A scavenger for removing insoluble protein aggregates in the brain, and a neurodegenerative disease treatment agent containing the same.

Abstract

The present invention aims to provide a drug that exhibits a fundamental therapeutic effect against neurodegenerative diseases. Specifically, it aims to provide a novel drug that can remove protein aggregates that characterize neurodegenerative diseases, particularly insoluble protein aggregates that are considered to be the direct cause of the disease. [Solution] The present invention, which solves the above problem, is a brain insoluble protein aggregate remover containing a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55. The insoluble protein is preferably tau.

Description

TECHNICAL FIELD

[0001] The present invention relates to an insoluble protein aggregate remover in the brain and a therapeutic agent for neurodegenerative diseases containing the same. BACKGROUND ART

[0002] Neurodegenerative diseases cover a wide range, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and the like. For most of these diseases, no fundamental therapeutic method has been established, and treatments that delay the progression of symptoms or alleviate symptoms are the mainstay. For example, for Alzheimer-type dementia, drugs such as donepezil, memantine, galantamine, and rivastigmine that act through mechanisms of action including acetylcholinesterase inhibition and NMDA receptor antagonism are used. Lecanemab is also known, which can delay the progression of Alzheimer's disease by removing amyloid β (oligomers and protofibrils), which are insoluble intermediate species from soluble precursors, via anti-amyloid β antibodies. However, all of these drugs only delay the progression of symptoms and cannot be called fundamental therapeutic agents.

[0003] On the other hand, it is known that in the brains of many patients with neurodegenerative diseases, protein aggregates that characterize the disease appear in nerve cells and glial cells. The main constituent protein of the aggregates differs depending on the disease: tau for Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), α-synuclein for Parkinson's disease (PD) and dementia with Lewy bodies (DLB), and TDP-43 for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), which have each been identified as the main constituent protein of disease-specific aggregates. However, almost nothing has been clarified regarding the mechanism by which specific proteins aggregate in the brain.

[0004] In previous research, the inventors discovered LPG (lysophosphatidylglucoside / LysoPtdGlc), a glucose lipid present in trace amounts in central nervous tissue, and identified the G protein-coupled receptor GPR55 as its receptor. With the aim of clarifying the activation mechanism of GPR55 by LysoPtdGlc and elucidating its pathophysiological significance, the inventors conducted research and revealed that in frontotemporal lobar degeneration, the LysoPtdGlc-GPR55 signaling axis exacerbates brain inflammation and neurodegeneration, and that blocking the LysoPtdGlc-GPR55 signaling axis may alleviate brain lesions (see Non-Patent Literature 1-3). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Post-evaluation results for the project selected in FY2016 under the Innovative Advanced Research and Development Support Program, "Elucidation of the physiological activity and function of lipids aiming to create groundbreaking pharmaceuticals, etc." (AMED CREST) ​​research and development area unit type, https: / / www.amed.go.jp / content / 000118070.pdf [Non-Patent Document 2] AMED-CREST Interim Report, FY2021, https: / / amedfind.amed.go.jp / amed / search / task_search_details?tid=7790011530 [Non-Patent Document 3] 2023 Grant-in-Aid for Scientific Research Performance Report, https: / / kaken.nii.ac.jp / ja / report / KAKENHI-PROJECT-23K27273 / 23K272732023jisseki, or https: / / kaken.nii.ac.jp / ja / grant / KAKENHI-PROJECT-23K27273 / [Overview of the project] [Problems that the invention aims to solve]

[0006] The present invention aims to provide a drug that exhibits a fundamental therapeutic effect against neurodegenerative diseases. Specifically, it aims to provide a novel drug that can remove protein aggregates that characterize neurodegenerative diseases, particularly insoluble protein aggregates that are considered to be the direct cause of the disease. [Means for solving the problem]

[0007] As described above, in previous research, the inventors discovered LPG (lysophosphatidylglucoside / LysoPtdGlc), a glucose lipid present in trace amounts in central nervous system tissue, and identified the G protein-coupled receptor GPR55 as its receptor. With the aim of clarifying the activation mechanism of GPR55 by LysoPtdGlc and elucidating its pathophysiological significance, research using a frontotemporal lobar degeneration model animal (PS19 mouse) revealed that glial activation mediated by the LysoPtdGlc-GPR55 signaling pathway is involved in brain inflammation and tau pathology. Specifically, it was demonstrated that administering an anti-LysoPtdGlc IgM antibody into the brains of PS19 mice, or knocking out GPR55 in PS19 mice, significantly suppressed abnormal tau phosphorylation. Furthermore, it was confirmed that a similar effect could be obtained with oral administration of auranofin, an existing rheumatoid arthritis treatment. Furthermore, in PS19 GPR55 knockout mice, in addition to suppression of abnormal tau phosphorylation, glial cell proliferation, inflammatory cytokine production, and neuronal cell death were also suppressed. Moreover, most notably, the accumulation of insoluble tau protein aggregates was significantly reduced in PS19 GPR55 knockout mice (an effect of removing insoluble tau protein aggregates). It was also confirmed that oral administration of auranofin to mice produced a similar effect of removing insoluble tau protein aggregates.

[0008] In other words, the present invention relates to a novel agent that can remove insoluble protein aggregates in the brain by blocking the LysoPtdGlc-GPR55 signaling axis. That is, the gist of the present invention is as follows.

[0009] [1] A brain-clearing agent for insoluble protein aggregates containing a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55. [2] The insoluble protein aggregate remover according to [1], wherein the insoluble protein is tau. [3] Substances that inhibit the binding of LPG and GPR55 i) Antisense nucleic acids or siRNA against genes involved in LPG biosynthesis or the GPR55 gene, ii) Antibodies specific to LPG or GPR55, iii) Chimeric proteolytic drugs (PROTACs) targeting GPR55, and iv) Low molecular weight compounds An insoluble protein aggregate remover according to [1] or [2], which is at least one selected from the group consisting of the following. [4] iv) The insoluble protein aggregate remover described in [3], wherein the low molecular weight compound is auranofin. A neurodegenerative disease treatment agent containing an insoluble protein aggregate remover as described in any of [5][1] to [4]. [6] The neurodegenerative disease described in [5] is a disease characterized by tau pathology in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration. [Effects of the Invention]

[0010] According to the present invention, by blocking the LysoPtdGlc-GPR55 signaling axis in the brain, it is possible to suppress not only brain inflammation but also abnormal phosphorylation of proteins such as tau and neuronal cell death, as well as reduce the accumulation of insoluble tau protein aggregates and remove insoluble tau protein aggregates in the brain. Thus, we have demonstrated that selectively blocking the LysoPtdGlc-GPR55 signaling axis can reduce the accumulation of insoluble tau protein aggregates in a frontotemporal lobar degeneration model animal (PS19 mouse). This strongly suggests that a drug that can selectively and potently block the LysoPtdGlc-GPR55 signaling axis can remove insoluble tau protein aggregates in the brain and has the potential to be an excellent fundamental treatment for neurodegenerative diseases such as Alzheimer's disease and frontotemporal lobar degeneration. [Brief explanation of the drawing]

