WO2023228701A1 - Agent préventif, inhibiteur de la progression d'une maladie, ou agent thérapeutique pour des maladies démyélinisantes centrales - Google Patents

Agent préventif, inhibiteur de la progression d'une maladie, ou agent thérapeutique pour des maladies démyélinisantes centrales Download PDF

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WO2023228701A1
WO2023228701A1 PCT/JP2023/017205 JP2023017205W WO2023228701A1 WO 2023228701 A1 WO2023228701 A1 WO 2023228701A1 JP 2023017205 W JP2023017205 W JP 2023017205W WO 2023228701 A1 WO2023228701 A1 WO 2023228701A1
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ffar1
acid
receptor agonist
cells
disease
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里衣子 池田
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国立研究開発法人国立精神・神経医療研究センター
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the present invention relates to a preventive agent, a disease progression inhibitor, or a therapeutic agent for central demyelinating diseases.
  • ALS Amyotrophic lateral sclerosis
  • ALS is a neurodegenerative disease in which upper and lower motor neurons selectively degenerate as the disease progresses.
  • the main symptoms are paralysis of the limbs and motor dysfunction such as difficulty swallowing, and in many cases death occurs due to respiratory muscle paralysis 3 to 5 years after onset.
  • riluzole or edaravone are on the market as therapeutic agents for ALS, they only show the effect of slightly prolonging survival time.
  • SOD1 G93A mice mutant SOD1 transgenic mice (SOD1 G93A mice) are widely used as ALS pathology model mice, as they exhibit significant motor dysfunction similar to that of ALS patients, as well as motor neuron degeneration and glial cell activation. (For example, see Non-Patent Document 1).
  • a central demyelinating disease develops, demyelination occurs in the central nervous system (CNS), and the main symptom is a decline in motor function.
  • CNS central nervous system
  • remyelination progresses and a mechanism works to restore nerve impulse transmission, but the mechanism of remyelination is still unclear, and there are ways to actively promote remyelination. is unknown (for example, see Non-Patent Document 6).
  • the decline in motor function associated with aging is caused by a decline in remyelination efficiency.
  • Age-related decline in remyelination results from impaired accumulation of oligodendrocyte progenitor cells (OPCs) in lesions and reduced differentiation of OPCs into remyelinating oligodendrocytes.
  • OPCs oligodendrocyte progenitor cells
  • Oligodendrocytes are a type of central nervous system glial cells that form myelin, and promote rapid conduction of action potentials by covering axons with myelin. Furthermore, oligodendrocytes support the survival and function of axons by providing metabolic support for neurons. Abnormal myelin morphology and decreased myelin amount are also observed in the motor cortex of the cerebral cortex of ALS patients. In addition, in SOD1 G93A mice, morphological changes and cell death of oligodendrocytes are observed even before the onset of symptoms, and at the same time, oligodendrocyte precursor cells (OPCs) proliferate excessively (for example, see Non-Patent Documents 2 to 4). ).
  • OPCs oligodendrocyte precursor cells
  • the present invention provides, for example, the following (1) to (50).
  • a preventive agent, a disease progression inhibitor, or a therapeutic agent is a therapeutic agent.
  • the pharmaceutical composition according to (7) further comprising a pharmaceutically acceptable carrier.
  • the pharmaceutical composition according to (7) or (8) for preventing or suppressing the progression of motor dysfunction associated with central demyelinating disease, or for preventing or treating the motor dysfunction.
  • the FFAR1 receptor agonist is oleic acid or linoleic acid.
  • the method according to (13), wherein administering the FFAR1 receptor agonist is administering a composition comprising the FFAR1 receptor agonist and a pharmaceutically acceptable carrier.
  • the FFAR1 receptor agonist is a saturated or unsaturated fatty acid.
  • (30) The FFAR1 receptor agonist for use according to (28) or (29), which is oleic acid or linoleic acid.
  • (31) The FFAR1 receptor agonist for use according to any one of (28) to (30), wherein the central demyelinating disease is amyotrophic lateral sclerosis.
  • (33) The use according to (32), wherein the FFAR1 receptor agonist is a saturated or unsaturated fatty acid.
  • (42) A method for preventing or suppressing the progression of motor dysfunction, or for preventing or treating motor dysfunction, which comprises administering a FFAR1 receptor agonist.
  • FFAR1 receptor agonist is oleic acid or linoleic acid.
  • a method for suppressing cell death of oligodendrocytes which comprises adding an FFAR1 receptor agonist to oligodendrocytes.
