Anti-inflammatory agents for the central nervous system, therapeutic agents for central nervous system diseases accompanied by neuroinflammation, and screening methods for therapeutic agents for central nervous system diseases accompanied by neuroinflammation.

Abstract

To provide anti-inflammatory agents for the central nervous system, therapeutic agents for central nervous system diseases accompanied by neuroinflammation, and screening methods for therapeutic agents for central nervous system diseases accompanied by neuroinflammation. [Solution] An anti-inflammatory agent for the central nervous system or a therapeutic agent for central nervous system diseases accompanied by neuroinflammation, comprising as an active ingredient a compound that inhibits the function of ceramide kinase, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein.

Description

[Technical Field]

[0001] The present invention relates to an anti-inflammatory agent for the central nervous system, a therapeutic agent for central nervous system diseases accompanied by neuroinflammation, and a screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation. [Background technology]

[0002] Neuroinflammation is caused by the activation of glial cells in the brain, such as microglia and astrocytes, and chronic neuroinflammation is known to contribute to the pathogenesis of various central nervous system diseases. It is hoped that suppressing glial cell activation will lead to the treatment of central nervous system diseases. However, the mechanisms regulating glial cell activity are complex, and disease treatments targeting glial cells have not yet been established. Non-patent document 1 describes a clinical trial evaluating the efficacy and safety of NP001 in patients with evidence of amyotrophic lateral sclerosis (ALS) and systemic inflammation. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Miller, R. et al. (2018). Neurology, 90, 15_supplement.S38.004. [Overview of the project] [Problems that the invention aims to solve]

[0004] Traditionally, attempts have been made to develop methods to suppress the activation of glial cells. For example, drug development targeting CD14 and CD169, which are involved in microglia activation, has progressed. However, these have not yet reached clinical application. Furthermore, NP001 was developed as a treatment for amyotrophic lateral sclerosis (ALS) targeting microglia. NP001 is a biomolecule that downregulates NF-κB within microglia, suppressing the production of inflammatory cytokines. While NP001 improved the symptoms of ALS in mice, it worsened the disease in human clinical trials. Thus, drugs targeting microglia have not yet reached clinical application. Because microglia have the ability to phagocytose unwanted substances in the brain, lowering their activity below their steady-state level may lead to side effects. Furthermore, neuroinflammation can lead to abnormal activation of microglia and astrocytes through their interaction and mutual activation.

[0005] The present invention aims to provide an anti-inflammatory agent for the central nervous system, a therapeutic agent for central nervous system diseases accompanied by neuroinflammation, and a screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation. [Means for solving the problem]

[0006] The inventors of this invention have discovered that inhibiting the function of ceramide kinase can suppress neuroinflammation and improve the pathological symptoms of central nervous system diseases, and have completed this invention.

[0007] The present invention encompasses the following embodiments [1] to [6]. [1] An anti-inflammatory agent for the central nervous system comprising as an active ingredient a compound that inhibits the function of ceramide kinase, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein. [2] A therapeutic agent for central nervous system disorders accompanied by neuroinflammation, comprising as an active ingredient a compound that inhibits the function of ceramide kinase, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein. [3] The agent according to [2], wherein the central nervous system disorder accompanied by neuroinflammation is Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, or depression. [4] The agent according to any one of [1] to [3], wherein the compound that inhibits the function of ceramide kinase is an siRNA, shRNA, dsRNA, miRNA, or antisense oligonucleotide (ASO) that targets the ceramide kinase gene. [5] Compounds that inhibit the function of ceramide kinase, NVP-231(N-[2-(Benzoylamino)-6-benzothiazolyl]tricyclo[3.3.1.1 3,7 ]decane-1-carboxamide) or Ceramide Kinase Inhibitor,K1((6aR,12aR,12bS)-10-Hydroxy-4,4,6a,12b-tetramethyl-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-2H-benzo[a]xanthene-8,11-dione) The agent described in any one of [1] to [3]. [6] A screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation, comprising the step of searching for a compound that inhibits the function of ceramide kinase and suppresses inflammation of the central nervous system, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein. [Effects of the Invention]

[0008] The present invention provides an anti-inflammatory agent for the central nervous system, a therapeutic agent for central nervous system diseases accompanied by neuroinflammation, and a screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 shows the results of Test Example 1. Inhibition of CERK suppresses the activation of human microglia (HMC3). [Figure 2] Figure 2 shows the results of Test Example 2. Inhibition of CERK suppresses the activation of mouse microglia (BV-2). [Figure 3] Figure 3 shows the results of Test Example 3. Inhibition of CERK suppresses astrocyte activation. [Figure 4-1] Figure 4-1 shows the results of Test Example 4. (A) The results in the hippocampus are shown. In mice, Cerk deficiency suppresses LPS-induced neuroinflammation. [Figure 4-2] Figure 4-2 shows the results of Test Example 4. (B) The results in the cerebellum are shown. In mice, Cerk deficiency suppresses LPS-induced neuroinflammation. [Figure 4-3] Figure 4-3 shows the results of Test Example 4. (C) The results in the prefrontal cortex are shown. In mice, Cerk deficiency suppresses LPS-induced neuroinflammation. [Figure 5] Figure 5 shows the results of Test Example 5. In mice, Cerk deficiency suppresses LPS-induced depression-like symptoms. [Figure 6] Figure 6 shows the results of Test Example 6. In mice, Cerk deficiency suppresses MPTP-induced Parkinson's disease symptoms. MODE FOR CARRYING OUT THE INVENTION

