WO2017130933A1 - Agent thérapeutique pour maladies neurodégénératives - Google Patents

Agent thérapeutique pour maladies neurodégénératives Download PDF

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WO2017130933A1
WO2017130933A1 PCT/JP2017/002244 JP2017002244W WO2017130933A1 WO 2017130933 A1 WO2017130933 A1 WO 2017130933A1 JP 2017002244 W JP2017002244 W JP 2017002244W WO 2017130933 A1 WO2017130933 A1 WO 2017130933A1
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therapeutic agent
lsd1
tranylcypromine
preventive
disease
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PCT/JP2017/002244
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Japanese (ja)
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秀信 谷原
圭一郎 岩尾
光善 中尾
林　秀樹
信次朗 日野
孝之 堤
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国立大学法人熊本大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
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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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • 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

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  • the present invention relates to a therapeutic agent for neurodegenerative diseases, and particularly relates to a therapeutic agent for neuroprotection.
  • Glaucoma is a major cause of blindness in the world. Glaucoma is well known as a major optic neuropathy characterized by retinal ganglion cell (RGC) loss, and RGC death causes visual field impairment. The major risk factor in most cases of visual field progression is elevated intraocular pressure (IOP). However, in normal-tension glaucoma patients, RGC suffers even if the IOP is within the normal range (10-20 mmHg).
  • IOP intraocular pressure
  • Epigenetic mechanisms affect gene expression and its function by DNA methylation, histone modifications, and non-coding RNA without changing the underlying DNA sequence. It is well known that histones are specifically modified by various mechanisms, such as N-terminal acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation of histones, and histone modifications are chromatin structures. Is a switch that changes the expression of a downstream “effector” protein, enabling transcriptional activation or repression. Recently, there have been several reports suggesting that multiple epigenetic factors play an important role in RGC survival and the development of glaucoma.
  • HDAC histone deacetylase
  • trichostatin A a major player in histone deacetylation
  • inhibition of retinal HDAC activity by trichostatin A is a representative RGC-specific gene.
  • Can be maintained and cell loss following optic nerve injury can be attenuated (Non-patent Document 1).
  • valproic acid another typical HDAC inhibitor, exhibits a neuroprotective effect, and leads to axonal regeneration after crushing the optic nerve by regulating the activity of the transcription factor CREB (cAMP response element binding protein).
  • Patent Document 3 gene removal of Hdac3 in retinal ganglion cells results in significant improvement in the characteristics of nuclear atrophy and significant suppression of RGC death in the acute phase after optic nerve injury.
  • Lysine-specific demethylase 1 (LSD1) is known as one of the flavin-containing amine oxidase family and is known to suppress transcription by monomethylation of histone H3 and removal of the methyl group from dimethylated lysine 4. ing. LSD1 has already been reported to have effects on cell proliferation, survival promotion, neurite morphogenesis, and neuronal differentiation and development (Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6; Non-patent document 7).
  • Non-Patent Document 4 Non-Patent Document 8
  • tranylcypromine an inhibitor of LSD1 regulates the proliferation of neural stem cells in the mouse brain and the development of zebrafish lateral sensation hills.
  • Non-Patent Document 8 Non-Patent Document 8
  • tranylcypromine an inhibitor of LSD1 regulates the proliferation of neural stem cells in the mouse brain and the development of zebrafish lateral sensation hills.
  • tranylcypromine an inhibitor of LSD1
  • Tranylcypromine has been reported to be a powerful dual action agent that inhibits LSD1 and MAO.
  • MAO catalyzes the oxidative deamination of food amines and monoamine neurotransmitters and produces by-products of MAO-related reactions including 3,4-dihydroxyphenylglycolaldehyde (DOPEGAL) or hydrogen peroxide.
  • DOPEGAL 3,4-dihydroxyphenylglycolaldehyde
  • HOPEGAL 3,4-dihydroxyphenylglycolaldehyde
  • These potential neurotoxic metabolites cause neuronal apoptosis and are considered to be one of the causes of neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. Therefore, inactivation of MAO is considered one of the excellent strategies for protecting nerve cells not only in vitro but also in vivo.
  • MAO inhibitors including L-deprenyl (Selegiline®) has become a novel pharmacotherapy for human neuropsychiatric disorders such as Parkinson's disease, treatment-resistant depression, anxiety disorders, and Alzheimer's disease. It has been reported.
