Agent for protecting and / or regenerating neuromuscular junction

Abstract

To provide a medicine for protecting and / or regenerating a neuromuscular junction, or an agent for protecting and / or regenerating a nervous system cell.SOLUTION: In one embodiment, the present invention provides an agent for protection and / or regeneration of a neuromuscular junction, or a therapeutic and / or prophylactic agent for a disease accompanied by a disorder of a neuromuscular junction, comprising an FF-MAS metabolism inhibitor. In addition, in one embodiment, the present invention provides a method for protecting and / or regenerating a neuromuscular junction, or a method for treating and / or preventing a disease associated with a disorder of neuromuscular junction, comprising administering an effective amount of FF-MAS metabolism inhibitor to a mammal. Further, in one aspect, the present invention provides an FF-MAS metabolism inhibitor for use in the protection and / or regeneration of a neuromuscular junction, or for use in the treatment and / or prevention of a disease associated with a disorder of a neuromuscular junction. In addition, in one aspect, the present invention provides use of an FF-MAS metabolism inhibitor for producing an agent for protecting and / or regenerating a neuromuscular junction, or for producing an agent for treating and / or preventing a disease associated with a disorder of a neuromuscular junction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to the use of an FF-MAS (Follicular fluid meiosis-activating sterol) metabolism inhibitor for protecting neuromuscular junctions and / or nervous system cells. [Background technology]

[0002] The nervous system is broadly divided into the central nervous system and the peripheral nervous system, and is composed of neurons, Schwann cells, and glial cells such as oligodendrocytes and microglia. Furthermore, in the peripheral nervous system, motor nerves form neuromuscular junctions with muscle tissue, and acetylcholine released from motor nerve terminals stimulates acetylcholine receptors present in muscle cells, resulting in muscle contraction. Amyotrophic lateral sclerosis (AMLS) is a disease in which muscle function declines due to degeneration of motor nerves and neuromuscular junctions. Although there are treatments aimed at improving symptoms and slowing progression, their effectiveness is not always sufficient, and the development of new therapeutic agents is eagerly awaited (Non-Patent Document 1). Meanwhile, it has been reported that neuromuscular junction damage occurs early in the onset of this disease, and improving this damage may be expected to have a therapeutic effect (Non-Patent Documents 2 and 3).

[0003] Incidentally, Patent Document 1 and Non-Patent Documents 4 and 5 report that inhibitors of sterol metabolic enzymes, such as CYP51 (Cytochrome P450 family 51), sterol C14-reductase, or EBP (emopamil binding protein), have the effect of promoting oligodendrocyte differentiation. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 022904 Brochure [Non-patent literature]

[0005] [Non-Patent Document 1] Amyotrophic Lateral Sclerosis Treatment Guidelines 2013 [Non-patent document 2] EMBO Molecular Medicine, Vol. 9, (2017) 880-889 [Non-patent document 3] Experimental Neurology, 185 (2004) 232-240 [Non-patent document 4] Nature, Vol. 560 (2018), 372-376 [Non-Patent Document 5] Cell Chemical Biology 26, (2019) 593-599 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a pharmaceutical agent for protecting and / or regenerating neuromuscular junctions or an agent for protecting and / or regenerating nervous system cells. [Means for solving the problem]

[0007] As a result of intensive research to solve the above-mentioned problems, the present inventors have found that FF-MAS metabolism inhibitors, sterol C14 reductase inhibitors, or TM7SF2 (Transmembrane 7 superfamily member 2) inhibitors (hereinafter sometimes referred to as FF-MAS metabolism inhibitors, etc.) have protective and / or regenerative effects on neuromuscular junctions, and further have protective and / or regenerative effects on nervous system cells.

[0008] The present invention has the following aspects, for example. [1] An agent for protecting and / or regenerating neuromuscular junctions or nervous system cells, which contains a metabolic inhibitor of sterols that constitute the cholesterol synthesis pathway.

[0009] [2] The agent according to [1], wherein the metabolic inhibitor of sterols constituting the cholesterol synthesis pathway is an FF-MAS metabolic inhibitor.

[0010] [3] The agent according to [1] or [2], which is an agent for protecting and / or regenerating neuromuscular junctions.

[0011] [4] The agent according to [1] or [2], which is an agent for protecting and / or regenerating nervous system cells.

[0012] [5] The agent described in [4], wherein the protection and / or regeneration of nervous system cells is mediated by neurite outgrowth activity.

[0013] [6] The agent described in [4], wherein the protection and / or regeneration of nervous system cells is mediated by the effect of promoting Schwann cell differentiation.

[0014] [7] An agent for protecting and / or regenerating neuromuscular junctions, comprising an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0015] [8] A neurite outgrowth agent containing an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0016] [9] A Schwann cell differentiation promoter containing an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0017]

[10] The agent according to any one of [2] to [9], wherein the FF-MAS metabolism inhibitor is a sterol C14 reductase inhibitor.

[0018]

[11] The agent described in

[10] , wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor and / or an LBR inhibitor.

[0019]

[12] The agent described in

[10] , wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0020]

[13] The agent according to

[10] , wherein the sterol C14 reductase inhibitor is an LBR inhibitor (preferably a selective LBR inhibitor).

[0021]

[14] The agent described in

[10] , wherein the sterol C14 reductase inhibitor is a TM7SF2 and LBR dual inhibitor (preferably a selective TM7SF2 and LBR dual inhibitor).

[0022]

[15] The agent described in

[10] , wherein the sterol C14 reductase inhibitor is a substance that selectively inhibits sterol C14 reductase (e.g., a selective TM7SF2 inhibitor, a selective LBR inhibitor, or a selective TM7SF2 and LBR dual inhibitor, preferably a selective TM7SF2 inhibitor).

[0023]

[16] An agent for protecting and / or regenerating neuromuscular junctions, or an agent for protecting and / or regenerating nervous system cells, comprising FF-MAS or 14-dehydrozymosterol or a derivative thereof.

[0024]

[17] An agent for protecting and / or regenerating a neuromuscular junction, or an agent for protecting and / or regenerating nervous system cells, comprising an agent having the effect of increasing FF-MAS concentration and / or 14-dehydrozymosterol concentration.

[0025]

[18] The agent according to any one of [1] to

[17] for treating and / or preventing a neurodegenerative disease.

[0026]

[19] The agent according to any one of [1] to [3], [7], or

[10] to

[17] for the treatment and / or prevention of a disease accompanied by a disorder of the neuromuscular junction.

[0027]

[20] The agent described in

[19] , wherein the disease accompanied by a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

[0028]

[21] The agent described in

[20] , wherein the disease accompanied by impairment of the neuromuscular junction is a neurodegenerative disease.

[0029]

[22] The agent according to

[18] or

[21] , wherein the neurodegenerative disease is peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

[0030]

[23] The agent described in

[20] , wherein the disease accompanied by a disorder of the neuromuscular junction is a myogenic disease.

[0031]

[24] The agent according to

[23] , wherein the myogenic disease is myasthenia gravis, muscular dystrophy, sarcopenia or myopathy.

[0032]

[25] An agent for protecting and / or regenerating nervous system cells, comprising an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0033]

[26] The agent described in

[25] , wherein the protection and / or regeneration of nervous system cells is mediated by neurite outgrowth activity.

[0034]

[27] The agent described in

[25] , wherein the protection and / or regeneration of nervous system cells is mediated by the effect of promoting Schwann cell differentiation.

[0035]

[28] An agent for protecting and / or regenerating a neuromuscular junction, comprising a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0036]

[29] The agent according to

[28] for the treatment and / or prevention of a neurodegenerative disease.

[0037]

[30] The agent according to

[28] for treating and / or preventing a disease associated with a disorder of the neuromuscular junction.

[0038]

[31] The agent described in

[30] , wherein the disease accompanied by a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

[0039]

[32] The agent described in

[31] , wherein the disease accompanied by impairment of the neuromuscular junction is a neurodegenerative disease.

[0040]

[33] The agent according to

[29] or

[32] , wherein the neurodegenerative disease is peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

[0041]

[34] The agent described in

[31] , wherein the disease accompanied by a disorder of the neuromuscular junction is a myogenic disease.

[0042]

[35] The agent according to

[34] , wherein the myogenic disease is myasthenia gravis, muscular dystrophy, sarcopenia or myopathy.

[0043] [2-1] A therapeutic and / or preventive agent for neurodegenerative diseases, comprising an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0044] [2-2] A therapeutic and / or preventive agent for myogenic diseases, comprising an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0045] [2-3] A therapeutic and / or preventive agent for diseases associated with disorders of the neuromuscular junction, comprising an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0046] [2-4] The agent according to [2-3] for protecting and / or regenerating neuromuscular junctions.

[0047] [2-5] The agent according to [2-3] or [2-4], wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

[0048] [2-6] The agent according to [2-5], wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease.

[0049] [2-7] The agent according to [2-1] or [2-6], wherein the neurodegenerative disease is peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

[0050] [2-8] The agent according to [2-6] or [2-7], further for protecting and / or regenerating nervous system cells.

[0051] [2-9] The agent described in [2-8], wherein the protection and / or regeneration of nervous system cells is neurite outgrowth.

[0052] [2-10] The agent described in [2-8], wherein the protection and / or regeneration of nervous system cells is promotion of Schwann cell differentiation.

[0053] [2-11] The agent according to [2-5], wherein the disease associated with a disorder of the neuromuscular junction is a myogenic disease.

