WO2020009248A1 - 眼組織の線維化抑制用組成物 - Google Patents

眼組織の線維化抑制用組成物 Download PDF

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WO2020009248A1
WO2020009248A1 PCT/JP2019/027233 JP2019027233W WO2020009248A1 WO 2020009248 A1 WO2020009248 A1 WO 2020009248A1 JP 2019027233 W JP2019027233 W JP 2019027233W WO 2020009248 A1 WO2020009248 A1 WO 2020009248A1
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tissue
fibrosis
pharmaceutical composition
eye
obp
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French (fr)
Japanese (ja)
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盛夫 上野
千恵 外園
羽室　淳爾
木下　茂
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Oncolys Biopharma Inc
Kyoto Prefectural PUC
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Oncolys Biopharma Inc
Kyoto Prefectural PUC
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a composition for suppressing fibrosis of eye tissue.
  • Fibrosis is a disease that occurs widely in important organs, such as the lungs, heart, liver, kidneys, and skin. Fibrosis in the parenchymal organs (liver, heart, lungs, kidneys, gastrointestinal tract, etc.) is a serious chronic illness that, if left unchecked, can lead to death. Despite the disease requiring improved life prognosis, research has lagged significantly and there are no effective medicines. Until now, research on organ fibrosis has focused mainly on the acquisition of immune function and collagen-producing ability, mainly on the action of tissue growth factor TGF ⁇ .
  • Tissue fibrosis is caused by excessive production and accumulation of extracellular matrix (ECM), mainly collagen.
  • ECM extracellular matrix
  • the cells are repaired by replacing the damaged tissues with extracellular matrix.
  • the stress stimulus such as chronic inflammation becomes chronic over a long period of time, the accumulation of extracellular matrix becomes excessive, resulting in a pathological state in which the original function of the tissue falls below a threshold for maintaining physiological balance.
  • Chronic inflammation is a condition in which inflammation persists and fibrosis at the site of inflammation (tissue remodeling), new blood vessels, and accumulation of specific immune cells are remarkable.
  • Chronic inflammation of various causes and pathological conditions is present, and internal and external environmental stress on an individual causes inflammation as a biological defense through the immune system and endocrine system.
  • the inflammation persists or repeats, it passes through a non-disease state without subjective symptoms, and the adaptive state as abnormal chronic inflammation with dysfunction in cells and tissues is established, and tissue fibrosis occurs due to the continuation of this chronic inflammatory state And the dysfunction of the organs is irreversible.
  • tissue / organ level changes in the interaction between tissue constituent cells and infiltrating immune cells and heterogeneous and diverse cells with different active states, non-physiological metabolic responses, extracellular matrix -The breakdown of tissue homeostasis occurs due to the involvement of various factors such as disturbance of the humoral factor network.
  • Immune privilege is a self-defense mechanism provided by the living body in the first place. Immune privilege can be interpreted as a homeostasis-maintaining mechanism that exists to protect the function of organs in which tissue damage and dysfunction become stronger when a normal immune inflammatory reaction occurs. However, once inflammation is exceeded, the immune privilege mechanism is lost and eye inflammation exacerbates. Damaged eye tissue is difficult to recover its function.
  • Immune reactions are broadly classified into “acquired immunity”, which mainly involves T lymphocytes, and “innate immunity”, which responds earlier. Innate immune cells are important proinflammatory cells in various eye diseases. In recent years, it has been recognized that Mps, NKT cells, ⁇ -type T cells, etc., which are responsible for so-called “innate immunity”, are indispensable for maintaining homeostasis and transparency of the eye.
  • CNV choroidal neovascularization
  • Age-related macular degeneration is a disease in which retinal pigment epithelial cells (RPE) in the macula degenerate due to environmental factors such as aging and oxidative stress, causing choroidal neovascularization (CNV). It is one of the serious visual impairment diseases of the elderly.
  • RPE retinal pigment epithelial cells
  • CNV choroidal neovascularization
  • Non-Patent Document 3 As a therapeutic target for choroidal neovascular disease, the dysfunction (scar healing) process of the macula, which occurs after bleeding and exudation from CNV, is also considered to be important. In addition to the pathogenesis of CNV, it is also important to suppress the pathology of retinochoroidal scars formed secondary to bleeding and exudation of blood components from CNV.
  • Non-Patent Document 1 The clinical condition of choroidal neovascular disease such as AMD progresses as shown in FIG. Proliferating and migrating RPE and Mps are mixed in the subretinal proliferating tissue of an AMD patient, and it is considered that Mps and RPE are important for pathogenesis.
  • Non-Patent Document 2 Intracellular ⁇ SMA is elevated in RPE co-cultured with Mps and subretinal injection leads to subretinal scar formation.
  • the present inventor has long sought a substance that suppresses the production of Mps, an inflammatory cytokine produced by a fellow dendritic cell, and developed a substance that retains its effect even in the presence of a fibrosis-related pathological exacerbation factor related to tissue fibrosis.
  • the headline was the starting point of the present invention.
  • Glaucoma which is another important ocular tissue disease targeted by the present invention, is a disease in which the optic nerve is damaged and has a characteristic change such as a narrowed visual field.
  • Trabeculectomy TLE
  • trabeculectomy to reduce intraocular pressure when intraocular pressure is insufficient even with anti-glaucoma drugs
  • Operation to lower intraocular pressure.
  • trabeculotomy and implant insertion for glaucoma treatment are also performed. Improving the prognosis of glaucoma surgery is an important medical need.
  • aqueous humor is discharged from the trabeculectomy below the scleral flap to the outside of the eye, and a filtration bleb is formed under the conjunctiva.
  • intraocular pressure may increase again and glaucoma may worsen.
  • Trabeculectomy involves the risk of infection, difficulties in the formation and maintenance of filtration blebs, and difficulties in long-term control of intraocular pressure.
  • MMC Mitomycin C
  • Histone deacetylase (HDAC) inhibitors have been studied in basic research to suppress fibrosis, but they are far from practical use and have a large bottleneck, and have not been developed.
  • HDAC Histone deacetylase
  • An object of the present invention is to provide a substance having an effect of suppressing fibrosis of eye tissue.
  • the present invention also provides, as one aspect, a method for treating ocular hypertension or glaucoma, and more specifically, to provide a treatment method in which the effect is synergistically improved and side effects can be reduced. It is an object of the present invention to provide a treatment method for improving exacerbation of AMD and a treatment method for a patient expected to develop AMD.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and as a result, have developed a glaucoma surgery technique for suppressing excessive fibrosis, promoting healthy conjunctival tissue repair, and maintaining a long-term low intraocular pressure maintenance effect.
  • it is effective in extremely low concentrations and doses of fibrosis of choroidal tissue including RPE in a laser-irradiated animal model, and has a comprehensive inhibitory effect on multiple genes related to tissue fibrosis.