  • Mitogen-activated protein kinase including ERK1 / 2, JNK and p38 is known as a molecule involved in survival, proliferation and differentiation in neurons and non-neuronal cells (Non-patent document 9; Non-patent document) 10; Non-Patent Document 11).
  • MAPK Mitogen-activated protein kinase
  • NF- ⁇ B activated B cell nuclear factor kappa light chain enhancer
  • the p38 MAPK family consists of four major isoforms, p38 ⁇ , p38 ⁇ , P38 ⁇ and p38 ⁇ , which are encoded by independent genes and functional redundancy among the p38 isoforms as well as isoform-specific functions. It is considered that there may be (Non-Patent Document 13). In central nervous system cells, there are multiple reports that the p38 ⁇ MAPK isoform contributes more significantly to the production and neurotoxicity of stress-induced proinflammatory cytokines than the isoform of p38 ⁇ (Non-Patent Literature). 14; Non-patent document 15; Non-patent document 16). However, the role of other types of p38 isoforms in neurons remained unclear. And it is reported that p38 (alpha) isoform causes the death of a neuron (nonpatent literature 17).
  • An object of the present invention is to provide a therapeutic agent for neurodegenerative diseases. More specifically, an object of the present invention is to provide a neuroprotective therapeutic agent. For example, it aims at providing the new therapeutic agent with respect to the neurodegenerative disease of an eye as one of the neurodegenerative diseases.
  • tranylcypromine an LSD1 inhibitor
  • tranylcypromine exhibits an eye neuroprotective effect, thereby completing the present invention.
  • the molecular target involved in RGC protection mediated by tranylcypromine is p38MAPK ⁇ and / or p38MAPK ⁇ , and completed further invention.
  • the present invention includes the following. [1] A preventive or therapeutic agent for a neurodegenerative disease comprising a substance that inhibits lysine-specific demethylase 1 (LSD1) activity as an active ingredient.
  • LSD1 lysine-specific demethylase 1
  • the substance is the following compound group:
  • a neuroprotective therapeutic agent comprising a substance that inhibits lysine-specific demethylase 1 (LSD1) activity as an active ingredient.
  • An inhibitor of retinal neuronal cell death comprising a substance that inhibits lysine-specific demethylase 1 (LSD1) activity as an active ingredient.
  • the present invention provides new therapeutic agents for neurodegenerative diseases, particularly neuroprotective therapeutic agents.
  • the present invention also provides a novel therapeutic agent for neurodegenerative diseases of the eye as one of neurodegenerative diseases.
  • FIG. 1 shows the decaying effect of tranylcypromine on glutamate neurotoxicity and apoptosis induced by oxidative stress.
  • FIG. 6 shows the result of detecting a fragmented or aggregated nucleus by Hoechst staining 24 hours after glutamic acid (Glu) -induced stress or oxidative (H 2 O 2 ) stress in the presence or absence of tranylcypromine (TC).
  • HBSS Hops Balanced Salt Solution
  • FIGS. A and B show RGC photographic images (upper figure) and fluorescent images (lower figure) after Glu stimulation.
  • FIG. 2 shows the result of confirming the protective effect of RGC from apoptosis by LSD1 inhibition.
  • FIG. A shows the results of Western blot analysis
  • FIG. B shows the results of band density ratios expressed as relative ratios.
  • FIG. C shows the results of confirming the effect of LSD1 siRNA on glutamate (Glu) -loaded apoptosis.
  • HBSS represents the control (Glu ( ⁇ ), S2101 ( ⁇ )), and Control represents only Glu.
  • LSD1 siRNA significantly suppressed apoptosis.
  • FIG. D shows the results of confirming the effect of LSD1 inhibitor S2101 on glutamate (Glu) -induced apoptosis.
  • HBSS represents a control.
  • S2101 shows a significant suppression effect.
  • Data in (C) and (D) represent mean ⁇ standard error.
  • FIG. 3 shows the results of confirming the promotion of p38MAPK ⁇ expression by tranylcypromine (TC) under glutamic acid (Glu) stress.
  • 3A, B and C are the results of Western blot analysis and densitometric evaluation in which phosphorylation of Akt by glutamic acid was confirmed.
  • FIGS. 3D to G show the results of Western blot analysis and densitometry evaluation for confirming the expression and phosphorylation of p38MAPK by glutamic acid and the expression of p38MAPK ⁇ .
