Treatment of HTLV-1-related myelopathy using nrf2 activator

Abstract

The present invention provides an antiviral agent (preferably an anti-HTLV-1 agent) and a therapeutic or prophylactic agent for viral infections (preferably HTLV-1 infection, more preferably HTLV-1-related myelopathy (HAM)), each containing a nuclear factor erythroid 2-related factor 2 (Nrf2) activator.

Description

Treatment of HTLV-1-associated myelopathy using Nrf2 activators

[0001] The present invention relates to a therapeutic agent for HTLV-1-associated myelopathy containing an Nrf2 activator.

[0002] HTLV-1-associated myelopathy (HAM) is a chronic inflammatory neurological disease that develops in some individuals infected with human T-cell leukemia virus type 1 (HTLV-1). It presents with symptoms such as spastic paraplegia, urinary dysfunction, and sensory impairment, and as it progresses, it can lead to wheelchair use or bedriddenness, severely impairing daily life. The pathogenesis of HAM is thought to involve the HTLV-1 virus infecting and proliferating primarily in CD4-positive T lymphocytes. These proliferating infected cells infiltrate the central nervous system, triggering an excessive inflammatory response centered on CD8-positive cytotoxic T lymphocytes (CTLs), leading to the destruction and degeneration of surrounding nerve tissue. Therefore, treatment strategies for HAM require the removal of HTLV-1 infected cells, suppression of the excessive immune response and inflammation in the spinal cord, and regeneration of damaged and degenerated nerve tissue.

[0003] Existing treatments for HAM have involved the use of steroids and interferon (IFN) as drugs to suppress immune responses and inflammation. However, the effects of these drugs are limited to the short term, and in the long term, they are often discontinued in clinical practice due to side effects and the progression of symptoms due to decreased therapeutic effect. Furthermore, because HTLV-1 increases the viral load in the body through the proliferation of infected cells rather than viral replication, antiviral drugs such as reverse transcriptase inhibitors are not very effective. In recent years, clinical trials of anti-CCR4 antibody drugs aimed at eliminating virus-infected cells have been conducted, but their effect has been limited to a transient reduction in infected cells and has not led to improvement in neurological symptoms. Nerve regeneration therapy is still in the research stage, and it is considered necessary to eliminate infected cells and suppress inflammation before regeneration therapy can be performed.

[0004] As such, current treatment options for HAM are limited, and the effectiveness of existing drugs is also limited, resulting in insufficient improvement in patients' quality of life (QOL). Consequently, many patients do not receive adequate treatment, and the management of HAM remains difficult. Therefore, the development of new drugs for HAM is urgently needed.

[0005] Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor belonging to the CNC (Cap'n'Collar) transcription factor group, possessing a basic-leucine zipper structure. Normally located in the cytoplasm, it binds to a regulatory protein called Keap1 (kelch-like ECH associated protein 1). However, upon oxidative stress stimulation, it dissociates from Keap1 and translocates to the nucleus, where it forms a heterodimer with small Maf group transcription factors. There, it binds to the ARE (Antioxidant Response Element) sequence located within the regulatory region of target genes, activating the transcription of various target genes involved in the oxidative stress response.

[0006] Therefore, Nrf2 activators are expected to be effective in treating various oxidative stress-related diseases such as liver disease, bile duct disease, lung disease, kidney disease, heart disease, central nervous system disease, mitochondrial disease, inflammatory diseases, and cancer (Non-Patent Literature 1), and many Nrf2 activators have been reported, with some receiving manufacturing and marketing approval or progressing to clinical trials (Non-Patent Literature 2).

[0007] However, there have been no reports whatsoever regarding the antiviral effects of Nrf2 activators on HTLV-1 or the therapeutic effects on HAM.

[0008] Al-Sawaf, O. et al., Clinical Science (2015) 129: 989-999Lu, MC. et al., Medicinal Research Reviews (2016) 36: 924-963

[0009] The objective of this invention is to provide a novel and effective therapeutic agent for HAM.