  • tissue fibrosis not only fibrosis, but also expression of multiple genes involved in tissue fibrosis, angiogenesis, and tissue scarring, such as VEGF and PDGF involved in angiogenesis, and LOX involved in cross-linking of Collagen and scar formation.
  • the aim of the present invention is to develop a novel therapeutic agent for AMD having both a novel mechanism of suppressing CNV and a suppressive effect on scar tissue formation based on a unique theory of AMD molecular pathology, and has conducted intensive studies to complete one of the present invention.
  • the inventor has completed the present invention in response to the above two large Unmet @ Needs, but the present invention may be applied to other eye tissue diseases and other organ diseases beyond the examples shown in this specification. I will provide a.
  • the present invention is as follows. (1) A pharmaceutical composition comprising a substance that suppresses fibrosis of eye tissue. (2) A substance that suppresses fibrosis of eye tissue inhibits in vivo at least one pathological exacerbation factor gene expression relating to each of the three stages of fibrosis, angiogenesis and scar formation in the eye tissue in vivo. 2.
  • the pharmaceutical composition according to 1 which is a substance that does (3)
  • the disease exacerbation factor genes are collagen 1A, collagen 3A1, collagen 4A1, TIMP2, TIMP3, TIMP4, Thrombospondin 1, Thrombospondin2, LOX, Loxl2, TGF3P, GF from the GF from the group of GFs, TGFb2T, bGFBT from the GFs, TGFb2Ts, TGFb2Ts, and TGFb2Ts.
  • the pharmaceutical composition according to any one of claims 1 to 3 comprising a substance that suppresses fibrosis of eye tissue at a dose of 100 pg / kg to 3000 pg / kg.
  • the glaucoma-related tissue is a trabecular meshwork or a tissue capable of controlling intraocular pressure.
  • the retinal-related tissue is a tissue relating to retinal pigment epithelium, choroidal neovascularization, or age-related macular degeneration.
  • the conjunctiva-related tissue is a filtration bleb tissue.
  • a compound having properties capable of suppressing excessive fibrosis, promoting healthy conjunctival tissue repair, and maintaining a long-term effect of maintaining low intraocular pressure Further, on the other hand, a compound having both a fibrosis-suppressing effect and / or angiogenesis-suppressing effect and a scar formation-suppressing effect, which is required but not provided for the current treatment of AMD, has been provided for the first time, Leads to the teaching of useful means for effective treatment.
  • Such compounds promote the wound healing process after glaucoma surgery on trabecular meshwork cells and normalize aqueous humor dynamics.
  • it is effective at extremely low concentrations and doses for fibrosis of peritoneal tissue, and has a comprehensive inhibitory effect on multiple genes related to tissue fibrosis and precursor lesions.
  • Many existing candidate substances for suppressing fibrosis are effective for TGF ⁇ -induced fibrosis but are ineffective for fibrosis due to the combined action of TGF ⁇ + TNF ⁇ in chronic inflamed tissues.
  • the pharmaceutical composition of the present invention is also remarkably effective against this complex condition, and may directly inhibit damage (including fibrosis) of photoreceptor cells and retinal pigment epithelial cells, which is most important for maintaining visual acuity in patients. Is high.
  • FIG. 10 is a diagram showing the measurement results of intraocular pressure up to 30 days when 200 ⁇ l of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery.
  • FIG. 10 shows blebs on days 7 and 30 when 200 ⁇ l of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. It is a figure which shows the bleb (filtration bleb) on the 7th day and the 30th day when the BSS was administered on the 0,1,3,5,7,9 day after operation as a control. It is a figure which shows Bleb on the 14th day when OBP-801 is administered on the 0,1,3,5,7,9 days after operation.
  • FIG. 10 is a graph showing the expression level of ⁇ SMA on day 14, when 200 ⁇ l of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery.
  • FIG. 9 is a graph showing the expression level of collagen on day 14, when 200 ⁇ l of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery.
  • FIG. 3 is a graph showing the expression level of collagen on day 30 when 200 ⁇ l of 10 nM OBP-801 was administered on days 0, 1, 3, 5, 7, and 9 after surgery. It is a figure which shows the alphaSMA expression suppression effect when OBP-801 is administered to HconF induced to fibrosis by TGF + TNF. It is a figure which shows the collagen and LOX expression suppression effect when OBP-801 is administered to HconF induced to fibrosis by TGF + TNF. It is a figure which shows the expression suppression effect of (alpha) SMA, col1, col4 at the time of administering OBP-801 before and / or after induction of fibrosis of HconF by TGF + TNF.
  • FIG. 9 is a diagram showing that OBP-801 inhibits HTMC myofibroblast formation.
  • FIG. 4 is a view showing the effect of OBP-801 on CD44 expression. It is a figure which shows the AMD pathological progress of Drusen and a laser-induced CNV model. It is a figure which shows that the expression of collagen-1 on the 30th day after operation was suppressed by OBP-801.
  • FIG. 3 shows the expression of ECM and ECM remodeling enzymes. It is a figure which shows the expression of an inflammatory cytokine and a chemokine.
  • FIG. 4 is a view showing expression of the TGF ⁇ superfamily. It is a figure which shows the expression of a transcription factor. It is a figure showing a result of real-time RT-PCR. It is a figure showing a result of real-time RT-PCR. It is a figure which shows the intraocular pressure suppression result by instillation of OBP-801. It is a figure which shows the expression of the gene group considered to be involved in maintenance of intraocular pressure.
  • FIG. 10 shows the results of ⁇ SMA expression on day 30 after surgery by immunostaining of rabbit filtration bleb tissue.
  • FIG. 7 shows the results of expression of Collagen I 30 days after surgery by rabbit filter vesicle tissue immunostaining.
  • FIG. 3 is a diagram showing the results of analysis of genes considered to be involved in maintaining intraocular pressure in human conjunctival tissue.
  • FIG. 3 is a diagram showing the results of analysis of genes considered to be involved in maintaining intraocular pressure in human conjunctival tissue.
  • FIG. 3 is a diagram showing the results of analysis of genes considered to be involved in maintaining intraocular pressure in human conjunctival tissue.
  • FIG. 3 is a diagram showing the results of analysis of genes considered to be involved in maintaining intraocular pressure in human conjunctival tissue.
  • FIG. 3 is a diagram showing the results of analysis of genes considered to be involved in maintaining intraocular pressure in human conjunctival tissue.
  • the present invention relates to a pharmaceutical composition and a method for restoring normal eye tissue from eye-related fibrotic tissue and for suppressing normal eye tissue from losing its original function due to fibrosis.