  • HBSS represents a control. Glutamic acid had no effect on Akt expression.
  • tranylcypromine had no effect on Akt expression and Akt phosphorylation.
  • glutamic acid did not affect the expression of total p38 MAPK and its phosphorylated state.
  • Tranylcypromine did not affect the expression of total p38 MAPK and its phosphorylation.
  • tranylcypromine significantly promoted the expression of p38MAPK ⁇ .
  • FIG. 4 shows the results of confirming contribution of RGC to survival through p38MAPK ⁇ activity of tranylcypromine. RGC apoptosis was evaluated by changes in nuclear morphology.
  • FIG. 4A shows the result of confirming the effect of Waltmannin.
  • FIG. 5 shows the results of morphological confirmation of retinal protection from NMDA-induced stress by intravitreal administration of tranylcypromine.
  • FIG. 5A is a light microscopic image of a retinal section specimen after treatment with PBS (control) and tranylcypromine in a NMDA stress state.
  • FIG. 6 shows the results of confirming whether or not tranylcypromine suppresses caspase 3 activity in the retina after NMDA-induced damage.
  • FIG. 7 shows the results of confirming the neuroprotective effect of tranylcypromine (TC) against NMDA-induced apoptosis in vivo. Retrograde labeling of retinal ganglion cells (RGC) with fluorogold was performed.
  • FIG. 7A shows flat mount specimens under four conditions, ie, whether NMDA was administered intravitreally and whether TC was administered.
  • FIGS. 7B and C show the actual number of RGCs (intermediate retina (B), peripheral retina (C)).
  • the TC administration group shows a statistically larger number than the TC non-administration group.
  • LSD1 lysine-specific demethylase 1
  • LSD1 is an important regulator of neuronal survival, and new molecular targets for the treatment of neurodegenerative diseases I found out.
  • the present invention relates to a preventive or therapeutic agent for a neurodegenerative disease comprising a substance that inhibits LSD1 activity as an active ingredient.
  • to inhibit LSD1 activity means to inhibit the enzyme activity of LSD1, which is an enzyme, and to inhibit the expression of LSD1 activity in cells by suppressing the expression of LSD1 itself. It is.
  • the substance that inhibits the enzyme activity of LSD1, which is an enzyme, is not limited to this.
  • tranylcypromine (2-PCPA), 2-PFPA, S2101, S1310, S1401, S1402, S1502, S1601, S1602, S1603, S1603, S1603 , S2101, S2107, S2111 and S2206, and preferred are tranylcypromine and S2101, particularly preferred is tranylcypromine.
  • the structural formulas of these compounds are shown below.
  • the IC 50 for LSD1 inhibition of these compounds is shown in the following table.
  • Tranylcypromine is an LSD1 inhibitor having the following structure, but is a pharmaceutical approved in Europe and the United States as an antidepressant based on monoamine oxidase inhibitory activity.
  • Tranylcypromine has (1R, 2S) tranylcypromine and (1S, 2R) tranylcypromine as optical isomers.
  • any optical isomer may be used as long as it has LSD1 inhibitory activity.
  • a mixture thereof may be used.
  • it may be used in the form of a pharmacologically acceptable salt.
  • S2101 is an LSD1 inhibitor having the following structure.
  • S2101 has an optical isomer, but in the present invention, any optical isomer or a mixture thereof may be used as long as it has LSD1 inhibitory activity.
  • Examples of substances that inhibit the expression of LSD1 activity in cells by suppressing the expression of LSD1 itself include siRNA, shRNA, etc. of LSD1, and these genes are introduced into cells according to conventional methods. By knocking down the LSD1 gene in cells, the expression of LSD1 itself can be suppressed and the expression of LSD1 activity can be inhibited.
  • LSD1-specific siRNA can be designed based on known information and synthesized according to a conventional method, or is commercially available and can be used.
  • the neurodegenerative disease targeted by the prophylactic or therapeutic agent of the present invention is not limited thereto, and examples thereof include optic neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, and multiple sclerosis, and preferably the optic nerve. It is a degenerative disease.
  • the optic neurodegenerative disease targeted by the present invention includes optic neuropathy associated with glaucoma, ischemic optic neuropathy, traumatic optic neuropathy, label hereditary optic neuropathy, and preferably optic neuropathy associated with glaucoma. is there.
