Composition for treating and / or preventing granuloma or disease associated with granuloma, and method and kit for determining whether subject has granuloma or not

Abstract

Provided is a composition for treating and / or preventing granuloma, the composition containing an inhibitor of an enzyme involved in the pentose phosphate pathway. Also provided is a composition for treating and / or preventing a disease associated with granuloma, the composition containing an inhibitor of an enzyme involved in the pentose phosphate pathway. Further provided are a method and a kit for determining whether a subject has granuloma or not on the basis of the level of an enzyme involved in the pentose phosphate pathway in a sample collected from the subject.

Description

Composition for treating and / or preventing granulomas or diseases associated with granulomas, and method and kit for determining whether a subject has granulomas

[0001] This patent application claims priority to Japanese Patent Application No. 2023-218324, the entire contents of which are incorporated herein by reference. The present disclosure relates to compositions for treating and / or preventing granulomas or diseases associated with granulomas, and methods and kits for determining whether a subject has granulomas.

[0002] Granulomas are a type of chronic inflammation that occurs as a defense response against pathogenic microorganisms and insoluble foreign bodies, and are formed by the accumulation of various inflammatory cells, primarily macrophage cells. Diseases known to involve granulomas include mycobacterial infections such as sarcoidosis, tuberculosis, and leprosy, as well as granuloma annulare and Crohn's disease.

[0003] Sarcoidosis is a disease that causes granulomas in the skin, lungs, eyes, heart, and other organs, and can be fatal if it causes pulmonary fibrosis or arrhythmia. The cause is currently unknown, and no radical treatment is available. JAK inhibitors are a potential new therapeutic agent for sarcoidosis, and clinical trials are currently underway (Non-Patent Document 1). However, JAK inhibitors have systemic side effects, such as carcinogenicity and immunosuppression, making them unsuitable for long-term use.

[0004] Granulomas are diagnosed histopathologically, and while granulomas in the skin are relatively easy to diagnose, it is difficult to diagnose granulomas in the lungs, eyes, heart, liver, kidneys, lymph nodes, nerves, muscles, etc. Angiotensin-converting enzyme (ACE) is used as a marker in blood tests for sarcoidosis.

[0005] William Damsky, Durga Thakral, Nkiruka Emeagwali, Anjela Galan, Brett King, Tofacitinib Treatment and Molecular Analysis of Cutaneous Sarcoidosis, N Engl J Med. 2018 Dec 27;379(26):2540-2546

[0006] One object of the present disclosure is to provide a composition for treating and / or preventing granulomas or diseases associated with granulomas. One object of the present disclosure is to provide a method or kit for determining whether a subject has a granuloma.

[0007] The present inventors have found that the levels of enzymes related to the pentose phosphate pathway are high in macrophages contained in granulomas and in the blood of sarcoidosis patients, and further found that inhibition of enzymes related to the pentose phosphate pathway suppresses granuloma formation.

[0008] Thus, in one aspect, a composition comprising an inhibitor of an enzyme associated with the pentose phosphate pathway for treating and / or preventing granulomas is provided.

[0009] In one aspect, a composition for treating and / or preventing a disease associated with granulomas is provided, comprising an inhibitor of an enzyme associated with the pentose phosphate pathway.

[0010] In one aspect, a method is provided for determining whether a subject has a granuloma based on the level of an enzyme associated with the pentose phosphate pathway in a sample taken from the subject.

[0011] In one aspect, a kit for determining whether a subject has a granuloma is provided, comprising a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway or a reagent for measuring gene expression of an enzyme associated with the pentose phosphate pathway.

[0012] The present disclosure is useful for treating and / or preventing granulomas or diseases associated with granulomas, or for determining whether a subject has granulomas.

[0013] Figure 1 shows clustering of antigen-presenting cells in normal skin and sarcoidosis skin by gene expression. Figure 2 shows the fractional proportions of antigen-presenting cells in normal skin and sarcoidosis skin. Figure 3 shows fractional gene expression of antigen-presenting cells in sarcoidosis skin. Figure 4 shows immunostaining images of CD68, FBP1, and CD163 in normal skin and sarcoidosis skin lesions. Figure 5 shows the expression of ACE, FBP1, and STAT1 in normal skin, atopic dermatitis, psoriasis, granuloma annulare, leprosy, and sarcoidosis skin lesions. Figure 6 shows immunostaining images of CD163 and FBP1 in normal skin, atopic dermatitis, psoriasis, sarcoidosis, and granuloma annulare skin lesions. Figure 7 shows immunostaining images of CD163, FBP1, and CD68 in sarcoidosis lesions of the lung, heart, and lymph nodes. Figure 8 shows a schematic diagram of the glucose metabolic pathway. Figure 1 shows gene expression (pentose phosphate pathway-related genes) in antigen-presenting cells of sarcoidosis skin. Figure 2 shows gene expression (G6PD, FBP1, TXNRD1) in antigen-presenting cells of sarcoidosis skin. Figure 3 shows immunostaining images of CD163, FBP1, and G6PD in sarcoidosis lesions of the skin, lung, heart, and lymph nodes. Figure 4 shows immunostaining images of FBP1 and TXNRD1 in sarcoidosis lesions of the skin. Figure 5 shows serum FBP1 concentrations in sarcoidosis patients and healthy controls. Figure 6 shows Giemsa-stained images of human monocytes after culturing for 3 days in the presence of cytokines. Figure 7 shows FBP1 and G6PD gene expression in human monocytes after culturing for 3 days in the presence of cytokines. Figure 8 shows Giemsa-stained images and giant cell counts in human monocytes after culturing for 3 days in the presence of cytokines and FBP inhibitors, 6AN and tofatinib. Figure 1 shows Giemsa-stained images and the number of giant cells after human monocytes were cultured for 3 days in the presence of cytokines and MB05032 or MB07229. Figure 2 shows Giemsa-stained images after human monocytes were cultured for 3 days in the presence of cytokines and auranofin or TRi-1. Figure 3 shows Giemsa-stained images, FBP1 gene expression, and fusion rate after human monocytes were cultured for 3 days in the presence of cytokines and siRNA against the FBP gene. Figure 4 shows Giemsa-stained images and the number of giant cells after human monocytes were cultured for 3 days in the presence of cytokines and an additional 3 days in the presence of an FBP inhibitor, 6AN, or tofatinib.Giemsa-stained images of human monocytes after culturing them in the presence of cytokines for 3 days and then culturing them in the presence of auranofin or TRi-1 for another 3 days are shown. Representative images of skin tissue from 6AN-administered and -unadministered groups of inflammation-induced mice are shown. The ear thickness of 6AN-administered and -unadministered groups of inflammation-induced mice is shown. CD4 infiltrating into the ear of inflammation-induced mice. ＋ T cells, CD8 ＋ The numbers of T cells, macrophages, and monocytes were compared between the 6AN-treated and untreated groups. Giemsa-stained images and the number of giant cells are shown after human monocytes were cultured for 3 days in the presence of cytokines and various concentrations of MB07803. Arrows indicate giant cells. Giemsa-stained images and the number of giant cells are shown after human monocytes were cultured for 3 days in the presence of cytokines and various concentrations of MB06322. Giemsa-stained images and the number of giant cells are shown after human monocytes were cultured for 3 days in the presence of cytokines and 100 nM MB05032, MB07229, or MB07803, and the number of giant cells after 3 days in the presence of various concentrations of each inhibitor. Representative skin histological images are shown for inflammation-induced mice in the MB07803-treated and untreated groups.