[0010] To achieve the above objective, the inventors conducted extensive research and found that several Nrf2 activators suppressed the spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) specifically observed in HAM patients in a concentration-dependent manner. This spontaneous proliferation of PBMCs is caused by the proliferation of HTLV-1 infected cells and the accompanying excessive immune response. The Nrf2 activators significantly suppressed the proliferation of both HTLV-1 infected CD4-positive T lymphocytes and CTLs, which play a central role in the excessive immune response. When CD4-positive T lymphocytes and HTLV-1 infected cells were isolated from PBMCs derived from HAM patients and the direct action of DMF was verified, it was shown that it acted directly on the HTLV-1 infected cells themselves, without the involvement of other immune cells such as CTLs. The inventors also confirmed that the Nrf2 activators have the effect of reducing the amount of HTLV-1 provirus. Furthermore, it was found that Nrf2 activators also suppressed the production of pro-inflammatory cytokines (interleukin-6 (IL-6), IFN-γ, and tumor necrosis factor-α (TNF-α)) that cause inflammation in HAM in a concentration-dependent manner. On the other hand, Nrf2 activators were shown to have no adverse effects on the survival of PBMCs derived from healthy individuals at therapeutically effective concentrations, and to have a low risk of side effects.

[0011] Based on these findings, the inventors conducted further research and have now completed the present invention. Specifically, the present invention provides the following:

[0012] [Claim 1] An antiviral agent comprising a Nuclear factor erythroid 2-related factor 2 (Nrf2) activator. [Claim 2] The agent according to Claim 1, wherein the virus is human T-cell leukemia virus type 1 (HTLV-1). [Claim 3] The agent according to Claim 2, for the treatment or prevention of HTLV-1-associated myelopathy (HAM). [Claim 4] The agent according to any one of Claims 1 to 3, wherein the Nrf2 activator is a derivative of monomethyl fumarate. [Claim 5] The agent according to Claim 4, wherein the derivative is dimethyl fumarate (DMF) or diloximel fumarate (DRF). [Claim 6] A method for reducing the virus in a subject infected with the virus, preferably HTLV-1, comprising administering an effective amount of the Nrf2 activator to the subject. [Clause 7] An Nrf2 activator for use in reducing viruses, preferably HTLV-1. [Clause 8] The use of an Nrf2 activator in the manufacture of a virus, preferably HTLV-1 reducing agent. [Clause 9] The method according to Claim 6, the Nrf2 activator according to Claim 7, or the use according to Claim 8, wherein the reduction of HTLV-1 is for the treatment or prevention of HAM.

[0013] According to the present invention, the Nrf2 activator acts specifically on pathological conditions related to HAM, suppressing the proliferation of HTLV-1 infected cells that cause symptoms and CTLs that lead to excessive immune responses, calming inflammation, and further eliminating the HTLV-1 virus.

[0014] This figure shows the concentration-dependent inhibitory effect of DMF on the spontaneous proliferation (SP) activity of PBMCs derived from HAM patients. ***: p < 0.001, ****: p < 0.0001 This figure shows the concentration-dependent inhibitory effect of DRF on the SP activity of PBMCs derived from HAM patients. This figure shows the inhibitory effect of DMF on the SP activity of a subset of T lymphocytes (CD3+, CD4+, CD8+) derived from HAM patients. This figure shows the concentration-dependent inhibitory effect of DMF on the SP activity of a subset of T lymphocytes (CD3+, CD4+, CD8+) derived from HAM patients. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001 This figure shows the concentration-dependent inhibitory effect of DMF on the SP activity of HTLV-1 infected cells (CD4+CADM1+) derived from HAM patients. *: p < 0.05, **: p < 0.01, ****: p < 0.0001 This figure shows the concentration-dependent inhibitory effect of DMF on the production of inflammatory cytokines (IL-6, IFN-γ, TNF-α) in PBMCs derived from HAM patients. *: p < 0.05, **: p < 0.01, ****: p < 0.0001 This figure shows the concentration-dependent HTLV-1 provirus elimination effect of DMF on PBMCs derived from HAM patients. This figure shows that DMF, at therapeutically effective concentrations for HAM, does not adversely affect the survival of PBMCs derived from healthy individuals. This figure shows the concentration-dependent inhibitory effect of DMF on the SP activity of CD4+ T cells and HTLV-1 infected cells (CD4+CADM1+) derived from HAM patients. *: p < 0.05, ****: p < 0.0001

