WO2024172043A1 - 網膜色素上皮裂孔の治療薬 - Google Patents

網膜色素上皮裂孔の治療薬 Download PDF

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WO2024172043A1
WO2024172043A1 PCT/JP2024/004888 JP2024004888W WO2024172043A1 WO 2024172043 A1 WO2024172043 A1 WO 2024172043A1 JP 2024004888 W JP2024004888 W JP 2024004888W WO 2024172043 A1 WO2024172043 A1 WO 2024172043A1
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retinal pigment
cells
human
therapeutic agent
pigment epithelial
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French (fr)
Japanese (ja)
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清子 坂東
耕一郎 真鍋
達也 亀井
直樹 高村
仁 渡邉
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Sumitomo Pharma Co Ltd
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Sumitomo Pharmaceuticals Co Ltd
Sumitomo Pharma Co Ltd
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Priority to EP24756885.0A priority patent/EP4659755A1/en
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    • 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
    • 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
    • A61K31/4353—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 ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—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 ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • 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
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • 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
    • 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
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins

Definitions

  • the present invention relates to a drug for treating retinal pigment epithelial tears.
  • Retinal pigment epithelial (RPE) cells are pigment epithelial cells that exist in the outermost layer of the retina, and play an important role in maintaining photoreceptors, such as phagocytosis of photoreceptor outer segments and recycling of visual pigment.
  • Retinal pigment epithelial tears are known to be pathological conditions related to RPE cells. Retinal pigment epithelial tears are thought to be caused by a tear in the retinal pigment epithelial cell layer, which then shrinks in the torn area, and in the long term, this can lead to a decrease in vision.
  • retinal pigment epithelial tears there is currently no treatment for retinal pigment epithelial tears.
  • surgical treatments such as macular relocation and autologous RPE transplantation have been performed in the past, but both methods are highly difficult and invasive, and have a high risk of complications.
  • retinal pigment epithelial cell sheets have been considered for age-related macular degeneration and other conditions, but problems have been pointed out with the use of RPE sheets, such as the need to make a large retinal incision near the macula, which can result in visual field defects in the same area. Furthermore, even in transplants in which a suspension of RPE cells is administered subretinal with minimal invasiveness, complications such as insufficient engraftment and premacular membranes have been reported. Therefore, the formulation and transplantation techniques for retinal pigment epithelial cells are still in the process of trial and error and have not yet been perfected.
  • the present invention aims to provide a therapeutic agent for treating retinal pigment epithelial breaks in human subjects.
  • a drug for treating retinal pigment epithelial breaks in a human subject, comprising a suspension of dispersed human retinal pigment epithelial cells.
  • [2-3] The therapeutic agent according to [2-1] or [2-2], wherein the pharma- ceutically acceptable aqueous liquid is a balanced salt solution.
  • the therapeutic agent described in [5] or [6] which is used in a treatment comprising replacing the vitreous cavity of the human subject with air after administration of the therapeutic agent.
  • HLA human leukocyte antigen
  • the at least one immunosuppressant is at least one selected from the group consisting of triamcinolone acetonide, cyclosporine, tacrolimus, prednisolone, and dexamethasone.
  • a method for treating a retinal pigment epithelium break in a human subject comprising administering to the human subject in need thereof an effective amount of a suspension in which human retinal pigment epithelial cells are dispersed.
  • a frozen preparation comprising retinal pigment epithelial cells for use as a therapeutic agent according to any one of [1] to [14].
  • the frozen preparation according to [20] which is thawed, replaced with a medium exhibiting a viscosity of 4 mPa ⁇ s or less at a shear rate of 2 (1/s) at 25°C, and administered to the human subject as a suspension in which the human retinal pigment epithelial cells are dispersed.
  • the present invention provides a therapeutic agent for treating a retinal pigment epithelial break in a human subject.
  • the therapeutic agent is administered (or transplanted) under the retina of the eyeball of a human subject having a retinal pigment epithelial break, thereby reducing the burden and side effects on the human subject and engrafting at the administration site in the human subject, thereby filling at least a portion of the retinal pigment epithelial break.
  • FIG. 1 is a schematic diagram showing a procedure for administering RPE cells subretinally via the transvitreal method.
  • 1 shows images obtained by fluorescent observation of RPE cells administered subretinaly into an excised pig eye in Example 1.
  • 1 shows a low-magnification fluorescent immunostained image of a representative retina including an administration site in the fluorescent immunostaining of Example 2.
  • 1 shows a low-magnification fluorescent immunostained image of a representative retina including an administration site in the fluorescent immunostaining of Example 2.
  • 1 shows a high-magnification fluorescent immunostained image of a representative retina including an administration site in the fluorescent immunostaining of Example 2.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using HBSS(+) medium in the fluorescent immunostaining of Example 3.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using HBSS(+) medium in the fluorescent immunostaining of Example 3.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using HBSS(+) medium in the fluorescent immunostaining of Example 3.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using HBSS(+) medium in the fluorescent immunostaining of Example 3.
  • 13 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using a 100-fold diluted Provisc medium in the fluorescent immunostaining of Example 3.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using a 100-fold diluted Provisc medium in the fluorescent immunostaining of Example 3.
  • Example 13 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using a 100-fold diluted Provisc medium in the fluorescent immunostaining of Example 3.
  • 1 shows a representative fluorescent immunostained image of the retina including the administration site when a suspension of human iPS cell-derived RPE cells was administered using a 100-fold diluted Provisc medium in the fluorescent immunostaining of Example 3.
  • 1 shows the results of an electroretinogram (ERG) test carried out using RCS rats in Example 6.
  • EDP visual evoked potential
  • Example 6 shows the results of an optokinetic reflex (OKT) test carried out using RCS rats in Example 6.
  • 1 shows a representative immunofluorescence staining image of an RCS rat retina transplanted with human iPS cell-derived RPE cells in Example 6.
  • 1 shows a representative immunofluorescence staining image of an RCS rat retina transplanted with human iPS cell-derived RPE cells in Example 6.
  • 1 shows a representative immunofluorescence staining image of an RCS rat retina transplanted with human iPS cell-derived RPE cells in Example 6.
  • 1 shows a representative immunofluorescence staining image of an RCS rat retina transplanted with human iPS cell-derived RPE cells in Example 6.
  • 1 shows representative immunofluorescence staining images of human iPS cell-derived RPE cells and rat photoreceptor outer segments after transplantation into RCS rat retina in Example 6.
  • Example 1 shows representative fluorescent immunostained images of human iPS cell-derived RPE cells and tight junction-constituting proteins after transplantation into the retina of an RCS rat in Example 6.
  • 13 shows representative fluorescent immunostaining images of synapses between photoreceptors and bipolar cells present in the outer plexiform layer in Example 6.
  • 13 shows the results of evaluating cell adhesion of human iPS cell-derived RPE cells onto a laminin-coated substrate in Example 7.
  • Example 7 shows an image of a simulated retinal pigment epithelium tear site formed by peeling off a portion of an RPE sheet onto a plate in Example 8.
  • 13 shows the results of evaluating cell adhesion of human iPS cell-derived RPE cells added as a suspension to a simulated retinal pigment epithelium break site in Example 8.
  • 13 shows the results of evaluating cell adhesion of human iPS cell-derived RPE cells added as a suspension to a simulated retinal pigment epithelium break site in Example 8.
  • 1 shows the results of observation of an untreated simulated retinal pigment epithelium tear site in Example 8.
  • 13 shows the results of evaluating cell adhesion of human iPS cell-derived RPE cells added as a suspension to a simulated retinal pigment epithelium break site in Example 8.
  • Example 13 shows the results of evaluating cell adhesion of human iPS cell-derived RPE cells added as a suspension to a simulated retinal pigment epithelium break site in Example 8.
  • 1 shows representative immunofluorescence staining images of rat retina when vehicle alone was intraperitoneally administered to F344/NJcl nude rats in Example 10.
  • 1 shows representative immunofluorescence staining images of rat retina when sodium iodate was administered intraperitoneally to F344/NJcl nude rats in Example 10.
  • 1 shows the results of an ERG test carried out using F344/NJcl nude rats in Example 10.
  • 1 shows fundus images of F344/NJcl nude rats taken with a fundus camera in Example 10.
  • 1 shows representative fluorescent immunostaining of the retina in a model animal prepared using F344/NJcl nude rats in Example 10, in which only a vehicle was administered.
  • 1 shows representative fluorescent immunostaining of the retina in a model animal prepared using F344/NJcl nude rats in Example 10, in which human iPS cell-derived RPE cells were transplanted.
  • 1 shows representative fluorescent immunostaining of the retina in a model animal prepared using F344/NJcl nude rats in Example 10, in which human iPS cell-derived RPE cells were transplanted.
  • 1 shows representative fluorescent immunostaining of the retina in a model animal prepared using F344/NJcl nude rats in Example 10, in which only a vehicle was administered.
  • 1 shows representative fluorescent immunostaining of the retina in a model animal prepared using F344/NJcl nude rats in Example 10, in which human iPS cell-derived RPE cells were transplanted.
  • retinal pigment epithelial (RPE) cells refers to epithelial cells present outside the neural retina in a living retina. Whether a cell is a retinal pigment epithelial cell can be easily confirmed by a person skilled in the art, for example, by the expression of cell markers (RPE65, MITF, CRALBP, MERTK, BEST1, TTR, PMEL17, etc.), the presence of melanin granules (black-brown), tight junctions between cells, characteristic polygonal/cobblestone cell morphology, etc. Whether a cell has the function of a retinal pigment epithelial cell can be easily confirmed by the secretion ability of cytokines such as VEGF and PEDF, etc.
  • the retinal pigment epithelial cell is an RPE65-positive cell, an MITF-positive cell, or an RPE65-positive and MITF-positive cell.
  • the RPE cells to be transplanted are not particularly limited, and are preferably RPE cells derived from rodents, carnivores, or primates, and are particularly preferably RPE cells derived from humans.
  • RPE cells derived from pluripotent stem cells are preferred, and are particularly preferably derived from in vitro differentiation of human pluripotent stem cells (particularly iPS cells).
  • pluripotent stem cells refer to cells that have the ability to self-replicate and pluripotency, can be cultured in vitro, and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm).
  • pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells).
  • the pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent, carnivorous or primate pluripotent stem cells, and more preferably human pluripotent stem cells.
  • mammals include primates such as humans and monkeys, rodents such as mice, rats, hamsters and guinea pigs, carnivorous animals such as dogs and cats, and other animals such as pigs, cows, goats, horses, sheep and rabbits.
