A therapeutic agent for advanced diseases caused by an increase in Eomes-positive CD4-positive T cells

Abstract

The present invention provides a therapeutic agent for a progressive disease caused by an increase in Eomes-positive CD4-positive T cells, said therapeutic agent comprising a compound represented by general formula (I) or a salt thereof as an active ingredient. In the formula: R1 represents an aldopyranose residue; R2 represents a hydrogen atom or a hydroxyl group; R3 represents -CH2-, -CH(OH)-CH2- or -CH=CH-; R4 represents a hydrogen atom or CH3; x is 0-35; and y and z are integers satisfying the relationship 0≤y+z≤3.

Description

[Technical Field]

[0001] The present invention relates to a therapeutic agent for progressive diseases caused by an increase in Eomes-positive CD4-positive T cells. [Background technology]

[0002] It is known that the production and release of perforin and granzyme B is controlled by the Eomes gene expressed on the cell surface in cytotoxic T cells (CTL: CD8-positive T cells) and NK cells (see Non-Patent Document 1). It has also been reported that the expression of the Eomes gene in some CD4-positive T cells is associated with antitumor immunity or chronic viral diseases (see Non-Patent Documents 2 and 3).

[0003] In recent years, it has been reported that when CD4+ T cells collected from patients with progressive diseases such as secondary progressive multiple sclerosis are examined, the expression levels of the Eomes gene and protein are increased.

[0004] The present inventors have found that in NR4A2-deficient mice in which monophasic experimental autoimmune encephalomyelitis (EAE) has been induced, EAE pathology accompanied by the usual limb paralysis is not observed in the early stage of induction, but EAE pathology (hereinafter also referred to as "late EAE pathology") is observed in the late stage of induction (approximately 28 days after induction or later), and that late EAE pathology serves as a model for progressive MS pathology (Patent Document 1). Furthermore, the present inventors have hypothesized that late EAE pathology, including neurodegeneration, is caused by persistent neuronal damage due to the release of granzyme B in response to stimulation, and have found that inhibition of the PAR1 receptor using a PAR1 receptor antagonist or the like ameliorates late EAE pathology (Patent Document 2). Furthermore, the inventors have newly discovered that in the late EAE pathology of NR4A2-deficient mice, stimulation by CNS-derived antigen-presenting cells induces the expression of Eomes molecules in Th cells, and that prolactin produced by antigen-presenting cells promotes the induction of Eomes molecule expression, and have found that inhibiting prolactin improves the late EAE pathology (Patent Document 3). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2016 / 002827 [Patent Document 2] International Publication No. 2016 / 114386 [Patent Document 3] International Publication No. 2018 / 101261 [Non-patent literature]

[0006] [Non-Patent Document 1] Science 302: 1041-1043, 2003. [Non-patent document 2] Eur. J. Immunol. 2019, 49: 38-41. [Non-patent document 3] J Leukoc Biol. 2014 May; 95(5): 705-713. [Non-patent document 4] Nature Reviews Neurology 2012, 8, 647-656. [Non-patent document 5] Nature Reviews Neurology 2013, 9, 496-503. [Non-patent document 6] Multiple Sclerosis Journal 2013,19: 1428-1436. Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, an object of the present invention is to provide a therapeutic agent for progressive diseases caused by an increase in Eomes-positive CD4-positive T cells. [Means for solving the problem]

[0008] The present inventors have newly discovered that the expression of Eomes-positive T cells is induced in the late stage of EAE pathology in NR4A2-deficient mice, and that a compound represented by general formula (I) or a salt thereof suppresses the induction of Eomes molecule expression.

[0009] The present invention provides the following (1) to (8). (1) A therapeutic agent for a progressive disease caused by an increase in Eomes-positive T cells, comprising a compound represented by general formula (I) or a salt thereof as an active ingredient. [ka] [In the formula, R 1 represents an aldopyranose residue, and R 2 represents a hydrogen atom or a hydroxyl group, and R 3 represents -CH2-, -CH(OH)-CH2-, or -CH=CH-, and R 4 represents a hydrogen atom or CH3, x is 0 to 35, and y and z are integers that satisfy the relationship y+z=0 to 3.] (2) R 1 The agent for treating progressive disease according to (1), wherein the compound is represented by the following formula (II): [ka] (3) R 2 and R 4 represents a hydrogen atom; x is 11 to 23; and z is 0. (4) The agent for treating progressive disease according to any one of (1) to (3), wherein the compound represented by general formula (I) or a salt thereof is a compound represented by general formula (III) or a salt thereof. [ka] [In the formula, R 2 represents a hydrogen atom, x is an integer of 12 to 23, and y is an integer of 0 to 3. (5) The agent for treating a progressive disease according to any one of (1) to (4), wherein the progressive disease is relapsing-remitting multiple sclerosis, progressive-relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis. (6) A method for treating a progressive disease caused by an increase in Eomes-positive CD4-positive T cells, which comprises administering a composition containing a compound represented by general formula (I) or a salt thereof. (7) A method for treating the progressive disease according to (5), which comprises orally administering a composition containing a compound represented by general formula (I) or a salt thereof. (8) The method for treating a progressive disease according to (6) or (7), wherein the progressive disease is relapsing-remitting multiple sclerosis, progressive-relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a therapeutic agent for progressive diseases caused by an increase in Eomes-positive CD4-positive T cells. [Brief explanation of the drawings]

