WO2023199828A1 - Particule fine, médicament prophylactique ou médicament thérapeutique pour une maladie oculaire néovasculaire, et méthode d'amélioration d'une maladie oculaire néovasculaire - Google Patents

Particule fine, médicament prophylactique ou médicament thérapeutique pour une maladie oculaire néovasculaire, et méthode d'amélioration d'une maladie oculaire néovasculaire Download PDF

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WO2023199828A1
WO2023199828A1 PCT/JP2023/014155 JP2023014155W WO2023199828A1 WO 2023199828 A1 WO2023199828 A1 WO 2023199828A1 JP 2023014155 W JP2023014155 W JP 2023014155W WO 2023199828 A1 WO2023199828 A1 WO 2023199828A1
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hsa
mir
microparticles
stem cells
microparticle
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Japanese (ja)
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祥嗣 古賀
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Ｄｅｘｏｎファーマシューティカルズ株式会社
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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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • the present invention relates to microparticles, a preventive or therapeutic agent for angiogenic eye diseases, and a method for improving angiogenic eye diseases.
  • the present invention relates to preventive or therapeutic agents for angiogenic eye diseases and methods for improving angiogenic eye diseases, where the angiogenic eye disease is age-related macular degeneration.
  • Eye diseases associated with pathological neovascularization such as age-related macular degeneration (AMD), age-related macular degeneration, diabetic retinopathy, diabetic macular edema (DME), central retinal vein occlusion (CRVO), and corneal neovascularization.
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • CRVO central retinal vein occlusion
  • corneal neovascularization is known.
  • therapeutic drugs for angiogenic eye diseases and their mechanisms of action have also been known (for example, see Patent Document 1 and Non-Patent Document 1).
  • Methods are known to inhibit vascular endothelial growth factor (VEGF), which is responsible for inappropriate new blood vessel growth.
  • Patent Document 1 describes a method in which ranibizumab and an anti-VEGF antibody are administered monthly by intravitreal injection, and a method in which multiple doses of a VEGF antagonist are administered to a patient once every eight weeks or more. is listed.
  • Non-Patent Document 1 states that the infiltration of M2 macrophages into the retina that causes pathological angiogenesis is dependent on leukotriene B4, and that BLT1 antagonists and leukotriene B4-producing enzyme inhibition are used.
  • the drug has been described to suppress pathological angiogenesis, leading to a new treatment for age-related macular degeneration.
  • New drugs other than those described in Patent Document 1 and Non-Patent Document 1 are required as preventive or therapeutic agents for neovascular eye diseases.
  • the problem to be solved by the present invention is to provide novel microparticles that can control (suppress, inhibit, etc.) the expression of proteins and/or genes associated with neovascular eye diseases such as age-related macular degeneration. .
  • microparticles containing specific microRNAs can control (suppress or inhibit, etc.) the expression of proteins and/or genes associated with angiogenic eye diseases such as age-related macular degeneration.
  • Microparticles containing miRNA that targets at least one gene among the gene related to the expression of vascular endothelial growth factor (VEGF-A) protein and the leukotriene A4 hydrolase (LTA4H) gene [2] The microparticle according to [1], wherein the microparticle is an exosome. [3] The microparticles contain miRNA that targets at least one gene among a gene related to the expression of VEGF-A and LTA4H at a higher concentration than the culture supernatant of dental pulp-derived stem cells, as described in [1] microparticles.
  • VEGF-A vascular endothelial growth factor
  • LTA4H leukotriene A4 hydrolase
  • a VEGF-A expression inhibitor The microparticle according to [1], wherein the microparticle contains miRNA whose target gene is a gene related to the expression of VEGF-A.
  • the microparticle contains at least one type of the following VEGF-A suppression-related miRNA group; VEGF-A suppression related miRNA group: hsa-miR-1-3p, hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-107, hsa-miR-126-3p, hsa-miR-134-5p, hsa-miR- 140-5p, hsa-miR-145-5p, hsa-miR-150-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miRNA
  • Microparticles contain VEGF-A suppression-related miRNAs, hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-423-3p, and hsa- The microparticle according to claim 5, comprising miR-93-5p.
  • An expression inhibitor of LTA4H The microparticle according to claim 1, wherein the microparticle contains miRNA that targets LTA4H.
  • LTA4H suppression-related miRNA group LTA4H suppression-related miRNA group: hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-miR-124-3p, hsa-miR-125b-5p, hsa-miR-26b-5p, hsa- miR-338-3p.
  • microparticle according to claim 8 wherein the microparticle contains hsa-let-7a-5p, hsa-let-7b-5p, and hsa-miR-125b-5p among the LTA4H suppression-related miRNA group.
  • the microparticles are microparticles purified and isolated from the culture supernatant of dental pulp-derived stem cells, The microparticle according to [1], wherein the microparticle does not contain components other than the culture supernatant of dental pulp-derived stem cells except for exosomes.
  • a prophylactic or therapeutic agent for angiogenic eye diseases comprising the microparticle according to [1].
  • novel microparticles can be provided that can control the expression of proteins related to angiogenic eye diseases such as age-related macular degeneration.
  • FIG. 1 is a flowchart showing the developmental mechanism of age-related macular degeneration.
  • FIG. 2 is a schematic diagram showing the mechanism of action of a leukotriene production inhibitor and an LTB4 inhibitor.
  • Figure 3 shows the expression level of miRNA expressed in exosomes (microparticles of Example 1; SGF exosome) purified from the culture supernatant of dental pulp-derived stem cells targeting VEGFA, and the expression level of miRNA in the cerebral cortex. This is a heat map shown in comparison with the amount.