  • Embryonic stem cells can be produced, for example, by culturing the inner cell mass present in a blastocyst-stage embryo prior to implantation on feeder cells or in a medium containing LIF. Specific methods for producing embryonic stem cells are described, for example, in WO96/22362, WO02/101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from designated institutions, and also commercially available products can be purchased. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. EB5 cells, which are all mouse embryonic stem cells, are available from the National Research and Development Agency RIKEN, and the D3 strain is available from the ATCC.
  • Human embryonic stem cells are established from human embryos within 14 days of fertilization.
  • Nuclear transfer ES cells a type of ES cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.
  • “Artificial pluripotent stem cells” are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, examples of such cells include cells in which pluripotency has been induced by reprogramming somatic cells, such as fibroblasts, skin cells, and peripheral blood mononuclear cells, by introducing any combination of multiple reprogramming factors selected from a group of genes such as Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, and Esrrb.
  • Preferred combinations of reprogramming factors include (1) Oct3/4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3/4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466).
  • Yamanaka et al. first established induced pluripotent stem cells in mouse cells (Cell, 2006, 126(4) pp.663-676), and in 2007 they were also established in human fibroblast cells (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106).
  • Various improvements have been made to the methods of inducing induced pluripotent stem cells since then, and specific production methods are described, for example, in Cell. 2006 Aug 25; 126(4): 663-76 for mouse induced pluripotent stem cells and in Cell. 2007 Nov 30; 131(5): 861-72 for human induced pluripotent stem cells.
  • induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341 pp. 651-654).
  • induced pluripotent stem cell lines for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University, are available from Kyoto University or iPS Academia Japan Inc. Also, as established induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01 cells established at Kyoto University are available from Kyoto University.
  • the somatic cells used in producing induced pluripotent stem cells are not particularly limited, and specific examples include fibroblasts, blood cells (e.g., peripheral blood mononuclear cells or T cells, and cells derived from umbilical cord blood), etc.
  • fibroblasts include those derived from the dermis.
  • the means for gene expression there is no particular limitation on the means for gene expression, and any gene transfer method well known to those skilled in the art or a direct protein injection method can be used.
  • the gene transfer method include an infection method using a viral vector (e.g., a retroviral vector, a lentiviral vector, a Sendai virus vector, an adenoviral vector, an adeno-associated virus vector), a calcium phosphate method using a plasmid vector (e.g., a plasmid vector, an episomal vector) or an RNA vector, a lipofection method, a retronectin method, or an electroporation method.
  • a viral vector e.g., a retroviral vector, a lentiviral vector, a Sendai virus vector, an adenoviral vector, an adeno-associated virus vector
  • a calcium phosphate method using a plasmid vector (e.g., a plasmid vector, an episomal vector
  • induced pluripotent stem cells When producing induced pluripotent stem cells, they can be produced in the presence or absence of feeder cells (feeder-free). When producing induced pluripotent stem cells in the presence of feeder cells, they can be produced in the presence of undifferentiated maintenance factors by a known method.
  • the medium used when producing induced pluripotent stem cells in the absence of feeder cells is not particularly limited, but a known maintenance medium for embryonic stem cells and/or induced pluripotent stem cells, or a medium for establishing induced pluripotent stem cells in a feeder-free manner can be used.
  • Examples of media for establishing induced pluripotent stem cells in a feeder-free manner include feeder-free media such as Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit (registered trademark).
  • feeder-free media such as Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit (registered trademark).
  • the pluripotent stem cells used in the present invention are preferably induced pluripotent stem cells from rodents, carnivores, or primates, and more preferably human induced pluripotent stem cells.
  • Pluripotent stem cells can be maintained and expanded by methods known to those skilled in the art, but from the standpoint of safety in the production of transplant cells, it is preferable to maintain and expand pluripotent stem cells under serum-free conditions and in the absence of feeder cells.
  • Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques.
  • genes on chromosomes that can be modified include cell marker genes, genes for histocompatibility antigens, and disease-related genes caused by disorders of retinal pigment epithelial cells.
  • Target genes on chromosomes can be modified using methods described in Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Production of Mutant Mice Using ES Cells, Yodosha (1995); etc.
  • genomic DNA containing the target gene to be modified e.g., a cell marker gene, a gene for a histocompatibility antigen, or a disease-related gene
  • a target vector for homologous recombination of the target gene is prepared using the isolated genomic DNA.
  • the target vector thus prepared is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the target vector are selected, thereby producing stem cells in which genes on chromosomes have been modified.
  • Genomic DNA containing a target gene can also be isolated using a genomic DNA library screening system (manufactured by Genome Systems) or Universal GenomeWalker Kits (manufactured by CLONTECH).
  • a polynucleotide encoding a target protein can also be used instead of genomic DNA.
  • the polynucleotide can be obtained by amplifying the corresponding polynucleotide by PCR.
  • a target vector for homologous recombination of a target gene and efficient selection of a homologous recombinant can be performed according to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells, Yodosha (1995); etc. Either a replacement type or an insertion type target vector can be used. Selection methods that can be used include positive selection, promoter selection, negative selection, and polyA selection.
  • Methods for selecting the desired homologous recombinant from the selected cell lines include Southern hybridization and PCR for genomic DNA.
  • the method for producing retinal pigment epithelial cells from pluripotent stem cells is not particularly limited as long as it is a known method, and examples thereof include those described in WO2005/070011, WO2006/080952, WO2009/051671, WO2011/063005, WO2012/173207, WO2015/053375, WO2014/087244, WO2014/121077, WO2015/053376, WO2015/068505, WO2017/043605, Stem Cell Reports, 2(2), 205-218 (2014) and Cell Stem Cell, 10(6), 771-785 (2012).
  • the method may be a method for producing retinal pigment epithelial cells, which includes the following steps (1) to (2) disclosed in WO2017/043605.
  • the method for producing retinal pigment epithelial cells may also include the following steps (1) and (2). (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and/or a MEK inhibitor for a period sufficient to induce gene expression of at least one of eye formation transcription factors and not exceeding 30 days; and (2) a second step of culturing the cells obtained in the first step in the presence of a Nodal signaling pathway inhibitor and/or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
  • the method for producing retinal pigment epithelial cells may further include the following steps (1) and (2). (1) a first step of culturing pluripotent stem cells in a medium containing an FGF receptor inhibitor and/or a MEK inhibitor for a period sufficient to induce expression of at least one of the genes PAX6, LHX2, and SIX3 and not exceeding 30 days; and (2) a second step of culturing the cells obtained in the first step in the presence of a Nodal signaling pathway inhibitor and/or a Wnt signaling pathway inhibitor to form retinal pigment epithelial cells.
  • the FGF receptor inhibitor is not particularly limited as long as it is a substance capable of suppressing signal transduction mediated by FGF, and may be any of proteins, nucleic acids, and low molecular weight compounds.
  • FGFs form a family consisting of at least 22 types.
  • Representative FGF receptors include FGFR1, FGFR2, FGFR3, and FGFR4, and an FGF receptor inhibitor is a substance that inhibits one, several, or all of these.
  • Such substances include, but are not limited to, substances that act directly on FGF or FGF receptors (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding FGF or FGF receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of FGF receptors to FGF (e.g., soluble FGF receptors, FGF antagonists, etc.), and substances that inhibit physiological activities resulting from signal transduction by FGF receptors (e.g., small molecular weight compounds such as PD173074 (N-[2-[[4-(Diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea) or SU5402 (2-[(1,2-Dihydro-2-oxo-3H-indol-3
  • PD173074 and SU5402 are known FGF receptor inhibitors, and are available as commercial products, etc. as appropriate.
  • PD173074 or SU5402 is a preferred FGF receptor inhibitor.
  • the MEK inhibitor there are no particular limitations on the MEK inhibitor, so long as it is a substance that inhibits the expression or activity of the MEK family, and it may be any of a protein, a nucleic acid, or a low molecular weight compound.
  • Representative examples of the MEK family include MEK1, MEK2, MEK3, etc., and a MEK inhibitor is a substance that inhibits the expression or activity of one, multiple, or all of these MEK family members.
  • Such substances include substances that suppress the expression of genes encoding various MEKs (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the enzymatic activity of various MEKs [PD0325901 (N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide), PD184352 ((2-[(2-Chloro-4-iodophenyl)amino]-N-cyclopropylmethoxy)-3,4-difluorobenzamide), PD98059 (2'-Amino-3'-Methoxyflavone), U0126 (1,4-Diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene), MEK162 (5-[(4-Bromo-2-fluorophenyl)amino]
  • PD0325901, PD184352, PD98059, U0126, MEK162, SL327, TAK-733 and AZD-8330 are known MEK inhibitors and are available as commercial products, etc.
  • Preferred examples of MEK inhibitors include PD0325901, PD184352, U0126, TAK-733 and AZD-8330.
  • the Nodal signaling pathway inhibitor is not particularly limited as long as it is capable of suppressing signaling mediated by Nodal, and may be any of proteins, nucleic acids, and low molecular weight compounds. Signals mediated by Nodal are transmitted via Nodal receptors. Nodal receptors exist as heterodimers of type I receptors (ALK (activin receptor-like kinase)-4, ALK-5, ALK-7) and type II receptors (ActRII).
  • inhibitors of the Nodal signaling pathway include substances that act directly on Nodal or Nodal receptors (anti-Nodal antibodies, anti-Nodal receptor antibodies, etc.), substances that suppress the expression of genes encoding Nodal or Nodal receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Nodal receptors to Nodal (Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, etc.), and substances that inhibit physiological activity caused by signal transduction by Nodal receptors [small molecular weight compounds such as SB-431542 (SB431542) (4-[4-(1,3-Benzodioxol-5-yl)-5-(pyridin-2-yl)-1H-imidazol-2-yl]benzamide), which inhibits the kinase activity of type I receptors by competing for ATP], but are not limited to these. SB-431542 is a
  • the Wnt signaling pathway inhibitor is not particularly limited as long as it can suppress signaling mediated by Wnt, and may be any of proteins, nucleic acids, low molecular weight compounds, etc.
  • Wnt-mediated signals are transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and LRP5/6 (low-density lipoprotein receptor-related protein 5/6).
  • Wnt signaling pathway inhibitors examples include substances that act directly on Wnt or Wnt receptors (anti-Wnt antibodies, anti-Wnt receptor antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus proteins, etc.), and substances that inhibit physiological activities caused by signaling via Wnt receptors [casein kinase I inhibitors CKI-7 (N-(2-Aminoethyl)-5-chloroisoquinoline-8-sulfonamide) and D4476 (4-[4-(2,3-Dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]beta] n
  • CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, and the like are known Wnt signal inhibitors, and are commercially available products, etc., as appropriate.