[0011] [Figure 1-1] This figure shows the device and administration site for administering an anti-Lyso-PtdGlc function inhibitor antibody to the brain of a PS19 mouse. [Figure 1-2] This is an immunohistochemical image (phosphorylated tau) of PS19 mouse brains after administration of an anti-Lyso-PtdGlc function inhibitor antibody or a control antibody. [Figure 1-3] Figure 1-2 shows the quantified and graphed phosphorylated tau intensity in immunohistochemical images. [Figure 1-4-1] This figure shows the gene expression of various inflammatory cytokines (CCL2, CCL3, CCL5, CXCL10) in the hippocampus after continuous administration of an anti-Lyso-PtdGlc function inhibitor antibody to PS19 mice. [Figure 1-4-2] This figure shows the gene expression of various inflammatory cytokines (IL-1β, TNF-α, Lipocalin-2) in the hippocampus after continuous administration of an anti-Lyso-PtdGlc function inhibitor antibody to PS19 mice. [Figure 2-1]It is a figure showing the gene expression of various inflammatory cytokines (CCL2, CCL3, CCL5, CXCL10) in the hippocampus of wild-type mice (C57BL / 6J), PS19 mice, GPR55 knockout mice and double mutant mice (PS19 / GPR55- / -). [Figure 2-2] It is a figure showing the gene expression of various inflammatory cytokines (IL-1β, TNF-α, Lipocalin-2) in the hippocampus of wild-type mice (C57BL / 6J), PS19 mice, GPR55 knockout mice and double mutant mice (PS19 / GPR55- / -). [Figure 3-1] It is an immunostaining image of brains (AT8 (anti-phosphorylated tau antibody)) from PS19 mice administered auranofin (Auranofin(+)) and control PS19 mice (Auranofin(-)), along with a graph quantifying the results. [Figure 3-2] It is an immunostaining image of brains (AT8 (anti-phosphorylated tau antibody)) from PS19 mice administered auranofin until 9 months of age (Auranofin(+)) and control PS19 mice (Auranofin(-)). [Figure 3-3] It is an immunostaining image of brains (AT8 (anti-phosphorylated tau antibody)) from PS19 mice administered auranofin until 12 months of age (Auranofin(+)) and control PS19 mice (Auranofin(-)). [Figure 3-4] It is an immunostaining image of brains (anti-Neurofibrillary Tangles antibody) from PS19 mice administered auranofin (Auranofin(+)) and control PS19 mice (Auranofin(-)). [Figure 3-5-1] It is an immunostaining image of brains (anti-NeuN antibody) from PS19 mice administered auranofin (Auranofin(+)), control PS19 mice (Auranofin(-)) and wild-type mice. [Figure 3-5-2] It is a graph obtained by quantifying and plotting the analysis results of the immunostaining images in Figure 3-5-1. [Figure 3-6]These are immunostained images (stained with anti-Iba1 antibody) of the brains of PS19 mice administered auranofin (Auranofin(+)) and control PS19 mice (Auranofin(-)), and a graph obtained by quantifying the images. [Figure 3-7] These are immunostained images (stained with anti-GFAP antibody) of the brains of PS19 mice administered auranofin (Auranofin(+)) and control PS19 mice (Auranofin(-)), and a graph obtained by quantifying the images. [Figure 4-1-1] It is a figure showing the effect of GPR55 knockout on soluble tau accumulation in PS19 mice. [Figure 4-1-2] It is a graph obtained by quantifying and graphing the results of Figure 4-1-1. [Figure 4-1-3] It is a figure showing the reducing effect of GPR55 knockout on insoluble tau accumulation in PS19 mice. [Figure 4-1-4] It is a graph obtained by quantifying and graphing the results of Figure 4-1-3. [Figure 4-2-1] It is a figure showing the effect of auranofin administration on soluble tau accumulation in PS19 mice. [Figure 4-2-2] It is a graph obtained by quantifying and graphing the results of Figure 4-2-1. [Figure 4-2-3] It is a figure showing the reducing effect of auranofin administration on insoluble tau accumulation in PS19 mice. [Figure 4-2-4] It is a graph obtained by quantifying and graphing the results of Figure 4-2-3. DETAILED DESCRIPTION OF THE INVENTION

[0012] The present invention is described in detail below. Unless otherwise specified, molecular biological procedures can be carried out by methods described in general experimental manuals known to those skilled in the art or methods equivalent thereto. In addition, terms used in the present specification, unless otherwise specified, shall be construed to have meanings commonly used in the relevant technical field.

[0013] <Agent for removing insoluble protein aggregates in the brain> The present invention provides a brain-insoluble protein aggregate remover containing a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55. Hereinafter, the brain-insoluble protein aggregate remover of the present invention may simply be referred to as the insoluble protein aggregate remover, aggregate remover, or remover.

[0014] In neurodegenerative diseases such as Alzheimer's disease, aggregates composed of specific proteins are known to accumulate within brain cells, but the main proteins constituting these aggregates differ depending on the disease. Specifically, tau is known as the main constituent protein of disease-specific aggregates in Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD), alpha-synuclein in Parkinson's disease (PD) and Lewy body dementia (DLB), and TDP-43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Furthermore, it is thought that these protein aggregates are relatively stable within cells when they are soluble, and their impact on nerve cell function is limited. However, it is said that the formation of insoluble protein aggregates (fibrils and neurofibrillaries: NFTs) accelerates the progression of the disease.

[0015] In this invention, insoluble protein aggregates refer to substances formed when specific proteins appearing in the brain during neurodegenerative diseases bind to each other, forming soluble oligomers, which then aggregate and become fibrillated, resulting in insoluble proteins. As mentioned above, these specific proteins include tau in diseases where tau pathology is observed in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration; alpha-synuclein in Parkinson's disease (PD) and Lewy body dementia (DLB); and TDP-43 in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD).

[0016] Here, we will explain the aggregation process of tau protein in Alzheimer's disease as an example. Tau protein is a type of microtubule-associated protein (MAP) that plays a role in stabilizing microtubules in nerve cells. This maintains normal intracellular transport and synaptic function. However, in Alzheimer's disease, abnormalities occur in tau protein, leading to hyperphosphorylation. As a result, tau detaches from microtubules, begins abnormal aggregation, and forms soluble oligomers. It is believed that at this stage, synaptic function in nerve cells is severely impaired, and symptoms such as memory impairment begin to appear. These soluble oligomers further aggregate, undergo fibrillation, and accumulate, forming insoluble neurofibrillary twins (NFTs). When these insoluble neurofibrillaries accumulate inside cells, nerve cells are stressed, leading to apoptosis (programmed cell death). Neuronal cell death progresses in brain regions such as the hippocampus and cerebral cortex, causing atrophy of the entire brain. Furthermore, if abnormal tau is released extracellularly and spreads to surrounding nerve cells, tau pathology spreads throughout the brain, leading to a worsening of Alzheimer's disease.