[0010] [Anti-inflammatory agent for central nervous system] [Therapeutic agent for central nervous system diseases accompanied by neuroinflammation] The anti-inflammatory agent for the central nervous system of the present invention comprises, as an active ingredient, a compound that inhibits the function of ceramide kinase (hereinafter also referred to as "the compound of the present invention"). Further, the therapeutic agent for central nervous system diseases accompanied by neuroinflammation of the present invention comprises, as an active ingredient, a compound that inhibits the function of ceramide kinase. Specifically, compounds that inhibit the function of ceramide kinase include compounds that suppress the expression of the ceramide kinase gene and compounds that inhibit the activity of the ceramide kinase protein.

[0011] <Neuroinflammation of the central nervous system> Neuroinflammation is an activation of the immune response that occurs primarily in the central nervous system, including the brain and spinal cord, and specifically refers to the activation of an abnormal immune response. In neuroinflammation, the activation of the immune response typically occurs in glial cells such as microglia and astrocytes. Neuroinflammation is known to occur due to a variety of causes, including damage to the central nervous system, infections caused by microorganisms and viruses, neurotoxins, and autoimmune diseases. Neuroinflammation can be transient or chronic. The neuroinflammation targeted by this invention is not limited to transient or chronic, but is preferably chronic. Neuroinflammation can be detected, for example, by the excessive production of inflammatory cytokines such as IL-1β, IL-6, and TNF-α.

[0012] <Central nervous system disorders accompanied by neuroinflammation> Central nervous system diseases accompanied by neuroinflammation include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and depression. Furthermore, the causes of neuroinflammation in these diseases are not particularly limited.

[0013] <Anti-inflammatory agents for the central nervous system> The anti-inflammatory agent of the present invention targets neuroinflammation of the central nervous system. The anti-inflammatory agent of the present invention has excellent anti-inflammatory properties that can suppress the excessive inflammatory response occurring in neuroinflammation of the central nervous system. The suppression of the inflammatory response can be evaluated, for example, by measuring the production levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α, and inflammatory markers such as COX-2.

[0014] <Therapeutic agent> The therapeutic agent of the present invention is intended for central nervous system diseases accompanied by neuroinflammation. The treatment of a disease primarily refers to alleviating symptoms or preventing their worsening. The diagnosis of a disease and the evaluation of the effectiveness of treatment can be carried out based on publicly known guidelines, etc.

[0015] <Compound> The present invention provides an anti-inflammatory agent for the central nervous system and a therapeutic agent for central nervous system diseases accompanied by neuroinflammation, which contains a compound that inhibits the function of ceramide kinase as an active ingredient. Hereinafter, the embodiment of inhibiting the function of ceramide kinase will also be simply referred to as "inhibiting ceramide kinase (CERK)".

[0016] (Ceramide kinase) Ceramide kinase (CERK) is an enzyme that catalyzes the reaction in living organisms in which the substrate ceramide is phosphorylated to produce ceramide-1-phosphate (C1P). Ceramide kinase is known to exist in a wide variety of organisms, including non-human mammals such as mice and rats, in addition to humans. In this invention, "ceramide kinase" is used as a term encompassing both the "ceramide kinase gene (CERK gene)" and the "ceramide kinase protein (CERK protein)." Furthermore, in this invention, the terms "ceramide kinase gene," "CERK gene," "ceramide kinase protein," and "CERK protein" are used without limiting the species from which they originate. The nucleotide sequence of the ceramide kinase gene and the amino acid sequence of the ceramide kinase protein are publicly known. For example, the nucleotide sequence of the human ceramide kinase gene cDNA and the amino acid sequence of the human ceramide kinase protein are registered in GenBank, provided by the National Center for Biotechnology Information (NCBI), under the following accession numbers (if multiple revisions are registered, it is understood that the latest revision is referred to): cDNA (mRNA) base sequence: NM_022766 Amino acid sequence: NP_073603

[0017] (Specific aspects of the compound) Compounds that inhibit the function of ceramide kinase include compounds that suppress the expression of the ceramide kinase gene and compounds that inhibit the activity of the ceramide kinase protein. Methods of suppressing CERK gene expression include disruption of mRNA encoding the CERK protein through RNA interference (RNAi), suppression of translation into the CERK gene protein, and degradation or splicing of mRNA by ASO. Embodiments of inhibiting the activity of the CERK protein include embodiments that specifically bind to the CERK protein and reduce or completely inhibit its enzymatic activity, and embodiments that competitively inhibit the binding of the CERK protein to its substrate, ceramide.