  • Figure 4 shows the expression level of miRNA expressed in exosomes (microparticles of Example 1; SGF exo) purified from the culture supernatant of dental pulp-derived stem cells targeting LTA4H, and the expression level of miRNA in the cerebral cortex.
  • FIG. 5 is a heat map showing the expression level of miRNA expressed in exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells targeting VEGFA or LTA4H.
  • the microparticles of the present invention contain miRNA that targets at least one of a gene related to the expression of vascular endothelial growth factor (VEGF-A) protein and a leukotriene A4 hydrolase (LTA4H) gene.
  • VEGF-A vascular endothelial growth factor
  • LTA4H leukotriene A4 hydrolase
  • the microparticles of the invention are capable of regulating the expression of proteins and/or genes associated with neovascular eye diseases such as age-related macular degeneration.
  • the microparticles of the present invention can improve, and more preferably prevent or treat, angiogenic eye diseases such as age-related macular degeneration.
  • preferred embodiments of the microparticles of the present invention will be explained.
  • angiogenic eye disease refers to eye disease associated with pathological angiogenesis.
  • Neovascular eye diseases include age-related macular degeneration (AMD), age-related macular degeneration, diabetic retinopathy, diabetic macular edema (DME), central retinal vein occlusion (CRVO), and corneal neovascularization.
  • AMD age-related macular degeneration
  • DME diabetic macular edema
  • CRVO central retinal vein occlusion
  • corneal neovascularization corneal neovascularization.
  • CNV choroidal neovascularization
  • Leakage from CNV causes macular edema and fluid collection under the macula, leading to vision loss.
  • Diabetic macular edema (DME) is another eye disease that involves neovascularization.
  • DME is the most common cause of moderate vision loss in diabetic patients and is a common complication of diabetic retinopathy, a disease that affects blood vessels in the retina. DME occurs when fluid leaks into the center of the macula, a light-sensitive part of the retina. Fluid within the macula can cause severe vision loss, or blindness. Yet another eye disease associated with abnormal angiogenesis is central retinal vein occlusion (CRVO). CRVO is caused by occlusion of central veins that lead to a backup of blood and fluid in the retina. The retina can also become ischemic, leading to inappropriate new blood vessel growth, which can lead to further vision loss and more severe complications.
  • CRVO central retinal vein occlusion
  • the microparticles of the present invention are preferably capable of preventing or treating age-related macular degeneration among these angiogenic eye diseases.
  • Types of age-related macular degeneration include, for example, precursor lesions (no effect on visual acuity, but a slight distortion is noticed), atrophic type (slow retinal function weakens, and often only mild visual loss), Examples include exudative type (new blood vessels develop from the choroid; early treatment is required).
  • the age-related macular degeneration to which the microparticles of the present invention are administered is preferably wet type age-related macular degeneration.
  • age-related macular degeneration will be explained in detail as a representative example of angiogenic eye diseases. However, the description herein also applies to neovascular eye diseases other than age-related macular degeneration.
  • FIG. 1 A flowchart showing the developmental mechanism of pathological angiogenesis in age-related macular degeneration is shown in FIG. 1 (see JCI Insight., 2018; 3(18): e96902).
  • FIG. 1 The flowchart of FIG. 1 will be explained.
  • Retinal damage occurs.
  • Leukotriene B4 (LTB4) is produced.
  • leukotriene B4 binds to the leukotriene B4 receptor (BLT1).
  • BLT1 leukotriene B4 receptor
  • BLT1 leukotriene B4 receptor
  • M2 macrophages M2 macrophages.
  • Macrophages produce vascular endothelial growth factor (VEGF).
  • VEGF Vascular endothelial growth factor
  • VEGF Vascular endothelial growth factor
  • macrophages are not a homogeneous cell population, but a group of cells with different characteristics.
  • M1 macrophages are considered to be inflammation-inducing cells
  • M2 macrophages are considered to be inflammation-containing cells.
  • M2 macrophages have the effect of promoting angiogenesis.
  • the microparticles of the present invention are preferably capable of improving angiogenic eye diseases such as age-related macular degeneration.
  • the therapeutic agent for age-related macular degeneration and its mechanism of action will be explained together with the mechanism of action of the microparticles of the present invention.
  • Therapeutic drugs and mechanisms of action for age-related macular degeneration include the following: (a) Treatment with VAGF-A inhibitor
  • VAGF-A inhibitor inhibits the production of VAGF and suppresses the production of VAGF.
  • Examples include SPRED1 and PIK3R2, which are VEGF signal suppressing proteins.
  • the microparticles of the present invention when the microparticles contain miRNA that targets a gene related to the expression of VEGF-A, preferably a gene expression regulator, more preferably a VAGF expression inhibitor, particularly preferably a VAGF-A expression inhibitor. Functions as an inhibitor.
  • Leukotriene production inhibitor inhibits the production of leukotriene B4 (LTB4) or the production of compounds that are raw materials for LTB4, thereby suppressing the production of LTB4.
  • LTB4 leukotriene B4
  • the mechanisms of action of the leukotriene production inhibitor and LTB4 inhibitor are shown in FIG.
  • the inhibitor (Zileuton) of the protein 5-lipoxygenase (5-LO) involved in the production of leukotriene A4 (LTA4) shown in Figure 2 and the inhibitor (MK-886) of the protein 5-lipoxygenase activating protein (FLAP) are Used as a leukotriene production inhibitor.
  • an inhibitor of the LTA4H gene (Bestatin; Ubenimesku), which is involved in the production of an enzyme that converts leukotriene A4 (LTA4) to leukotriene B4 (LTB4), is used as a leukotriene production inhibitor.