[0017] As mentioned above, tau aggregation is closely related to the progression of Alzheimer's disease, and it is known that as insoluble neurofibrillary fibers (NFTs) begin to accumulate, the disease progresses from early symptoms such as memory impairment and decreased attention and judgment, to mid-stage symptoms such as speech disorders, visuospatial cognitive impairment, and personality changes accompanied by depression, to terminal symptoms such as decreased motor function, decreased level of consciousness, and loss of life-sustaining functions.

[0018] Based on the above, drugs that can remove insoluble protein aggregates (fibrils and neurofibrillary tangles: NFTs), such as the brain insoluble protein aggregate remover of the present invention, are expected to be a fundamental treatment for neurodegenerative diseases.

[0019] The brain insoluble protein aggregate remover of the present invention refers to a drug that, when administered to a patient with a neurodegenerative disease, removes some or all of the insoluble protein aggregates that characterize the disease, for example, within nerve cells or glial cells in the brain of the patient. The invention also includes drugs that, when administered to a patient with a neurodegenerative disease before the appearance of insoluble protein aggregates that characterize the disease, prevent the aggregation and formation of insoluble proteins by suppressing further aggregation of soluble oligomers or removing soluble oligomers.

[0020] Lysophosphatidylglucoside (LPG) is a metabolite of phosphatidiglucoside (PtdGlc) and is a potent extracellular bioactive lipid that regulates the construction of neural circuits. It is also written as LysoPtdGlc.

[0021] [ka]

[0022] GPR55 (G-protein coupled receptor 55) is a G protein-coupled receptor (GPCR) that responds specifically to LPG. GPR55 is a molecule cloned as a G protein-coupled receptor expressed in the brain (Molecular Brain Research, Volume 64, Issue 2, 5 February 1999, Pages 193-198). The inventors have found that insoluble protein aggregates in the brain can be removed by inhibiting the binding of LPG to GPR55. That is, a composition containing a substance that inhibits the binding of LPG to GPR55 functions as an insoluble protein aggregate remover in the brain and can be effectively used as a preventive and / or therapeutic agent for neurodegenerative diseases. GPR55 is known to exist in humans (EntrezGene:9290), mice (EntrezGene:227326), and rats (EntrezGene:501177), and the species of GPR55 in the present invention is preferably human or mouse, and more preferably human. The amino acid sequence of human GPR55 is shown as Sequence ID 1, the nucleic acid sequence as Sequence ID 2, the amino acid sequence of mouse GPR55 as Sequence ID 3, the nucleic acid sequence as Sequence ID 4, the amino acid sequence of rat GPR55 as Sequence ID 5, and the nucleic acid sequence as Sequence ID 6 in the sequence listing.

[0023] In the present invention, the substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55 is not particularly limited as long as it can inhibit some or all of the binding between LPG and GPR55, or can reduce the frequency of binding between LPG and GPR55, but examples include the following i) to iv).

[0024] i) Antisense nucleic acids or siRNA against genes involved in LPG biosynthesis or the GPR55 gene, ii) Antibodies specific to LPG or GPR55, iii) Chimeric proteolytic drugs (PROTACs) targeting GPR55, iv) Low molecular weight compounds Each of the points i) through iv) is explained in detail below.

[0025] i) Antisense nucleic acids or siRNA against genes involved in LPG biosynthesis or the GPR55 gene Antisense nucleic acids or siRNAs targeting genes involved in LPG biosynthesis can indirectly suppress LPG biosynthesis by inhibiting the gene expression of various enzymes involved in LPG biosynthesis. This, in turn, can reduce the binding frequency between LPG and GPR55. Similarly, antisense nucleic acids or siRNAs targeting the GPR55 gene can suppress GPR55 expression, thereby also reducing the binding frequency between LPG and GPR55.

[0026] Since LPG is a metabolite of phosphatidiglucoside (PtdGlc), the glycosyltransferase UGGT2 (UDP-glucose: glycoprotein glucosyltransferase 2), which is responsible for the biosynthesis of PtdGlc, can be said to be an enzyme involved in LPG biosynthesis. Therefore, antisense nucleic acids or siRNAs targeting the gene encoding UGGT2, which is a gene involved in LPG biosynthesis, are preferred examples.

[0027] The GPR55 gene has the nucleic acid sequences shown in SEQ ID NO: 2 (human), SEQ ID NO: 4 (mouse), and SEQ ID NO: 6 (rat).

[0028] The "antisense nucleic acid" in this invention is an antisense nucleic acid complementary to the enzyme involved in LPG biosynthesis or the transcript of the DNA encoding GPR55. There are several factors that contribute to the suppression of target gene expression by antisense nucleic acids, including: degradation by RNase activity due to RNA double-strand recognition; inhibition of transcription initiation by triple-strand formation; transcription repression by hybridization with sites where locally open loop structures are formed by RNA polymerase; transcription inhibition by hybridization with RNA that is being synthesized; splicing repression by hybridization at intron-exon junctions; splicing repression by hybridization with splicosome formation sites; suppression of translocation from the nucleus to the cytoplasm by hybridization with mRNA; translation repression by hybridization with translation initiation factor binding sites; inhibition of peptide chain elongation by hybridization with mRNA translation regions or polysome binding sites; and gene expression repression by hybridization with nucleic acid-protein interaction sites. Among these, the expression of target genes is suppressed by inhibiting the processes of transcription, splicing, or translation.

[0029] The antisense sequence used in this invention may suppress the expression of the target gene by any of the above-described mechanisms. In one embodiment, designing an antisense sequence complementary to the untranslated region near the 5' end of the mRNA of the enzyme involved in LPG biosynthesis or GPR55 is considered effective in inhibiting gene translation. However, sequences complementary to the coding region or the 3' untranslated region can also be used. Thus, sequences complementary to the untranslated region as well as the coding region of a gene can be used. In this way, nucleic acids containing antisense sequences of the untranslated region as well as the coding region of a gene are also included in the antisense nucleic acids used in this invention. The antisense nucleic acid preferably has 90% or more, most preferably 95% or more, complementarity with the transcript of the target gene. The length of the antisense RNA that effectively inhibits the expression of the target gene using the antisense sequence is not particularly limited.

[0030] In this invention, "siRNA" refers to double-stranded RNA consisting of short chains that do not exhibit toxicity in cells, and can be, for example, 15 to 49 base pairs, preferably 15 to 35 base pairs, and more preferably 21 to 30 base pairs. Alternatively, shRNA, which can obtain the same effect as siRNA, may be used. shRNA is RNA in which single-stranded RNA forms a double helix via a hairpin structure.

[0031] siRNA and shRNA do not need to be completely identical to the target gene, but they should have at least 70%, preferably 80%, more preferably 90%, and most preferably 95% or more sequence homology.

