WO2022025239A1 - Procédé d'amélioration de l'effet thérapeutique d'une thérapie par injection de cellules endothéliales cornéennes - Google Patents

Procédé d'amélioration de l'effet thérapeutique d'une thérapie par injection de cellules endothéliales cornéennes Download PDF

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WO2022025239A1
WO2022025239A1 PCT/JP2021/028299 JP2021028299W WO2022025239A1 WO 2022025239 A1 WO2022025239 A1 WO 2022025239A1 JP 2021028299 W JP2021028299 W JP 2021028299W WO 2022025239 A1 WO2022025239 A1 WO 2022025239A1
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corneal
cells
composition
minutes
disorders
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English (en)
Japanese (ja)
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範子 小泉
直毅 奥村
靖史 松岡
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学校法人同志社
アクチュアライズ株式会社
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue

Definitions

  • the present disclosure relates to techniques for treating symptoms, disorders or diseases of the corneal endothelium.
  • the light taken in from the cornea which is the transparent tissue in the foreground of the eyeball, reaches the retina and excites the nerve cells of the retina, and the generated electrical signal is transmitted to the visual cortex of the cerebral via the optic nerve. It is recognized by doing.
  • the cornea needs to be transparent. The transparency of the cornea is maintained by keeping the water content constant by the pumping function and barrier function of the corneal endothelial cells.
  • the present disclosure provides new techniques for treating symptoms, disorders or diseases of the corneal endothelium.
  • the present inventors can obtain a high therapeutic effect by pre-administering a Rho-kinase (ROCK) inhibitor (also referred to as “ROCK inhibitor” in the present specification) before cell infusion.
  • a Rho-kinase (ROCK) inhibitor also referred to as “ROCK inhibitor” in the present specification
  • the present disclosure provides: (Item 1) A composition comprising a ROCK inhibitor for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the composition comprises corneal endothelial cells and /.
  • the composition is characterized by being administered prior to injection of the corneal endothelial cells and / or corneal endothelium-like cells into the anterior chamber of the subject. .. (Item 1-1)
  • a composition comprising a ROCK inhibitor for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the composition comprises corneal endothelial cells and /.
  • the composition is characterized by being ophthalmic administration prior to injection of the corneal endothelial cells and / or corneal endothelium-like cells into the anterior chamber of the subject.
  • the composition (Item 1-2) The composition according to item 1, wherein the administration comprises an ophthalmic administration.
  • the composition is administered so as to be applied to the conjunctival sac.
  • the composition is an eye drop or an eye ointment.
  • the composition is an eye drop.
  • composition according to any one of the above items wherein the composition is administered twice or more every about 1 minute to about 180 minutes.
  • the ROCK inhibitor is ripasudil or a pharmaceutically acceptable salt thereof.
  • the symptoms, disorders or diseases of the corneal endothelium include decreased corneal endothelium density, formation of guttae, thickening of the desmeal membrane, thickening of the corneal thickness, corneal epithelial disorder, opacity, scars, parenchymal opacity of the cornea, glare, and fog.
  • One of the above items selected from the group consisting of visual impairment, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, halo, glare and edema of the corneal stroma. The composition described.
  • the symptoms, disorders or diseases of the corneal endothelium include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorders, corneal endothelitis, trauma, post-ophthalmic surgery disorders, post-ophthalmic laser surgery disorders, aging, posterior polymorphic corneal dystrophy,
  • An eye drop for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or diseases in a subject which comprises lipasdil or a pharmaceutically acceptable salt thereof.
  • the eye drops are about 5 minutes prior to injection of the corneal endothelial cells and / or corneal endothelium-like cells into the subject's anterior chamber.
  • An eye drop characterized by being administered 3 times every other time.
  • (Item 1A) A method of enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the method comprises administering a ROCK inhibitor to the subject and the corneal endothelium.
  • a method comprising the step of administering cells and / or corneal endothelium-like cells to the subject, wherein the ROCK inhibitor is administered prior to administration of the corneal endothelial cells and / or corneal endothelium-like cells.
  • (Item 1A-1) A method of enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the method comprises administering a ROCK inhibitor to the subject and the corneal endothelium.
  • a method comprising the step of administering cells and / or corneal endothelium-like cells to the subject, wherein the ROCK inhibitor is administered ocularly prior to administration of the corneal endothelial cells and / or corneal endothelium-like cells.
  • the administration comprises ophthalmic administration.
  • (Item 1A-3) The method of item 1, wherein the administration comprises administration to the external eye.
  • (Item 2A) The method of item 1A or 1A-1, wherein the ROCK inhibitor is selected from the group consisting of ripasudil, Y-27632, fasdil, netalszil and pharmaceutically acceptable salts thereof.
  • (Item 2A-1) The method according to any one of the above items, wherein the ROCK inhibitor is selected from the group consisting of ripasudil, Y-27632, faszil, netalszil, veloszil, vermosdil and pharmaceutically acceptable salts thereof.
  • (Item 2A-2) The method according to any one of the above items, wherein the composition is administered so as to be applied to the conjunctival sac.
  • (Item 2A-3) The method according to any one of the above items, wherein the composition is an eye drop or an eye ointment.
  • (Item 3A) The method according to any one of the above items, wherein the ROCK inhibitor is formulated as an eye drop.
  • (Item 4A) The method according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 180 minutes.
  • (Item 5A) The method according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 30 minutes.
  • (Item 6A) The method according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 10 minutes.
  • the symptoms, disorders or diseases of the corneal endothelium include decreased corneal endothelium density, formation of guttae, thickening of the desmeal membrane, thickening of the corneal thickness, corneal epithelial disorder, opacity, scars, parenchymal opacity of the cornea, glare, and fog.