  • the target disease of the prophylactic or therapeutic agent of the present invention is particularly preferably a disease based on a disorder of retinal nerve cells, for example, a disease caused by retinal ganglion cell death.
  • the preventive agent or therapeutic agent of the present invention when used for a neurodegenerative disease based on glaucoma, it can be used in combination with other therapeutic agents or treatment methods for glaucoma.
  • a combination therapy with a therapeutic agent for reducing intraocular pressure and a combination therapy with a therapeutic agent for reducing intraocular pressure can be given.
  • the prophylactic or therapeutic agent of the present invention is a compound
  • the compound can be administered either orally or parenterally.
  • the dosage form include, but are not limited to, tablets, capsules, granules, powders, injections, suspensions, eye drops, eye ointments, sustained-release intraocular implants, and the like. it can. Formulation can be performed using techniques widely used in the field.
  • the target disease is an optic neurodegenerative disease, an intraocular injection or eye drop is preferable.
  • the dose of the compound is appropriately selected depending on the type of disease, the symptom of the administration subject, age, administration method, and the like.
  • the dose of the compound is appropriately selected depending on the type of disease, the symptom of the administration subject, age, administration method, and the like.
  • 0.1 to 5000 mg per day preferably 1 to 2000 mg, more preferably 5 to 1000 mg may be administered in one or several divided doses.
  • a solution of 0.01 to 5%, preferably 0.05 to 1% may be instilled several times per day.
  • a topical ocular injection for example, 0.1 mg to 150 mg, preferably 1 mg to 100 mg, more preferably 10 mg to 50 mg of a drug dissolved in physiological saline is used as a symptom. Accordingly, the injection may be performed intraocularly, preferably intravitreally. In addition, preparation of an injection can be performed based on a conventional method.
  • the prophylactic or therapeutic agent of the present invention is a gene such as siRNA
  • the gene can be administered by injection.
  • siRNA in the treatment of glaucoma, for example, siRNA can be administered directly or indirectly into the vitreous of the eye.
  • Another preferred embodiment of the present invention is ocular neurodegeneration by preventing and / or restoring loss of retinal ganglion cells (RGC) comprising as an active ingredient a substance that inhibits lysine-specific demethylase 1 (LSD1) activity. It is a preventive and / or therapeutic drug for diseases. Thereby, it is possible to prevent and / or treat an RGC disorder seen in glaucoma patients, for which a sufficient effect cannot be expected with a therapeutic agent or a treatment method aimed at lowering the elevated intraocular pressure.
  • RGC retinal ganglion cells
  • Tranylcypromine is also known as 2-phenylcyclopropylamine. Tranylcypromine was purchased from Sigma-Aldrich. S2101 (LSD1 inhibitor II, also known as 489477) was obtained from Merck Millipore. Two day old Sprague Dawley (SD) rats obtained from Kudo were used for isolation of RGCs. The experimental procedure was performed according to the Statement for the Use of Animals in Ophthalmic and Vision Research of Association for Research in Vision and Ophthalmology (ARVO). All experimental procedures were approved by the Animal Experiment Committee of Kumamoto University.
  • the cell suspension was first incubated on a panning plate (150 mm Petri dish) coated with goat anti-rabbit IgG.
  • Non-adherent cells were incubated on a second panning plate (100 mm Petri dish) coated with goat anti-mouse IgM ⁇ and mouse anti-Thy1.1 antibody secreted from T11D7e2 cells.
  • PBS phosphate buffered saline
  • the isolated RGC was added to 1 mM glutamine, 5 ⁇ g / ml insulin, 60 ⁇ g / ml N-acetylcysteine, 62 ng / ml progesterone, 16 ⁇ g / ml putrescine, 40 ng / ml sodium selenite, 0.1 mg / Ml bovine serum albumin, 40 ng / ml triiodothyronine, 0.1 mg / ml transferrin, 1 mM sodium pyruvate, 2% B-27 supplement (# 17504-044, Invitrogen), 10 ⁇ M false Choline, 50 ng / ml brain-derived neurotrophic factor (BDNF) (PeproTech), 50 ng / ml ciliary neurotrophic factor (CNTF) (PeproTech), and 50 ng / ml basic fibroblast growth factor (PeproTech) And suspended in Neurobasal medium containing Culture plates (96 wells
  • Immunoblotting was performed according to the method of Hayashi et al. Proteins were separated by SDS polyacrylamide gel electrophoresis, transferred to PVDF membrane, and detected with primary and peroxidase-conjugated secondary antibodies. Immunoreactive proteins were visualized with SuperSignal West Pico, Dura or Femoto (Thermo Fisher Scientific).