[0032] The double-stranded RNA regions in siRNA and shRNA that are paired together are not limited to perfectly paired regions; they may also contain unpaired regions due to mismatches (where the corresponding bases are not complementary) or bulges (where one strand lacks a corresponding base). In the present invention, both bulges and mismatches may be present in the double-stranded RNA region in dsRNA where RNAs are paired together.

[0033] ii) Antibodies specific to LPG or GPR55 The "antibodies specific to LPG or GPR55" in the present invention can be obtained as polyclonal or monoclonal antibodies using known means. The origin of the antibodies used in the present invention is not particularly limited, but they are preferably mammalian in origin, and more preferably human in origin. Mammalian monoclonal antibodies include those produced in hybridomas and those produced in hosts transformed with expression vectors containing antibody genes using genetic engineering techniques. There are also antibodies obtained by Kaiom's antibody production technology (ADLib® system), which utilizes the mechanism by which chicken B cell-derived DT40 cells produce various types of antibodies. These antibodies inhibit the binding of LPG to GPR55 by binding to LPG or GPR55.

[0034] Antibody-producing hybridomas can be produced using basically known techniques. Specifically, they can be produced by using LPG or GPR55 as a sensitizing antigen, immunizing with it according to a standard immunization method, fusing the resulting immune cells with known parent cells using a standard cell fusion method, and then screening for monoclonal antibody-producing cells using a standard screening method.

[0035] For example, an anti-GPR55 antibody can be produced as follows: The GPR55 used as the antigen for antibody acquisition can be obtained by using the GPR55 gene / amino acid sequence disclosed in known literature. After inserting the GPR55 gene sequence into a known expression vector system and transforming a suitable host cell, the target human GPR55 protein can be purified from the host cell or culture supernatant using a known method, and this purified human GPR55 protein can be used as the sensitization antigen. Alternatively, a human GPR55 protein produced by chemical synthesis can also be used as the sensitization antigen. Furthermore, a fusion protein of human GPR55 protein and another protein can also be used as the sensitization antigen.

[0036] While there are no particular limitations on the mammals that can be immunized with the sensitizing antigen, it is preferable to select them considering their compatibility with the parent cells used for cell fusion. Generally, rodents such as mice, rats, and hamsters are used.

[0037] Immunizing animals with sensitizing antigens is carried out according to known methods. For example, a common method involves injecting the sensitizing antigen into the peritoneal cavity or subcutaneously of mammals. Specifically, it is preferable to dilute and suspend the sensitizing antigen in an appropriate amount with PBS (Phosphate-Buffered Saline) or physiological saline, and, if desired, mix it with an appropriate amount of a standard adjuvant, such as Freund's complete adjuvant, emulsify it, and then administer it to mammals several times every 4 to 21 days. A suitable carrier can also be used during sensitizing antigen immunization.

[0038] After immunization and confirmation that the desired antibody levels in the serum have risen, immune cells are extracted from the mammal and subjected to cell fusion. Splenocytes are particularly preferred immune cells for cell fusion.

[0039] The mammalian myeloma cells used as the other parent cells to be fused with the aforementioned immune cells are various already known cell lines, such as P3X63Ag8.653 (J.Immunol.123,1548-1550(1979)), which can be used as appropriate.

[0040] The aforementioned cell fusion of immune cells and myeloma cells can be carried out basically by known methods, such as the method of Milstein et al. (Methods Enzymol., 73, 3-46 (1981)).

[0041] The hybridomas obtained by cell fusion are selected by culturing them in a standard selective culture medium, such as HAT culture medium (a culture medium containing hypoxanthine, aminopterin, and thymidine). Culturing in the HAT culture medium is continued for a sufficient amount of time, usually several days to several weeks, to eliminate cells other than the target hybridoma (non-fused cells). Then, standard limiting dilution methods are used to screen and clone hybridomas that produce the target antibody.

[0042] The hybridomas that produce monoclonal antibodies in this manner can be subcultured in a normal culture medium and can also be stored for long periods in liquid nitrogen.

[0043] Methods for obtaining monoclonal antibodies from the hybridoma include culturing the hybridoma according to standard procedures and obtaining the antibody as the culture supernatant, or administering the hybridoma to a compatible mammal to allow it to grow and obtaining the antibody as ascites fluid. The former method is suitable for obtaining high-purity antibodies, while the latter method is suitable for mass production of antibodies.

[0044] In the present invention, recombinant antibodies can be used as monoclonal antibodies, which are produced by cloning an antibody gene from a hybridoma, incorporating it into a suitable vector, introducing it into a host, and using genetic recombination technology (see, for example, Borrebaeck CAK and Larrick JW THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0045] In this invention, genetically modified recombinant antibodies, such as chimeric antibodies, humanized antibodies, and human antibodies, which are artificially modified for purposes such as reducing heterologous antigenicity against humans, can be used. These modified antibodies can be manufactured using known methods.

[0046] The antibodies of the present invention include not only bivalent antibodies represented by IgG, but also monovalent antibodies or polyvalent antibodies represented by IgM, as long as they bind to LPG or GPR55. The polyvalent antibodies of the present invention include polyvalent antibodies having all the same antigen-binding sites, or polyvalent antibodies having some or all different antigen-binding sites.

[0047] Furthermore, the antibody of the present invention may be a bispecific antibody, as long as it binds to LPG or GPR55. A bispecific antibody is an antibody that has variable regions that recognize different epitopes within the same antibody molecule, but these epitopes may be located in different molecules or in the same molecule. In other words, in the present invention, a bispecific antibody may have antigen-binding sites that recognize different epitopes of LPG or GPR55. It is also possible to have a bispecific antibody in which one recognition site recognizes LPG or GPR55 and the other recognition site recognizes an antigen other than LPG or GPR55.

[0048] Methods for producing bispecific antibodies are well known. For example, bispecific antibodies can be produced by conjugating two antibodies that recognize different antigens. The antibodies to be conjugated may be half a molecule of an antibody that each has a heavy chain and a light chain, or a quarter of a molecule of an antibody that consists only of a heavy chain. Alternatively, bispecific antibody-producing fusion cells can be created by fusing hybridomas that produce different monoclonal antibodies. Furthermore, bispecific antibodies can be produced using genetic engineering techniques.

[0049] The antibody of the present invention may be a low-molecular-weight antibody, as long as it binds to LPG or GPR55. A low-molecular-weight antibody includes an antibody fragment in which a portion of a full-length antibody (whole antibody, e.g., whole IgG) is missing. Partial deletion of the antibody molecule is acceptable as long as it binds to LPG or GPR55. The antibody fragment in the present invention preferably contains either or both of the heavy chain variable region (VH) and the light chain variable region (VL). The amino acid sequence of VH or VL may include additions, deletions, and / or substitutions. Furthermore, as long as it binds to LPG or GPR55, a portion of either or both of VH and VL may be deleted. The antibody fragment may also be chimeric or humanized. Specific examples of antibody fragments include, for example, Fab, Fab', F(ab')2, Fv, etc. Furthermore, specific examples of low-molecular-weight antibodies include, for example, Fab, Fab', F(ab')2, Fv, scFv (single-chain Fv), Diabody, and sc(Fv)2 (single-chain (Fv)2). Multimers of these antibodies (e.g., dimers, trimers, tetramers, polymers) are also included in the antibodies of the present invention.