[0014] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0015] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" is intended to include "18 to 22." A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. Thus, for example, "about 20 to 30" is intended to include "18 to 33."

[0016] In the present disclosure, the subject may be of any species, and is typically a mammal (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), preferably a human.

[0017] Granulomas are nodular infiltrations of mononuclear phagocytes of the monocyte and macrophage system, and can be infectious or non-infectious. Granulomas are histopathologically classified into foreign body, suppurative, sarcoid, tuberculoid, palisade, and interstitial types. In the present disclosure, granulomas may be of any classification, such as sarcoid, tuberculoid, or palisade granulomas. Granulomas can occur in a variety of diseases, including sarcoidosis, granuloma annulare, mycobacterial infections (tuberculosis, leprosy, nontuberculous mycobacteriosis, etc.), and Crohn's disease. In some embodiments, the granuloma is a granuloma associated with granuloma annulare, mycobacterial infection, or Crohn's disease. Sites where granulomas form include, but are not limited to, the skin, lungs, eyes, heart, liver, kidneys, lymph nodes, nerves, and muscles. In some embodiments, the granuloma is formed in the skin, lungs, heart, or lymph nodes, particularly the skin.

[0018] The pentose phosphate pathway is a pathway in the glucose metabolic pathway that metabolizes glucose 6-phosphate to ribose 5-phosphate or xylulose 5-phosphate. In the previous step, fructose 1,6-bisphosphate is converted to glucose 6-phosphate. In addition, NADPH, which is produced in the process of converting glucose 6-phosphate to 6-phosphogluconic acid, is converted to NADP. ＋A portion of the glucose metabolic pathway, including these reactions, is shown in Figure 8. In the present disclosure, "enzymes associated with the pentose phosphate pathway" refers to enzymes that act in the process of metabolizing fructose 1,6-bisphosphate to ribose 5-phosphate or xylulose 5-phosphate in the glucose metabolic pathway, and includes fructose-1,6-bisphosphatase 1 (FBP-1), glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconolactonase (PGLS), 6-phosphogluconate dehydrogenase (6PGD), ribulose phosphate 3-epimerase (RPE), ribose phosphate isomerase (RPI), and thioredoxin reductase 1 (TXNRD1). Preferably, the enzyme is FBP-1, GPI, G6PD, 6PGD or TXNRD1, more preferably FBP-1, G6PD or TXNRD1, and even more preferably FBP-1 or TXNRD1.

[0019] Fructose-1,6-bisphosphatase 1 (FBP-1) is an enzyme that converts fructose-1,6-bisphosphate to fructose-6-phosphate. Representative amino acid sequences of FBP-1 are registered under GenBank accession numbers NP_001121100.1 (human, SEQ ID NO: 1) and NP_062268.1 (mouse, SEQ ID NO: 2). In the present disclosure, FBP-1 includes products of all alleles.

[0020] Glucose phosphate isomerase (GPI) is an enzyme that converts fructose-6-phosphate to glucose-6-phosphate. Representative amino acid sequences of GPI are registered under GenBank accession numbers NP_001171651.1 (human, SEQ ID NO: 3) and NP_032181.2 (mouse, SEQ ID NO: 4). In this disclosure, GPI includes the products of all alleles.

[0021] Glucose-6-phosphate dehydrogenase (G6PD) is an enzyme that converts glucose-6-phosphate to glucono-1,5-lactone-6-phosphate. Representative amino acid sequences of G6PD are registered under GenBank accession numbers NP_000393.4 (human, SEQ ID NO: 5), NP_032088.1 (mouse, SEQ ID NO: 6), and NP_062341.2 (mouse, SEQ ID NO: 7). In the present disclosure, G6PD includes products of all alleles.

[0022] 6-Phosphogluconolactonase (PGLS) is an enzyme that converts glucono-1,5-lactone-6-phosphate to 6-phosphogluconic acid. Representative amino acid sequences of PGLS are registered under GenBank accession numbers NP_036220.1 (human, SEQ ID NO: 8) and NP_079672.1 (mouse, SEQ ID NO: 9). In this disclosure, PGLS includes the products of all alleles.

[0023] 6-phosphogluconate dehydrogenase (6PGD) is an enzyme that converts 6-phosphogluconate to ribulose-5-phosphate. Representative amino acid sequences of 6PGD are registered under GenBank accession numbers NP_002622.2 (human, SEQ ID NO: 10) and NP_001074743.1 (mouse, SEQ ID NO: 11). In this disclosure, 6PGD includes products of all alleles.

[0024] Ribulose phosphate 3-epimerase (RPE) is an enzyme that converts ribulose-5-phosphate to xylulose-5-phosphate. Representative amino acid sequences of RPE are registered under GenBank accession numbers NP_001265211.1 (human, SEQ ID NO: 12) and NP_001297571.1 (mouse, SEQ ID NO: 13). In this disclosure, RPE includes products of all alleles.

[0025] Ribose phosphate isomerase (RPI) is an enzyme that converts ribulose-5-phosphate to ribose-5-phosphate. Representative amino acid sequences of RPI are registered under GenBank accession numbers NP_653164.2 (human, SEQ ID NO: 14) and NP_033101.2 (mouse, SEQ ID NO: 15). In this disclosure, RPI includes products of all alleles.

[0026] Thioredoxin reductase 1 (TXNRD1) reduces oxidized thioredoxin to reduced thioredoxin, converting NADPH to NADP ＋ Representative amino acid sequences of TXNRD1 are registered under GenBank accession numbers NP_877393.1 (human, SEQ ID NO: 16) and NP_001035978.1 (mouse, SEQ ID NO: 17). In the present disclosure, TXNRD1 includes the products of all alleles.

[0027] In the present disclosure, enzymes associated with the pentose phosphate pathway may contain sequences in which one or several amino acids have been deleted, substituted, or added from the original amino acid sequence, so long as their function is maintained. "Several" preferably means 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Amino acid substitutions are preferably conservative substitutions between similar amino acid residues. Amino acid residues can be classified into groups based on their side chains, such as acidic amino acids (e.g., glutamic acid, aspartic acid, 2-aminoadipic acid), basic amino acids (e.g., histidine, lysine, arginine), aliphatic amino acids (e.g., methionine, alanine, leucine, isoleucine, valine, 2-aminoheptanoic acid, norleucine), aromatic amino acids (e.g., phenylalanine, tryptophan, tyrosine), and polar, uncharged amino acids (e.g., asparagine, cysteine, glutamine, serine, threonine). Preferably, amino acid substitutions are conservative substitutions between amino acid residues within the same group.