[0015] The present invention provides an antiviral agent (hereinafter sometimes abbreviated as "the antiviral agent of the present invention") containing an Nrf2 activator as an active ingredient (antiviral agent). In this specification, "antiviral agent" means a drug that can reduce the amount of virus in a person infected with a virus compared to when the drug is not administered, and its mechanism of action is not limited. Preferably, the virus targeted by the antiviral agent of the present invention is a virus whose mode of infection spread is mainly through clonal proliferation of infected cells rather than novel infection, and therefore, the amount of virus can be reduced by suppressing the proliferation of infected cells. Examples of such viruses include HTLV-1, HTLV-2, EBV, HPV, HBV, etc. Particularly preferably, the virus targeted by the antiviral agent of the present invention is HTLV-1.

[0016] The antiviral agent of the present invention can be used for the treatment or prevention of viral infections. As described above, one of the preferred viruses targeted by the antiviral agent of the present invention is HTLV-1, so in a preferred embodiment, the present invention provides a therapeutic or prophylactic agent for HTLV-1 infection (hereinafter sometimes abbreviated as "the therapeutic / prophylactic agent of the present invention") containing an Nrf2 activator. Examples of HTLV-1 infections include HAM, HTLV-1-associated uveitis (HU), adult T-cell leukemia / lymphoma (ATLL), etc., but HAM is preferred.

[0017] In this specification, "Nrf2 activator" can be any substance that can consequently enhance the transcriptional activation activity of Nrf2 target genes. Examples include, but are not limited to, substances that promote the release of Nrf2 from keep1 and / or inhibit the association of Nrf2 and keep1, substances that promote the nuclear translocation of Nrf2 (e.g., kinases), substances that inhibit the degradation of Nrf2 via the ubiquitin-proteasome pathway (e.g., GSK3β inhibitors), substances that promote autophagy of keep1 (e.g., p62 / SQSTM1), substances that promote the binding of Nrf2 to the ARE sequence (e.g., small Maf protein), and substances that enhance the expression of the Nrf2 gene (e.g., lansoprazole).

[0018] Substances that promote the release of Nrf2 from keep1 include, for example, substances that target cysteine ​​residues in keep1. Such substances include, for example, (i) Michael acceptors (e.g., fumarate derivatives, maleate derivatives, curcumin, etc.), (ii) isothiocyanates (e.g., sulforaphane, PEITC, etc.), (iii) organosulfur compounds (e.g., ortiplasm, D3T, etc.), (iv) electrophiles with leaving groups (e.g., IAB, AI-1, etc.), (v) selenium compounds (e.g., SFN-isoSe, Ebselen, etc.), (vi) trivalent arsenic (e.g., ATO, MMA, etc.), (vii) heavy metals (e.g., Hg, Cd, Zn, etc.), (viiii) hydroperoxides and nitrosates (e.g., Spermine). Examples include, but are not limited to, (NONOate, etc.), (ix) compounds having a terminal dithiol group (e.g., 1,2-ethanediol, etc.), (x) polyphenol compounds (e.g., EGCG, tBHQ, etc.), and (xi) triterpenoid derivatives of oleic acid (e.g., bardoxolone methyl (CDDO-Me)).

[0019] In another embodiment, for example, the compounds described in Non-Patent Documents 1 and 2 above can be used as the Nrf2 activator. Alternatively, compounds described in WO2006 / 043671, WO2006 / 073042, JP 2011-256126, JP 2008-208038, JP 2008-110962, JP 2007-031315, etc. can be used.

