Use of IRAP aminopeptidase inhibitors for the treatment of inflammatory diseases - Patents.com

Abstract

Upon activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules through regulated exocytosis. This acute degranulation is followed by a late activation phase involving the synthesis and secretion of cytokines, growth factors and other inflammatory molecules through poorly defined homeostatic pathways. Here we describe a role for insulin-responsive vesicle-like endosomal compartments characterized by insulin-regulated aminopeptidase (IRAP) in the secretion of TNF-α and IL-6 in mast cells and macrophages. IRAP-deficient mice are protected from TNF-dependent renal injury and inflammatory arthritis. In the absence of IRAP, TNF is not efficiently exported from the Golgi apparatus. Chemical targeting of IRAP+ endosomes reduces proinflammatory cytokine secretion, highlighting this compartment as a promising target for therapeutic control of inflammation. Thus, the present invention relates to the use of IRAP inhibitors for the treatment of inflammatory diseases.

Description

[Technical field]

[0001] Field of the invention: The present invention is in the field of medicine, particularly inflammatory diseases. [Background technology]

[0002] Background of the invention: Mast cells are phylogenetically ancient innate immune cells present in most connective and mucosal tissues. Their hallmark is the large intracytoplasmic granules filled with proteases, histamine, serotonin, cytokines, and inflammatory mediators that are rapidly released upon signaling through specific cell surface receptors, including Fcε receptors, complement receptors, Toll-like receptors, and G protein-coupled receptors. Furthermore, in the second phase after activation, mast cells synthesize and secrete large amounts of pro- and anti-inflammatory cytokines, chemokines, and growth factors, prostaglandins, and leukotrienes. Interestingly, certain stimuli, such as lipopolysaccharide and interleukin-1β, exclusively activate the secretion of newly synthesized mediators without triggering the release of preformed granules. 1~3 Although our understanding of the processes involved in the biogenesis and triggered release of preformed granule contents in anaphylactic reactions has progressed in recent years, there remains much to be explored regarding the immunomodulatory role of mast cell-derived cytokines, chemokines and growth factors, including the regulation of their secretion. 3、4 .

[0003] Mast cell-derived cytokines, chemokines and growth factors can act autocrine, paracrine, locally and systemically and are involved in physiological and defensive processes such as angiogenesis, wound healing and immune defense against bacteria and viruses, as well as in pathological processes such as autoimmune, metabolic and neurological disorders, fibrosis and cancer. 5Regarding the role of mast cells in disease-related situations, pro-inflammatory cytokines play a major role. In particular, mast cell-derived TNF-α and interleukin-6 have been the focus of much research. They chemotactically attract neutrophils and macrophages and upregulate adhesion molecules on endothelial cells. 6~8 , regulating dendritic cell function 9、10 , promotes colitis 11 , mediating cisplatin-induced renal injury 12 , involved in inflammatory arthritis 13 , and regulates airway hyperresponsiveness, inflammation, Th2 recruitment, and cytokine production in a mouse antigen-induced asthma model. 14 It has been reported that:

[0004] Therefore, targeting cytokine synthesis and secretion during the late or chronic activation phase of mast cells may represent a promising therapeutic strategy to prevent the deleterious inflammatory reactions associated with mast cells and actively shape the immunoregulatory responses of these versatile cells.

[0005] Remarkably, hundreds of biological compounds with diverse functions have been identified in the mast cell secretome. 15、16 Some of these have opposing physiological functions, suggesting that mast cell secretory pathways and products may be temporally and spatially regulated. Exocytosis mechanisms, active vesicular transport leading to the release of compounds, are present in all eukaryotic cells. Although the products and biological functions of exocytosis vary widely between cell types, the underlying pathways and transport mechanisms are highly conserved. Two major pathways can be distinguished mechanistically, termed regulated and homeostatic secretion, respectively. Several recent studies have begun to elucidate the mechanistic aspects that separate homeostatic cytokine secretion from regulatory granule exocytosis. 17~20This distinction has been refined by the identification of distinct soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins that are involved in either pathway. SNARE proteins confer membrane identity and control the fusion of lipid bilayers with distinct compartments because each v(vesicle)-SNARE family member can complex only with a limited set of t(target)-SNARE pairs.

[0006] In the best-studied homeostatic pathway of cytokine secretion in macrophages, cytokines are post-translationally transported from the endoplasmic reticulum to the Golgi stack, from where they are exported into vesicles identified by the syntaxin (Stx)6 SNARE. 21 These post-Golgi carriers then fuse with Rab11-positive VAMP3-positive recycling endosomes and are further transported to the plasma membrane (PM). 21、22 In mouse mast cells and macrophages, the major SNARE complex required for fusion of VAMP3 vesicles with the plasma membrane is composed of SNAP23 and Stx (syntaxin) 4. 19、23 Because the specific sorting signals that route cargo from the trans-Golgi network (TGN) to homeostatic secretion have yet to be identified, we consider this pathway the "default" pathway. 24 .

[0007] In contrast, proteins destined for loading into lysosome-associated secretory granules are actively sorted in the Golgi apparatus to form immature secretory granules. These pregranules undergo a series of fusion and fission events that result in the removal and condensation of missorted cargo into mature granules that are stored in the cytoplasm. 25 .

[0008] The release of secretory granules in mast cells requires activation through inflammatory cell surface receptors, including Fcε receptors, Fcγ receptors, Toll-like receptors, complement receptors and other G protein-coupled receptors, and this is mediated by the release of intracellular Ca 2+Mediated through increased levels and activation of protein kinase C 26、27 The main SNARE protein mediating the attachment and fusion of secretory granules with the plasma membrane is VAMP8. Notably, the same SNARE complex as in the homeostatic pathway, consisting of SNAP23 and Stx4, is used for the attachment and fusion of secretory granules with the plasma membrane. 19、28、29 .

[0009] Interestingly, small amounts of preformed cytokines, including TNF-α, were found in secretory granules. 6 , suggesting that, at least in human mast cell lines, they are transported there by re-endocytosis from the extracellular space rather than after sorting directly from the trans-Golgi network into these granules. 30 .

[0010] Furthermore, distinct recycling vesicle compartments in mast cells have been described. 31 These vesicles are identified by the expression of insulin-regulated aminopeptidase (IRAP), which in resting cells displays a ubiquitous cytosolic distribution near the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), from where they undergo slow recycling to the plasma membrane. Upon signaling through Fcε receptors, IRAP rapidly translocates to the plasma membrane, where it can participate in signaling events. Importantly, recruitment of IRAP to endosomes is mechanistically separate from exocytosis of secretory granules. 31This suggests that IRAP and granule-contained mediators, such as histamine, exist in separate compartments, although the exact relationship of IRAP to the various secretory pathways in mast cells remains unclear. For example, the possible positive and negative regulation of the degranulation process by IRAP endosomes was not elucidated by Liao et al. due to the absence of IRAP-null cells or animals in their study. Therefore, given the importance of the intersection of exocytosis with endocytic and recycling pathways and their coordination in mast cell physiology, we decided to investigate the communication between IRAP-containing endosomes and the exocytic pathways mentioned above.

[0011] IRAP endosomes have been described primarily as glucose transporter (Glut) 4 storage vesicles (GSVs) in adipocytes and muscle cells, where they have been extensively studied for their function in insulin-stimulated Glut4 transport. 32、33 In insulin-responsive cells, upon activation signaling through the insulin receptor, the dynamic retention of GSVs in the cytosol is released, allowing them to translocate to the cell surface, where Glut4, IRAP, and other transmembrane GSV proteins are inserted into the plasma membrane. 34、35 From here, IRAP and Glut4 are re-internalized into sorting endosomes where they have been shown to interact with the retromer complex, which promotes their exit from the late degradative endosome / lysosome pathway and retrieves them for retrograde transport to the trans-Golgi network, assembly of GSVs, and budding sites.

[0012] Notably, in contrast to the relatively restricted expression pattern of Glut4 in insulin-responsive tissues, IRAP-containing endosomes are broadly expressed across cell types and tissues, where they are recruited by cell-specific surface receptor signaling and used for a variety of cell type-specific functions. 37~39Thus, with respect to immune cells, IRAP endosomal trafficking may be involved in phagosome maturation and MHC-I cross-presentation in dendritic cells. 40~43 , activation of Toll-like receptor 9 44 and endocytic and exocytic trafficking in T cells for delivery of T cell receptor signaling components and optimal T cell receptor signaling. 45 It has recently been recognized that it crosses and regulates Summary of the Invention [Problem to be solved by the invention]

[0013] Summary of the invention: The invention is defined by the claims. In particular, the invention relates to the use of IRAP inhibitors for the treatment of inflammatory diseases. [Means for solving the problem]

[0014] Detailed description of the invention: Upon activation, mast cells rapidly release preformed inflammatory mediators from large cytoplasmic granules through regulated exocytosis. This acute degranulation is followed by a late activation phase involving the synthesis and secretion of cytokines, growth factors and other inflammatory molecules through poorly defined homeostatic pathways. Here we describe a role for insulin-responsive vesicle-like endosomal compartments characterized by insulin-regulated aminopeptidase (IRAP) in the secretion of TNF-α and IL-6 in mast cells and macrophages. IRAP-deficient mice are protected from TNF-dependent renal injury and inflammatory arthritis. In the absence of IRAP, TNF is not efficiently exported from the Golgi apparatus. Subsequently, a decrease in colocalization of Stx4 and VAMP3-positive endosomes was observed, whereas VAMP8-dependent exocytosis of secretory granules was promoted. Chemical targeting of IRAP+ endosomes reduced pro-inflammatory cytokine secretion, highlighting this compartment as a promising target for therapeutic control of inflammation.

[0015] Accordingly, a first object of the present invention relates to a method for treating an inflammatory disease in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an IRAP inhibitor. [Brief description of the drawings]

[0016] [Figure 1A] IRAP endosomes are required for proinflammatory cytokine secretion in mast cells. (A) Peritoneal mast cells were stimulated with ionomycin / PMA (phorbol myristate acetate) for 18 h, and cytokines secreted into the culture supernatant were quantified by ELISA. Graphs show the mean ± SEM of more than three experiments. [Figure 1B] (B) Peritoneal mast cells were stimulated with ionomycin / PMA and TAPI-I for 4 hours, and plasma membrane-bound TNF-α on live cells was detected via flow cytometry. Graphs show the mean ± SEM of four experiments. *p<0.05, **p<0.01, ***p<0.001. [Figure 2-1]IRAP endosomes are required for the secretion of inflammatory cytokines in vivo. (A-E) One ear of IRAP wild-type and knockout mice (A-C) or Wsh mice lacking mast cells reconstituted with IRAP wild-type and knockout mouse bone marrow-derived mast cells (D, E) was challenged with 30 mg / ml arachidonic acid, while control ears were left untreated. Cytokine concentrations in ear tissue homogenates were quantified and normalized to total protein concentration. (F) Arthritis scores of IRAP wild-type and knockout mice 8 days after induction of collagen-induced arthritis. Graphs show pooled data from two independent experiments. (G) Kidney injury scores of IRAP wild-type and knockout mice treated with cisplatin. (H) Plasma TNF-α concentrations of IRAP wild-type and knockout mice treated with cisplatin 24 hours after cisplatin injection from one experiment out of three. (I) Damage scores of paraffin kidney sections from kit-Wsh / sh mice reconstituted with cisplatin-treated IRAP wild-type or knockout mouse bone marrow-derived mast cells from three independent experiments. *p<0.05, **p<0.01, ***p<0.001. [Figure 2-2] (Same as Figure 2-1) [Figure 2-3] (Same as Figure 2-1) [Diagram 3] The IRAP inhibitor HFI-419 blocks cytokine secretion via destabilization of IRAP and VAMP3 positive endosomes. IRAP wild-type mice were intravenously injected with 6 μg HFI-419 or vehicle 24 hours and 15 minutes before ear challenge. Cytokine concentrations in ear tissue homogenates were quantified and normalized to total protein concentration. Graph shows one of two similar experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The term "inflammatory disease" as used herein has its general meaning in the art and refers to diseases associated with inflammation mediated by at least one pro-inflammatory cytokine.

[0018] The term "proinflammatory cytokine" as used herein has its general meaning in the art and refers to a cytokine that promotes inflammation, including, for example, IL-6, IL-8, TNF-α, IL1-α, IL1-β, interferon α, interferon β, interferon γ, IL-10, IL12, IL-23, IL17, and IL18, IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-11, IL-12, TNFα, TNFβ, interferon γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, or macrophage colony-stimulating factor.

[0019] The term "treatment" or "treating" as used herein refers to both prophylactic or preventative treatments, as well as curative or disease-modifying treatments (including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition), including the suppression of clinical recurrence. Treatments may be administered to subjects with a medical disorder or who may eventually develop a disorder to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond the expected survival in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment for a disease, e.g., a dosing pattern used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. An induction regimen may use (partially or entirely) a "loading regimen", which may involve administering a higher dose of drug than the physician would use during a maintenance regimen, administering a drug more frequently than the physician would administer during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) that is used to maintain a patient during disease treatment, for example, to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon reaching a certain predetermined criterion (e.g., symptoms of disease, etc.).

[0020] In particular, the IRAP inhibitors of the present invention are particularly suitable for reducing the secretion of pro-inflammatory cytokines, in particular by mast cells.