[0028] Furthermore, as long as the function of the enzyme associated with the pentose phosphate pathway is maintained, the enzyme may comprise an amino acid sequence that has an identity to the original amino acid sequence of at least about 60% or more, preferably about 70% or more, more preferably about 80% or more, even more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97% or more, about 98% or more, or about 99% or more, when calculated using BLAST or the like (for example, when using the default, i.e., initial condition, parameters of BLAST).

[0029] As demonstrated in the Examples below, granulomas can be treated and / or prevented by administering an inhibitor of an enzyme associated with the pentose phosphate pathway to a subject. In this disclosure, "treating a granuloma" or "treatment of a granuloma" means slowing or stopping the progression of a granuloma and / or reducing, alleviating, ameliorating, or eliminating the granuloma in a subject with a granuloma.

[0030] In this disclosure, "preventing granulomas" or "preventing granulomas" means preventing granuloma formation or reducing the likelihood of granuloma formation in a subject, particularly in a subject at risk of forming granulomas. Subjects at risk of forming granulomas include, for example, patients with granuloma-associated diseases such as sarcoidosis, mycobacterial infections, and Crohn's disease.

[0031] Furthermore, by administering an inhibitor of an enzyme associated with the pentose phosphate pathway to a subject, diseases associated with granulomas can be treated and / or prevented. In this disclosure, "treating a disease" or "treatment of a disease" means slowing or halting the progression of the disease and / or alleviating, ameliorating, ameliorating, or eliminating the disease in a subject having the disease.

[0032] In this disclosure, "preventing disease" or "prevention of disease" means preventing the onset of disease or reducing the likelihood of developing disease in a subject, particularly in a subject who is likely to develop the disease but has not yet done so, where onset of disease includes recurrence.

[0033] In the present disclosure, an "inhibitor of an enzyme associated with the pentose phosphate pathway" refers to a substance that inhibits the activity or expression of an enzyme associated with the pentose phosphate pathway. The inhibitor may be, for example, a compound, peptide, antibody, or nucleic acid that inhibits the activity or expression of an enzyme associated with the pentose phosphate pathway. Substances known as inhibitors of enzymes associated with the pentose phosphate pathway, for example, substances sold as inhibitors, may be used. Substances that inhibit the activity or expression of enzymes associated with the pentose phosphate pathway may be identified and used in in vitro or in vivo experimental systems.

[0034] Examples of FBP-1 inhibitors include 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032 (P-[5-[2-amino-5-(2-methylpropyl)-4-thiazolyl]-2-furanyl]phosphonic acid, CAS number: 261365-11-1) or its prodrug MB06322 (ethyl(2S)-2-[[[5-[2-amino-5-(2-methylpropyl)-1,3-thiazol-4-yl]furan-2-yl]-[[(2S)-1-ethoxy-1-oxopropan-2-yl]amino]phosphanyl]amino]phosphonic acid). propanoic acid, CAS number: 280782-97-0), or MB07229 ((5-(2-amino-5-pivaloylthiazol-4-yl)furan-2-yl)phosphonic acid, CAS number: 882755-95-5) or its prodrug MB07803 (ethyl 2-[({5-[2-amino-5-(2,2-dimethylpropanoyl)-1,3-thiazol-4-yl]furan-2-yl}[(1-ethoxy-2-methyl-1-oxopropan-2-yl)amino]phosphoryl)amino]-2-methylpropanoic acid, CAS number: 882757-24-6). As an inhibitor of G6PD and 6PGD, for example, 6-aminonicotinamide may be used. As an inhibitor of GPI, for example, 2-deoxy-D-glucose may be used. Inhibitors of TXNRD1 may include, for example, auranofin, TRi-1, chaetocin, or LCS3. In some embodiments, the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB06322, MB07229, MB07803, 6-aminonicotinamide, auranofin, or TRi-1. In some embodiments, the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB07229, MB07803, 6-aminonicotinamide, auranofin, or TRi-1.In certain embodiments, the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB07229, 6-aminonicotinamide, auranofin, or TRi- 1. In certain embodiments, the inhibitor of an enzyme associated with the pentose phosphate pathway is MB06322, MB07803, or auranofin.

[0035] In one embodiment, the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07229 or MB07803, preferably MB07803. MB07803 has advantages such as being able to be used at lower doses compared to other FBP1 inhibitors and / or having a gentler concentration-dependent activity curve, allowing greater flexibility in dose setting. For example, MB07803 may be administered at about 100-1000 mg / day, about 150-500 mg / day, or about 200-400 mg / day. For example, about 1000 mg of MB07803 may be administered as a single dose. For example, about 400 mg of MB07803 may be administered once daily, or about 200 mg of MB07803 may be administered twice daily. For example, MB07803 may be administered orally or parenterally (e.g., intravenously).

[0036] Nucleic acids that inhibit the activity or expression of enzymes associated with the pentose phosphate pathway may inhibit any stage of the genes for these enzymes, such as transcription, post-transcriptional regulation, translation, or post-translational modification, or may inhibit enzymatic activity. Examples of such nucleic acids include siRNA, antisense nucleic acids, ribozymes, nucleic acid aptamers, and decoy nucleic acids. An example of a nucleic acid that inhibits the expression of an enzyme associated with the pentose phosphate pathway is siRNA.

[0037] siRNA refers to double-stranded RNA that specifically destroys target mRNA. Single-stranded RNA (shRNA) in which such double strands are linked via a loop region is also included in siRNA. Those skilled in the art can design appropriate siRNA based on the nucleotide sequence of the target mRNA. siRNAs targeting various mRNAs are commercially available, and appropriate siRNAs may be used. In one embodiment, the FBP-1 inhibitor is an siRNA against FBP-1. In one embodiment, the TXNRD1 inhibitor is an siRNA against TXNRD1.

[0038] A vector containing a nucleic acid that inhibits the activity or expression of an enzyme associated with the pentose phosphate pathway can also be used. Examples of vectors include viral vectors such as adenovirus, adeno-associated virus, retrovirus, lentivirus, and herpes simplex virus vectors.

[0039] The method of administration of the inhibitor of an enzyme associated with the pentose phosphate pathway is not particularly limited, and can be via common administration routes such as oral administration, parenteral administration, injection, and infusion. Parenteral administration can be systemic administration or local administration. Examples include intravenous administration, intraarterial administration, intradermal administration, subcutaneous administration, transdermal administration, intramuscular administration, intraperitoneal administration, and intranasal administration. When the granuloma is located on the skin, local administration is preferred, and more specifically, examples include application, spraying, or injection to the site of the granuloma.

[0040] The inhibitor of an enzyme involved in the pentose phosphate pathway can be administered in a composition. The composition can be solid, liquid, or any form in between (e.g., semisolid), and can take various known formulations depending on the site of the granuloma, the administration method, and the dosage. Examples include tablets, powders, granules, granules, fine granules, capsules, suppositories, solid injections that dissolve when used, liquid injections, infusions, and drip infusions. The composition can also be an external preparation such as a spray, lotion, cream, patch, ointment, solution, emulsion, or suspension.