  • the microparticles when the microparticles contain miRNA that targets LTA4H, it preferably functions as a gene expression regulator, more preferably a leukotriene production inhibitor, and particularly preferably an LTA4H expression suppressor.
  • the bioactive lipid leukotriene B4 is a bioactive fatty acid enzymatically produced from arachidonic acid. It is also known as a chemotactic factor that causes neutrophils, eosinophils, and differentiated T cells to migrate to sites of inflammation.
  • BLT1 antagonist Antagonizes or inhibits the binding of LTB4 to the receptor BLT1. As shown in FIG. 2, CP105696 is known as an antagonist of the binding of LTB4 to the receptor BLT1.
  • BLT1 is a high affinity cell membrane receptor for leukotriene B4. BLT1 is expressed in neutrophils, eosinophils, and differentiated T cells, and transmits a signal into the cells that leukotriene B4 has bound, resulting in cell chemotaxis.
  • BLT1 antagonists are expected to be therapeutic agents for inflammatory diseases such as rheumatoid arthritis and bronchial asthma. Note that BLT1 antagonists have little relevance to the microparticles of the present invention.
  • microparticles of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells such as dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium.
  • the microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably are microparticles derived from the culture supernatant of dental pulp-derived stem cells.
  • the microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily need to be obtained from the culture supernatant of dental pulp-derived stem cells.
  • the microparticles inside dental pulp-derived stem cells are isolated by any method, if they are the same as the microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, then It can be said that they are microparticles derived from water.
  • Microparticles derived from a culture supernatant such as dental pulp-derived stem cells may be used in a state contained in the culture supernatant, or may be used in a state purified from the culture supernatant.
  • the microparticles are microparticles purified from culture supernatant.
  • the origin of the microparticles can be determined by a known method. For example, it can be determined whether the microparticles are derived from stem cells such as dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, or umbilical cord-derived stem cells using the method described in J Stem Cell Res Ther (2016) 8:2. I can do it. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticle.
  • the microparticles contain miRNA that targets at least one of a gene related to the expression of vascular endothelial growth factor (VEGF-A) protein and a leukotriene A4 hydrolase (LTA4H) gene.
  • miRNA miRNA
  • miRNA is, for example, an RNA molecule of 21 to 25 bases (nucleotides). miRNAs can regulate gene expression by suppressing target gene (target) mRNA degradation or decoding steps.
  • miRNA may be, for example, single-stranded (monomer) or double-stranded (dimer).
  • the miRNA is preferably a mature miRNA that has been cleaved with a ribonuclease such as Dicer.
  • miRNAs described herein are registered in known databases (e.g., miRBase database) in association with accession numbers, and those skilled in the art will be able to identify the sequences. It can be defined uniquely.
  • the accession number of hsa-let-7a-5p is MI0000060, and the sequence is registered in miRBase database.
  • the accession number of each miRNA will be omitted.
  • miRNA in this specification also includes variants that differ by about 1 to 5 bases from mature miRNA such as hsa-let-7a-5p.
  • each miRNA in this specification refers to a polynucleotide consisting of a base sequence having identity with the base sequence of each miRNA (for example, hsa-let-7a-5p), or a polynucleotide consisting of a complementary base sequence thereof. It includes a polynucleotide having the function of miRNA in the present invention.
  • Identity refers to the degree of identity when the sequences to be compared are properly aligned, and refers to the incidence (%) of exact amino acid matches between the sequences. Alignment can be performed, for example, by using any algorithm such as BLAST.
  • Polynucleotides consisting of identical base sequences may have, for example, point mutations, deletions, and/or additions in the miRNA base sequence.
  • the number of bases in the point mutation, etc. is, for example, 1 to 5, 1 to 3, 1 to 2, or 1.
  • the polynucleotide consisting of a complementary base sequence is, for example, a polynucleotide that hybridizes under stringent conditions with a polynucleotide consisting of a base sequence of miRNA, and includes a polynucleotide having the function of miRNA in the present invention.
  • Stringent conditions are not particularly limited, and include, for example, the conditions described in [0028] of JP-A-2017-184642, the contents of which are incorporated herein by reference.
  • the microparticles contain miRNA targeting at least one gene among a gene related to the expression of VEGF-A and LTA4H at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
  • miRNA targeting at least one gene among a gene related to the expression of VEGF-A and LTA4H at a higher concentration than the culture supernatant of dental pulp-derived stem cells.
  • VEGF-A expression inhibitor The microparticles of the present invention are preferably VEGF-A expression inhibitors. In this case, it is preferable that the microparticles contain miRNA that targets a gene related to the expression of VEGF-A. In the present invention, it is more preferable that the microparticles contain at least one type of the following VEGF-A suppression-related miRNA group (58 types).
  • VEGF-A suppression related miRNA group hsa-miR-1-3p, hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-107, hsa-miR-126-3p, hsa-miR-134-5p, hsa-miR- 140-5p, hsa-miR-145-5p, hsa-miR-150-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17- 5p, hsa-miR-185-5p, hsa-miR-195-5p, hsa-miR-199a-5p, hsa-miR-200b-3p, hsa-miR-203a-3p, hsa-miR-205-5p,
  • the microparticles contain two or more types of miRNAs related to VEGF-A suppression, more preferably five or more types, particularly preferably 20 or more types, and even more particularly 30 or more types. preferable.
  • the microparticles can be used to detect hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-423-3p, and hsa-miR-93. It is more preferable to contain any one of -5p, and particularly preferably to contain any one of hsa-miR-16-5p and hsa-miR-21-5p.
  • the microparticles contain both hsa-miR-16-5p and hsa-miR-21-5p; It is particularly preferable to include all of 29a-3p, hsa-miR-423-3p and hsa-miR-93-5p.