[0021] In some embodiments, the inflammatory disease is selected from the group consisting of arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, degenerative arthritis. deformans), chronic primary polyarthritis, reactive arthritis, and ankylosing arthritis), inflammatory hyperproliferative skin diseases, psoriasis (e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis), dermatitis (including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis), x-linked hyper IgM syndrome, urticaria (e.g., chronic allergic urticaria and chronic idiopathic urticaria (chronic (including autoimmune urticaria)), polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma, systemic sclerosis, sclerosis, systemic sclerosis, multiple sclerosis (MS), spinal optic MS, primary progressive MS (PPMS), Relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis colitis, ulcerative colitis (colitis ulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, sudden hearing loss, IgE-mediated disorders (e.g., anaphylaxis, and allergic and atopic rhinitis), encephalitis, Rasmussen's encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, nongranulomatous uveitis, phacoantigenic uveitis, uveitis), posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranous proliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis,Leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) or systemic lupus erythematodes (e.g., cutaneous SLE, subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritic, encephalitis, pediatric, non-renal, extra-renal, discoid, alopecia)), juvenile-onset (Type I) diabetes mellitus (including childhood insulin-dependent diabetes mellitus (IDDM)), adult-onset diabetes mellitus (Type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis vasculitis, agranulocytosis, vasculitis (including vasculitis, large vessel vasculitis), polymyalgia rheumatica, giant cell (Takayasu) arteritis, medium-sized vessel vasculitis, Kawasaki disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis, and ANCA-associated vasculitis (e.g., Churg-Strauss vasculitis or syndrome (CSS)), temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs positive anemia, Diamond-Blackfan anemia, hemolytic anemia or immune hemolytic anemia (autoimmune hemolytic anemia (AIHA), pernicious anemia anemia (including pernicious anemia (anemia perniciosa)), Addison's disease, pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory disorders, multiple organ injury syndromes (e.g., secondary to sepsis, trauma, or hemorrhage), diseases mediated by antigen-antibody complexes, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome, pemphigoid (e.g., bullous pemphigoid and cutaneous pemphigoid), pemphigus (occasionally pemphigus vulgaris), pemphigus foliaceus,Mucus-membrane pemphigoid pemphigoid), erythematous pemphigoid, autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody mediated nephritis, neuromyelitis optica, polyneuritis, chronic neuropathy, IgM polyneuropathy, IgM mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis); subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease, polyglandular syndromes (e.g., autoimmune polyglandular syndrome (or polyendocrinopathy syndrome)), paraneoplastic syndromes (including neurological paraneoplastic syndromes (e.g., Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome)), stiff-man or stiff-person syndrome syndrome), encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or clonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytoid interstitial pneumonia, bronchiolitis obliterans (non-transplant related) vs NSIP, Guillain-Barre syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease, coeliac disease disease), celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear disease (e.g., autoimmune inner ear disease (AGED), autoimmune hearing loss), opsoclonus-myoclonus syndrome (OMS), polychondritis (e.g., refractory or relapsing polychondritis), pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis, primary lymphocytosis (which includes monoclonal B-cell lymphocytosis),Monoclonal gammopathy or garnmopathy of undetermined significance, possibly benign, peripheral neuropathy, paraneoplastic syndromes, channelopathies (e.g., epilepsy, migraine, arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and CNS channelopathies), autism, inflammatory myopathy, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis retina, retinitis chorioretinitis, autoimmune hepatopathy, fibromyalgia, multiple endocrine deficiencies, Schmidt syndrome, adrenal inflammation, gastric atrophy, presenile dementia, demyelinating diseases (e.g., autoimmune demyelinating diseases), diabetic nephropathy, Dressler syndrome , alopecia areata, crest syndrome (calcinosis, Raynaud's phenomenon, esophageal hypoperistalsis, digital sclerosis), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird breeder's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis (e.g., allergic alveolitis and fibrosing alveolitis), interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome, syndrome, Kaplan's syndrome, dengue fever, endocarditis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Schulman's syndrome, Felty's syndrome, filariasis, cyclitis (e.g., chronic cyclitis, heterochromatic cyclitis, iridocyclitis, or Fuchs' cyclitis), Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans' syndrome, autoimmune gonadal dysfunction, Sydenham's chorea, poststreptococcal nephritis, thromboangitis ubiterans), thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephrosis, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphtha,Aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacosensitivity endophthalmitis, allergic enteritis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hamman-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Kerbain's disease Thyroiditis, acquired atrophic spleen, infertility due to antisperm antibodies, nonmalignant thymoma, vitiligo, SCID and diseases associated with Epstein-Barr virus, acquired immune deficiency syndrome (AIDS), parasitic diseases (e.g., leishmaniasis), toxic shock syndrome, food poisoning, conditions involving T cell infiltration, leukocyte adhesion deficiency, immune responses with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte extravasation, multiple organ injury syndrome, diseases mediated by antigen-antibody complexes, antiglomerular basement membrane disease, allergic neuritis, autoimmune polyglandular endocrinology thyroid disorders, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatoid diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, peripheral neuropathy, autoimmune polyendocrine syndrome type I, adult-onset idiopathic hypothyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid, frontal, maxillary, or sphenoid sinusitis, eosinophil-related disorders (e.g., eosinophilia, hypereosinophilia) pulmonary infiltrates with eosinophilia and myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, focal pulmonary eosinophilia, bronchopulmonary aspergillosis, aspergilloma, or eosinophil-containing granuloma), anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient hypogammaglobulinemia of infancy, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmune disorders associated with connective tissue diseases, rheumatism, neurological disorders, ischemia-reperfusion injury, decreased blood pressure response, vascular insufficiency, vasoectasia,tissue injury, cardiovascular ischemia, hyperalgesia, cerebral ischemia, and diseases involving angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-related syndromes, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelitis, pulmonary cirrhosis, diabetic retinopathy, diabetic aortopathy, intimal hyperplasia, peptic ulcer, valvulitis, and endometriosis.

[0022] In some embodiments, the patient suffers from an allergic disorder. As used herein, "allergic disorder" refers to any disorder resulting from the activation of mast cells by antigens, leading to an "allergic reaction", i.e., a state of hypersensitivity and influx of inflammatory and immune cells. Such disorders include, but are not limited to, systemic allergic reactions, systemic anaphylaxis or hypersensitivity reactions, anaphylactic shock, drug allergies, and sting allergies; respiratory allergic diseases, such as asthma, hypersensitivity lung disease, hypersensitivity pneumonitis, and interstitial lung disease (ILD), ILD associated with rheumatoid arthritis, or other autoimmune conditions; rhinitis, hay fever, conjunctivitis, and allergic rhinoconjunctivitis.

[0023] In some embodiments, the patient suffers from asthma. As used herein, the term "asthma" refers to an inflammatory respiratory airway disease characterized by airway obstruction, wheezing, and shortness of breath.

[0024] In some embodiments, the patient is suffering from anaphylaxis. As used herein, the term "anaphylaxis" refers to a life-threatening allergic reaction characterized by low blood pressure, respiratory failure with bronchoconstriction, and a skin rash, due to the release of mediators from cells, such as mast cells.

[0025] In some embodiments, the inflammatory disease is subsequent to therapeutic treatment, particularly treatment with an immune checkpoint inhibitor. As used herein, the term "immune checkpoint inhibitor" has its general meaning in the art and refers to any compound that inhibits the function of an immunosuppressive checkpoint protein. Inhibition includes a reduction or complete blockage of function. A preferred immune checkpoint inhibitor is an antibody that specifically recognizes an immune checkpoint protein. In some embodiments, the immune checkpoint inhibitor is an antibody selected from the group consisting of an anti-CTLA4 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-TIM-3 antibody, an anti-LAG3 antibody, an anti-B7H3 antibody, an anti-B7H4 antibody, an anti-BTLA antibody, and an anti-B7H6 antibody.

[0026] In some embodiments, the patient suffers from chemotherapy-induced inflammation. Chemotherapy is a category of cancer treatment that uses chemicals, particularly one or more anti-cancer drugs (chemotherapeutic agents), administered as part of a standard chemotherapy regimen. Chemotherapy may be administered with the intent of cure, or it may be aimed at extending life or reducing symptoms. Chemotherapy includes alkylating agent chemotherapy, antimetabolite chemotherapy, anti-microtubule chemotherapy, topoisomerase inhibitor chemotherapy, and cytotoxic antibiotic-based chemotherapy. In certain aspects, the chemotherapy is alkylating chemotherapy. Alkylating chemotherapy includes, but is not limited to, nitrogen mustard, nitrosoureas, tetrazines, aziridines, and cisplatin. In particular, the method of the present invention is particularly suitable for treating renal inflammation induced by cisplatin.

[0027] As used herein, the term "IRAP" has its general meaning in the art and refers to insulin-regulated membrane aminopeptidase. The term is also known as leucyl-cystinyl aminopeptidase, insulin-responsive aminopeptidase. An exemplary amino acid sequence is shown by SEQ ID NO:1. [ka]

[0028] As used herein, "IRAP inhibitor" has its general meaning in the art and refers to any compound that inhibits the activity or expression of IRAP. The compound may be a competitive inhibitor, a non-competitive inhibitor, an orthosteric inhibitor, an allosteric inhibitor, or a partial inhibitor. In some embodiments, the inhibitor is a molecule that inhibits the enzymatic activity of IRAP, for example, by binding to the active site or competing with an enzyme substrate or co-effector or signaling mechanism. The inhibitor may be specific for IRAP and have a somewhat lower level of inhibitory activity against other receptors (e.g., a Ki of about 50 μM or higher than 100 μM, preferably 1 mM, against other receptors, or, for example, a Ki of at least 10-fold higher than the Ki for IRAP, as measured using an assay as described herein). The enzymatic activity of IRAP can be determined by hydrolysis of the synthetic substrate Leu-MCA (Sigma-Aldrich, MO, USA) monitored by the release of the fluorogenic product methylcoumarinamide (MCA) at excitation and emission wavelengths of 380 and 440 nm, respectively, according to Albiston et al. 2008 The FASEB Journal 22:4209-4217 or other methods described herein. Inhibitors of IRAP are known in the art.For example, Albiston et al. (2008) The FASEB Journal 22:4209-4217; Albiston et al. (2011), British Journal of Pharmacology, 164:37-47, Albiston, et al. J. Biol. Chem. 276, 48263-48266; US Patent No. 6,066,672; Albiston, et al. al. Pharmacol. Ther. 1 16, 417-427; Axen, et al. (2006) J. Pept. Sci. 12, 705-713; Albiston et al. (2010) Molecular Pharmacology, 78(4): 600-607; Mountford, et al. (2014) J Med Chem 57(4): 1368-1377; Andersson et al. J Med Chem (2010) 53, 8059, Andersson et al. (201 1 ) J Med Chem 54(1 1 ):3779-3792; WO 2009065169; WO 2010001079; WO 2000 / 012544; U.S. Patent Application Publication No. 2004 / 0086510; WO 2003 / 011304; and WO 2006026832 may be useful in the present invention.

[0029] In some embodiments, the IRAP inhibitors of the present invention have formula (I): [ka] (A is aryl, heteroaryl carbocyclyl, or heterocyclyl (each of which is optionally substituted when R1 is NHCOR8); or quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridyl, phthalazinyl, or pteridinyl (each of which is optionally substituted when R1 is NR7R8, NHCOR8, N(COR8)2, N(COR7)(COR8), N=CHOR8, or N=CHR8); X is O, NR' or S, where R' is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted acyl, optionally substituted heteroaryl, optionally substituted carbocyclyl, or optionally substituted heterocyclyl; R7 and R8 are independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, or R7 and R8 together with the nitrogen atom to which they are attached form a 3-8 membered ring, which may be optionally substituted; R2 is CN, CO2R9, C(O)O(O)R9, C(O)R9, or C(O)NR9R10, where R9 and R10 are independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, each of which may be optionally substituted, and hydrogen; or R9 and R10, together with the nitrogen atom to which they are attached, form a 3- to 8-membered ring, which may be optionally substituted; R3-R6 are independently selected from hydrogen, halo, nitro, cyano, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, amino, acyl, acyloxy, carboxy, carboxy ester, methylenedioxy, amido, thio, alkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, heterocyclylthio, carbocyclylthio, acylthio, and azido, each of which may be optionally substituted where appropriate, or any two adjacent R3-R6 together with the atoms to which they are attached form a 3- to 8-membered ring, which may be optionally substituted; and Y is hydrogen or C1-C10 alkyl. or a pharma- ceutically acceptable salt or solvate thereof.

[0030] In some embodiments, A is optionally substituted heteroaryl when R1 is NHCOR8. In some embodiments, A is pyridinyl.

[0031] In some embodiments, X is 0.

[0032] In some embodiments, R2 is CO2R9.

[0033] In some embodiments, R5 is hydroxyl.

[0034] In some embodiments, the IRAP inhibitor has the structure: [ka] has.

[0035] In some embodiments, the IRAP inhibitors of the present invention have formula (II): [ka] (In the formula, A is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted; R and R are independently selected from hydrogen, alkyl, and acyl; R1 is selected from CN or CO2RC; R2 is selected from CORC and acyl; R3 is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted; or R2 and R3 together form a 5-6 membered saturated keto carbocyclic ring: [ka] (wherein n is 1 or 2; the ring is optionally substituted one or more times with C1-6 alkyl). or R2 and R3 are a 5-membered lactone ring (a) or a 6-membered lactone ring (b). [ka] (In the formula, [ka] is an optional double bond, and R' is alkyl. Forming Rc is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted. or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0036] In some embodiments, A is an optionally substituted aryl. In some embodiments, A is an aryl substituted with -COOH, or a salt, ester, or prodrug thereof. For example, A is -CO2 - NH4 + It can be aryl substituted with

[0037] In some embodiments, R1 is CN.

[0038] In some embodiments, R2 is acyl.

[0039] In some embodiments, the IRA inhibitors of the present invention have the structure: [ka] has.

[0040] In some embodiments, the IRAP inhibitors of the present invention include [ka] TIFF2024543981000010.tif215161 and / or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0041] In some embodiments, the IRAP inhibitors of the present invention have formula (III): [ka] (In the formula, R1 is H or CH2COOH; n is 0 or 1; m is 1 or 2; W is CH or N. or a pharma- ceutically acceptable salt, solvate or prodrug thereof.

[0042] In some embodiments, the IRA inhibitors of the present invention have the structure: [ka] has.