[0041] These dosage forms are produced by formulating them in a conventional manner. Furthermore, various pharmaceutical substances that are approved for use in pharmaceuticals can be blended as needed for the formulation. The pharmaceutical substances can be appropriately selected depending on the dosage form of the formulation, and examples thereof include buffering agents, surfactants, stabilizers, preservatives, excipients, diluents, additives, disintegrants, binders, coating agents, lubricants, flavoring agents, sweeteners, solubilizers, pigments, dyes, and fragrances.

[0042] The dosage and frequency of administration of an inhibitor of an enzyme associated with the pentose phosphate pathway can be appropriately determined by one skilled in the art depending on the animal species, health condition, age, body weight, administration route, administration form, etc., of the subject, so that an effective amount of the inhibitor is administered to the subject. The effective amount in a given situation can be easily determined by routine experimentation.

[0043] The inhibitor of an enzyme involved in the pentose phosphate pathway may be administered in a single dose, multiple doses, or continuously. When administered multiple times, it may be administered, for example, once to several times a day, for example, once, twice, or three times a day, every day or every few days, for example, every 1, 2, 3, or 7 days. The administration period is not limited, and administration may be continued, for example, until the granuloma is alleviated. A drug-free period may also be provided.

[0044] The inhibitor of an enzyme involved in the pentose phosphate pathway can be used alone or in combination with one or more additional active ingredients, particularly active ingredients for treating or preventing granulomas. "Combining" ingredients refers not only to the use of a dosage form containing all of the ingredients and the use of a combination of dosage forms containing each ingredient separately, but also to the simultaneous administration of each ingredient or the administration of any ingredient at a later time, as long as they are used for the treatment or prevention of granulomas. It is also possible to use two or more additional active ingredients in combination. For example, a composition containing one or more additional active ingredients in addition to an inhibitor of an enzyme involved in the pentose phosphate pathway can be used.

[0045] Suitable active ingredients for concomitant use include, for example, JAK inhibitors, nonsteroidal anti-inflammatory drugs, steroids, antihistamines, antiallergic drugs, immunosuppressants, antibiotics, antifungal agents, antiviral agents, etc. A therapeutic agent for a disease accompanied by granulomas can also be used in combination.

[0046] In addition to administering inhibitors of enzymes associated with the pentose phosphate pathway, non-pharmacological medical interventions, such as surgical resection, can also be performed.

[0047] In one aspect, a composition for treating and / or preventing granulomas is provided, the composition comprising an inhibitor of an enzyme associated with the pentose phosphate pathway. In one aspect, a method for treating and / or preventing granulomas is provided, the method comprising administering an effective amount of an inhibitor of an enzyme associated with the pentose phosphate pathway to a subject in need of such treatment and / or prevention. In one aspect, an inhibitor of an enzyme associated with the pentose phosphate pathway for treating and / or preventing granulomas is provided. In one aspect, the use of an inhibitor of an enzyme associated with the pentose phosphate pathway for treating and / or preventing granulomas is provided. In one aspect, the use of an inhibitor of an enzyme associated with the pentose phosphate pathway in the manufacture of a composition for treating and / or preventing granulomas is provided.

[0048] In one aspect, a composition for treating and / or preventing a disease associated with granulomas is provided, comprising an inhibitor of an enzyme associated with the pentose phosphate pathway. In one aspect, a method for treating and / or preventing a disease associated with granulomas is provided, comprising administering an effective amount of an inhibitor of an enzyme associated with the pentose phosphate pathway to a subject in need of such treatment and / or prevention. In one aspect, an inhibitor of an enzyme associated with the pentose phosphate pathway for treating and / or preventing a disease associated with granulomas is provided. In one aspect, the use of an inhibitor of an enzyme associated with the pentose phosphate pathway for treating and / or preventing a disease associated with granulomas is provided. In one aspect, the use of an inhibitor of an enzyme associated with the pentose phosphate pathway in the manufacture of a composition for treating and / or preventing a disease associated with granulomas is provided.

[0049] Furthermore, as demonstrated in the Examples below, the levels of enzymes associated with the pentose phosphate pathway are high in macrophages contained in granulomas and in the blood of sarcoidosis patients. Therefore, whether or not a subject has granulomas can be determined based on the level of an enzyme associated with the pentose phosphate pathway in a sample collected from the subject. The enzyme level may be the amount or concentration of the enzyme in the sample, or the expression level of the enzyme.

[0050] The sample may be blood, plasma, or serum collected from a subject. Blood samples may be collected by conventional methods, for example, from a vein or artery. Plasma or serum samples may be prepared by appropriately treating the blood using methods well known to those skilled in the art. This treatment is not particularly limited and may be any clinically acceptable treatment. For example, addition of an anticoagulant, centrifugation, etc. may be performed. The sample may also be other body fluids collected from a subject, such as cerebrospinal fluid, saliva, nasal discharge, sputum, pleural effusion, or ascites. The collected sample may be appropriately concentrated or diluted as needed for use. The sample may be stored at a low temperature, for example, frozen, during or after its preparation prior to use.

[0051] The level of the enzymes involved in the pentose phosphate pathway in the sample can be measured using a reagent that specifically binds to the enzymes involved in the pentose phosphate pathway.The reagent that specifically binds to the enzymes involved in the pentose phosphate pathway can be, for example, a compound, a peptide, an antibody, or an aptamer.These reagents can also be fragments, derivatives, or analogs, as long as they can specifically bind to the enzymes involved in the pentose phosphate pathway.

[0052] For example, the level of an enzyme involved in the pentose phosphate pathway in a sample can be measured by an immunological technique using an antibody that binds to the enzyme. Examples of immunological techniques include enzyme-linked immunosorbent assays (ELISAs, e.g., direct, indirect, sandwich, or competitive), immunochromatography, Western blotting, flow cytometry, and radioisotope immunoassays (RIAs), with ELISA being preferred.

[0053] In the context of this disclosure, antibody means an affinity ligand based on an immunoglobulin scaffold, and includes monoclonal and polyclonal antibodies of any origin, including murine, rat, rabbit, goat, human, and other antibodies, as well as chimeric antibodies containing sequences from multiple species, e.g., partially humanized antibodies, e.g., partially humanized mouse antibodies. The antibody may be a fragment or derivative thereof, provided that it is capable of specifically binding to an enzyme associated with the pentose phosphate pathway.

[0054] Antibodies can be produced by existing general production methods using an enzyme associated with the pentose phosphate pathway or a partial peptide thereof having antigenicity as an immunogen. For example, polyclonal antibodies can be produced by immunizing animals with an antigen, and monoclonal antibodies can be produced using hybridoma technology. Alternatively, commercially available antibodies may be used.

[0055] The reagent can be labeled with a detectable substance. Examples of detectable substances include radioisotopes, fluorescent labels, luminescent labels, bioluminescent labels, enzyme labels, and biotin. A substance that specifically binds to the reagent and has a label, such as a secondary antibody, can also be used.

[0056] The reagent may be bound to a suitable support. The support is not particularly limited as long as it can immobilize the reagent, and may be of any shape or material. Examples of such supports include membranes such as nylon membranes, beads, glass, plastic, and metal supports.