  • the present inventors have continued intensive studies on (1) spatiotemporally what kind of gene expression is related to a disease state in a fibrosis model in ocular tissue, and Not only the fibrosis stimulus but also (2) a substance in which the multiple genes are simultaneously suppressed by many stimuli including chronic inflammation related to fibrosis have been sought.
  • the selection criterion is that, in the presence of a factor well known as a fibrosis-inducing action, the action of inflammatory cytokines also acts under cell stress similar to chronic inflamed tissue in which the action of inflammatory cytokines is overlaid.
  • a hydrophobic compound that penetrates into a cell and exerts an action to suppress gene expression has an extremely weak effect in vivo in terms of drug accessibility unless the action concentration is too low. Its effectiveness as a fibrotic agent cannot be expected at all. In fact, as seen in the above-mentioned limitations of SAHA, no antifibrotic agent that can be practically used has been found so far. The inventors also pay attention to this point, and (4) the action concentration is effective at a concentration of one-thousandth of the analogous compound being studied, that is, a dosage route that can be practically used even in vivo. We have been keenly searching for compounds that meet the strict selection criteria that can be expected.
  • Compounds satisfying the above conditions (1) to (4) can be used to evaluate ECM such as Collagen accumulated in tissues in ophthalmic tissues, which have been continuously subjected to various fibrotic stimulations, in experimental model animals and human eye tissues.
  • ECM such as Collagen accumulated in tissues in ophthalmic tissues, which have been continuously subjected to various fibrotic stimulations, in experimental model animals and human eye tissues.
  • the present inventors have found that it can be reduced in related cells and completed the present invention.
  • the ability to reduce ECM related to cell sclerosis in eye-related fibrotic tissue chronically exposed to multiple fibrotic stress stimuli has not been known so far and is a novel finding.
  • a compound having an effect of suppressing tissue fibrosis occurring before CNV formation enables early treatment before CNV formation, and is expected to avoid a decrease in visual acuity.
  • a therapeutic effect can be expected for the above-mentioned anti-VEGF resistance through the effect of inhibiting fibrosis formation.
  • the present invention focuses on fibrosis, a functional phase transition triggered by the disruption of the epigenetic regulatory mechanism of retinal pigment epithelial cells (RPE) during the progression of AMD pathology.
  • the active ingredient of the pharmaceutical composition of the present invention is effective at extremely low concentrations and doses for fibrosis of choroid tissue containing RPE, and has a comprehensive inhibitory effect on a plurality of genes related to tissue fibrosis.
  • the pharmaceutical composition of the present invention uses a mouse model of fibrosis and angiogenesis of retinal tissue, and suppresses Collagen fiber cross-linking in the late stage, surpasses the anti-angiogenic effect, and correlates with anti-VEGF therapy resistance. It possesses a novel action characteristic of inhibiting the transformation of cells into myofibroblasts, confirming its superiority to existing therapy. Simultaneous suppression of the expression of multiple genes related to fibrosis such as LOX, THBS1, Serpin, MMP, etc. characterizes the drug properties of the present compounds.
  • the first reason for using AMD as a model target disease is that the limit of anti-VEGF antibody therapy as an anti-angiogenesis inhibitory effect has been clinically clarified.
  • This antibody therapy only targets the suppression of angiogenesis, which is only one of the phenotypes after the onset and exacerbation of the disease, and is most important for maintaining the visual acuity of the patient.
  • Disorders including fibrosis
  • Suppression of fibrosis by the pharmaceutical composition of the present invention can maintain and repair retinal pigment epithelial cells and photoreceptor cell functions normally.
  • the present invention is a medicine containing a compound that suppresses fibrosis of a plurality of eye tissues such as a retinal tissue and a conjunctival tissue. Tissues attempt to repair tissues by accumulating extracellular matrix by stimulating various cells, such as oxidative stress, hypoxia, inflammation, and apoptosis.
  • the pharmaceutical composition of the present invention can be used to suppress such stress on tissues and treat diseases caused by fibrosis of eye tissues such as retina and conjunctival tissues. Further, the pharmaceutical composition of the present invention can be used to suppress the production of extracellular matrix substances such as collagen caused by various stresses on such cells.
  • fibrosis-like metastasis of ocular tissue cells refers to external or internal cellular stress in ocular tissues such as retina and conjunctival tissue (depending on aging, etc.) that alters cell functions and disrupts tissue homeostasis Indicates the situation.
  • Cytopathy includes metastasis to fibroblast-like cell morphology and changes in cell function called epithelial-mesenchymal transition (EMT), enhanced apoptosis, abnormal autophagy, enhanced production of extracellular matrix components,
  • EMT epithelial-mesenchymal transition
  • the substance of the present invention inhibits hard tissue formation due to a change in the function of the cells constituting the tissue, such as abnormal cross-linking between proteins such as collagen and elastin that constitute the substance.
  • a substance having an effect of maintaining a filtration bleb or improving the prognosis of glaucoma surgery is also included in the present invention.
  • Pharmaceuticals for the treatment of the above-mentioned pathological conditions include compounds having an action such as inhibition of collagen and ⁇ -SMA production, TGF or TNF production inhibitor, TGF or TNF signal transduction inhibition, and HDAC inhibition.
  • a related gene in a fibrosis model of an eye tissue, (1) the expression level of a related gene is spatiotemporally changed, (2) the plurality of gene groups caused by many stimuli related to fibrosis are simultaneously suppressed, (3) At the same time, it is a compound that acts under cell stress similar to chronic inflamed tissue in which the action of inflammatory cytokines is superimposed in the presence of a well-known factor that induces fibrosis.
  • the depsipeptide compound or a pharmaceutically acceptable salt thereof, and more preferably, OBP-801 is (4) has good penetration into cells in each tissue, and has a sufficiently low action concentration. It is suitable as a compound having a fibrotic action and an anti-scarring action.
  • the present invention is a medicament containing a compound capable of regulating or controlling the expression of a disease exacerbation factor gene.
  • a disease exacerbation factor gene ⁇ Conjunctiva-related tissues, trabecular meshwork cells, retinal pigment epithelial cells or pathological aggravation factor genes expressed in choroidal neovessels, choroidal tissues in mammals
  • Pathogenic hate factor genes expressed in choroidal neovascular choroid tissue ⁇ Human or rabbit fibrosis-related genes
  • Pathogenic hate factor genes which fluctuate in expression due to human or rabbit glaucoma surgery
  • the pathological hate factor genes include fibrosis-inducing genes, angiogenesis-related genes, scarring-related genes, ECM-related genes, and the like.
  • fibrosis-inducing genes By controlling the expression of the gene and controlling the fibrosis of eye tissue and the accumulation of ECM, it can be expected to maintain the filtration bleb, maintain the low intraocular pressure, inhibit the angiogenesis, and suppress the scar formation.