  • the administered FFAR1 receptor agonist can suppress cell death of oligodendrocytes and suppress the onset or progression of motor dysfunction.
  • the treatment of central demyelinating diseases e.g., neuromyelitis optica, spinal cord injury, cerebral infarction, multiple sclerosis, autism, amyotrophic lateral sclerosis
  • central demyelinating diseases e.g., neuromyelitis optica, spinal cord injury, cerebral infarction, multiple sclerosis, autism, amyotrophic lateral sclerosis
  • (a) is a schematic diagram showing the procedure of a test for evaluating free fatty acids in SOD1 G93A mouse plasma.
  • (b) is a heat map showing the content of various free fatty acids (FFAs) in the plasma of each mouse.
  • (c) is a graph showing the relative concentrations of oleic acid (OA) and linoleic acid (LA) with respect to the blood free fatty acid content of WT mice.
  • (a) is a schematic diagram showing the procedure of a test to evaluate the effect of suppressing oligodendrocyte cell death by administering a mixed solution of oleic acid and linoleic acid (OA+LA).
  • (b) is a micrograph of cells stained with CC1, Olig2, or CC-3.
  • (c) is a graph showing the number of CC1 + Olig2 + CC-3 + cells in the lumbar spinal cord as a density per mm 2 .
  • (a) is a photomicrograph of immunohistochemical staining of ChAT + motor neurons in SOD1 G93A mice at P140, and a graph comparing the number of motor neurons in the Vehicle group and the OA + LA administration group.
  • (b) is a photomicrograph of immunohistochemical staining of Iba1 in the lumbar spinal cord of SOD1 G93A mice at P140, and a graph comparing the Iba1 positive rate between the Vehicle group and the OA+LA administration group.
  • (a) is a schematic diagram showing the procedure of a test to evaluate the effect of suppressing cell death on oligodendrocytes by administering a mixed solution of oleic acid and linoleic acid (OA+LA).
  • (b) is a micrograph showing immune cell staining obtained by staining primary cultured oligodendrocytes with PI, DAPI, or MBP.
  • (c) is a graph showing a comparison of the percentage of PI + MBP + cells to MBP + cells.
  • (a) is a schematic diagram showing the procedure of a test to evaluate the effect of suppressing cell death on oligodendrocytes by administering a mixed solution of oleic acid and linoleic acid (OA+LA).
  • (b) is a graph showing the ratio of the number of PI + cells to MBP + PI + cells.
  • (a) is a plot showing -log 10 (p-value) and log 2 (fold change) of the percentage of PI + MBP + cells in each siRNA treatment compared to control siRNA.
  • (b) is a graph showing the relative expression level of Ffar1 mRNA in primary oligodendrocytes after siControl or siFfar1 treatment.
  • (c) is a graph showing the percentage of PI + MBP + cells in MBP + cells after siControl or siFfar1 treatment.
  • (a) is a graph showing the percentage of PI + MBP + cells in MBP + cells for four groups with and without glutamate and with and without OA+LA treatment.
  • (b) is a graph showing the influence of OA+LA treatment on glutamate-induced oligodendrocyte cell death in oligodendrocytes introduced with siControl or siFfar1.
  • (c) is a graph showing the percentage of CC-3 + MBP + cells in MBP + cells for the four groups with and without glutamate and with and without OA+LA treatment.
  • (d) is a graph showing the influence of OA+LA treatment on glutamate-induced oligodendrocyte cell death in oligodendrocytes introduced with siControl or siFfar1.
  • (a) and (b) are photographs showing immunohistochemistry for CC1 (green), Olig2 (red), and Ffar1 (cyan) in the spinal cord of SOD1 G93A mice at P100, and Merge is a superimposed photograph. It is.
  • (c) is a graph quantifying the number of CC-3 + CC1 + Olig2 + cells per 1 mm 2 from the photograph in (b).
  • (d) is a graph quantifying the number of CC1 + Olig2 + cells per 1 mm 2 from the photograph in (b). It is a graph showing changes in grip strength in the Vehicle group and the OA+LA treatment group.
  • a first embodiment of the present invention is a preventive agent, an agent for suppressing disease progression, or a therapeutic agent for central demyelinating disease, which includes an FFAR1 receptor agonist.
  • the preventive agent, disease progression inhibitor, or therapeutic agent for central demyelinating disease according to this embodiment may be in the form of a pharmaceutical composition.