[0011] [Figure 1] 1 is a graph showing EAE scores in NR4A2-deficient mice and control mice in which EAE was induced by sensitization with MOG35-55 peptide. [Figure 2] 1 is a graph showing EAE scores in NR4A2-deficient mice and B6 mice in which EAE was induced by sensitization with MOG35-55 peptide. [Figure 3] 1 is a graph showing EAE scores in NR4A2-deficient mice and B6 mice in which EAE was induced by sensitization with MOG35-55 peptide. [Figure 4] 1 is a graph showing EAE scores in B6 mice in which EAE was induced by sensitization with MOG35-55 peptide. [Figure 5] 1 shows graphs showing EAE scores in NR4A2-deficient mice in which EAE was induced by MOG35-55 peptide sensitization, where (a) is a graph showing the results after administration of 400 μg / kg of OCH, and (b) is a graph showing the results after administration of 40 μg / kg of OCH. [Figure 6]1 is a graph showing the amount of IFNγ produced when MOG35-55 peptide was added to spleen-derived T cells from mice sensitized with the MOG35-55 peptide. [Figure 7] 10 is a graph showing the amount of IL-17 produced when the MOG35-55 peptide was added to the spleen-derived T cells. DETAILED DESCRIPTION OF THE INVENTION

[0012] [Definition] As used herein, the term "progressive disease caused by an increase in Eomes-positive CD4-positive T cells" refers to a progressive disease caused by an increase in CD4-positive T cells in which the Eomes gene and the protein encoded by it are expressed intracellularly. Examples of progressive diseases caused by an increase in Eomes-positive CD4-positive T cells include progressive multiple sclerosis (e.g., primary progressive multiple sclerosis (PP-MS) and secondary progressive multiple sclerosis (SP-MS)). )) These include autoimmune optic neuritis, chronic inflammatory demyelinating polyneuropathy (CIDP), and myalgic encephalomyelitis-chronic fatigue syndrome.

[0013] Multiple sclerosis (MS) is an autoimmune disease caused by multiple inflammations in the central nervous system targeting myelin sheaths and axons, resulting in widespread demyelination and impaired nerve conduction. As the pathology of multiple sclerosis progresses, severe neurological symptoms such as motor disorders, higher brain dysfunction, ataxia, sensory impairment, and visual impairment appear. Multiple sclerosis is classified into relapsing-remitting MS (RR-MS), which is characterized by repeated acute exacerbations and remissions, and progressive MS. Progressive MS is classified into primary progressive MS (PP-MS), secondary progressive MS (SP-MS), which transitions to a progressive form after a certain period of RR-MS symptoms, and progressive-relapsing MS (PR-MS), which progresses with repeated relapses (see Non-Patent Documents 4 to 6).

[0014] Known disease-modifying drugs (DMDs) for RR-MS include type 1 interferon, anti-inflammatory drugs, and immunosuppressants. Currently, anti-CD20 antibodies are known to be used to treat progressive MS, and S1P receptor modulators are known to be used to treat RR-MS and SP-MS. However, many aspects of the detailed pathology and mechanisms of progressive MS remain unknown.

[0015] [NR4A2 gene] The NR4A2 gene, also known as the Nurr1 gene, NOT gene, or RNR1 gene, is an orphan nuclear receptor. The NR4A2 gene is primarily expressed in the central nervous system, with particularly high expression in the ventral midbrain, brainstem, and spinal cord. NR4A2 expression is induced in response to prostaglandins, growth factors, inflammatory cytokines, and T cell receptor crosslinking, and regulates transcription by directly binding to DNA in a ligand-dependent or ligand-independent manner. The NCBI Reference Sequence accession number for the human NR4A2 gene transcript is NM_006186.3.

[0016] The present inventors investigated changes in gene expression levels in T cells isolated from patients with multiple sclerosis or healthy adults and found that NR4A2 gene expression was significantly increased in T cells isolated from patients with multiple sclerosis. They also demonstrated that NR4A2 is important for the function of Th17 cells, which produce interleukin-17 and are involved in the pathology of various autoimmune diseases, including multiple sclerosis (Raveney et al., PLoS One, 2013, 8(2): e56595). Furthermore, when experimental autoimmune encephalomyelitis (EAE) was induced in mice lacking the NR4A2 gene specifically in CD4+ T cells, i.e., NR4A2 conditional knockout mice (NR4A2cKO mice), they found that, unlike conventional EAE models, clinical scores did not increase approximately 14 days after EAE induction, but did increase 28 days after induction (see Patent Documents 1 and 2). Thus, the experimental autoimmune encephalomyelitis (EAE) model using NR4A2cKO mice can be used as a pathological model of progressive immune-mediated demyelinating diseases that is independent of Th17 cells, particularly as an animal model that shows the late (progressive) pathology of progressive MS.