[0018] One preferred embodiment of the compounds of the present invention is a nucleic acid such as siRNA, shRNA, dsRNA, or miRNA that targets the CERK gene. siRNA, shRNA, and dsRNA are known to have RNA interference (RNAi) activity targeting protein-coding mRNA. siRNA may be single-stranded or double-stranded. RNAi activity typically refers to the phenomenon in which a double-stranded nucleic acid molecule induces sequence-specific degradation of the target mRNA. siRNA typically has an antisense strand complementary to the target sequence and a sense strand complementary to the antisense strand, with both strands forming at least partially double-stranded structures. miRNA is known to be able to suppress the translation of protein-coding mRNA. Another preferred embodiment of the compounds of the present invention is an antisense oligonucleotide (ASO) targeting the CERK gene. The ASO is a single-stranded molecule and is not essentially double-stranded. The nucleic acids are preferably ribonucleic acids containing the native bases adenine (A), guanine (G), cytosine (C), thymine (T), uracil (U), and hypoxanthine (I), as well as their non-native modified bases. Modified bases include modified adenines such as 1-methyladenine, 2-methyladenine, N6-methyladenine, and 2-methylthio-N6-isopentenyladenine; modified guanines such as 2,2-dimethylguanine, 2-methylguanine, and 7-methylguanine; modified cytosines such as 5-methylcytosine, 4-acetylcytosine, 3-methylcytosine, and 2-thiocytosine; uracil-5-oxyacetic acid, pseudouracil, 3-methyluracil, dihydrouracil, 5-ethyluracil, 5-bromouracil, 6-methyluracil, 2-thiouracil, 4-thiouracil, and 5-methyluracil. Modified uracils such as -(carboxyhydroxymethyl)uracil, 5'-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, 5-methylaminomethyluracil, 5-methylcarbonylmethyluracil, 5-methyloxyuracil, and 5-methyl-2-thiouracil; modified hypoxanthines such as 1-methylhypoxanthine; and other examples include 6-azapyrimidine, purines, 2,6-diaminopurines, 2-aminopurines, indoles, imidazoles, and xanthines. ASO may be an antisense oligonucleotide (ASO) other than ribonucleic acid, such as morpholino oligomers, peptide nucleic acids (PNA), or glycol nucleic acids (GNA).

[0019] Furthermore, vectors such as plasmids and viral vectors capable of expressing the above-mentioned nucleic acid molecules are also preferred embodiments of the compounds of the present invention.

[0020] The design of the above-mentioned nucleic acids (siRNA, shRNA, dsRNA, miRNA, antisense oligonucleotides) can be carried out based on publicly known reports, guidelines, etc., and / or using open-source or commercially available software. The nucleic acids and vectors containing them can be prepared using known methods based on the nucleotide sequence of the CERK gene. Specifically, for example, nucleic acids and antisense oligonucleotides can be chemically synthesized using conventional RNA automated synthesizers, with nucleic acids or antisense oligonucleotides complementary to a pre-defined target sequence of approximately 18-29 nucleotides. The target sequence can be determined, for example, based on the description in Ui-Tei, K. (2004). Nucleic Acids Research, 32, 936-948.

[0021] Another preferred embodiment of the compound of the present invention is an antibody that specifically binds to the CERK protein. The antibody may be either a polyclonal antibody or a monoclonal antibody. If the antibody is a monoclonal antibody, it may also be a chimeric antibody, a humanized antibody, a human antibody, etc. The antibody can be produced by known methods. For example, serum containing a polyclonal anti-CERK protein antibody can be obtained by immunizing an immunized animal such as a rabbit, rat, mouse, or goat with the full-length or fragment of the CERK protein as an antigen. Monoclonal antibodies can be produced, for example, by the hybridoma method, the phage display method, etc.

[0022] Another preferred embodiment of the compounds of the present invention is the compound represented by the following formula (A) and its analogues, disclosed in International Publication No. 2007 / 112914.

[0023] [ka]

[0024] [In the formula, R A1 This is a linear, branched, or cyclic aliphatic, aromatic, or heterocyclyl group containing at least 8, for example, 8 to 22 carbon atoms. R A2 is a linear, branched, or cyclic aliphatic, aromatic, or heterocyclyl group containing 1 to 12 carbon atoms, and, Ring A3 is a 5- or 6-membered heterocyclic ring containing 1 to 4 heteroatoms selected from N, S and O, and is fused to the phenyl ring to which Ring A3 is attached.]

[0025] Among these, a compound represented by the following formula (A1) called "NVP-231" is more preferred. The systematic name is N-[2-(Benzoylamino)-6-benzothiazolyl]tricyclo[3.3.1.1 3,7 decane-1-carboxamide.

[0026]

Chemical Formula

[0027] One of another preferred embodiments of the compound of the present invention is a compound represented by the following formula (B) disclosed in Japanese Patent Application Laid-Open No. 2009-051743.

[0028]

Chemical Formula

[0029] [wherein R B1 is a hydroxyl group, a lower alkoxy group having 1 to 4 carbon atoms or an amino acid group; R B3 is a lower alkyl group having 1 to 4 carbon atoms; R B9 is a hydrogen atom; and R B2 , R B4 to R B8 and R B10 to R B11 are the same or different and each represent a hydrogen atom or a lower alkyl group having 1 to 4 carbon atoms, provided that when R B2 is a lower alkyl group having 1 to 4 carbon atoms, R B1 is not a hydroxyl group.]