  • One of the above items selected from the group consisting of visual impairment, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, halo, glare and edema of the corneal stroma.
  • the symptoms, disorders or diseases of the corneal endothelium include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorders, corneal endothelitis, trauma, post-ophthalmic surgery disorders, post-ophthalmic laser surgery disorders, aging, posterior polymorphic corneal dystrophy, The method according to any one of the above items, selected from the group consisting of congenital hereditary corneal endothelial dystrophy, idiopathic corneal endothelial injury, and cytomegalovirus corneal endothelitis.
  • (Item 10A) A method of enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the method comprises eye drops containing ripasudil or a pharmaceutically acceptable salt thereof.
  • a method comprising three doses approximately every 5 minutes prior to intratumoral injection.
  • a ROCK inhibitor for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject wherein the ROCK inhibitor is corneal endothelial cells and / or corneal endothelium-like. Used in combination with cells, the ROCK inhibitor is characterized by being administered prior to injection of the corneal endothelial cells and / or corneal endothelial-like cells into the anterior chamber of the subject.
  • (Item 1B-1) A ROCK inhibitor for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, wherein the ROCK inhibitor is corneal endothelial cells and / or corneal endothelium-like. Used in combination with cells, the ROCK inhibitor is characterized by being administered ocularly prior to injection of the corneal endothelial cells and / or corneal endothelial-like cells into the anterior chamber of the subject.
  • the ROCK inhibitor according to item 1 wherein the administration comprises ophthalmic administration.
  • (Item 1B-3) The ROCK inhibitor according to item 1, wherein the administration comprises administration to the external eye.
  • (Item 2B-3) The ROCK inhibitor according to any one of the above items, wherein the composition is an eye drop or an eye ointment.
  • (Item 3B) The ROCK inhibitor according to any one of the above items, wherein the ROCK inhibitor is an eye drop.
  • (Item 4B) The ROCK inhibitor according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every 1 minute to about 180 minutes.
  • (Item 5B) The ROCK inhibitor according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 30 minutes.
  • (Item 6B) The ROCK inhibitor according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 10 minutes.
  • the ROCK inhibitor according to any one of the above items, wherein the ROCK inhibitor is ripasudil or a pharmaceutically acceptable salt thereof.
  • the symptoms, disorders or diseases of the corneal endothelium include decreased corneal endothelium density, formation of guttae, thickening of the desmeal membrane, thickening of the corneal thickness, corneal epithelial disorder, opacity, scars, parenchymal opacity of the cornea, glare, and fog.
  • One of the above items selected from the group consisting of visual impairment, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, halo, glare and edema of the corneal stroma.
  • the ROCK inhibitor described. (Item 9B)
  • the symptoms, disorders or diseases of the corneal endothelium include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorders, corneal endothelitis, trauma, post-ophthalmic surgery disorders, post-ophthalmic laser surgery disorders, aging, posterior polymorphic corneal dystrophy,
  • the ROCK inhibitor according to any one of the above items, selected from the group consisting of congenital hereditary corneal endothelial dystrophy, idiopathic corneal endothelial injury, and cytomegalovirus corneal endothelitis.
  • Ripasudil or a pharmaceutically acceptable salt thereof for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or diseases in a subject, the ripasudil or a pharmaceutically acceptable salt thereof.
  • the salt to be used is used as an eye drop in combination with corneal endothelial cells and / or corneal endothelium-like cells, and the ripasudil or its pharmaceutically acceptable salt is the corneal endothelial cells and / or corneal endothelium-like cells.
  • Ripasudil or a pharmaceutically acceptable salt thereof characterized in that it is administered three times approximately every 5 minutes prior to injection into the anterior chamber of the subject.
  • a ROCK inhibitor in the manufacture of a pharmaceutical to enhance the therapeutic effect of cell infusion therapy to treat or prevent corneal endothelium symptoms, disorders or disorders in a subject, wherein the ROCK inhibitor is a corneal endothelial cell.
  • the ROCK inhibitor Used in combination with and / or corneal endothelium-like cells, the ROCK inhibitor is characterized by being administered prior to infusion of the corneal endothelial cells and / or corneal endothelium-like cells into the subject's anterior chamber. ,use.
  • (Item 1C-1) The use of a ROCK inhibitor in the manufacture of a pharmaceutical to enhance the therapeutic effect of cell infusion therapy to treat or prevent corneal endothelium symptoms, disorders or disorders in a subject, wherein the ROCK inhibitor is a corneal endothelial cell. Used in combination with and / or corneal endothelium-like cells, the ROCK inhibitor is characterized by ocular administration prior to injection of the corneal endothelial cells and / or corneal endothelium-like cells into the anterior chamber of the subject. To use. (Item 1C-2) The use according to item 1, wherein the administration comprises ophthalmic administration. (Item 1C-3) The use according to item 1, wherein the administration comprises administration to the external eye.
  • (Item 2C) The use according to any one of the above items, wherein the ROCK inhibitor is selected from the group consisting of ripasudil, Y-27632, fasdil, netalszil and pharmaceutically acceptable salts thereof.
  • (Item 2C-1) The use according to any one of the above items, wherein the ROCK inhibitor is selected from the group consisting of Ripasudil, Y-27632, Fasdil, Netaluszil, Veloszil, Belmosdil and pharmaceutically acceptable salts thereof.
  • (Item 2C-2) The use according to any one of the above items, wherein the composition is administered so as to be applied to the conjunctival sac.