[0017] Here, conditional knockout (cKO) refers to the deletion of a target gene in a specific tissue or specific cell of a non-human animal, rather than the deletion of the entire desired gene. For example, CD4 + These are mice in which the NR4A2 gene is specifically deleted in T cells.

[0018] NR4A2cKO mice can be established, for example, by Cre-loxP site-specific recombination technology. Specifically, NR4A2cKO mice can be produced by crossbreeding a mouse into which two loxP genes have been introduced so as to sandwich the NR4A2 gene, which is the target gene to be deleted, with a mouse into which a cre gene has been introduced downstream of the promoter region of the target cell so that the Cre enzyme is expressed in the cell in which the target gene is to be deleted. Those skilled in the art can select tissues or cells in which the target gene is to be deleted depending on the purpose. For example, when the target cell is a CD4+ For T cells, CD4 + It is possible to generate mice in which the NR4A2 gene is deleted only in T cells.

[0019] Recent studies have shown that Th17 cells (especially NR4A2-positive Th17 cells) are involved in early EAE pathology, but IL-17 production, which could explain EAE pathology, was not observed in late EAE pathology. Furthermore, CNS-infiltrating CD4 + Examination of T cells revealed that expression of the Eomes gene was significantly increased around 28 days after EAE induction. + CD4 + The lack of IL-17 expression in T cells suggests that they are a distinct subset from Th17 cells. Furthermore, the intravenous administration of Eomes-specific siRNA to NR4A2cKO mice with EAE significantly improved the late-stage pathology of EAE, and NR4A2 / Eomes-deficient mice did not develop the late-stage pathology of EAE, suggesting that suppression of Eomes gene expression is related to the improvement of the late-stage pathology of EAE.

[0020] [Eomes gene] The Eomes gene, also known as Eomesodermin or Tbr2, is a member of the T-box transcription factor family and is a protein involved in the development and differentiation of vertebrates. + It is known to be expressed in T cells (cytotoxic T cells, CTL) and NK cells, and to directly induce the expression of perforin and granzyme B. The NCBI Reference Sequence accession numbers for the human Eomes gene transcripts are NM_001278182.1 (variant 1), NM_005442.3 (variant 2), and NM_001278183.1 (variant 3).

[0021] [Treatment agent for progressive immune-mediated demyelinating diseases] A first embodiment of the present invention is a therapeutic agent for a progressive disease caused by an increase in Eomes-positive T cells, which comprises a compound represented by general formula (I) or a salt thereof as an active ingredient.

[0022] The compound represented by general formula (I) is a kind of α-galactosylceramide. The compound represented by general formula (I) includes all stereoisomers (e.g., enantiomers, diastereomers) derived from quaternary carbon atoms. The compound represented by general formula (I) may be a mixture thereof (e.g., racemate). [ka]

[0023] In general formula (I), R 1 represents an aldopyranose residue. Examples of aldopyranose residues include α-D-glucosyl, α-D-galactosyl, α-D-mannosyl, β-D-glucosyl, β-D-galactosyl, β-D-mannosyl, 2-deoxy-2-amino-α-D-galactosyl, 2-deoxy-2-amino-β-D-galactosyl, 2-deoxy-2-acetylamino-α-D-galactosyl, 2-deoxy-2-acetylamino-β-D-galactosyl, β-D-allopyranosyl, β-D-altropyranosyl, and β-D-idosyl. 1 The aldopyranose group represented by the formula (II) is preferably an α-form, and more preferably an α-D-galactopyranosyl group represented by the formula (II) below. [ka]

[0024] In general formula (I), R 2 represents a hydrogen atom (-H) or a hydroxyl group (-OH), preferably a hydrogen atom.

[0025] In general formula (I), R 3is -CH2-, -CH(OH)-CH2-, or -CH=CH-, preferably -CH2- or -CH(OH)-CH2-, and more preferably -CH(OH)-CH2-.

[0026] In general formula (I), R 4 represents a hydrogen atom (-H) or CH3, preferably a hydrogen atom.

[0027] In general formula (I), x is an integer of 0 to 35, preferably an integer of 0 to 26, more preferably an integer of 11 to 26, still more preferably an integer of 11 to 23, and particularly preferably an integer of 18 to 23.