[0054] [2-12] The agent according to [2-2] or [2-11], wherein the myogenic disease is myasthenia gravis, muscular dystrophy, sarcopenia or myopathy.

[0055] [2-13] The agent according to any one of [2-1] to [2-12], wherein the FF-MAS metabolism inhibitor is a sterol C14 reductase inhibitor.

[0056] [2-14] The agent according to [2-13], wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor and / or an LBR inhibitor.

[0057] [2-15] The agent according to [2-13], wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0058] [2-16] The agent according to [2-13], wherein the sterol C14 reductase inhibitor is an LBR inhibitor (preferably a selective LBR inhibitor).

[0059] [2-17] The agent according to [2-13], wherein the sterol C14 reductase inhibitor is a TM7SF2 and LBR dual inhibitor (preferably a selective TM7SF2 and LBR dual inhibitor).

[0060] [2-18] The agent described in [2-13], wherein the sterol C14 reductase inhibitor is a substance that selectively inhibits sterol C14 reductase (e.g., a selective TM7SF2 inhibitor, a selective LBR inhibitor, or a selective TM7SF2 and LBR dual inhibitor, preferably a selective TM7SF2 inhibitor).

[0061] A therapeutic and / or preventive agent for diseases associated with disorders of the transmuscular junction, comprising [2-19]FF-MAS or 14-dehydrozymosterol or a derivative thereof.

[0062] [2-20] A therapeutic and / or preventive agent for diseases associated with disorders of the neuromuscular junction, comprising an agent having the effect of increasing FF-MAS concentration and / or 14-dehydrozymosterol concentration.

[0063] [2-21] A therapeutic and / or preventive agent for a neurodegenerative disease, comprising a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0064] [2-22] A therapeutic and / or preventive agent for a myogenic disease, comprising a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0065] [2-23] A therapeutic and / or preventive agent for a disease associated with a disorder of the neuromuscular junction, comprising a TM7SF2 inhibitor (preferably a selective TM7SF2 inhibitor).

[0066] [2-24] The agent according to [2-23] for protecting and / or regenerating neuromuscular junctions.

[0067] [2-25] The agent according to [2-23] or [2-24], wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

[0068] [2-26] The agent according to [2-25], wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease.

[0069] [2-27] The agent according to [2-21] or [2-26], wherein the neurodegenerative disease is peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

[0070] [2-28] The agent according to [2-26] or [2-27], further for protecting and / or regenerating nervous system cells.

[0071] [2-29] The agent according to [2-28], wherein the protection and / or regeneration of nervous system cells is neurite outgrowth.

[0072] [2-30] The agent according to [2-28], wherein the protection and / or regeneration of nervous system cells is promotion of Schwann cell differentiation.

[0073] [2-31] The agent according to [2-25], wherein the disease associated with a disorder of the neuromuscular junction is a myogenic disease.

[0074] [2-32] The agent according to [2-22] or [2-31], wherein the myogenic disease is myasthenia gravis, muscular dystrophy, sarcopenia or myopathy.

[0075] [3-1] A method for determining or predicting the effectiveness of an FF-MAS metabolic inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for a neurodegenerative disease in a patient, using the concentration of sterols (e.g., FF-MAS, T-MAS, 14-dehydrozymosterol, or 14-dehydrozymostenol) that constitute the cholesterol synthesis pathway in a biological sample from the patient as an indicator.

[0076] [3-2] A method for determining or predicting the effectiveness of an FF-MAS metabolic inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for a disease associated with a disorder of the neuromuscular junction in a patient, using as an indicator the concentration of sterols (e.g., FF-MAS, T-MAS, 14-dehydrozymosterol, or 14-dehydrozymostenol) that constitute the cholesterol synthesis pathway in a biological sample from the patient with the disease associated with a disorder of the neuromuscular junction.

[0077] [3-3] A method for determining or predicting the effectiveness of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for a neurodegenerative disease in a patient, using the expression level of TM7SF2 in a biological sample from the patient as an indicator.

[0078] [3-4] A method for determining or predicting the effectiveness of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for a disease associated with a disorder of the neuromuscular junction in a patient, using the expression level of LBR or TM7SF2 (preferably TM7SF2 expression level) in a biological sample of the patient with the disease as an indicator.

[0079] [4-1] A method for determining or predicting the onset, severity, or prognosis of a neurodegenerative disease in a patient, using the concentration of sterols that constitute the cholesterol synthesis pathway in a biological sample from the patient as an indicator.

[0080] [4-2] A method for determining or predicting the onset, severity, or prognosis of a disease associated with neuromuscular junction disorders in a patient, using the concentration of sterols that constitute the cholesterol synthesis pathway in a biological sample from the patient as an indicator.

[0081] [4-3] A method for determining or predicting the onset, severity, or prognosis of a neurodegenerative disease in a patient using the expression level of TM7SF2 in a biological sample from the patient as an indicator.

[0082] [4-4] A method for determining or predicting the onset, severity, or prognosis of a disease associated with neuromuscular junction disorders in a patient, using the LBR expression level or TM7SF2 expression level (preferably TM7SF2 expression level) in a biological sample from the patient as an indicator.

[0083] [5-1] A method for protecting and / or regenerating neuromuscular junctions or nervous system cells, comprising administering an effective amount of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) to a mammal.

[0084] [5-2] A method for treating and / or preventing a neurodegenerative disease or a myopathic disease, comprising administering an effective amount of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) to a mammal (a patient in need thereof).

[0085] [5-3] A method for treating and / or preventing a disease associated with a disorder of the neuromuscular junction, comprising administering an effective amount of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) to a mammal (a patient in need thereof).

[0086] [5-4] An FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for use in protecting and / or regenerating neuromuscular junctions or protecting and / or regenerating nervous system cells.

[0087] [5-5] An FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for use in the treatment and / or prevention of a neurodegenerative or myogenic disease.

[0088] [5-6] An FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for use in the treatment and / or prevention of a disease associated with disorders of the neuromuscular junction.

[0089] [5-7] Use of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for producing an agent for protecting and / or regenerating neuromuscular junctions or an agent for protecting and / or regenerating nervous system cells.

[0090] [5-8] Use of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for the manufacture of an agent for the treatment and / or prevention of a neurodegenerative disease or a myogenic disease.

[0091] [5-9] Use of an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for the manufacture of an agent for treating and / or preventing a disease associated with a disorder of the neuromuscular junction. [Effects of the Invention]

[0092] The agents of the present invention (eg, agents containing an FF-MAS metabolism inhibitor) can be used to protect and / or regenerate neuromuscular junctions and nervous system cells. [Brief explanation of the drawings]

[0093] [Figure 1] This figure shows the FF-MAS concentration in C2C12 cells, a mouse myoblast cell line containing human mutant SOD1 (G93A), 3 hours after treatment with a TM7SF2 inhibitor (results for each group are shown as the mean ± standard deviation). [Figure 2] This figure shows the FF-MAS concentration in C2C12 cells, a mouse myoblast cell line containing human mutant SOD1 (G93A), 24 hours after treatment with a TM7SF2 inhibitor (results are shown as the mean ± standard deviation for each group). [Figure 3]This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after amorolfine treatment in a co-culture system of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and mouse primary cultured motor neurons. (The results are shown as the mean ± standard error for each group, with the mean value of the control group set to 100%. Control group: N = 6 wells, Amorolfine-treated group: N = 3 wells each. *: p < 0.05, **: p < 0.01 (Dunnett's test, versus control)). [Figure 4] This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after Ziprasidone treatment in a co-culture system of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and mouse primary motor neurons. (The results are shown as the mean ± standard error for each group, with the mean value for the control group set to 100%. Control group: N = 6 wells, Ziprasidone-treated group: N = 3 wells each. ***; p < 0.001 (Dunnett's test, versus control)). [Figure 5] This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after treatment with ifenprodil in a co-culture system of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and mouse primary cultured motor neurons. (The results are shown as the mean ± standard error for each group, with the mean value of the control group set to 100%. Control group: N = 6 wells, Ifenprodil-treated group: N = 3 wells each. **; p < 0.01, ***; p < 0.001 (Dunnett's test, versus control)). [Figure 6] This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after FF-MAS treatment in a co-culture system of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and mouse primary cultured motor neurons. (The results are shown as the mean ± standard error of the six wells for each group, with the mean value of the control group set to 100%. ***; p<0.001 (Dunnett's test, versus control)). [Figure 7] This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after treatment with 14-dehydrozymosterol in a co-culture system of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and mouse primary motor neurons. (The results are shown as the mean ± standard error for each group, with the mean value of the control group set to 100%. Control group: N = 6 wells, EtOH and 14-dehydrozymosterol group: N = 3 wells each. *; p < 0.05 (Dunnett's test, versus control)). [Figure 8] This shows the total process length (sum of neurite lengths) on day 4 after treatment of mouse primary cultured motor neurons with amorolfine. (The mean value of the control group was set to 100%, and the results are shown as the mean ± standard error for each group. Control group: N = 6 wells, Amorolfine-treated group: N = 3 wells each. ***; p < 0.001 (Dunnett's test, versus control)). [Figure 9] This figure shows the total process length (sum of neurite lengths) on day 4 after treatment of mouse primary cultured motor neurons with Ziprasidone. (The mean value of the control group was set to 100%, and the results are shown as the mean ± standard error for each group. Control group: N = 6 wells, Ziprasidone-treated group: N = 3 wells each. ***; p < 0.001 (Dunnett's test, versus Control)). [Figure 10] This shows the total process length (sum of neurite lengths) on day 4 after treatment of mouse primary cultured motor neurons with ifenprodil. (The mean value of the control group was set to 100%, and the results are shown as the mean ± standard error for each group. Control group: N = 6 wells, Ifenprodil-treated group: N = 3 wells each. ***; p < 0.001 (Dunnett's test, versus control)). [Figure 11]This shows the total process length (sum of neurite lengths) on day 4 after treatment of mouse primary cultured motor neurons with FF-MAS (the results are expressed as the mean ± standard error of the six wells in each group, with the mean value of the control group set at 100%. ***; p<0.001 (Dunnett's test, versus control)). [Figure 12] The figure shows the ratio of the MAG-positive area to the number of cell nuclei (MAG+area / nuclei) calculated from the MAG-positive area and the number of cell nuclei on day 3 after treatment of rat primary Schwann cells with FF-MAS (the results are shown as the mean ± standard error for each group. Control group: N = 6 wells, FF-MAS-treated group: N = 3 wells for each group. **; p < 0.01 (Dunnett's test, versus Control)). [Figure 13] This figure shows the α-Bungarotoxin (BTX)-positive area on day 11 after treatment with amorolfine in a co-culture system of mouse primary motor neurons and mouse myoblast cell line C2C12 cells overexpressing either human mutant SOD1 (G93A) alone or both human mutant SOD1 (G93A) and the human TM7SF2 gene. (The results are shown as the mean ± standard error for each group, with the mean value for the control group set to 100%. Control group: N = 6 wells, Amorolfine-treated group: N = 3 wells each. *; p < 0.05 (Dunnett's test, versus control)). [Figure 14] This figure shows the effectiveness of the TM7SF2 inhibitor (Ifenprodil) in myasthenia gravis model rats (the results show grip strength 96 hours after challenge with anti-acetylcholine receptor antibodies, and individual and average values ​​for each group are shown. *: p<0.05, **: p<0.01 (Dunnett's test, versus Control)). In the figure, mpk represents mg / kg. DETAILED DESCRIPTION OF THE INVENTION