  • (Item 2C-3) The use according to any one of the above items, wherein the composition is an eye drop or an eye ointment.
  • (Item 3C) The use according to any one of the above items, wherein the ROCK inhibitor is an eye drop.
  • (Item 4C) The use according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every 1 minute to about 180 minutes.
  • (Item 5C) The use according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 30 minutes.
  • (Item 6C) The use according to any one of the above items, wherein the ROCK inhibitor is administered twice or more every about 1 minute to about 10 minutes.
  • the symptoms, disorders or diseases of the corneal endothelium include decreased corneal endothelium density, formation of guttae, thickening of the desmeal membrane, thickening of the corneal thickness, corneal epithelial disorder, opacity, scars, parenchymal opacity of the cornea, glare, and fog.
  • One of the above items selected from the group consisting of visual impairment, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, decreased contrast, halo, glare and edema of the corneal stroma.
  • the symptoms, disorders or diseases of the corneal endothelium include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorders, corneal endothelitis, trauma, post-ophthalmic surgery disorders, post-ophthalmic laser surgery disorders, aging, posterior polymorphic corneal dystrophy, The use according to any one of the above items, selected from the group consisting of congenital hereditary corneal endothelial dystrophy, idiopathic corneal endothelial injury, and cytomegalovirus corneal endothelitis.
  • ripasudil or a pharmaceutically acceptable salt thereof in the manufacture of a drug to enhance the therapeutic effect of cell infusion therapy to treat or prevent corneal endothelium symptoms, disorders or disorders in a subject.
  • the pharmaceutically acceptable salt thereof is used as an eye drop in combination with the corneal endothelial cells and / or the corneal endothelium-like cells
  • the ripasudil or the pharmaceutically acceptable salt thereof is used as the corneal endothelial cells and / or the corneal endothelial cells.
  • use characterized in that the corneal endothelium-like cells are administered three times approximately every 5 minutes prior to injection into the subject's anterior chamber.
  • a high therapeutic effect can be obtained by administering a ROCK inhibitor in advance before cell injection.
  • FIG. 1 shows observation images of the anterior segment of the right eye of a rabbit corneal endothelial injury model by a slit lamp microscope in the control group, the Y-27632 combination group, and the Ripasudil instillation group.
  • FIG. 2 shows the results of central corneal thickness measurement in the control group, the Y-27632 combination group, and the Ripasudil eye drop group. The results were measured by Pentacam and calculated as 1200 ⁇ m for all 1200 ⁇ m and above.
  • FIG. 3 shows the results of intraocular pressure measurement in the control group, the Y-27632 combination group, and the Ripasudil instillation group.
  • FIG. 4 shows the results of phalloidin staining (green) in an immunohistological test. Blue indicates DAPI.
  • FIG. 5 shows the results of staining (green) of ZO-1 (upper), N-cadherin (middle), and Na + / K + -ATPase (lower). Blue indicates DAPI.
  • FIG. 6 shows the results of phalloidin staining (green) in an immunohistological test. Blue indicates DAPI.
  • FIG. 7 shows the measurement results of the Ripasudil concentration in the aqueous chamber water over time after instillation of 0.4% Granatec. Data are shown as mean ⁇ standard deviation ( ⁇ M). The quantification limit for ripasudil is 0.25 ⁇ M.
  • FIG. 8 shows a graph of the measurement results of the Ripasudil concentration in the aqueous chamber water over time after instillation of 0.4% Granatec.
  • FIG. 9 shows the measurement results of the Y-27632 concentration in the aqueous chamber water over time after Y-27632 instillation. Data are shown as mean ⁇ standard deviation ( ⁇ M). The quantification limit of Y-27632 is 0.1 ⁇ M.
  • FIG. 10 shows a graph of the measurement result of the Y-27632 concentration in the aqueous chamber water over time after Y-27632 instillation. Data are shown as mean ⁇ standard deviation ( ⁇ M). The quantification limit of Y-27632 is 0.1 ⁇ M.
  • FIG. 11 shows the measurement results of the anterior chamber concentration after single administration and frequent administration of Netaluszil instillation. Data are shown as mean ⁇ standard deviation ( ⁇ M). The limit of quantification of netaluszil is 0.22 ⁇ M.
  • FIG. 12 shows the measurement results of the concentration in the anterior chamber after three instillations of Netaluszil. Data are shown as mean ⁇ standard deviation ( ⁇ M). The limit of quantification of netaluszil is 0.22 ⁇ M.
  • FIG. 13 shows a graph of the measurement results of the concentration in the anterior chamber after three instillations of Netaluszil.
  • cornea endothelial cell is used in the usual sense as used in the art.
  • the cornea is one of the layered tissues that make up the eye, is transparent, and is located in the part closest to the outside world. In humans, the cornea is said to be composed of five layers in order from the outside (body surface), and is composed of the corneal epithelium, Bowman's membrane, eigenlayer, Descemet's membrane (corneal endothelial basement membrane), and corneal endothelium from the outside.
  • the epithelium and parts other than the endothelium may be collectively referred to as "stroma", which is also referred to herein.
  • HCEC human corneal endothelial cells
  • HCEC human corneal endothelial cells
  • corneal endothelium-like cell refers to a cell differentiated from a stem cell, for example, an iPS cell or the like, and has substantially the same function as a corneal endothelial cell.
  • stem cells such as ES cells and iPS cells
  • Methods for differentiating stem cells, such as ES cells and iPS cells, into corneal endothelium-like cells are well known in the art (McCave et al., PLoS One. 2015 Dec 21; 10 (12): e014256; Ali et. al., Invest Opthalmol Vis Sci. 2018 May 1; 59 (6): 2437-2444).