[0030] Among these, the compound represented by the following formula (B1), called "Ceramide Kinase Inhibitor, K1," is more preferred. Its systematic name is (6aR,12aR,12bS)-10-Hydroxy-4,4,6a,12b-tetramethyl-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-2H-benzo[a]xanthene-8,11-dione.

[0031] [ka]

[0032] [Pharmaceutical composition] The anti-inflammatory agent for the central nervous system and the therapeutic agent for central nervous system diseases accompanied by neuroinflammation of the present invention may be provided as a pharmaceutical composition. <Active ingredients> The pharmaceutical composition of the present invention contains as an active ingredient a compound that suppresses the expression of the ceramide kinase gene (CERK gene) or inhibits the activity of the ceramide kinase protein (CERK protein). The active ingredient may be one or more of the above compounds.

[0033] <carrier> The pharmaceutical composition is provided as a pharmaceutical composition comprising, as necessary, one or more pharmaceutically acceptable carriers. Examples of carriers include pH adjusters such as aqueous buffer solutions, acids, and bases; stabilizers such as ascorbic acid and p-aminobenzoic acid; excipients such as D-mannitol; isotonic agents; and preservatives. If the above compound is a nucleic acid or the like, the support may be lipid nanoparticles (LNPs).

[0034] <Dosage form> There are no particular restrictions on the dosage form, but examples include various injectable preparations, oral preparations, intravenous preparations, inhalants, ointments, lotions, sprays, etc. Furthermore, the pharmaceutical composition can be provided in the form of an aqueous solution, a frozen solution, or a lyophilized product.

[0035] <Content of active ingredients> The content of the active ingredient in the pharmaceutical composition is not particularly limited. It can be appropriately set depending on the type of active ingredient, its use, dosage form, and the type of carrier. For example, the content of the active ingredient in the pharmaceutical composition is 0.0001% by mass or more, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and 100% by mass or less, preferably 90% by mass or less, more preferably 50% by mass or less.

[0036] <Target recipients> The pharmaceutical composition can be administered to patients with neuroinflammation of the central nervous system; patients with central nervous system diseases accompanied by neuroinflammation, such as Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, and depression. The target population is preferably humans or non-human mammals. Humans are not particularly limited in race, sex, or age, and non-human mammals include pet animals such as dogs and cats.

[0037] <Route of administration> Routes of administration for pharmaceutical compositions include parenteral administration such as transdermal, subcutaneous, intramuscular, intravenous, and intra-arterial administration, as well as oral administration.

[0038] <Dosage> The dosage can be appropriately determined by those skilled in the art. The dosage of the active ingredient is preferably 0.1 mg / kg or more, more preferably 1 mg / kg or more, even more preferably 10 mg / kg or more, and preferably 5000 mg / kg or less, more preferably 500 mg / kg or less, and even more preferably 100 mg / kg or less. Furthermore, it can be administered once or several times a day, or with an interval of one or several days between doses.

[0039] [Treatment method] The present invention also provides a method for treating central nervous system disorders accompanied by neuroinflammation, comprising the step of administering an effective amount of the above-mentioned anti-inflammatory agent for the central nervous system or therapeutic agent for central nervous system disorders accompanied by neuroinflammation, or the above-mentioned pharmaceutical composition, to a patient with a central nervous system disorder accompanied by neuroinflammation. The specific active ingredients, target recipients, dosage, etc., are as described above.

[0040] [Screening methods for therapeutic agents for central nervous system disorders accompanied by neuroinflammation] The present invention also provides a method for screening therapeutic agents for central nervous system diseases accompanied by neuroinflammation. The present invention provides a screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation, comprising the step of searching for compounds that inhibit the function of ceramide kinase and suppress inflammation of the central nervous system, wherein the compounds are compounds that suppress the expression of ceramide kinase genes or inhibit the activity of ceramide kinase proteins. The test substances to be screened are not particularly limited.

[0041] Whether a test substance is a compound that inhibits the function of ceramide kinase can be evaluated, for example, by one or more of the following steps (ai) to (a-iii), steps (bi) to (b-iii), and steps (ci) to (c-iii).

[0042] -Process (ai)~(a-iii): (ai) The process of bringing the test substance into contact with glial cells. (a-ii) A step to detect the expression level of the CERK gene in glial cells, (a-iii) A step of selecting the test substance as a compound that inhibits the function of ceramide kinase when the expression level of the CERK gene is reduced compared to control glial cells. -Process (bi)~(b-iii): (bi) The process of bringing the test substance into contact with glial cells. (b-ii) A step to detect the expression level of CERK protein in glial cells, (b-iii) A step of selecting the test substance as a compound that inhibits the function of ceramide kinase when the expression level of CERK protein is reduced compared to control glial cells. -Process (ci)~(c-ii): (ci) A step of bringing the CERK protein into contact with the substrate in the presence of the test substance. (c-ii) A step of detecting the enzymatic activity of the CERK protein based on the contact described above. (c-iii) A step of selecting the test substance as a compound that inhibits the function of ceramide kinase when the enzymatic activity of the CERK protein is reduced compared to contact with the control.

[0043] The glial cells used in steps (ai) and (bi) are cells other than nerve cells that make up the central nervous system, specifically including microglia and astrocytes. Either in vitro systems such as established glial cell lines or primary cultured cells, or in vivo systems, can be used as glial cells. For simplicity, it is preferable to use established glial cell lines. Examples of established glial cell lines include human microglia cells HMC3, mouse microglia cells BV-2, and human astrocyte cells HASTR / ci35.