[0028] In the general formula (I), y and z represent integers that satisfy y+z=0 to 3. Preferably, z is 0 and y is 0 to 3. More preferably, z is 0 and y is 1 to 3. In addition, -(CH2) y (CH(CH3)) z The - does not mean that the order of (CH2) and (CH(CH3)) follows the order of the descriptions, but merely indicates the quantitative relationship between (CH2) and (CH(CH3)). For example, when y=2 and z=1, -(CH2) y (CH(CH3)) z This means that there are two (CH2)s and one (CH(CH3)) within the -, and the order of the two (CH2)s and one (CH(CH3)) does not matter. Specifically, it may be any of -CH(CH3)CH2CH2-, -CH2CH(CH3)CH2-, or -CH2CH2CH(CH3)-.

[0029] The compound represented by the general formula (I) or a salt thereof is preferably a compound represented by the general formula (III) or a salt thereof. [ka] In the formula, R 2represents a hydrogen atom or a hydroxyl group, and is preferably a hydrogen atom. x is 12 to 23, preferably 15 to 23, and more preferably 18 to 23. y is 1, 2 or 3, and preferably 2 or 3.

[0030] Specific examples of the compound represented by general formula (I) include the following compounds (1) to (48). Compounds (3) to (9), (15) to (21), (27) to (33), and (39) to (45) are more preferred as the active ingredient according to this embodiment.

[0031] (1) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-triacontanoylamino)-1,3,4-heptanetriol (2) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonacosanoylamino)-1,3,4-heptanetriol (3) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-octacosanoylamino)-1,3,4-heptanetriol, (4) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heptacosanoylamino)-1,3,4-heptanetriol (5) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-hexacosanoylamino)-1,3,4-heptanetriol (6) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-pentacosanoylamino)-1,3,4-heptanetriol (7) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-heptanetriol (8) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tricosanoylamino)-1,3,4-heptanetriol (9) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-docosacosanoylamino)-1,3,4-heptanetriol (10) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heneicosanoylamino)-1,3,4-heptanetriol (11) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-eicosanoylamino)-1,3,4-heptanetriol (12) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonadecanoylamino)-1,3,4-heptanetriol (13) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-triacontanoylamino)-1,3,4-octanetriol (14) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonacosanoylamino)-1,3,4-octanetriol (15) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-octacosanoylamino)-1,3,4-octanetriol (16) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heptacosanoylamino)-1,3,4-octanetriol (17) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-hexacosanoylamino)-1,3,4-octanetriol (18) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-pentacosanoylamino)-1,3,4-octanetriol (19) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-octanetriol (20) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tricosanoylamino)-1,3,4-octanetriol (21) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-docosacosanoylamino)-1,3,4-octanetriol, (22) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heneicosanoylamino)-1,3,4-octanetriol (23) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-eicosanoylamino)-1,3,4-octanetriol (24) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonadecanoylamino)-1,3,4-octanetriol (25) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-triacontanoylamino)-1,3,4-nonanetriol (26) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonacosanoylamino)-1,3,4-nonanetriol, (27) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-octacosanoylamino)-1,3,4-nonanetriol (28) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heptacosanoylamino)-1,3,4-nonanetriol (29) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-hexacosanoylamino)-1,3,4-nonanetriol (30) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-pentacosanoylamino)-1,3,4-nonanetriol (31) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-nonanetriol (32) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tricosanoylamino)-1,3,4-nonanetriol (33) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-docosacosanoylamino)-1,3,4-nonanetriol (34) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heneicosanoylamino)-1,3,4-nonanetriol (35) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-eicosanoylamino)-1,3,4-nonanetriol (36) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonadecanoylamino)-1,3,4-nonanetriol (37) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-triacontanoylamino)-1,3,4-hexanetriol (38) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonacosanoylamino)-1,3,4-hexanetriol (39) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-octacosanoylamino)-1,3,4-hexanetriol (40) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heptacosanoylamino)-1,3,4-hexanetriol (41) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-hexacosanoylamino)-1,3,4-hexanetriol (42) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-pentacosanoylamino)-1,3,4-hexanetriol (43) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-hexanetriol (44) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tricosanoylamino)-1,3,4-hexanetriol (45) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-docosacosanoylamino)-1,3,4-hexanetriol (46) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-heneicosanoylamino)-1,3,4-hexanetriol (47) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-eicosanoylamino)-1,3,4-hexanetriol (48) (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-nonadecanoylamino)-1,3,4-hexanetriol Examples include:

[0032] "Salt of a compound represented by general formula (I)" means a salt prepared by mixing a compound represented by general formula (I) with a base or an acid to form a salt between a specific substituent in the chemical structure represented by general formula (I) and the base or the acid. Depending on the base or the acid used, salts can be classified into base addition salts and acid addition salts. The base or the acid is preferably a pharmaceutically acceptable base or acid. In other words, the salt of a compound represented by general formula (I) is preferably a pharmaceutically acceptable salt of a compound represented by general formula (I).