[0043] In some embodiments, the IRAP inhibitor of the present invention is the compound [ka] It has a structure according to:

[0044] In some embodiments, the IRAP inhibitor of the present invention is (±)-ethyl-2-acetamido-7-hydroxy-4-(pyridin-3-yl)-4H-chromene-3-carboxylate, also known as HFI-419 and described in Mountford, SJ, et al. 2014. J. Med. Chem. 57, 1368; Albiston, AL, et al. 2011. Br. J. Pharmacol. 164, 37, Albiston, AL, et al. 2010. Mol. Pharmacol. 78, 600; and Albiston, AL, et al. 2008. FASEB J. 22, 4209. The compound is [ka] It has the formula:

[0045] As used herein, the term "alkyl" or "alk" refers to a straight-chain or branched alkyl, preferably a C-2o alkyl, e.g., C-MO or C 1~6Examples of straight-chain and branched alkyls include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, f-butyl, n-pentyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, hexyl, 4-methylpentyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1,3 ... propyl, 1,2-trimethylpropyl, heptyl, 5-methylhexyl, 1-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3-tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyl-octyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- and 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-methyldecyl, 1-, 2-, 3-, 4-, 5 Examples of suitable alkyl groups include 1-, 6-, or 7-ethylnonyl, 1-, 2-, 3-, 4-, or 5-propyloctyl, 1-, 2-, or 3-butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5-, or 6-propylnonyl, 1-, 2-, 3-, or 4-butyloctyl, 1-, or 2-pentylheptyl, and the like. Although alkyl groups are commonly referred to as "propyl," "butyl," and the like, it is understood that this can refer to either straight-chain or branched isomers, as appropriate. Alkyl groups may be optionally substituted by one or more optional substituents as defined herein.

[0046] The term "alkenyl" as used herein refers to a group formed from a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon double bond, including ethylenically mono-, di-, or poly-unsaturated alkyl groups as previously defined, preferably 02-20 alkenyl (e.g., 02-10 or 02-Θ). Examples of alkenyl include vinyl, allyl, 1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl, 1-pentenyl, 1-hexenyl, 3-hexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3-hexadienyl, and 1,4-hexadienyl. Alkenyl groups may be optionally substituted with one or more optional substituents as herein defined.

[0047] The term "alkynyl" as used herein refers to a group formed from a straight-chain or branched hydrocarbon residue containing at least one carbon-carbon triple bond, including ethylenically mono-, di-, or poly-unsaturated alkyl groups as previously defined. When the number of carbon atoms is not specified, the term preferably refers to 02-20 alkynyl (e.g., C 2~10 Or C 2~6 ). Examples include ethynyl, 1-propynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers. Alkynyl groups may be optionally substituted by one or more optional substituents as defined herein.

[0048] As used herein, the term described as "oxy group" refers to a particular group when linked to oxygen, for example, the terms "alkoxy", "alkenoxy", "alkynoxy", "aryloxy" and "acyloxy" refer to an alkyl group, an alkenyl group, an alkynyl group, an aryl group and an acyl group, as previously defined herein, respectively, when linked to an oxygen atom. The term described as "thio group" refers to a particular group when linked to sulfur, for example, the terms "alkylthio", "alkenylthio", "alkynylthio" and "arylthio" refer to an alkyl group, an alkenyl group, an alkynyl group and an aryl group, as previously defined herein, respectively, when linked to a sulfur atom. Similarly, the term described as "[group A] group B" is intended to refer to group A when linked to group B in its divalent form, for example, "hydroxyalkyl" is a hydroxy group when linked to an alkylene group.

[0049] As used herein, the term "halogen" ("halo") refers to fluorine, chlorine, bromine, or iodine (fluoro, chloro, bromo, or iodo).

[0050] The term "ar" shorthand form used in compound words such as "aryl" (or "carboaryl") or "aralkyl" refers to any monocyclic, bicyclic, or polycyclic (e.g., conjugated and fused) hydrocarbon ring system containing an aromatic residue. Examples of aryl include phenyl, biphenyl, terphenyl, quaterphenyl, naphthyl, tetrahydronaphthyl (tetralinyl), anthracenyl, dihydroanthracenyl, benzanthracenyl, dibenzanthracenyl, phenanthrenyl, fluorenyl, pyrenyl, idenyl, isoindenyl, indanyl, azulenyl, and chrysenyl. Specific examples of aryl include phenyl and naphthyl. An aryl group may be optionally substituted by one or more optional substituents as defined herein.

[0051] The term "carbocyclyl" as used herein includes non-aromatic monocyclic, bicyclic, and polycyclic (e.g., fused, bridged, or conjugated) hydrocarbon residues, e.g., any of C-3-10, C-8, or Cs-6. The rings may be saturated, e.g., cycloalkyl, or may have one or more double bonds (cycloalkenyl) and / or one or more triple bonds (cycloalkynyl). Specific examples of carbocyclyls are monocyclic 5-6 membered ring systems or bicyclic 9-10 membered ring systems. Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl, and decalinyl. A carbocyclyl group may be optionally substituted by one or more optional substituents as defined herein. In particular, a monocarbocyclyl group may be substituted by a bridging group to form a bicyclic bridged group.

[0052] As used herein, the term "carbocyclyl" refers to non-aromatic monocyclic, bicyclic, and polycyclic (e.g., fused, bridged, or conjugated) hydrocarbon residues, such as C 3~20 (For example, C 3~10 , C 3~8, or Cs^). The ring may be saturated, e.g., cycloalkyl, or may have one or more double bonds (cycloalkenyl) and / or one or more triple bonds (cycloalkynyl). Examples of carbocyclyls include monocyclic 5-6 membered ring systems or bicyclic 9-10 membered ring systems. Suitable examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, cyclopentenyl, cyclohexenyl, cyclooctenyl, cyclopentadienyl, cyclohexadienyl, cyclooctatetraenyl, and decalinyl. Carbocyclyl groups may be optionally substituted by one or more optional substituents as defined herein. Monocarbocyclyl groups may be substituted by a bridging group to form a bicyclic bridging group.

[0053] The term "heterocyclyl" when used alone or in compound words refers to a monocyclic, bicyclic, or polycyclic (e.g., fused, bridged, or conjugated) hydrocarbon residue, such as C 3~20 (For example, C 3~10 Or C 3~8), where one or more carbon atoms are independently replaced by a heteroatom to provide a non-aromatic heteroatom-containing ring-containing group. Suitable heteroatoms include O, N, S, P and Se, particularly O, N and S. When two or more carbon atoms are replaced, this may be by two or more of the same heteroatoms or by different heteroatoms. The heterocyclyl group may be saturated or partially saturated, e.g., may have one or more double bonds. Particularly preferred heterocyclyls are monocyclic 5-6 membered and bicyclic 9-10 membered heterocyclyls. Examples of heterocyclyl groups include azuridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 2H-pyrrolyl, pyrrolidinyl, 1-, 2- and 3-pyrrolinyl, piperidyl, piperazinyl, morpholinyl, indolinyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, thiomorpholinyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, tetrahydrothiophenyl (tetramethylene sulfide), pyrazolinyl, dioxalanyl, thiazolidinyl, isopropyl, ethyl ... Examples of heterocyclyl groups include soxazolidinyl, dihydropyranyl, oxazinyl, thiazinyl, thiomorpholinyl, oxathiyl, dithianyl, trioxanyl, thiadiazinyl, dithiazinyl, trithianyl, azepinyl, oxepinyl, thiepinyl, indenyl, indanyl, 3H-indolyl, isoindolinyl, 4H-quinolazinyl, chromenyl, chromanyl, isochromanyl, benzoxazinyl (21-1-1,3, 21-1-1,4-, IH-2,3-, 41-1-3, 1-4H-1,4)pyranyl and dihydropyranyl. Heterocyclyl groups may be optionally substituted by one or more optional substituents as defined herein.

[0054] The term "heteroaryl" as used herein includes any monocyclic, bicyclic, polycyclic, fused, bridged, or conjugated hydrocarbon residue, in which one or more carbon atoms are replaced by a heteroatom to provide a residue having at least one aromatic heteroatom-containing ring. Examples of heteroaryls have rings containing 3 to 20 atoms, for example 3 to 10 atoms. Particularly preferred heteroaryls are 5-6 monocyclic and 9-10 membered bicyclic ring systems. Suitable heteroatoms include O, N, S, P, and Se, especially O, N, and S. When two or more carbon atoms are replaced, this may be by two or more of the same heteroatoms or by different heteroatoms. Suitable examples of heteroaryl groups may include pyridyl, pyrrolyl, thienyl, imidazolyl, furanyl, benzothienyl, isobenzothienyl, benzofuranyl, isobenzofuranyl, indolyl, isoindolyl, pyrazolyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolizinyl, quinolyl, isoquinolyl, phthalazinyl, 1,5-naphthyridinyl, quinozalinyl, quinazolinyl, quinolinyl, oxazolyl, thiazolyl, isothiazolyl, isoxazolyl, triazolyl, oxadiazolyl, oxatriazolyl, triazinyl, tetrazolyl, and furazanyl. Heteroaryl groups may be optionally substituted by one or more optional substituents as defined herein.