[0050] The following anti-LPG antibodies can be specifically listed as preferred antibodies in the present invention. The following antibodies are anti-LPG antibodies that the inventors have produced for uses other than the present invention, as antibodies having the activity to suppress the repulsive effect of LPG on axon extension of nerve cells expressing TrkA, as described in Patent 5920726.

[0051] This antibody binds to LPG and has the activity to suppress the repulsive effect of LPG on axonal extension of nerve cells expressing TrkA. It possesses a light chain variable region containing the amino acid sequences described in SEQ ID NOs. 7 to 9 as CDR1 to 3, respectively, and a heavy chain variable region containing the amino acid sequences described in SEQ ID NOs. 10 to 12 as CDR1 to 3, respectively.

[0052] An antibody that retains a light chain variable region containing the amino acid sequence described in SEQ ID NO: 13 and a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 14.

[0053] This antibody binds to LPG and has the activity to suppress the repulsive effect of LPG on axonal extension of nerve cells expressing TrkA, and possesses a light chain variable region containing the amino acid sequences described in SEQ ID NOs. 15 to 17 as CDR1 to 3, respectively, and a heavy chain variable region containing the amino acid sequences described in SEQ ID NOs. 18 to 20 as CDR1 to 3, respectively.

[0054] An antibody that retains a light chain variable region containing the amino acid sequence described in SEQ ID NO: 21 and a heavy chain variable region containing the amino acid sequence described in SEQ ID NO: 22.

[0055] iii) Chimeric proteolytic drugs (PROTACs) that target GPR55 Proteolysis Targeting Chimeras (PROTACs) are a novel therapeutic strategy aimed at selectively degrading target proteins. PROTACs consist of bifunctional molecules that simultaneously bind to both the target protein and an E3 ubiquitin ligase. This binding leads to ubiquitination of the target protein, followed by degradation via the proteasome pathway.

[0056] In the present invention, a chimeric proteolytic drug (PROTAC) targeting GPR55 is composed of three elements: a GPR55-binding motif (a ligand that specifically binds to GPR55), an E3 ubiquitin ligase-binding motif (a ligand that binds to E3 ubiquitin ligase), and a linker (a spacer that links the two ligands). This brings GPR55 and E3 ubiquitin ligase into close proximity, inducing ubiquitination and subsequent degradation by the proteasome. This process is repeated within cells, and since the PROTAC molecule is reusable, effective degradation can be induced with small amounts.

[0057] Ligands that specifically bind to GPR55 are not particularly limited as long as they are substances that specifically bind to GPR55, but can be found by screening from, for example, single substances such as natural compounds, organic compounds, inorganic compounds, nucleic acids, proteins (including antibodies), and peptides; or from compound libraries, nucleic acid libraries, peptide libraries, gene library expression products, etc.

[0058] iv) Low molecular weight compounds In the present invention, the "low molecular weight compound" is a relatively small molecular weight compound that can partially or completely inhibit the binding of LPG and GPR55, and that exhibits the effect of removing insoluble protein aggregates in the brain. It may be derived from a natural product or a chemically synthesized product. From a library of low molecular weight compounds, compounds that can partially or completely inhibit the binding of LPG and GPR55 can be found using screening methods known to those skilled in the art. Furthermore, the degree of the effect of removing insoluble protein aggregates in the brain can be confirmed for the low molecular weight compounds found by the above screening methods, and a low molecular weight compound effective as an insoluble protein aggregate remover in the brain can be selected. A preferred example of a low molecular weight compound that inhibits the binding of LPG and GPR55 and has the effect of removing insoluble protein aggregates in the brain is auranofin.

[0059] Auranofin Auranofin (CAS number 34031-32-8) is an organogold compound with the following structure and is known as an anti-rheumatic drug.

[0060] [ka]

[0061] Auranofin exerts its effect of removing insoluble protein aggregates in the brain by inhibiting the binding of LPG to GPR55. Furthermore, because auranofin can suppress the accumulation of insoluble protein aggregates in the brain, it also has a preventive effect against Alzheimer's disease. As shown in the present invention's examples, oral administration of auranofin to Alzheimer's model mice can almost completely remove insoluble tau protein aggregates in the brain.

[0062] (Other ingredients) The brain-insoluble protein aggregate removal agent of the present invention may contain formulation-acceptable materials such as preservatives and stabilizers. Formulation-acceptable means that the material itself may have the above-mentioned brain-insoluble protein aggregate removal effect, or it may not have such effect, and it means a formulation-acceptable material that can be administered together with the removal agent. Furthermore, the material may not have the above effect, but may have a synergistic or additive stabilizing effect when used in combination with a substance that inhibits the binding of LPG and GPR55 as described above.

[0063] Examples of materials that are acceptable in formulations include sterile water, physiological saline, preservatives, stabilizers, excipients, buffers, antiseptics, surfactants, chelating agents (such as EDTA), and binders.

[0064] When the removal agent in the present invention is an aqueous solution for injection, it may be used in combination with, for example, physiological saline, an isotonic solution containing glucose or other adjuvants (e.g., D-sorbitol, D-mannose, D-mannitol, sodium chloride), a suitable solubilizer, for example, alcohol (ethanol, etc.), polyalcohol (propylene glycol, PEG, etc.), a nonionic surfactant (polysorbate 80, HCO-50), etc. Optionally, it may further contain diluents, solubilizers, pH adjusters, analgesics, sulfur-containing reducing agents, antioxidants, etc.

[0065] (Formulation form, etc.) If necessary, the scavenging agent in the present invention may be encapsulated in microcapsules (such as hydroxymethylcellulose, gelatin, or poly[methylmethacrylate] microcapsules) or in a colloidal drug delivery system (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules). Furthermore, methods for making the scavenging agent in the present invention a sustained-release agent are also known and applicable to the present invention (Langer et al., J.Biomed.Mater.Res. 1981, 15: 167-277; Langer, Chem. Tech. 1982, 12: 98-105; U.S. Patent No. 3,773,919; European Patent Application Publication (EP) No. 58,481; Sidman et al., Biopolymers 1983, 22: 547-556; EP No. 133,988).