[0057] Alternatively, the sample may be tissue suspected of having a granuloma collected from a subject. The level of the enzyme associated with the pentose phosphate pathway can be measured by contacting the sample with a reagent that binds to the enzyme associated with the pentose phosphate pathway and measuring the amount of the bound reagent on an image. For example, the tissue may be sectioned and observed, or the tissue may be fixed on a substrate such as a glass slide and observed. General techniques for detecting substances in a sample, such as sample fixation and permeabilization, are well known in the art. The reagent that binds to the enzyme associated with the pentose phosphate pathway is as described above, and a label that can be detected by image processing is used.

[0058] The label is visualized under a microscope such as an upright microscope or a fluorescence microscope, and the image is photographed using a CCD camera or the like to measure the signal intensity of the label. Image processing software such as ImageJ software (NIH, Bethesda, MD, USA) can be used to measure the signal intensity.

[0059] The level of enzymes involved in the pentose phosphate pathway can be measured by automated imaging and image analysis technology, such as high content analysis (HCA) technology.HCA technology is known in the art and involves the automated imaging of a large number of cells followed by quantitative image analysis.Synonyms for HCA include high content imaging and high content screening.To speed up measurement, robots or automated devices or microfluidic devices can be used.

[0060] Alternatively, tissue suspected of being a granuloma may be collected from a subject, lysed, and the level of an enzyme associated with the pentose phosphate pathway in the sample may be measured by the immunological method described above. The gene expression level of the enzyme may also be measured by known methods such as quantitative PCR, microarray analysis, RNA sequencing, Northern blotting, and SAGE.

[0061] In this method, if the level of an enzyme associated with the pentose phosphate pathway in a sample taken from the subject is higher than a cutoff value, the subject is determined to have a granuloma, and if the level is lower than the cutoff value, the subject is determined to not have a granuloma.

[0062] When the level of the enzyme associated with the pentose phosphate pathway is equal to the cutoff value, the subject is determined to have or not have a granuloma, and this can be arbitrarily set depending on the purpose of the determination, etc. Thus, in one embodiment, when the level of the enzyme associated with the pentose phosphate pathway is equal to or greater than the cutoff value, the subject is determined to have a granuloma, and when the level of the enzyme associated with the pentose phosphate pathway is less than the cutoff value, the subject is determined to not have a granuloma. In another embodiment, when the level of the enzyme associated with the pentose phosphate pathway is higher than the cutoff value, the subject is determined to have a granuloma, and when the level of the enzyme associated with the pentose phosphate pathway is equal to or less than the cutoff value, the subject is determined to not have a granuloma.

[0063] The cutoff value is a value that can statistically significantly separate a group of subjects with granulomas from a group of subjects without granulomas. The cutoff value can be set by known methods using various statistical analysis techniques. For example, the levels of enzymes associated with the pentose phosphate pathway in samples obtained from a group of subjects with granulomas and in samples obtained from a group of subjects without granulomas can be statistically analyzed to set the cutoff value. Statistical significance can be analyzed using known testing methods such as the chi-square test, generalized Wilcoxon test, Wilcoxon signed-rank test, Mann-Whitney test, log-rank test, and Cox proportional hazards. Statistical analysis software such as Prism can be used to set the cutoff value.

[0064] The cutoff value may be set based on sensitivity and / or specificity. Preferably, the cutoff value exhibits both high sensitivity and high specificity. Here, sensitivity refers to the true positive rate, and specificity refers to the true negative rate. For example, the level of an enzyme associated with the pentose phosphate pathway that exhibits a high positive rate in a group of subjects with granulomas and a high negative rate in a group of subjects without granulomas may be set as the cutoff value.

[0065] For example, a cutoff value can be set using receiver operating characteristic (ROC) analysis, a commonly used method for examining the usefulness of diagnostic tests. In ROC analysis, an ROC curve is created by plotting the sensitivity at each cutoff value on the vertical axis and the false positive rate (1 - specificity) on the horizontal axis. For tests without diagnostic ability, the ROC curve is a diagonal straight line, but as the diagnostic ability improves, the curve becomes more arched upward to the left. The cutoff value that gives the point on the ROC curve with the smallest distance from the upper left corner is said to have excellent sensitivity and specificity. The cutoff value can also be set based on the Youden index. Specifically, sensitivity and specificity are determined from the levels of enzymes related to the pentose phosphate pathway in a group of subjects with granulomas and a group of subjects without the disease, and an ROC curve is created based on these values ​​using commercially available analytical software. The values ​​at which sensitivity and specificity are as close to 100% as possible are then determined, and these values ​​can be used as the cutoff value.

[0066] Furthermore, for example, the diagnostic efficiency (i.e., the ratio of the total number of cases in which subjects with granulomas were correctly diagnosed as having "granulomas" and the total number of cases in which subjects without granulomas were correctly diagnosed as not having "granulomas" to the total number of cases) can be calculated, and the level of an enzyme related to the pentose phosphate pathway that gives the highest diagnostic efficiency can be used as the cutoff value.

[0067] Cutoff values ​​can also be set for patient subgroups according to characteristics, for example, cutoff values ​​may be set according to gender, age group, or race.

[0068] In some embodiments, the results of the determination by this method can be provided as information for diagnosis. In some embodiments, the method includes collecting a sample from a subject. In some embodiments, the method includes measuring the level of an enzyme associated with the pentose phosphate pathway in the sample collected from the subject. In some embodiments, the method includes treating the granuloma in a subject determined to have the granuloma. For example, an inhibitor of an enzyme associated with the pentose phosphate pathway is administered according to the present disclosure.

[0069] In another aspect, a kit for determining whether a subject has granulomas is provided, comprising a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway or a reagent for measuring gene expression of an enzyme associated with the pentose phosphate pathway. The reagent may be dissolved in water or a suitable buffer, such as phosphate-buffered saline (PBS), or lyophilized and provided in a suitable container. Suitable containers include bottles, vials, syringes, test tubes, plates, membranes, etc. The container may be made of a variety of materials, such as glass or plastic. The kit may further include other components or reagents necessary for detecting the enzyme associated with the pentose phosphate pathway. For example, the kit may further include a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, etc. The kit may further include other materials desirable from a commercial and user standpoint, such as a package insert containing instructions for use.

[0070] In one aspect, a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway or a reagent for measuring gene expression of an enzyme associated with the pentose phosphate pathway is provided for determining whether a subject has a granuloma. In one aspect, a use of a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway or a reagent for measuring gene expression of an enzyme associated with the pentose phosphate pathway is provided for manufacturing a kit for determining whether a subject has a granuloma.