  • iPS cells are seeded on a 35 mm Matrigel-coated plate (Corning) at 1:12 dilution on day 0 using a cell dissociation buffer (Life Technologies) (80% confluent plate). Divide into 12 plates). iPS cells are grown in medium (mTeSR1; STEMCELL Technologies Inc.) for 4 days. On the 4th day, mTeSR1 medium was added to 80% DMEM-F12 (Life Technologies), 20% KSR (Life Technologies), 1% non-essential amino acids (Life Technologies), 1 mM L-glutamine (STEMCELL Technolog).
  • 0.1 mM ⁇ -mercaptoethanol (MilliporeSigma), and 8 ng / mL ⁇ FGF (MilliporeSigma) in the basal medium of 0.1 ⁇ B27 supplement (Life Technologies), 10 ng / mL recombinant human platelet-derived growth factor-BB ( Replace with corneal medium containing PDGF-BB; PeproTech, Rocky Hill, NJ, USA) and 10 ng / mL recombinant human Dickkopf-related protein-2 (DKK-2; R & D Systems).
  • DKK-2 Dickkopf-related protein-2
  • the "corneal endothelial cells” and “corneal endothelium-like cells” may contain a magnetic substance (for example, iron).
  • a magnetic substance for example, iron
  • corneal endothelial cells containing a magnetic substance are injected into the anterior chamber, it is possible to attract and promote adhesion to the inside of the cornea (for example, Descemet's membrane) by magnetic force (Patel et al., Invest Opthalmol Vis Sci. 2009 May; 50 (5): 2123-31; Mimura et al., Exp Eye Res. 2003 Jun; 76 (6): 745-51; and Mimura et al., Exp Eye Res. 2005 Feb; 80 (2) 149-57).
  • the "magnetic material” refers to a substance magnetized by a magnetic field, and examples thereof include iron, cobalt, nickel, and ferrite.
  • Ophthalmic Administration refers to administration to the external eye. According to the definition of the route of administration defined by the US Food and Drug Administration (FDA) (https:: //www.fda.gov/drugs/data-standards-manual-monographs/route-administration). Administration to the external eye includes application to the conjunctival sac, typically eye drops and eye ointment.
  • FDA US Food and Drug Administration
  • the "outer eye portion" refers to the outside of the eyeball.
  • the term "conjunctival sac” refers to the mucous membrane that connects the eyeball and the eyelid, and refers to the anterior sclera and the sac-like part that covers the inner surface of the eyelid.
  • the present disclosure provides a composition comprising a ROCK inhibitor for enhancing the therapeutic effect of cell infusion therapy for treating or preventing corneal endothelium symptoms, disorders or diseases in a subject.
  • This composition is used in combination with corneal endothelial cells and / or corneal endothelial-like cells, and the composition is administered prior to injection of corneal endothelial fibrils and / or corneal endothelial-like cells into the anterior chamber of the subject. It is characterized by that.
  • the compositions of the present disclosure may be referred to as pharmaceuticals, pharmaceuticals, pharmaceutical compositions, pharmaceuticals, etc., which are interchangeably used for the purpose of administration to a subject and are collectively referred to herein as "(the present). Although it may be referred to as "composition, etc. (disclosed)", the term “composition” also applies to terms such as pharmaceuticals, pharmaceuticals, pharmaceutical compositions, and drugs, unless otherwise specified.
  • the present disclosure is a composition
  • a composition comprising a ROCK inhibitor for treating or preventing a symptom, disorder or disease of the corneal endothelium in a subject, wherein the composition is a corneal endothelial cell and / or a corneal membrane.
  • the corneal endothelial cells and / or corneal endothelium-like cells are injected into the anterior chamber of the subject and the composition is the subject of the corneal endothelial cells and / or corneal endothelium-like cells.
  • a composition characterized by being administered prior to injection into the anterior chamber.
  • compositions of the present disclosure can be administered ocularly.
  • ocular administration may be application to the conjunctival sac, eg, administration of eye drops or application of ointment.
  • the compositions of the present disclosure can be eye drops or eye ointments.
  • the present inventors have found that in cell injection therapy, a high therapeutic effect can be obtained by pre-administering a ROCK inhibitor before cell injection.
  • a ROCK inhibitor When the ROCK inhibitor and the cells are mixed in advance, the cells adhere to the wall surface of the container or form a cell mass after a certain period of time, so that it is necessary to mix the cells and the ROCK inhibitor immediately before the cell injection.
  • such mixing work is not easy in the operating room. That is, it takes time and it is difficult to mix accurately without skill.
  • compositions and the like make it possible to use a immediately injectable (so-called "ready-to-use") preparation in cell injection therapy without complicated procedures.
  • ROCK inhibitor examples include the following documents: US Pat. No. 4,678,873, Pat. No. 3,421,217, International Publication No. 95/28387, International Publication 99/20620, International Publication 99/61403, International Publication 02/076976, International Publication 02/076977, International Publication No.
  • Such compounds can be produced by the methods described in the disclosed documents, respectively.
  • Specific examples include 1- (5-isoquinoline sulfonyl) homopiperazine or a salt thereof (eg, fasdyl (1- (5-isoquinolinsulfonyl) homopiperazine)), (+)-trans-4- (1-aminoethyl)-.
  • ROCK inhibitors that can be used include Y-27632 ((+)-trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane), ribasudil (4-).
  • the ROCK inhibitor is ripasudil, Y-27632, faszil, netalszil, veloszil, vermosdil or a pharmaceutically acceptable salt thereof, more preferably ripasudil, Y-27632 or a drug thereof. It may be a pharmaceutically acceptable salt, more preferably ripasudil or a pharmaceutically acceptable salt thereof.