[0066] The brain-insoluble protein aggregate removal agent of the present invention can be administered in pharmaceutical form and can be administered orally or parenterally, systemically or locally. For example, intravenous injection such as infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, oral preparation, suppositories, enema, oral enteric-coated preparation, etc., can be selected, and the method of administration can be appropriately selected depending on the patient's age and symptoms. The effective dose is selected in the range of 0.001 mg to 100 mg per kg of body weight per dose, as the amount of the substance that inhibits the binding of LPG and GPR55, such as auranofin. Alternatively, a dose of 0.1 to 1000 mg, preferably 0.1 to 50 mg, can be selected per patient. The preferred dosage and method of administration is, for example, 0.1 mg to 40 mg per kg of body weight per month (4 weeks), preferably 1 mg to 20 mg, divided into one to several doses, administered on a schedule such as daily, every other day, twice a week, once a week, once every two weeks, or once every four weeks, by oral administration, intravenous injection such as infusion, or subcutaneous injection. To obtain excellent effects, it may be administered daily for a certain period, for example, continuously for about 1 week, 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, or 100 days. This continuous administration schedule can be adjusted by extending the administration interval while observing the patient's condition after administration and the trends in blood test values.

[0067] The present invention's agent for removing insoluble protein aggregates in the brain is suitably used for the treatment of neurodegenerative diseases such as Alzheimer's disease. Of these, from the viewpoint of therapeutic effect, it is more suitably used for diseases in which tau pathology is observed in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration, and is even more suitably used for Alzheimer's disease and frontotemporal lobar degeneration.

[0068] <Therapeutic agents for neurodegenerative diseases> The present invention also includes a neurodegenerative disease treatment agent containing the above-described insoluble protein aggregate remover. According to the neurodegenerative disease treatment agent of the present invention, by blocking the LysoPtdGlc-GPR55 signaling axis in the brain, it is possible to suppress not only brain inflammation but also abnormal phosphorylation of proteins such as tau and neuronal cell death, further reducing the accumulation of insoluble tau protein aggregates and removing insoluble tau protein aggregates in the brain. Therefore, it is expected to be an excellent fundamental treatment agent for neurodegenerative diseases in which tau pathology is observed in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration. The neurodegenerative disease treatment agent of the present invention contains the above-described insoluble protein aggregate remover, and is essentially a specification of using an insoluble protein aggregate remover for the treatment of neurodegenerative diseases; therefore, the description in the section on insoluble protein aggregate removers can be applied directly for specific explanations.

[0069] <Method for removing insoluble protein aggregates in the brain> The present invention also includes a method for removing insoluble protein aggregates in the brain, comprising the step of administering to a subject a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55.

[0070] In the method of the present invention, the insoluble protein is preferably tau. Furthermore, the substance that inhibits the binding of LPG and GPR55 is i) Antisense nucleic acids or siRNA against genes involved in LPG biosynthesis or the GPR55 gene, ii) Antibodies specific to LPG or GPR55, iii) Chimeric proteolytic drugs (PROTACs) targeting GPR55, and iv) Low molecular weight compounds It is preferable that it be at least one selected from the group consisting of the following:

[0071] The present invention relates to a method for removing insoluble protein aggregates in the brain by administering a brain insoluble protein aggregate removal agent containing a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55. Therefore, a specific explanation can be applied to the section on brain insoluble protein aggregate removal agents.

[0072] <Treatment methods for neurodegenerative diseases> The present invention also includes a method for treating neurodegenerative diseases, comprising the step of administering to a subject a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55.

[0073] In the method of the present invention, the neurodegenerative disease is preferably a disease in which tau pathology is observed in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration.

[0074] The present invention's method for treating neurodegenerative diseases includes the same steps as the above-described method for removing insoluble protein aggregates, and since it is an invention of a method for treating neurodegenerative diseases by removing insoluble protein aggregates, the explanation in the section on the method for removing insoluble protein aggregates in the brain, and furthermore, the explanation in the section on the agent for removing insoluble protein aggregates in the brain, can be applied to the specific explanation. [Examples]

[0075] The present invention will be specifically described in the following embodiments, but the present invention is not intended to be limited to these embodiments.

[0076] The materials and methods used in this embodiment are as follows: <Experimental animals> P301S mutant human tau gene-transfer mice (PS19 mice) were purchased from Jackson Laboratory, and GPR55 knockout mice were obtained from EMMA. Both were backcrossed to C57BL / 6J. Male PS19 mice were crossed with female GPR55 knockout mice to create double mutant mice (PS19 / GPR55- / -). Some PS19 mice were also fed CRF-1 (a basal diet based on the Charles River formula) supplemented with 0.01% auranofin from weaning until 5, 6, 9, and 12 months of age. All mice used in this experiment were male.

[0077] <Section preparation> Mice aged 5, 9, and 12 months were deeply anesthetized with isoflurane and perfused and fixed with 25 ml PBS and 4% paraformaldehyde. The brains were then harvested and post-fixed overnight in 4% paraformaldehyde at 4°C. After replacing with 10% sucrose / PBS for half a day at 4°C and then 20% sucrose / PBS overnight, the brains were embedded in OCT compound (Sakura FineTech Japan) and 10 μm thick frozen sections were prepared.

[0078] <Immunostaining> The prepared sections were thoroughly air-dried and replaced with high salt buffer (500 mM NaCl, 12.5 mM Na2HPO4, 9.2 mM NaH2PO4), and incubated in blocking solution (10% normal horse serum / 0.3% Triton X-100 / high salt buffer or 10% normal goat serum / 0.3% Triton X-100 / high salt buffer) for 1 hour at room temperature. Subsequently, the primary antibody diluted in the blocking solution was incubated overnight at 4°C. The primary antibody and dilution ratios used are as follows.

[0079] AT8 (absolute antibody) 1 / 500, RTM49 (Fujifilm Wako Pure Chemical Industries) 1 / 250, anti-Neurofibrillary Tangles antibody (Millipore) 1 / 500, anti-NeuN antibody (Cell Signaling Technology) 1 / 500, anti-Iba1 antibody (Fujifilm Wako Pure Chemical Industries) 1 / 500, anti-GFAP antibody (abcam) 1 / 500.

[0080] After incubation with the primary antibody described above, the secondary antibody was washed five times for 10 minutes with high salt buffer, then diluted with blocking solution and incubated at room temperature for 2 hours. The secondary antibodies used are as follows, all at a dilution ratio of 1 / 1000.

[0081] Alexa fluor 488 goat anti rabbit IgG (invitrogen), Alexa fluor 546 goat anti rat IgG (invitrogen), Alexa fluor 594 goat anti rabbit IgG (invitrogen), Alexa fluor 488 donkey anti rabbit IgG (invitrogen), Alexa fluor 594 donkey anti goat IgG (invitrogen), DAPI(sigma).

[0082] After incubation with the secondary antibody described above, the samples were washed five times in high salt buffer for 10 minutes, and then mounted on coverslips. Observation was performed using a confocal microscope (Leica TCS SP8 or Olympus FV3000), and images were taken with 2x, 4x, 20x, and 40x objective lenses.

[0083] <Methods for analyzing immunohistochemical staining> Brightness values ​​for AT8 in the RTM49-positive area and Iba1 and GFAP in the hippocampal area were calculated, and graphs were created using relative values ​​with control IgM or auranofin(-) set to 1. The thickness of the pyramidal cell layer in the hippocampal CA1 region was measured at three locations, and the average value was calculated. Additionally, pyramidal cells in the hippocampal CA3 region within the captured images were counted and graphs were created.