[0071] For example, the following embodiments are provided: [1] A composition comprising an inhibitor of an enzyme associated with the pentose phosphate pathway for preventing and / or treating granulomas. [2] The composition according to item 1, wherein the granuloma is a granuloma associated with sarcoidosis, granuloma annulare, mycobacterial infection, or Crohn's disease. [3] A composition for treating and / or preventing a disease accompanied by granulomas, comprising an inhibitor of an enzyme associated with the pentose phosphate pathway. [4] The composition according to item 3, wherein the disease accompanied by granulomas is sarcoidosis, granuloma annulare, mycobacterial infection, or Crohn's disease. [5] The composition according to any of items 1 to 4, wherein the granuloma is a sarcoid-type, tuberculoid-type, or palisade granuloma. [6] The composition according to any of items 1 to 5, wherein the granuloma is a granuloma formed in the skin, lungs, eyes, heart, liver, kidneys, lymph nodes, nerves, or muscles. [7] The composition according to any one of items 1 to 6, wherein the granuloma is formed in the skin, lung, heart, or lymph node. [8] The composition according to any one of items 1 to 7, wherein the granuloma is formed in the skin.

[0072] [9] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, RPI, or TXNRD1.

[10] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, or RPI.

[11] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, 6PGD, or TXNRD1.

[12] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, or 6PGD.

[13] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, G6PD or TXNRD1.

[14] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1 or G6PD.

[15] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1 or TXNRD1.

[16] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is FBP-1.

[17] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is G6PD.

[18] The composition according to any one of items 1 to 8, wherein the enzyme associated with the pentose phosphate pathway is TXNRD1.

[0073]

[19] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB06322, MB07229, MB07803, 2-deoxy-D-glucose, 6-aminonicotinamide, auranofin, TRi-1, chaetocin, or LCS3.

[20] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB06322, 2-deoxy-D-glucose, or 6-aminonicotinamide.

[21] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB06322, MB07229, MB07803, 6-aminonicotinamide, auranofin, or TRi-1.

[22] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB07229, MB07803, 6-aminonicotinamide, auranofin, or TRi-1.

[23] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide, MB05032, MB07229, 6-aminonicotinamide, auranofin, or TRi-1.

[24] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide or 6-aminonicotinamide.

[25] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB05032, MB06322, MB07229, MB07803, auranofin, or TRi-1.

[26] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB05032, MB07229, MB07803, auranofin, or TRi-1.

[27] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB05032, MB07229, auranofin, or TRi-1.

[28] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07229, MB07803, auranofin, or TRi-1.

[29] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07229, auranofin, or TRi-1.

[30] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07229 or MB07803.

[31] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07229.

[32] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB07803.

[33] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is auranofin or TRi-1.

[34] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is MB06322, MB07803 or auranofin.

[35] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is an siRNA against FBP-1, GPI, G6PD, 6PGD, or TXNRD1.

[36] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is an siRNA against FBP-1, GPI, G6PD, or 6PGD.

[37] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is an siRNA against FBP-1.

[38] The composition according to any one of items 1 to 8, wherein the inhibitor of an enzyme associated with the pentose phosphate pathway is an siRNA against TXNRD1.

[0074]

[39] A method for determining whether a subject has a granuloma, comprising comparing the level of an enzyme associated with the pentose phosphate pathway in a sample collected from the subject with a cutoff value.

[40] The method of item 39, comprising measuring the level of an enzyme associated with the pentose phosphate pathway in a sample collected from the subject.

[41] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, RPI, or TXNRD1.

[42] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, or RPI.

[43] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, 6PGD, or TXNRD1.

[44] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD or 6PGD.

[45] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is FBP-1.

[46] The method of item 39 or 40, wherein the enzyme associated with the pentose phosphate pathway is TXNRD1.

[47] The method of any of items 39 to 46, wherein the subject is determined to have a granuloma if the level of the enzyme associated with the pentose phosphate pathway in a sample collected from the subject is higher than a cutoff value.

[48] The method of any of items 39 to 46, wherein the subject is determined to not have a granuloma if the level of the enzyme associated with the pentose phosphate pathway in a sample collected from the subject is lower than a cutoff value.

[49] The method of any of items 39 to 48, wherein the sample collected from the subject is blood, plasma, serum or tissue suspected of having a granuloma.

[50] The method according to any one of items 39 to 49, wherein the sample collected from the subject is blood, plasma, or serum.

[51] The method according to any one of items 39 to 49, wherein the sample collected from the subject is tissue suspected of having a granuloma.

[0075]

[52] A kit for determining whether a subject has a granuloma, comprising a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway or a reagent for measuring gene expression of an enzyme associated with the pentose phosphate pathway.

[53] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, RPI, or TXNRD1.

[54] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, PGLS, 6PGD, RPE, or RPI.

[55] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, 6PGD, or TXNRD1.

[56] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is FBP-1, GPI, G6PD, 6PGD, or TXNRD1.

[57] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is FBP-1.

[58] The kit according to item 49, wherein the enzyme associated with the pentose phosphate pathway is TXNRD1.

[59] The kit according to any of items 49 to 55, comprising a reagent that specifically binds to an enzyme associated with the pentose phosphate pathway.

[60] The kit according to any of items 49 to 56, wherein the reagent is an antibody.

[0076] All documents cited in this specification are incorporated herein by reference. The above descriptions are all non-limiting, and the present invention is defined in the appended claims, and various modifications are possible within the scope of the technical idea. The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0077] Materials and Methods: Patient Sample Collection. Human samples were obtained under a protocol approved by the Ethics Committee of the Kyoto University Graduate School of Medicine (R0743). Sarcoidosis patients had not used topical steroids or immunomodulators in the past two weeks, nor had they received systemic immunosuppressant therapy in the past four weeks. After biopsy, skin samples were placed in Bambanker (Nihon Genetics) solution and frozen at -80°C. Tissues from atopic dermatitis, psoriasis, granuloma annulare, cutaneous sarcoidosis, and pulmonary sarcoidosis used for histological staining were immersed in 10% neutral-buffered formalin for 24 hours after biopsy and surgery, then embedded in paraffin. Serum was collected using a serum separator-containing dish and collected by centrifugation.

[0078] Single-cell RNA sequencing library construction. Cryopreserved biopsy specimens were thawed and treated for 2 hours with the Whole Skin Dissociation Kit, human (Miltenyi Biotec) to prepare a cell suspension. The cell suspension was then subjected to library construction using the Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics). The library was sequenced on a DNBSEQ-G400 (MGI Tech) platform, yielding approximately 150 to 200 million reads. Sequencing data were analyzed using CellRanger 3.1.0 (10x Genomics).

[0079] Clustering analysis using Seurat. Clustering analysis was performed using the Seurat package (version 4.0.6) at both a first-level analysis across all cells and a second-level, high-resolution analysis within cell types. Single-cell RNA-seq datasets were combined into a single Seurat object and normalized using the SCTransform function to remove batch effects. Cells with fewer than 100 genes, more than 5000 genes, or mitochondrial content greater than 20% were excluded. Differentially expressed genes (DEGs) were identified in each cluster using a Wilcoxon rank-sum test, which was repeated for each resulting cell type to define broad cell types and further characterize subpopulations.

[0080] Creation of a sarcoidosis score: The top 100 genes that were elevated in lesions compared to non-lesion areas were extracted from the bulk RNA sequencing data of sarcoidosis (GSE32887). The average expression of these genes was used as the sarcoidosis score.