  • compositions and the like of the present disclosure may be administered by eye drops. Therefore, the compositions and the like of the present disclosure may be eye drops.
  • compositions and the like of the present disclosure may be administered multiple times prior to cell infusion.
  • the number of administrations is 2, 3, 4, 5, 6 or more, preferably 2 to 4 times, more preferably 3 times.
  • compositions and the like of the present disclosure may be prepared, for example, from about 1 minute to about 180 minutes, from about 1 minute to about 60 minutes, from about 2 minutes to about 180 minutes, from about 2 minutes to about 60 minutes. It can be administered about 2 minutes to about 50 minutes, about 2 minutes to about 40 minutes, about 2 minutes to about 30 minutes, about 2 minutes to about 20 minutes, and about every 2 minutes to about 10 minutes. In certain embodiments, the compositions and the like of the present disclosure may be administered three times approximately every 5 minutes.
  • the timing of administration of the ROCK inhibitor is not particularly limited as long as it is before injection of corneal endothelium and / or corneal endothelium-like cells into the anterior chamber of the subject, but preferably about 4 before cell injection. It can be within hours, within about 2 hours, within about 1 hour, within about 30 minutes, within about 15 minutes, within about 10 minutes, within about 5 minutes, etc. It is preferably within about 4 hours, within about 2 hours, within about 1 hour, within about 30 minutes, within about 15 minutes, within about 10 minutes, within about 5 minutes, etc. before cell injection. In certain embodiments, the timing of administration of the ROCK inhibitor is immediately prior to cell injection, eg, within about 5 minutes, within about 4 minutes, within about 3 minutes, within about 2 minutes, within about 1 minute before cell injection. Or it can be within about 30 seconds.
  • the concentration of the ROCK inhibitor in the anterior chamber water is equal to or greater than the IC50 value.
  • the reference values of the IC50 values of Ripasudil, Y-27632, Faszil, Netalszil, Veloszil, and Belmosdil are Ripasudil: 51 nM (ROCK1), 19 nM (ROCK2); Y-27632: 110 nM (ROCK1), 170 nM (ROCK2); Faszil, respectively.
  • the number and timing of instillation of the ROCK inhibitor may be appropriately adjusted so that the concentration of the ROCK inhibitor in the anterior chamber water after instillation of the ROCK inhibitor achieves a concentration equal to or higher than the IC50 value. For example, at least 60 minutes, 120 minutes, or 180 minutes after instillation of the ROCK inhibitor, the concentration of the ROCK inhibitor in the anterior chamber water is at least about 1 time, at least about 2 times, and at least about 3 times the IC50 value.
  • Double, at least about 4 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 100 times, at least about 150 times or at least about 200 times Can be double.
  • the concentration of the ROCK inhibitor in the anterior chamber water should be about 5-10 times or higher than the IC50 value, as typically about 5-10 times the IC50 value results in near complete inhibition. However, it may be lower than 5 times if a therapeutic effect is obtained.
  • the corneal endothelium symptoms, disorders or diseases covered by the compositions of the present disclosure include decreased corneal endothelium density, formation of guttae, thickening of the desmeh membrane, thickening of the corneal thickness, and the like.
  • Corneal epithelial disorder, opacity, scar, corneal stroma, dizziness, fog, visual impairment, eye pain, tearing, congestion, pain, bullous keratopathy, eye discomfort, reduced contrast, halo, glare and stroma Examples include, but are not limited to, edema.
  • the corneal endothelium symptoms, disorders or diseases targeted by the compositions of the present disclosure include Fuchs corneal endothelial dystrophy, post-corneal transplantation disorders, corneal endothelitis, trauma, post-ophthalmic disorders, ophthalmic lasers.
  • Postoperative disorders, aging, posterior polymorphic corneal dystrophy, congenital hereditary corneal endothelial dystrophy, idiopathic corneal endothelial disorders, and cytomegalovirus corneal endothelitis include, but are not limited to.
  • the present disclosure is an eye drop for treating or preventing corneal endothelium symptoms, disorders or disorders in a subject, comprising ripasudil or a pharmaceutically acceptable salt thereof, wherein the eye drop is a corneal.
  • the corneal endothelial cells and / or corneal endothelium-like cells are injected into the anterior chamber of the subject and the eye drops are the corneal endothelial cells and / or corneal membrane.
  • an eye drop characterized by being administered three times about every 5 minutes prior to injection of the endothelial-like cells into the anterior chamber of the subject.
  • Corneal endothelial cells can be cells derived from mammals (humans, mice, rats, hamsters, rabbits, cats, dogs, cows, horses, sheep, monkeys, etc.), but are preferably derived from primates, especially from humans. preferable.
  • the number of cells to be administered is too small, the therapeutic effect will be low, so it can be at least about 40,000. If the disorder, disease or symptom of the corneal endothelium is partial, it may be less than usual, for example at least 40,000, at least about 100,000, preferably at least about 200,000. In some embodiments, the number of cells administered is from about 40,000 to about 4 million, preferably from about 100,000 to about 2 million, more preferably from about 200,000 to about 1.4 million. Most preferably, it may be about 400,000 to about 1 million.
  • the amount of liquid to be administered can be appropriately set in consideration of an appropriate viscosity, an allowable amount that can be administered to the site to be administered (for example, in the anterior chamber), and the like.