  • at least one of each of the three stages of fibrosis, angiogenesis, and scar formation is inhibited in vivo by the disease exacerbation factor gene expression.
  • pathogenesis and aggravation factor genes are collagen 1A, collagen 3A1, collagen 4A1, TIMP 2, TIMP 3, TIMP 4, Thrombospondin 1, Thrombospondin 2, GOX, LOX, Loxl2, TGFb, TGFb, TGFb, TGFb, TGFbT, TGFbT
  • the expression can be controlled by the pharmaceutical composition of the present invention.
  • the present invention provides a pharmaceutical composition that suppresses at least two of the stages of fibrosis-suppressing effect and / or angiogenesis-inhibiting effect and scar formation in ocular tissue cells in vivo.
  • a pharmaceutical composition comprising a substance that inhibits the expression of at least one of the exacerbation factor genes.
  • a substance controlling the activity of a hub gene related to the expression of a disease state exacerbation factor-related gene is also included in the present invention.
  • ⁇ ⁇ A more preferred form of such a substance is a depsipeptide compound, more preferably, OBP-801.
  • OBP-801 has been found to be the first compound showing a comprehensive inhibitory effect on a plurality of genes related to tissue fibrosis. Furthermore, OBP-801 can be confirmed to have an inhibitory effect not only on fibrosis but also on the expression of a plurality of genes such as VEGF and PDGF involved in angiogenesis and LOX involved in cross-linking Collagen and scar formation, High therapeutic effects are expected for various conditions of AMD patients.
  • an inhibitory nucleic acid against these genes for example, an antisense nucleic acid, a decoy nucleic acid, a microRNA, a shRNA or an siRNA can also be used.
  • the nucleotide sequence of the gene to be inhibited is known, and sequence information can be obtained for each.
  • GenBank accession numbers for each gene are shown below.
  • COL1A1 NM_000088
  • COL4A2 NM_001846
  • COL16A1 NM_001856
  • ITGA2 NM_002203
  • ITGA5 NM_002205
  • ITGB3 NM_000212
  • ITGAV NM_002210
  • LAMA1 NM_005559
  • VCAN NM_004385
  • TIMP1 NM_003254
  • CTGF NM_001901
  • Anti-fibrotic chemotherapy is known as a pathological significance of LOX expression inhibition. There is a report that LOX and LOX2 gene expression levels are increased in scar formation after CNV by laser irradiation surgery, and that fibrosis can be suppressed by introducing both antibodies.
  • the present invention is a medicament containing a compound having a filtration bleb maintenance effect.
  • Wound healing occurs in glaucoma-related tissues and conjunctiva-related tissues of the eye tissue after glaucoma surgery, and fibrosis or scarring of trabecular meshwork cells (HTMC) and conjunctival fibroblasts (HconF) occurs.
  • HTMC trabecular meshwork cells
  • HconF conjunctival fibroblasts
  • conjunctival fibroblasts are stimulated by TGF + TNF for fibrosis after glaucoma surgery, which hinders maintenance of filtration bleb.
  • fibrosis-related genes include the genes of collagen 1A, collagen 3A1, collagen 4A1, TIMP 2, TIMP 3, TIMP 4, Thrombospondin 1, Thrombospondin 2, LOX, Loxl2, TGFb2, TGFbE, GF, and GFB3, TGFbE, GFVE, and the like.
  • the present invention is a medicament containing a compound having an effect of improving the prognosis of glaucoma surgery.
  • Glaucoma is a disease that has findings with characteristic changes in the optic nerve and visual field and is characterized by functional and structural abnormalities. Usually, optic nerve damage can be improved or suppressed by sufficiently lowering intraocular pressure.
  • the following surgical treatments are usually performed.
  • surgical operations such as outflow tract reconstruction such as trabeculotomy, filtration surgery such as trabeculectomy, and implant insertion for glaucoma treatment (with or without plate) are performed, and ocular tissues are removed.
  • Treatment to return the structure to a normal state is usually performed.
  • Trabeculectomy is a method of reconstructing an artificial outflow tract while absorbing aqueous humor from the conjunctiva and evaporating it from the surface.
  • TLE has the disadvantage that the filtration bleb is easily localized and easily avascular. Under such circumstances, long-term control of intraocular pressure, healthy formation of filtration bleb, and reduction of infection risk of the filtration bleb are desired. There is a need for a TLE that has a wide range of formation of filter vesicles, maintains the vascular properties of the conjunctiva, and has an intraocular pressure lowering effect for a long time after surgery.
  • the glaucoma operation includes Ex-PRESS, INFOCUS, Baerveldt Glaucoma Implant, Ahmed Glaucoma Valve, XEN Implant, Hydrous Microstent, and the like.
  • composition comprising a depsipeptide compound represented by the following formula I or II or a pharmaceutically acceptable salt thereof.
  • R1 to R3 independently represent a hydrogen atom, a methyl group, an ethyl group
  • R4 represents a hydrogen atom, a methyl group, an ethyl group, an n-propyl group, an isopropyl group, a sec-butyl group or an isobutyl group
  • R5 to R8 Each independently represents a hydrogen atom, a methyl group, an ethyl group or an isopropyl group
  • R8 represents a hydrogen atom, a methyl group or a protecting group
  • R10 and R11 independently represent a hydrogen atom, a methyl group or a protecting group.
  • the invention also provides a compound of formula III: (In the formula, R4 represents an isopropyl group, a sec-butyl group, or an isobutyl group.)
  • a pharmaceutical composition comprising the depsipeptide compound represented by or a pharmaceutically acceptable salt thereof is also provided.
  • R4 represents an isopropyl group
  • OBP-801 is a compound in which R4 is an isopropyl group.
  • OBP-801 above, fibrosis inhibitor, disease state aggravation factor gene expression suppressor, substance having filter bleeding maintenance effect, medical treatment for suppression of pathological exacerbation of AMD patients, prevention of pathology leading to blindness starting from neovascularization in the choroid, glaucoma surgery
  • a drug containing OBP-801 as a substance having an effect of improving prognosis is preferable as an embodiment.
  • OBP-801 has been found to be the first compound showing a comprehensive inhibitory effect on a plurality of genes related to tissue fibrosis. Furthermore, OBP-801 can not only inhibit fibrosis but also inhibit the expression of multiple genes, such as VEGF, PDGF, and LOX involved in scar formation by cross-linking Collagen. High therapeutic effects are expected for various disease states. OBP-801 has a stronger antifibrotic effect than SAHA, and may further suppress postoperative fibrosis. In addition, OBP-801 is one of the compounds that penetrates into cells and exhibits an anti-fibrotic effect at an action concentration low enough to exert the action. According to the present invention, it was revealed that OBP-801 exhibited an effect at a concentration 1/1000 of that of SAHA.