[0094] FF-MAS (follicular fluid meiosis activating sterol) is a type of sterol that is part of the cholesterol synthesis pathway. It refers to 4,4-dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol and has the following structure: [ka] The compound is represented by the formula:

[0095] 14-dehydrozymosterol is a type of sterol that is part of the cholesterol synthesis pathway, and refers to 5alpha-cholesta-8,14,24-trien-3beta-ol, which is biosynthesized from FF-MAS by demethylating the dimethyl group at position 4. It has the following structure: [ka] The compound is represented by the formula:

[0096] Sterol C14-reductase (sometimes referred to as Δ14 sterol reductase) is an enzyme that metabolizes FF-MAS to T-MAS (testis meiosis-activating sterol, 4,4-dimethyl-5 alpha-cholesta-8,24-diene-3 beta-ol), and examples thereof include TM7SF2 (transmembrane 7 superfamily member 2) and LBR (lamin B receptor).

[0097] In the present invention, the term "FF-MAS metabolism inhibitor" refers to a substance that has the function of directly or indirectly (preferably directly) inhibiting the metabolism of FF-MAS in a living body. The FF-MAS metabolism inhibitor is preferably a substance that has the effect of increasing the intracellular or extracellular concentrations of FF-MAS and / or 14-dehydrozymosterol through its inhibitory effect, and examples of such an inhibitor include sterol C14 reductase inhibitors, TM7SF2 inhibitors, and LBR inhibitors.

[0098] In the present invention, the term "sterol C14-reductase inhibitor" refers to a substance that directly or indirectly (preferably directly) inhibits the function of sterol C14-reductase in vivo. Preferred sterol C14-reductase inhibitors are those that have the effect of increasing intracellular or extracellular FF-MAS concentrations and / or 14-dehydrozymosterol levels through their inhibitory activity. Examples of such inhibitors include TM7SF2 inhibitors and LBR inhibitors. Preferred sterol C14-reductase inhibitors are TM7SF2 inhibitors and / or LBR inhibitors, with TM7SF2 inhibitors being more preferred. Examples of sterol C14-reductase inhibitors include amorolfine, ifenprodil, and ziprasidone. The term "sterol C14-reductase inhibitor" as used herein includes not only those that have been discovered to date but also those that will be discovered in the future.

[0099] In the present invention, the term "TM7SF2 inhibitor" refers to a substance that directly or indirectly (preferably directly) inhibits the function of TM7SF2 in vivo. A TM7SF2 inhibitor is preferably a substance that increases intracellular or extracellular FF-MAS concentrations through its inhibitory effect, and its inhibitory activity is preferably, for example, 10 μmol / L or less, more preferably 1 μmol / L or less, and even more preferably 0.1 μmol / L or less, in terms of IC50 value. Some embodiments of TM7SF2 inhibitors include amorolfine, ifenprodil, and ziprasidone. TM7SF2 inhibitors of the present invention include not only those that have been discovered to date, but also those that will be discovered in the future. For example, TM7SF2 inhibitors can be screened using the screening method (evaluation system) described in Example 8 below. Examples of TM7SF2 inhibitors include compounds that exhibit an IC50 value of 10 μmol / L or less (preferably 1 μmol / L or less, more preferably 0.1 μmol / L or less) in the evaluation system described in Example 8.

[0100] In certain embodiments, the TM7SF2 inhibitor is a selective TM7SF2 inhibitor that selectively inhibits TM7SF2.

[0101] An example of a selective TM7SF2 inhibitor is a compound that has selectivity for CYP51 and / or EBP (preferably CYP51, preferably EBP, more preferably CYP51 and EBP). In one aspect, the selective TM7SF2 inhibitor is a compound that exhibits 10-fold or more, 20-fold or more, 50-fold or more, or 100-fold or more selectivity for CYP51 and / or EBP. In one embodiment, selectivity can be determined by performing affinity selection mass spectrometry (ASMS) for each enzyme or by comparing IC50 values ​​in a binding test. The inhibitory activity (IC50 value) for CYP51 and / or EBP can be determined, for example, by adjusting the evaluation system described in Example 8 below for CYP51 or EBP, or by using a known method, such as the method described in Non-Patent Document 4.

[0102] In the present invention, the term "LBR inhibitor" refers to a substance that directly or indirectly (preferably directly) inhibits the function of LBR in vivo. Preferred LBR inhibitors are those that have the effect of increasing intracellular or extracellular FF-MAS concentrations through their inhibitory action, and their inhibitory activity is, for example, preferably 10 μmol / L or less, more preferably 1 μmol / L or less, and even more preferably 0.1 μmol / L or less, in terms of IC50 value. Some embodiments of LBR inhibitors include amorolfine, ifenprodil, and ziprasidone.

[0103] It is described in Non-Patent Document 4, Patent Document 1, etc. that amorolfine, ifenprodil, ziprasidone, etc. have LBR or TM7SF2 inhibitory activity.

[0104] The LBR inhibitors of the present invention include not only those that have been discovered to date but also those that will be discovered in the future. For example, LBR inhibitors can be screened using the evaluation system described in Example 8 below. Examples of LBR inhibitors include compounds that show an IC50 value of 10 μmol / L or less (preferably 1 μmol / L or less, more preferably 0.1 μmol / L or less) in the evaluation system described in Example 8.

[0105] In certain embodiments, the LBR inhibitor is a selective LBR inhibitor that selectively inhibits LBR.

[0106] An example of a selective LBR inhibitor is a compound that has selectivity for CYP51 and / or EBP. In one embodiment, a selective LBR inhibitor is a compound that exhibits 10-fold or more, 20-fold or more, 50-fold or more, or 100-fold or more selectivity for CYP51 and / or EBP (preferably CYP51, preferably EBP, more preferably CYP51 and EBP). In one embodiment, selectivity can be determined by performing ASMS for each enzyme or comparing IC50 values ​​in a binding test.

[0107] In some embodiments, the selective TM7SF2 inhibitor is a compound that also has selectivity for LBR, or in some embodiments, a compound that also has inhibitory activity against LBR (selective TM7SF2 and LBR dual inhibitor).

[0108] In some embodiments, a selective LBR inhibitor is a compound that also has selectivity for TM7SF2. In other embodiments, the selective LBR inhibitor is a compound that also has inhibitory activity against TM7SF2 (a selective TM7SF2 and LBR dual inhibitor).

[0109] In one embodiment, the sterol C14-reductase inhibitor is a substance that selectively inhibits sterol C14-reductase. In one embodiment, the selective sterol C14-reductase inhibitor is a selective TM7SF2 inhibitor, a selective LBR inhibitor, or a selective TM7SF2 and LBR dual inhibitor. A selective TM7SF2 and LBR dual inhibitor is a substance that selectively inhibits TM7SF2 and LBR, for example, a compound that has selectivity for CYP51 and / or EBP (preferably CYP51, more preferably CYP51 and EBP).

[0110] In the present invention, the sterol C14-reductase inhibitor may be any combination of two or more. Specific examples include a combination of a TM7SF2 inhibitor and an LBR inhibitor. Furthermore, the selective sterol C14-reductase inhibitor may be any combination of two or more. Specific examples include a combination of a selective TM7SF2 inhibitor and a selective LBR inhibitor.

[0111] In the present invention, FF-MAS metabolism inhibitors, sterol C14-reductase inhibitors, TM7SF2 inhibitors, or LBR inhibitors include, but are not limited to, small molecular weight compounds (e.g., amorolfine, ifenprodil, ziprasidone), polypeptides (e.g., antibodies (preferably antagonistic antibodies), antibody fragments, etc.), and nucleic acids (e.g., antisense, siRNA, etc.).