[0055] The term "acyl," as used herein, either alone or in compound words, refers to a group containing a C=O moiety. In some embodiments, acyls do not include carboxylic acids, esters, or amides. Acyl includes C(O)-Z, where Z is hydrogen or an alkyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, arylalkyl, heteroarylalkyl, carbocyclylalkyl, or heterocyclylalkyl residue. Examples of acyl include formyl, straight-chain or branched alkanoyl (e.g., Ci-2o), such as acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, and icosanoyl; cycloalkylcarbonyl, such as cyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl, and cyclohexylcarbonyl; aroyl, such as benzoyl, toluoyl, and naphthoyl; aralkanoyl, such as phenylalkanoyl (e.g., phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutyryl, phenylpentanoyl, and phenyl). arylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl); aralkenoyl, for example, phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylmethacryloyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aryloxyalkanoyl, for example, phenoxyacetyl and phenoxypropionyl; arylthiocarbamoyl, for example, phenylthiocarbamoyl; arylglyoxyloyl, for example, phenylglyoxyloyl and naphthylglyoxyloyl; arylsulfonyl, for example, phenylsulfonyl and naphthylsulfonyl; heterocyclic carbonyl; heterocyclic alkanoyl, for example, thienylacetyl, thienylpropanoyl, thienylbutanoyl,Thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl, and tetrazolylacetyl; heterocyclic alkenoyl, such as heterocyclic propenoyl, heterocyclic butenoyl, heterocyclic pentenoyl, and heterocyclic hexenoyl; and heterocyclic glyoxyloyl, such as thiazolyglyoxyloyl and thienylglyoxyloyl. The R and Z residues may be optionally substituted as described herein. As used herein, "optionally substituted" means that the group is unsubstituted or substituted with an alkyl, alkenyl, alkynyl, carbocyclyl, aryl, heterocyclyl, heteroaryl, acyl, aralkyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, alkylcarbocyclyl, halo, haloalkyl, haloalkenyl, haloalkynyl, haloaryl, halocarbocyclyl, haloheterocyclyl, haloheteroaryl, haloacyl, haloarylalkyl, hydroxy, hydroxyalkyl, hydroxyalkenyl, hydroxyalkynyl, hydroxycarbocyclyl, hydroxyaryl, hydroxyheterocyclyl, hydroxyheteroaryl, hydroxyacyl, hydroxyaralkyl, alkoxyalkyl, alkoxyalkenyl, alkoxyalkynyl, alkoxycarbocyclyl, alkoxyaryl, alkoxyheterocyclyl , alkoxyheteroaryl, alkoxyacyl, alkoxyaralkyl, alkoxy, alkenyloxy, alkynyloxy, aryloxy, carbocyclyloxy, aralkyloxy, heteroaryloxy, heterocyclyloxy, acyloxy, haloalkoxy, haloalkenyloxy, haloalkynyloxy, haloaryloxy, halocarbocyclyloxy, haloaralkyloxy, haloheteroaryloxy, haloheterocyclyloxy, haloacyloxy, nitro, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl, nitrocarbocyclyl, nitroacyl, nitroaralkyl, amino(NH2), alkylamino, dialkylamino, alkenylamino, alkynylamino, arylamino, diarylamino, aralkylamino, diaralkylamino, acylamino, diacylamino,Heterocyclic amino, heteroaryl amino, carboxy, carboxy ester, amido, alkylsulfonyloxy, arylsulfenyloxy, alkylsulfenyl, arylsulfenyl, thio, alkylthio, alkenylthio, alkynylthio, arylthio, aralkylthio, carbocyclylthio, heterocyclylthio, heteroarylthio, acylthio, sulfoxide, sulfonyl, sulfonamide, aminoalkyl, aminoalkenyl, aminoalkynyl, aminocarbocyclyl, aminoaryl, aminoheterocyclyl, aminoheteroaryl, aminoacyl, aminoaralkyl, thioalkyl, thioalkenyl, thioalkynyl, thiocarbocyclyl, thioaryl, thioheterocyclyl, thioheteroaryl, thioacyl, thioaralkyl, carboxyalkyl, carboxyalkenyl, carboxyalkynyl, carboxycarbocyclyl, carboxyaryl, carboxyheterocyclyl, carboxyheteroaryl, carboxyacyl, carboxyaralkyl, carboxyester alkyl, carboxyester alkenyl, carboxyester alkynyl, carboxyester capryl ... aryl, carboxyester heterocyclyl, carboxyester heteroaryl, carboxyester acyl, carboxyester aralkyl, amido alkyl, amido alkenyl, amido alkynyl, amido carbocyclyl, amido aryl, amido heterocyclyl, amido heteroaryl, amido acyl, amido aralkyl, formyl alkyl, formyl alkenyl, formyl alkynyl, formyl carbocyclyl, formyl aryl, formyl heterocyclyl, formyl heteroaryl, formyl acyl, formyl rumylaralkyl, acylalkyl, acylalkenyl, acylalkynyl, acylcarbocyclyl, acylaryl, acylheterocyclyl, acylheteroaryl, acylacyl, acylaralkyl, sulfoxidealkyl, sulfoxidealkenyl, sulfoxidealkynyl, sulfoxidecarbocyclyl, sulfoxidearyl, sulfoxideheterocyclyl, sulfoxideheteroaryl, sulfoxideacyl, sulfoxidearalkyl, sulfonylalkyl, sulfonylalkenyl, sulfoxidealkynyl, sulfoxidecarbocyclyl,It is taken to mean that the ring structure may be further substituted or fused (forming a fused bicyclic or polycyclic group) with one, two, three or more organic and inorganic groups including groups selected from sulfonylaryl, sulfonylheterocyclyl, sulfonylheteroaryl, sulfonylacyl, sulfonylaralkyl, sulfonamidoalkyl, sulfonamidoalkenyl, sulfonamidoalkynyl, sulfonamidocarbocyclyl, sulfonamidoaryl, sulfonamidoheterocyclyl, sulfonamidoheteroaryl, sulfonamidoacyl, sulfonamidoaralkyl, nitroalkyl, nitroalkenyl, nitroalkynyl, nitrocarbocyclyl, nitroaryl, nitroheterocyclyl, nitroheteroaryl, nitroacyl, nitroaralkyl, cyano, sulfate, sulfonate, phosphonate, and phosphate groups. Optional substitutions also include those in which a CH group in a chain or ring is replaced by a carbonyl group (C=O) or a thiocarbonyl group (C=S), and those in which two adjacent or non-adjacent carbon atoms (e.g. 1,2- or 1,3) are replaced by one end of each of the -O-(CH)SO- groups or -NRx-(CH)S-NRx- groups, where s is 1 or 2 and each R, x are independently H or C h Alkyl (Chalkl!) and two adjacent or non-adjacent atoms independently selected from C and N are C 1~5 Alkylene group or C 2~5 The term may be taken to refer to an alkenylene group substituted at each end (thereby forming a bridging group). Exemplary optional substituents include alkyl, (e.g., CH alkyl, e.g., methyl, ethyl, propyl, butyl), cycloalkyl (e.g., C 3~6 Cycloalkyl (e.g., cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl), hydroxyalkyl (e.g., hydroxyC 1~6 Alkyl (e.g., hydroxymethyl, hydroxyethyl, hydroxypropyl), alkoxyalkyl (e.g., C 1~6 Alkoxy C 1~6Alkyl, e.g., methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl), alkoxy (e.g., C 1~6 Alkoxy, e.g., methoxy, ethoxy, propoxy, butoxy), alkoxyalkoxy (e.g., C 1~6 Alkoxy C 1~6 Alkoxy, for example, methoxymethoxy, methoxyethoxy, methoxypropoxy, ethoxymethoxy, ethoxyethoxy, ethoxypropoxy, propoxymethoxy, propoxyethoxy, propoxypropoxy), cycloalkoxy (for example, cyclopropoxy, cyclobutoxy, cyclopentoxyl, cyclohexyloxy), halo, haloalkyl (for example, haloC 1~6 Alkyl, for example, chloromethyl, difluoromethyl, trifluoromethyl, trichloromethyl, tribromomethyl), haloalkoxy (for example, haloC 1~6 alkoxy), hydroxy, thio (-SH), sulfonyl, sulfonamide, phenyl (per se, e.g., C 1~6 Alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), benzyl (wherein benzyl itself can be, for example, CH alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), phenoxy (wherein phenyl itself may be further substituted by, for example, CH alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alki Lu, Haro C 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), benzyloxy (wherein benzyl itself can be, for example, C 1~6 Alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), NH, alkylamino (e.g., -NHC 1~6 Alkyl, for example, methylamino, ethylamino, propylamino, etc.), dialkylamino (for example, -NH(C 1~6alkyl) 2, for example, dimethylamino, diethylamino, dipropylamino), acylamino (for example, -NHC(O)C 1~6 alkyl, e.g., -NHC(O)CH), phenylamino (i.e., -NHphenyl, where phenyl itself can be, e.g., CH alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, Hydroxy C 1~6 Alkoxy C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), nitro, cyano, formyl, -C(O)-alkyl (e.g., -C(O)C 1~6 alkyl, e.g., acetyl), OC(O)-alkyl (e.g., -OC(O)C 1~6 alkyl, e.g., acetyloxy), benzoyl (wherein benzyl itself can be, e.g., CH alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), benzoyloxy (wherein benzyl itself can be, for example, 1~6Alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 may be further substituted by one or more of CO2H, CO2alkyl (e.g., CO2C 1~6 alkyl, e.g., methyl ester, ethyl ester, propyl ester, butyl ester), COphenyl (wherein phenyl is itself, e.g., C 1~6 Alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 benzyl (wherein benzyl itself can be, for example, CH alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), CONH, C(O)NHphenyl (wherein phenyl itself can be, for example, CHalkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 alkyl), C(O)NHbenzyl (wherein benzyl itself can be, for example, C 1~6 Alkyl, halo, hydroxy, hydroxyC 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Alkoxy C 1~6 Alkyl, C 1~6 Alkoxy C 1~6 Alkoxy, HaloC 1~6 Alkyl, haloC 1~6 Alkoxy, cyano, nitro, OC(O)C 1~6 Alkyl, NH2, NHC 1~6 Alkyl, NHC(O)C 1~6 Alkyl and NC 1~6 Alkyl C 1~6 C(O)NH alkyl (e.g., C(O)NHC 1~6 Alkyl, e.g., methylamido, ethylamido, propylamido, butylamido), C(O)N dialkyl (e.g., C(O)N(C |~6 alkyl) 2) aminoalkyl (e.g., HNC 1~6 Alkyl-, C1~6 AlkylHN-C 1~6 Alkyl and (C 1~6 Alkyl)2N-C 1~6 alkyl-), thioalkyl (e.g., HSC 1~6 alkyl-), carboxyalkyl (e.g., HO2CC 1~6 alkyl-), carboxy ester alkyl (e.g., C 1~6 Alkyl O2CC 1~6 alkyl-), carboxy ester alkyl (e.g., C 1~6 Alkyl O2CC 1~6 alkyl-), amidoalkyl (e.g., HN(O)CC 1~6 Alkyl-H(C 1~6 Alkyl)N(O)CC 1~6 alkyl-), formyl alkyl (e.g., OHCC 1~6 alkyl-), acylalkyl (e.g., C 1~6 Alkyl(O)CC 1~6 alkyl-), nitroalkyl (e.g., ONC 1~6 alkyl), replacement of CH2 with C=O, replacement of CH2 with C=S, -O-(CH2) S -O- group or -NR'-(CH2) S replacement of two adjacent or non-adjacent carbon atoms (e.g., 1,2 or 1,3) by one end of each of the -NR'- groups (where s is 1 or 2 and each R' is independently H or CH alkyl); and 2~5 Alkylene group or C 2~5 replacement of two adjacent or non-adjacent atoms independently selected from C and N with an alkenylene group.

[0056] The term "sulfoxide" either alone or in compound words refers to the group -S(O)R, where R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, and aralkyl. Examples of R include hydrogen, C 1~20The term "sulfonyl" either alone or in compound words refers to the group S(O)2-R, where R is selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples include alkyl, phenyl, and benzyl.

[0057] As used herein, the term "sulfonamide" either alone or in compound words, or the "sulfonamylin" of "sulfonamide" refers to the group S(O)2NRR, where each R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples of R include alkyl, phenyl and benzyl. In one embodiment, at least one R is hydrogen. In another embodiment, both R are hydrogen.

[0058] The term "sulfamate" as used herein, either alone or in compound words, refers to the group OS(O)2NRR, where each R is independently selected from hydrogen, alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples of R include alkyl, phenyl and benzyl. In one embodiment, at least one R is hydrogen. In another embodiment, both R are hydrogen.

[0059] As used herein, the term "sulfamide" either alone or in compound words refers to the group -NRS(O)2NRR, where each R is independently selected from hydrogen, alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~20 Examples of R include alkyl, phenyl and benzyl. In one embodiment, at least one R is hydrogen. In another embodiment, both R are hydrogen.

[0060] As used herein, the term "sulfate" group refers to an -OS(O)2OR group, where each R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o As used herein, the term "sulfonate" refers to the group SO3R, where each R is independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples include alkyl, phenyl and benzyl.

[0061] The term "thio" as used herein is intended to include groups of the formula "-SR", where R can be hydrogen (thiol), alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, aralkyl, and acyl. Examples of R include hydrogen, C i~2o Examples include alkyl, phenyl and benzyl.

[0062] As used herein, the term "amino" is used herein in its broadest sense as understood in the art and refers to a group of the formula -NR A R B group, where R A and R B R may be independently selected from hydrogen, hydroxyalkyl, alkoxyalkyl, aryl, heteroaryl, carbocyclyl, heterocyclyl, arylalkyl, heteroarylalkyl, carbocyclylalkyl, heterocyclylalkyl, acyl, and amido, each of which may be optionally substituted as described herein. A and R Bcan also form, together with the nitrogen atom to which they are attached, monocyclic or fused polycyclic ring systems, e.g., 3-10 membered rings, particularly 5-6 and 9-10 membered ring systems. Examples of "amino" include -NH2, -NHalkyl (e.g., -NHC i~2o alkyl), -NHalkoxyalkyl, -NHaryl (e.g., -NHphenyl), -NHaralkyl (e.g., -NHbenzyl), -NHacyl (e.g., -NHC(O)C 1~20 alkyl, -NHC(O)phenyl), -NH amide, (e.g., NHC(O)NHC 1~6 alkyl, NHC(O)NHphenyl), -Ndialkyl (wherein each alkyl, e.g., C i~2o may be the same or different), and optionally contain one or more of the same or different heteroatoms (e.g., O, N, and S). References to groups described as "amino [groups]" include R A Groups and R B The meaning of the radical is intended to reflect the nature of the radical. For example, "alkylamino" is -NR A R B where R A or R B One of the radicals is alkyl. "Dialkylamino" is -NR A R B where R A and R B are each (independently) an alkyl group.

[0063] As used herein, the term "amide" is used herein in its broadest sense as understood in the art and refers to an amide of the formula C(O)NR A R B where R A and R B is as defined above. Examples of amides include C(O)NH, C(O)NH alkyl (e.g., C 1~20 alkyl), C(O)NH aryl (e.g., C(O)NH phenyl), C(O)NH aralkyl (e.g., C(O)NH benzyl), C(O)NH acyl (e.g., C(O)NHC(O)C 1~20alkyl, C(O)NHC(O)phenyl), C(O)Nalkylalkyl (wherein each alkyl, e.g., C 1~20 Alkyl includes 5- or 6-membered rings, which may be the same or different, and optionally containing one or more of the same or different heteroatoms (eg, O, N and S).

[0064] As used herein, the term "carboxy ester" is used in its broadest sense as understood in the art and includes groups having the formula -COR, where R can be selected from the group including alkyl, alkenyl, alkynyl, aryl, carbocyclyl, heteroaryl, heterocyclyl, arylalkyl, heteroarylalkyl, carbocyclylalkyl, heterocyclylalkyl, aralkenyl, heteroarylalkenyl, carbocyclylalkenyl, heterocyclylalkenyl, aralkynyl, heteroarylalkynyl, carbocyclylalkynyl, heterocyclylalkynyl, and acyl, each of which can be optionally substituted. Some examples of carboxy esters include -COC 1~20 Alkyl, -CO2aryl (e.g., -CO2phenyl), -CO2arC 1~20 Alkyl (eg, -CO2 benzyl) is included.

[0065] As used herein, the term "phosphonate" refers to the group -P(O)(OR), where R is independently selected from hydrogen, alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples include alkyl, phenyl and benzyl.

[0066] As used herein, the term "phosphate" refers to the group -OP(O)(OR), where R is independently selected from hydrogen, alkyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, acyl, and aralkyl. Examples of R include hydrogen, C i~2o Examples include alkyl, phenyl, and benzyl.

[0067] Carboxyclic isosteres are groups that may exhibit the same or similar properties as carboxylic acid groups. Some examples of carboxylic isosteres include -SO3H, -SONHR, -PO2R2, -CN, -PO2R2, -OH, -OR, -SH, -SR, -NHCOR, -NR2, -CONR2, -CONH(O)R, -CONHNHS02R, -COHNS02R, and -CONR-CN, where R is selected from H, alkyl (e.g., d-6 alkyl), phenyl, and benzyl. Other carboxylic isosteres include carbocyclic and heterocyclic groups, such as: [ka] TIFF2024543981000016.tif95161 Includes.

[0068] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, and the like, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. Examples of such inorganic acids are hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, sulfuric acid, and phosphoric acid. Suitable organic acids may be selected from the aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic acid classes of organic acids, examples of which are formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, gluconic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, glucuronic acid, fumaric acid, maleic acid, pyruvic acid, alkylsulfonic acids, arylsulfonic acids, aspartic acid, glutamic acid, benzoic acid, anthranilic acid, mesylic acid, salicylic acid, p-hydroxybenzoic acid, phenylacetic acid, mandelic acid, ambonic acid, pamoic acid, pantothenic acid, sulfanilic acid, cyclohexylaminosulfonic acid, stearic acid, algenic acid, β-hydroxybutyric acid, galactaric acid and galacturonic acid. Suitable pharma- ceutically acceptable base addition salts of the compounds of the present invention include metal salts made from lithium, sodium, potassium, magnesium, calcium, aluminum, and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine, etc. Alternatively, organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine), procaine, ammonium salts, quaternary salts, such as tetramethylammonium salts, amino acid addition salts, such as salts containing glycine and arginine. For example, alkali metal salts (K, Na) and alkaline earth metal salts (Ca, Mg) may be used as deemed appropriate for the structure, but again any pharma- ceutically acceptable non-toxic salt may be used where appropriate. Sodium and calcium salts are preferred. Pharmaceutically acceptable solvates (including hydrates) of such compounds and such salts are also intended to be included within the scope of the present invention.