[0044] If the glial cells are a line of glial cells or primary cultured glial cells, steps (ai) to (a-iii) and steps (bi) to (b-iii) preferably include the following steps (a-0) / (b-0). Step (a-0) / Step (b-0): A step in which glial cells are cultured in a culture medium. The culture medium and culture conditions for culturing glial cells can be appropriately set by those skilled in the art. An example is the culture medium and culture conditions used in the examples described below.

[0045] In steps (ai) and (bi), for example, the test substance can be brought into contact with glial cells by adding it to the culture medium. The amount of the test substance to be added is not particularly limited.

[0046] Methods for detecting the expression level of the CERK gene in process (a-ii) include quantitative PCR (Polymerase Chain Reaction) such as real-time PCR and digital PCR, and microarray analysis. Methods for detecting the expression level of CERK protein in process (b-ii) include Western blotting, ELISA (Enzyme-Linked ImmunoSorbent Assay), immunohistochemistry, and proteomic analysis.

[0047] In steps (a-iii) and (b-iii), the control is, for example, glial cells that have not been in contact with the test substance. Alternatively, glial cells that have been in contact with a compound that inhibits the function of a known ceramide kinase can be used as the control. For example, specific compounds listed under <Compound> in the [Anti-inflammatory agents for the central nervous system] and [Therapeutic agents for central nervous system diseases accompanied by neuroinflammation] sections above, particularly NVP-231, Ceramide Kinase Inhibitor, K1, etc., can be used as compounds that inhibit the function of a known ceramide kinase.

[0048] In steps (a-iii) and (b-iii), it is preferable to select a compound to inhibit the function of ceramide kinase, for example, when the expression level of the detected CERK gene or CERK protein is below a predetermined threshold. The predetermined threshold can be set, for example, based on an ROC curve.

[0049] For simplicity, it is preferable to use purified CERK protein as the CERK protein in step (ci). The substrate is preferably ceramide. The ratio of CERK protein, substrate, and test substance can be appropriately determined by those skilled in the art. The means for detecting the enzymatic activity of the CERK protein in step (c-ii) can be carried out in accordance with known kinase assays.

[0050] In steps (c-iii), the control contact is, for example, the contact between the CERK protein and the substrate in the absence of the test substance. Alternatively, the control can be the contact between the CERK protein and the substrate in the presence of a compound that inhibits the function of a known ceramide kinase. In step (c-iii), for example, if the enzyme activity of the detected CERK protein is below a predetermined threshold, it is preferable to select a compound to inhibit the function of ceramide kinase. The predetermined threshold can be set, for example, based on an ROC curve.

[0051] In the screening method of the present invention, for example, a test substance identified as a compound that inhibits the function of ceramide kinase by one or more of the above steps (ai) to (a-iii), steps (bi) to (b-iii), and steps (ci) to (c-iii) can be further evaluated by, for example, the following steps (di) to (d-iv) to select a compound that inhibits the function of ceramide kinase and also suppresses inflammation of the central nervous system. Furthermore, for example, a test substance identified as a compound that suppresses inflammation of the central nervous system by the following steps (di) to (d-iv) can be evaluated by one or more of the above steps (ai) to (a-iii), steps (bi) to (b-iii), and steps (ci) to (c-iii) to select a compound that inhibits the function of ceramide kinase and also suppresses inflammation of the central nervous system.

[0052] The fact that the test substance is a compound that suppresses inflammation in the central nervous system can be evaluated, for example, by the following steps (di) to (d-iv). -Process (di)~(d-iv): (di) A process to activate glial cells, (d-ii) A step of bringing the test substance into contact with activated glial cells. (d-iii) A step to detect the expression level of an activation marker in activated glial cells. (d-iv) A step in which the above test substance is selected as a compound that suppresses inflammation of the central nervous system when the expression level of the activation marker is reduced compared to the control.

[0053] The glial cells used in step (di) are the same as those used in steps (ai) and (bi) above, and for simplicity, it is preferable to use a cell line of glial cells. If the glial cells are a line of glial cells or primary cultured glial cells, steps (di) to (d-iii) preferably include step (d-0) below. Step (d-0): Step of culturing glial cells in culture medium. The culture medium and culture conditions for culturing glial cells can be appropriately set by those skilled in the art. An example is the culture medium and culture conditions used in the examples described below.

[0054] Activation of glial cells in step (di) can be achieved, for example, by adding lipopolysaccharide (LPS) to the culture medium. The amount of LPS to be added can be appropriately determined by those skilled in the art.

[0055] In step (d-ii), for example, the test substance can be brought into contact with activated glial cells by adding it to the culture medium. The amount of the test substance to be added is not particularly limited.

[0056] In step (d-iii), examples of activation markers include inflammatory cytokines such as IL-1β, IL-6, and TNF-α; inflammatory markers other than inflammatory cytokines such as COX-2; microglial activation markers such as Iba1, C1q, and CXCR1; and astrocyte activation markers such as GFAP, LCN2, and C3. It is preferable to use the microglia activation marker when the glial cells are microglia, and the astrocyte activation marker when the glial cells are astrocytes. In step (d-iii), means for detecting the expression level of the activation marker include, for example, quantitative PCR such as real-time PCR and digital PCR, analysis of gene expression levels by microarray analysis, etc., and analysis of protein expression levels by Western blotting, ELISA, immunohistochemistry, proteomic analysis, etc.