  • central demyelinating disease refers to a disease in which myelin sheaths are lost from myelinated nerves present in the central nervous system (CNS), and symptoms of decreased motor function are generally observed.
  • central demyelinating diseases include multiple sclerosis, neuromyelitis optica, spinal cord injury, and cerebral infarction.
  • diseases that may cause demyelination of the central nervous system include genetic diseases (e.g., phenylketonuria, Tay-Sachs disease, Niemann-Pick disease, Gaucher disease, Hurler syndrome, Krabbe disease, and other diseases).
  • ALS amyotrophic lateral sclerosis
  • hypoxia and ischemia e.g. carbon monoxide poisoning and other delays
  • hypoxic cerebral demyelination syndrome e.g., osmotic demyelination syndrome, Marchiafava-Bignami disease, vitamin B12 deficiency
  • viral infection of the central nervous system e.g., osmotic demyelination syndrome, Marchiafava-Bignami disease, vitamin B12 deficiency
  • Direct invasion e.g., progressive multifocal leukoencephalopathy, subacute sclerosing panencephalitis, tropical spastic paraplegia/HTLV-1 associated myelopathy
  • primary demyelinating disease e.g., recurrent and progressive disease ( multiple sclerosis and its subtypes)
  • monophasic diseases such as optic neuritis, acute transverse myelitis, acute disseminated encephalomyelitis, acute hemorrhagic leukoencephalopathy, neuromyelitis optica
  • absorption of toxic substances e.g. alcohol , ethambutol.
  • FFAR1 receptor stands for free fatty acid receptor 1 (also called GPR40), and was discovered as one of the G protein-coupled receptors (GPCRs) that is highly expressed in pancreatic ⁇ cells, which are insulin-secreting cells, and is highly expressed in glucose concentration. It is known to exhibit an insulin secretagogue effect in a dependent manner.
  • GPCRs G protein-coupled receptors
  • the FFAR1 receptor is known to be abundantly expressed in central nervous system regions such as the brain and spinal cord, but its physiological significance and role are unknown.
  • capric acid (FA (10:0)), lauric acid (FA (12:0)), tridecylic acid (FA (13:0)), myristic acid (FA (14:0)) , pentadecyl acid (FA (15:0)), palmitic acid (FA (16:0)), margaric acid (FA (17:0)), stearic acid (FA (18:0)), nonadecyl acid (FA ( 19:0)), arachidic acid (FA (20:0)), henicosylic acid (FA (21:0)), eicosadienoic acid (FA (21:1)), behenic acid (FA (22:0)), Saturated fatty acids such as lignoceric acid (FA (24:0)), myristoleic acid (FA (14:1)), pentadecenoic acid (FA (15:1)), sapienoic acid or palmitoleic acid (FA (16:1)) ), hexadecadienoic acid
  • the unsaturated fatty acids may be any of ⁇ (FA (20:1)), eicosadienoic acid (FA (20:2)), eicosatrienoic acid, dihomo- ⁇ -linolenic acid, or mead acid (FA (20:3)), eicosatetraenoic acid or arachidonic acid (FA (20:4)), eicosapentaenoic acid (FA (20:5)), erucic acid or docosenoic acid (FA (22:1)), docosadienoic acid (FA (22:2)), docosatriene acid (FA (22:3)), adrenic acid (FA (22:4)), sardine acid or ozbond acid (FA (22:5)), docosahexaenoic acid (FA (22:6)), tricosylic acid (FA (23:0)), trichosenoic acid (FA (23:1)), nervonic acid or tetracosenoic acid (FA (24:1)).
  • FFAR1 receptor agonists include AM-1638 (see Med.Chem.Lett.2012,3,726-730.), AM6226 (see PLoSOne. 2012;7(10):e46300.), AS2034178 (J.Pharmacol.Exp Ther.2013, 346(3):443-452.), AS2575959 (Life Sci.
  • the FFAR1 receptor agonist is a saturated or unsaturated fatty acid; in a more preferred embodiment, the FFAR1 receptor agonist is oleic acid or linoleic acid.
  • the pharmaceutical composition according to the present embodiment may be composed of only the FFAR1 agonist, or may contain a pharmaceutically acceptable carrier in addition to the above-mentioned FFAR1 agonist.
  • pharmaceutically acceptable carriers include excipients, buffers, stabilizers, antioxidants, binders, disintegrants, fillers, emulsifiers, flow additives, etc. commonly used in the field of pharmaceutical technology. It will be done.