[0020] In one embodiment, various compounds approved as pharmaceuticals or undergoing clinical trials can be used as Nrf2 activators. These compounds can be incorporated into the therapeutic / preventive agents of the present invention to the extent that their usage and dosage have already been clinically evaluated and their safety has been confirmed. Examples of Nrf2 activators that are undergoing clinical application include the compounds described in Non-Patent Documents 1 and 2 above.

[0021] In one preferred embodiment, a derivative of monomethyl fumarate can be used as the Nrf2 activator. Preferably, examples of monomethyl fumarate derivatives include prodrug compounds that, when administered to human subjects, are metabolized in vivo to produce monomethyl fumarate. Examples of such monomethyl fumarate derivatives include dimethyl fumarate (DMF) and the compounds described in Japanese Patent Publication No. 2016-520524 and Japanese Patent Publication No. 2017-149735. DMF is marketed by Biogen as a treatment for multiple sclerosis (trade name: Tecfidera). Furthermore, the monomethyl fumarate derivatives described in Japanese Patent Publication No. 2016-520524 and Japanese Patent Publication No. 2017-149735 have improved properties compared to DMF, and diloximel fumarate (DRF) is said to have a lower methanol concentration in the small intestine than DMF, and can reduce the severity and frequency of gastrointestinal adverse events. Preferably, the derivative of monomethyl fumarate used in the present invention is DMF or DRF.

[0022] Each Nrf2 activator can be manufactured by a method known to the public.

[0023] When administered to HAM patients or asymptomatic HTLV-1 carriers, Nrf2 activators suppress the proliferation of CD4-positive HTLV-1 infected cells and CD8-positive CTLs, which play a central role in the excessive immune response. They also reduce the proviral load of HTLV-1 and enhance the expression of Nrf2 target genes such as HO-1, thereby promoting the production of anti-inflammatory cytokines. Furthermore, they suppress the production of inflammatory cytokines by inhibiting the NF-κB pathway, thereby mitigating the inflammatory response that causes the neurological symptoms of HAM. Thus, they can alleviate the symptoms of HAM and suppress its progression (prevent or delay the onset of the disease) in a multifaceted way.

[0024] The Nrf2 activator has low toxicity and can be safely administered orally or parenterally (e.g., topical, rectal, intravenous administration, etc.) to humans or other mammals as a pharmaceutical composition, which is either in its original form or mixed with a pharmacologically acceptable carrier according to a method known per se, such as tablets (including sugar-coated tablets and film-coated tablets), powders, granules, capsules (including soft capsules), orally disintegrating tablets, liquids, injections, suppositories, sustained-release agents, patches, and other formulations.

[0025] In the pharmaceutical composition of the present invention, the content of the Nrf2 activator is about 0.01% to 100% by weight of the total composition. The dosage of the pharmaceutical composition varies depending on the type of the Nrf2 activator, the body weight, age, gender, severity, administration route, etc. of the administration subject. For example, when DMF is orally administered to a HAM patient (60 kg), it can be administered at about 120 to about 240 mg / time, twice a day as DMF. The AUC ,

[0026] , ｍａｘ , ｍａｘ , １／２ , ０-２４ｈｒ , ｍａｘ , ｍａｘ , 1 / 2 , ｍａｘ 4915 ± 981 (hour·ng / mL), C ｍａｘ 1347 ± 638 (ng / mL), T ｍａｘ 4.00 (hour), T 1 / 2 0.86 ± 0.85 (hour), and the AUC when administered at 240 mg ０-２４ｈｒ 8,325 ± 2,911 (hour·ng / mL), C ｍａｘ 2,366 ± 1,353 (ng / mL), T ｍａｘ 5.00 (hour), t １／２ 0.66 ± 0.22 (hour). When DRF is orally administered to a HAM patient (60 kg), it can be administered at about 220 to about 480 mg / time, twice a day as DRF. The blood concentration at steady state is 8,32 (hour·mg / L), C ｍａｘ 2.11 (mg / L), T ｍａｘ 2.5 - 3.0 (hour).