[0057] In steps (d-iv), the control is, for example, activated glial cells that have not been in contact with the test substance. Alternatively, activated glial cells that have been in contact with a known central nervous system inflammation inhibitor can be used as the control.

[0058] If the test substance is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein, and is also a compound that suppresses inflammation in the central nervous system, then the test substance is selected as a candidate for the treatment of central nervous system diseases accompanied by neuroinflammation. [Examples]

[0059] Examples are given below to illustrate the present invention in detail, but the present invention is not limited to these examples. In the following examples, human gene names are written in uppercase, while mouse gene names are written with the first letter capitalized and the rest of the letters lowercase.

[0060] Test Example 1: Inhibition of human microglia activation (method) Human microglia cells (HMC3, supplied from the American Type Culture Collection (ATCC), Cat# CRL-3304) were placed in a 12-well plate in 0.5 × 10⁶ layers. 5 Cells were seeded in a cell / well and cultured at 37°C under conditions of 5% CO2 / 95% air in Dulbecco's modified eagle medium (DMEM) containing 10% (v / v) fetal bovine serum (FBS) and 1% (v / v) penicillin-streptomycin. For the NVP-treated group, the following day, a DMSO solution of NVP-231 (Cayman Chemicals, Cat# 13858) was added to the culture medium at a final concentration of 200 nM, either at a ratio of 2 / 1000 (NVP) or 2 / 1000 (Ctrl) of DMSO alone, and the culture was incubated for 24 hours. For the siRNA-treated group, the following day, transfection was performed with siRNA against the CERK gene (siCERK) or control siRNA (siCtrl) to a final concentration of 10 nM, and the cells were cultured for 48 hours. Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the instructions for use. The sequences of the siRNAs are shown in Table 1 below.

[0061] [Table 1]

[0062] Subsequently, lipopolysaccharide (LPS) (Merck, Sigma-Aldrich brand, Cat# 12190105) was added to the culture medium to a final concentration of 100 ng / mL, and the cells were incubated for 4 hours. LPS treatment activates microglial cells and induces neuroinflammation. Cells were washed twice with ice-cold PBS on ice and recovered with 100 μL of IsogenII (Nippon Gene Co., Ltd.). RNA extraction was performed according to the instructions, and cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd.). Quantitative PCR was performed on the GAPDH, IL-6, IL-1B, and CERK genes using THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Ltd.) and the CFX Duet real-time PCR system (Bio-Rad). The sequences of the primers used are shown in Table 2 below. The thermal profiles for quantitative PCR were as follows: 1. 95°C, 1 min; 2. 95°C, 10 sec; 3. 60°C, 15 sec (2-3 for 40 cycles). Quantification was performed using the ΔΔCq method.

[0063] [Table 2]

[0064] (result) The results are shown in Figure 1. As shown in Figures 1(A) and (B), pretreatment with the CERK inhibitor NVP-231 (200 nM) for 24 hours suppressed LPS-induced microglial activation. Furthermore, as shown in Figures 1(C) and (D), LPS-induced microglial activation was also suppressed under conditions where CERK was knocked down by pretreatment with siRNA against CERK for 48 hours. Figure 1(E) shows the knockdown efficiency of CERK in terms of relative mRNA expression. Experimental results are shown as mean ± standard error (SEM). The obtained data were tested using unpaired two-tailed Student's t-test or one-way ANOVA. A statistically significant difference was considered to exist if P < 0.05. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

[0065] Test Example 2: Inhibition of mouse microglia activation (method) Place mouse microglia cells BV-2 in a 12-well plate in a 0.35 × 10⁶ arrangement. 5 Cells were seeded per well and cultured at 37°C under conditions of 5% CO2 / 95% air in DMEM containing 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin. The mouse microglia cell BV-2 is described in the literature: Blasi, E. et al. (1990). Journal of Neuroimmunology, 27, 229-237. and was provided by Dr. Eui-Ju Choi of Korea University, South Korea. For the NVP-treated group, the following day, 2 / 1000 of a DMSO solution of NVP-231 (NVP) or 2 / 1000 of DMSO alone (Ctrl) was added to the culture medium to achieve a final concentration of 200 nM, and the culture was incubated for 24 hours. For the siRNA-treated group, the following day, transfection was performed with either siRNA against the Cerk gene (siCerk) or control siRNA (siCtrl) to a final concentration of 10 nM, followed by 48 hours of incubation. Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific) according to the instructions for use. The siRNA sequences are shown in Table 3 below.

[0066] [Table 3]

[0067] Subsequently, LPS was added to the culture medium to a final concentration of 100 ng / mL, and the cells were incubated for 4 hours. The cells were washed twice with ice-cold PBS on ice and harvested with 100 μL of Isogen II. RNA extraction was performed according to the instructions, and cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover. Quantitative PCR was performed on the Gapdh, Il-6, Il-1b, and Cerk genes using THUNDERBIRD SYBR qPCR Mix and the CFX Duet real-time PCR system. The sequences of the primers used are shown in Table 4 below. The thermal profiles for quantitative PCR were as follows: 1. 95°C, 1 min; 2. 95°C, 10 sec; 3. 55°C, 30 sec; 4. 72°C, 60 sec (2-4 for 40 cycles). Quantification was performed using the ΔΔCq method.