[0084] <Data Analysis> All graph creation and statistical analysis were performed using GraphPad Software Prism. Data are shown as mean ± SEM. Statistical significance was assessed as needed using the Mann-Whitney U test, Student's t-test, one-way ANOVA, and Tukey's multiple comparison post-hoc test. Statistical significance was considered to be P<0.05, and is indicated as *P<0.05, **P<0.01, and ***P<0.001.

[0085] The specific experiment will be described below.

[0086] 1. Continuous administration of anti-LysoPtdGlc function inhibitory antibody to PS19 mice. Three-month-old PS19 mice were induced with inhalation anesthesia (1.5% isoflurane (Viatris Pharmaceuticals)), and an iPRECIO pump (PrimeTec) connected to a 30G diameter ICV guide cannula was implanted dorsally. The cannula was fixed to the skull with dental cement and cyanoacrylate adhesive, and the tip of the cannula was positioned at a stereotactic coordinate of "0.5 mm posterior, 1.1 mm lateral, and 2.5 mm deep from the bregma" (Figure 1-1). Anti-Lyso-PtdGlc function inhibitory antibody (0.5 mg / ml) or control IgM antibody (0.5 mg / ml) was injected into the iPRECIO pump and continuously administered at a flow rate of 0.5 μl / h for 40 days.

[0087] 1-1.Immunostaining Frozen sections of mouse brain were prepared using the methods described above under <Section Preparation> and <Immunostaining>, and stained with AT8, an antibody against phosphorylated tau. The results of the immunostaining are shown in Figure 1-2, and the analysis results are shown in Figure 1-3.

[0088] As shown in Figures 1-2 and 1-3, continuous administration of an anti-Lyso-PtdGlc function inhibitor antibody to PS19 mice resulted in a significant decrease in phosphorylated tau in the brain. Since blocking the LPG-GPR55 signaling pathway by administration of the anti-Lyso-PtdGlc function inhibitor antibody reduced phosphorylated tau in the brain, it was revealed that the LPG-GPR55 signaling pathway is involved in the increase of phosphorylated tau.

[0089] 1-2. Digital PCR Mice were deeply anesthetized with isoflurane, and their hippocampi were collected and stored in RNALater RNA Stabilization Solution (QIAGEN). Total RNA was extracted from the hippocampi stabilized in RNALater RNA Stabilization Solution using the RNeasy mini kit (QIAGEN). The extraction method followed the protocol of the RNeasy mini kit.

[0090] Reverse transcription was performed by reacting 500 ng of total RNA purified from the hippocampus using SuperScript IV VILO Master mix (Thermo Fisher Scientific). The reaction conditions followed the protocol for SuperScript IV VILO Master mix. For digital PCR, the reaction mixture was prepared using cDNA reaction mixture reverse-transcribed from hippocampal RNA, QIAcuity Probe Kit (QIAGEN), and Taqman Gene Expression Assay (Thermo Fisher) with duplicates of each sample. Details of the Taqman Gene Expression Assay are as follows.

[0091] ccl2 Mm00441242_m1, ccl3 Mm00441259_g1, ccl5 Mm01302427_m1, cxcl10 Mm00445235_m1, IL-1b Mm00434228_m1, TNF Mm00443258_m1, LCN2 Mm01324470_m1

[0092] The reaction and analysis were performed using the QIAcuity Digital PCR System (QIAGEN QIAcuity One, QIAcuity Nanoplate 8.5k 24-well, and QIAcuity Software Suite). The reaction conditions were 95°C for 2 min, 40 cycles (95°C for 15 sec, 60°C for 30 sec). The results are shown in Figures 1-4-1 (CCL2, CCL3, CCL5, CXCL10) and 1-4-2 (IL-1β, TNF-α, Lipocalin-2).

[0093] As shown in Figures 1-4-1 and 1-4-2, continuous administration of an anti-Lyso-PtdGlc inhibitory antibody to PS19 mice significantly reduced the mRNA gene expression of various inflammatory cytokines in the hippocampus. This result suggests that the LPG-GPR55 signaling pathway is involved in brain inflammation.

[0094] 2. Analysis of double mutant mice (PS19 / GPR55- / -) Using the method described in the "Experimental Animals" section above, GPR55 knockout mice and double mutant mice (PS19 / GPR55- / -) were generated.

[0095] Digital PCR Using the same method as described in 1-2 above, total RNA was extracted from the hippocampus of each mouse, and the gene expression of various inflammatory cytokines was examined by PCR. The results are shown in Figures 2-1 (CCL2, CCL3, CCL5, CXCL10) and 2-2 (IL-1β, TNF-α, Lipocalin-2).

[0096] As shown in Figures 2-1 and 2-2, gene expression of various inflammatory cytokines was enhanced in the hippocampus of PS19 mice compared to wild-type mice. On the other hand, in GPR55 knockout mice, expression of all inflammatory cytokines was almost absent, similar to the wild-type mice. Furthermore, in double mutant mice (PS19 / GPR55- / -), the expression of all inflammatory cytokines was significantly reduced compared to PS19 mice. These results indicate that the LPG-GPR55 signaling pathway is involved in brain inflammation.

[0097] 3. Effects of auranofin administration on PS19 mice 3-1. Tau pathology and neuronal cell death PS19 mice were fed CRF-1 (a basal diet based on the Charles River formula) supplemented with 0.01% auranofin from weaning until 5, 9, or 12 months of age. Mice fed the above diet until 5, 9, or 12 months of age were used for immunostaining with AT8 (anti-phosphorylated tau antibody), mice fed the above diet until 9 months of age were used for immunostaining with anti-Neurofibrillary Tangles antibody, and mice fed the above diet until 12 months of age were used for immunostaining with anti-NeuN antibody.

[0098] Frozen brain sections were prepared from auranofin-treated mice (Auranofin(+)), control mice (Auranofin(-)), and wild-type mice using the methods described in the sections on section preparation and immunostaining above. Immunostaining was then performed using AT8 (antiphosphorylated tau antibody), anti-neurofibrillary Tangles antibody, and anti-NeuN antibody. The results are shown in Figures 3-1 to 3-4, 3-5-1, and 3-5-2.

[0099] As shown in Figures 3-1 to 3-4, auranofin administration significantly reduced tauopathy (phosphorylated tau) and neurofibrillary tangles in the brains of PS19 mice. Furthermore, as shown in Figures 3-5-1 and 3-5-2, auranofin administration reduced neuronal cell death in the brains of PS19 mice.

[0100] 3-2. Neuroinflammation PS19 mice were fed CRF-1 (a basal diet based on the Charles River formula) with 0.01% auranofin added internally from weaning until 9 months of age.

[0101] Frozen brain sections were prepared from mice given auranofin (Auranofin(+)) and control mice (Auranofin(-)) using the methods described in the sections on section preparation and immunohistochemistry above. Immunostaining was then performed using anti-Iba1 antibody (microglia marker) and anti-GFAP antibody (astrocytocyte marker). The results are shown in Figures 3-6 (Iba1) and 3-7 (GFAP).