  • a fluid volume (cell suspension) of about 20 ⁇ L to about 500 ⁇ L, preferably about 30 ⁇ L to about 400 ⁇ L, more preferably about 50 ⁇ L to about 400 ⁇ L, most preferably about 200 ⁇ L to about 300 ⁇ L is administered. obtain.
  • Example 1 Cultured rabbit corneal endothelium transplantation combined with eye drop administration of ROCK inhibitor in a rabbit corneal endothelium disorder model
  • 1. Experimental method (primary culture of rabbit corneal endothelial cells) The research rabbit eyeball (Oriental Bioservice) was washed with 1 ⁇ PBS (-) (Nissui Pharmaceutical Co., Ltd., 203802). After washing, the research rabbit eyeball was excised with a spring shear blade along the corneal ring under a stereomicroscope (Leica, ES2-A). The crystalline lens and iris were removed from the research rabbit eyeball from which the sclera was resected to prepare a piece of sclera.
  • the detached corneal endothelial cells were collected in Dulbecco's modified Eagle's medium (DMEM, Nacalai Tesque, 08456-36) and then centrifuged at 1200 rpm for 3 minutes.
  • Corneal endothelial cells were seeded in 1 well per eye on a 6-well plate (CORNING, 3516) coated with Cell matrix TypeIC (Fujifilm Wako Pure Chemical Industries, Ltd., KP-4020). After seeding, the seeded cells were observed with a phase-contrast microscope. Further, the 6-well plate was shaken so that the seeded cells did not become spotted.
  • fetal bovine serum SIGMA, 13H467
  • 2 ng / ml basic FGF Fujifilm Wako Pure Chemical Industries, Ltd., 062-06661
  • P / S penicillin / streptomycin, Nacalai Tesque, 26252-94
  • Ketalal registered trademark
  • Ceractal Bayer Yakuhin, KP0DVTH
  • 2 ml of the prepared mixed anesthesia was injected intramuscularly.
  • white rabbits were anesthetized with Benokiseal ophthalmic solution 0.4% (Santen Pharmaceutical Co., Ltd., B2036).
  • the nictitating membrane of the white rabbit was excised with Cooper scissors.
  • Sandor P ophthalmic solution (NIHON TENGAN, GS1) was instilled into a white rabbit to widen the pupil.
  • the crystalline lens was removed using a 20000 Legacy Surgical system (Alcon, 203002075) to deepen the anterior chamber depth of the white rabbit.
  • a white rabbit was incised about 1.5 mm in the corneal ring under general anesthesia.
  • the corneal endothelium was peeled off using a 20-gauge silicon needle (Inami and Co, S-218C).
  • Rabbit corneal endothelium disorder model was euthanized by intravenous administration of about 6 ml / animal of potassium chloride 3 hours and 24 hours after the operation, 1 animal from each group, and 3 animals from each group on the 14th day after the operation. I let you.
  • the right eye of the euthanized rabbit corneal endothelium disorder model was removed using a utility scissors.
  • the removed eyeball was washed with 1 ⁇ PBS ( ⁇ ).
  • the sclera of the washed eyeball was excised along the corneal ring using a spring scissors blade. After excision, the crystalline lens, vitreous body, and iris were removed to prepare a strong corneal piece.
  • Blocking was performed by immersing a strong corneal piece washed with PBS in 2% BSA (Nacalai Tesque, 01863-77) for 1 hour. Next, three solutions used for the primary antibody treatment were prepared. With 2% BSA as the solvent, ZO-1 (Life Technologies, 33-9100), N-cadherin (BD Biosciences, 610921), Na + / K + -ATPase (MILLIPORE, 05-369) are 300 for each solvent. It was added so as to be double diluted. Next, the strong corneal pieces washed with 1 ⁇ PBS ( ⁇ ) were immersed in these solutions and allowed to stand for 1 hour to perform primary antibody treatment.
  • the strong corneal piece was washed 3 times with 1 ⁇ PBS ( ⁇ ) for 5 minutes. Then, a solution used for the secondary antibody treatment was prepared. Using 2% BSA as a solvent, Alexa Fluor® 488-conjugated goat anti-mouse (Life Technologies, A11001) was added so as to be diluted 1000-fold with respect to the solvent. In addition, 4', 6-diamidino-2-phenylindole (DAPI, Dojindo, KP116) was added to this solution so as to be diluted 1000-fold with respect to the solvent for nuclear staining.
  • Alexa Fluor® 488-conjugated goat anti-mouse (Life Technologies, A11001) was added so as to be diluted 1000-fold with respect to the solvent.
  • 4', 6-diamidino-2-phenylindole DAPI, Dojindo, KP116
  • Alexa Fluor® 488-conjugated phalloidin (Phalloidin, Life Technologies, A12381) was added to the solvent in a 1000-fold dilution to observe cell morphology.
  • the strong corneal piece was immersed in this solution and allowed to stand at room temperature for 1 hour in the dark.
  • the strong corneal piece was washed 3 times with 1 ⁇ PBS ( ⁇ ) for 5 minutes.
  • the stained strong keratotic membrane pieces were encapsulated using MICRO SLIDE GLASS (Matsunami Glass Industry, S2441) and Matsunami Cover Glass NEO 24 ⁇ 60 (Matsunami Glass Industry, 83-0217).
  • the enclosed strong corneal piece was observed with an inverted confocal fluorescence microscope (Leica, DMI4000 B).
  • the central corneal thickness of the right eye of the rabbit corneal endothelial injury model was measured over time using an ultrasonic pachymeter.
  • the central corneal thickness of the control group exceeded the measurement limit of 1200 ⁇ m of the ultrasonic pachymeter in most of the individuals during the course, and was at least 1200 ⁇ m or more.