[0112] [Medicinal uses] In some embodiments, the FF-MAS metabolism inhibitors of the present invention (e.g., sterol C14 reductase inhibitors or TM7SF2 inhibitors) are useful for protecting and / or regenerating neuromuscular junctions or protecting and / or regenerating nervous system cells.

[0113] In the present invention, "protection of the neuromuscular junction" refers to the action of suppressing disorders (including functional and morphological disorders and hypoplasia) of the neuromuscular junction.

[0114] In the present invention, "regeneration of neuromuscular junction" refers to the action of regenerating a damaged neuromuscular junction.

[0115] In the present invention, "protection and / or regeneration of the neuromuscular junction" can be evaluated by staining the neuromuscular junction formed by co-culturing nerve cells and muscle cells for acetylcholinesterase or acetylcholine receptor, which are markers of the neuromuscular junction, and then performing image analysis.

[0116] In the present invention, "protection of nervous system cells" refers to the action of protecting nervous system cells (for example, nerve cells, Schwann cells, etc.) from damage and / or degeneration.

[0117] In the present invention, "regeneration of nervous system cells" refers to the action of regenerating damaged and / or degenerated nervous system cells (for example, nerve cells, Schwann cells, etc.).

[0118] In the present invention, the "neurite outgrowth effect" refers to the effect of extending the length from the cell body of a neuron to the terminal end of a neurite, and can be evaluated, for example, by staining the nucleus and neurite of a neuron and performing image analysis.

[0119] In the present invention, the "Schwann cell differentiation promoting effect" refers to the effect of promoting the change in state from undifferentiated Schwann cells to differentiated Schwann cells that form myelin sheaths, or the effect of suppressing the change in state from differentiated Schwann cells to undifferentiated Schwann cells that occurs upon nerve injury, etc., and can be evaluated, for example, by staining proteins such as MAG (myelin associated glycoprotein), which is involved in myelin formation, and performing image analysis.

[0120] In the present invention, "treatment" means, for example, curing or ameliorating a certain disease or its symptoms, and "prevention" means preventing the onset of a certain disease or symptom or delaying its onset for a certain period of time. Treatment and / or prevention includes halting the progression of the pathology by suppressing the progress or worsening of symptoms, and preventing or reducing the possibility of recurrence of a certain disease or symptom.

[0121] The FF-MAS metabolism inhibitors (e.g., sterol C14 reductase inhibitors or TM7SF2 inhibitors) of the present invention are useful for treating and / or preventing diseases involving disorders of the neuromuscular junction (e.g., neurodegenerative diseases, myogenic diseases).

[0122] Therefore, one embodiment of the present invention is a therapeutic and / or preventive agent for diseases associated with disorders of the neuromuscular junction, which contains an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0123] Examples of neurodegenerative diseases include facial paralysis, peripheral neuropathy (e.g., diabetic peripheral neuropathy, peripheral neuropathy associated with anticancer drug administration, etc.), spinal canal stenosis, glaucoma, age-related macular degeneration, Alzheimer's disease, dementia with Lewy bodies, progressive supranuclear palsy, corticobasal degeneration, Parkinson's disease, multiple system atrophy, amyotrophic lateral sclerosis, spinal-bulbar muscular atrophy, primary lateral sclerosis, spinal muscular atrophy, spinocerebellar degeneration, frontotemporal degeneration, Huntington's disease, Charcot-Marie-Tooth disease, and traumatic neurodegenerative diseases, with peripheral neuropathy, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis being preferred. Another example of neurodegenerative diseases is a neurodegenerative disease associated with damage to the neuromuscular junction or nervous system cells (e.g., nerve cells (e.g., peripheral nerve cells), Schwann cells, etc.).

[0124] Some aspects of myopathic diseases include, for example, myasthenia gravis, muscular dystrophy, sarcopenia, and myopathy.

[0125] Therefore, one embodiment of the present invention is an agent for protecting and / or regenerating neuromuscular junctions, which contains an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0126] Another embodiment of the present invention is an agent for protecting and / or regenerating nervous system cells, which contains an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0127] Another aspect of the present invention is an agent for protecting and / or regenerating nervous system cells through neurite outgrowth, which contains an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0128] Another aspect of the present invention is an agent for protecting and / or regenerating nervous system cells through promoting Schwann cell differentiation, which contains an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0129] Another embodiment of the present invention is a neurite outgrowth agent containing an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0130] Another embodiment of the present invention is an agent for promoting Schwann cell differentiation, which contains an FF-MAS metabolism inhibitor (for example, a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0131] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) containing an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0132] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) that contains an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) and that acts to protect and / or regenerate the neuromuscular junction.

[0133] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases mediated by neurite outgrowth (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis), which comprises an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0134] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) mediated by promoting Schwann cell differentiation, which comprises an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0135] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases associated with disorders of the neuromuscular junction (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis), which contains an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0136] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (preferably involving disorders of the neuromuscular junction) (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) containing an FF-MAS metabolic inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for protecting and / or regenerating the neuromuscular junction.

[0137] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) accompanied by damage to nervous system cells (e.g., neurites or Schwann cells), which comprises an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0138] Another aspect of the present invention is a therapeutic and / or preventive agent for neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) containing an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for protecting and / or regenerating neuromuscular junctions and / or for protecting and / or regenerating nervous system cells (for promoting neurite outgrowth and / or Schwann cell differentiation).

[0139] Another aspect of the present invention is a therapeutic and / or preventive agent for myogenic diseases (e.g., myasthenia gravis, muscular dystrophy, sarcopenia, myopathy) containing an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) through the protective and / or regenerative effect of the neuromuscular junction.

[0140] Another aspect of the present invention is a therapeutic and / or preventive agent for myogenic diseases associated with disorders of the neuromuscular junction (e.g., myasthenia gravis, muscular dystrophy, sarcopenia, myopathy), which contains an FF-MAS metabolism inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0141] Another aspect of the present invention is a therapeutic and / or preventive agent for myogenic diseases (preferably involving disorders of the neuromuscular junction) (e.g., myasthenia gravis, muscular dystrophy, sarcopenia, myopathy) containing an FF-MAS metabolic inhibitor (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor) for protecting and / or regenerating the neuromuscular junction.

[0142] Another aspect of the present invention is an agent for protecting and / or regenerating neuromuscular junctions, or an agent for protecting and / or regenerating nervous system cells, which contains FF-MAS or 14-dehydrozymosterol, or a derivative thereof.

[0143] Another aspect of the present invention is an agent for protecting and / or regenerating neuromuscular junctions, or an agent for protecting and / or regenerating nervous system cells, which contains an agent having the effect of increasing FF-MAS and / or 14-dehydrozymosterol concentrations (e.g., a sterol C14 reductase inhibitor or a TM7SF2 inhibitor).

[0144] Furthermore, sterols that constitute the cholesterol synthesis pathway, such as FF-MAS, and their metabolic enzymes (e.g., TM7SF2, LBR, EBP, DHCR7) are deeply related to neurodegenerative diseases (e.g., peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis) and diseases involving disorders of the neuromuscular junction, and therefore can be used as therapeutic and / or diagnostic biomarkers for these diseases. Examples of sterols that make up the cholesterol synthesis pathway include lanosterol, dihydro lanosterol, FF-MAS, T-MAS, dihydro FF-MAS, 14-dehydrozymosterol, 14-dehydrozymostenol, zymosterol, dehydrolathosterol, zymostenol, lathosterol, 7-DHC, 7-DHD, desmosterol, cholesterol, cholecalciferol, and 25-hydroxycholecalciferol2, and preferred are FF-MAS, T-MAS, 14-dehydrozymosterol, and 14-dehydrozymostenol.

[0145] Therefore, another aspect of the present invention is a method for determining or predicting the effectiveness of a therapeutic agent (e.g., an FF-MAS metabolism inhibitor, a sterol C14 reductase inhibitor, or a TM7SF2 inhibitor) for a neurodegenerative disease or a disease accompanied by damage to the neuromuscular junction in a patient, using the concentration of sterols that constitute the cholesterol synthesis pathway in a biological sample from the patient as an indicator.

[0146] Another aspect is a method for determining or predicting the effectiveness of a therapeutic drug (e.g., an FF-MAS metabolism inhibitor, a sterol C14 reductase inhibitor, or a TM7SF2 inhibitor) for a neurodegenerative disease or a disease accompanied by damage to the neuromuscular junction, using the TM7SF2 expression level or LBR expression level (preferably TM7SF2 expression level) in a biological sample from the patient as an indicator.

[0147] In another aspect of the present invention, the concentrations of each sterol constituting the cholesterol synthesis pathway, the ratio of sterols, TM7SF2 expression level and / or LBR expression level are biomarkers (the present biomarkers) that predict (suggest) the effectiveness of an FF-MAS metabolism inhibitor.

[0148] Another aspect of the present invention is a pharmaceutical composition for neurodegenerative diseases containing an FF-MAS metabolism inhibitor, which is administered to a patient with a neurodegenerative disease for whom an FF-MAS metabolism inhibitor has been determined or predicted to be effective by the above-mentioned method.

[0149] Another aspect of the present invention is a pharmaceutical composition for treating a disease accompanied by a disorder of the neuromuscular junction, which contains an FF-MAS metabolism inhibitor and is administered to a patient suffering from a disease accompanied by a disorder of the neuromuscular junction for whom an FF-MAS metabolism inhibitor has been determined or predicted to be effective by the above-described method.