  • the pharmaceutical composition may also contain a medicament for treating central demyelinating diseases or alleviating symptoms of central demyelinating diseases. Examples of such drugs include riluzole (glutamate antagonist).
  • the agent for treating or preventing central demyelinating diseases according to this embodiment may be administered orally or parenterally.
  • a specific dosage for example, when administered to an adult human male (body weight 60 kg), the daily dosage of the agent for treating or preventing central demyelinating disease is usually calculated in terms of the amount of active ingredient. 0.0001 ⁇ g to 10000 mg/day/person.
  • the pharmaceutical composition according to this embodiment may be in any dosage form, such as a powder, pill, granule, tablet, syrup, troche, capsule, or injection.
  • the agent for treating or preventing a central demyelinating disease according to the present embodiment is administered in a different dosage form from the medicine for treating the central demyelinating disease or alleviating the symptoms of the central demyelinating disease. You may.
  • the pharmaceutical composition according to this embodiment prevents or suppresses the progression of motor dysfunction, or contains a sufficient amount (therapeutically effective amount) of a FFAR1 receptor agonist to prevent or treat the motor dysfunction. obtain.
  • neuronal homeostasis is maintained. It may be effective to prevent or suppress not only motor dysfunction associated with central demyelinating disease but also motor dysfunction due to other factors, or to prevent or treat motor dysfunction.
  • motor dysfunction develops, symptoms such as muscle atrophy, muscle weakness (e.g., decreased grip strength), spasticity of the limbs, paralysis of the limbs, decreased grip strength, dysarthria, and difficulty swallowing appear, leading to poor quality of life (QOL). ) decreases markedly.
  • ALS amyotrophic lateral sclerosis
  • the main symptoms are muscle atrophy and muscle weakness (e.g., decreased grip strength) in the upper limbs, and upper limb-type symptoms showing spasticity in the lower limbs, as well as bulbar symptoms such as dysarthria and difficulty swallowing.
  • the symptoms include progressive bulbar palsy, mainly consisting of lower extremity tendon reflexes, reduced or absent tendon reflexes, and lower extremity symptoms accompanied by secondary motor neuron damage.
  • Other symptoms of ALS include respiratory muscle paralysis, trunk muscle disorders, and dementia. According to this embodiment, it may be effective in preventing the onset of, suppressing the progression of, and treating central demyelinating diseases (particularly motor dysfunction).
  • Another aspect of this embodiment is a method for preventing or suppressing the progression of motor dysfunction associated with a central demyelinating disease, or for preventing or treating motor dysfunction, the method comprising administering a FFAR1 receptor agonist. It is.
  • a "subject in need” is a patient (e.g., human) who is suggested to have a possibility of developing motor dysfunction, such as a central demyelinating disease or its early symptoms.
  • patients who have been observed to have cell death in oridodendrocytes or shrinkage of myelin sheath, patients who have been diagnosed with mild motor dysfunction, patients whose blood fatty acid concentration is lower than the normal range, etc. patients with confirmed SOD1 mutations (for example, point mutations such as H46R and L84V).
  • this study revealed that even before the onset of amyotrophic lateral sclerosis, the concentration of fatty acids in the blood is decreased, so the concentration of fatty acids in the blood is lower than the normal range. It is expected that the administration of a FFAR1 receptor agonist to a subject will have a preventive effect on motor dysfunction.
  • FFAR1 receptor agonist when administering the FFAR1 receptor agonist, it may be administered in the form of a composition containing a pharmaceutically acceptable carrier in addition to the FFAR1 receptor agonist.
  • FFAR1 receptor and pharmaceutically acceptable carrier can refer to the above definitions.
  • a sufficient amount of the FFAR1 receptor agonist is administered to the above subject to prevent the onset of motor dysfunction, suppress the progression of pathological conditions, or treat the disease.
  • the administration method may be oral administration or injection (intravenous, intramuscular, subcutaneous, intraperitoneal). It may also be administered by mixing it into an intravenous drip.
  • Another aspect of the present embodiment is the FFAR1 receptor for preventing, suppressing disease progression, or treating central demyelinating disease, or for producing an agent for preventing, inhibiting disease progression, or treating central demyelinating disease.
  • a second embodiment of the invention is a composition comprising an FFAR1 receptor agonist for inhibiting oligodendrocyte cell death.
  • the definition in the first embodiment can be referred to.
  • the composition according to the present embodiment may contain additives such as a buffer, a preservative, a stabilizer, an antioxidant, a solvent, and an emulsifier. These additives can be used as appropriate within the range that allows oligodendrocytes to survive.