[0068] [Table 4]

[0069] (result) The results are shown in Figure 2. Similar to the above example 1, in mouse microglia (BV-2), LPS-induced microglial activation was suppressed by 24-hour pretreatment with NVP-231 (200 nM), as shown in Figures 2(A) and (B), and by 48-hour pretreatment with siRNA targeting the Cerk gene, as shown in Figures 2(C) and (D). Figure 2(E) shows the knockdown efficiency of the Cerk gene as measured by relative mRNA expression. Experimental results are shown as mean ± SEM. The obtained data were tested using one-way ANOVA. A statistically significant difference was considered to exist when P < 0.05. **: P < 0.01, ****: P < 0.0001

[0070] Test Example 3: Inhibition of astrocyte activation (method) Human astrocyte cells HASTR / ci35 were placed in a 12-well collagen-coated dish in a 6.0 × 10⁻¹⁶ arrangement. 4 Cells were seeded in cells / well and cultured in DMEM (hereinafter referred to as complete astrocyte medium; CAM) containing 1% (v / v) N-2 Supplement (manufactured by Fujifilm Wako Pure Chemical Corporation), 10% (v / v) FBS, 4 μg / mL blastcidin S, and 1% (v / v) penicillin-streptomycin at 33°C under conditions of 5% CO2 / 95% air. The human astrocyte HASTR / ci35 is described in the reference: Furihata, T. et al. (2015). Journal of Neurochemistry, 136, 92-105., and was provided by Dr. Tomomi Furihata of Tokyo University of Pharmacy and Life Sciences. The day after sowing, the culture medium was washed twice with FBS-free DMEM and then replaced with FBS-free CAM. Simultaneously, the culture medium was transfected with either the siRNA against CERK (siCERK) used in Test Example 1 above or the control siRNA (siCtrl) to a final concentration of 10 nM. Transfection was performed using Lipofectamine RNAiMAX Transfection Reagent, following the instructions for use. After 48 hours, the samples were washed twice with FBS-free DMEM and treated with IL-1α (Recombinant Human IL-1α, PeproTech, Cat# 200-01A) and TNF-α (Recombinant Human TNF-α, PeproTech, Cat# 300-01A) at 3 ng / mL and 50 ng / mL, respectively. After 24 hours, the samples were washed twice with ice-cold PBS on ice and collected in 100 μL of Isogen II. Total RNA was extracted according to the instructions, and cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover. Quantitative PCR was performed on the GAPDH gene, IL-6 gene, PTGS2 gene encoding COX-2, LCN2 gene, and CERK gene using THUNDERBIRD SYBR qPCR Mix and the CFX Duet real-time PCR system. The sequences of the primers used are shown in Table 5 below. The thermal profiles for quantitative PCR are as follows: 1. 95°C, 1 min; 2. 95°C, 10 sec; 3. 60°C, 15 sec (2-3 repeated for 40 cycles). Quantification was performed using the ΔΔCq method.

[0071] [Table 5]

[0072] (result) The results are shown in Figure 3. As shown in Figure 3(A), in human astrocyte cells HASTR / ci35, IL-1α (3 ng / mL) / TNF-α (50 ng / mL) stimulation (24 h) induced elevated gene expression levels of IL-6, PTGS2, and LCN2, which are astrocyte activation markers. Under conditions where CERK was knocked down by pretreatment with siRNA against CERK for 48 hours, astrocyte activation induced by IL-1α / TNF-α stimulation was suppressed. Figure 3(B) shows the knockdown efficiency of CERK in terms of mRNA expression level (Relative mRNA expression). Experimental results are shown as mean ± SEM. The obtained data were tested using unpaired two-tailed Student's t-test or one-way ANOVA. A statistically significant difference was considered to exist if P < 0.05. *: P < 0.05, **: P < 0.01, ****: P < 0.0001

[0073] Test Example 4: Suppression of neuroinflammation in living mice (method) Brains were removed from wild-type mice (WT, C57BL / 6 strain) and ceramide kinase-deficient mice (Cerk KO) three hours after intraperitoneal administration of physiological saline or 1 mg / kg LPS. The ceramide kinase-deficient mouse (Cerk KO) is described in the literature: Mitsutake, S. et al. (2007). Biochemical and Biophysical Research Communications, 363, 519-524. and was provided by Dr. Yasuyuki Igarashi of Hokkaido University. The hippocampus, cerebellum, and prefrontal cortex were homogenized with Isogen II. Total RNA was extracted according to the instructions, and cDNA was prepared using ReverTra Ace qPCR RT Master Mix with gDNA Remover. Quantitative PCR was performed on the Gapdh, Il-6, Il-1b, Tnf-α, and Ptgs2 genes using THUNDERBIRD SYBR qPCR Mix and the CFX Duet real-time PCR system. The sequences of the primers used are shown in Table 6 below. The thermal profiles for quantitative PCR were as follows: 1. 95°C, 1 min; 2. 95°C, 10 sec; 3. 55°C, 30 sec; 4. 72°C, 1 min (2-4 for 40 cycles). Quantification was performed using the ΔΔCq method.