  • a preferred example of a Wnt signal inhibitor is CKI-7.
  • Eye Field Transcription Factors refer to genes that are expressed in the eye-forming region during early development, and ET (Tbx3), Rx1 (Rax), Pax6, Six3, Lhx2, Tlx (Nr2e1), Optx2 (Six6), etc. have been identified. These eye-forming transcription factors can be used as markers for early eye formation.
  • the number of days for the first step is not particularly limited as long as it is within the period during which the cells generated by exposing pluripotent stem cells to the above-mentioned FGF receptor inhibitor and/or MEK inhibitor retain the ability to differentiate into retinal pigment epithelial cells, but the pluripotent stem cells in the first step are cultured for a period not exceeding 30 days. This period may vary depending on the strain of pluripotent stem cells used, but is usually 2 days or more, preferably 3 days or more, and more preferably 4 days or more.
  • the pluripotent stem cells in the first step are more preferably cultured for 2 to 13 days or 2 to 6 days, and even more preferably 4 to 6 days.
  • the culture period in the second step is not particularly limited as long as it is a period during which the desired retinal pigment epithelial cells can be induced, but an example of such a culture period is one in which retinal pigment epithelial cells can usually be generated on the 14th to 40th day counting from the start of the second step.
  • retinal pigment epithelial cells based on the expression of cell markers (RPE65 (retinal pigment epithelial cells), Mitf (retinal pigment epithelial cells), BEST1 (retinal pigment epithelial cells), CRALBP (retinal pigment epithelial cells), etc.), the presence of melanin granules (black-brown), tight junctions between cells, and characteristic polygonal/pavement-like cell morphology, and can set the culture period by confirming these.
  • cell markers RPE65 (retinal pigment epithelial cells), Mitf (retinal pigment epithelial cells), BEST1 (retinal pigment epithelial cells), CRALBP (retinal pigment epithelial cells), etc.
  • a maintenance medium for retinal pigment epithelial cells may be, for example, that described in IOVS, March 2004, Vol. 45, No. 3, Masatoshi Haruta, et. al., IOVS, November 2011, Vol. 52, No. 12, Okamoto and Takahashi, J. Cell Science 122 (17), Fumitaka Osakada, et. al., IOVS, February 2008, Vol. 49, No. 2, Gamm, et. al., and is composed of a basal medium, serum and/or serum substitute, and other components.
  • retinal pigment epithelial cells by omitting the first step and performing only the second step.
  • the sheet of retinal pigment epithelial cells can be prepared as a suspension that can be transplanted into a patient by dispersing the retinal pigment epithelial cells using a method well known to those skilled in the art, such as using an enzyme such as trypsin, recovering the retinal pigment epithelial cells as independent, single cells, and suspending these in a dispersion medium.
  • Adhesion culture refers to culture carried out under conditions that allow cells or cell aggregates to adhere to culture equipment, etc.
  • cell adhesion means that strong cell-substrate bonds are formed between the cells or cell aggregates and the culture equipment.
  • adhesion culture refers to culture under conditions that allow strong cell-substrate bonds to be formed between the cells or cell aggregates and the culture equipment, etc.
  • Those skilled in the art can set the culture conditions for the culture equipment, etc. as appropriate and carry out adhesion culture.
  • Retinal pigment epithelial cells can also be produced by suspension culture. That is, embryonic bodies (also known as EBs) can be produced from pluripotent stem cells, and these can be induced to differentiate into retinal pigment epithelial cells to produce cell aggregates containing retinal pigment epithelial cells.
  • the cell aggregates can be dispersed by a method well known to those skilled in the art, such as using an enzyme such as trypsin, to recover the retinal pigment epithelial cells as independent, single cells, which can then be suspended in a dispersion medium to prepare a suspension that can be transplanted into a patient.
  • the retinal pigment epithelial cells After dispersing the cell aggregates/sheets of retinal pigment epithelial cells produced by the above-mentioned method, the retinal pigment epithelial cells can be suspended in a freezing medium and frozen to prepare a frozen preparation containing retinal pigment epithelial cells that can be stored for a long period of time.
  • the freezing medium used here is not particularly limited as long as it does not adversely affect the function or survival of the retinal pigment epithelial cells, and may be any medium containing a cryoprotectant.
  • the cryoprotectant may be any substance that is effective in protecting cells and organelles (e.g., capable of preventing ice crystals from forming within cells when they are frozen), and examples of such substances include dimethyl sulfoxide (DMSO), glycerol, polyethylene glycol, propylene glycol, glycerin, sorbitol, dextran, and trehalose.
  • DMSO dimethyl sulfoxide
  • glycerol polyethylene glycol
  • propylene glycol glycerin
  • sorbitol dextran
  • trehalose trehalose
  • a commercially available cell freezing preservation solution e.g., StemCellBanker (Zenoaq)
  • StemCellBanker Zenoaq
  • a suspension that can be transplanted into a patient can be prepared.
  • retinal pigment epithelium tear refers to a condition in which a tear occurs in the retinal pigment epithelium cell layer on the living retina, and refers to a pathological condition in which there is a partial defect or loss of the retinal pigment epithelium cell layer.
  • a retinal pigment epithelium tear is a pathological condition in which the retinal pigment epithelium cell layer tears, contracts and rolls from the tear, and the retinal pigment epithelium is partially lost. It also exposes Bruch's membrane and choroid that are located below the retinal pigment epithelium cell layer.
  • the most classic shape of a retinal pigment epithelium tear is a crescent-shaped tear, but there are various shapes, such as a concentric tear with an island of rolled-up retinal pigment epithelium in the center of the tear.
  • retinal pigment epithelium tear there is no particular limitation on the cause of a "retinal pigment epithelium tear,” and it includes both retinal pigment epithelium tears caused by retinal, choroidal, or other eye-related diseases, and retinal pigment epithelium tears that are not caused by retinal-related diseases such as trauma.
  • the layered structure of the retinal pigment epithelium and photoreceptor cells is destroyed, resulting in a breakdown of the visual cycle and a loss of the dark curtain function of the retinal pigment epithelium in the short term, and in the long term in damage to or degeneration of the photoreceptor cells.
  • the size of the tear correlates with the decline in visual function, and if the tear overlaps with the fovea, it often leads to severe decline in visual function. In addition, if the tear occurs near the fovea, it can cause a paracentral scotoma, resulting in severe visual impairment.
  • Retinal pigment epithelial tears are often associated with age-related macular degeneration (AMD). They may also occur in patients with a pigment epithelial detachment (PED), a condition in which the retinal pigment epithelium is detached from Bruch's membrane and protrudes into a dome shape, creating a space that can contain serous fluid, blood, fibrovascular membrane, drusenoid material, or a combination of these.
  • PED pigment epithelial detachment
  • a tear forms in the periphery of a pigment epithelial detachment (PED) caused by choroidal neovascularization (CNV), either naturally, after photodynamic therapy, or after intravitreal injection of an anti-vascular endothelial growth factor (VEGF) drug.
  • PED pigment epithelial detachment
  • CNV choroidal neovascularization
  • VEGF anti-vascular endothelial growth factor
  • Diagnosis of retinal pigment epithelial tears is made by various imaging modalities.
  • OCT optical coherence tomography
  • the torn part of the retinal pigment epithelium rolls to the edge of the tear, forming a brown raised lesion.
  • the defective part of the retinal pigment epithelium is slightly depressed, and the choroidal vessels are more clearly visible than in normal areas.
  • the area where the rolled retinal pigment epithelium is invaginated is hypofluorescent because the fluorescence from the choroid is blocked.
  • the defective part of the retinal pigment epithelium strong hyperfluorescence of the choriocapillaris tissue is seen.
  • fundus autofluorescence the defective part of the retinal pigment epithelium is hypofluorescent, and the rolled part is hyperfluorescent in the early stage of onset.
  • the therapeutic agent of the present invention is a therapeutic agent for retinal pigment epithelial breaks in a human subject, and comprises a suspension in which human retinal pigment epithelial cells are dispersed in a dispersion medium.
  • the therapeutic agent of the present invention can be used to treat retinal pigment epithelial breaks in a human subject by administering (or transplanting) human retinal pigment epithelial cells under the retina of the eyeball.
  • the human retinal pigment epithelial cells in the therapeutic agent of the present invention may be the human retinal pigment epithelial cells described in 1. Definition, and more preferably are derived from in vitro differentiation of human pluripotent stem cells (particularly human iPS cells).
  • the retinal pigment epithelial cells may be dispersed retinal pigment epithelial cells, for example, dispersed cells obtained by collecting a sheet of retinal pigment epithelial cells and dispersing them into single cells and/or cell clusters of 2 to 5 cells.
  • human retinal pigment epithelial cells are preferably contained at a concentration of 0.1 ⁇ 10 7 cells/mL to 1 ⁇ 10 7 cells/mL, more preferably at a concentration of 0.5 ⁇ 10 7 cells/mL to 1 ⁇ 10 7 cells/mL.
  • the administration volume of the therapeutic agent may be 20 ⁇ L to 80 ⁇ L, preferably 20 ⁇ L to 50 ⁇ L, more preferably 30 ⁇ L to 50 ⁇ L. That is, the administration volume may be, for example, 0.2 ⁇ 10 5 cells to 5 ⁇ 10 5 cells, 1 ⁇ 10 5 cells to 5 ⁇ 10 5 cells.
  • the diameter of the bleb formed under the retina of cynomolgus monkeys by administration of 50 ⁇ L is a sufficient volume to cover an average retinal pigment epithelial break lesion (e.g., 1-15 mm 2 , 3-10 mm 2 ) or a human fovea and parafovea (diameter about 2-4 mm (e.g., 3 mm), area about 6-8 mm 2 (e.g., 7.07 mm 2 )).
  • an average retinal pigment epithelial break lesion e.g., 1-15 mm 2 , 3-10 mm 2
  • a human fovea and parafovea diameter about 2-4 mm (e.g., 3 mm)
  • area about 6-8 mm 2 e.g., 7.07 mm 2
  • the cells can be engrafted over an area of, for example, about 3 to 5 mm wide. Therefore, compared to transplanting sheet-like retinal pigment epithelial cells, transplanting retinal pigment epithelial cells in a suspension form makes it easier to cover a wider range of pathological sites and also reduces the burden of surgery. Furthermore, when human retinal pigment epithelial cells are administered as a suspension, they can be engrafted at sites in the retina of the recipient where retinal pigment epithelial breaks exist and transplantation is required. Therefore, it is possible to accommodate any shape of break.