  • mice anti- ⁇ -actin # A5441, Sigma-Aldrich
  • rabbit anti-LSD1 C69G12
  • rabbit anti-phosphorylated-p38 MAPK Thr180 / Tyr182
  • D3F9 # 4511, Cell Signaling Technology
  • rabbit anti-p38 MAPK # 9212, Cell Signaling Technology
  • rabbit anti-phosphorylated SAPK3 Thr183 + Tyr185
  • rabbit anti-p38 ⁇ MAPK # 2307, Cell Signaling Technology
  • Rabbit anti-phosphorylated Akt Ser473
  • 9271, Cell Signaling Technology rabbit anti-Akt
  • 9272 Cell Signaling Technology
  • rabbit anti-cleaved Caspase3 Asp175)
  • RGC apoptosis of retinal ganglion cells The RGC apoptosis was evaluated as described in Hayashi et al. (2012). Primary RGCs were washed twice with Hanks balanced salt solution (HBSS, Invitrogen) containing 2.4 mM CaCl 2 , 20 mM HEPES (no magnesium) (15 minutes incubation at 37 ° C.). Magnesium was removed from the wash solution to avoid blocking the NMDA receptor. Subsequently, RGCs in HBSS containing 2.4 mM CaCl 2 , 20 mM HEPES (without magnesium), together with 300 ⁇ M glutamate and 10 ⁇ M glycine, which are coactivators of NMDA receptors, at 37 ° C.
  • HBSS Hanks balanced salt solution
  • Invitrogen Hanks balanced salt solution
  • Magnesium was removed from the wash solution to avoid blocking the NMDA receptor.
  • RGCs were cultured at 37 ° C. for 22 hours in the same medium without neurotrophic factors such as forskolin, BDNF, CNTF and bFGF.
  • Apoptosis was also induced by adding a nutrient additive containing “B27 Supplement AO depleted of antioxidant” (Invitrogen) and 50 ⁇ M hydrogen peroxide for 0.5 hours, and the cells were cultured for 24 hours.
  • Tranylcypromine is added simultaneously with the addition of glutamic acid or hydrogen peroxide, while S2101 (# 489477, Merck Millipore), BIRB796 (# S1574, Selleck Chemical), SB203580 (# 199-16551, Wako) induce apoptosis.
  • S2101 # 489477, Merck Millipore
  • BIRB796 # S1574, Selleck Chemical
  • SB203580 # 199-16551, Wako
  • Apoptosis detection using Hoechst 33342 was performed by incubating RGC with 1.0 g / ml Hoechst 33342 for 15 minutes. Fluorescence images were observed using an IX71 fluorescence microscope (Olympus) and 96 well plates were used to obtain at least 6 images / well. Fragmented or reduced nuclei stained with Hoechst dye were counted as apoptotic neurons and round / smooth nuclei were counted as healthy neurons according to Hayashi et al. In order to minimize measurement bias, the software MetaMorph (Molecular Devices) was used to automatically measure over 200 neurons for each condition.
  • siRNA Non-silencing small interfering RNA (siRNA) (1 ⁇ M) (Accell non-targeting siRNA # 1, Thermo Fisher Scientific) or specific siRNA of LSD1 (E-105863-00-0010, Accell) Rat Kdm1a [Gene ID: 500569] siRNA SMARTpool, Thermo Fisher Scientific) was added to the medium according to the manufacturer's instructions and incubated with RGC for 6 days. The knockdown effect by LSD1 siRNA was confirmed using the immunoblotting method.
  • RNA was amplified, labeled and hybridized to Rat GE 4x44K v3 Microarray Kit (Agilent Technologies) according to the manufacturer's instructions. All hybridized microarrays were scanned with an Agilent scanner and all probe signals were calculated using Feature Extraction Software (Agilent Technologies). Using the procedure recommended by Agilent, the raw signal intensity and flag for each probe was calculated from the hybridization intensity and spot information. In order to make a comparison between control and experimental samples to identify up-regulated and down-regulated genes, select a probe with the P flag registered in at least one sample, and use an intensity-based Z-score And the ratio (non-log scale fold change) was calculated from the normalized signal intensity of each probe.