  • the values were lower than those in the control group throughout most of the observation period. No significant difference was observed between the Y-27632 combination group and the ripasudil instillation group (Fig. 2).
  • Tonovet® was used to measure the intraocular pressure of the right eye of a rabbit corneal endothelial injury model over time. Measurements 14 days after surgery did not exceed the normal value limit of 20 mmHg in all individuals (Fig. 3). Therefore, it was shown that by transplanting corneal endothelial cells after instilling ripasudil, an abnormal increase in intraocular pressure due to abnormal deposition of the transplanted cells in the eye did not occur.
  • the fibroblast-like cells observed by phalloidin staining in the control group were N-cadherin and ZO-1, which are indicators of barrier function, and Na + / K + -, which are indicators of pump function. Almost no expression of ATPase was observed.
  • these function-related molecules were observed to be expressed between cells as in the living body (Fig. 5). From these results, it was shown that the corneal endothelium can be regenerated in vivo and the cornea can be made transparent by continuously transplanting the corneal endothelial cells derived from rabbits into Ripasudil eye drops.
  • Example 2 Cultured human corneal endothelium transplantation combined with eye drop administration of ROCK inhibitor in a rabbit corneal endothelium disorder model
  • 1. Experimental method (culture of human corneal endothelial cells) Corneal endothelial cells were collected from human corneal tissue and subjected to primary culture and subculture. First, 0.5% trypan blue staining solution (Nacalai Tesque, 29853-34) was added to the center of the donor cornea for staining.
  • the trypan blue staining solution was washed away with Opti MEM TM -I (invitrogen, 31985-088) to which gentamicin (invitrogen, 15710-064) was added to a final concentration of 50 ⁇ g / L.
  • the cornea was observed using a microscope and a monitor installed in a clean bench, and the Descemet's membrane stained with trypan blue was peeled from the cornea with tweezers.
  • the exfoliated Descemet's membrane was transferred to a 15 mL centrifuge tube containing 1 mg / mL collagenase A (Roche, 101356001) and incubated at 37 ° C. (5% CO2) for 16 hours.
  • the centrifuge tube was centrifuged at 300 G for 5 minutes. After centrifugation, the supernatant was removed and the pellet was resuspended using medium supplemented with Y-27632 (WAKO, 253-00513) to a final concentration of 10 ⁇ M.
  • the resuspended cell suspension was seeded on a 6-well plate coated with a laminin E8 fragment (iMatrix-511; Nippi, Inc., 381-07363). After 24 hours, the medium was replaced with a medium containing no Y-27632, and then the medium was replaced every two days, and the cells were cultured until they became confluent.
  • fetal bovine serum FBS
  • Thermo Fisher Scientific GVJ0081
  • 50 ⁇ g / mL gentamicin 200 mg / L calcium chloride
  • 0.08% chondroitin sulfate C sodium in OptiMEMTM-I as a medium.
  • WAKO 032-14613
  • 5 ng / mL epidermal growth factor EGF; invitrogen, PHG0311
  • 20 ⁇ g / mL ascorbic acid Nakalitesk, M9K6113
  • 10 ⁇ M SB431542 WAKO, 192-16541
  • 10 ⁇ M SB203580 Cayman Ce
  • Ketalal registered trademark
  • Ceractal Bayer Yakuhin, KP0DVTH
  • 2 ml of the prepared mixed anesthesia was injected intramuscularly.
  • white rabbits were anesthetized with Benokiseal ophthalmic solution 0.4% (Santen Pharmaceutical Co., Ltd., B2036).
  • the nictitating membrane of the white rabbit was excised with Cooper scissors.
  • Sandor P ophthalmic solution (NIHON TENGAN, GS1) was instilled into a white rabbit to widen the pupil.
  • the crystalline lens was removed using a 20000 Legacy Surgical system (Alcon, 203002075) to deepen the anterior chamber depth of the white rabbit.
  • a white rabbit was incised about 1.5 mm in the corneal ring under general anesthesia.
  • the corneal endothelium was peeled off using a 20-gauge silicon needle (Inami and Co, S-218C).
  • Permeation treatment was performed by immersing the strong corneal piece washed with 1 ⁇ PBS ( ⁇ ) in 1% Toriton X-100 (Nacalai Tesque, 28229-25) for 5 minutes. After the permeation treatment, the corneal pieces were washed 3 times with 1 ⁇ PBS ( ⁇ ). Blocking was performed by immersing the corneal piece 2% BSA (Nacalai Tesque, 01863-77) washed with PBS for 1 hour. Next, a solution used for the secondary antibody treatment was prepared.
  • 2% BSA was used as a solvent, and 4', 6-diamidino-2-phenylindole (DAPI, Dojindo, KP116) was added to this solution in a 1000-fold dilution with respect to the solvent for nuclear staining.
  • Alexa Fluor® 488-conjugated phalloidin Phalloidin, Life Technologies, A12381 was added to the solvent in a 1000-fold dilution to observe cell morphology.
  • the strong corneal piece was immersed in this solution and allowed to stand at room temperature for 1 hour in the dark. After the secondary antibody treatment, the strong corneal piece was washed 3 times with 1 ⁇ PBS ( ⁇ ) for 5 minutes.
  • a aqueous humor pipette Nipro Co., Ltd., 19B18
  • the aqueous humor collected at each time was diluted with methanol (Nacalai Tesque, 21929-23) at a ratio of 3: 7. Then, centrifugation was performed at 4 ° C., 3000 rpm, and 10 minutes, and about 200 ⁇ l of the supernatant was taken out, and the drug concentration in the anterior chamber water was measured by HPLC according to the following conditions.