[0069] In some embodiments, the IRAP inhibitor leads to destabilization and / or degradation of IRAP. In some embodiments, the IRAP inhibitor is a compound that targets the degradation of IRAP. For example, the compound can be a proteolysis-inducing chimeric molecule (PROTAC). PROTACs are heterobifunctional compounds composed of a targeting compound-binding ligand and an E3 ubiquitin ligase ligand, which induce proteasome-mediated degradation of selected proteins through their recruitment to E3 ubiquitin ligase, followed by ubiquitination. These drug-like molecules offer the possibility of temporal control over protein expression. Such compounds can induce the inactivation of a protein of interest when added to cells or administered to animals or humans, and can be useful for the degradation of pathogenic or oncogenic proteins (Crews C, Chemistry & Biology, 2010, 17(6):551-555; Schnnekloth JS Jr., Chembiochem, 2005, 6(l):40-46).

[0070] In some embodiments, the IRAP inhibitor is an inhibitor of IRAP expression. "Expression inhibitor" refers to a natural or synthetic compound that has the biological effect of inhibiting gene expression. In a preferred embodiment of the present invention, the gene expression inhibitor is a small interfering RNA, an antisense oligonucleotide, or a ribozyme. For example, an antisense oligonucleotide (including antisense RNA molecules and antisense DNA molecules) will act to directly block the translation of IRAP mRNA by binding to IRAP mRNA, thereby preventing protein translation or increasing the degradation of the mRNA, thereby reducing the level of IRAP in the cell, thereby reducing its activity. For example, an antisense oligonucleotide that is at least 15 bases long and complementary to a unique region of the mRNA transcript sequence encoding IRAP can be synthesized, for example, by conventional phosphodiester technology. Methods for using antisense technology to specifically inhibit gene expression of genes whose sequences are known are well known in the art (see, for example, U.S. Patent Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as expression inhibitors for use in the present invention. IRAP gene expression can be reduced by contacting a subject or cell with small double-stranded RNA (dsRNA), or a vector or construct that causes the production of small double-stranded RNA, such that IRAP gene expression is specifically inhibited (i.e., RNA interference or RNAi). The antisense oligonucleotides, siRNAs, small hairpin RNAs, and ribozymes of the present invention can be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the introduction of an antisense oligonucleotide, siRNA, shRNA, or ribozyme nucleic acid into a cell, typically a cell expressing IRAP. Typically, the vector transports the nucleic acid into the cell with reduced degradation compared to the extent of degradation that would occur in the absence of the vector.Generally, vectors useful in the present invention include, but are not limited to, plasmids, phagemids, viruses, antisense oligonucleotides, siRNA, shRNA or other vehicles derived from viral or bacterial sources that are engineered by inserting or incorporating ribozyme nucleic acid sequences.Viral vectors are a preferred type of vector, including, but not limited to, the nucleic acid sequences derived from the following viruses: retroviruses, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, mouse mammary tumor virus, and Rous sarcoma virus; adenoviruses, adeno-associated viruses; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes viruses; vaccinia viruses; polio viruses; and RNA viruses, such as retroviruses.Other vectors that are not named but are known in the art can also be easily used.

[0071] According to the present invention, the IRAP inhibitor is administered to the patient in a therapeutically effective amount. By "therapeutically effective amount" is meant an amount of active ingredient sufficient to treat or reduce symptoms at a reasonable benefit / risk ratio applicable to any medical treatment. It is understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the patient; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination with the active ingredient; and similar factors well known in the medical field. For example, it is well within the skill of the skilled artisan to start the dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. However, the daily dose of the product may vary over a wide range, from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of active ingredient, for adjustment of dosage according to the symptoms of the patient to be treated. The pharmaceutical preparation typically contains about 0.01 mg to about 500 mg of active ingredient, typically 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of 0.0002 mg / kg (body weight) / day to about 20 mg / kg / day, particularly about 0.001 mg / kg (body weight) / day to 7 mg / kg / day.

[0072] Typically, the IRAP inhibitor is formulated with a pharma- ceutically acceptable excipient and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The term "pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other undesirable reactions when administered appropriately to a mammal, particularly a human. A pharma- ceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any kind. The carrier may also be a solvent or dispersion medium, containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition a substance that delays absorption, such as aluminum monostearate and gelatin. In the pharmaceutical composition of the present invention, the active ingredient of the present invention may be administered in a unit dosage form as a mixture with a conventional pharmaceutical support. Suitable unit dosage forms include oral route dosage forms, such as tablets, gel capsules, powders, granules, and oral suspensions or solutions, sublingual and buccal dosage forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal dosage forms, and rectal dosage forms.

[0073] The present invention is further illustrated by the following figures and examples, which should not, however, be construed as limiting the scope of the present invention in any way. EXAMPLES

[0074] method Study design We used IRAP knockout and wild-type mice to study the role of IRAP endosomes in mast cell function. To this end, we examined bone marrow-derived mast cells and peritoneal mast cells in vitro for degranulation and cytokine secretion. We investigated the role of IRAP endosomes in mast cell function in wild-type vs. IRAP knockout mice, and in mast cell-deficient kit-W mice reconstituted with IRAP knockout or wild-type mast cells. sh / sh Inflammatory models of TNF-dependent autoantibody-induced arthritis and cisplatin-mediated nephropathy in mice were used. To elucidate the underlying cell biological mechanisms, we performed immunofluorescence staining and image stream colocalization experiments with relevant endosomal markers to analyze the Golgi export kinetics of TNF. Experiments with the IRAP chemical inhibitor HFI-419 completed the study.

[0075] Reagents and antibodies The following antibodies were used in this study: mouse monoclonal IRAP antibody clone 3E, rabbit monoclonal anti-IRAP XP clone D7C5, rabbit anti-EEA1 antibody (all from Cell Signaling); rat anti-mouse lysosome-associated membrane protein (LAMP) 1 clone 1D4B, mouse monoclonal anti-STX6, mouse monoclonal anti-GM130 antibody (BD Pharmingen); rabbit polyclonal anti-STX6 antibody (ProteinTech Group, Chicago, IL, USA); mouse monoclonal anti-Stx4 antibody clone QQ-17 (Santa Cruz); rabbit polyclonal anti-TNF antibody (Abcam 34674 for confocal imaging and imaging flow cytometry), PE-PerCP5.5 anti-mouse TNF antibody clone MP6-XT22 (eBiosciences, for fluorescence-activated cell sorting (FACS)); rat anti-IL-6 and rat anti-IL-10 antibodies (eBiosciences); rabbit polyclonal anti-VAMP3 antibody (Abcam 2102); rabbit polyclonal anti-VAMP8 antibody (novus); goat anti-serotonin antibody (Abcam 66047), rabbit polyclonal anti-Rab14 antibody (Sigma-Aldrich). All secondary reagents were Alexa-conjugated highly cross-adsorbed antibodies from Molecular Probes (Life Technologies). Alexa 647-Transferrin was from Life Technologies. IL-3 and stem cell factor (top grade) were purchased from Miltenyi Biotec.

[0076] Mouse cytokine detection Duoset enzyme-linked immunosorbent assay (ELISA) kits were from R&D Systems (mouse IL-6, mouse TNF) or BioLegend (mouse IL-10). Easysep™ anti-mouse CD117 positive selection kit was from StemCell. TNF-α converting enzyme (TACE) inhibitors TAPI-1, ionomycin, PMA (phorbol myristate acetate), HFI-419, Dynasoar, GDC-0941 were all from Calbiochem. p-Nitrophenyl-N-acetyl-β-D-glucosaminide (pNAG) was from Sigma. IRAP inhibitors 4u and 11b were gifts from E. Stratikos (Democritus Research Center, Athens).

[0077] mouse A previously described IRAP study on the Sv129 background obtained from S. Keller - / - Mice were backcrossed up to 10 times to C57BL / 6 mice obtained from Janvier (Saint-Quentin-Fallavier, France). Control wild-type mice were C57BL / 6 mice bred in our facility or purchased from Charles River. Kit-W sh / sh was purchased from Jackson Laboratories (strain no. 30764). Animal experiments were performed in accordance with the guidelines of the local authorities and approved by the Animal Experimentation Ethics Committee of the University of Paris V (Comite d'Ethique pour l'Experimentation Animale).

[0078] Mast cell isolation and culture Murine bone marrow-derived mast cells (BMMCs) were produced in vitro by culturing extruded cells from large bones for 4-6 weeks in complete medium [Iscove's modified Dulbecco's medium (IMDM) supplemented with 10% fetal calf serum (FCS), 25 mM HEPES (pH 7.4), 2 mM glutamine, 100 U / ml penicillin, 100 g / ml streptomycin, 50 μM β-mercaptoethanol, 1% non-essential amino acids, and 1 mM sodium pyruvate] supplemented with 10 ng / ml IL-3.

[0079] Culture medium was changed every 5-7 days. All cell cultures were grown at 37°C in a humidified atmosphere containing 5% carbon dioxide. Differentiation of mouse bone marrow-derived mast cells after 4 weeks was >98% as confirmed by staining with CD117 and Fcε receptor antibodies. Mouse peritoneum-derived mast cells (PCMCs) were obtained by peritoneal lavage with 5 mL ice-cold PBS / 0.1% bovine serum albumin and cultured in complete medium (see above) supplemented with 10 ng / ml IL-3 and 10 ng / ml stem cell factor.

[0080] Non-adherent cells, including mast cells, were separated from adherent macrophages after 3 hours of culture. Cultured cells were enriched for mast cells (>90%) after 7 days of culture. For use after shorter culture times, mast cells were purified via anti-CD117 antibody beads (StemCell).

[0081] kit-W sh / sh Reconstitution of mouse mast cells Mouse bone marrow-derived mast cells from wild-type and IRAP knockout mice were cultured in the presence of mouse IL-3 and mouse stem cell factor for 4 weeks as described above. 6 Mouse bone marrow-derived mast cells were cultured using kit-W sh / sh Mice were injected intravenously and allowed to reconstitute for 8-12 weeks before functional experiments. Reconstituted mice that yielded less than 50 nM histamine per µg of total protein in untreated ear tissue homogenates were deemed not successfully reconstituted and were excluded from analysis.

[0082] Flow cytometry assay For TNF-α surface staining, mouse peritoneum-derived mast cells were stimulated with 1 μM ionomycin / 10 nM PMA (phorbol myristate acetate) or 100 ng / ml lipopolysaccharide in the presence of TAPI-I at 37° C., washed with ice-cold PBS, and incubated with Fcblock (Miltenyi) followed by fluorochrome-conjugated CD117, FcεRI, and TNF-α antibodies diluted in PBS-1% bovine serum albumin at 4° C. Intracellular staining of cytokines, IRAP, and VAMP3 was performed using BD intracellular staining kits and appropriate species-specific fluorescent secondary antibodies (Life Technologies).

[0083] For degranulation experiments, mouse peritoneum-derived mast cells were stimulated with 1 μM ionomycin / 10 nM PMA or 48 / 80 for 30 min at 37° C., placed on ice, surface stained with AlexaFluor488 anti-LAMP1BD Canto™, and a Gallios flow cytometer was used for cell analysis.

[0084] Mouse Ear Challenge 20 μl of arachidonic acid (AA) (30 mg / ml in acetone) was applied to the inner and outer surfaces of one mouse ear, while the other ear was left untreated.

[0085] One hour after application of arachidonic acid, mice were sacrificed and ears were harvested. Ear biopsies were dissociated in 800 μl of ice-cold homogenization buffer (PBS containing 0.4 M NaCl, 0.05% Tween-20, 10 mM EDTA and complete protease inhibitor cocktail (Roche)) using the preset "protein" protocol on a gentleMACS™ Octo Dissociator (Miltenyi Biotec). Homogenates were clarified by centrifugation at 5000×g for 10 min, and total protein concentrations were determined by BCA (bicinchoninic acid) assay. Histamine or cytokines in the supernatants were quantified as described below.

[0086] Cytokine and histamine measurements Mouse peritoneum-derived mast cells were stimulated with 1 μM ionomycin / 10 nM PMA or 100 ng / ml lipopolysaccharide for 6 h at 37°C for cytokine secretion, or with ionomycin / PMA or 10 μg / ml 48 / 80 for 30 min for histamine measurement.

[0087] Supernatants were collected and histamine was quantified using a Histamine Dynamic HTRF kit (Cysbio). TNF-α, IL-6, or IL-10 were quantified using specific cytokine ELISA kits. The kits were used according to the manufacturer's instructions.

[0088] Release of β-hexosaminidase Mouse peritoneal derived mast cells were stimulated with 1 μM ionomycin / 10 nM PMA or 10 μg / ml 48 / 80 in Tyrode's buffer for 30 min. After stimulation, the cell suspension was centrifuged, placed on ice, and the supernatant was collected. The cell pellet of unstimulated cells was lysed with 0.5% Triton X-100 to determine the maximum enzyme activity of β-hexosaminidase. 5 × 10 3A volume of 25 μl of supernatant or lysate corresponding to 10 cells was incubated with 50 μl of a 1.3 mg / ml p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNAG) solution in 50 mM citrate buffer, pH 4.5, for 90 min at 37° C. The reaction was stopped with 150 μl of 0.2 M glycine buffer, pH 10.7, and the absorbance was read at 405 nm. The rate of degranulation was expressed as the ratio of the absorbance of a given supernatant to the absorbance measured in the lysate of unstimulated cells.

[0089] Cisplatin-induced renal injury model Mice were injected intraperitoneally with 10 mg / kg cisplatin. Blood samples for measurement of plasma TNF-α levels were taken 24 hours after cisplatin injection. Mice were sacrificed at 96 hours and kidneys were processed for histological analysis as described in the histology section below. Renal tubular damage was scored independently in a blinded manner by three investigators.