[0150] The predetermined thresholds corresponding to each of the above biomarkers can be set appropriately. The predetermined thresholds can be set, for example, based on data on the biomarkers in biological samples (tissues, blood, etc.) collected from patients with neurodegenerative diseases or patients with diseases accompanied by neuromuscular junction disorders. For example, the predetermined thresholds can be set as follows: First, biological samples are collected from multiple patients with neurodegenerative diseases or patients with diseases accompanied by neuromuscular junction disorders who have not been administered an FF-MAS metabolism inhibitor. After collection of the biological samples, the FF-MAS metabolism inhibitor is administered to the patients with neurodegenerative diseases or patients with diseases accompanied by neuromuscular junction disorders, and the progress is observed. The efficacy of the FF-MAS metabolism inhibitor is confirmed based on known evaluation indices. The biological samples are then classified into samples from patients who responded to the FF-MAS metabolism inhibitor and samples from patients who did not respond to the FF-MAS metabolism inhibitor. The biomarkers are measured for each sample to obtain measured values. Then, from the obtained measured values, a value that can distinguish between patients who responded to the FF-MAS metabolism inhibitor and patients who did not respond to the FF-MAS metabolism inhibitor is calculated, and this value is set as a predetermined threshold. When setting the predetermined threshold, it is preferable to also take into consideration the sensitivity, specificity, positive predictive value, and negative predictive value of the determination.

[0151] Yet another aspect is a method for determining or predicting the onset, severity, or prognosis of a neurodegenerative disease or a disease accompanied by damage to the neuromuscular junction in a patient, using as an indicator the concentration of sterols that constitute the cholesterol synthesis pathway in a biological sample from the patient.

[0152] Yet another aspect is a method for determining or predicting the onset, severity, or prognosis of a neurodegenerative disease or a disease associated with damage to the neuromuscular junction in a patient, using the TM7SF2 expression level or LBR expression level (preferably TM7SF2 expression level) in a biological sample from the patient as an indicator.

[0153] formulation The FF-MAS metabolism inhibitors and the like used in the present invention are usually administered systemically or locally, orally or parenterally. Examples of oral preparations include liquid preparations for oral administration (e.g., elixirs, syrups, pharmaceutically acceptable solutions, suspensions, and emulsions), solid preparations for oral administration (e.g., tablets (including sublingual tablets and orally disintegrating tablets), pills, capsules (including hard capsules, soft capsules, gelatin capsules, and microcapsules), powders, granules, and lozenges). Examples of parenteral preparations include liquid preparations (e.g., injections (intravitreal injections, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, instillations, etc.), eye drops (e.g., aqueous eye drops (aqueous eye drops, aqueous suspension eye drops, viscous eye drops, solubilized eye drops, etc.), non-aqueous eye drops (non-aqueous eye drops, non-aqueous suspension eye drops, etc.)), topical preparations (e.g., ointments (ophthalmic ointments, etc.)), ear drops, etc. These preparations may be controlled-release preparations such as immediate-release preparations and sustained-release preparations. These preparations can be produced by known methods, for example, methods described in the Japanese Pharmacopoeia.

[0154] Oral liquid preparations are prepared by dissolving, suspending, or emulsifying the active ingredient in a commonly used diluent (e.g., purified water, ethanol, or a mixture thereof), and may further contain a wetting agent, suspending agent, emulsifier, sweetener, flavoring agent, fragrance, preservative, buffer, etc.

[0155] Oral solid preparations for internal use are formulated in accordance with conventional methods by mixing the active ingredient with excipients (e.g., lactose, mannitol, glucose, microcrystalline cellulose, starch, etc.), binders (e.g., hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium aluminometasilicate, etc.), disintegrants (e.g., calcium cellulose glycolate, etc.), lubricants (e.g., magnesium stearate, etc.), stabilizers, solubilizers (glutamic acid, aspartic acid, etc.), etc. If necessary, the solid preparations may be coated with a coating agent (e.g., sucrose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose phthalate, etc.), or may be coated with two or more layers.

[0156] External preparations as parenteral agents are prepared by known methods or commonly used formulations. For example, ointments are prepared by triturating or dissolving the active ingredient in a base. The ointment base is selected from known or commonly used bases. Examples include higher fatty acids or higher fatty acid esters (e.g., adipic acid, myristic acid, palmitic acid, stearic acid, oleic acid, adipate esters, myristic acid esters, palmitate esters, stearic acid esters, oleate esters, etc.), waxes (e.g., beeswax, spermaceti, ceresin, etc.), surfactants (e.g., polyoxyethylene alkyl ether phosphate esters, etc.), higher alcohols (e.g., cetanol, stearyl alcohol, cetostearyl alcohol, etc.), silicone oils (e.g., cetearyl alcohol, cetearyl alcohol, etc.), and the like. These agents may be used alone or in combination of two or more selected from the group consisting of organic solvents (e.g., dimethylpolysiloxane, etc.), hydrocarbons (e.g., hydrophilic petrolatum, white petrolatum, refined lanolin, liquid paraffin, etc.), glycols (e.g., ethylene glycol, diethylene glycol, propylene glycol, polyethylene glycol, macrogol, etc.), vegetable oils (e.g., castor oil, olive oil, sesame oil, turpentine oil, etc.), animal oils (e.g., mink oil, egg yolk oil, squalane, squalene, etc.), water, absorption enhancers, and anti-rash agents. Furthermore, these agents may contain moisturizers, preservatives, stabilizers, antioxidants, fragrances, etc.

[0157] Injectable parenteral preparations include solutions, suspensions, emulsions, and solid injectable preparations that are dissolved or suspended in a solvent before use. Injectable preparations are used, for example, by dissolving, suspending, or emulsifying the active ingredient in a solvent. Examples of solvents that can be used include distilled water for injection, physiological saline, vegetable oil, propylene glycol, polyethylene glycol, alcohols such as ethanol, and combinations thereof. Furthermore, these injectable preparations may contain stabilizers, solubilizers (e.g., glutamic acid, aspartic acid, polysorbate 80 (registered trademark), etc.), suspending agents, emulsifiers, soothing agents, buffers, preservatives, etc. These are sterilized in the final process or produced by aseptic procedures. Alternatively, sterile solid preparations, such as lyophilized products, can be produced and then dissolved in sterilized or sterile distilled water for injection or other solvents before use.

[0158] When the compound of the present invention or a combination of the compound of the present invention and other drugs is used for the above-mentioned purposes, it is usually administered systemically or locally, orally or parenterally. The dosage varies depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc., but is usually orally administered in the range of 1 ng to 1000 mg per dose per adult, once to several times a day, or parenterally administered in the range of 0.1 ng to 10 mg per dose per adult, once to several times a day, or continuously administered intravenously for 1 to 24 hours a day. Of course, as mentioned above, the dosage varies depending on various conditions, so in some cases a smaller dosage than the above-mentioned dosage is sufficient, and in other cases a dosage exceeding the range is necessary.

[0159] Concomitant or combination drugs Furthermore, the FF-MAS metabolism inhibitors and the like used in the present invention may be used in combination with other drugs.

[0160] When the present invention is used in combination with other drugs (combined use), the dosage form may be a combination formulation in which both components are combined in a single formulation, or may be a separate formulation. Such combination can complement the preventive, symptom progression suppression, recurrence suppression, and / or therapeutic effects of the other drug, and can maintain or reduce the dosage or frequency of administration. When the FF-MAS metabolism inhibitor or other drug used in the present invention is administered separately from the other drug, they may be administered simultaneously for a certain period of time, followed by administration of only the FF-MAS metabolism inhibitor or other drug alone. Alternatively, the FF-MAS metabolism inhibitor or other drug used in the present invention may be administered first, followed by administration of the other drug after completion of that administration, or the other drug may be administered first, followed by administration of the FF-MAS metabolism inhibitor or other drug after completion of that administration. The administration methods for each drug may be the same or different. A formulation containing the FF-MAS metabolism inhibitor or other drug used in the present invention and a formulation containing the other drug may also be provided as a kit. The dosage of the other drug can be appropriately selected based on clinically used doses. Any two or more of the other drugs may be administered in combination in an appropriate ratio. The other drugs include not only those that have been discovered so far but also those that will be discovered in the future.

[0161] For example, the FF-MAS metabolism inhibitors and the like used in the present invention may be used in combination with riluzole, edaravone, mecobalamin, epalrestat, pregabalin, gabapentin, duloxetine, prostaglandin derivatives (latanoprost, etc.), donepezil, rivastigmine, galantamine, memantine, levodopa, dopamine agonists (pergolide, ropinirole, etc.), anticholinergics (trihexyphenidyl, etc.), amantadine hydrochloride, zonisamide, adenosine receptor antagonists (istradefylline, etc.), MAO-B inhibitors (selegiline hydrochloride, etc.), catechol-O-methyltransferase inhibitors (entacapone, etc.), droxidopa, risdiplam, nucleic acid drugs (nusinersen sodium, etc.), and / or gene therapy (onasemnogene abeparvovec, etc.), and the like.