[0026] Examples of pharmacokinetically acceptable carriers that may be used in the manufacture of the pharmaceutical composition of the present invention include various organic or inorganic carrier substances commonly used as pharmaceutical materials, such as excipients, lubricants, binders, disintegrants, water-soluble polymers, and basic inorganic salts in solid formulations; and solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. In addition, conventional additives such as preservatives, antioxidants, colorants, sweeteners, acidulants, foaming agents, and fragrances may be used as needed.

[0027] Examples of excipients include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, light anhydrous silicic acid, and titanium dioxide. Examples of lubricants include magnesium stearate, sucrose fatty acid ester, polyethylene glycol, talc, and stearic acid.

[0028] Examples of binders include hydroxypropylcellulose, hydroxypropylmethylcellulose, crystalline cellulose, starch, polyvinylpyrrolidone, acacia powder, gelatin, pullulan, and low-substituted hydroxypropylcellulose.

[0029] Examples of disintegrants include (1) crospovidone, (2) disintegrants referred to as super disintegrants such as croscarmellose sodium (FMC - Asahi Kasei) and carmellose calcium (Gotoku Pharmaceutical), (3) carboxymethyl starch sodium (e.g., manufactured by Matsutani Chemical Co., Ltd.), (4) low-substituted hydroxypropyl cellulose (e.g., manufactured by Shin-Etsu Chemical Co., Ltd.), and (5) corn starch. The "crospovidone" may be any of the polymers that have the chemical name 1-ethenyl-2-pyrrolidinone homopolymer and are crosslinked, including those referred to as polyvinylpolypyrrolidone (PVPP) and 1-vinyl-2-pyrrolidinone homopolymer. Specific examples include Coridone CL (manufactured by BASF), Polyplasdone XL (manufactured by ISP), Polyplasdone XL-10 (manufactured by ISP), Polyplasdone INF-10 (manufactured by ISP), etc.

[0030] Examples of water-soluble polymers include ethanol-soluble water-soluble polymers [e.g., cellulose derivatives such as hydroxypropyl cellulose (hereinafter sometimes referred to as HPC), polyvinylpyrrolidone, etc.] and ethanol-insoluble water-soluble polymers [e.g., cellulose derivatives such as hydroxypropyl methylcellulose (hereinafter sometimes referred to as HPMC), methylcellulose, sodium carboxymethylcellulose, sodium polyacrylate, polyvinyl alcohol, sodium alginate, guar gum, etc.].

[0031] Examples of basic inorganic salts include basic inorganic salts of sodium, potassium, magnesium, and / or calcium. Preferably, it is a basic inorganic salt of magnesium and / or calcium. More preferably, it is a basic inorganic salt of magnesium. Examples of basic inorganic salts of sodium include sodium carbonate, sodium bicarbonate, disodium hydrogen phosphate, etc. Examples of basic inorganic salts of potassium include potassium carbonate, potassium bicarbonate, etc. Examples of basic inorganic salts of magnesium include heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, magnesium aluminometasilicate, magnesium silicate, magnesium aluminate, synthetic hydrotalcite [Mg ６ Al ２ (OH) １６ CO ３ 4H ２ Examples of calcium include calcium O and alumina magnesium hydroxide, preferably heavy magnesium carbonate, magnesium carbonate, magnesium oxide, magnesium hydroxide, etc. Examples of basic inorganic salts of calcium include precipitated calcium carbonate and calcium hydroxide.

[0032] Examples of solvents include, for example, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, olive oil, and the like. Examples of solubilizers include, for example, polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, and the like.

[0033] Examples of suspending agents include, for example, surfactants such as stearyl triethanolamine, sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; hydrophilic polymers such as, for example, polyvinyl alcohol, polyvinyl pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, and the like.