[0090] Collagen-antibody induced arthritis model Mice were injected intravenously with 4 mg of antibody cocktail against collagen II (Chondrex) per mouse on day 0, followed by an intraperitoneal injection of lipopolysaccharide (25 μg / mouse) on day 3. Severity of arthritis was assessed on day 8 according to a quantitative scoring system as follows: 0-normal, 1-mild but distinct redness of the ankle or wrist, or apparent redness and swelling limited to individual digits, 2-moderate redness and swelling of the ankle or wrist, 3-severe redness and swelling of the entire paw including digits, 4-maximally inflamed limb with involvement of multiple joints. Mice were sacrificed and hind paws were collected and processed for histological analysis as described below.

[0091] Histological examination Mouse ears, kidneys, or hind paws were harvested and fixed in 10% formalin for 24 hours. Hind paws were decalcified in 1M EDTA solution for 1 week. After paraffin embedding, 4 μm sagittal (paws), transverse (ears), or coronal (kidneys) sections were cut and stained with hematoxylin / eosin or periodic acid-Schiff stain as indicated. Tissue sections were imaged using a Leica DM2000 microscope equipped with an MC160HD camera using 5x and 20x objectives.

[0092] Confocal microscopy Mouse bone marrow-derived mast cells were seeded in complete medium containing IL-3 on IBIDI poly-lysine-coated microscope chambers for 16 h at 37°C in a humidified atmosphere containing 5% carbon dioxide, stimulated as indicated, washed with PBS, and fixed in PBS-4% paraformaldehyde for 15 min at room temperature. Permeabilization, blocking, washing, and incubation with antibodies were performed in PBS-0.1% saponin / 0.2% bovine serum albumin at 18°C. Image acquisition was performed on a Zeiss LSM700 with a 63x oil immersion objective. Images were analyzed and constructed using FIJI with the FigureJ plugin.

[0093] Imaging flow cytometry One million mouse bone marrow-derived mast cells were stimulated as indicated, fixed with 4% paraformaldehyde for 10 min, permeabilized with permeabilization buffer (Invitrogen), and stained for the indicated markers for 30 min at room temperature, followed by a washing step in permeabilization buffer and secondary staining with fluorescently labeled antibodies for 30 min at room temperature. Cells were washed and resuspended in PBS-2% fetal bovine serum. Image acquisition was performed using an ImageStreamXMkII multispectral imaging flow cytometer (Amnis, Seattle, USA) at 60x magnification, and acquired images were analyzed using IDEAS software (version 6.2; Amnis). For SNARE protein analysis, a Stx4-positive mask was defined and the mean pixel intensity of VAMP8 or VAMP3 was measured within the mask. For Golgi export assays, the Golgi mask was defined by staining for GM130, and the mean pixel intensity of TNF within the Golgi mask was quantified.

[0094] statistical analysis Values ​​are expressed as mean ± standard error unless otherwise stated. Statistical significance between two groups was analyzed using unpaired t-test with Welch's correction or one-sample t-test, where replicates were expressed as a proportion of the control group. P values ​​are indicated as follows: * p < 0.05; ** p < 0.01; *** p<0.001; **** p<0.0001, ns=not significant. Statistical analysis was performed using GraphPad Prism version 9.0.

[0095] result IRAP endosomes are not required for exocytosis of secretory granules To shed light on the role of IRAP endosomes in the exocytic trafficking pathway in mast cells, we first colocalized IRAP with various endosomal markers of early endosomes and GSV-like endosomes (data not shown). As in dendritic cells, IRAP colocalized well with the early endosome marker EEA1 and mast cell endocytosed transferrin, as well as with the GSV markers Rab14 and Stx6, which are involved in Golgi-to-endosome trafficking, confirming a high level of conservation of IRAP-associated vesicular trafficking machinery among various cell types. Interestingly, in activated cells, IRAP strongly colocalized with the granule-containing monoamine serotonin at the plasma membrane (data not shown), which is consistent with earlier studies. 31 Similar to the observations with histamine in , this prompted us to re-examine the role of IRAP in mast cell degranulation.

[0096] Physiologically, ligation of mast cell surface receptors, including cross-linking of cognate antigens with Fcε receptors through immunoglobulin E, activates a signaling cascade, the majority of which releases Ca from intracellular stores. 2+ Converging to release Ca 2+ Whether and how secretion of prestored granules versus newly synthesized mediators is regulated upon signal transduction is unclear.

[0097] Since we aimed to analyze the possible involvement of IRAP endosomes in exocytosis, apart from their hypothesized “upstream” role in the Fcε receptor-associated signaling cascade, 31 Therefore, we have exclusively used Fcε receptor-independent activation of mast cells throughout our studies.

[0098] The present inventors stimulated peritoneal mast cells with ionomycin / PMA or with the G protein-coupled receptor-dependent compound 48 / 80. 46In this study, degranulation was measured either as the release of the major granule component β-hexosaminidase into the culture supernatant (data not shown) or as the exocytosis of the lysosomal marker LAMP-1 in flow cytometry assays (data not shown). Degranulation was significantly increased in IRAP knockout (IRAPko) cells upon 48 / 80 activation, whereas stimulation with ionomycin / PMA led to a strong degranulation response without significant differences between IRAP-expressing and IRAP-deficient cells. As saturation effects could mask differences upon stimulation with ionomycin / PMA, we initiated an in vitro assay for the measurement of histamine release. This FRET (fluorescence resonance energy transfer)-based technique is quantitative over a wide range of histamine concentrations and detected significantly increased degranulation in the absence of IRAP for both types of stimulation (data not shown).

[0099] Exocytosis of secretory granules depends on the SNARE VAMP8. We observed no or low colocalization of IRAP with VAMP8 and preformed TNF-α stored in secretory granules of resting mast cells (data not shown). Prior to release, granule-bound VAMP8 associates with t-SNAREs Stx4 and SNAP23 at the plasma membrane or at intracellular degranulation channels (Moon et al., 2014). Even during the degranulation process, we could not observe any colocalization of IRAP with VAMP8 (data not shown), confirming their localization to distinct endosomal compartments.

[0100] However, we wondered whether the increased release of granule contents in IRAP knockout mast cells was reflected by increased complex formation between VAMP8 and Stx4 upon activation. Therefore, we quantified the colocalization of VAMP8-positive granules and Stx4 by imaging flow cytometry after stimulation with ionomycin / PMA. As expected, we observed more colocalization of Stx4 and VAMP8 in IRAP knockout mast cells than in wild-type (wt) mast cells (data not shown).

[0101] Next, we sought to assess degranulation in vivo. To this end, we challenged one ear of IRAP wild-type and knockout mice with arachidonic acid to degranulate, while leaving the other ear untreated. Arachidonic acid induces degranulation and cytokine production in mast cells through the prostaglandin EP receptor. 47 In line with our in vitro results, we detected significantly more histamine in crude homogenates of stimulated IRAP knockout mouse ears than in wild-type ears (data not shown), which was not due to a different density of mast cells in tissues of wild-type compared to IRAP knockout mice (data not shown).

[0102] To confirm the mast cell specificity of this test, we used mast cell-deficient kit-W sh / shMice (Wsh) were reconstituted with bone marrow-derived mast cells (BMMCs) from wild-type or IRAP knockout donor mice and challenged with unreconstituted Wsh mice. As expected, no histamine was detected in ear homogenates from mast cell-deficient, unreconstituted Wsh mice, but histamine secretion was increased in challenged ears of Wsh mice reconstituted with IRAP knockout mouse bone marrow-derived mast cells compared to wild-type mouse bone marrow-derived mast cells (data not shown). It is important to note that this assay does not distinguish between the source of the detected histamine, from an extracellular location after degranulation versus an intracellular reservoir. However, the histamine epitopes recognized in the antibody-based detection assay are likely more exposed after exocytosis, explaining the net increase in histamine detectable in challenged ears containing IRAP knockout mast cells, whereas the smaller amount of molecules released in wild-type ears may not have been detectable using this protocol due to the strong background signal generated by histamine from intracellular stores.

[0103] In conclusion, we show that IRAP endosomes are dispensable for the VAMP8-dependent regulated secretory pathway in mast cells and, furthermore, that in their absence, degranulation is increased in vitro and in vivo.

[0104] Homeostatic cytokine secretion relies on IRAP endosomes in mast cells Next to regulated secretion of stored granule contents, mast cells produce and secrete newly synthesized cytokines via the homeostatic secretory pathway. Although secretory vesicles of both species originate from the Golgi apparatus and bind Stx4 and SNAP23 for docking and fusion at the plasma membrane, they follow distinct post-Golgi trafficking pathways. Thus, regulated secretion is dependent on VAMP8, whereas newly synthesized cytokines in the homeostatic pathway in mouse mast cells co-stain with VAMP3.19 We observed that IRAP endosomes colocalized well with VAMP3 in mast cells (data not shown). VAMP3 is associated with the Golgi apparatus, transports to and from the recycling compartment, and is involved in the secretion of TNF-α in macrophages. 21 In mast cells, TNF-α is stored in small amounts in secretory granules and is produced and secreted de novo via a homeostatic pathway at late activation. TNF-α is transported throughout the cell as a transmembrane pro-cytokine. Release of soluble TNF-α into the extracellular space requires the activity of the TNF-α cleaving enzyme TACE. In the presence of the TACE inhibitor TAPI-I, TNF-α accumulated at the surface of activated cells starting 1 h after activation, where it strongly colocalized with IRAP (data not shown). Therefore, we hypothesized that IRAP may be involved in a homeostatic cytokine secretion pathway in mast cells.

[0105] Indeed, secretion of TNF-α and IL-6 was reduced by approximately 50% in IRAP knockout peritoneal mast cells compared to wild-type peritoneal mast cells after stimulation with ionomycin / PMA, as determined by ELISA (Fig. 1A) or flow cytometric analysis following TAPI-I treatment and TNF-α surface staining (Fig. 1B). Of note, secretion of the regulatory cytokine IL-10 was unaffected by the absence of IRAP (Fig. 1A).

[0106] To confirm that the observed secretory defects were due to transport defects in IRAP knockout cells, rather than reduced synthesis rates, we compared intracellular cytokine levels 4 h after activation under inhibition of Golgi / post-Golgi transport with Brefeldin A. Since we could not detect any significant difference in the amount of cytokines produced intracellularly between IRAP wild-type and knockout mast cells under these conditions (data not shown), we concluded that the absence of IRAP endosomes results in a transport defect of newly synthesized IL-6 and TNF within or across the Golgi. This defect also reduces the amount of VAMP3-containing vesicles colocalized with Stx4 at the plasma membrane of IRAP knockout cells, detectable by confocal imaging (data not shown) and imaging flow cytometry (data not shown).

[0107] Macrophages increase VAMP3 expression upon stimulation with lipopolysaccharide, presumably to accommodate the need for more transport machinery during increased cytokine synthesis. 21 To test whether the same was true for IRAP expression, we stimulated mast cells with lipopolysaccharide for various times and measured IRAP expression by intracellular flow cytometry. Indeed, IRAP was induced over time (data not shown), which is consistent with a role in the transport of proinflammatory cytokines.

[0108] IRAP endosomes are required for TNF-α secretion in vivo To quantify cytokine secretion by mast cells in vivo, we performed the mouse ear challenge experiment described above. Increased TNF-α and IL-6 levels were detected in wild-type ears within 45 min after challenge, whereas no cytokine release was observed in the ears of IRAP knockout animals (Figures 2A and B). Confirming our in vitro results, IL-10 secretion was not affected by the lack of IRAP endosomes in vivo (Figure 2C). In an effort to monitor the contribution of mast cells to the observed effects, we repeated the study in Wsh mice that had been reconstituted with wild-type or IRAP knockout mouse bone marrow-derived mast cells. Wsh mice reconstituted with IRAP knockout mast cells showed abnormal secretion of TNF-α and IL-6, indicating that the cytokines measured in this experimental setting could indeed be attributed to mast cells (Figures 2D and E).

[0109] The role of TNF-α in the pathogenesis of collagen-induced arthritis (CAIA) is well documented. 48、49 To investigate the relevance of IRAP endosomes to the secretion of TNF-α in this disease model, we challenged wild-type and IRAP knockout mice with an antibody cocktail directed against arthritic collagen. Eight days after induction of arthritis, wild-type mice showed signs of joint inflammation characterized by intense redness, swelling of the paws and joints, and difficulty in walking, whereas the majority of IRAP knockout mice showed no or only mild symptoms (Figure 2F). Histological analysis of the knee (data not shown) and ankle (data not shown) revealed joint swelling accompanied by intense infiltration of inflammatory cells into the synovial cavity and bone erosion in wild-type animals. IRAP knockout mice showed less or no infiltration, less swelling, and no bone damage. We conclude that IRAP is required for the intense inflammatory disease phenotype observed in wild-type mice.

[0110] The role of mast cells in this model is Kit-Wv / v Mice may have a higher phenotype, possibly due to differences in their megakaryocyte populations. 51 , but Wsh mice were not protected from collagen-induced arthritis. 13、50 As an alternative approach, we turned to a cisplatin-induced renal inflammation model that was previously reported to be dependent on mast cell-derived TNF-α. 12 Cisplatin is an efficient and widely used cytostatic agent for cancer therapy, but its tolerability is limited by the frequent adverse effect of acute kidney injury. We hypothesized that TNF-α-dependent kidney injury after cisplatin administration, characterized by tubular apoptosis, necrosis, and inflammation, would be attenuated in IRAP knockout mice. To confirm this, we histologically analyzed the kidneys of IRAP wild-type and knockout mice 96 hours after intraperitoneal injection of cisplatin. HE (hematoxylin and eosin) staining (data not shown) and PAS (periodic acid-Schiff reagent) staining (data not shown) of paraffin-embedded kidney samples revealed visibly reduced tubular injury in IRAP knockout animals, resulting in significantly lower injury scores, which was determined independently by blinded evaluation in three different experiments (Figure 2G). These observations were consistent with the significantly decreased plasma TNF-α levels in cisplatin-treated IRAP knockout mice (Figure 2H). Cisplatin-induced inflammation and nephrotoxicity are mediated via Toll-like receptor 4. 52 To exclude the possibility that different expression levels of Toll-like receptor 4 in wild-type versus IRAP knockout cells were responsible for the observed effects, we confirmed equivalent surface expression of Toll-like receptor 4 in wild-type and IRAP knockout mast cells (data not shown).