  • Another aspect of this embodiment is a method for suppressing cell death of oligodendrocytes, which includes adding an FFAR1 receptor agonist to oligodendrocytes.
  • FFAR1 receptor agonist when adding a FFAR1 receptor agonist, it may be added in the form of a composition containing the above-mentioned additives in addition to the FFAR1 receptor agonist.
  • FFAR1 receptor and additives can refer to the definitions above.
  • Oligodendrocytes are oligodendrocytes obtained from brain tissue collected from target animals (e.g., mice, rats, rabbits, chickens, dogs, monkeys) by known methods such as enzyme treatment and centrifugation. Alternatively, the cells may be primary cultured oligodendrocytes into which a specific gene has been artificially introduced.
  • target animals e.g., mice, rats, rabbits, chickens, dogs, monkeys
  • the cells may be primary cultured oligodendrocytes into which a specific gene has been artificially introduced.
  • a FFAR1 receptor agonist or a composition containing the same is added to the medium in which primary cultured oligodendrocytes are cultured, and cultured for a predetermined period of time. Oligodendrocyte cell death can be inhibited by the addition of FFAR1 receptor agonists. Thereafter, depending on the purpose, propidium iodide may be added to remove dead cells and further immunocytochemical staining may be performed.
  • the method according to the present embodiment is useful for screening for new drug candidates related to the prevention or onset of oligodendrocyte cell death and motor dysfunction associated with a reduction in fatty acid content.
  • mice and genotyping C57BL/6J mice on the first day after birth were purchased from Tokyo Experimental Animals Co., Ltd.
  • B6SJL-Tg SOD1 G93A 1Gur/J (catalog number: 002726, purchased from Jackson Laboratory) was used.
  • Genotyping of SOD1 G93A mice determined the genotype of DNA collected from the tail by the alkaline method by PCR using the following primers and Tks Glex DNA polymerase (manufactured by Takara Bio Inc.). PCR was performed under the conditions of 35 cycles of 95°C for 30 seconds, 95°C for 5 seconds, and 60°C for 45 seconds.
  • mice were housed in an air-conditioned room at 22-24°C with a 12-hour light/dark cycle, with free access to drinking water and food, and under SPF (specific pathogen free) conditions.
  • the mice were anesthetized using a triple anesthetic mixture (medetomidine hydrochloride (0.3 mg/kg, manufactured by ZENOAQ), midazolam (4 mg/kg, manufactured by Maruishi Pharmaceutical Co., Ltd.), and vetorfal tartrate (5 mg/kg, manufactured by Meiji Seika Pharma Co., Ltd.). (manufactured by )) was administered intraperitoneally. All animal experiments were conducted under the approval of the Animal Experiment Ethics Committee of the National Center of Psychiatry and Neurology.
  • qRT-PCR Quantitative reverse transcription PCR
  • Trizol reagent catalog number: 10296010, manufactured by Thermo Fisher Scientific.
  • Relative mRNA expression was normalized to Gapdh mRNA levels and calculated by the ⁇ / ⁇ -Ct method.
  • FIG. 1(b) is a heat map showing the content of various free fatty acids (FFA) in the plasma of each mouse, and shows the standardized value of the relative area of the detected peak with respect to FFA.
  • FFA free fatty acids
  • FIG. 1(c) is a graph showing the relative concentrations of oleic acid (OA) and linoleic acid (LA) with respect to the blood free fatty acid content of WT mice.
  • the content of FFA in the plasma of SOD1 G93A mice was lower at P60, P100, and P140 compared to the content in the plasma of WT mice at P100. Furthermore, the content of LPC tended to be lower than that of WT mice in the pathological condition after P100, and the content of LPA did not change even as the pathological condition progressed.
  • mice were perfused transcardially with 4% paraformaldehyde-phosphate buffered saline (PFA-PBS), and the lumbar spinal cord was post-fixed with 4% PFA-PBS at 4°C overnight. % sucrose-PBS overnight at 4°C.
  • the lumbar spinal cord was embedded in frozen sectioning embedding medium (Tissue-Tek, manufactured by Sakura Finetek), and 30 ⁇ m sections were prepared using a cryostat (Leica). Five sections were placed at 300 ⁇ m intervals on a Matsunami slide glass. (Matsunami Glass).