  • the microparticle preferably contains at least one type of miRNA group related to VEGF-A suppression, as a Log2 Ratio of read counts obtained by analysis using IMOTA, preferably 4.0 or more, more preferably 10.0 or more. , 15.0 or more is particularly preferred.
  • Microparticles are hsa-miR-16-5p, hsa-miR-21-5p, hsa-miR-29a-3p, hsa-miR-423-3p as Log2 Ratio of read count number obtained by analysis using IMOTA.
  • hsa-miR-93-5p preferably 4.0 or more, more preferably hsa-miR-16-5p and hsa-miR-21-5p, 10.0 or more, hsa-miR- It is particularly preferable to contain 15.0 or more of 16-5p and hsa-miR-21-5p.
  • microparticles of the present invention have hsa-miR-16-5p and hsa-miR-21-5p compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.
  • the expression level of at least one of them is preferably 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more.
  • the microparticles of the present invention are VEGF-A expression inhibitors
  • the microparticles of the present invention are preferably LTA4H expression inhibitors.
  • the microparticles contain miRNA that targets LTA4H (ie, a gene related to the expression of a protein encoded by LTA4H).
  • the microparticles contain at least one type of the following LTA4H suppression-related miRNA group (seven types).
  • LTA4H suppression-related miRNA group hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-miR-124-3p, hsa-miR-125b-5p, hsa-miR-26b-5p, hsa- miR-338-3p.
  • the microparticles contain two or more types of the LTA4H suppression-related miRNA group, more preferably three or more types, particularly preferably five or more types, and even more preferably seven types.
  • the microparticles contain any one of hsa-let-7a-5p, hsa-let-7b-5p, and hsa-miR-125b-5p among the LTA4H suppression-related miRNA group; It is particularly preferable that one of -7b-5p and hsa-miR-125b-5p is included. Furthermore, it is more preferable that the microparticles contain both hsa-let-7b-5p and hsa-miR-125b-5p; It is particularly preferable to include both 125b-5p.
  • the microparticle preferably contains at least one type of miRNA group related to VEGF-A suppression, as a Log2 Ratio of read counts obtained by analysis using IMOTA, preferably 4.0 or more, more preferably 10.0 or more. , 15.0 or more is particularly preferred.
  • the microparticles contain hsa-let-7a-5p, hsa-let-7b-5p, and hsa-miR-125b-5p all at least 4.0 as Log2 Ratio of read count number obtained by analysis using IMOTA.
  • the content of hsa-let-7b-5p and hsa-miR-125b-5p is 10.0 or more, and it is more preferable that the content of hsa-let-7b-5p and hsa-miR-125b-5p is 15.0 or more. It is particularly preferred to include.
  • microparticles of the present invention have hsa-let-7b-5p and hsa-miR-125b-5p compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.
  • the expression level of at least one of them is preferably 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more.
  • the expression of the LTA4H gene in any cell can be suppressed to 0.8 times or less of normal (in the case of untreated cells), and preferably 0.6 It is more preferable that it can be suppressed to 0.4 times or less, and it is particularly preferable that it can be suppressed to 0.4 times or less.
  • the microparticles derived from the culture supernatant of dental pulp-derived stem cells contain about 2600 types of small RNA. Approximately 1800 of these are miRNAs. Among these miRNAs, there are 180 to 200 types of miRNAs that are abundant.
  • the high content of miRNA in microparticles derived from dental pulp-derived stem cells is characterized by the fact that there are many microRNAs related to treatment of cranial nerve diseases and eye diseases. This is my knowledge. This feature is significantly different from the types of miRNA that are contained in large amounts in other mesenchymal stem cell microparticles. For example, miRNAs that are abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to treatment of cranial nerve diseases or eye diseases.
  • the microparticles preferably contain two or more types of microRNAs (hereinafter also referred to as angiogenic eye disease-related microRNAs) that can control the expression of proteins related to angiogenic eye diseases such as age-related macular degeneration, and five types. It is more preferable to include the above, more preferably to include 20 or more types, particularly preferably to include 30 or more types, and even more preferably to include 50 or more types.
  • angiogenic eye disease-related microRNAs proteins related to angiogenic eye diseases such as age-related macular degeneration
  • the microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles. , more preferably exosomes.
  • the diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, particularly preferably 50 to 150 nm.
  • molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles, and it may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three thereof. good.
  • exosomes are used as microparticles may be described, but the microparticles used in the present invention are not limited to exosomes.
  • exosomes are extracellular vesicles released from cells upon fusion of multivesicular bodies with the plasma membrane.
  • the surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.
  • the interior of the exosome preferably contains intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.
  • Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another. Exosomes are easily traceable and can be targeted to specific regions.
  • the content of microparticles in the microparticle composition is not particularly limited.
  • the microparticle composition preferably contains 0.5 x 10 8 or more microparticles, more preferably 1.0 x 10 8 or more, particularly preferably 2.0 x 10 8 or more, It is particularly preferable to contain 2.5 ⁇ 10 8 or more, and even more particularly preferably to contain 1.0 ⁇ 10 9 or more. Further, there is no particular restriction on the concentration of fine particles contained in the fine particle composition.
  • the microparticle composition preferably contains 1.0 ⁇ 10 8 particles/mL or more, more preferably 2.0 ⁇ 10 8 particles/mL or more, and 4.0 ⁇ 10 8 particles/mL or more.
  • a preferred embodiment of the microparticles of the present invention is that by containing the microparticles in such a large amount or at a high concentration, miRNAs whose expression is suppressed in neovascular eye diseases such as age-related macular degeneration or blood vessels affected by age-related macular degeneration, etc.