[0033] Examples of basic addition salts include alkali metal salts such as sodium salts or potassium salts, alkaline earth metal salts such as calcium salts or magnesium salts, aliphatic amine salts such as trimethylamine salts, triethylamine salts, dicyclohexylamine salts, ethanolamine salts, diethanolamine salts, triethanolamine salts, or brocaine salts, aralkylamine salts such as N,N-dibenzylethylenediamine, heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts, or isoquinoline salts, basic amino acid salts such as arginine salts or lysine salts, ammonium salts, or quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylammonium salts, benzyltributylammonium salts, methyltrioctylammonium salts, or tetrabutylammonium salts.

[0034] Examples of acid addition salts include inorganic acid salts such as hydrochloride, sulfate, nitrate, phosphate, carbonate, hydrogencarbonate, and perchlorate; organic acid salts such as acetate, propionate, lactate, maleate, fumarate, tartrate, malate, citrate, and ascorbate; sulfonates such as methanesulfonate, isethionate, benzenesulfonate, and p-toluenesulfonate; and acidic amino acid salts such as aspartate and glutamate.

[0035] In the present specification, the compound represented by general formula (I) may be a compound that does not itself exhibit a pharmacological action, but that can provide a desired pharmacological activity by changing its chemical structure with an enzyme or the like after administration to a living body. That is, the compound represented by general formula (I) may also include a prodrug form.

[0036] The agent for treating progressive disease according to this embodiment may consist solely of the compound represented by general formula (I) or a salt thereof as the active ingredient, or may further contain a pharmaceutically acceptable carrier and / or solvent.

[0037] The term "pharmaceutically acceptable carrier" refers to a substance that is approved for use in the field of pharmaceutical technology because it has no or very little adverse effects, such as side effects, on animals including humans. For example, it refers to non-toxic excipients, binders, disintegrants, fillers, emulsifiers, flow control agents, etc. that are commonly used in the field of pharmaceutical technology.

[0038] Excipients include, for example, sugars (including, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium phosphate, calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, and high molecular weight polyethylene glycols (PEGs), pluronics, kaolin, silicic acid, or combinations thereof.

[0039] Examples of binders include starch paste, syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone.

[0040] Disintegrants include, for example, starch, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

[0041] Examples of fillers include the sugars and / or calcium phosphate (eg, tricalcium phosphate or calcium hydrogen phosphate).

[0042] Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

[0043] Flow regulators and lubricants include, for example, silicates, talc, stearates or polyethylene glycol.

[0044] In addition to the above, pharmaceutically acceptable carriers may also include, as necessary, isotonicity agents, lubricants, flavorings, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption promoters (e.g., quaternary ammonium salts, sodium lauryl sulfate), bulking agents, pH adjusters, humectants (e.g., glycerin, starch), adsorbents (e.g., starch, lactose, kaolin, bentonite, colloidal silicic acid), disintegration inhibitors (e.g., sucrose, stearin, cocoa butter, hydrogenated oil), coating agents, coloring agents, preservatives, antioxidants, fragrances, flavorings, sweeteners, buffers, soothing agents, and the like.

[0045] The term "pharmaceutically acceptable solvent" refers to a solvent that can be approved for use in the field of pharmaceutical formulation technology because it has no or very little harmful effects, such as side effects, on animals including humans. Examples of such solvents include non-toxic solvents that are commonly used in the field of pharmaceutical formulation technology, such as water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and polyoxyethylene sorbitan fatty acid esters. These solvents are preferably adjusted to be isotonic with blood.

[0046] The above-mentioned carriers and solvents are mainly used to facilitate formulation and administration, and to maintain the dosage form and drug efficacy, and may be used appropriately as needed.

[0047] [Method for producing the compound represented by formula (I)] The compound represented by formula (I) can be produced by various methods known in the art, for example, according to the method described in Japanese Patent No. 4,064,346 or Japanese Patent No. 4,742,220.

[0048] [Method for producing a therapeutic agent for progressive diseases caused by an increase in Eomes-positive CD4-positive T cells] The therapeutic agent for progressive disease according to this embodiment can be prepared using a compound represented by general formula (I) or a salt thereof by a method known in the art, and is intended to improve or treat progressive disease caused by an increase in Eomes-positive CD4-positive T cells. For formulation, for example, the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.) may be used.

[0049] The dosage form of the therapeutic agent for progressive disease caused by an increase in Eomes-positive CD4-positive T cells is selected appropriately depending on the administration method and / or prescription conditions. The administration method can be broadly divided into oral administration and parenteral administration.

[0050] Examples of dosage forms suitable for oral administration include tablets, pills, granules, powders, capsules, drops, sublingual tablets, troches, and liquids.