[0111] To evaluate the contribution of mast cells to these effects, we administered cisplatin to Wsh mice reconstituted with wild-type or IRAP knockout bone marrow-derived mast cells. Histological scoring of injury levels showed that the mean renal injury in mice reconstituted with IRAP knockout mast cells was reduced compared to mice reconstituted with wild-type mast cells (Figure 2I), but the difference was less pronounced than that between wild-type mice vs. systemic IRAP knockout mice. The involvement of other TNF-α-producing cell types in the tested cisplatin model may explain the slight differences in the experiments with mast cell-reconstituted mice. Therefore, we addressed the secretion of TNF-α in peritoneal macrophages using the confocal imaging and TAPI-based flow cytometry assays described above. IRAP strongly colocalized with TNF-α in ionomycin-activated macrophages (data not shown). Furthermore, IRAP knockout macrophages showed reduced surface staining of TNF-α after 4 h activation with ionomycin / PMA or lipopolysaccharide (data not shown). We conclude that macrophages also depend on IRAP endosomes for efficient secretion of TNF-α via the constitutive pathway.

[0112] In sum, IRAP knockout mast cells, and possibly other immune cell types, secrete less TNF-α in vivo, leading to a milder phenotype in TNF-α-dependent disease models.

[0113] IRAP is required for export of TNF-α transport vesicles by the Golgi apparatus We next sought to determine at what stage exocytic cytokine transport was impaired in the absence of IRAP. To this end, we analyzed the colocalization of Stx6 and VAMP3 in activated mast cells. Stx6 decorates IRAP vesicles in various cell types and is present on TNF-α carriers after budding from the Golgi apparatus in macrophages. 21、22Stx6 colocalized well with VAMP3 at the plasma membrane in activated mast cells, but due to the overall reduced staining of Stx6 in the periphery, significantly less Stx6 was detected in the VAMP3-stained areas in IRAP knockout cells (data not shown). Total VAMP3 levels are also reduced in IRAP knockout mast cells (data not shown).

[0114] These observations prompted us to investigate whether TNF-α carriers require IRAP for budding from the Golgi apparatus. Therefore, we adapted a previously published Golgi egress assay. 53 Lipopolysaccharide-preactivated cells were incubated at 20°C for 3 h to enrich for Golgi cytokines. A subsequent temperature shift to 37°C reactivated the budding of exocytic transport vesicles from the Golgi, allowing the analysis of Golgi export kinetics of cytokines in homeostatic pathways.

[0115] After 3 h at 20°C, TNF-α strongly colocalized with the Golgi marker GM130 in both wild-type and IRAP knockout cells (data not shown), indicating successful inhibition of Golgi export under these conditions. Reactivation of exocytic transport led to progressive export of TNF-α from the Golgi in wild-type cells, whereas a net accumulation was observed over the first 30 min in IRAP knockout cells, indicating that the translation rate exceeds the export rate in these cells (data not shown). At 50 min, IRAP-expressing cells had almost emptied TNF-α from the Golgi, whereas colocalization of TNF-α with GM130 persisted in IRAP knockout cells (data not shown).

[0116] Considering that the cytosolic pool of IRAP vesicles upon activation has been proposed to traffic to the plasma membrane without passing through the Golgi apparatus, 38、54However, under prolonged activating signaling, IRAP is reinternalized and retrieved from sorting endosomes to the Golgi apparatus via the action of retromer. 36 Therefore, we speculated that inhibition of endocytosis might alter the intracellular localization of IRAP and ultimately alter the secretion of TNF-α. Indeed, in the presence of the dynamin inhibitor Dynasore, IRAP showed strong plasma membrane staining 3 hours after activation with lipopolysaccharide (data not shown), indicating efficient inhibition of re-internalization of IRAP. Consistently, both Dynasore and the phosphatidylinositol 3 kinase I inhibitor GDC-0941 were able to specifically reduce the secretion of TNF-α in wild-type cells (data not shown). These results suggest that internalization of IRAP is essential for normal TNF-α transport (data not shown).

[0117] Inhibition of IRAP by HFI-419 destabilizes IRAP endosomes Given the aminopeptidase function of IRAP, we wondered whether its catalytic activity is required for efficient cytokine secretion. To test this, we treated mast cells with the IRAP inhibitor HFI-419, 4u prior to activation with ionomycin / PMA. 55 and 22b (a gift from E. Stratikos) for 24 h. Although all three inhibitors showed a tendency to inhibit IRAP-dependent cytokine secretion in vitro, only HFI-419 mediated significant inhibition (data not shown) and was therefore selected for further in vivo studies.

[0118] Vehicle or inhibitors at a dose of 1 μmol / kg were administered intravenously 24 hours and 15 minutes before ear challenge. HFI-419-treated animals secreted significantly less TNF-α and IL-6 than vehicle-treated animals, whereas IL-10 secretion was unaffected, indicating that availability of the catalytic domain of IRAP was required, directly or indirectly, for the transport of these proinflammatory cytokines (FIG. 3). This is somewhat surprising given previous studies in various cell types in which reconstitution with a protease-ablated IRAP mutant fully restored vesicle distribution and endosomal trafficking in IRAP knockout cells, suggesting that enzymatic activity is not required for IRAP-mediated transport function. 43、45 Therefore, we hypothesized that in addition to blocking its catalytic activity, HFI-419 might also affect the stability of IRAP. In accordance with this hypothesis, using intracellular flow cytometry staining, we detected decreased expression levels of IRAP and VAMP3 starting from 24 h after treatment with HFI-419 (data not shown), suggesting that ligation of the inhibitor HFI-419 induced the degradation of IRAP and ultimately prevented the secretion of TNF-α.

[0119] Consideration: In this study, we demonstrate that IRAP controls the secretion of late pro-inflammatory cytokines in mast cells in vitro and in vivo. 2+ We found that in IRAP-knockout mast cells activated by IL-1, secretion of TNF-α and IL-6 was reduced compared to wild-type cells. This was due to abnormal transport rather than decreased cytokine synthesis. Because intracellular Ca 2+Intracellular cytokine levels in response to triggers were comparable between wild-type and IRAP knockout cells. The observed inhibition of cytokine secretion was of the order of 50%, a reduction that was physiologically relevant, since IRAP knockout mice displayed milder disease phenotypes in two experimental models of TNF-α-dependent pathologies: collagen-induced arthritis and cisplatin-induced acute kidney injury.

[0120] Previous reports have demonstrated that recycling of the endosome-associated SNARE VAMP3 occurs in mast cells. 17、19 and macrophages 21 We extend these findings by showing that in the absence of IRAP, the amount of VAMP3 colocalized with Stx4, a SNARE involved in vesicle fusion with the plasma membrane, was reduced, most likely due to the observed decreased formation or stabilization of Stx6-positive post-Golgi carriers in IRAP knockout cells.

[0121] Consistently, Golgi export assays confirmed that newly synthesized TNF-α remained in the Golgi for a longer period in IRAP knockout mast cells. Collectively, these results suggest a role for IRAP in the formation of Stx6-positive carriers responsible for the transport of TNF-α and IL-6 in the trans-Golgi network.

[0122] The widespread localization of IRAP to a sequestered pool of cytoplasmic vesicles at steady state is difficult to reconcile with a role as a sorting effector in the trans-Golgi network. However, IRAP vesicles are recruited in response to specific activation signals that trigger cleavage of the cytoplasmic retention protein TUG and transport of IRAP to the cell surface. 34 Importantly, under prolonged stimulation, IRAP is suggested to be recycled back to the plasma membrane through endosomes and the Golgi apparatus without trafficking through a storage pool. 38、54In this study, we identify this IRAP exocytosis / recycling pathway as overlapping with homeostatic cytokine secretion. Furthermore, the abnormal secretion of TNF-α in the presence of endocytosis inhibitors, specifically observed in IRAP wild-type cells, suggests that endocytosis of IRAP is required for efficient post-Golgi cytokine trafficking. In summary, we suggest that mast cell activation leads to the recruitment of IRAP to the plasma membrane, re-internalization, and retrieval to the trans-Golgi network, where it functions as a sorting receptor for cytokines and possibly other molecules secreted along the homeostatic pathway.

[0123] Post-Golgi transport vesicles containing TNF-α and IL-6 form through fission of the luminal compartment from the trans-Golgi network. Budding of these luminal carriers occurs from different trans-Golgi network subdomains and depends on different coiled-coil golgins. 56 For example, the transporter involved in the exit of TNF-α from the Golgi apparatus is golgin-245 / p230 positive. 57 In addition, sorting and export of Glut4 and IRAP to the adipocyte-sequestered GSV pool is golgin-160 dependent. Importantly, upon golgin-160 depletion, Glut4 is targeted to the plasma membrane. 58 We therefore speculate that under sustained activation, the interaction between IRAP and golgin-160 is abolished, possibly through posttranslational modifications of IRAP, altering the post-Golgi trafficking of IRAP and sorting it into a distinct, most likely golgin-245-dependent pathway to the plasma membrane.

[0124] Interestingly, IL-10 secretion was unaffected by loss of IRAP. Studies in macrophages, where the secretory pathways of TNF-α, IL-6, and IL-10 have been studied in detail, have revealed that these three cytokines may use a common pathway from the trans-Golgi network to recycling endosomes, but that IL-10 instead uses a distinct post-Golgi pathway that overlaps with the trafficking of the lipoprotein apolipoprotein E. 59 Based on our results, we suggest that, at least in mast cells, a small proportion of IL-10 is transported along the same IRAP-dependent pathway as IL-6 and TNF-α.

[0125] With regard to regulated exocytosis, we observed increased secretory granule release in mast cells in the absence of IRAP. Consistently, more VAMP8 staining was observed on Stx4-positive membrane domains, indicating that fusion events between secretory granules and the plasma membrane were increased in IRAP knockout cells compared to wild-type cells.

[0126] This dichotomy between impaired constitutive trafficking and enhanced trafficking of secretory lysosomes / granules is reminiscent of reports in sortilin knockout cytotoxic T cells and natural killer cells, in which sortilin has been suggested to regulate both the targeting of VAMP7 to lysosomes and the constitutive secretion of interferon-γ (but not TNF-α). 53 .

[0127] Although a direct role of IRAP in targeting to lysosomes or degradation of Vamp8 seems unlikely given the lack of colocalization between these two proteins, we cannot exclude IRAP-dependent protein trafficking that negatively regulates VAMP8 degradation. Alternatively, given that the same SNARE docking and fusion mechanisms are used for exocytosis of VAMP3-positive vesicles and VAMP8-positive granules, a reduced amount of VAMP3-positive carriers at the plasma membrane could leave more Stx4-SNAP23 molecules available for the formation of VAMP8-SNARE complexes, ultimately increasing the rate of VAMP8-dependent degranulation. Furthermore, the activity of some VAMP family members, including VAMP8, could be regulated via phosphorylation through protein kinase C, which terminates the degranulation response. 60 This regulation likely prevents the dangerous consequences of excessive degranulation from mast cells, particularly anaphylactic shock. In contrast, this level of regulation is absent in VAMP3 due to the absence of phosphorylation motifs. 60 This suggests that other regulatory mechanisms may exist, and the involvement of signal-responsive IRAP endosomes in VAMP3-dependent exocytosis may constitute such a mechanism, i.e., linking extracellular cues to cytokine transport.

[0128] Expression of IRAP protein was induced by lipopolysaccharide, which is consistent with previous reports showing induction of IRAP mRNA by lipopolysaccharide and interferon-γ, but not by tumor growth factor-β, in macrophages. 61 These findings suggest that IRAP endosomes are part of a transcriptionally regulated trafficking mechanism induced by proinflammatory environmental cues. In particular, in light of the recently emerging polarization concept of mast cell function in inflammation and cancer, similar to the M1 vs. M2 polarization of macrophages, the transcriptional regulation of IRAP endosomes merits further investigation.

[0129] We also showed that macrophages depend on IRAP expression for secretion of TNF. Given the broad expression profile of IRAP among immune cells, IRAP may regulate cytokine secretion in other cell types, particularly those that require maintaining temporal or spatial separation between regulatory and homeostatic secretion of storage granules, such as platelets, cytotoxic T cells, natural killer cells, and basophils.

[0130] Finally, IRAP expression was reduced using the chemical inhibitor HFI-419, which binds to a substrate-binding pocket within the intraluminal region of IRAP. 62 The reduction in protein levels is essential for the regulated transport of IRAP, as well as for several proteins involved in vesicular transport, e.g., formins. 44、64 , Tankiraze 65 and p115 66 These results strongly suggest a trans-acting conformational effect on the cytoplasmic tail of IRAP, which contains a specific motif for its interaction with β-lactamase. 63 We previously showed that loss of IRAP tethering to the actin cytoskeleton promoted destabilization and degradation of IRAP endosomes through rapid retrograde dynein-mediated transport and fusion with lysosomes. 42、44 .

[0131] In summary, our results identify IRAP as a central transcriptionally regulated late cytokine secretion center in mast cells and a promising target for the development of anti-inflammatory drugs.

[0132] References: Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into this disclosure. [Table 1] TIFF2024543981000018.tif249165 TIFF2024543981000019.tif248165 TIFF2024543981000020.tif241165 TIFF2024543981000021.tif249165 TIFF2024543981000022.tif59165

Claims

1. A pharmaceutical composition for treating an inflammatory disease in a patient, comprising a therapeutically effective amount of an IRAP inhibitor.