[0034] Examples of isotonic agents include, for example, glucose, D-sorbitol, sodium chloride, glycerin, D-mannitol, and the like. Examples of buffers include, for example, buffer solutions such as phosphates, acetates, carbonates, citrates, and the like.

[0035] Examples of pain relievers include, for example, benzyl alcohol and the like. Examples of preservatives include, for example, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and the like.

[0036] Examples of antioxidants include, for example, sulfites, ascorbic acid, α-tocopherol, and the like. Examples of coloring agents include, for example, food colors such as Food Yellow No. 5, Food Red No. 2, Food Blue No. 2; food lake pigments, red iron oxide, and the like.

[0037] Examples of sweeteners include, for example, sodium saccharin, dipotassium glycyrrhizinate, aspartame, stevia, thaumatin, and the like. Examples of acidifiers include, for example, citric acid (anhydrous citric acid), tartaric acid, malic acid, and the like.

[0038] As the foaming agent, for example, sodium bicarbonate etc. can be mentioned. As the fragrance, either a synthetic or a natural product may be used, and for example, lemon, lime, orange, menthol, strawberry etc. can be mentioned.

[0039] Hereinafter, the present invention will be described more specifically with reference to examples, but these are merely illustrative and do not limit the scope of the present invention in any way.

[0040] Example 1 Suppression of SP activity of PBMC derived from HAM patients by DMF PBMC was isolated from a blood sample collected from a HAM patient by Ficoll-Hypaque density gradient centrifugation. PBMC was suspended in Roswell Park Memorial Institute (RPMI) 1640 medium containing 10% fetal bovine serum (FBS), 100 μg / mL penicillin, and 100 μg / mL streptomycin, and various concentrations of DMF (0, 10, 25, 50, 100 μM) were added to a 96-well round-bottom microplate and seeded at a concentration of 2.0×10 ５ / well. After culturing PBMC in an incubator at 37.0 °C and 5% CO ２ +95% atmosphere for 72 hours, an XTT labeling reagent (Cell Proliferation Kit, manufactured by Roche) was added, and further cultured in an incubator under the same conditions as described above for 5 hours, and the absorbance was measured with an Enzyme-Linked Immunosorbent Assay (ELISA) reader. The absorbance of the produced Formazan was set at 492 nm, and the reference wavelength was set at 690 nm. The effect of DMF on the spontaneous proliferation (SP) activity of PBMC characteristic of HAM was examined by the XTT assay. As a result, DMF suppressed the SP activity in a concentration-dependent manner (Figure 1).

[0041] Example 2 Suppression of SP activity of PBMC derived from HAM patients by DRF In the same manner as in Example 1, various concentrations of DRF (0, 10, 25, 50, 100 μM) were added to the medium of PBMC derived from HAM patients and cultured, and the effect of DRF on the SP activity of PBMC was examined by the XTT assay. As a result, DRF suppressed the SP activity in a concentration-dependent manner (Figure 2).

[0042] Example 3: Suppression of SP activity in HAM patient-derived T lymphocyte subsets by DMF (1) PBMCs derived from HAM patients were cultured in the presence and absence of DMF (50 μM). Cell proliferation activity of each T lymphocyte subset was analyzed by multicolor flow cytometry (CytoFLEX flow cytometer, Beckman Coulter) using fluorescently labeled antibodies against CD3, CD4, and CD8 (BD Biosciences) and CFSE fluorescent dye (CellTrace CFSE Cell Profitation Kit, Invitrogen). As a result, DMF suppressed the SP activity of all T lymphocyte subsets (Figure 3).

[0043] Example 4: Suppression of SP activity in HAM patient-derived T lymphocyte subsets by DMF (2) The same test as in Example 3 was performed by varying the concentration of DMF added (0, 5, 10, 25, 50, 100 μM). As a result, DMF suppressed SP activity in a concentration-dependent manner in all T lymphocyte subsets (Figure 4).