[0051] Tablets may be coated as needed as known in the art, e.g., sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double-layered tablets, or multi-layered tablets. For example, capsules may be prepared by mixing a powdered active ingredient with excipients such as lactose, starch or its derivatives, or cellulose derivatives, and then filling a gelatin capsule. Tablets may be prepared by kneading the above-mentioned excipients with binders such as sodium carboxymethylcellulose, alginic acid, or gum arabic, and water, optionally forming granules, followed by the addition of lubricants such as talc or stearic acid, and then using a conventional compression tablet press. For oral administration, the shape and size of each dosage form are not particularly limited, as long as they are within the ranges known in the art.

[0052] Dosage forms suitable for parenteral administration include, for example, solutions (including suspensions), emulsions (creams), gels, ointments (including pastes), plasters, powders, and suppositories. These can be made into dosage forms suitable for the administration method, such as systemic administration, local administration, or rectal administration. Dosage forms suitable for systemic administration include, for example, liquids for injection. In the case of injection, the active ingredient is dissolved in sterile distilled water or sterile physiological saline together with a solubilizer and sealed in an ampoule to form an injectable preparation. If necessary, a stabilizer and a buffer substance may be added. Dosage forms suitable for local administration include, for example, solutions such as eye drops or nasal drops, emulsions, powders such as nasal drops, pastes, gels, ointments, and plasters. Dosage forms suitable for rectal administration include, for example, suppositories.

[0053] Preferably, an effective amount of the active ingredient is contained in one dosage unit of the therapeutic agent for progressive disease. As used herein, "effective amount" refers to the amount of the active ingredient required to exert its function, i.e., the amount of the compound represented by general formula (I) or a salt thereof required to suppress the proliferation of Eomes-positive CD4-positive T cells or reduce the intracellular Eomes expression level in T cells, and which does not cause any or no harmful side effects in the subject to which it is administered. This effective amount may vary depending on various conditions, such as information about the subject, dosage form, and route of administration. "Information about the subject" refers to the stage or severity of the disease, general health condition, age, weight, sex, diet, drug sensitivity, presence or absence of concomitant medications, and tolerance to treatment. As a specific example of the effective amount of the therapeutic agent for progressive disease according to this embodiment, when the therapeutic agent for progressive disease according to this embodiment is orally administered to an adult male (weight 60 kg), the active ingredient may be contained in an amount of 0.01% to 100% by weight, preferably 0.1% to 100% by weight, per dosage unit. Furthermore, when the agent for treating progressive disease according to this embodiment is administered as an injection, the injection may contain 0.01% (w / v) to 20% (w / v), preferably 0.1% (w / v) to 10% (w / v) of the active ingredient per dosage unit. In the case of dosage forms such as tablets, pills, or capsules, the effective amount for Eomes-positive CD4-positive T cells can be administered in divided doses adjusted depending on the number of doses, and therefore it is not necessary for one tablet to contain an effective amount.

[0054] A second embodiment of the present invention is a method for treating a progressive disease caused by an increase in Eomes-positive CD4-positive T cells, which comprises administering a composition containing a compound represented by general formula (I) or a salt thereof.

[0055] In this embodiment, the definitions of "a compound represented by general formula (I) or a salt thereof" and "a progressive disease caused by an increase in Eomes-positive CD4-positive T cells" can be referred to those described in the first embodiment. A composition containing a compound represented by general formula (I) or a salt thereof may be the agent for treating a progressive disease described in the first embodiment.

[0056] [Administration method] The living body to which the composition containing the compound represented by general formula (I) or a salt thereof (for example, the agent for treating progressive disease shown in the first embodiment) is administered is a vertebrate, preferably a mammal, more preferably a human.

[0057] Specific administration routes include oral administration and parenteral administration, as described above. Parenteral administration can be further divided into systemic administration and local administration (e.g., subcutaneous administration, transdermal administration, transmucosal administration, rectal administration, etc.).

[0058] The administration method can be appropriately selected depending on the site of disease onset or the progression of the disease, and may be either systemic or local administration. Oral administration, which is less invasive, is preferred. Furthermore, when the active ingredient is to be rapidly distributed via the circulatory system, such as the bloodstream, systemic administration via intravascular injection is suitable. If the target disease is localized, local administration, in which the active ingredient is administered directly to the site of disease onset and its surroundings via local injection, can also be employed. The injection site of the pharmaceutical composition by injection is not particularly limited. Examples include the circulatory system, such as within a blood vessel or ventricle, or within an organ or tissue, such as the liver, intramuscularly, intraarticularly, intramedullary, intrathecal, transdermally, subcutaneously, intradermally, intraperitoneally, intranasally, intestine, or sublingually.