[0162] The present invention will be described in more detail by the following examples, but the scope of the present invention is not limited thereto. [Example]

[0163] Example 1: Effect of sterol C14 reductase inhibitor (TM7SF2 inhibitor) on FF-MAS and 14-dehydrozymosterol accumulation

[0164] <Experimental Method> After differentiation of mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) into myotube-like cells, we treated them with sterol C14 reductase inhibitors (TM7SF2 inhibitors) (amorolfine, ziprasidone, and ifenprodil) to evaluate intracellular sterol changes. Specifically, to differentiate C2C12 cells into myotubes, the cells were cultured in a culture flask using 15% FBS-DMEM (Dulbecco's Modified Eagle's Medium containing 15% fetal bovine serum, 100 units / mL penicillin, and 100 μg / mL streptomycin). The medium was then replaced with low-serum medium (Dulbecco's Modified Eagle's Medium containing 0.5% FBS, 1% insulin-transferrin-selenium, 100 units / mL penicillin, and 100 μg / mL streptomycin) and cultured for two days. After this, the medium was replaced with low-serum medium containing 10 μmol / L arabinocytidine (AraC) to remove proliferative undifferentiated cells and cultured for another two days. C2C12 cells differentiated into myotubes were detached from the culture flask by treatment with 0.25% Trypsin-1 mmol / L EDTA and plated at 2.4 × 10 cells per well onto a 3.5 cm culture dish coated with Matrigel. 5Cells were seeded at 1000 cells / dish and cultured for 3 days. After treatment with a sterol C14 reductase inhibitor (TM7SF2 inhibitor) or 0.03% DMSO, cells were harvested at 3 and 24 hours. After removing the medium and washing with phosphate buffered saline (PBS), the cells were harvested with 200 μL of 90% methanol containing 1 μmol / L d7-cholesterol (internal standard), followed by another 200 μL of 90% methanol solution, for a total of approximately 400 μL of cell harvest solution. 160 μL of this cell harvest solution was pretreated by solid-phase extraction using an OASIS PRiME HLB μElution Plate. The sample eluted with methanol / IPA (1:1) was analyzed by high-performance liquid chromatography / atmospheric pressure chemical ionization tandem mass spectrometry (LC / APCI-MS / MS), and the concentrations of each component in the harvested solution were calculated using an internal standard calibration curve.

[0165] <Results / Discussion> The results of FF-MAS are shown in Figures 1 and 2. In the DMSO-treated group, FF-MAS concentrations in the mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) were below the detection limit. However, treatment with amorolfine, ziprasidone, and ifenprodil, which have sterol C14 reductase inhibitory activity (TM7SF2 inhibitory activity), increased intracellular FF-MAS concentrations under all conditions. Furthermore, similar treatments also increased 14-dehydrozymosterol concentrations. Therefore, these compounds were confirmed to increase intracellular concentrations of FF-MAS and 14-dehydrozymosterol, which are substrates of this enzyme, by inhibiting sterol C14 reductase (TM7SF2 inhibition).

[0166] Example 2: Amelioration of neuromuscular junction disorders by sterol C14 reductase inhibitors (TM7SF2 inhibitors), FF-MAS, or 14-dehydrozymosterol

[0167] <Experimental Method> We evaluated the effects of sterol C14 reductase inhibitors (TM7SF2 inhibitors), FF-MAS, and 14-dehydrozymosterol on neuromuscular junction formation using a cell assay system that assesses neuromuscular junction formation by co-culturing mouse myoblast cell line C2C12 cells transfected with human mutant SOD1 (G93A) and primary mouse motor neurons. First, to differentiate C2C12 cells into myotubes, cultured in 15% FBS-DMEM in a culture flask, the medium was changed to low-serum medium and cultured for 2 days. To remove proliferative undifferentiated cells, the medium was changed to low-serum medium containing 10 μmol / L AraC and cultured for another 2 days. Myotube-like differentiated C2C12 cells were detached from the culture flask by treatment with 0.25% trypsin-1 mmol / L EDTA and plated at 1.5 × 10 cells onto a Matrigel-coated 96-well imaging plate. 4 Cells were seeded at 1000 cells / well and cultured in low-serum medium containing AraC for 3 days before co-culture with primary mouse motor neurons. Primary mouse motor neurons were prepared from embryonic day 13 mouse fetuses. Pregnant mice were euthanized by cervical dislocation, and the fetuses were removed through an abdominal incision. The fetuses were decapitated and their spinal cords were isolated under a stereomicroscope. The spinal cords were treated with HBSS containing 0.25% trypsin for 15 minutes at 37°C. The supernatant was removed, the cells were washed three times with HBSS, and 2 mL of dispersion solution (HBSS containing 0.125 mg / mL DNase) was added. A cell suspension was prepared by repeated pipetting with a fine-tipped Pasteur pipette. The cell suspension was centrifuged (200g, 5 minutes), and 2 mL of motor neuron medium (Neurobasal medium containing 2% B27 Supplement, 1 μmol / L AraC, 5 mmol / L Creatine, 10 ng / mL BDNF, 10 ng / mL GDNF, 10 ng / mL CNTF, and 0.125 mmol / L L-Glutamine) was added to the sediment, and the cells were resuspended and counted. All medium was removed from the 96-well imaging plate in which the myotube-like differentiated C2C12 cells had been cultured, and 3.0 × 10 primary cultured mouse motor neurons suspended in motor neuron medium were added. 4Cells were seeded at 100 μL / well and co-culture was initiated. Four hours after the initiation of co-culture, 50 μL of culture supernatant was removed, and 50 μL of Matrigel solution diluted 20-fold with motor neuron medium was added. After 5 minutes at room temperature, the cells were incubated in a CO2 incubator for 1 hour, and then 100 μL of motor neuron medium was added. Sterol C14 reductase inhibitors (TM7SF2 inhibitors) (amorolfine, ziprasidone, and ifenprodil) were added at concentrations of 0.01, 0.03, 0.1, 0.3, 1, and 3 μmol / L, FF-MAS at concentrations of 0.4, 1.1, 3.3, 10, and 30 μmol / L, or 14-dehydrozymosterol at concentrations of 0.1, 0.3, 1, 3, and 10 μmol / L. The cells were cultured for 11 days until neuromuscular junction formation. Neuromuscular junctions were assessed by staining acetylcholine receptors, a marker of neuromuscular junctions in muscle, with α-Bungarotoxin. Specifically, cells were fixed in 1% paraformaldehyde for 20 minutes at room temperature. The 96-well plates were then washed three times with 100–150 μL / well of PBS. 50 μL / well of PBS containing 2 μg / mL α-Bungarotoxin, Alexa Fluor 488 Conjugate was added. The plates were incubated at room temperature for 1 hour in the dark, and then washed three times with 100–150 μL / well of PBS. The stained plates were photographed using an Opera Phenix high-throughput, high-content imaging system (PerkinElmer). Images of α-Bungarotoxin were taken using an Alexa Fluor 488 wavelength filter, with three fields of view per well. The α-Bungarotoxin-positive area per field was calculated as the area of ​​the neuromuscular junction using an analysis program. The average area of ​​the neuromuscular junction in the vehicle group (0.03% DMSO or 0.2% ethanol) evaluated per plate was set as 100%, and the area of ​​the neuromuscular junction in the sterol C14 reductase inhibitor (TM7SF2 inhibitor), FF-MAS, and 14-dehydrozymosterol-treated groups was calculated.

[0168] <Results / Discussion> The results are shown in Figures 3 to 7. Amorolfine, ziprasidone, and ifenprodil significantly increased the neuromuscular junction-positive area at concentrations of 0.3 μmol / L, 3 μmol / L, and 1 μmol / L, respectively. FF-MAS and 14-dehydrozymosterol significantly increased the neuromuscular junction-positive area at concentrations of 10 μmol / L and above. These results suggest that sterol C14 reductase inhibitors (TM7SF2 inhibitors) promote or protect the formation of neuromuscular junctions through increases in FF-MAS and / or 14-dehydrozymosterol.

[0169] Furthermore, when the EBP inhibitor TASIN-1 was added at 0.1 μmol / L and the same evaluation was performed as above, no significant increase in the neuromuscular junction-positive area was observed.Furthermore, when zymosterol was added at 0.05 to 10 μmol / L, no significant increase in the neuromuscular junction-positive area was observed.

[0170] Example 3: Neurite outgrowth effect of sterol C14 reductase inhibitor (TM7SF2 inhibitor) or FF-MAS