[0044] Example 5: Suppression of SP activity in HTLV-1 infected cells from HAM patients by DMF. HTLV-1 infected cells were identified from PBMCs derived from HAM patients using CD4 antibody (BD Biosciences) and CADM1 antibody (MBL). The effect of DMF on their SP activity was investigated using the same method as in Example 3. As a result, DMF suppressed the SP activity of HTLV-1 infected cells in a concentration-dependent manner (Figure 5).

[0045] Example 6: Suppression of inflammatory cytokine production by DMF Patient-derived PBMCs from HAM were suspended in RPMI1640 medium containing 10% FBS, 100 μg / mL penicillin, and 100 μg / mL streptomycin. Various concentrations of DMF (0, 10, 25, 50, 100 μM) were added to 96-well round-bottom microplate to form 2.0 × 10⁻⁶ ５ Seeds were sown at a concentration of / well. PBMC was treated at 37.0°C with 5% CO2. ２After culturing in an incubator at +95% air for 6 days, the supernatant was collected after centrifugation at 1300 rpm for 5 minutes. The concentrations of IL-6, IFN-γ, and TNF-α in the supernatant were measured using a Milliplex@Human Cytokine / Chemokine Magnetic Bead Panel (Millipore). As a result, DMF suppressed the production of these inflammatory cytokines in a concentration-dependent manner (Figure 6).

[0046] Example 7: Removal of HTLV-1 Provirus by DMF PBMCs derived from HAM patients were cultured with DMF at various concentrations (0, 5, 10, 25, 50, 100 μM). After culturing, DNA was extracted from the PBMCs (using a DNeasy Blood and Tissue kit, Qiagen), and the amount of HTLV-1 provirus was measured using real-time polymerase chain reaction. The results showed that DMF reduced the amount of provirus in a concentration-dependent manner (Figure 7).

[0047] Example 8: Effect of DMF on the survival rate of PBMCs derived from healthy individuals. DMF was added to PBMCs derived from healthy individuals at various concentrations and cultured for 6 days to investigate the effect of the drug. As a result, at therapeutically effective concentrations, DMF did not reduce the survival rate of PBMCs derived from healthy individuals (Figure 8).

[0048] Example 9 DMF directly suppresses SP activity in CD4-positive T cells and HTLV-1 infected cells derived from HAM patients. In the SP process in PBMCs of HAM patients, CD4-positive T cells are known to express the viral protein Tax and become targets of HTLV-1-specific cytotoxic T lymphocytes (CTLs). Therefore, it was unclear whether the suppression of CD4-positive T cell proliferation by DMF was a result of cytotoxicity by CD8-positive T cells or a direct effect of DMF. To investigate this, CD4-positive T cells and HTLV-1 infected cells were isolated from PBMCs derived from HAM patients using a cell sorter (SH800 cell sorter, Sony Biotechnology) with CD4 antibody (BD Biosciences) and CADM1 antibody (MBL), and the direct effect of DMF was verified. As a result, DMF directly suppressed the SP activity of both cells in a concentration-dependent manner (Figure 9). Therefore, it was suggested that DMF may directly act on HTLV-1 infected cells themselves to suppress proliferation, without the involvement of other immune cells such as CD8-positive T cells.

[0049] The therapeutic and preventive agent of the present invention, which contains an Nrf2 activator as an active ingredient, is extremely useful as a novel, effective, and safe means of treating and preventing HAM.

[0050] This application is based on Japanese Patent Application No. 2025-008095, filed in Japan on January 20, 2025, the contents of which are incorporated herein by reference.

Claims

1. An antiviral agent comprising a Nuclear Factor Erythroid 2-related Factor 2 (Nrf2) activator.

2. The agent according to claim 1, wherein the virus is human leukemia virus type 1 (HTLV-1).

3. The agent according to claim 2, for the treatment or prevention of HTLV-1-associated myelopathy (HAM).

4. The agent according to any one of claims 1 to 3, wherein the Nrf2 activator is a derivative of monomethyl fumarate.

5. The agent according to claim 4, wherein the derivative is dimethyl fumarate (DMF) or diloximel fumarate (DRF).

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