  • OBP-801 exhibits an effect at a lower concentration than SAHA, so there is no concern about toxicity and there is a possibility that OBP-801 is safer from the viewpoint of protecting the conjunctiva.
  • the production method of this compound is according to a known method (Patent Document 1).
  • the embodiments also include the above-mentioned patent documents.
  • composition The pharmaceutical composition of the present invention can be used in the form of eye drops, coatings, sustained release agents, inserts, injections, ointments and the like.
  • Eye drops include isotonic agents such as sodium chloride and concentrated glycerin; buffering agents such as sodium phosphate and sodium acetate; interfaces such as polyoxyethylene sorbitan monooleate, polyoxyl stearate 40, and polyoxyethylene hydrogenated castor oil.
  • Activator such as sodium citrate, sodium edetate
  • Preservative such as benzalkonium chloride, paraben, etc.
  • the pH is acceptable for ophthalmic preparation. Although it may be within a certain range, usually a range of 4 to 8 is preferable.
  • the eye ointment can be prepared using a commonly used base such as white petrolatum or liquid paraffin.
  • the compound or a pharmaceutically acceptable salt thereof can be used in the form of gel, cream and lotion.
  • it can be formulated for topical or topical application to the skin and mucous membranes in the eye, and for application to the eye.
  • the form of the topical pharmaceutical composition is not limited, and examples thereof include a solution, cream, ointment, gel, lotion, emulsion, detergent, humectant, spray, and skin patch. Solutions are formulated as 0.01% to 10% isotonic solutions, pH 5 to 7, with appropriate salts.
  • the compounds of the present invention or pharmaceutically acceptable salts thereof can be formulated as a transdermal patch for transdermal administration.
  • topical pharmaceutical compositions containing a compound or a pharmaceutically acceptable salt thereof include, for example, water, alcohol, aloe vera gel, allantoin, glycerin, vitamin A and E oils, mineral oil, propylene glycol, PPG- Various carrier materials known in the art, such as 2 myristyl propionate, can be mixed.
  • Other materials suitable for use in topical carriers include, for example, emollients, solvents, water retention agents, thickeners, and powders. Examples of each of these types of materials that can be used alone or as a mixture of one or more materials are as follows.
  • Typical application or skin ointments include stearyl alcohol, glyceryl monoricinoleate, glyceryl monostearate, propane-1,2-diol, butane-1,3-diol, mink oil, cetyl alcohol, iso-isostearate.
  • the sustained-release agent or the intercalating agent is obtained by pulverizing and mixing a biodegradable polymer such as hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxyvinyl polymer, and polyacrylic acid with the present compound, and compression-molding this powder. And, if necessary, an excipient, a binder, a stabilizer, and a pH adjuster can be used. Preparations for intraocular implants can be prepared using biodegradable polymers such as polylactic acid, polyglycolic acid, lactic acid / glycolic acid copolymer, and hydroxypropyl cellulose.
  • the injection is selected and used as necessary from a tonicity agent such as sodium chloride; a buffering agent such as sodium phosphate; a surfactant such as polyoxyethylene sorbitan monooleate; a thickener such as methyl cellulose; Can be prepared.
  • a tonicity agent such as sodium chloride
  • a buffering agent such as sodium phosphate
  • a surfactant such as polyoxyethylene sorbitan monooleate
  • a thickener such as methyl cellulose
  • the compositions of the present invention can be in the form of liquids, such as solutions, emulsions or suspensions, or semi-solids, such as gels, eye ointments, and the like.
  • Diluents for aqueous solutions and suspensions include distilled water and physiological saline.
  • Non-aqueous diluents for solutions and suspensions include vegetable oils, liquid paraffin, mineral oil, propylene glycol, p-octyldodecanol and the like.
  • an isotonic agent such as sodium chloride, boric acid, sodium citrate, and the like, for the purpose of keeping the pH constant at about 5.0 to 8.0, for example, Buffers such as boric acid, buffers, phosphate buffers and the like can be added.
  • a stabilizer such as sodium sulfite and propylene glycol
  • a chelating agent such as sodium edetate
  • a thickener such as glycerin, carboxymethylcellulose, and carboxyvinyl polymer
  • a preservative such as methylparapene and propylpyrparapene.
  • O ointment is based on petrolatum, selenium 50, plastibase, macrogol or the like, and may contain a surfactant for the purpose of enhancing hydrophilicity. Further, it may contain a jelly agent such as carboxymethylcellulose, methylcellulose, and carboxyvinyl polymer.
  • Dosage and Administration The dose of the compound can be appropriately changed depending on the dosage form, the severity of the condition of the patient to be administered, age, weight, judgment of a physician, and the like. On the other hand, per day is as follows. Injection: 10 nM 200 ⁇ l subconjunctival injection Once a day (0, 1, 3, 5 days) In the case of eye drops or intercalators: 100 nM 80 ⁇ l twice daily instillation (0, 1, 2, 3, 4, 5, 6, 7 days)
  • the pharmaceutical composition of the present invention is, for example, 2 pg / eye to 9000 pg / eye or 100 pg / kg to 3000 pg / kg in the case of OBP-801, and is used in the following dosage and administration depending on the form of conjunctival injection and eye drops. can do.
  • the amount of OBP-801 injected is 100 pg / kg to 3000 pg / kg, preferably 100 pg / kg to 500 pg / kg, and further The dose is preferably 200 pg / kg to 400 pg / kg, and more preferably 315 pg / kg, and is administered by subconjunctival injection 30 minutes before operation, 1, 3 and 5 days after operation (total of 4 times).
  • 2 pg / eye to 9000 pg / eye preferably 2 pg / eye to 1500 pg / eye, more preferably 4 pg / eye to 1200 pg / eye, more preferably 944 pg / eye, one time before and 30 minutes before and after the operation Administer by subconjunctival injection on days 1, 3 and 5 (4 administrations in total).
  • the amount of OBP-801 injected is 100 pg / kg to 3000 pg / kg, preferably 2000 pg / kg to 3000 pg / kg, and more preferably.
  • the dose is 2500 pg / kg to 3000 pg / kg, more preferably 2517 pg / kg once, and a total of 15 times in the morning and evening 30 minutes before the operation, 1, 2, 3, 4, 5, 6, and 7 days after the operation. Dosing once.
  • 6 pg / eye to 27000 pg / eye preferably 5000 pg / eye to 10000 pg / eye, more preferably 7000 pg / eye to 8000 pg / eye, more preferably 7552 pg / eye, one time before and 30 minutes before and after the operation On the 1, 2, 3, 4, 5, 6, and 7 days, a total of 15 doses in the morning and evening are administered.