[0081] Analysis of single-cell RNA-seq data using public databases. HLA-DRA-positive antigen-presenting cells were extracted from the databases for atopic dermatitis, psoriasis vulgaris (HUMAN CELL ATLAS DEVELOPMENTAL), granuloma annulare (GSE158924), and leprosy (GSE151528) using the subset function in Seurat, and the proportions of FBP-1-, ACE-, and STAT1-positive cells were examined using the Dotplot function.

[0082] Immunostaining was performed on formalin-fixed, paraffin-embedded (FFPE) tissues using a BOND RX automated immunostainer (Leica). Antigen retrieval was performed with ER solution 2 for 40 minutes. Sections were stained with CD163 (clone 10D6, 1:200; Leica), FBP-1 (clone EPR4619, 1:100; Abcam, Cambridge, MA, USA), CD68 (clone PG-M1, 1:200; Dako), G6PD (1:2000; Abcam), and TXNRD1 (clone B-2, 1:100; SANTA CRUZ). The secondary antibody used was an Opal tyramide signaling amplification kit (Akoya Biosciences, Marlborough, MA, USA). Images were taken using a fluorescence microscope BZ9000 (Keyence) and analyzed using ImageJ software (NIH).

[0083] Measurement of Serum FBP1 Serum FBP1 concentrations were measured using a Human FBP1 ELISA Kit (LSBio).

[0084] In vitro sarcoidosis model: Peripheral blood mononuclear cells were isolated from peripheral blood of healthy individuals using lymph node isolation solution (Nacalai). CD14-positive monocytes were isolated from peripheral blood mononuclear cells using CD14 microbeads (Miltenyi Biotec). Monocytes were placed in RPMI containing 10% fetal bovine serum, supplemented with concanavalin A (Sigma, 5 μM), CD40L (Peprotech, 300 ng / ml) or anti-human CD40 antibody (BioLegend, 1 μg / ml), and IFNγ (Peprotech, 10 ng / ml), and 0.5 × 10 ５ Cells were seeded at 1 / well in a 96-well flat-bottom plate. For inhibitor experiments, fructose-1,6-bisphosphatase-1 inhibitor (FBP-1 inhibitor, 2,5-dichloro-N-(5-chloro-2-benzoxazolyl)-benzenesulfonamide) (Cayman, 100 or 500 μM), tofacitinib (Sigma, 1 μM), 6-aminonicotinamide (6AN) (Sigma, 100 μM), MB05032 (Cayman, 10 μM), MB07729 (TargetMol, 10 μM), auranofin (Fujifilm Wako Pure Chemical Industries, 0.1 or 10 μM), and TRi-1 (Selleck, 1 or 10 μM) were added. Alternatively, Horizon's siRNA against the FBP-1 gene (product number E-008725-00-0010) and control siRNA were used (1 μM). After fixation with methanol, the cells were stained with Giemsa stain, photographed under a microscope (Olympus CKK53), and analyzed using ImageJ software (NIH).

[0085] Gene Expression Analysis Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA was reverse transcribed from total RNA samples using the Prime Script RT reagent kit (Takara Bio). Quantitative RT-PCR was performed using SYBR Green I (Roche, Basel, Switzerland) and a LightCycler real-time PCR system (Roche). All primers were obtained from Greiner Japan. The primer sequences are as follows: PCR conditions included an initial enzyme activation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 10 seconds and 60°C for 20 seconds. Gene-specific fluorescence was measured at 60°C. Gene expression was analyzed using duplicate test reactions for each sample, and results were normalized to the expression level of the housekeeping gene, ACTB.

[0086] All mice were maintained under specific pathogen-free conditions at the Kyoto University Graduate School of Medicine Laboratory Animal Research Facility. Female mice aged 7–10 weeks were used for all experiments. All experimental procedures were approved by the Kyoto University Animal Care and Use Committee.

[0087] Murine skin sarcoidosis model: Seven-week-old C57BL / 6 mice (Charles River) were intraperitoneally administered 6AN (20 mg / kg) or PBS (Nacalai Tesque). Seven hours later, under anesthesia, 2,000 beads (Bio-Gel P1000, Bio-Rad) and 25 μg of concanavalin A were injected intradermally into the ear. Ear thickness was measured daily using a constant pressure thickness gauge (Teclock). Mice were euthanized on day 4 and analyzed.

[0088] Mouse skin tissue staining method Ear skin was immersed in 10% neutral buffered formalin for 24 hours and then embedded in paraffin. 5 μm thick sections were prepared and stained with hematoxylin and eosin (Sigma).

[0089] Analysis of mouse skin infiltrating cells. Mouse ears were separated into dorsal and ventral sheets and incubated at 37°C for 60 minutes in RPMI 1640 containing 10% fetal calf serum, 1% penicillin-streptomycin, 1% sodium pyruvate, 1% MEM non-essential amino acids (Thermo Fisher Scientific), 0.25 mg / ml Liberase TL (Roche, Basel, Switzerland), and 0.3 mg / ml DNase I (Sigma-Aldrich, St. Louis, MO). The digested skin sheets were filtered through a 40 μm cell strainer (BD Biosciences) to obtain single-cell suspensions.

[0090] Single cell suspensions obtained from the skin were stained with Fixable Viability Dye eFluor. （商標） Cells were stained with 780 (Thermo Fisher Scientific) to exclude dead cells. Nonspecific Ab binding was then blocked with anti-CD16 / 32 Ab (BD Biosciences), and cells were stained for surface antigens. After surface staining with the antibodies listed below, cells were fixed with Cytofix / Cytoperm solution (BD Biosciences). Samples were evaluated using a BD LSRFortessa cell analyzer (BD Biosciences), and data were analyzed using FlowJo software (BD Biosciences).

[0091] Antibody List

[0092] [Results] Activation of the pentose phosphate pathway in sarcoidosis lesions. Single-cell RNA sequencing of sarcoidosis skin lesions revealed macrophages (TREM2 macrophages) that were increased in sarcoidosis (Figures 1 and 2). Sarcoidosis-specific macrophages expressed sarcoidosis-specific genes, such as angiotensin-converting enzyme (ACE), lysozyme (LYZ), and STAT1 (Figure 3). Sarcoidosis-specific macrophages also showed increased expression of a previously undescribed molecule, fructose-1,6-bisphosphatase 1 (FBP-1) (Figure 3).

[0093] Immunostaining of sarcoidosis skin lesions with FBP-1 revealed staining consistent with sarcoidosis granulomas, but no staining at all in normal skin (Fig. 4). Analysis of previously reported single-cell RNA sequencing data revealed FBP-1 expression in skin macrophages of granuloma annulare (GA) and leprosy patients, but not in healthy skin, atopic dermatitis, or psoriasis (Fig. 5). Next, protein expression was confirmed in GA and sarcoidosis granulomas in the lung, heart, and lymph nodes, but not in atopic dermatitis or psoriasis (Figs. 6 and 7).