  • Permeabilization was performed twice for 10 minutes in 0.1% Triton X-100-PBS, and sections were treated with blocking solution (3% normal donkey serum (NDS)-PBS) for 1 hour at room temperature. Primary antibodies diluted in blocking solution were added to the sections and incubated overnight at 4°C. The sections were then incubated with secondary antibodies diluted in blocking solution for 1 hour at room temperature.
  • the secondary antibody dilution solution included Alexa Fluor 488, 568, 647-conjugated antibody (Thermo Fisher Scientific, 1:500) and 4',6-diamidino-2-phenylindole (1 mg/ml) for counterstaining. , Dojindo Laboratories).
  • the anterior horn of the lumbar spinal cord was photographed using a confocal laser scanning microscope (catalog number: FV3000, manufactured by Olympus) equipped with an objective lens of ⁇ 20/0.75 magnification.
  • the primary antibodies used were anti-cleaved caspase-3 (CC-3) antibody (catalog number: 9661, manufactured by Cell Signaling Technology, 1:1000) and anti-oligodendrocyte transcription factor 2 (Olig2) antibody (catalog number: AF2418, manufactured by R&D Systems, 1:1000), anti-Olig2 antibody (catalog number: MABN50, manufactured by Sigma-Aldrich, 1:1000), anti-CC1 antibody (catalog number: OP80, manufactured by Calbiochem, 1:1000), Anti-ionized calcium binding adapter molecule 1 (Iba1) antibody (Catalog number: 019-19741, manufactured by Wako Pure Chemical Industries, Ltd., 1:1000), anti-glial fibrillary acidic protein (GFAP) antibody (G3893, Sigma-Aldrich (manufactured by Millipore, 1:1000), anti-choline acetyltransferase (ChAT) antibody (catalog number: AB144P, manufactured by Millipore, 1:1000), anti
  • CC1 + Olig2 + CC-3 + cells Number of CC1 + Olig2 + CC-3 + cells, number of CC1 + Olig2 + cells, number of ChAT + cells, fluorescence intensity of Iba1 + cells, fluorescence intensity of GFAP + cells and of the ventral horn region of the lumbar spinal cord.
  • the area was measured using ImageJ software (manufactured by National Institute of Health).
  • CC1 is a marker for mature oligodendrocytes
  • Olig2 is a marker for oligodendrocyte cells.
  • FIG. 2(b) is a micrograph of cells stained with CC1, Olig2, or CC-3 in the vehicle group and the OA+LA administration group (scale bar indicates 50 ⁇ m). CC1 + Olig2 + CC-3 + cells are indicated by white arrows.
  • FIG. 2(c) is a graph showing the number of CC1 + Olig2 + CC-3 + cells in the lumbar spinal cord as a density per mm 2 ( * p ⁇ 0.05). Furthermore, when the total number of oligodendrocytes in the lumbar spinal cord was quantified, no difference was found in the number of CC1 + Olig2 + cells between the two groups.
  • Figure 3(a) shows the number of ChAT + cells per section in the lumbar spinal cord. There was a tendency for the number of ChAT + motor neurons to be larger in the OA + LA administration group than in the Vehicle group.
  • FIG. 3(b) shows a micrograph of immunohistochemical staining of Iba1 in the lumbar spinal cord of SOD1 G93A mice at P140 (scale bar indicates 100 ⁇ m), and a comparison of the number of motor neurons in the Vehicle group and the OA + LA administration group. It is a graph.
  • FIG. 3(b) shows the fluorescence intensity per section of GFAP in the lumbar spinal cord. Although no difference was observed in the fluorescence intensity of Iba1 + in the ventral horn of the spinal cord, the fluorescence intensity of GFAP was increased in the OA+LA administration group.
  • oligodendrocytes were prepared by the following method. Whole brains of P1 mice were collected and minced with fine scissors in ice-cold phosphate buffered saline (PBS). The finely minced tissue was incubated for 37 minutes using Dulbecco's Modified Eagle Medium (DMEM) (catalog number: 12800082, manufactured by Thermo Fisher Scientific) containing 0.25% trypsin (catalog number: 15090-046, manufactured by Thermo Fisher Scientific). The reaction was carried out at °C for 10 minutes.
  • DMEM Dulbecco's Modified Eagle Medium
  • the cells were washed with PBS, and the remaining cells were treated with 0.05% trypsin-PBS at 37°C for 3 minutes.
  • the detached cells were filtered using a 40 ⁇ m nylon cell strainer (catalog number: 353440, manufactured by FALCON), and the cells were seeded on a 10 cm dish not coated with PLL and incubated at 37° C. for 30 minutes.