  • a retinal incision is required to insert the sheet, which is at least as wide as the sheet or the device used to insert the sheet.
  • Retinotomy is an iatrogenic retinal tear, which can be said to artificially create a tear in the retina.
  • the risk of postoperative retinal detachment increases.
  • it may be necessary to perform photocoagulation around the incision which can cause irreversible damage to the normal retina and normal retinal pigment epithelial cell layer and affect visual function.
  • the area around the lesion generally has normal visual function, so photocoagulation around the injection site is considered inappropriate as it may reduce visual function in the normal area more than the recovery achieved by treatment.
  • the medium used for dispersing human retinal pigment epithelial cells in the therapeutic agent of the present invention (hereinafter also referred to as "dispersion medium”) is not particularly limited, but the upper limit of the viscosity at a shear rate of 2 (1/s) at 25°C may be 4 mPa ⁇ s or less, 3.5 mPa ⁇ s or less, or 3 mPa ⁇ s or less.
  • the lower limit of the viscosity at a shear rate of 2 (1/s) at 25°C is not particularly limited, but may be, for example, 0.5 mPa ⁇ s or more, 0.6 mPa ⁇ s or more, 0.7 mPa ⁇ s or more, or 0.8 mPa ⁇ s or more.
  • the continuation of the retinal detachment state prolongs the time during which no direct effect occurs on the photoreceptor cells of the retinal pigment epithelium layer, which may further worsen the pathology, so the above viscosity can be set as a viscosity that particularly prevents the prolongation of retinal detachment.
  • the dispersion medium may have a viscosity of 1.3 to 3.8 mPa ⁇ s, 1.5 to 3.5 mPa ⁇ s, or 2 to 3 mPa ⁇ s at a shear rate of 2 (1/s) at 25°C.
  • the dispersion medium may contain a pharma- ceutically acceptable aqueous liquid, or may be a pharma-ceutically acceptable aqueous liquid.
  • pharmaceutical-ceutically acceptable aqueous liquid is not particularly limited as long as it is an aqueous solution that can be administered to a living body and has physical properties suitable for transplantation, and may be, for example, an aqueous solution containing a buffer solution, infusion solution, physiological saline, water for injection, perfusion solution, etc. A buffer solution is preferred.
  • the dispersion medium may contain a substance that affects viscosity (a substance added for the purpose of increasing viscosity, such as a thickener). As described above, examples of substances that affect viscosity include hyaluronic acid and chondroitin sulfate, and preferably the dispersion medium does not contain any other substances that affect viscosity (such as a thickener).
  • the dispersion medium may contain hyaluronic acid.
  • the dispersion medium may contain chondroitin sulfate, but preferably does not contain chondroitin sulfate. Both hyaluronic acid and chondroitin sulfate are mucopolysaccharides present in living organisms, and function as thickening components for the dispersion medium.
  • hyaluronic acid is a linear polymeric polysaccharide with a molecular weight of tens of thousands to millions, which has a structure in which D-glucuronic acid and D-N-acetylglucosamine are alternately linked by ⁇ -1,4 and ⁇ -1,3 glycosidic bonds.
  • Hyaluronic acid has high water retention capacity and viscosity, and is a substance that is widely used in pharmaceuticals, cosmetics, and the like.
  • “hyaluronic acid” refers to a concept that includes both free hyaluronic acid and its salts.
  • salts of hyaluronic acid include alkali metal salts and alkaline earth metal salts of hyaluronic acid, and specifically, commercially available sodium hyaluronate, potassium hyaluronate, and the like can be used.
  • Chondroitin sulfate is a mucopolysaccharide with a structure in which sulfate is bound to a glycan chain in which the disaccharide units D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) are repeated.
  • chondroitin sulfate refers to a concept that includes both free chondroitin sulfate and its salts.
  • salts of chondroitin sulfate include alkali metal salts and alkaline earth metal salts of chondroitin sulfate, and specifically, commercially available sodium chondroitin sulfate, potassium chondroitin sulfate, etc. can be used.
  • hyaluronic acid or its salt a commercially available aqueous solution of hyaluronic acid or its salt can be used.
  • Viscoat registered trademark
  • ocular viscoelastic agent containing 3% sodium hyaluronate, 4% sodium chondroitin sulfate, sodium dihydrogen phosphate monohydrate, anhydrous sodium dihydrogen phosphate, and an isotonic agent as ingredients
  • Provisc registered trademark
  • an isotonic agent as ingredients, 0.6% sodium hyaluronate, sodium chloride, dihydrogen phosphate
  • suitable ophthalmic solutions include OPEGAN (registered trademark) 0.6 ophthalmic viscoelastic agent, which contains sodium and sodium dihydrogen phosphate as
  • aqueous solutions of hyaluronic acid or its salts preferably do not contain any substance that affects viscosity (e.g., thickeners) other than hyaluronic acid and chondroitin sulfate, and more preferably do not contain any substance that affects viscosity (e.g., thickeners) other than hyaluronic acid.
  • “Pharmaceutically acceptable aqueous liquids” include aqueous solutions containing balanced salts (i.e., balanced salt solutions).
  • Aqueous solutions containing balanced salts can contain buffers, isotonicity agents, pH adjusters, antioxidants, chelating agents, etc., as appropriate, within the range that does not affect the survival rate of the transplanted retinal tissue.
  • buffers include phosphate buffers, borate buffers, citrate buffers, tartrate buffers, acetate buffers, amino acids, and epsilon-aminocaproic acid.
  • Isotonicity agents include sugars such as sorbitol, glucose, and mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, and boric acid.
  • Chelating agents include sodium edetate and citric acid.
  • pH adjusters examples include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or its salts (borax), hydrochloric acid, citric acid or its salts (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or its salts (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or its salts (sodium acetate, ammonium acetate, etc.), tartaric acid or its salts (sodium tartrate, etc.), etc.
  • antioxidants examples include ascorbic acid, glutathione, sodium bisulfite, dry sodium sulfite, sodium pyrosulfite, tocopherol, etc.
  • aqueous solutions containing one or more components selected from sugars such as glucose, inorganic salts such as calcium chloride, sodium chloride, magnesium chloride, potassium chloride, and magnesium sulfate, inorganic substances such as sodium bicarbonate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, anhydrous sodium monohydrogen phosphate, and hydrochloric acid, and chelating agents such as edetate salts (e.g., sodium edetate).
  • sugars such as glucose
  • inorganic salts such as calcium chloride, sodium chloride, magnesium chloride, potassium chloride, and magnesium sulfate
  • inorganic substances such as sodium bicarbonate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, anhydrous sodium monohydrogen phosphate, and hydrochloric acid
  • chelating agents such as edetate salts (e.g., sodium edetate).
  • a commercially available liquid as an intraocular irrigation/cleaning solution can be used.
  • an aqueous solution containing 1.5 mg of glucose, 6.6 mg of sodium chloride, 0.36 mg of potassium chloride, 0.18 mg of calcium chloride hydrate, 0.3 mg of magnesium sulfate hydrate, 2.1 mg of sodium bicarbonate per mL, and sodium citrate hydrate, sodium acetate hydrate, and hydrochloric acid as additives, or oxyglutathione ocular irrigation solution, etc. commercially available as Opeguard (registered trademark) MA ocular irrigation solution (Senju Pharmaceutical Co., Ltd.), can be used as an intraocular irrigation/cleaning solution.
  • HBSS Hanks' balanced salt solution
  • EBSS Eagle's balanced salt solution
  • PBS phosphate buffered saline
  • DPBS Dulbecco's phosphate buffered saline
  • the dispersion medium is a medium containing a balanced salt solution that contains hyaluronic acid and/or chondroitin sulfate as a thickening component, and also contains one or more components selected from the above-mentioned buffers, isotonicity agents, pH adjusters, antioxidants, chelating agents, etc., in an amount that does not affect the viability of retinal tissue.
  • the dispersion medium is a medium containing only hyaluronic acid as a thickening component.
  • it can be prepared by diluting ocular viscoelastic agent Provisc (registered trademark) 0.85 ocular viscoelastic agent 1% (Nihon Alcon Co., Ltd.), which contains 10 mg/mL sodium hyaluronate and does not contain chondroitin sulfate, 20 to 200 times (preferably 50 to 150 times, about 100 times) with a balanced salt solution such as HBSS.
  • the dispersion medium can be prepared by diluting Viscoat (registered trademark) 0.5 ophthalmic viscoelastic agent (Nihon Alcon Co., Ltd.), which contains 30 mg/mL sodium hyaluronate and 40 mg/mL sodium chondroitin sulfate, 20 to 200 times (preferably 50 to 150 times, about 100 times) with a balanced salt solution such as HBSS.
  • Viscoat registered trademark
  • ophthalmic viscoelastic agent Nahon Alcon Co., Ltd.
  • a balanced salt solution such as HBSS.
  • Opegan registered trademark
  • 0.6 ophthalmic viscoelastic agent can be prepared by diluting it 20 to 200 times with an aqueous liquid such as Opeguard.
  • Hyalein Mini registered trademark
  • eye drops 0.3% can be prepared by diluting it 6 to 60 times with an aqueous liquid such as Opeguard.
  • the dispersion medium is a medium containing hyaluronic acid as a thickening component. It may further contain chondroitin sulfate. There is no particular limitation on the concentration of hyaluronic acid and chondroitin sulfate, and it is preferable that the viscosity of the dispersion medium at a shear rate of 2 (1/s) at 25°C can be maintained at 0.5 to 4 mPa ⁇ s, preferably 2 to 3 mPa ⁇ s.
  • the dispersion medium is a dispersion medium containing 0.005 w/v% to 0.15 w/v% (e.g., 0.007 w/v% to 0.04 w/v%, 0.008 w/v% to 0.035 w/v%, 0.009 w/v% to 0.03 w/v%, about 0.01 w/v%) of hyaluronic acid having an average molecular weight of 500,000 to 3.9 million (preferably 1.5 million to 3.9 million).
  • examples of the dispersion medium include a dispersion medium containing more than 0 w/v% (e.g., 0.001 w/v%) to 0.20 w/v% (or 0.1 w/v%, 0.05 w/v%) of chondroitin sulfate having a molecular weight of 20,000 to 24,000 or a salt thereof.
  • the dispersion medium more preferably does not contain chondroitin sulfate.
  • the dispersion medium preferably does not contain any thickeners (thickening components) other than hyaluronic acid and chondroitin sulfate, or any thickeners (thickening components) other than hyaluronic acid.