  • the amount of miRNA used in the treatment of neoplastic eye diseases can be maintained at a high level.
  • the microparticle composition may contain other components in addition to the microparticles, depending on the type of animal to be administered and the purpose, to the extent that the effects of the present invention are not impaired.
  • Other ingredients include nutritional ingredients, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, etc. It will be done.
  • nutritional components include fatty acids, vitamins, and the like.
  • antibiotics include penicillin, streptomycin, and gentamicin.
  • the carrier may include materials known as pharmaceutically acceptable carriers.
  • the microparticle composition may be the culture supernatant of dental pulp-derived stem cells itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, and the like.
  • the purpose of the pharmaceutical composition is to facilitate administration of microparticles to the recipient.
  • the pharmaceutically acceptable carrier is a carrier (including diluents) that does not cause significant irritation to the administered subject and does not inhibit the biological activity and properties of the administered compound.
  • carriers are propylene glycol; (physiological) saline; emulsions; buffers; media, such as DMEM or RPMI; cryopreservation media containing components that scavenge free radicals.
  • the microparticle composition may contain active ingredients of conventionally known therapeutic agents for angiogenic eye diseases such as age-related macular degeneration. Those skilled in the art can make appropriate changes depending on the intended use, administration target, etc.
  • the microparticle composition does not contain the predetermined substance.
  • the microparticle composition preferably does not contain dental pulp-derived stem cells.
  • the microparticle composition preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Other cytokines include those described in [0014] to [0020] of JP 2018-023343A.
  • the microparticle composition does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.
  • the microparticle composition preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.).
  • the microparticle composition is substantially free of conventional serum replacements, such as Knockout serum replacement (KSR).
  • KSR Knockout serum replacement
  • the content (solid content) of the other components described above is preferably 1% by mass or less, more preferably 0.1% by mass or less, and 0.01% by mass or less. It is particularly preferable that
  • the microparticles of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells, etc., and then purifying microparticles from the culture supernatant of dental pulp-derived stem cells.
  • the microparticles of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells.
  • the microparticles of the present invention are prepared by receiving a composition containing the culture supernatant of dental pulp-derived stem cells that had been disposed of (or purifying the composition as appropriate) and refining the microparticles therefrom. You may.
  • the culture supernatant of dental pulp-derived stem cells there are no particular limitations on the culture supernatant of dental pulp-derived stem cells or the like. It is preferable that the culture supernatant of dental pulp-derived stem cells and the like is substantially free of serum.
  • the serum content of the culture supernatant of dental pulp-derived stem cells is preferably 1% by mass or less, more preferably 0.1% by mass or less, and preferably 0.01% by mass or less. Particularly preferred.
  • Dental pulp-derived stem cells may be derived from humans or animals other than humans.
  • animals other than humans include those similar to the animals (species) to which the microparticles of the present invention are administered, which will be described later, and mammals are preferred.
  • dental pulp-derived stem cells used in the culture supernatant.
  • Stem cells from exfoliated deciduous teeth can be used.
  • dental pulp stem cells from deciduous teeth obtained by other methods can be used.
  • dental pulp-derived stem cells derived from animals other than humans such as pig deciduous tooth pulp stem cells, can be used.
  • dental pulp-derived stem cells contain vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-).
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • IGF insulin-like growth factor
  • PDGF platelet-derived growth factor
  • TGF- transforming growth factor-beta
  • the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are dental pulp-derived stem cells that contain many proteins, and it is preferable to use dental pulp stem cells of deciduous teeth. That is, in the present invention, it is preferable to use a culture supernatant of deciduous tooth pulp stem cells.
  • the dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as the desired treatment can be achieved.
  • immortalized stem cells derived from dental pulp can be used.
  • the immortalized stem cells are preferably immortalized stem cells that have not become cancerous.
  • Immortalized stem cells of dental pulp-derived stem cells can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them.
  • the TGF ⁇ receptor inhibitor is not particularly limited as long as it has the effect of inhibiting the function of the transforming growth factor (TGF) ⁇ receptor, and for example, 2-(5-benzo[1,3 ] Dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1- Phenylthiocarbamoyl-1H-pyrazole (A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridine -2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine( Merck & Co.), SB431542 (S
  • the ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-binding kinase.
  • ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • Y-27632 Preferred is Y-27632.
  • GSK3 inhibitors are not particularly limited as long as they inhibit GSK-3 (Glycogen Synthase Kinase 3), and include A 1070722, BIO, BIO-acetoxime (TOCRIS), etc. can be mentioned.
  • MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), such as AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86 -9766), SL327, U0126-EtOH (all sold by Selleck), PD98059, U0124, and U0125 (all sold by Cosmo Bio Inc.).
  • MEK MAP kinase-ERK kinase
  • AZD6244 CI-1040
  • PD0325901 PD0325901
  • RDEA119 BAY86 -9766
  • SL327 U0126-EtOH (all sold by Selleck)
  • PD98059 U0124
  • U0125 all sold by Cosmo Bio Inc.
  • microparticles of the present invention When using the microparticles of the present invention in regenerative medicine, in accordance with the requirements of the Regenerative Medicine Safety Act, dental pulp-derived stem cells, culture supernatants of these immortalized stem cells, and compositions containing microparticles derived therefrom must be This embodiment does not contain any other somatic stem cells other than derived stem cells.
  • the microparticle composition may contain mesenchymal stem cells and other somatic stem cells other than dental pulp-derived stem cells, it is preferable not to contain them. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc.
  • Examples of dermal somatic stem cells include epithelial stem cells, hair follicle stem cells, and the like.