  • the OBP-801 concentration is 10 nM and the liquid volume is 0.5 ⁇ l
  • the injected OBP-801 volume is 100 pg / kg to 3000 pg / kg, preferably 100 pg / kg to 500 pg / kg, more preferably 100 pg / kg to 200 pg / kg, More preferably, the dose is 118 pg / kg once, and the administration is performed 1 to 10 times a day.
  • 2 pg / eye to 9000 pg / eye preferably 2 pg / eye to 1500 pg / eye, more preferably 2 pg / eye to 10 pg / eye, and further preferably 2.36 pg / eye once, and a total of 1 to Administer 10 times.
  • ⁇ In the case of eye tissue culture cells: Inhibits fibrosis-like metastasis of eye tissue culture cells at a concentration of 10 nM or less.
  • Eye tissue conjunctival-related tissue, glaucoma-related tissue, retinal-related tissue, corneal tissue, conjunctival tissue, scleral tissue, lens tissue, angle trabecular tissue, retinochoroidal tissue, optic nerve tissue, vitreous tissue
  • Fibrosis conjunctival fiber Cell proliferation of blast cells (HconF) and trabecular meshwork cells (HTMC) progresses, and abnormal expression of extracellular matrix due to excessive expression of fibrosis-inducing genes such as TGF and TNF and col1, col3, col4, and col6 Occurs. As a result, the fibers have reduced cellular and parenchymal physiology.
  • inhibitors anti-TGFb2 antibody, siRNA (anti-TGFb), siRNA (anti-TGFb2 receptor), tranilast, genstein, Suramin, angiotensin-converting enzyme inhibitor, Chemase inhibitor, Smad7 gene transfer, ROCK (Rho-associated @ kinase) Inhibitor, Decorin, Ribozymes, Aptamers (ARC # 126 and ARC # 127), dominant negative of p38 MAPK gene by adenovirus, Simvastatin, HMG-CoA reductase inhibitor, Lovastatin, Folistatin, Folistatin, Folistatin, Mulberry Cell, Folistatin, Folistatin , Bleomycin, Thiotepa (alkylating agent), retinoic acid and its Derivatives (vitamin A), IFN-alpha, lectin, saporin, cytostatic genes p21, bevacizumab, ranibizumab, MMC, steroids,
  • Glaucoma-related tissue is a tissue composed of trabecular meshwork and trabecular meshwork cells (HTMC), and can control intraocular pressure by controlling the flow channel of aqueous humor. Including organization. Types of glaucoma-related tissues: trabecular meshwork, Schlemm's canal, collecting duct, episcleral vein
  • (2-1) Trabecular meshwork The trabecular meshwork is located in the form of a mesh on the outflow channel of aqueous humor accumulated in the anterior chamber of the eye, and has a role of filtering.
  • the aqueous humor is exchanged in about 0.3 mL for 1-2 hours to maintain nutrition of avascular tissue, transportation of waste products, and homeostasis of intraocular pressure. .
  • Intraocular pressure refers to the pressure of the intraocular fluid filling the eyeball. It is slightly higher than the atmospheric pressure, and the difference from the atmospheric pressure is expressed as the value of the intraocular pressure. The unit is expressed in mmHg.
  • Intraocular pressure is controlled by the amount of aqueous humor circulating in front of the eye. Aqueous humor is made of the ciliary body and flows out through the gap between the iris and the lens (posterior chamber) into the space just below the cornea (anterior chamber). After that, it passes through the trabecular meshwork at the corner called the corner of the cornea and the base of the iris, and is excreted by Schlemm's canal.
  • a high intraocular pressure is determined when the intraocular pressure is 21 mmHg or more.
  • normal tension glaucoma in which the optic disc is impaired even though the intraocular pressure is lower than that, is high.
  • Conjunctiva-related tissue has a structure composed of filter bleb tissue, connective tissue around the filter bleb, and aqueous humor tissue. Its function is to absorb aqueous humor from the conjunctiva, evaporate water from the surface, and at the same time, form an artificial aqueous humor outflow channel to control the outflow of water in the eye and maintain intraocular pressure normally. I do.
  • Types of conjunctiva-related tissues include conjunctival epithelium, lamina basement, Tenon's capsule, episclera, and sclera.
  • Retina-related tissue The retina exists inside the choroid, and more than 100 million photoreceptors form a thin membrane of 0.2 to 0.5 mm. It is considered the most important part to sense light and darkness and color and to see things.
  • the light coming from the pupil in the retina hits the front of the fundus, and the portion that looks slightly darker yellow than the surrounding retina is the macula.
  • the macula one point that is slightly thinner in the central part than the surrounding retina is called a fovea. It is one point where the visual acuity is the most sensitive because there are no blood vessels except for densely packed cone cells.
  • the optic nerve head is located on the fundus slightly inside (nasally) the macula, where the nerve fibers connected to the photoreceptors on the retina are gathered. Light information received by the retina exits the eyeball from here and is sent to the brain to become an image.
  • the optic disc is also a gathering point of blood vessels in the retina, from which retinal arteries and veins spread throughout the retina.
  • the retinal pigment epithelium is located at the outermost layer of the ten layers of the retina and is a monolayer epithelial cell.
  • the tip, called the outer segment, is constantly phagocytosed by the retinal pigment epithelium and replaced by a new one.
  • the retinal pigment epithelium has photoreceptor phagocytosis and the ability to regenerate visual substances (such as retinal), and constitutes the blood-retinal barrier. It is also the main focus of age-related macular degeneration.
  • Photoreceptors are one of the cells that make up the retina.
  • the retinal pigment epithelium is called a photoreceptor and converts light energy into electric energy.
  • Fibrosis of the retinal pigment epithelial tissue is a serious condition common to intraocular proliferative diseases, and a common treatment called fibrosis suppression can be used as a therapeutic intervention for multiple diseases.
  • Age-related macular degeneration (AMD), proliferative hyaline retinopathy, and proliferative diabetic retinopathy (PDR) are all age-related diseases and are considered to be disease states caused by acquired epigenetic gene changes.
  • One of them is fibrotic pathology of retinal pigment epithelial cell tissue (RPE).
  • the prognosis of the visual acuity is deteriorated due to fibrosis of the posterior interstitial fibrosis, the malignancy of the disease progresses, and a functional phase transition of cells occurs. Subsequently, as the cell senescence of the retinal pigment epithelial cell tissue (RPE) further progresses, fibrosis progresses from epithelial to mesenchymal transition, leading to age-related macular degeneration.
  • RPE retinal pigment epithelial cell tissue
  • a tissue that develops age-related macular degeneration As a tissue that develops age-related macular degeneration, a retinal-related tissue, a retina constituting the blood-retinal barrier, a retinal pigment epithelium, a photoreceptor cell which is a cell constituting the retina, etc. There is.