[0102] As shown in Figures 3-6 and 3-7, auranofin administration significantly reduced the accumulation of microglia and astroglia in the brains of PS19 mice. This result indicates that neuroinflammation in the brains of PS19 mice was improved by auranofin administration.

[0103] 4. Analysis of the effect on insoluble tau accumulation 4-1. Reduction of insoluble tau accumulation by knockout of GPR55 in PS19 mice. Six-month-old PS19 mice and double mutant mice (PS19 / GPR55- / -) were deeply anesthetized with isoflurane, and their hippocampi were collected. The hippocampi were homogenized with 500 μl of TBS (50 mM Tris-HCl, 274 mM NaCl, 5 mM KCl), and then centrifuged at 25,000 rpm for 20 minutes at 4°C. The supernatant was collected (soluble fraction), and the precipitate was homogenized with 500 μl of high salt & sucrose buffer (10 mM Tris-HCl, 0.8 M NaCl, 10% sucrose, 1 mM EGTA), and then centrifuged at 25,000 rpm for 20 minutes at 4°C. The supernatant was collected, 50 μl of sarcosyl (Fujifilm Wako Pure Chemical Industries) was added, and the mixture was incubated at 37°C for 1 hour, followed by centrifuging at 59,000 rpm for 1 hour at 4°C. The supernatant was discarded, and 100 μl of TE was added to the precipitate and used as the insoluble fraction.

[0104] Insoluble fraction (0.1 μg / well) was added to Extra PAGE One Precast Gel 5-10% (Nacalai Tesque), and soluble fraction (5 μg / well) was added to Extra PAGE One Precast Gel 5-20% (Nacalai Tesque). Electrophoresis was performed at 30 mA, 300 V, 60 minutes, and the samples were transferred to a PVDF membrane (bio-rad) at 1.3 A, 25 V, 7 minutes. Blocking was performed at room temperature for 1 hour with 5% skim milk / TBST, and the primary antibody diluted with 1% skim milk / TBST was incubated overnight at 4°C. The primary antibodies used are as follows, all at a dilution ratio of 1 / 5000.

[0105] AT8 (absolute antibody), AT270 (Thermo Fisher Scientific), tau5 (Thermo Fisher Scientific), HT7 (Thermo Fisher Scientific), RTM47 (Fujifilm Wako Pure Chemical Industries), anti-β-actin antibody (SIGMA). The binding affinity of various tau antibodies to mouse tau, human tau, unphosphorylated tau, and phosphorylated tau is shown in the table below. In the table, "+++" indicates strong binding affinity, "+" indicates slight binding affinity, and "-" indicates no binding affinity.

[0106] [Table 1]

[0107] After incubation with the primary antibody described above, the samples were washed five times with TBST for 10 minutes, and then the secondary antibody, diluted with 1% skim milk / TBST, was incubated at room temperature for 1 hour. The secondary antibodies used are as follows, all at a dilution ratio of 1 / 10000.

[0108] Sheep anti mouse IgG-HRP (cytiva), donkey anti rabbit IgG-HRP(cytiva), goat anti rat IgG-HRP(abcam)

[0109] After incubation with the secondary antibody described above, the samples were washed five times for 10 minutes with TBST, and then luminescence was achieved using Immunostar zeta (Fujifilm Wako Pure Chemical Industries) or Immunostar LD (Fujifilm Wako Pure Chemical Industries). Bands were then detected using ImageQuant LAS4000 (GE Healthcare) (Figures 4-1-1 and 4-1-3). After calculating the volume value of each band using ImageQuant TL, the values ​​were corrected with β-actin, and graphs were created using relative values ​​with PS19 set to 1 (Figures 4-1-2 and 4-1-4).

[0110] As shown in Figures 4-1-1 to 4-1-4, knocking out GPR55 in PS19 mice significantly reduced the accumulation of insoluble tau in the hippocampus, making it almost undetectable. On the other hand, while soluble phosphorylated tau decreased, the decrease in soluble tau itself was slight.

[0111] 4-2. Reduction of insoluble tau accumulation in PS19 mice by auranofin administration. PS19 mice were fed CRF-1 (a basal diet based on the Charles River formula) with 0.01% auranofin added internally from weaning until 6 months of age. Soluble and insoluble fractions were prepared from the hippocampus of mice given auranofin (Auranofin(+)) and control mice (Auranofin(-)) in the same manner as in 4-1 above, and analyzed by Western blotting using the various antibodies listed in Table 1 (Figures 4-2-1 to 4-2-4).

[0112] As shown in Figures 4-2-1 to 4-2-4, auranofin administration significantly reduced the accumulation of insoluble tau in the hippocampus, bringing it to an almost undetectable level. On the other hand, while auranofin administration reduced soluble phosphorylated tau, the reduction in soluble tau itself was slight. [Industrial applicability]

[0113] According to the present invention, by blocking the LysoPtdGlc-GPR55 signaling axis in the brain, it is possible to suppress not only brain inflammation but also abnormal phosphorylation of proteins such as tau and neuronal cell death, as well as reduce the accumulation of insoluble tau protein aggregates and remove insoluble tau protein aggregates in the brain. Thus, we have demonstrated that selectively blocking the LysoPtdGlc-GPR55 signaling axis can reduce the accumulation of insoluble tau protein aggregates in a frontotemporal lobar degeneration model animal (PS19 mouse). This strongly suggests that a drug that can selectively and potently block the LysoPtdGlc-GPR55 signaling axis can remove insoluble tau protein aggregates in the brain and has the potential to be an excellent fundamental treatment for neurodegenerative diseases such as Alzheimer's disease and frontotemporal lobar degeneration.

Claims

1. A brain-clearing agent for removing insoluble protein aggregates, containing a substance that inhibits the binding of lysophosphatidylglucoside (LPG) to GPR55.

2. The insoluble protein aggregate remover according to claim 1, wherein the above-mentioned insoluble protein is tau.

3. Substances that inhibit the binding of LPG and GPR55 are i) Antisense nucleic acids or siRNA against genes involved in LPG biosynthesis or the GPR55 gene, ii) Antibodies specific to LPG or GPR55, iii) Chimeric proteolytic drugs (PROTACs) targeting GPR55, and iv) Low molecular weight compounds An insoluble protein aggregate remover according to claim 1 or 2, which is at least one selected from the group consisting of the following.

4. iv) The insoluble protein aggregate remover according to claim 3, wherein the low molecular weight compound is auranofin.

5. A neurodegenerative disease treatment agent containing an insoluble protein aggregate remover according to any one of claims 1 to 4.

6. The neurodegenerative disease treatment agent according to claim 5, wherein the neurodegenerative disease is a disease characterized by tau pathology in the brain, selected from the group consisting of Alzheimer's disease, frontotemporal lobar degeneration, progressive supranuclear palsy, and corticobasal degeneration.

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