  • Example 4 Comparison of anterior chamber concentration between 10 mM Y-27632 eye drops and injection of Y-27632 into the eye
  • aqueous humor pipette Napro Co., Ltd., 19B18
  • the aqueous humor collected at each time was diluted with methanol (Nacalai Tesque, 21929-23) at a ratio of 3: 7. Then, centrifugation was performed at 4 ° C., 3000 rpm, and 10 minutes, and about 200 ⁇ l of the supernatant was taken out, and the drug concentration in the anterior chamber water was measured by HPLC according to the following conditions.
  • the concentrations of Y-27632 in the aqueous humor after 100 ⁇ M Y-27632 injection were 68.19 ⁇ 13.95 ⁇ M and 17.07 ⁇ 4.15 ⁇ M, 5.18 ⁇ 2 at 0, 15, 30, and 60 minutes after injection, respectively. It was .88 ⁇ M and 0.91 ⁇ 0.37 ⁇ M. After 120 and 180 minutes, it was below the limit of quantification.
  • the concentration of Y-27632 in the aqueous humor of 10 mM Y-27632 ophthalmic solution was 25.94 ⁇ 2.47, 11.66 ⁇ 0.65, 9.60 ⁇ 0. It was 91, 2.33 ⁇ 0.75 and 0.50 ⁇ 0.27 ⁇ M. After 180 minutes, it was below the limit of quantification (Fig. 9).
  • Example 5 Cultured rabbit corneal endothelium transplantation combined with instillation of netalszil in a rabbit corneal endothelium disorder model
  • cultured rabbit corneal endothelium transplantation was performed in combination with instillation of Netaluszil.
  • Primary culture of rabbit corneal endothelial cells, primary culture of rabbit corneal endothelial cells, cultured corneal endothelial transplantation, and immunohistological studies were performed based on Example 1.
  • Example 6 Concentration in anterior chamber water after instillation of Netaluszil
  • concentration in the anterior chamber water after instillation of Netaluszil was measured. The experiment was performed based on Example 3. In this example, 0.02% Netaluszil instillation was used.
  • the drug concentration in the anterior chamber water was measured based on Example 3.
  • Example 7 Eye drops of other ROCK inhibitors
  • the ROCK inhibitor As an eye drop of the ROCK inhibitor, 0.01% to 0.2% of Veloszil, Belmosdil, or Fasdil (solvent: physiological saline) is used. Similar to Example 1, primary culture of rabbit corneal endothelial cells, primary culture of rabbit corneal endothelial cells, cultured corneal endothelial transplantation, and immunohistological tests are performed. Twenty-four hours after the operation, the area where the cells adhere to the corneal endothelium is measured. Also, confirm that the morphology of the cells is a polygonal morphology similar to the corneal endothelium.
  • Example 3 the concentration in the anterior chamber water after instillation of veloszil, vermoszil, or fasdil is confirmed. Check the concentration in the anterior chamber water 60 minutes after instillation, and confirm that it exceeds each IC 50 value.
  • a composition for treating a symptom, disorder or disease of the corneal endothelium is provided and can be used in the fields of pharmaceuticals and the like.

Abstract

La présente divulgation concerne une composition destinée à améliorer l'effet thérapeutique d'une thérapie par injection de cellules, ladite thérapie visant à traiter ou à prévenir un symptôme, un trouble ou une maladie endothélial(e) cornéen(ne). Dans un mode de réalisation, la présente divulgation concerne une composition, qui comprend un inhibiteur de ROCK, destinée à améliorer l'effet thérapeutique d'une thérapie par injection de cellules, ladite thérapie visant à traiter ou à prévenir un symptôme, un trouble ou une maladie endothélial(e) cornéen(ne) d'un sujet. Cette composition, qui doit être utilisée en combinaison avec des cellules endothéliales cornéennes et/ou des cellules de type endothéliales cornéennes, est caractérisée en ce que la composition est administrée avant l'injection des cellules endothéliales cornéennes et/ou des cellules de type endothéliales cornéennes dans la chambre antérieure du sujet. Dans certains modes de réalisation, la composition, etc. de la présente divulgation sont formulés en gouttes ophtalmiques.
PCT/JP2021/028299 2020-07-31 2021-07-30 Procédé d'amélioration de l'effet thérapeutique d'une thérapie par injection de cellules endothéliales cornéennes WO2022025239A1 (fr)

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JP2019510509A (ja) * 2016-02-15 2019-04-18 京都府公立大学法人 ヒト機能性角膜内皮細胞およびその応用

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JP2019510509A (ja) * 2016-02-15 2019-04-18 京都府公立大学法人 ヒト機能性角膜内皮細胞およびその応用

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Title
KOIZUMI, NORIKO: "Development of Corneal Endothelium Regenerative Treatment Using Somatic Stem Cells", JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 241, 30 November 2011 (2011-11-30), JP , pages 765 - 770, XP009533726, ISSN: 0039-2359 *
YUJI SAKAMOTO, NAOKI OKUMURA, NORIKO KOIZUMI, RYOUHEI NUMATA, JUNJI KITANO, MAYUMI YAMAMOTO, TAKAFUMI KOMADA, NOBUHIKO HOSHI: "Ferret animal model of corneal endothelial dysfunction for evaluation of drug effect on corneal endothelial wound healing", ANIMAL EYE RESEARCH, vol. 32, 1 January 2013 (2013-01-01), JP , pages 15 - 21, XP055421934, ISSN: 0286-7486, DOI: 10.11254/jscvo.32.15 *

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