  • thickeners include gum arabic, karaya gum, xanthan gum, casein, agar, alginic acid, ⁇ -cyclodextrin, dextrin, dextran, carrageenan, gelatin, collagen, pectin, starch, chitin and its derivatives, chitosan and its derivatives, elastin, heparin, heparinoid, heparin sulfate, heparan sulfate, hyaluronic acid, polysaccharides such as methylcellulose and hydroxyethylcellulose or their derivatives, ceramide, macrogol, glycerin, povidone, polyvinyl methacrylate, polyvinyl alcohol, polyacryl
  • the dispersion medium is preferably free of antibacterial agents and preservatives.
  • the dispersion medium contains fewer additives, as long as the function and stability of the retinal pigment epithelial cells are ensured. Furthermore, when a medium that does not contain a thickening component is used, it is expected that the bleb will be restored as soon as possible at the time of administration, thereby protecting the photoreceptor cells and maintaining and improving visual function.
  • one embodiment of the dispersion medium is a medium that does not contain a thickening component and contains one or more components selected from the above-mentioned buffers, isotonicity agents, pH adjusters, antioxidants, chelating agents, etc., in a range that does not affect the viability of retinal tissue and exhibits physical properties suitable for administration.
  • physical properties suitable for administration include pH and osmotic pressure.
  • pH there are no particular limitations on the pH of the dispersion medium as long as it is in the neutral range, and the dispersion medium may be adjusted to pH 6.5 to 8.0, preferably pH 7.0 to 7.5.
  • the osmotic pressure of the dispersion medium may be hypotonic, isotonic, or hypertonic, but is preferably close to isotonic.
  • the osmotic pressure ratio may be adjusted to 0.7 to 1.3, preferably 0.9 to 1.1.
  • the dispersion medium can be preferably sterilized, such as by filtration sterilization using a membrane filter, before use.
  • the therapeutic agent of the present invention is one in which, when the viscosity of the medium used for dispersing human retinal pigment epithelial cells is within the above-mentioned specified range, particularly when combined with the administration method described below (air replacement of the vitreous cavity, etc.), after administration to a human subject, no preretinal membrane formation is observed or the degree of preretinal membrane formation is reduced.
  • the degree of preretinal membrane formation means the degree of preretinal membrane formation when compared with the case where a disease or pathology based on a disorder of the retinal pigment epithelium is treated with a therapeutic agent using a dispersion medium with a viscosity of more than 4 mPa ⁇ s (shear rate 2 (1/s) at 25°C) (postoperative air replacement of the vitreous cavity may not be performed), or the case where a disease or pathology based on a disorder of the retinal pigment epithelium is treated with a surgical procedure of subretinal administration via the vitreous without postoperative air replacement of the vitreous cavity using the same dispersion medium.
  • the degree of preretinal membrane formation being reduced may mean, for example, that the degree of preretinal membrane formation is reduced by 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more compared to the degree of preretinal membrane formation when a disease or condition based on damage to the retinal pigment epithelium is treated with a therapeutic drug using a dispersion medium with a viscosity of more than 4 mPa ⁇ s (shear rate 2 (1/s) at 25°C) (postoperative air replacement of the vitreous cavity may not be performed), or when a disease or condition based on damage to the retinal pigment epithelium is treated with the same dispersion medium by a procedure of subretinal administration via the vitreous without postoperative air replacement of the vitreous cavity.
  • the reduction in the degree of preretinal membrane formation can be confirmed, for example, by the results of fundus examination or optical coherence tomography (OCT) findings.
  • OCT optical coherence tomography
  • the therapeutic agent of the present invention when the viscosity of the medium used for dispersing human retinal pigment epithelial cells is within the above-mentioned specified range, does not cause retinal detachment to be prolonged or reduces the degree of retinal detachment to be prolonged after administration to a human subject.
  • the degree of retinal detachment prolongation means the degree of retinal detachment prolongation compared to when a disease or pathology based on retinal pigment epithelium damage is treated with a therapeutic agent using a dispersion medium with a viscosity of more than 4 mPa ⁇ s (shear rate 2 (1/s) at 25°C).
  • the degree of retinal detachment prolongation being reduced may mean, for example, that the degree of retinal detachment prolongation is reduced by 70% or more, 80% or more, 90% or more, or 95% or more compared to when a disease or pathology based on retinal pigment epithelium damage is treated with a therapeutic agent using a dispersion medium with a viscosity of more than 4 mPa ⁇ s (shear rate 2 (1/s) at 25°C).
  • the absence of persistent retinal detachment can be confirmed, for example, by the results of fundus examination, fundus photography, fluorescein angiography, or optical coherence tomography (OCT) findings.
  • OCT optical coherence tomography
  • the human subject to which the therapeutic agent of the present invention is administered is not particularly limited as long as it is a human subject with a retinal pigment epithelium break, but may be appropriately determined based on, for example, the extent and size of the retinal pigment epithelium break, the degree of visual function, etc., or a combination of these.
  • the degree and size of the retinal pigment epithelium tear may be used to determine who should receive the treatment, for example, according to the grading system of Sarraf et al. (Retina 30(7)1039 45, 2010). The classification is shown in Table 1 below.
  • the human subject to which the therapeutic agent of the present invention is administered may be classified as any of the above grades 1 to 4, but is preferably classified as grades 3 to 4.
  • the human subject to which the therapeutic agent of the present invention is administered is preferably one who is affected by a retinal pigment epithelium tear but whose visual function is maintained to a certain level or who has the potential to maintain or recover.
  • "Visual function maintained to a certain level” may mean that photoreceptor cells survive to a certain level in the human subject, or the state of ocular tissues is maintained to a certain level in the human subject. Visual function can be evaluated based on the number of days since onset or diagnosis, findings from an ophthalmologic examination, etc.
  • retinal pigment epithelial tears occur suddenly, and if a tear occurs in the macula, the patient can notice it as a sudden change in vision (such as decreased vision or a scotoma). If the tear occurs in a place other than the macula, it is difficult to notice, but if the patient regularly visits a medical institution for retinal, choroidal, or other eye-related diseases, it can be easily detected by fundus examination, OCT, etc. It is preferable that the time of onset of the human subject to which the therapeutic drug of the present invention is administered is specified to some extent, and the human subject may be one who has not yet passed a long time since onset.
  • human subjects who have been onset or diagnosed within 1 year, 10 months, 8 months, 6 months, 4 months, 2 months, 1 month, 2 weeks, or 1 week since onset or diagnosis are preferable as subjects whose visual function is maintained to a certain level or there is a possibility of maintaining or recovering.
  • retina-choroidal and other eye-related diseases include macular degeneration (e.g., atrophic and exudative age-related macular degeneration), hereditary retinal diseases, central serous chorioretinopathy, choroidal tumors, degeneration of the retinal pigment epithelium (including retinitis pigmentosa and crystalline retinopathy), macular dystrophies (including Stargardt's disease), cone dystrophies, rod dystrophies, cone-rod dystrophies, high myopia, posterior staphyloma, Vogt-Koyanagi-Harada disease, panuveitis, scleral surgery, and complications of retinal detachment surgery and glaucoma surgery.
  • the therapeutic agent for retinal pigment epithelium breaks in this specification includes a therapeutic agent for retinal pigment epithelium breaks that is transplanted into a patient diagnosed with any of the above diseases (e.g., macular degeneration).
  • Examples of ophthalmologic examinations include visual acuity tests (tests using Landolt rings, ETDRS visual acuity charts, picture targets, stripe targets, etc., visual behavior, etc.), color vision tests, contrast tests, OCT tests, dynamic visual field tests, static visual field tests, fundus photography, fluorescent fundus photography, fundus autofluorescence photography, electroretinogram (ERG) tests, full-field stimulus testing (FST), visual evoked potential (VEP) tests, etc.
  • visual acuity tests tests using Landolt rings, ETDRS visual acuity charts, picture targets, stripe targets, etc., visual behavior, etc.
  • color vision tests contrast tests
  • OCT tests dynamic visual field tests
  • static visual field tests fundus photography
  • fluorescent fundus photography fluorescent fundus photography
  • fundus autofluorescence photography electroretinogram (ERG) tests
  • FST full-field stimulus testing
  • VEP visual evoked potential
  • Human subjects who maintain a certain level of visual function may be, for example, subjects with a best corrected visual acuity (BCVA) using the ETDRS visual acuity chart of 0 to 85 letters (equivalent to a minor acuity of 0.02 to 1.0) or 24 to 78 letters in letter scores. Even if a letter score is 100 letters or less (equivalent to a minor acuity of 2.0), if there is a visual field defect or the like, they may be included as human subjects.
  • BCVA visual acuity
  • a human subject whose visual function is maintained to a certain level may be, for example, a human subject whose central visual field angle is 28 degrees or more when measured with a Goldmann perimeter I/2 target.
  • Human subjects in which the state of ocular tissues is maintained to a certain level or may be so may be, for example, human subjects with no damage other than the retinal pigment epithelium cell layer, human subjects in which at least the outer limiting membrane (ELM) and inner segment-outer segment junction (IS/OS-line) are maintained, and human subjects in which the outer nuclear layer (ONL) at the site of a retinal pigment epithelium break is not thinned compared to the surrounding normal area.
  • ELM outer limiting membrane
  • IS/OS-line inner segment-outer segment junction
  • HLA mainly consists of six antigen loci: A, B, C, DR, DQ, and DP, each of which is made up of complex combinations of dozens of different types (alleles), with tens of thousands of possible combinations.
  • A, B, C, DR, DQ, and DP antigen loci
  • the human subject to which the therapeutic agent of the present invention is administered may or may not have a human leukocyte antigen (HLA) type that is compatible with the human retinal pigment epithelial cells, but it is preferable that the HLA type is compatible.
  • HLA human leukocyte antigen
  • all of the HLA antigens HLA-A, -B, -C, -DR, -DQ and -DP of the human subject and the retinal pigment epithelial cells are the same.
  • the HLA type of the human subject is not compatible with the retinal pigment epithelial cells of the present invention, it is sufficient that at least one of the HLA antigens HLA-A, -B, -C, -DR, -DQ and -DP differs between the HLA type of the human subject and the HLA type of the retinal pigment epithelial cells, or all of them may differ.
  • a human subject in which the genotype of at least one allele at a locus corresponding to the locus of the retinal pigment epithelial cells matches the genotype of the allele at the locus of the retinal pigment epithelial cells, respectively, may be included in cases where the human subject has an HLA type compatible with the retinal pigment epithelial cells of the present invention.
  • the retinal pigment epithelium break is preferably a state in which the retinal pigment epithelium cell layer of the human subject is partially torn and the exposed Bruch's membrane, which is not covered by the retinal pigment epithelium cells of the human subject, remains under the retina as a basement membrane.