  • the non-adherent cells were collected and placed in a PLL-coated 96-well plate (catalog number: 5866-960, manufactured by IWAKI) at a density of 5 ⁇ 10 cells/well in oligodendrocyte progenitor cell (OPC) medium.
  • PLL-coated 96-well plate catalog number: 5866-960, manufactured by IWAKI
  • Non-adherent cells were seeded and cultured for 3 days.
  • the OPC medium contains 1mM sodium pyruvate (catalog number: S8636, manufactured by Sigma-Aldrich), 0.1% BSA (manufactured by Sigma-Aldrich), 50 ⁇ g/ml apotransferrin (catalog number: T5391, manufactured by Sigma-Aldrich).
  • RNAiMAX Lipofectamine RNAiMAX
  • the differentiation medium was 1mM sodium pyruvate (catalog number: S8636, manufactured by Sigma-Aldrich), 0.1% BSA (manufactured by Sigma-Aldrich), 50 ⁇ g/ml apotransferrin (T5391, manufactured by Sigma-Aldrich), 5 ⁇ g/ml ml insulin (catalog number: I1882, manufactured by Sigma-Aldrich), 30 nM sodium selenite (catalog number: S9133, manufactured by Sigma-Aldrich), 10 nM biotin (manufactured by Sigma-Aldrich), 10 nM hydrocortisone (catalog number: H6909) DMEM/F12 containing 40 ng/ml triiodothyronine (Sigma-Aldrich) and 40 ng/ml triiodothyronine (Sigma-Aldrich).
  • oligodendrocytes Sixteen days after culturing the primary cultured oligodendrocytes, glutamic acid (100 ⁇ M) and OA+LA (30 ⁇ M each) were added and further cultured for 24 hours. Thereafter, a 1 ⁇ g/mL propidium iodide aqueous solution (PI, catalog number: 169-26281, manufactured by Wako Pure Chemical Industries, Ltd.) was added and cultured for 30 minutes, followed by immunocytochemical staining.
  • PI propidium iodide aqueous solution
  • Figure 4(b) is a micrograph showing immune cell staining obtained by staining primary cultured oligodendrocytes supplemented with Glu, OA+LA, or Glu+OA+LA with PI, DAPI, or MBP (scale bar is 50 ⁇ m). ).
  • Glu addition group the percentage of MBP + oligodendrocytes that took up PI was significantly increased compared to the Vehicle group.
  • Glu + OA + LA addition group the proportion of MBP + oligodendrocytes that took up PI decreased compared to the Glu addition group.
  • Introduction of siRNA against free fatty acid receptor 1 (FFAR1) resulted in a higher proportion of oligodendrocytes that took up PI compared to control siRNA.
  • FFAR1 siRNA was introduced, a decrease in the expression of FFAR1 was observed, and no change was observed in the PI positive rate when only FFAR1 siRNA was introduced.
  • FFAR1 was confirmed to be expressed in CC1 + Olig2 + oligodendrocytes in the spinal cord of SOD1 G93A mice.
  • siRNA libraries include Mukherjee et al. Cell Metab. 32, 259-272. e10, Allan et al. Cell Stem Cell 28, 257-272. e11., Simoneschi et al. Nature 592, 789-793. Described in Uyeda et al. Glia 69, 2591-2604.
  • siRNA targeting Ffar1 also referred to as siFfar1
  • OPCs Oligodendrocyte precursor cells
  • PDGFR ⁇ CD140a
  • PLL-coated 96-well glass plate together with a microbead kit (catalog number: 130-101-547, manufactured by Miltenyi Biotec). It was placed in A high purity culture (90.26 ⁇ 0.93%) of Olig2 + cells was obtained by purifying and culturing PDGFR ⁇ + OPCs using magnetic activated cell sorting (MACS).
  • MCS magnetic activated cell sorting
  • siRNA siControl or siFfar1
  • siRNA siControl or siFfar1
  • Glu or Glu+OA+LA was added to oligodendrocytes introduced with each siRNA, and the ratio of PI + MBP + cells to MBP + cells was examined.
  • FIG. 7(b) is a graph showing the influence of OA+LA treatment on glutamate-induced oligodendrocyte cell death in oligodendrocytes introduced with siControl or siFfar1.
  • oligodendrocytes introduced with siFfar1 glutamate-induced cell death was not significantly suppressed even when treated with OA+LA.