  • somatic stem cells of the digestive system include pancreatic (general) stem cells, hepatic stem cells, and the like.
  • Examples of myeloid somatic stem cells (other than mesenchymal stem cells) include hematopoietic stem cells and the like.
  • Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, and the like.
  • the microparticle composition may contain stem cells other than somatic stem cells, but preferably does not contain them.
  • Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).
  • a culture supernatant such as dental pulp-derived stem cells is a culture solution obtained by culturing dental pulp-derived stem cells.
  • a culture supernatant that can be used in the present invention can be obtained by separating and removing cell components after culturing dental pulp-derived stem cells.
  • a culture supernatant that has been appropriately subjected to various treatments for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc. may be used.
  • Dental pulp-derived stem cells for obtaining a culture supernatant of dental pulp-derived stem cells can be selected by conventional methods, and can be selected based on cell size and morphology, or as adherent cells. Dental pulp cells collected from fallen baby teeth or permanent teeth can be sorted as adherent cells or their successor cells. As the culture supernatant of dental pulp-derived stem cells, a culture supernatant obtained by culturing selected stem cells can be used.
  • the "culture supernatant of dental pulp-derived stem cells, etc.” is preferably a culture solution that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc.
  • the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain any cells (regardless of the type of cells) as a whole. Due to this feature, the composition of this embodiment is clearly distinguished from dental pulp-derived stem cells themselves as well as from various compositions containing dental pulp-derived stem cells.
  • a typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of a culture supernatant of dental pulp-derived stem cells.
  • the culture supernatant of dental pulp-derived stem cells used in the present invention may contain culture supernatants of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells.
  • the culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of deciduous dental pulp-derived stem cells as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the culture supernatant of dental pulp-derived stem cells used in the present invention is a composition composed only of the culture supernatant of dental pulp-derived stem cells of deciduous teeth.
  • a basic medium or a basic medium to which serum or the like is added can be used.
  • DMEM Dulbecco's modified Eagle's medium
  • IMDM Iscove's modified Dulbecco's medium
  • Ham F12 medium Ham F12, SIGMA, GIBCO, etc.
  • RPMI1640 medium etc.
  • a serum-free medium may be used throughout the entire process or during the last or several subcultures from the end. For example, by culturing dental pulp-derived stem cells in a serum-free medium (serum-free medium), a serum-free culture supernatant of dental pulp-derived stem cells can be prepared.
  • a serum-free culture supernatant of dental pulp-derived stem cells can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.
  • culturing dental pulp-derived stem cells For culturing dental pulp-derived stem cells to obtain a culture supernatant, commonly used conditions can be applied as they are.
  • the method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the stem cell isolation and selection steps are appropriately adjusted depending on the type of stem cells.
  • Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells according to the type of the dental pulp-derived stem cells.
  • special conditions may be applied to culturing dental pulp-derived stem cells in order to produce a large amount of microparticles such as exosomes. Examples of special conditions include conditions for co-cultivation with some stimulus, such as low temperature conditions, hypoxic conditions, microgravity conditions, and the like.
  • the culture supernatant of dental pulp-derived stem cells used in the preparation of microparticles such as exosomes in the present invention may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it does not substantially contain other components. Preferably not. However, various types of additives used for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.
  • Microparticles can be prepared by purifying microparticles from culture supernatants such as dental pulp-derived stem cells.
  • microparticles are preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles.
  • Microparticles can be isolated by separating them from unassociated components based on the properties of the microparticles. For example, microparticles can be isolated based on molecular weight, size, morphology, composition, or biological activity.
  • microparticles can be purified by separating a specific fraction (eg, precipitate) containing many microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) in fractions other than the predetermined fractions may be removed.
  • microparticles can be purified by filtering a culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration treatment. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed at the same time.
  • the material, pore size, etc. of the filtration membrane used for the filtration treatment are not particularly limited. Filtration can be carried out using filtration membranes of appropriate molecular weight or size cut-off using known methods.
  • the pore size of the filtration membrane is preferably from 10 to 1000 nm, more preferably from 30 to 500 nm, and particularly preferably from 50 to 150 nm, from the viewpoint of easy separation of exosomes.
  • a culture supernatant of dental pulp-derived stem cells, a centrifuged product thereof, or a filtered product thereof can be separated using an additional separation means such as lamb chromatography.
  • additional separation means such as lamb chromatography.
  • HPLC high performance liquid chromatography
  • the column can be a size exclusion column or a binding column.
  • One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing step.
  • light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track microparticles.
  • specific enzyme activities, etc. can be used to track the activity in each fraction.
  • As a method for purifying microparticles the method described in [0034] to [0064] of Japanese Patent Publication No. 2019-524824 may be used, and the contents of this publication are incorporated herein by reference.
  • the final form of the microparticle composition is not particularly limited.
  • the microparticle composition can be formed by filling a container with microparticles together with a solvent or dispersion medium; by gelling the microparticles together with a gel and filling the container; by freezing and/or drying the microparticles.
  • it may be solidified into a formulation or filled into a container.
  • containers include tubes, centrifuge tubes, bags, etc. suitable for cryopreservation.
  • the freezing temperature can be, for example, -20°C to -196°C.
  • the microparticles of the present invention are easier to mass produce than conventional compositions that can be used as therapeutic or preventive agents for angiogenic eye diseases such as age-related macular degeneration, and are conventionally discarded as industrial waste.
  • There are advantages such as being able to utilize the stem cell culture solution that was previously used, and reducing the cost of disposing of the stem cell culture solution.
  • the culture supernatant of dental pulp-derived stem cells is the culture supernatant of human dental pulp-derived stem cells
  • the microparticles of the present invention are highly safe from an immunological standpoint when applied to humans. Another advantage is that there are fewer ethical issues.