[0171] <Experimental Method> The effects of a sterol C14 reductase inhibitor (TM7SF2 inhibitor) and FF-MAS on the neurite outgrowth were evaluated in a monoculture system of mouse primary motor neurons. Primary mouse motor neurons were prepared from mouse fetuses on embryonic day 13. Pregnant mice were euthanized by cervical dislocation on day 13 of pregnancy, and the fetuses were removed through an abdominal incision. The fetuses were decapitated and their spinal cords were isolated under a stereomicroscope. The spinal cords were treated with HBSS containing 0.25% trypsin for 15 minutes at 37°C. The supernatant was removed, the cells were washed three times with HBSS, and a cell suspension was prepared by adding 2 mL of dispersion solution and pipetting repeatedly with a fine-tipped Pasteur pipette. The cell suspension was centrifuged (200 g, 5 minutes). The sediment was resuspended in 2 mL of motor neuron medium, and the cells were counted. Mouse primary motor neurons suspended in motor neuron medium were collected at 1.5 × 10 4Cells were seeded onto a poly-D-lysine-coated 96-well imaging plate at 100 μL / well. Five hours after the start of culture, a sterol C14 reductase inhibitor (TM7SF2 inhibitor) was added at 0.01, 0.03, 0.1, 0.3, 1, or 3 μmol / L, or FF-MAS was added at 0.4, 1.1, or 3.3 μmol / L, and the plate was cultured for 4 days. Neurite length was assessed by immunocytochemical staining of neuronal nuclei and processes. Specifically, cells were fixed with 4% paraformaldehyde at room temperature for 30 minutes. The 96-well plate was then washed three times with 100–150 μL / well of PBS. Then, 100 μL / well of blocking solution (PBS containing 5% fetal bovine serum and 0.3% Triton® X-100) was added and the plate was allowed to stand at room temperature for 1 hour. Primary antibodies, anti-neuron-specific beta-III tubulin antibody (R&D Systems, MAB1195, 1 / 1000 dilution) and anti-NeuN antibody (Merck Millipore, ABN78, 1 / 500 dilution) diluted in blocking solution, were added at 50 μL / well and incubated overnight at 4°C. After washing three times with 100–150 μL / well of PBS, secondary antibodies, Alexa Fluor 647 goat anti-mouse IgG (H+L) (Invitrogen, A-21236, 1 / 1000 dilution) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Invitrogen, A-11034, 1 / 1000 dilution), were added at 50 μL / well and incubated at room temperature for 1 hour in the dark, followed by three washes with 100–150 μL / well of PBS. The stained plates were photographed using an Opera Phenix high-throughput, high-content imaging system (PerkinElmer Japan). Five fields of view were acquired per well using an Alexa Fluor 647 wavelength filter to photograph neurites and an Alexa Fluor 488 wavelength filter to photograph neuronal nuclei. An analysis program was used to analyze the neuronal nuclei and neurites per field, and the total neurite length per field was calculated.The average value of the total neurite length in the vehicle group (0.03% DMSO or 0.2% ethanol) evaluated for each plate was set as 100%, and the total neurite length in the sterol C14 reductase inhibitor (TM7SF2 inhibitor) and FF-MAS treatment groups was calculated.

[0172] <Result discussion> The results are shown in Figures 8 to 11. Amorolfine, ziprasidone, and ifenprodil significantly increased total neurite length from 0.3 μmol / L, 1 μmol / L, and 1 μmol / L, respectively. FF-MAS also significantly increased total neurite length from 1.1 μmol / L. These results suggest that sterol C14 reductase inhibitors (TM7SF2 inhibitors) exert neurite outgrowth or protective effects via an increase in FF-MAS.

[0173] Example 4: Effect of sterol C14 reductase inhibitor (TM7SF2 inhibitor) or FF-MAS on promoting Schwann cell differentiation

[0174] <Experimental Method> The Schwann cell differentiation-promoting effect of FF-MAS was evaluated in a rat primary Schwann cell culture system. Neonatal rats (0–2 days old) were decapitated and sterilized by immersion in disinfectant ethanol. They were then transferred to a clean bench and washed with collection medium (phenol red-free Dulbecco's Modified Eagle Medium (Sigma-Aldrich, Product No. D5921) containing 1 vol% Antibiotic-Antimycotic (Antibiotic-Antimycotic 100x, Thermo Fisher Scientific, Product No. 15240062)). DRGs were then excised under a microscope and collected in a 15 mL tube filled with collection medium. The tubes were centrifuged at 400 x g for 3 minutes at room temperature, and the supernatant was removed. Three mL of 0.25% collagenase (Worthington Biochemical, product number LS004196) dissolved in recovery medium was added to the sediment, which was then disrupted by pipetting and incubated in an incubator at 37°C for 30 minutes. After incubation, the mixture was centrifuged at 400 xg for 3 minutes at room temperature, and the supernatant was removed. Three mL of 0.25% Trypsin / EDTA solution (Fujifilm Wako Pure Chemical Industries, product number 209-16941) and 1 μL of DNase I (ThermoFisher Scientific, product number 18047-019) were added to the sediment, which was then disrupted by pipetting and incubated in an incubator at 37°C for 30 minutes. After incubation, trypsin was inactivated by adding culture medium (phenol red-containing DMEM (Sigma Aldrich, product number D6546) containing inactivated 10 vol% fetal bovine serum (fetal bovine serum, qualified, EU-approved, South American origin, Thermo Fisher Scientific, product number 10270106) and 1 vol% antibiotic-antimycotic (Thermo Fisher Scientific)). The mixture was centrifuged at 400 xg for 3 minutes at room temperature, and the supernatant was removed. Culture medium was added to the pellet to prepare a cell suspension, which was then passed through a filter (70 μm diameter).Count the number of cells in the filtered cell suspension to 2.0 x 10. 5After adjusting the concentration to cells / mL, 100 μL / well of the cells were seeded onto a poly-D-lysine-coated 96-well plate and cultured statically in a CO2 incubator under conditions of 5% CO2, 95% air, and 37°C. To evaluate the effect of FF-MAS on Schwann cell differentiation, the medium in the cell culture plate was aspirated and replaced with evaluation medium (phenol red-containing DMEM (Sigma-Aldrich, Product No. D6546) containing heat-inactivated 1 vol% dialyzed fetal bovine serum (fetal bovine serum, dialyzed, US origin, Thermo Fisher Scientific, Product No. 26400044), 1 vol% antibiotic-antimycotic (Thermo Fisher Scientific), and 100 μmol / L db-cAMP (Sigma-Aldrich, Product No. D0627). FF-MAS was added at concentrations of 0.03, 0.1, 0.3, 1, 3, or 10 μmol / L and cultured for 3 days. The vehicle group contained 0.1% DMSO and 0.1% ethanol. The Schwann cell differentiation-promoting activity was evaluated based on the expression level of myelin-associated glycoprotein (MAG). After removing the medium, 100 μL of 4% paraformaldehyde (Fujifilm Wako Pure Chemical Industries, Ltd., 163-20145) was added to each well and allowed to stand at room temperature for at least 20 minutes to fix the cells. After removing the supernatant, 100 μL of Block Ace solution containing 0.1% Tween®-20 was added to each well and allowed to stand at room temperature for at least 30 minutes. After removing the supernatant, 60 μL of 5 μg / mL anti-MAG antibody (Millipore, product number MAB1567) solution was added to each well and allowed to stand at room temperature for at least 60 minutes. After washing three times with PBS, 70 μL of 10 μg / mL Alexa fluor 488 anti-mouse IgG (Thermo Fisher Scientific, product number A11029) solution was added to each well and allowed to stand at room temperature for at least 60 minutes. After washing three times with PBS, 100 μL of a 10 μg / mL Hoechst33342 (Invitrogen, product number H-3570) solution was added to each well to stain the nuclei.Fluorescence images were then acquired from nine fields per well using Opera Phenix (PerkinElmer). The MAG-positive area and the number of cell nuclei were calculated, and the sum of the nine fields per well was used to calculate the MAG-positive area and the number of cell nuclei for each well. The MAG-positive area for each well was divided by the number of cell nuclei (MAG / number of cell nuclei). Furthermore, by adding a sterol C14 reductase inhibitor (TM7SF2 inhibitor) instead of FF-MAS in the above method, the effect of the sterol C14 reductase inhibitor (TM7SF2 inhibitor) in promoting Schwann cell differentiation could be evaluated.

[0175] <Result discussion> The results of the Schwann cell differentiation promoting effect of FF-MAS are shown in Figure 12. FF-MAS significantly increased the MAG / cell nucleus value from 3 μmol / L onwards, suggesting that FF-MAS has the effect of promoting Schwann cell differentiation.

[0176] Example 5: Effect of TM7SF2 overexpression on the improvement of neuromuscular junction dysfunction by sterol C14 reductase inhibitors (TM7SF2 inhibitors)

[0177] <Experimental Method> We evaluated the effect of amorolfin, a sterol C14 reductase inhibitor (TM7SF2 inhibitor), on neuromuscular junction formation using a cell assay system in which mouse myoblast cell line C2C12 cells overexpressing human mutant SOD1 (G93A) alone or both human mutant SOD1 (G93A) and human TM7SF2 genes were co-cultured with mouse primary motor neurons. First, to differentiate C2C12 cells into myotubes, the medium was changed to low-serum medium containing 15% FBS-DMEM in a culture flask. After 2 days of culture, the medium was changed to low-serum medium containing 10 μmol / L AraC to remove proliferative undifferentiated cells, and the cells were cultured for another 2 days. C2C12 cells differentiated into myotubes were detached from the culture flask by treatment with 0.25% Trypsin-1mmol / L EDTA and plated onto a 96-well imaging plate coated with Matrigel at 1.5 × 10 4Cells were seeded at 1000 cells / well and cultured in low-serum medium containing AraC for 3 days before co-culture with mouse primary motor neurons. Mouse primary motor neurons were prepared from mouse fetuses at embryonic day 13. Pregnant mice were euthanized by cervical dislocation, and the fetuses were removed through an abdominal incision. After decapitation, the spinal cords were extracted under a stereomicroscope. The spinal cords were treated with HBSS containing 0.25% trypsin for 15 minutes at 37°C. The supernatant was removed, and the cells were washed three times with HBSS. 2 mL of dispersion solution (HBSS containing 0.125 mg / mL DNase) was added, and the cells were suspended by repeated pipetting with a fine-tipped Pasteur pipette. The cell suspension was centrifuged (200g, 5 minutes), and 2 mL of motor neuron medium (Neurobasal medium containing 2% B27 Supplement, 1 μmol / L AraC, 5 mmol / L Creatine, 10 ng / mL BDNF, 10 ng / mL GDNF, 10 ng / mL CNTF, and 0.125 mmol / L L-Glutamine) was added to the sediment, and the cells were resuspended and counted. All medium was removed from the 96-well imaging plate in which the myotube-like differentiated C2C12 cells had been cultured, and 3.0 × 10 primary cultured mouse motor neurons suspended in motor neuron medium were resuspended. 4Cells were seeded at 100 μL per well and co-culture was initiated. Four hours after the start of co-culture, 50 μL of culture supernatant was removed, and 50 μL of Matrigel solution diluted 20-fold with motor neuron medium was added. The cells were incubated at room temperature for 5 minutes, then incubated in a CO2 incubator for 1 hour, after which 100 μL of motor neuron medium was added. Amorolfine was added at concentrations of 0.1, 0.3, 1, or 3 μmol / L, and the cells were cultured for 11 days until neuromuscular junction formation. Neuromuscular junctions were assessed by staining acetylcholine receptors, a marker of neuromuscular junctions in muscle, with α-bungarotoxin. Specifically, cells were fixed in 1% paraformaldehyde for 20 minutes at room temperature in a 96-well plate. After washing three times with 100–150 μL / well of PBS, 50 μL / well of 2 μg / mL α-Bungarotoxin, Alexa Fluor 488 Conjugate in PBS was added. The plate was then incubated at room temperature for 1 hour in the dark and washed three times with 100–150 μL / well of PBS. Images of the stained plate were captured using an Opera Phenix high-throughput, high-content imaging system (PerkinElmer). Images of α-Bungarotoxin were taken using an Alexa Fluor 488 wavelength filter, with three fields of view per well. The α-Bungarotoxin-positive area per field was calculated as the area of ​​the neuromuscular junction using an analysis program. The area of ​​the neuromuscular junction in the sterol C14 reductase inhibitor (TM7SF2 inhibitor) treatment group was calculated based on the average area of ​​the neuromuscular junction in the vehicle group (0.03% DMSO) evaluated for each plate, which was set as 100%.