2. Inflammatory diseases include arthritis, rheumatoid arthritis, acute arthritis, chronic rheumatoid arthritis, gouty arthritis, acute gouty arthritis, chronic inflammatory arthritis, degenerative arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, spondyloarthritis, and juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, arthritis deformans, chronic primary polyarthritis, reactive arthritis, and ankylosing arthritis), inflammatory hyperproliferative skin diseases; Psoriasis, such as plaque psoriasis, guttate psoriasis, pustular psoriasis, and nail psoriasis; Dermatitis, including contact dermatitis, chronic contact dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, and atopic dermatitis; systemic sclerosis, sclerosis, systemic sclerosis, multiple sclerosis (MS), spino-optic MS, primary progressive MS (PPMS), relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, and ataxic sclerosis, inflammatory bowel disease (IBD), Crohn's disease, colitis, ulcerative colitis colitis), ulcerative colitis (colitis) ulcerosa), microscopic colitis, collagenous colitis, polypoid colitis, necrotizing enterocolitis, transmural colitis, autoimmune inflammatory bowel disease, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, episcleritis, respiratory distress syndrome, adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune blood disorders, rheumatoid spondylitis, sudden hearing loss; IgE-mediated diseases such as anaphylaxis and allergic and atopic rhinitis; encephalitis, Rasmussen's encephalitis, limbic and / or brainstem encephalitis, uveitis, anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, phacoantigenic uveitis uveitis), posterior uveitis, autoimmune uveitis, glomerulonephritis (GN), idiopathic membranous GN or idiopathic membranous nephropathy, membranous or membranoproliferative GN (MPGN), rapidly progressive GN, allergic conditions, autoimmune myocarditis, leukocyte adhesion deficiency; systemic lupus erythematosus (SLE) or systemic lupus erythematodes, e.g., cutaneous SLE; subacute cutaneous lupus erythematosus, neonatal lupus syndrome (NLE), disseminated lupus erythematosus, lupus (including nephritic, encephalitis, pediatric, non-renal, extrarenal, discoid, alopecia); juvenile-onset (Type 1) diabetes, including pediatric insulin-dependent diabetes mellitus (IDDM);Adult-onset diabetes mellitus (type II diabetes), autoimmune diabetes, idiopathic diabetes insipidus, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis; vasculitis, including large-vessel vasculitis; polymyalgia rheumatica, giant cell (Takayasu) arteritis, medium-sized vasculitis, Kawasaki disease, polyarteritis nodosa, microscopic polyarteritis, CNS vasculitis, necrotizing, cutaneous, hypersensitivity vasculitis, systemic necrotizing vasculitis; and ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS); temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs-positive anemia, Diamond-Blackfan anemia; autoimmune hemolytic anemia (AIHA), pernicious anemia anemia) (hemolytic anemia or immune hemolytic anemia, including anemia perniciosa; Addison's disease, pure red cell anemia or aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory disorders; multiple organ injury syndromes, for example, secondary to sepsis, trauma, or hemorrhage; diseases mediated by antigen-antibody complexes, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Bechet's or Behcet's disease, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjogren's syndrome, Stevens-Johnson syndrome; pemphigoid, for example, bullous pemphigoid and cutaneous pemphigoid; pemphigus, optionally pemphigus vulgaris;Pemphigus foliaceus, mucous membrane pemphigoid pemphigoid), pemphigus erythematosus, autoimmune polyendocrinopathy, Reiter's disease or syndrome, immune complex nephritis, antibody-mediated nephritis, neuromyelitis optica, polyneuritis, chronic neuropathy, IgM polyneuropathy, IgM-mediated neuropathy, thrombocytopenia, thrombotic thrombocytopenic purpura (TTP), idiopathic thrombocytopenic purpura (ITP), autoimmune orchitis and oophoritis, primary hypothyroidism, hypoparathyroidism, autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, autoimmune thyroid disease, idiopathic hypothyroidism, Graves' disease; polyglandular syndromes, e.g., autoimmune polyglandular syndrome (or polyendocrinopathy syndrome); paraneoplastic syndromes, including neurological paraneoplastic syndromes (e.g., Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome); stiff-man or stiff-person syndrome syndrome), encephalomyelitis, allergic encephalomyelitis, experimental allergic encephalomyelitis (EAE), myasthenia gravis, thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or clonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia, bronchiolitis obliterans (non-transplant related) vs. NSIP, Guillain-Barré syndrome, Berger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, primary biliary cirrhosis, pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease, Coeliac disease disease), celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease; autoimmune ear diseases, e.g., autoimmune inner ear disease (AGED), autoimmune hearing loss; opsoclonus-myoclonus syndrome (OMS); polychondritis, e.g., refractory or relapsing polychondritis; pulmonary alveolar proteinosis, amyloidosis, scleritis, non-cancerous lymphocytosis; primary lymphocytosis, including monoclonal B-cell lymphocytosis;Monoclonal gammopathy or garnmopathy of undetermined significance, possibly benign; peripheral neuropathy, paraneoplastic syndromes; channelopathies, e.g., epilepsy, migraine, cardiac arrhythmias, myopathy, hearing loss, blindness, periodic paralysis, and channelopathies of the CNS; autism, inflammatory myopathies, focal segmental glomerulosclerosis (FSGS), endocrine ophthalmopathy, uveitis, chorioretinitis, autoimmune liver disease, fibromyalgia, multiple endocrine deficiencies, Schmidt's syndrome, adrenal inflammation, gastric atrophy, presenile dementia; demyelinating diseases, e.g., autoimmune demyelinating diseases; diabetic nephropathy, Dressler's syndrome , alopecia areata, crest syndrome (calcinosis, Raynaud's phenomenon, esophageal hypoperistalsis, digital sclerosis), and telangiectasia), male and female autoimmune infertility, mixed connective tissue disease, Chagas' disease, rheumatic fever, recurrent abortions, farmer's lung, erythema multiforme, post-cardiotomy syndrome, Cushing's syndrome, bird breeder's lung, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport's syndrome; alveolitis, e.g., allergic alveolitis and fibrosing alveolitis; interstitial lung disease, transfusion reactions, leprosy, malaria, leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Sampter's syndrome syndrome), Kaplan's syndrome, dengue fever, endocarditis, endophthalmitis, persistent erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Schulman's syndrome, Felty's syndrome, filariasis; Cyclitis, e.g., chronic cyclitis, heterochromatic cyclitis, iridocyclitis or Fuchs' cyclitis;Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, echovirus infection, cardiomyopathy, Alzheimer's disease, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, mumps, Evans syndrome, autoimmune dysgonadism, Sydenham's chorea, post-streptococcal nephritis, thromboangitis ubiterans), thyrotoxicosis, tabes dorsalis, choroiditis, giant cell polymyalgia, endocrine ophthalmopathy, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephrosis, benign familial and ischemia-reperfusion injury, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway disease, silicosis, aphthous stomatitis, arteriosclerotic disorders, aspermiogenese, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, phacosensitivity endophthalmitis, allergic enteritis, erythema nodularis plaques, idiopathic facial paralysis, chronic fatigue syndrome, rheumatic fever, Hammann-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, focal ileitis, leukopenia, infectious mononucleosis, transverse myelitis, primary idiopathic myxedema, nephrosis, sympathetic ophthalmia, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired atrophic spleen, infertility due to antisperm antibodies, nonmalignant thymoma, vitiligo, SCID and Epstein-Barr virus related diseases, acquired immune deficiency syndrome (AIDS); parasitic diseases such as leishmaniasis;Toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte extravasation, multiple organ injury syndrome, diseases mediated by antigen-antibody complexes, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmia, rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine deficiency, peripheral nerve menstrual disorders, autoimmune polyendocrine syndrome type I, adult-onset idiopathic hypothyroidism (AOIH), alopecia totalis, dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, maxillary sinusitis, or sphenoid sinusitis; eosinophil-related disorders, e.g., eosinophilia, pulmonary infiltrates with eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, focal pulmonary eosinophilia, Bronchopneumonic aspergillosis, aspergilloma, or eosinophil-containing granuloma; anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, or chronic mucocutaneous candidiasis, Bruton's syndrome, transient infantile hypogammaglobulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia, autoimmune disorders associated with connective tissue disease, rheumatism, neurological disorders, ischemia-reperfusion injury, decreased blood pressure response, vascular insufficiency, vasodilation, tissue damage, cardiovascular ischemia, hyperalgesia, and cerebral ischemia 2. The pharmaceutical composition of claim 1, wherein the therapeutic agent is selected from the group consisting of diseases involving blood and angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, reperfusion injury of the myocardium or other tissues, skin diseases with an acute inflammatory component, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, acute severe inflammation, chronic refractory inflammation, pyelitis, pulmonary cirrhosis, diabetic retinopathy, diabetic aortopathy, intimal hyperplasia, peptic ulcer, valvulitis, and endometriosis.

3. 2. The pharmaceutical composition of claim 1, wherein the inflammatory disease is an allergic disorder, asthma, anaphylaxis, or an inflammatory disease subsequent to treatment with an immune checkpoint inhibitor.

4. The pharmaceutical composition according to claim 1, wherein the inflammatory disease is chemotherapy-induced inflammation.

5. 1. The IRAP inhibitor of formula (I): 【Chemical 1】 A is aryl, heteroaryl, carbocyclyl, or heterocyclyl, each of which is selected from the group consisting of aryl, heteroaryl, carbocyclyl, and heterocyclyl. ８ or quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridyl, phthalazinyl, or pteridinyl (each of which is optionally substituted when R1 is NR ７ R ８ , NHCOR ８ , N(COR ８ ) ２ , N(COR ７ ) (COR ８ ), N=CHOR ８ or N=CHR ８ (optionally substituted when X is O, NR', or S, where R' is hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted acyl, optionally substituted heteroaryl, optionally substituted carbocyclyl, or optionally substituted heterocyclyl; R ７ and R ８ are independently selected from hydrogen, optionally substituted alkyl, optionally substituted aryl, or R ７ and R ８ together with the nitrogen atom to which they are attached form a 3- to 8-membered ring, which may be optionally substituted; R2 is CN, CO ２ R ９ ,C(O)O(O)R ９ , C(O)R ９ , or C(O)NR ９ R １０ where R ９ and R １０ is independently selected from alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocyclyl, heterocyclyl (each of which is optionally substituted), and hydrogen; or R ９ and R １０ together with the nitrogen atom to which they are attached form a 3- to 8-membered ring, which may be optionally substituted; R ３ ~R ６ are independently selected from hydrogen, halo, nitro, cyano alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, carbocyclyl, hydroxy, alkoxy, alkenyloxy, alkynyloxy, aryloxy, heteroaryloxy, heterocyclyloxy, amino, acyl, acyloxy, carboxy, carboxyester, methylenedioxy, amido, thio, alkylthio, alkenylthio, alkynylthio, arylthio, heteroarylthio, heterocyclylthio, carbocyclylthio, acylthio, and azido, each of which is optionally substituted, where appropriate, or any two adjacent R ３ ~R ６ together with the atoms to which they are attached form a 3- to 8-membered ring, which may be optionally substituted; and Y is hydrogen or C １ ~C １０ alkyl) 10. The pharmaceutical composition of claim 1, having a structure according to: or a pharmaceutically acceptable salt or solvate thereof.

6. 1. An IRAP inhibitor having the structure: 【Chemistry 2】 2. The pharmaceutical composition of claim 1, comprising:

7. The IRAP inhibitor has the formula (II): 【Chemistry 3】 (In the formula, A is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted; R A and R B are independently selected from hydrogen, alkyl, and acyl; R1 is selected from CN or CO2RC; R2 is selected from CO2RC and acyl; R3 is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted; or R2 and R3 together form a 5-6 membered saturated keto carbocyclic ring: 【Chemistry 4】 wherein n is 1 or 2; the ring is optionally substituted one or more times with C1-6 alkyl. or R2 and R3 together form a 5-membered lactone ring (a) or a 6-membered lactone ring (b). 【Chemistry 5】 (In the formula, 【Chemistry 6】 is an optional double bond, and R' is alkyl. Forming Rc is selected from alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, carbocyclyl, carbocyclylalkyl, each of which is optionally substituted.

10. The pharmaceutical composition of claim 1, having a structure according to: or a pharmaceutically acceptable salt, solvate or prodrug thereof.

8. 5. The method of claim 1, wherein the IRA inhibitor has the structure: 【Chemistry 7】 2. The pharmaceutical composition of claim 1, comprising:

9. The IRAP inhibitor is 【Chemistry 8】 【change】 10. The pharmaceutical composition of claim 1, having a structure selected from the group consisting of: and / or a pharmaceutically acceptable salt, solvate, or prodrug thereof.

10. The IRAP inhibitor is represented by formula (III): 【Chemistry 9】 (In the formula, R1 is H or CH ２ COOH; n is 0 or 1; m is 1 or 2; W is CH or N.

10. The pharmaceutical composition of claim 1, having a structure according to: or a pharmaceutically acceptable salt, solvate or prodrug thereof.

11. 5. The method of claim 1, wherein the IRA inhibitor has the structure: 【Chemistry 10】 2. The pharmaceutical composition of claim 1, comprising:

12. 1. An IRAP inhibitor having the structure: 【Chemistry 11】 2. The pharmaceutical composition of claim 1, comprising:

13. 2. The pharmaceutical composition of claim 1, wherein the IRAP inhibitor is (±)-ethyl-2-acetamido-7-hydroxy-4-(pyridin-3-yl)-4H-chromene-3-carboxylate (HFI-419).

14. The pharmaceutical composition of claim 1 , wherein the IRAP inhibitor is an inhibitor of IRAP expression, such as an siRNA or an antisense oligonucleotide.

15. The pharmaceutical composition of claim 1 , wherein the IRAP inhibitor causes destabilization and / or degradation of IRAP.

Images