  • Bruch's membrane is a thin membrane that exists between the retinal pigment epithelium cell layer and the choroid, and is composed of type IV collagen, laminin, etc.
  • Bruch's membrane remaining under the retina as a basement membrane means that there is no loss or defect of Bruch's membrane under the retina of the ocular tissue, and there is a scaffold to which the administered retinal pigment epithelial cells can adhere.
  • Bruch's membrane is preferably undamaged, but may be damaged as long as there is no loss or defect.
  • Bruch's membrane is composed of five layers, in order from the retina side: the basal membrane of the retinal pigment epithelium cells, the inner collagen fiber layer, the elastic fiber layer, the outer collagen fiber layer, and the basal membrane of the choriocapillary endothelial cells.
  • the loss or deficiency of Bruch's membrane specifically refers to the loss or deficiency of the superficial layer on the retinal side (the basal membrane of the retinal pigment epithelium).
  • the therapeutic effect of the therapeutic agent of the present invention is high when Bruch's membrane remains as a basement membrane under the retina.
  • human retinal pigment epithelial cells exhibit polarity, form an epithelial layer to cover the exposed Bruch's membrane, and exert a barrier function.
  • “Exhibiting polarity” means that the cells exhibit a characteristic protein localization pattern, and the function is biased between the apical membrane side and the basement membrane side. “Exhibiting a barrier function” means that retinal pigment epithelial cells adhere firmly to each other through tight junctions, strictly regulating the movement of substances and maintaining the intraocular environment necessary for neural activity.
  • a retinal pigment epithelium tear is a state in which the retinal pigment epithelium cell layer of a human subject is partially torn and exposed Bruch's membrane remains under the retina as a basement membrane without being covered by the retinal pigment epithelium cells of the human subject, which may mean, for example, that the grade of the retinal pigment epithelium tear in the human subject is 3 to 4, that the number of days since the onset or diagnosis of the retinal pigment epithelium tear in the human subject is within the above range, that the neural retina (photoreceptors) of the human subject is not in contact with the retinal pigment epithelium cell layer, and that tearing or disappearance of Bruch's membrane has not been clearly confirmed by imaging tests such as OCT.
  • the method of administering (transplanting) the therapeutic agent of the present invention subretinally is not particularly limited as long as it is a known method, and examples thereof include a method of injecting using a syringe of an appropriate size, or a transplantation device including a needle or resin part. More specifically, examples of the method include a transvitreal method and a transchoroidal method. The transvitreal method is outlined in FIG.
  • blebs hydraulically debridement retinal detachments
  • a method for forming a bleb in addition to a method in which the therapeutic agent of the present invention itself is injected to form the bleb, there is also a method in which an ocular irrigation solution (such as HBSS) is first injected to form a bleb, and the liquid in the bleb is aspirated as necessary, and then the therapeutic agent of the present invention is injected.
  • an ocular irrigation solution such as HBSS
  • the therapeutic agent of the present invention is preferably administered via the vitreous cavity, and after subretinal implantation via the vitreous cavity, the vitreous is preferably replaced with air.
  • the therapeutic agent of the present invention can be administered (injected) using a needle, syringe, etc., and therefore can be performed as a minimally invasive method among transvitreal cavity methods.
  • the therapeutic agent of the present invention may be administered through a hole of about 0.4 mm to about 0.65 mm on the sclera while forming a bleb in the area surrounding the retinal pigment epithelial tear.
  • the hole on the sclera may be about 0.4 mm (27 gauge) to about 0.63 mm (23 gauge) (e.g., about 0.5 mm (25 gauge)) depending on the size of the needle. It is preferable that the bleb is formed so as to cover the major axis of the retinal pigment epithelial tear.
  • the area surrounding the retinal pigment epithelium break is preferably as close as possible to the edge of the retinal pigment epithelium break, and may be, for example, within one optic disc diameter (approximately 1.5 mm) of the edge of the retinal pigment epithelium break.
  • the agent of the present invention When the therapeutic agent of the present invention is administered to the area surrounding the retinal pigment epithelium break through a hole of about 0.4 mm to about 0.65 mm in the sclera while forming a bleb, it is preferable that the agent be used in a treatment that includes air replacement of the vitreous body after administration.
  • the transchoroidal approach is an approach that approaches the eye from the outside, in which part of the sclera is incised and peeled off, a catheter is inserted into the choroidal cavity, and an appropriate amount of the therapeutic agent of the present invention is injected under the retina for administration.
  • the administration needle is not particularly limited, but examples include metal bevel needles and polytip needles. Polytip needles are particularly preferred. In one embodiment, the outer diameter of the administration needle may be 38 gauge, 39 gauge, or 40 gauge.
  • the transplant composition or the retinal pigment epithelial cells for transplantation may leak from the bleb into the vitreous body, and the leaked retinal pigment epithelial cells may form a preretinal membrane.
  • the transplant composition hardly leaks into the vitreous cavity unless a hole is made on the retinal side.
  • the retinal pigment epithelial cells leaking from the bleb may form a preretinal membrane.
  • both the transvitreal cavity method and the transchoroidal cavity method cause retinal detachment.
  • the formation of a preretinal membrane and the prolongation of retinal detachment are considered.
  • the dispersion medium in the therapeutic agent of the present invention has a specific viscosity, which can suppress the cell leakage of the therapeutic agent of the present invention into the vitreous body and the prolongation of retinal detachment.
  • the therapeutic agent of the present invention may be administered in combination with an immunosuppressant.
  • the immunosuppressant administered in combination may be one type or multiple types.
  • immunosuppressants include steroidal anti-inflammatory drugs, calcineurin inhibitors, metabolic antagonists, and mTOR inhibitors.
  • steroidal anti-inflammatory drugs include dexamethasone and prednisolone
  • calcineurin inhibitors include cyclosporine and tacrolimus
  • metabolic antagonists include mycophenolic acid
  • mTOR inhibitors include sirolimus (rapamycin).
  • betamethasone sodium phosphate and/or fluorometholone may be combined.
  • the immunosuppressant is used for the purpose of rejection of the therapeutic agent of the present invention (retinal pigment epithelial cells).
  • the steroidal anti-inflammatory drugs dexamethasone, betamethasone sodium phosphate, and/or fluorometholone may be used for the purpose of suppressing postoperative inflammation.
  • triamcinolone acetonide can be administered topically (e.g., subtenon or intravitreal), and cyclosporine, tacrolimus, dexamethasone, and prednisolone can be administered systemically (e.g., orally, intravenously, etc.).
  • Dexamethasone can be administered subconjunctivally, and betamethasone sodium phosphate and fluorometholone can be administered as eye drops.
  • prednisolone and cyclosporine or tacrolimus are administered before surgery. Administration may begin up to 7 days (6, 5, 4, 3, 2 or 1 day) before surgery. Triamcinolone acetonide, dexamethasone, prednisolone, and cyclosporine or tacrolimus may be administered after surgery. Betamethasone sodium phosphate and fluorometholone may be administered after surgery.
  • the therapeutic agent of the present invention may be prepared as a suspension at a medical institution using the above-mentioned dispersion medium.
  • the retinal pigment epithelial cells may be frozen and transported to the medical institution as a frozen preparation containing the retinal pigment epithelial cells.
  • the retinal pigment epithelial cells are preferably frozen in a dispersed state.
  • the method for cryopreserving the retinal pigment epithelial cells is not particularly limited as long as it is a method generally known as a method for cryopreserving cells.
  • the retinal pigment epithelial cells may be frozen at -80°C using a commercially available cell cryopreservation solution StemCellBanker (Zenoaq), and then cryopreserved using liquid nitrogen or the like.
  • the frozen preparation may be thawed at the time of use at a medical institution, suspended in a dispersion medium or the like, and used in transplantation medicine. After thawing the frozen preparation, washing may be performed using a buffer solution such as a dispersion medium before suspending it in a dispersion medium or the like.
  • a frozen preparation containing retinal pigment epithelial cells is also provided for use as a therapeutic agent of the present invention by preparing the preparation into a suspension using the above-mentioned dispersion medium after thawing.
  • a frozen preparation containing retinal pigment epithelial cells is provided for use in treating retinal pigment epithelial breaks in a human subject as a suspension in which human retinal pigment epithelial cells are dispersed after thawing.
  • the method of the present invention for treating a retinal pigment epithelial break in a human subject comprises administering to a human subject in need thereof an effective amount of a suspension in which human retinal pigment epithelial cells are dispersed.
  • the suspension in which human retinal pigment epithelial cells are dispersed may be any of the therapeutic agents described in 2. Therapeutic Agents above.
  • the method of administering the suspension in which human retinal pigment epithelial cells are dispersed is not particularly limited as long as it is a known method, and examples of such methods include the transvitreal method and the transchoroidal method.
  • the administration of the suspension in which human retinal pigment epithelial cells are dispersed preferably includes administration by the transvitreal method.
  • a human subject is administered local anesthesia (sub-Tenon anesthesia/retrobulbar anesthesia) or general anesthesia.
  • a vitrectomy is performed using an appropriate vitreous surgery device (e.g., Constellation Vision System).
  • an appropriate vitreous surgery device e.g., Constellation Vision System
  • triamcinolone acetonide 0.5 to 4 mg
  • the suspension in which human retinal pigment epithelial cells are dispersed may be administered through a hole of about 0.4 mm to about 0.65 mm on the sclera so as to form a bleb in the area surrounding the retinal pigment epithelial tear.
  • the hole on the sclera may be a hole of about 0.4 mm (27 gauge) to about 0.63 mm (23 gauge) (e.g., about 0.5 mm (25 gauge)) depending on the size of the needle. It is preferable that the bleb is formed so as to cover the long axis of the retinal pigment epithelial tear.
  • the method when administering a suspension in which human retinal pigment epithelial cells are dispersed includes forming a bleb in the area surrounding the retinal pigment epithelial break through a hole of about 0.4 mm to about 0.65 mm on the sclera, the method may further include washing the vitreous cavity after administration of the suspension and replacing the vitreous cavity of the human subject with air.
  • the above-mentioned immunosuppressants may be administered in combination.
  • the therapeutic method of the present invention can be applied in the same manner as the therapeutic agent of the present invention described above.
  • retinal pigment epithelial cells are engrafted at the administration site of the human subject, and an adhesion structure with the photoreceptor layer is reconstructed.
  • the transplanted retinal pigment epithelial cells function, and visual function is restored, or further deterioration is prevented and visual function is maintained, compared to before the administration of the therapeutic agent or before the implementation of the treatment method.