[0074] [Table 6]

[0075] (result) The results are shown in Figures 4-1, 4-2, and 4-3. When mice (C57BL / 6) were intraperitoneally administered LPS (1 mg / kg), their brains were removed 3 hours later, and mRNA levels of the inflammatory markers Il-1β, Il-6, Tnf-α, and Ptgs2 were measured in the hippocampus, cerebellum, and prefrontal cortex. mRNA levels of these inflammatory markers were elevated. In Cerk KO mice, the increase in mRNA levels of inflammatory markers induced by LPS administration was significantly suppressed. Experimental results are shown as mean ± SEM. The obtained data were tested using one-way ANOVA. A statistically significant difference was considered to exist when P < 0.05. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

[0076] Test Example 5: Suppression of depressive symptoms (method) Wild-type mice (WT) and Cerk KO mice used in Test Example 4 above were subjected to a forced swimming test in accordance with the description in the reference: Karl, T. et al. (2004). Proc. Natl. Acad. Sci. USA, 101, 12742-12747. Specifically, wild-type mice (WT) or Cerk KO mice were intraperitoneally administered physiological saline or 1 mg / kg LPS. 24 hours later, the mice were allowed to move around for 6 minutes in a cylindrical water tank (20 cm high x 15 cm in diameter) containing 2 L of water, and their immobility time (seconds) was measured. In this study, immobility time is an indicator of depressive behavior.

[0077] (result) The results are shown in Figure 5. In wild-type mice, prolonged immobility, i.e., depressive symptoms, were observed, but this was not observed in Cerk KO mice. Experimental results are shown as mean ± SEM. The obtained data were tested using one-way ANOVA. A statistically significant difference was considered to exist if P < 0.05. *: P < 0.05, **: P < 0.01

[0078] Test Example 6: Suppression of Parkinson's disease symptoms (method) Wild-type mice (WT) and Cerk KO mice used in Test Example 4 were subjected to the Wire-hang test and Pole test according to the description in the reference: Karl, T. et al. (2004). Proc. Natl. Acad. Sci. USA, 101, 12742-12747. Specifically, wild-type mice (WT) or Cerk KO mice were administered intraperitoneally with physiological saline or 20 mg / kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, manufactured by Tokyo Chemical Industry Co., Ltd., Cat# M2690) at 2-hour intervals for four doses. MPTP is a neurotoxin that causes Parkinson's disease-like symptoms.

[0079] -Wire-hang test Seven days after MPTP administration, a wire-hang test was performed by placing a 2mm diameter, 25cm long metal rod horizontally 30cm above the ground and suspending both lower limbs of mice from the rod horizontally. The time (in seconds) until the mouse fell from the rod was measured twice, and the average value was calculated. In the wire-hang test, mice exhibiting a Parkinson's disease-like phenotype showed a shorter time until they fell from the metal rod.

[0080] -Pole test Seven days after MPTP administration, a pole test was performed on mice placed on a metal rod 60 cm high and 1 cm in diameter, with a grip wrapped around its circumference. The time (in seconds) from when the mouse was placed on the pole until it was lowered was measured three times, and the average value was calculated. In the pole test, mice exhibiting a Parkinson's disease-like phenotype took longer to lower themselves.

[0081] (result) The results are shown in Figure 6. Parkinson's disease symptoms were observed in wild-type mice, but not in Cerk KO mice. Experimental results are shown as mean ± SEM. The obtained data were tested using unpaired two-tailed Student's t-test or one-way ANOVA. A statistically significant difference was considered to exist if P < 0.05. *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001

Claims

1. An anti-inflammatory agent for the central nervous system comprising a compound that inhibits the function of ceramide kinase as an active ingredient, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein.

2. A therapeutic agent for central nervous system disorders accompanied by neuroinflammation, comprising as an active ingredient a compound that inhibits the function of ceramide kinase, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein.

3. The agent according to claim 2, wherein the central nervous system disorder accompanied by neuroinflammation is Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, or depression.

4. The agent according to claim 1 or 2, wherein the compound that inhibits the function of ceramide kinase is an siRNA, shRNA, dsRNA, miRNA, or antisense oligonucleotide (ASO) that targets the ceramide kinase gene.

5. Compounds that inhibit the function of ceramide kinase, NVP-231 (N-[2-(Benzoylamino)-6-benzothiazolyl]tricyclo[3.3.1.1 3,7 decane-1-carboxamide) or Ceramide Kinase Inhibitor, K1 ((6aR,12aR,12bS)-10-Hydroxy-4,4,6a,12b-tetramethyl-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-2H-benzo[a]xanthene-8,11-dione) The agent according to claim 1 or 2.

6. A screening method for therapeutic agents for central nervous system diseases accompanied by neuroinflammation, comprising the step of searching for a compound that inhibits the function of ceramide kinase and suppresses inflammation of the central nervous system, wherein the compound is a compound that suppresses the expression of the ceramide kinase gene or inhibits the activity of the ceramide kinase protein.

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