[0094] Because FBP-1 acts on the pentose phosphate pathway in the glucose metabolic pathway (Figure 8), we performed clustering analysis of the expression of enzymes related to the pentose phosphate pathway. The results confirmed that sarcoidosis-specific macrophages also exhibited elevated expression of glucose phosphate isomerase (GPI), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and thioredoxin reductase 1 (TXNRD1) (Figures 9 and 10). These enzymes act downstream of FBP-1 in the pentose phosphate pathway. Furthermore, immunohistochemistry revealed G6PD-positive cells in association with FBP-1-positive cells (Figure 11). Immunostaining for FBP1 and TXNRD1 in sarcoidosis lesions revealed staining consistent with sarcoid granulomas, with FBP1 specifically staining epithelioid cells and TXNRD1 specifically staining giant cells (Figure 12). Furthermore, we confirmed that serum FBP1 concentrations were high in sarcoidosis patients (Figure 13). These results suggest that the pentose phosphate pathway is activated in macrophages of granulomas and that it may be used as a new diagnostic marker.

[0095] Inhibition of the Pentose Phosphate Pathway in a Sarcoidosis Model A previously known in vitro sarcoidosis model involves culturing human peripheral blood monocytes with concanavalin A, IFNγ, and CD40L to induce giant cell formation (Figure 14). In this model, genes related to the pentose phosphate pathway, such as FBP-1 and G6PD, were elevated (Figure 15). Therefore, when giant cell formation was induced in the presence of an FBP-1 inhibitor, a G6PD and 6PGD inhibitor (6AN), or tofacitinib (a JAK inhibitor), giant cell formation was suppressed (Figure 16). However, no difference was observed between the control and a low concentration of the FBP-1 inhibitor (100 μM). Giant cell formation was also suppressed in the presence of FBP1 inhibitors (MB05032 (10 μM), MB07229 (10 μM)) or TXNRD inhibitors (auranofin (0.1 μM), TRi-1 (0.1 μM)), which have already been clinically tested in humans for other diseases and can be administered systemically (Figures 17 and 18). Furthermore, FBP-1 gene expression and giant cell formation were suppressed in the presence of siRNA against the FBP-1 gene (Figure 19). Giant cell formation was also suppressed by the use of 2-deoxy-D-glucose, a GPI inhibitor.

[0096] Furthermore, administration of the FBP-1 inhibitors, 6AN or tofacitinib, after giant cell formation and analysis 3 days later showed a reduction in giant cells (Figure 20). However, a low concentration of the FBP-1 inhibitor (100 μM) did not show any difference from the control. Auranofin (10 μM) or TRi-1 (10 μM) also reduced giant cells (Figure 21).

[0097] These results suggest that inhibitors of enzymes involved in the pentose phosphate pathway, similar to JAK inhibitors, which have been suggested to be effective in treating sarcoidosis, could be used to prevent and treat granuloma-forming diseases such as sarcoidosis.

[0098] Inhibition of the pentose phosphate pathway in mice with induced skin inflammation. Mice were intraperitoneally administered 20 mg / kg of 6AN suspended in dimethyl sulfoxide (DMSO) followed by intradermal injections of concanavalin A and beads, and skin inflammation and macrophage accumulation were examined. Subcutaneous granuloma formation was observed in both the DMSO (vehicle) and 6AN-treated groups. However, the size of the granulomas in the 6AN group was significantly reduced compared to the DMSO group (Figure 22). The 6AN-treated group exhibited reduced ear swelling compared to the control group (Figure 23). Furthermore, a comparison of cells infiltrating the ear revealed reduced macrophage accumulation in the 6AN-treated group (Figure 24). These results suggest that inhibition of the pentose phosphate pathway suppresses skin inflammation and granuloma formation, suggesting that inhibitors of pentose phosphate pathway-related enzymes may be useful for the prevention and treatment of granuloma-forming diseases such as sarcoidosis.

[0099] Inhibition of the pentose phosphate pathway in a sarcoidosis model Giant cell formation was induced in an in vitro sarcoidosis model in the presence of various concentrations of the FBP-1 inhibitor MB07803. The results are shown in Figure 25. Giant cell formation was inhibited in a dose-dependent manner, with IC ５０ was 12 nM.

[0100] In an in vitro sarcoidosis model, giant cell formation was induced in the presence of various concentrations of the FBP-1 inhibitor MB06322. The results are shown in Figure 26. Giant cell formation was inhibited in a dose-dependent manner, with IC ５０ was 6.8 μM.

[0101] Furthermore, the IC value of FBP-1 inhibitors MB05032, MB07229, and MB07803 on giant cell formation in an in vitro sarcoidosis model was evaluated. ５０ The results are shown in Figure 27. ５０ is 100 μM or more, and the IC of MB07803 ５０ was 10-20 nM.

[0102] Inhibition of the pentose phosphate pathway in mice with induced skin inflammation. Mice were intraperitoneally administered MB07803 (60 mg / kg) suspended in dimethyl sulfoxide (DMSO) followed by intradermal injection of concanavalin A and beads, and skin inflammation and macrophage accumulation were examined. Subcutaneous granuloma formation was observed in the DMSO (vehicle) and MB07803-treated groups. However, the size of the granulomas in the MB07803 group was reduced compared to the DMSO group (Figure 28). These results indicate that MB07803 suppresses granulomas in this mouse model.

[0103] INDUSTRIAL APPLICABILITY According to the present disclosure, it is possible to treat and / or prevent granuloma or a disease accompanied by granuloma, and to determine whether a subject has granuloma, which is useful in the medical field.

Claims

1. A composition for preventing and / or treating granuloma, comprising an inhibitor of an enzyme associated with the pentose phosphate pathway.

2. The composition according to claim 1, wherein the enzyme associated with the pentose phosphate pathway is fructose-1,6-bisphosphatase-1 (FBP-1) or thioredoxin reductase 1 (TXNRD1).

3. The composition according to claim 2, wherein the enzyme associated with the pentose phosphate pathway is TXNRD1.

4. The composition according to claim 3, wherein the inhibitor of the enzyme associated with the pentose phosphate pathway is auranofin, TRI-1, ketosine or LCS3.

5. The composition according to claim 3, wherein the inhibitor of the enzyme associated with the pentose phosphate pathway is auranofin or TRI-1.

6. The composition according to claim 3, wherein the inhibitor of the enzyme associated with the pentose phosphate pathway is siRNA against TXNRD1.

7. The composition according to claim 2, wherein the enzyme associated with the pentose phosphate pathway is FBP-1.

8. The composition according to claim 7, wherein the inhibitor of the enzyme associated with the pentose phosphate pathway is MB07229 or MB07803.

9. The composition according to claim 7, wherein the inhibitor of the enzyme associated with the pentose phosphate pathway is siRNA against FBP-1.

10. The composition according to any one of claims 1 to 9, wherein the granuloma is a granuloma in sarcoidosis, granuloma annulare, mycobacterial infection or Crohn's disease.

11. A composition for treating and / or preventing a disease accompanied by granuloma, comprising an inhibitor of an enzyme associated with the pentose phosphate pathway.

Images