  • FIG. 7(d) is a graph showing the influence of OA+LA treatment on glutamate-induced oligodendrocyte cell death in oligodendrocytes introduced with siControl or siFfar1.
  • Figure 8(a) is a photograph showing immunohistochemistry for CC1 (green), Olig2 (red), and Ffar1 (cyan) in the spinal cord of SOD1 G93A mice at P100, and Merge is a photograph of these superimposed images. . As shown in FIG. 8(a), it was revealed that FFAR1 was expressed in CC1 + Olig2 + oligodendrocytes of SOD1 G93A mice.
  • Figure 8(b) is a photograph showing immunohistochemistry for CC1 (green), Olig2 (red), and CC-3 (cyan) in the spinal cord of SOD1 G93A mice at P100, and Merge superimposes them. This is a photo.
  • White triangular arrows indicate CC1 + Olig2 + CC-3 + cells.
  • Immunohistochemical analysis showed that in the anterior column of OA+LA-treated SOD1 G93A spinal cord, CC-3 + apoptotic cells remained unchanged in the number of CC1 + Olig2 + oligodendrocytes, as shown in Figure 8(b). The number of oligodendrocytes was reduced compared to vehicle treatment (control).
  • FIG. 8(c) is a graph quantifying the photograph of FIG. 8(b), and the vertical axis indicates the number of CC-3 + CC1 + Olig2 + cells per 1 mm 2 .
  • FIG. 8(d) is a graph quantifying the photograph of FIG. 8(b), and the vertical axis indicates the number of CC1 + Olig2 + cells per 1 mm 2 .
  • OA+LA suppressed the progression of grip strength decline. Furthermore, it was suggested that the protective effect of OA+LA on oligodendrocytes also contributes to alleviating the pathology of ALS.

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Abstract

La présente invention concerne un agent préventif, un inhibiteur de la progression d'une maladie, ou un agent thérapeutique pour des maladies démyélinisantes centrales, comprenant un agoniste du récepteur FFAR1.
PCT/JP2023/017205 2022-05-24 2023-05-02 Agent préventif, inhibiteur de la progression d'une maladie, ou agent thérapeutique pour des maladies démyélinisantes centrales WO2023228701A1 (fr)

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KR20210054741A (ko) * 2019-11-06 2021-05-14 건국대학교 산학협력단 진토닌을 포함하는 희소돌기아교세포 분화용 조성물 및 탈수초성 질환의 예방 또는 치료용 조성물

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KR20210054741A (ko) * 2019-11-06 2021-05-14 건국대학교 산학협력단 진토닌을 포함하는 희소돌기아교세포 분화용 조성물 및 탈수초성 질환의 예방 또는 치료용 조성물

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CHO YEON-JIN, CHOI SUN-HYE, LEE RAMI, HWANG HONGIK, RHIM HYEWHON, CHO IK-HYUN, KIM HYOUNG-CHUN, LEE JEONG-IK, HWANG SUNG-HEE, NAH : "Ginseng Gintonin Contains Ligands for GPR40 and GPR55", MOLECULES, MDPI AG, CH, vol. 25, no. 5, CH , pages 1102, XP093112393, ISSN: 1420-3049, DOI: 10.3390/molecules25051102 *
MARUYAMA TAKASHI, TANABE SHOGO, UYEDA AKIKO, SUZUKI TATSUNORI, MURAMATSU RIEKO: "Free fatty acids support oligodendrocyte survival in a mouse model of amyotrophic lateral sclerosis", FRONTIERS IN CELLULAR NEUROSCIENCE, FRONTIERS RESEARCH FOUNDATION, CH, vol. 17, CH , XP093112402, ISSN: 1662-5102, DOI: 10.3389/fncel.2023.1081190 *
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NAM SUNG MIN, CHOI JONG HEE, CHOI SUN-HYE, CHO HEE-JUNG, CHO YEON-JIN, RHIM HYEWHON, KIM HYOUNG-CHUN, CHO IK-HYUN, KIM DO-GEUN, NA: "Ginseng gintonin alleviates neurological symptoms in the G93A-SOD1 transgenic mouse model of amyotrophic lateral sclerosis through lysophosphatidic acid 1 receptor", JOURNAL OF GINSENG RESEARCH, GO'RYEO INSAM HAGHOE, SEOUL,, KR, vol. 45, no. 3, 1 May 2021 (2021-05-01), KR , pages 390 - 400, XP093112386, ISSN: 1226-8453, DOI: 10.1016/j.jgr.2020.04.002 *

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