[0059] The term "therapeutic agent" refers to an agent that refers to either therapeutic treatment or prophylactic or preventative measures, the purpose of which is to treat, prevent, or slow (alleviate) an undesirable physiological change or disorder, such as the onset, spread, or progression of an advanced disease. [Example]

[0060] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0061] 1. EAE analysis of NR4A2cKO mice (1)Animals All mice used were 6-8 weeks old and raised under specific pathogen-free conditions. NR4A2 was transfected using a targeting vector containing the NR4A2 gene flanked by loxp sequences. fl / fl The NR4A2 transgene flanked by loxp sequences was introduced into C57BL / 6 embryonic stem cells by microinjection. The established strain was crossed with C57BL / 6 FLPe mice (RIKEN BioResource Center) to remove the neomycin cassette, and the resulting strains were crossed to generate homozygous NR4A2 fl / fl C57BL / 6 mice were generated. The resulting mice were crossed with C57BL / 6 CD4-Cre mice (Taconic) to generate CD4-specific NR4A2cKO C57BL / 6 mice (C57BL / 6 Cre-CD4 / NR4A2 fl\fl The resulting mice were backcrossed with SJL / J mice (female, Charles River Japan) for 10 generations to develop NR4A2 fl / fl SJL mice and CD4-specific NR4A2cKO SJL mice (SJL / J Cre-CD4 / NR4A2 fl\fl mouse) was established.

[0062] Eomes purchased from The Jackson Laboratory fl / fl Mice were crossed with C57BL / 6 CD4-Cre mice to generate Cre-CD4 Eomes fl / fl C57BL / 6 mice were obtained. fl / fl Mice were transfected with Cre-CD4 / NR4A2 fl / fl The mice were crossed with C57BL / 6 mice and transfected with Cre-CD4 / NR4A2 fl / fl Eomes fl / fl C57BL / 6 mice were obtained.

[0063] (2) EAE induction (monophasic EAE) 100 μg MOG 35-55The peptide corresponding to the residue (synthesized at Toray Research Center, Tokyo, Japan, hereinafter also referred to as "MOG peptide") and 1 mg of killed Mycobacterium tuberculosis H37Ra (Difco, Kansas, USA) emulsified with complete Freund's adjuvant were mixed in equal amounts and emulsified using a homogenizer to prepare a MOG emulsion. The obtained MOG emulsion was injected subcutaneously at one or two sites on the back of CD4-specific NR4A2 cKO C57BL / 6 mice (Cre-CD4 / NR4A2 fl / fl C57BL / 6 mice, NR4A2 cKO) and NR4A2 fl / fl C57BL / 6 mice (Control) as a control to confer immunity. Furthermore, on days 0 and 2 after immunization, 200 μL of a PBS solution containing 200 ng of pertussis toxin (List Biological Laboratories, USA) per mouse was injected intraperitoneally into the mice. After the injection, the EAE pathology of the mice was evaluated daily according to the EAE evaluation criteria shown below. <EAE Evaluation Criteria> 0: No clinical signs 1: Partial paralysis of the tail 2: Flaccid tail 3: Partial paralysis of the hind limbs 4: Paralysis of all hind limbs 5: Paralysis of the hind and forelimbs

[0064] The results are shown in Figure 1. As shown in Figure 1, in NR4A2-deficient mice (NR4A2 cKO), the EAE pathology during the onset, peak, and chronic phases (about 9 - 28 days after EAE induction) observed in control mice (Control) was improved. On the other hand, in NR4A2-deficient mice, a new pathology (late EAE pathology) appeared 28 days after EAE induction. In the following experiments, 4 - 9 mice were used per group, and the bars in Figures 1 - 7 indicate the standard error (SEM).

[0065] 2. The compound represented by the general formula (I) In the examples, (2S,3S,4R)-1-O-(α-D-galactosyl)-2-(N-tetracosanoylamino)-1,3,4-nonanetriol (hereinafter also referred to as "OCH") was used as the compound represented by general formula (I). OCH is represented by the following formula: [ka]

[0066] 3. Effect of OCH administration on EAE pathology (1) Effects of OCH administration EAE was induced in NR4A2-deficient mice as described in 1.(2) above, and OCH or PBS (phosphate-buffered saline) was administered at a dose of 400 μg / kg immediately after induction (day 0), and 7, 14, 21, and 28 days later. The EAE pathology of the mice was evaluated daily according to the EAE evaluation criteria described above. For comparison, OCH or PBS (phosphate-buffered saline) was also administered to B6 mice in which monophasic EAE had been induced, and the EAE pathology of the mice was evaluated daily. OCH was orally administered as a suspension in PBS, and PBS was also orally administered.

[0067] The results are shown in Figure 2. When the PBS-administered group and the OCH-administered group were compared in each model mouse, OCH administration improved both the early and late EAE pathology.