  • the effectiveness of the above treatments can be confirmed by the above-mentioned eye examination. It can also be evaluated using scales that measure health-related quality of life (QOL). These scales comprehensively evaluate the health state from physical, mental and social aspects, and some indices are specific to visual function. Examples include the Visual Function Questionnaire (VFQ-25), Euro-QOL 5 Dimension-5 Level (EQ-5D-5L), and Health Utilities Index Mark 3 (HUI3).
  • QOL health-related quality of life
  • the present invention provides use of a suspension of dispersed human retinal pigment epithelial cells in treating a retinal pigment epithelial break in a human subject.
  • the present invention also provides a suspension of dispersed human retinal pigment epithelial cells for use in treating a retinal pigment epithelial break in a human subject.
  • the present invention further provides use of a suspension of dispersed human retinal pigment epithelial cells in the manufacture of a medicament for treating a retinal pigment epithelial break in a human subject.
  • Example 1 Examination of the effect on cell leakage using isolated pig eyes Using pig eyes, whose eyeball size is close to that of humans, the effects of the viscosity of the dispersion medium and postoperative air replacement in the vitreous cavity on cell leakage were examined ex vivo.
  • a suspension of human iPS cell-derived RPE cells was administered (transplanted) subretinaly using the method described below.
  • subretinal injection of the RPE cell suspension was performed according to the procedure of general vitreous surgery (transvitreal cavity method).
  • a cataract and vitreous surgery device Constellation Vision System LTX, manufactured by Alcon Japan
  • triamcinolone acetonide (209-10961, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was injected into the vitreous to visualize the vitreous
  • a membrane peel was made from the macula to the posterior vitreous
  • peripheral vitrectomy was performed.
  • the ophthalmic surgery syringe and needle adaptor were connected to the vitreous surgery device using appropriate tubing, and the RPE cell suspension was filled.
  • a subretinal injection needle (Medone Cannula: Tapered Cannula Polytip 25G/38G, 3219, Medone Surgical, Inc./Nihon Surgery Co., Ltd.) was connected.
  • the subretinal injection needle was inserted through the port, and an appropriate amount of RPE cell suspension was injected subretinally.
  • a bleb was formed by subretinal injection of the RPE cell suspension.
  • the RPE cell suspensions administered were obtained by suspending RPE cells labeled with the fluorescent labeling reagent PKH26 Red Fluorescent Cell Linker Mini Kit (Sigma-Aldrich, MINI26) in two types of transplantation media at a concentration of 5.7 x 107 cells/mL (air exchange condition) or 6.0 x 106 cells/mL (without air exchange).
  • Viscous medium Viscoat 0.5 ocular viscoelastic agent (manufactured by Alcon Japan) and Hank's balanced salt solution HBSS(+) (manufactured by Thermo Fisher Scientific, 14025092) mixed in a ratio of 1:6 (7-fold dilution)
  • Buffer only medium HBSS(+) alone
  • the dilution ratio of Viscoat in the medium was varied in the range of 7 to 21 times, and subretinal administration was performed. It was found that when the concentration of Viscoat was high (the dilution ratio was low), the resistance during ejection was strong, making it difficult to form a bleb. It was also confirmed that the administration volume could be administered up to about 100 ⁇ L, but backflow from the injection port was observed when the administration volume was increased. It was confirmed that the RPE cell concentration could be administered without problems up to a suspension of 5.7 ⁇ 10 7 cells/mL.
  • Example 2 Transplantation experiment 1 into cynomolgus monkeys
  • cynomolgus monkeys which have a macula and anatomical characteristics of the eyeball that are very similar to humans.
  • Provisc registered trademark 0.85 ocular viscoelastic agent 1%
  • Viscoat registered trademark
  • Viscoat 0.5 ocular viscoelastic agent
  • Viscoat contains 30 mg/mL of sodium hyaluronate and 40 mg/mL of sodium chondroitin sulfate
  • Provisc contains 10 mg/mL of sodium hyaluronate and does not contain chondroitin sulfate.
  • Combined anesthesia was performed by intramuscular administration of ketamine hydrochloride (Ketalar 500 mg for intramuscular injection, Daiichi Sankyo Propharma Co., Ltd.) 10 mg/kg and medetomidine hydrochloride (Domitor, Orion Corporation) 0.08 mg/kg, followed by tracheal intubation and controlled respiration using a mixture of oxygen and isoflurane (isoflurane inhalation anesthetic solution "Pfizer", Mylan Pharmaceutical Co., Ltd.) 0.5-2.0%.
  • ketamine hydrochloride Ketalar 500 mg for intramuscular injection, Daiichi Sankyo Propharma Co., Ltd.
  • medetomidine hydrochloride Domitor, Orion Corporation
  • the animal was fixed on the operating table in a supine position, and an appropriate amount (50-100 ⁇ L) of RPE cell suspension (2.5 ⁇ 10 6 -1.5 ⁇ 10 7 cells/mL) was administered under the retina in the same manner as in Example 1. After administration, if any suspension leaked, the leaked liquid was aspirated. If necessary, the vitreous cavity was replaced with air as in Example 1. The supine position was maintained under inhalation anesthesia for a maximum of 3 hours in order to promote cell sedimentation and engraftment.
  • immunosuppression was performed using a combination of a once-daily intramuscular injection of cyclosporine (Sandimmune IV 250 mg, Novartis Pharma K.K.) and sub-Tenon administration of triamcinolone acetonide (Macuaid ophthalmic 40 mg, Wakamoto Pharmaceutical Co., Ltd.) during surgery.
  • cyclosporine Synchronization IV 250 mg
  • sub-Tenon administration of triamcinolone acetonide Macuaid ophthalmic 40 mg, Wakamoto Pharmaceutical Co., Ltd.
  • the administered eye was observed over time using a fundus camera (Kowa Optimed, VX-10 ⁇ ) and an OCT device (Heidelberg Engineering, Spectralis OCT2).
  • Tables 2 and 3 The results of OCT findings up to 4 weeks are shown in Tables 2 and 3. As can be seen from Tables 2 and 3, the formation of preretinal membrane was observed in 7 out of 8 cases in which the vitreous cavity was not replaced with air, and in 8 out of 23 cases in which the air was replaced, indicating that air replacement may be able to suppress leakage of the cell suspension and reduce the formation of the preretinal membrane.
  • the anti-CD147 antibody used showed a strong positive signal in some of the areas where human iPS cell-derived RPE cells were thought to have engrafted, but the entire retinal pigment epithelium layer of the cynomolgus monkey was stained, suggesting that it also cross-reacts with monkey antigens. Therefore, the anti-PMEL17 antibody positive signal was used as the main indicator of the engraftment and distribution of human iPS cell-derived RPE cells, and the anti-CD147 antibody was used in multiple staining to make it easier to identify the monkey retinal pigment epithelium layer during observation.
  • FIG. 3 The site indicated by the arrow in Figure 3 (A) indicates the site where human iPS cell-derived RPE cells were continuously engrafted. Five continuous engraftment sites were confirmed in this individual (individual number 1), and the result was grade 3.
  • Figure 5 shows high-magnification fluorescent immunostaining images (partial areas of Figures 3 and 4) of individual number 1 (4 weeks after surgery), where (A) is an image where the various stainings are superimposed, and (B) to (D) show images stained with DAPI, CD147, and PMEL17 alone, respectively.
  • the group containing hyaluronic acid-added medium (100-fold dilution), which showed a good effect of suppressing cell leakage, was scored for engraftment. Furthermore, because the administration volume (total number of administered cells) differed for each individual, a score per unit number of cells was calculated. The results are shown in Table 3. From Table 4, it can be seen that the administered cells engrafted over a wide area under the conditions using Provisc, whereas engraftment was observed only in a small area under the conditions using Viscoat.
  • Example 3 Transplantation experiment 2 into cynomolgus monkeys
  • the immunosuppression of cynomolgus monkeys was enhanced to evaluate the effect of transplantation media on the engraftment of RPE cells.
  • the protocol of Example 2 was used in combination with oral administration of mycophenolate mofetil (Cellcept suspension powder 31.8%, Chugai Pharmaceutical Co., Ltd.) once a day, and a double dose of cyclosporine was administered to the subretina of the right eye of a cynomolgus monkey for immunosuppression.
  • mycophenolate mofetil Cellcept suspension powder 31.8%, Chugai Pharmaceutical Co., Ltd.
  • a microtip beveled cannula 25 g/40 g (MedOne #3261) was used as the subretinal injection needle.
  • 50 ⁇ L of a cell suspension (1.0 ⁇ 10 7 cells/mL) prepared using HBSS(+) or Provisc diluted 100-fold with HBSS(+) was injected into the upper and lower parts of the same eye.
  • the vitreous cavity was replaced with air after surgery, and ophthalmological examinations were performed over time, after which the transplanted eyes were histologically analyzed by fluorescent immunostaining.
  • the eyeballs were enucleated, fixed, and embedded in paraffin after the end of the 4-week observation period.
  • Serial sections (3-4 ⁇ m thick) were prepared at intervals of approximately 0.28 mm to cover the entire bleb, and fluorescent immunostaining was performed according to the usual method using anti-human PMEL17 antibody (Novus Biologicals, NBP2-47780AF647, mouse) and anti-human CD147 antibody (R&D Systems, AF972, goat).
  • the area including the human PMEL17-positive site of the stained specimen was imaged using a slide scanner (Zeiss, Axio Scan.Z1), and the engraftment of human iPS cell-derived RPE cells was evaluated.
  • the axial diameter was 5.90 mm
  • the sagittal diameter was 5.93 mm
  • the axial diameter was 4.74 mm
  • the sagittal diameter was 4.30 mm
  • the average bottom area was 16.01 mm2 .
  • the total score for the Provisc 100-fold diluted media condition was 14 to 54, showing variation in engraftment between individuals, whereas the HBSS(+) media condition was 32 to 42, showing high engraftment in all four cases, confirming good engraftment.
  • the use of HBSS(+) as a medium showed superior engraftment of human iPS cell-derived RPE cells compared to Provisc 100-fold diluted.
  • the number of sections with a score of 1 or more in the HBSS(+) media condition was 14 to 19, and the engraftment range in the horizontal direction was a wide range of approximately 3.64 to 5.04 mm.
  • the number of positive sections in the Provisc 100-fold diluted media condition was 2.8 to 5.6 mm (number of positive sections: 11 to 21).
  • Example 4 Transplantation experiment 3 into cynomolgus monkeys Administration and evaluation were performed in the same manner as in Example 3.