[0178] <Results / Discussion> The results are shown in Figure 13. When C2C12 cells overexpressing human mutant SOD1(G93A) alone were combined with mouse primary motor neurons, amorolfine significantly increased the neuromuscular junction-positive area at concentrations of 1 μmol / L and 3 μmol / L. On the other hand, when C2C12 cells overexpressing both human mutant SOD1(G93A) and the human TM7SF2 gene were combined with mouse primary motor neurons, amorolfine treatment at the same concentration range did not result in an increase in the neuromuscular junction-positive area. These results indicate that the overexpression of TM7SF2 negates the amorolfine-induced increase in the neuromuscular junction-positive area. Therefore, amorolfine may promote or protect the formation of neuromuscular junctions by inhibiting TM7SF2.

[0179] Example 6: Evaluation of the efficacy of sterol C14 reductase inhibitors (TM7SF2 inhibitors) in myasthenia gravis

[0180] <Experimental Method> The efficacy of ifenprodil, a sterol C14 reductase inhibitor (TM7SF2 inhibitor), was evaluated in a rat myasthenia gravis model. This model involves treatment of rats with mAb35, an anti-acetylcholine receptor (ACHR) antibody, resulting in impaired neuromuscular junction (NMJ) function and impaired grip strength. Five-week-old female LEW / CrlCrlj rats were first induced by intraperitoneal administration of the anti-ACHR antibody mAb35 (BioXCell, product number BE0123) at a concentration of 0.29 mg / mL at a volume of 5 mL / kg. The test substance, ifenprodil (MedChemExpress, product number HY-12882A), was dissolved in water for injection (Otsuka Pharmaceutical Factory, Inc.) containing 2% DMSO to concentrations of 0.1, 0.3, and 1 mg / mL. Ifenprodil was orally administered repeatedly at doses of 1, 3, or 10 mL / kg once daily from 9 hours after challenge with mAb35 administration until day 4 after challenge, which was the final day of evaluation.

[0181] The animals used were those that showed no abnormalities during the one-week acclimation period. Grip strength was measured the day before challenge (Pre-value). Grip strength was measured again 9 hours after challenge to calculate the percentage decrease in grip strength from the Pre-value. Only animals that showed a certain degree of decrease in grip strength due to challenge were used for evaluation. Grip strength was quantitatively evaluated using a Traction Meter (Brain Science Idea Co., Ltd.).

[0182] <Results / Discussion> The results are shown in Figure 14. In myasthenia gravis model rats, ifenprodil significantly improved grip strength at 96 hours after induction, starting at 3 mg / kg. This indicates that sterol C14 reductase inhibitors (TM7SF2 inhibitors) are effective against myasthenia gravis.

[0183] Example 7: Evaluation of the efficacy of sterol C14 reductase inhibitors (TM7SF2 inhibitors) for ALS Mice transgenic for human mutant SOD1 (G93A) are used as a model mouse that reflects the pathology of ALS. One strain of this transgenic mouse, the B6SJL-Tg(SOD*G93A)1Gur / J strain, begins to show declines in grip strength, EMG, motor coordination, and fatigue resistance around 50 days after birth. They also begin to lose weight around 90 days after birth and begin to die around 120 days after birth. By observing the progression of these symptoms, which reflect the pathology of ALS, the efficacy of sterol C14 reductase inhibitors (TM7SF2 inhibitors) in ALS can be evaluated.

[0184] Example 8: Screening method for TM7SF2 inhibitors or LBR inhibitors <Experimental Method> Human TM7SF2 and LBR were expressed in Expi293F cells, and the IC50 values ​​of inhibitors were calculated using membrane fractions. DMSO solutions of inhibitor candidates were dispensed into a 96-well plate using a D300e micropipetter to achieve final concentrations of 0.0003, 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol / L (final DMSO concentration: 0.3 v / v%). A control group was prepared by adding DMSO to a final concentration of 0.3 v / v%. A substrate solution (3 μmol / L FF-MAS, pH 7.4) containing 0.1 w / v% BSA and a protein solution (0.01 mg / mL human TM7SF2 or human LBR-expressing membrane fraction) were added to the 96-well plate containing the inhibitor candidate. After preincubation at 37°C for 5 minutes, an NADPH solution (2 mmol / L, pH 7.4) containing 0.1 w / v% BSA and a protein solution (0.1 mol / L potassium phosphate buffer, pH 7.4) were added. The plate was then incubated at 37°C for 5 minutes, and the enzyme reaction was initiated.

[0185] One hour after the start of the enzyme reaction, methanol containing 1 μmol / L d7-cholesterol was added, the mixture was stirred with a pipette, and the reaction was stopped. The mixture was then sealed with an aluminum foil seal. After leaving the mixture to stand for 5 minutes, it was centrifuged at 2300 g for 5 minutes at 4°C. The amount of T-MAS produced in the enzyme reaction using FF-MAS as a substrate was measured by LC / MS / MS, and the IC50 value of each inhibitor candidate was calculated from the T-MAS concentration using Excelfit.

Claims

1. An agent for protecting and / or regenerating neuromuscular junctions, comprising an FF-MAS metabolism inhibitor.

2. The agent according to claim 1, wherein the FF-MAS metabolism inhibitor is a sterol C14 reductase inhibitor.

3. The agent according to claim 2, wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor and / or an LBR inhibitor.

4. The agent according to claim 2, wherein the sterol C14 reductase inhibitor is a TM7SF2 inhibitor.

5. The agent according to claim 2, wherein the sterol C14 reductase inhibitor is a selective sterol C14 reductase inhibitor.

6. The agent according to claim 5, wherein the selective sterol C14 reductase inhibitor is a selective TM7SF2 inhibitor.

7. The agent according to any one of claims 1 to 6 for the treatment and / or prevention of a disease accompanied by a disorder of the neuromuscular junction.

8. The agent according to claim 7, wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

9. The agent according to claim 8, wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease.

10. The agent according to claim 9, wherein the neurodegenerative disease is peripheral neuropathy, Alzheimer's disease, Parkinson's disease, or amyotrophic lateral sclerosis.

11. The agent according to claim 8, wherein the disease associated with a disorder of the neuromuscular junction is a myogenic disease.

12. The agent according to claim 11, wherein the myogenic disease is myasthenia gravis, muscular dystrophy, sarcopenia, or myopathy.

13. A therapeutic and / or preventive agent for diseases associated with disorders of the neuromuscular junction, comprising an FF-MAS metabolism inhibitor.

14. The agent according to claim 13 for protecting and / or regenerating neuromuscular junctions.

15. The agent according to claim 13 or 14, wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease or a myogenic disease.

16. The agent according to claim 15, wherein the disease associated with a disorder of the neuromuscular junction is a neurodegenerative disease.

17. The agent according to claim 16, further for the protection and / or regeneration of nervous system cells.

18. The agent according to claim 15, wherein the disease associated with a disorder of the neuromuscular junction is a myogenic disease.

19. A method for protecting and / or regenerating a neuromuscular junction, which comprises administering to a mammal an effective amount of an FF-MAS metabolism inhibitor.

20. A method for treating and / or preventing a disease associated with a disorder of the neuromuscular junction, which comprises administering to a mammal an effective amount of an FF-MAS metabolism inhibitor.

21. FF-MAS metabolism inhibitors for use in the protection and / or regeneration of neuromuscular junctions.

22. An FF-MAS metabolism inhibitor for use in the treatment and / or prevention of diseases associated with disorders of the neuromuscular junction.

23. Use of an FF-MAS metabolism inhibitor for producing an agent for protecting and / or regenerating neuromuscular junctions.

24. Use of an FF-MAS metabolism inhibitor for the manufacture of an agent for treating and / or preventing a disease associated with a disorder of the neuromuscular junction.

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