  • a gene having a Z score of 2.0 or less and a ratio of 1.5 times or more was regarded as an up-regulated gene, and a Z score of -2.0 or less and a ratio of 0.66 or less was regarded as a down-regulated gene.
  • the results were “control” vs. “glutamate stimulation” and “glutamate stimulation” vs. “glutamate stimulation + tranylcypromine”.
  • NMDA-induced retinal damage Intravitreal injection of NMDA was performed in the same manner as reported by Inomata (Inomata et al. (2006) Journal of Neurochemistry, 98, 372-385). Briefly, rats were anesthetized by intraperitoneal injection of a 1: 1 mixture of xylazine hydrochloride (4 mg / kg) and ketamine hydrochloride (10 mg / kg). The pupil was dilated with phenylephrine hydrochloride and tropicamide and 20 nanomolar NMDA was injected into the vitreous cavity.
  • Tranylcypromine was injected into SD rats simultaneously with NMDA administration.
  • NMDA was mixed with 500 mM tranylcypromine solution or phosphate buffered saline (PBS) as a control to make the total volume 2.0 ⁇ L, and injected into the vitreous cavity.
  • PBS phosphate buffered saline
  • Morphological analysis in vivo Morphological analysis for evaluating the protective effect was performed according to a report (Inomata et al., (2003) Brain Research, 991, 163-170). Briefly, 7 days after NMDA injection, the animals were euthanized by carbon dioxide asphyxiation and the eyes were removed. The eyes were fixed with 4% paraformaldehyde overnight at 4 ° C., followed by dehydration and paraffin embedding. A 4 ⁇ m-thick transverse section was prepared from the eye of a rat with an optic disc and stained with hematoxylin and eosin for morphological analysis. The thickness of the inner reticulated layer (IPL) was measured from 1.0 to 1.5 mm from the optic nerve head. Using a microscope BX51 (Olympus), the data of the minimum values of the three sections were averaged to obtain numerical values for each eye.
  • IPL inner reticulated layer
  • rats were deeply anesthetized by intraperitoneal injection of a 1: 1 mixture of xylazine hydrochloride (4 mg / kg) and ketamine hydrochloride (10 mg / kg), then their head hair was shaved and the skin was An incision was made at the midline to expose the skull and sutures (sagittal, coronal and lateral sutures). Both 2 mm diameter craniotomys were performed 0.5 mm on the posterior side of the sagittal and transverse suture lines. The brain material on the upper hill was then carefully removed with a vacuum pump. In each hole on the surface of the upper hill, a small piece of sterile sponge was pre-immersed in 6 ⁇ L of 6% FG solution.
  • the skin wound was then sutured closed. After surgery, the rats were kept warm and recovered themselves. Seven days after NMDA injection, the animals were euthanized and intracardiac perfused with PBS to flush the blood. After PBS irrigation, perfusion was performed with 4% paraformaldehyde. After perfusion fixation, the eyes were removed. The eyes were fixed with 4% paraformaldehyde overnight at 4 ° C.
  • the retina was removed from the sclera and divided into 4 quadrants (excellent, inferior, nasal, and temporal) and mounted on a slide. Each quadrant was subdivided into three regions (center, middle, periphery) that are 1.0, 2.0, and 3.0 mm from the optic nerve transcranial head. A total of 12 fields per retina were analyzed in a masked fashion by counting the number of FG-labeled RGCs using an all-in-one fluorescence microscope BZ-X710 (Keyence).
  • Tranylcypromine has a dual molecular effect of Lsd1 inhibitor and monoamine oxidase (MAO) inhibition.
  • Monoamine oxidase inhibitory activity may be related to anti-apoptotic and neuroprotective activity. Therefore, it was next confirmed whether or not tranylan cypromine exhibits a neuroprotective action through an anti-Lsd1 effect. First, after knocking down LSD1, the survival of neurons was confirmed, and the knockdown effect was verified. Immunoblotting results showed that Lsd1 (Kdm1a) specific siRNA significantly suppressed Lsd1 expression to 30.4 percent compared to non-silencing siRNA controls (FIGS.
  • Example 3 Analysis of target pathway and gene in survival of retinal ganglion cells by administration of tranylcypromine The signal transduction pathway involved in the neuroprotective effect of tranylcypromine was examined.