  • the macula is located in the center of the retina, and the macula is a site in the retina where important cells that control vision are concentrated, and pathologically fragile blood vessels (choroidal neovascularization) are newly formed behind the macula. Then blood seeps out from here and overflows into the fundus. As a result, the macula develops the above-mentioned disease due to degeneration or damage, and central vision is impaired, leading to symptoms such as a decrease in vision.
  • the target disease to which the pharmaceutical composition of the present invention is administered includes fibrosis-related diseases, inflammation-related diseases, It can be applied to any of the diseases related to angiogenesis.
  • glaucoma diabetic macular edema (DME), age-related macular degeneration (AMD) (especially wet or non-wet age-related macular degeneration), cataract, infectious or non-infectious uveitis Scleritis, corneal surgery, non-infectious keratitis, ulceris, choroidal retinal inflammation, inflammatory diseases that damage the retina of the eye, and retinopathy, especially diabetic retinopathy, arterial hypertension-induced hypertensive retinopathy, Radiation-induced retinopathy, sun-induced retinopathy, trauma-induced retinopathy, such as an inflammatory disease of the eye selected from Pulcher's retinopathy, retinopathy of prematurity (ROP) and hyperviscosity-related retinopathy.
  • DME diabetic macular edema
  • AMD age-related macular degeneration
  • cataract infectious or non-infectious uveitis Scleritis
  • corneal surgery non-infectious keratiti
  • Intraocular inflammatory disease examples include, for example, after anterior / posterior eye surgery, for example, cataract surgery, laser ophthalmic surgery, glaucoma surgery, refractive surgery, corneal surgery, vitreous-retinal surgery, ocular muscle surgery
  • cataract surgery laser ophthalmic surgery
  • glaucoma surgery refractive surgery
  • corneal surgery vitreous-retinal surgery
  • ocular muscle surgery After eye surgery, eye tumor surgery, conjunctival surgery involving pterygium, and / or surgery involving lacrimal organs, especially after complicated eye surgery, post-traumatic surgery and / or after uncomplicated eye surgery It can also be applied to diseases such as intraocular inflammation.
  • uveitis especially anterior, intermediate and / or posterior uveitis, sympathetic uveitis and / or panleitis
  • general scleritis especially Anterior segment scleritis, limbic scleritis, posterior scleritis, and scleritis with corneal disorders
  • general episcleritis especially transient episcleritis and Nodular episcleritis
  • Retinitis Corneal surgery
  • Mucopurulent conjunctivitis Atopic conjunctivitis, Toxic conjunctivitis, Pseudoconjunctival conjunctivitis, Serous conjunctivitis, Chronic conjunctivitis, Giant papillary conjunctivitis, Follicular conjunctivitis, Spring conjunctivitis, Eyelid conjunctivitis And / or foveal blepharitis
  • common non-infectious keratitis in particular,
  • retinopathy trauma-induced retinopathy, for example, Pulcher's retinopathy, retinopathy of prematurity (ROP) and / or hyperviscosity-related retinopathy, non-diabetic proliferative retinopathy, and / or proliferative vitreoretinopathy; Follicular inflammation; endophthalmitis; sympathetic ophthalmitis; stye; chalazion; blepharitis; eyelids Dermatitis and other inflammations of the lacrimal gland; lacrimal inflammation, especially acute and chronic lacrimal inflammation; lacrimal inflammation; orbital inflammation, particularly orbital inflammation, orbital periosteitis, orbital Tenon's capsule, orbital Granuloma and orbital myositis; inflammatory and non-inflammatory diseases of the eye selected from purulent endophthalmitis and parasitic endophthalmitis, but are not limited thereto.
  • ROP retinopathy of prematurity
  • Example 1 In vivo test for maintenance of filtration bleb after trabeculectomy
  • a semi-scleral tunnel was created using MVR @ Lance (20 g) from 4 mm posterior to the limbus until the corneal stroma of the anterior chamber was visible.
  • a venous cannula was inserted into the scleral tunnel and inserted into the anterior chamber.
  • the eyeball is enucleated, and the intraocular reflux fluid, MMC and OBP-801 are examined histologically for their maintenance effect on the filtration bleb. If abnormalities such as rapid weight loss are observed, the animals will be euthanized by pentobarbital overdose and used as humane endpoints.
  • the above-mentioned one-month experiment was defined as one course.
  • OBP-801 administration frequency OBP-801 administration frequency ranges from 6 times (immediately before operation, 1, 3, 5, 7, 9 days after operation) to 4 times (immediately before operation, 1, 3, 5 after operation). Day), it was possible to maintain the same low intraocular pressure until 30 days after the operation (FIG. 5).
  • Method 1 Cell culture, addition of OBP-801 (including pulse), observation Conjunctival fibroblasts (HconF) (P1) were purchased from ScienceCell (# 6570).
  • ⁇ Culture culture according to procedure: Medium: Fibroblast Medium (# 2301) was supplemented with a medium additive, an antibiotic, and FBS. The cells were seeded at 5 ⁇ 10 3 cells / cm 2 in a culture vessel coated with Poly-1-lysine. All experiments were performed at passage (P) 3.
  • “Drug treatment” OBP-801 was used as a stock solution in which DMSO (Dimethyl sulfoxide) was dissolved at a concentration of 10 ⁇ M, diluted with a medium, and prepared as a pharmaceutical composition for administration to an administration group. At the time of 80 to 90% confluent, the medium was replaced with a medium without FBS, OBP-801 (0 to 5 nM) was added, and after 0 to 24 hours, TGF ⁇ (20 ng / ml) and TNF ⁇ (10 ng / ml) were added and exposed for 24 hours. ⁇ Observation Live cells were observed with a phase contrast microscope.
  • DMSO Dimethyl sulfoxide
  • RNA extraction RNeasy mini kit (QIAGEN # 74104) Reverse transcription reaction: RT2 First Strand Kit (QIAGEN # 330401) PCR reaction: RT 2 SYBR Green ROX qPCR Mastermix (QIAGEN # 330522) RT 2 Profiler TM PCR Array Rabbit Fibrosis ] (QIAGEN # PANZ-120ZC)
  • OBP-801 Inhibition of HconF myofibrosis induction by OBP-801 It is shown that TGF + TNF stimulation induces expression of ⁇ SMA and Type IV Collagen (col4) in conjunctival fibroblasts (HconF). (FIGS. 12 and 13). It is considered that the HconF culture system can be used as a model of fibrotic tissue formation in the filtration bleb. OBP-801 confirmed the effect of inhibiting the expression of ⁇ SMA and Type IV Collagen LOX12 induced by TGF + TNF stimulation (FIGS. 12 and 13). This suggests that OBP-801 may be effective in maintaining filtration blebs in glaucoma surgery.