  • the microparticles of the present invention will be safer and more ethical when applied to that patient. There will be fewer problems.
  • the microparticles of the present invention are derived from the culture supernatant of dental pulp-derived stem cells, they can also be used in restorative medicine.
  • compositions containing microparticles derived from culture supernatants such as dental pulp-derived stem cells are preferably used for restorative medical applications.
  • stem cells are not the main actors in regeneration, but rather that the humoral components produced by stem cells repair organs together with one's own stem cells.
  • Difficult issues associated with conventional stem cell transplantation such as canceration, standardization, administration methods, storage stability, and culture methods, have been resolved, and restorations can be achieved using a composition using the culture supernatant of dental pulp-derived stem cells or microparticles derived therefrom. Medical care becomes possible.
  • stem cell transplantation when using the microparticles of the present invention, tumor formation is less likely to occur because cells are not transplanted, and it can be said to be safer.
  • the microparticles of the present invention have the advantage of being of uniformly standardized quality. Since mass production and efficient administration methods can be selected, it can be used at low cost.
  • the prophylactic or therapeutic agent for neovascular eye diseases of the present invention includes the microparticles of the present invention.
  • prevention refers to preventing the onset of a disease (in this specification, neovascular eye disease).
  • treatment refers to alleviating, suppressing, or preventing the progression of symptoms of a disease that has developed, and improving symptoms.
  • the method for improving angiogenic eye diseases of the present invention includes administering an effective amount of the microparticles of the present invention or an effective amount of the prophylactic or therapeutic agent for angiogenic eye diseases of the present invention to angiogenic eye diseases such as age-related macular degeneration. This includes administering to a subject who has developed symptoms.
  • Administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, dripping, local administration, nasal spray, etc., and minimally invasive methods are preferred. Injection is a preferred method of local administration. Also preferred is electroporation, which temporarily creates minute holes in cell membranes by applying voltage (electrical pulses) to the skin surface, allowing active ingredients to penetrate into the dermal layer, which cannot be reached with normal skin care.
  • examples include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc.; intraarterial administration, intravenous administration, subcutaneous administration or Intraperitoneal administration is more preferred.
  • various formulation techniques can be used to alter the in vivo distribution of microparticles. Many methods of altering in vivo distribution are known to those skilled in the art. Examples of such methods include protection of exosomes in vesicles composed of materials such as proteins, lipids (eg, liposomes), carbohydrates or synthetic polymers.
  • the microparticles of the present invention administered to a subject who has developed a neovascular eye disease such as age-related macular degeneration may circulate within the subject's body and reach a predetermined tissue.
  • the frequency of administration can be one or more times per week, preferably five or more times, more preferably six or more times, particularly preferably seven or more times.
  • the administration interval is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be adjusted as appropriate depending on the species to be administered and the symptoms of the subject.
  • the microparticles of the present invention are preferably used to administer the microparticles to a subject who has developed dementia at least once a week over a therapeutically effective period.
  • the number of administrations per week is preferably large, preferably 5 or more times per week over the effective therapeutic period, and preferably daily.
  • the amount is preferably 0.1 to 5 ml per mouse (about 25 g), and 0.3 to 5 ml per mouse (approximately 25 g).
  • the amount is more preferably 3 ml, and particularly preferably 0.5 to 1 ml.
  • the amount is preferably 1 to 50 ⁇ g, more preferably 3 to 30 ⁇ g, and more preferably 5 to 50 ⁇ g per mouse (approximately 25 g). More particularly preferred is ⁇ 25 ⁇ g.
  • the preferred range of dosage per body weight for other animals can be calculated from the dosage per body weight (about 25 g) for model mice using a proportional relationship. However, it can be adjusted as appropriate depending on the symptoms of the subject.
  • the animals to which the microparticles of the present invention are administered are preferably mammals, birds (chickens, quail, ducks, etc.), and fish (salmon, trout, tuna, bonito, etc.).
  • the mammal may be a human or a non-human mammal, but humans are particularly preferred. More preferably, the non-human mammal is a cow, pig, horse, goat, sheep, monkey, dog, cat, mouse, rat, guinea pig, or hamster.
  • microparticles of the present invention may be used in combination with conventionally known therapeutic agents for angiogenic eye diseases such as age-related macular degeneration. Specifically, it may be used in combination with conventionally known VAGF-A inhibitors, leukotriene production inhibitors such as LTA4H expression inhibitors, and BLT1 antagonists.
  • Example 1 ⁇ Preparation of culture supernatant of dental pulp-derived stem cells> Using DMEM medium instead of the DMEM/HamF12 mixed medium and otherwise following the method described in Example 6 of Patent No. 6296622, a culture supernatant of human deciduous dental pulp stem cells was prepared, and the culture supernatant was fractionated. did. In primary culture, fetal bovine serum (FBS) is added to the culture, and in subculture, the supernatant of the subculture medium is cultured using the primary culture medium, and the supernatant of the subculture medium is separated so that it does not contain FBS. culture supernatant was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.
  • FBS fetal bovine serum
  • exosomes of dental pulp-derived stem cells were purified by the following method. After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells through a filter with a pore size of 0.22 micrometers, the solution was centrifuged at 100,000 ⁇ g at 4° C. for 60 minutes. The supernatant was decanted and the exosome enriched pellet was resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 xg for 60 minutes.
  • PBS phosphate buffered saline
  • the pellet was again collected from the bottom of the centrifuge tube as a concentrated sample (approximately 100 ⁇ l). Protein concentration was determined by microBSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C. A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the microparticle composition sample of Example 1.