  • a microarray analysis including changes in 26,930 gene expression was performed. Since LSD1 has been previously reported to suppress gene transcription through the demethylation reaction of lysine 4 of histone H3, the present inventors have amplified expression by tranylcypromine and simultaneously administered glutamate. It was hypothesized that genes with reduced expression are good candidates for identifying gene targets that modulate neuronal viability.
  • KEGG route annotation analysis was performed using Database for annotation, Visualization and Integrated Discovery (DAVID). For the functional classification associated with 110 gene candidates, three enriched KEGG pathway terms were significantly detected. These were involved in fcgammaR-mediated phagocytosis, neurotrophin signaling pathway, and purine metabolism (shown in Table 2).
  • Example 4 Enhancement of RGC Survival through p38MAPK ⁇ Activity by Tranylcypromine
  • neurotrophin signaling molecule v-akt mouse thymoma virus oncogene homolog 1 (Akt) and mitogen activation
  • Akt v-akt mouse thymoma virus oncogene homolog 1
  • mitogen activation We focused on protein kinase 12 (P38 MAPK ⁇ ). This is because the PI3K / Akt / mTOR pathway and the MAP kinase pathway were well known as major signaling pathways for cell survival and anti-apoptotic activity.
  • SB203580 is reported to be a specific inhibitor of p38MAPK ⁇ and p38MAPK ⁇ , but not p38MAPK ⁇ or p38MAPK ⁇ . Therefore, these results on pharmacological action indicate that tranylipypromine contributes to RGC survival via p38 MAPK ⁇ activity.
  • Example 5 Confirmation of RGC Survival Promotion by Tranylcypromine In Vivo It was confirmed in vivo whether or not tranylcypromine regulates RGC survival under stress conditions. Materials and methods (7)-(10).
  • an in vivo NMDA-induced retinal injury model was used to evaluate morphological retinal changes after tranylcypromine administration. Intravitreal NMDA significantly reduced IPL thickness compared to PBS control.
  • the retina treated with tranylcypromine and injected with NMDA maintained the IPL thickness at the same level as the control retina. In other words, administration of tranylcypromine was able to completely recover glutamate-induced retinal damage. The results are shown in FIG.
  • NMDA-induced caspase 3 activity after administration of tranylcypromine was measured by Western blot analysis. As confirmed by eyes treated with NMDA and vehicle, cleaved caspase 3 was significantly induced in the retina 18 hours after NMDA injection, but this activity was significantly suppressed in the retina treated with tranylcypromine. It had been. The results are shown in FIG. These results indicate that intravitreal administration of tranylcypromine exerts a neuroprotective effect on the intracellular apoptosis signaling pathway and suppresses retinal morphological changes.
  • tranylcypromine protects RGC from NMDA-induced retinal neurotoxicity.
  • the actual number of RGCs retro-labeled with fluorogold was counted in the retina for each group of sham (control), tranylcypromine treatment, NMDA treatment, and NMDA and tranylcypromine treatment.
  • the number of RGCs in the NMDA administration group 7 days after NMDA intravitreal injection was reduced to 21.4% in the intermediate region of the retina and 23.4% in the peripheral region.
  • the number of RGCs was 48.7% and 50.9%, respectively, in the middle region and the peripheral region, which were significantly increased.
  • the results are shown in FIG. Therefore, intravitreal tranylcypromine treatment enhanced RGC survival after retinal damage due to NMDA neurotoxicity.
  • tranylcypromine not only maintains the thickness of the inner plexiform layer but also reduces RGC loss after NMDA administration, which is an overload on the retina of rats. It was. Thus, tranylcypromine targeting LSD1 activity could enhance the neuroprotective effect in vivo during retinal damage.
  • tranylcypromine a typical LSD1 inhibitor, is prominent in retinal ganglion cells by promoting the expression of neuronal P38 ⁇ isoforms that are considered main players to increase neuronal survival. Shows neuroprotective effect.
  • Topical administration of tranylcypromine also provided neuroprotection for retinal ganglion cells in a rat NMDA-induced excitotoxic stress model. This indicates that treatment of neurodegenerative diseases such as glaucoma is possible by suppressing LSD1.
  • the present invention is useful as a preventive or therapeutic agent for novel neurodegenerative diseases.
  • the present invention is also useful as a new preventive or therapeutic agent for ocular neurodegenerative diseases such as glaucoma.

