, or each of R1, R2, R3, R4 and R5 is H, or a pharmaceutically acceptable salt and / or solvate and / or complex thereof; 11. The H2S donor compound for use according to claim 9, wherein The compound has the general formula 1.3 where R 10 is a 5- to 10-membered heterocycle, preferably a 6-membered heterocycle, preferably a heterocycle containing O and / or N, in particular a morpholinyl group attached via N, R1, R2, R3, R4, R5 and R9 are as defined above.

12. The H2S donor compound for use according to claim 10, wherein: Preferably, the compound has the general formula 1.3.1 where R 11 is a C1-4 alkyl group, Preferably, the compound for use according to claim 9 is GYY4137.

13. The H2S donor compound for use according to any one of claims 1 to 7, said compound having the general formula II (preferably as moiety M if dependent on claim 6 or 7) wherein R1, R2, R3, R4 and R5 are as defined above (claim 7) wherein at least two, preferably three or four of R1, R2, R3, R4 and R5 are H, in, Preferably R1, R2, R3, R4 and R5 are independently selected from the group consisting of (preferably R1 is 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 , where R 16 、R 17 、R 18 and R 19 selected from H and substituted or unsubstituted C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C1-C8 alkoxy, C1-C8 alkylamide, C6-C10 aryl, C7-C12 alkylaryl (aralkyl), 5- to 10-membered heteroaryl, 6- to 12-membered alkylheteroaryl, C1-C5 amide C1-C8 carbonyl (preferably C2-C8 alkylcarbonyl, C3-C8 alkenylcarbonyl, C3-C8 -C8 carboxyl (preferably C2-C8 alkylcarboxyl, C3-C8 alkenylcarboxyl or C3-C8 alkynylcarboxyl), C2-C8 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), and the substituents, if any, are selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, ONO2, -NH2, -NHMe, Preferably, R1 is selected from -OCOR 16 、-COOR 17 、-OR 18 , where 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 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), and the substituents, if any, are selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, -NH2, -NHMe, More preferably, R2, R3, R4 and R5 are H, and R1 is as defined above, or Even more preferably, R1 is selected from -OH, -OCOR 16 , where R 16 is selected from H and substituted or unsubstituted C1-C8 alkyl (preferably methyl or ethyl), C2-C8 alkenyl, C2-C8 carboxylate (preferably C2-C8 alkyl ester, C3-C8 alkenyl ester or C3-C8 alkynyl ester), the substituents, if any, being selected from halides, pseudohalides, -OH, -SH, -OMe, -NO2, ONO2, -NH2, -NHMe, R 14 is a group having formula III.2 Wherein, in formula III.2: R1, R2, R4 and R5 are independently selected from 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 H or OCH3, R 13 Selected from H and OCH3, preferably 14. The H2S donor compound for use according to claim 12, wherein the compound is selected from 15. The H2S donor compound for use according to any one of claims 1 to 5, wherein The compound is a natural H2S donor compound selected from the following: allicin, alliin, propylene trisulfide, propylene disulfide, propylene tetrasulfide, allicene (such as E-allicene or Z-allicene), shiitake essence, egg mercaptan, S-allylthiocysteine ​​(SAMC), 3H-1,2-dithiole-3-thione, and α-lipoic acid.

16. The H2S donor compound for use according to any one of claims 1 to 5 or claim 14, wherein The compound comprises a substituted or unsubstituted 5-membered heterocyclic ring, wherein the 5-membered heterocyclic ring includes a dithiolene group (-SS-), preferably a 1,2-dithiolene group or a 1,2-dithiolene group, preferably a group having Formula 5: where R 13 is H or C1-8 alkyl.

17. The H2S donor compound for use according to any one of claims 1 to 15, wherein The composition is administered daily, preferably once, twice or three times a day, for at least one month, two months, three months, six months, one year or longer.

18. A pharmaceutical composition comprising the H2S donor compound for use according to any one of claims 1 to 15, preferably any one of claims 5 to 15, formulated for systemic administration.

19. The pharmaceutical composition comprising the H2S donor compound for use according to claim 17, The composition is formulated for oral administration, preferably in the form of capsules and / or tablets; wherein the composition is preferably protected from light, moisture and decay during storage to form H2S; Preferably, the composition is formulated in light-proof packaging.

20. The pharmaceutical composition comprising a H2S donor compound for use according to claim 17, with the proviso that said use is different from oral administration and said composition is formulated for oral administration. - for intravenous administration or for intraperitoneal administration; and The composition is formulated as an injection or infusion.

21. A pharmaceutical composition comprising a H2S donor compound for use according to any one of claims 1 to 15, preferably according to any one of claims 6 to 15, with the proviso that said use is different from oral administration, The composition is formulated for topical administration. Preferably in the form of an ointment, gel or lotion, The topical administration form comprises an excipient to protect the H2S donor compound from moisture and / or decay, preferably from light, during storage to form H2S; Preferably, the composition is formulated in light-proof packaging.

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