  • Comparison with HDAC inhibitor SAHA OBP-801 has a fibrosis inhibitory effect at 1 nM, which is comparable to that of SAHA 1 ⁇ M (FIG. 22). It was shown that OBP-801 has a cell growth inhibitory effect on HconF cells induced by administration of TGF ⁇ and TNF ⁇ to myofibrillation (FIG. 23). OBP-801 has a cell growth inhibitory effect at 0.25 nM similar to that of 0.25 ⁇ M SAHA. A low intraocular pressure maintenance effect can be expected due to excessive suppression of fibrotic scar formation in combination with the Col16 expression suppression effect (FIG. 23). [Example 3] (In vitro test for glaucoma suppression effect)
  • OBP-801 was used as a stock solution in which DMSO (Dimethyl sulfoxide) was dissolved at a concentration of 10 ⁇ M, diluted using a medium, and prepared as a pharmaceutical composition for administration to an administration group.
  • DMSO Dimethyl sulfoxide
  • Example 4 In vivo test on age-related macular degeneration inhibitory effect
  • ⁇ OBP-801 administration Vitreous injection was performed for the control (1 ml PBS + 1 ⁇ l DMSO) and the experimental group (1 ml PBS + 1 ⁇ l 10 ⁇ M OBP-801), and the sclera near the ciliary body was incised using a 22.5 ° slit knife ( The incision width was about 30 G needle diameter). 32G was beveled up into the incision and 1 ⁇ l solution was injected. In consideration of the amount of liquid and leakage at the bevel-up insertion point, the lens was injected slightly above the 0.5 ⁇ l scale at a position where the inserted needle was slightly visible and did not reach the retina.
  • “Drug treatment” OBP-801 was used as a stock solution in which DMSO (Dimethyl sulfoxide) was dissolved at a concentration of 10 ⁇ M, diluted with a medium, and prepared as a pharmaceutical composition for administration to an administration group. At the time of 80 to 90% confluent, the medium was replaced with a medium without FBS, and OBP-801 (0 to 1 nM) was added. Twenty-four hours later, TGF ⁇ (20 ng / ml) and TNF ⁇ (10 ng / ml) were added and exposed for 48 hours. ⁇ Observation Live cells were observed with a phase contrast microscope. ⁇ Immunostaining The medium was removed, and washing was performed three times for 5 minutes with PBS.
  • DMSO Dimethyl sulfoxide
  • Blocking The reaction was performed in a 1% bovine albumin solution at room temperature for about 30 to 60 minutes. A primary antibody reaction was performed, and the reaction was performed at 4 ° C. overnight, followed by washing three times for 5 minutes with PBS. The secondary antibody reaction was performed at room temperature for 60 minutes. Nuclear staining was performed at room temperature for 15 minutes using 5 ⁇ g / ml DAPI. Washing for 5 minutes with PBS was performed three times. The cells were observed with a fluorescence microscope in a state in which PBS was added.
  • PCR Array RNA extraction using the RNeasy mini kit (QIAGEN # 74104) , then performed using a reverse transcription reaction RT2 First Strand Kit (QIAGEN # 330401 ), PCR reactions RT 2 SYBR Green ROX qPCR Mastermix ( QIAGEN # with 330,522), it was performed using RT 2 Profiler TM PCR Array Human Fibrosis (QIAGEN # PAHS-120ZC).
  • Glaucoma filtration surgery rabbit model using cannula administration of mitomycin, administration of OBP-801, observation Mitomycin C (MMC) was dissolved in water for injection 30 minutes before operation and 100 ⁇ l of 0.02% (w / v) was administered. I do. Glaucoma surgery, OBP-801, and intraocular perfusion solution administration were performed as described above. For the purpose of collecting RNA, the bulbar conjunctival epithelium, the lamina intestinal and the Tenon's capsule were collected at 2, 5, 12, and 30 days after the operation.
  • RNA extraction was used for RNA extraction.
  • RT2 First Strand Kit was used for the reverse transcription reaction.
  • RT 2 SYBR Green ROX qPCR Mastermix was used for the PCR reaction.
  • RT 2 Profiler using (R) PCR Array Rabbit Fibrosis was used for the PCR reaction.
  • OBP-801 is used as a stock solution in which DMSO (Dimethyl sulfoxide) is dissolved at a concentration of 10 ⁇ M, and diluted with an eye perfusion solution (BSS: Balanced Salt Solution) to prepare a pharmaceutical composition for administration to an administration group. did.
  • the amount of OBP-801 was 100 nM, 20 ⁇ l ⁇ 4 times (30 seconds each time) as 1 cool, 1 cool 30 minutes before the operation, and 1, 2, 3, 4, 5, 6, 7 days after the operation.
  • OBP-801 inhibits expression in 30 days, and is effective for long-term intraocular pressure control. (Fig. 40, Fig. 45, Fig. 46).
  • Method 1 Western Blotting Conjunctival tissue and filtered vesicle tissue were collected from rabbit eyeballs, and protein extraction was performed using RIPA buffer. The tissue was dissolved in a 5 min (15 sec-10 sec pause) Sonication, 4 ° C. O / N rotation, and the undissolved tissue was removed by centrifugation (10,000 g, 10 min). The BCA kit was used for protein quantification. SDS-page electrophoresis (30 ⁇ g / lane [iBlot 4-12% Bis-Tris Plus Gel]) was performed, followed by transfer to a PVDF membrane [iBlot PVDF transfer stack regular]. After performing blocking and an antibody reaction [iBind Western System], ECL chemiluminescence [NOVEX ECL CHEMI SUBSTRATE] was performed and detected [LAS3000 (Fuji film)].
  • Tissue immunostaining Conjunctival tissue and filtered vesicle tissue were collected from rabbit eyeballs, embedded in a compound (SurgiPath FSC 22), and frozen with liquid nitrogen. Sections (10- ⁇ m thick) made with a cryostat (CM3050S: Leica) were collected on glass slides coated with 2% silane (3-aminopropyltriethoxysilane). After immobilization for 15 minutes using Cold Methanol (-30 ° C.), it was air-dried. Subsequently, blocking was carried out at room temperature for about 30 to 60 minutes using a reaction solution containing 1% bovine albumin. Primary antibody reaction: 4 ° C.

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CN113057142A (zh) * 2021-03-30 2021-07-02 四川大学华西医院 一种视网膜内和/或视网膜下纤维化动物模型的构建方法
CN113057142B (zh) * 2021-03-30 2022-12-09 四川大学华西医院 一种视网膜内和/或视网膜下纤维化动物模型的构建方法
WO2023196555A1 (en) * 2022-04-08 2023-10-12 University Of North Texas Health Science Center At Fort Worth Treatment for ocular fibrosis
US12440538B2 (en) 2022-04-08 2025-10-14 University Of North Texas Health Science Center At Fort Worth Treatment for ocular fibrosis

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