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  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

La présente invention a pour but de fournir un agent thérapeutique pour des maladies neurodégénératives, plus précisément de fournir un agent thérapeutique neuroprotecteur, par exemple un nouvel agent thérapeutique pour maladies neurodégénératives ophtalmiques faisant partie des maladies neurodégénératives. La présente invention concerne un agent thérapeutique ou un agent prophylactique destiné aux maladies neurodégénératives, qui contient, en tant que principe actif, une substance qui inhibe l'activité de la déméthylase spécifique de la lysine 1 (LSD1). La présente invention concerne également un agent thérapeutique neuroprotecteur contenant une substance inhibant l'activité de la LSD1 en tant que principe actif.
PCT/JP2017/002244 2016-01-25 2017-01-24 Agent thérapeutique pour maladies neurodégénératives WO2017130933A1 (fr)

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US10047086B2 (en) 2014-07-10 2018-08-14 Incyte Corporation Imidazopyridines and imidazopyrazines as LSD1 inhibitors
US10112950B2 (en) 2014-07-10 2018-10-30 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US10125133B2 (en) 2014-07-10 2018-11-13 Incyte Corporation Substituted [1,2,4]triazolo[1,5-a]pyridines and substituted [1,2,4]triazolo[1,5-a]pyrazines as LSD1 inhibitors
US10138249B2 (en) 2014-07-10 2018-11-27 Incyte Corporation Triazolopyridines and triazolopyrazines as LSD1 inhibitors
US10166221B2 (en) 2016-04-22 2019-01-01 Incyte Corporation Formulations of an LSD1 inhibitor
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US10676457B2 (en) 2014-02-13 2020-06-09 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
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US11247992B2 (en) 2014-02-13 2022-02-15 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US9994546B2 (en) 2014-02-13 2018-06-12 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10174030B2 (en) 2014-02-13 2019-01-08 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10300051B2 (en) 2014-02-13 2019-05-28 Incyte Corporation Cyclopropylamines as LSD1 inhibitors
US10968221B2 (en) 2014-07-10 2021-04-06 Incyte Corporation Substituted [1,2,4]triazolo[1,5-a]pyrazines as LSD1 inhibitors
US10556908B2 (en) 2014-07-10 2020-02-11 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US10640503B2 (en) 2014-07-10 2020-05-05 Incyte Corporation Imidazopyridines and imidazopyrazines as LSD1 inhibitors
US10112950B2 (en) 2014-07-10 2018-10-30 Incyte Corporation Substituted imidazo[1,2-a]pyrazines as LSD1 inhibitors
US10125133B2 (en) 2014-07-10 2018-11-13 Incyte Corporation Substituted [1,2,4]triazolo[1,5-a]pyridines and substituted [1,2,4]triazolo[1,5-a]pyrazines as LSD1 inhibitors
US10138249B2 (en) 2014-07-10 2018-11-27 Incyte Corporation Triazolopyridines and triazolopyrazines as LSD1 inhibitors
US10047086B2 (en) 2014-07-10 2018-08-14 Incyte Corporation Imidazopyridines and imidazopyrazines as LSD1 inhibitors
US10800779B2 (en) 2015-04-03 2020-10-13 Incyte Corporation Heterocyclic compounds as LSD1 inhibitors
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US10723700B2 (en) 2015-08-12 2020-07-28 Incyte Corporation Salts of an LSD1 inhibitor
US10166221B2 (en) 2016-04-22 2019-01-01 Incyte Corporation Formulations of an LSD1 inhibitor
US10968200B2 (en) 2018-08-31 2021-04-06 Incyte Corporation Salts of an LSD1 inhibitor and processes for preparing the same
US11512064B2 (en) 2018-08-31 2022-11-29 Incyte Corporation Salts of an LSD1 inhibitor and processes for preparing the same
KR20210001326A (ko) * 2019-06-27 2021-01-06 재단법인대구경북과학기술원 2-펜틸퓨란을 유효성분으로 함유하는 퇴행성 뇌질환 치료용 조성물
WO2020263012A1 (fr) * 2019-06-27 2020-12-30 재단법인대구경북과학기술원 Composition pour le traitement de maladies dégénératives du cerveau, contenant du 2-pentylfurane en tant que principe actif
JP2022540020A (ja) * 2019-06-27 2022-09-14 テグ・ギョンブク・インスティテュート・オブ・サイエンス・アンド・テクノロジー ２－ペンチルフランを有効成分として含有する退行性脳疾患の治療用組成物
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