  • the average particle diameter and concentration of the microparticles contained in the microparticle composition of Example 1 were evaluated using a nanoparticle analysis system NanoSight (manufactured by Nippon Quantum Design Co., Ltd.).
  • the average particle size of the microparticles contained in the microparticle composition of Example 1 was 50 to 150 nm.
  • the microparticle composition of Example 1 was a high concentration exosome solution of 1.0 ⁇ 10 9 particles/ml or more, specifically, a high concentration exosome solution of 2.0 ⁇ 10 9 particles/ml.
  • the components of the obtained microparticle composition of Example 1 were analyzed by a known method.
  • microparticle composition of Example 1 did not contain stem cells of dental pulp-derived stem cells, did not contain MCP-1, and did not contain Siglec-9. Therefore, it was found that an active ingredient different from MCP-1 and Siglec-9, which are the active ingredients of the culture supernatant of mesenchymal stem cells, and their analogs, was the active ingredient of the microparticle composition of Example 1. .
  • Example 1 MicroRNA expressed in exosomes Small RNA contained in the microparticle composition of Example 1 was analyzed by next generation sequencing (NGS) analysis. NGS analysis identified 1787 miRNAs contained in the microparticle composition (dental pulp-derived stem cell exosomes) of Example 1. The results obtained are shown in Table 1 below.
  • IMOTA Interactive Multi-Omics-Tissue Atlas
  • IMOTA is an interactive multi-omics atlas that allows you to investigate interactions and expression levels of miRNAs, mRNAs, and proteins in each tissue and cell (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770- D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions").
  • the Omic counts were 3071 proteins, 14182 mRNAs, and 1124 miRNAs.
  • the counts of Omics with a high expression rate in the cerebral cortex were 1119 for proteins, 1110 for mRNA, and 145 for miRNA.
  • the miRNAs expressed in the microparticle composition (exosomes of dental pulp-derived stem cells) of Example 1 and highly expressed in the cerebral cortex were 122 as shown in Tables 2 and 3 below.
  • Test Example 3 Search for microRNAs involved in diseases Based on the obtained analysis results, microRNAs involved in diseases were searched. Extraction of disease-related miRNAs was performed using IMOTA. Here, we will investigate whether the microRNAs contain microRNAs that suppress proteins associated with neovascular eye diseases such as age-related macular degeneration, or microRNAs that control proteins that are targets for therapeutic drugs. I explored. We searched on Imota for miRNAs that control VEGF, which promotes angiogenesis, which causes angiogenic eye diseases such as age-related macular degeneration. JCI Insight.
  • the protein encoded by the LTA4H gene (EC 3.3.2.6) is an enzyme that converts leukotriene A4 to leukotriene B4.
  • miRNAs related to LTA4H were searched using Imota, seven miRNAs were found to be involved in the regulation of this enzyme.
  • miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells targeting VEGFA were the following VEGF-A suppression-related miRNA group (58 types).
  • miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells targeting LTA4H were the following LTA4H suppression-related miRNA group (seven types). hsa-let-7a-5p, hsa-let-7b-5p, hsa-let-7c-5p, hsa-miR-124-3p, hsa-miR-125b-5p, hsa-miR-26b-5p, hsa- miR-338-3p.
  • FIG. 5 is a heat map showing the expression level of miRNA expressed in exosomes (microparticles of Example 1) purified from the culture supernatant of dental pulp-derived stem cells targeting VEGFA or LTA4H.

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Abstract

La présente invention concerne : une particule fine contenant des miARN qui ciblent au moins l'un d'un gène associé à l'expression d'une protéine du facteur de croissance vasculaire endothéliale (VEGF-A) ou du gène de la leucotriène A4 hydrolase (LTA4H), la particule fine étant capable de réguler l'expression d'une protéine et/ou d'un gène associé à une maladie oculaire néovasculaire telle que la dégénérescence maculaire liée à l'âge ; un médicament prophylactique ou un médicament thérapeutique pour une maladie oculaire néovasculaire ; et une méthode d'amélioration d'une maladie oculaire néovasculaire.
PCT/JP2023/014155 2022-04-11 2023-04-05 Particule fine, médicament prophylactique ou médicament thérapeutique pour une maladie oculaire néovasculaire, et méthode d'amélioration d'une maladie oculaire néovasculaire WO2023199828A1 (fr)

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ALSAEEDI HIBA AMER; KOH AVIN EE-HWAN; LAM CHENSHEN; RASHID MUNIRAH BINTI ABD; HARUN MOHD HAIRUL NIZAM; SALEH MUHAMAD FAKHRI BIN MO: "Dental pulp stem cells therapy overcome photoreceptor cell death and protects the retina in a rat model of sodium iodate-induced retinal degeneration", JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B: BIOLOGY, ELSEVIER SCIENCE S.A., BASEL., CH, vol. 198, 16 July 2019 (2019-07-16), CH , XP085769500, ISSN: 1011-1344, DOI: 10.1016/j.jphotobiol.2019.111561 *
HE GUANG-HUI, WEI ZHANG, YING-XUE MA, JING YANG, LI CHEN, JIAN SONG, SONG CHEN: "Mesenchymal stem cells-derived exosomes ameliorate blue light stimulation in retinal pigment epithelium cells and retinal laser injury by VEGF-dependent mechanism", INTERNATIONAL JOURNAL OF OPHTHALMOLOGY, INTERNATIONAL COUNCIL OF OPHTHALMOLOGY, CN, vol. 11, no. 4, 18 April 2018 (2018-04-18), CN , pages 559 - 566, XP093099062, ISSN: 2222-3959, DOI: 10.18240/ijo.2018.04.04 *
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