  • the subretinal injection needle used was the polytip tapered cannula manufactured by MedOne, as described in Example 1.
  • Human iPS cell-derived RPE cell suspension (1.0 x 107 cells/mL, 50 ⁇ L/bleb/eye) was administered subretinally to cynomolgus monkeys using HBSS(+) as a medium, and ophthalmological examinations were performed over time (4 weeks and 13 weeks of observation, 3 cases each) to evaluate the prolongation of retinal detachment and the presence or absence of a preretinal membrane on the macula.
  • engraftment was evaluated by fluorescent immunostaining.
  • OCT examination showed that retinal detachment associated with this administration method was no longer observed one week after administration, suggesting that the bleb had reattached in less than one week. Furthermore, no preretinal membrane was observed on the macula throughout the observation period in any individual, and no membranous tissue was observed on the retinal surface in histopathological evaluation using HE staining. This suggests that the administration medium and administration method were appropriate.
  • Example 5 Viscosity measurement of dispersion medium
  • the following dispersion media were prepared. Based on the results of Examples 1 and 2, the media (2) to (7) are suitable dispersion media, and among them, (3) and (6), especially (6), are preferred.
  • Example 6 Evaluation of efficacy using a rat model of RPE dysfunction
  • the preclinical efficacy of human iPS cell-derived RPE cells was examined by subretinal administration in pigmented RCS rats.
  • RCS rats lack the phagocytic ability of photoreceptor outer segments, which RPE cells inherently possess, due to a mutation in the MERTK gene, and photoreceptor cells are degenerated secondary to abnormal accumulation of outer segments.
  • RCS rats are widely used as a model of RPE dysfunction to evaluate the pharmacological efficacy of RPE cell replacement therapy (Hum Mol Genet. 2000; 9(4): 645-51., Stem Cells Int. 2017; 2017: 9428176.).
  • 1x105 cells of human iPS cell-derived RPE cells were administered subretinal to the left eye of pigmented RCS rats (4 weeks old) that had been immunosuppressed by administering cyclosporine in drinking water. After administration, visual function of the administered eye was evaluated over time by electroretinogram (ERG) tests, visual evoked potential (VEP) tests, and optokinetic reflex (OKT) tests. Engraftment and function of human iPS cell-derived RPE cells were evaluated by fluorescent immunostaining of the enucleated eye two months after administration.
  • ERP electroretinogram
  • VEP visual evoked potential
  • OKT optokinetic reflex
  • the phagocytic ability and barrier function of human iPS cell-derived RPE cells after transplantation were also evaluated by fluorescent immunostaining.
  • human iPS cell-derived RPE cells stained with anti-human PMEL17 antibody vesicles containing rat photoreceptor outer segments stained with anti-rhodopsin antibody were observed ( Figure 13(C), indicated by arrow), confirming that the transplanted human iPS cell-derived RPE cells have phagocytic ability.
  • Retinal whole mount staining revealed the expression of Zo-1, a component of tight junctions, overlapping with human iPS cell-derived RPE cells stained with anti-CD147 antibody, suggesting that the human iPS cell-derived RPE cells have taken root and are exerting barrier function (Figure 14).
  • the protective ability of photoreceptors was evaluated using the survival of the outer nuclear layer (ONL), which is composed of the cell bodies of photoreceptors.
  • ONL the outer nuclear layer
  • the ONL shown by the dotted line in Figure 11A
  • Figure 11A the ONL was either completely lost or only partially remained in a layer consisting of 1-2 cells, whereas in the human iPS cell-derived RPE cell-treated group, a significantly greater amount of ONL remained adjacent to the engraftment site than in the vehicle control group ( Figure 11A).
  • Example 7 Evaluation of RPE cell adhesion with and without laminin coating
  • Adhesion of RPE cells to wells coated with iMatrix511, an E8 fragment of laminin511, a substrate with properties similar to those of the components constituting Bruch's membrane (0.5 ⁇ g/cm 2 ), and uncoated wells was evaluated.
  • Human iPS cell-derived RPE cells were seeded at 2.5 ⁇ 10 7 cells/well in a 24-well plate and cultured in RPE maintenance medium (a mixed medium of 355 mL DMEM, 150 mL F-12 HAM, 5 mL L-Gln, and 10 mL B-27).
  • Example 8 Examination of the usefulness of RPE cell suspension using an in vitro retinal pigment epithelium tear model Human iPS cell-derived RPE cells were seeded on a 24-well plate (0.5 ⁇ g/cm 2 ) coated with iMatrix511, and after culturing, an RPE sheet was formed. Mitomycin C was treated at 6 ⁇ g/mL to suppress cell proliferation in the RPE sheet. After culturing for one day, a part of the RPE sheet (approximately 5 mm ⁇ 10 mm) was detached with a cell scraper to form a pseudo-retinal pigment epithelial tear ( FIG. 17 ).
  • a human iPS cell-derived RPE cell suspension expressing EGFP was seeded at 1 ⁇ 10 6 cells/well as a pseudo-transplantation RPE cell suspension.
  • the number of seeded cells was determined based on the number of cells per area, regarding the entire well as a bleb at the time of transplantation.
  • the day after seeding the pseudo-transplantation RPE cell suspension the EGFP-expressing human iPS cell-derived RPE cells adhered to the entire pseudo-retinal pigment epithelium break ( Figure 18).
  • the classical shape of a retinal pigment epithelial tear is a crescent-shaped tear, but various other shapes exist.
  • the usefulness of the RPE cell suspension for pseudo-retinal pigment epithelial tears of complex shapes was examined using a similar method using pseudo-retinal pigment epithelial tears. As a result, it was confirmed that suspension-derived RPE cells were able to attach to the detached site without any gaps, even for pseudo-retinal pigment epithelial tears of complex shapes, and restore a single RPE sheet ( Figures 21 and 22).
  • the RPE cell suspension has the potential to quickly restore the host RPE sheet without leaking cover for retinal pigment epithelial tears of various shapes.
  • Example 9 Phase I/II study of iPS cell-derived RPE cell product in patients with retinal pigment epithelial tears
  • the purpose of the study is to evaluate the safety and efficacy of subretinal administration of a suspension of iPS cell-derived RPE cell product in patients with retinal pigment epithelial tears (RPE tears), and is a randomized, unobstructed, multicenter study.
  • the efficacy of the suspension of iPS cell-derived RPE cells will be evaluated in terms of visual acuity, retinal sensitivity, etc.
  • Part 1 HLA-mismatched subjects are administered a suspension of iPS cell-derived RPE cell product.
  • Part 2 The subjects will be randomly assigned to an administration group or an observation group, and the administration group will be administered a suspension of the iPS cell-derived RPE cell product. After 48 weeks of observation, the observation group will be administered a suspension of the iPS cell-derived RPE cell product. Observation period: Both consist of two periods: a treatment period (48 weeks) and a continuation treatment period (48 weeks). Dosage: 50 ⁇ L of 1.0 ⁇ 10 7 cells/mL cell suspension is administered subretinally once to the treated eye.
  • Immunosuppressants such as triamcinolone acetonide, cyclosporine, and prednisolone are administered to suppress immune rejection reactions associated with cell transplantation or to suppress postoperative inflammation.
  • AMD age-related macular degeneration
  • idiopathic RPE tear longest axis greater than one optic disc diameter
  • Example 10 Evaluation of efficacy using retinal pigment epithelium degeneration model rats
  • the preclinical efficacy of human iPS cell-derived RPE cells was examined by administering human iPS cell-derived RPE cells subretinaly to rats with a retinal pigment epithelium degeneration model (sodium iodate-induced model). It is known that systemic administration of sodium iodate to rodents causes specific damage to RPE cells, and it is widely used as a retinal pigment epithelium degeneration model (J Photochem Photobiol B. 2019 Jul:196:111514.).
  • Sodium iodate was administered intraperitoneally to immunodeficient model F344/NJcl nude rats (7 or 9 weeks old) at 0 or 70 mg/kg (administration volume of 0.5 mL/kg). Seven days later, 1 ⁇ 10 5 cells of human iPS cell-derived RPE cells were administered subretinally to the left or right eye. Electrophysiological function was evaluated by electroretinogram (ERG) test about 1 month after administration, and fundus images were confirmed by fundus camera photography about 2 months after administration, and the engraftment area of human iPS cell-derived RPE cells was evaluated. Then, the engraftment of human iPS cell-derived RPE cells and the host retina were evaluated by fluorescent immunostaining of the excised eye about 2 months after administration.
  • ERP electroretinogram
  • Electrophysiological function was evaluated by ERG test about one month after administration. After the rats were dark-adapted for more than 3 hours, measurements were performed under light stimulation conditions of 10 cd x s/ m2 . The results are shown in Figure 25. In the vehicle-administered group after administration of 0 mg/kg sodium iodate (left, Figure 25, Sham), which is the control, the b-wave amplitude was about 350 ⁇ V. In the vehicle-administered group after administration of 70 mg/kg sodium iodate (in Figure 25, Sham), electrophysiological function decreased, which is thought to be due to the disorder of rat RPE cells, and a significant decrease in b-wave amplitude was observed.
  • FIG. 26 (A) to (C) A fundus image was taken with a fundus camera approximately two months after administration, and the engraftment area of human iPS cell-derived RPE cells was evaluated. The results are shown in Figures 26 (A) to (C).
  • the engrafted human RPE cells were recognized as black areas on the fundus due to melanin deposition. Note that nude rats have albino eyes, so there is no melanin deposition in RPE cells. Therefore, the observed black areas were determined to be melanin deposition of transplanted human RPE cells.
  • the engraftment area of human RPE cells in the sodium iodate administration group (Figure 26 (C)) was wider than the engraftment area of human RPE cells in the vehicle administration control group ( Figure 26 (A)). On the other hand, no black areas were observed in the vehicle administration group after administration of 70 mg/kg sodium iodate ( Figure 26 (B)).
  • Engraftment of human iPS cell-derived RPE cells and rat retina was evaluated by fluorescent immunostaining of the enucleated eyes about 2 months after administration. The results are shown in Figures 27 to 31.
  • the vehicle was administered subretinal after sodium iodate administration (sham group)
  • disappearance of rat RPE cells stained with an antibody that specifically recognizes rat/human RPE65 was observed ( Figure 27(B)).
  • human RPE cells stained with an antibody that specifically recognizes human PMEL17 were engrafted in the rat RPE layer in a single layer ( Figure 28(C)).

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PCT/JP2024/004888 2023-02-14 2024-02-13 網膜色素上皮裂孔の治療薬 Ceased WO2024172043A1 (ja)

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