[0068] (2) Effects of OCH administration Monophasic EAE was induced in NR4A2-deficient mice as described in 1.(2) above, and OCH or PBS (phosphate-buffered saline) was administered at a dose of 400 μg / kg on days 5, 10, 15, 20, 25, and 30 after induction. The mice were evaluated daily for EAE pathology according to the EAE evaluation criteria described above. For comparison, B6 mice with monophasic EAE were also administered OCH or PBS (phosphate-buffered saline) and the mice were evaluated daily for EAE pathology. OCH was orally administered as a suspension in PBS, and PBS was also orally administered.

[0069] The results are shown in Figure 3. When the PBS-administered group and the OCH-administered group were compared in each model mouse, OCH administration improved both the early and late EAE pathology.

[0070] (3) Effects of OCH administration on B6 mice Monophasic EAE was induced in B6 mice as described in 1.(2) above, and OCH was administered at a low dose (40 μg / kg) or a high dose (400 μg / kg) on ​​days 5, 10, 15, 20, 25, and 30 after induction. The EAE pathology of the mice was evaluated daily according to the EAE evaluation criteria described above. For comparison, the EAE pathology of B6 mice (untreated) in which monophasic EAE had been induced was evaluated daily. OCH was orally administered as a suspension in PBS, and PBS was also orally administered.

[0071] The results are shown in Figure 4. In the B6 mouse EAE model, early EAE pathology was improved in all administration groups, and the high-dose administration group also improved late EAE pathology.

[0072] (4) Effects of OCH administration on NR4A2-deficient mice Monophasic EAE was induced in NR4A2-deficient mice as described in 1.(2) above, and OCH was administered at 40 μg / kg or 400 μg / kg on days 5, 10, 15, 20, 25, and 30 after induction. The mice were evaluated daily for EAE pathology according to the EAE evaluation criteria described above. For comparison, monophasic EAE was induced in NR4A2-deficient mice (untreated) and the EAE pathology was evaluated daily. OCH was orally administered as a suspension in PBS, and PBS was also orally administered.

[0073] The results are shown in Figure 5. In the EAE model of NR4A2-deficient mice, early EAE pathology was not observed, and late EAE pathology was improved in all treatment groups, with the high-dose treatment group showing more significant improvement. These results suggest that OCH treatment ameliorates late EAE pathology in a dose-dependent manner.

[0074] (5) Effects of OCH administration on NR4A2-deficient mice Monophasic EAE was induced in NR4A2-deficient mice or B6 mice (control) as described in 1.(2) above. OCH was administered at 40 μg / kg or 400 μg / kg on days 5, 10, 15, 20, 25, and 30 after induction. On day 34 after induction, spleens were harvested from the mice, and T cells were extracted. Changes in cytokine (IFNγ and IL-17) production (MOG recall response) were examined in the T cells obtained by administration of a predetermined concentration of MOG peptide. Cytokine concentrations in the culture supernatant were measured using a standard ELISA. Significant IL-17 expression was not detected in NR4A2-deficient mice, confirming that the involvement of Th17 cells in the EAE model induced in these mice was negligible. OCH was administered orally as a suspension in PBS, and PBS was also administered orally.

[0075] The results are shown in Figure 6 (IFNγ) and Figure 7 (IL-17). As shown in Figure 6, OCH administration did not significantly affect IFNγ production in either B6 or NR4A2-deficient mice. As shown in Figure 7, IL-17 production was significantly increased in B6 mice, whereas IL-17 production was reduced in the OCH-treated B6 mice, indicating suppression of Th17 cell activity. In contrast, NR4A2-deficient mice did not show a significant increase in IL-17 production, and no significant changes were observed with OCH administration. This confirms that Th17 cell function is significantly impaired in these mice, suggesting that the ameliorative effect of OCH on EAE induced in NR4A2-deficient mice is not the result of suppression of Th17 cells.

Claims

1. A therapeutic agent for a progressive disease caused by an increase in Eomes-positive CD4-positive T cells, comprising a compound represented by general formula (I) or a salt thereof as an active ingredient, The agent for treating a progressive disease, wherein the progressive disease is progressive relapsing multiple sclerosis, primary progressive multiple sclerosis, or secondary progressive multiple sclerosis. 【Chemical 1】 [In the formula, R １ represents an aldopyranose residue selected from the group consisting of α-D-glucosyl, α-D-galactosyl, α-D-mannosyl, β-D-glucosyl, β-D-galactosyl, β-D-mannosyl, β-D-allopyranosyl, β-D-altropyranosyl, and β-D-idosyl; R ２ represents a hydrogen atom or a hydroxyl group, R ３ -CH(OH)-CH ２ represents -, and R ４ is a hydrogen atom or CH ３ wherein x is 11 to 35, and y and z are integers satisfying the relationship y+z=0 to 3.

2. The R １ The therapeutic agent for progressive disease according to claim 1, wherein the compound is represented by the following formula (II): 【Chemistry 2】

3. The R ２ and R ４ The agent for treating progressive disease according to claim 1 or 2, wherein represents a hydrogen atom, x is 11 to 23, and z is 0.

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