US20160339018A1 - Therapeutic agent for ocular fundus disease - Google Patents

Therapeutic agent for ocular fundus disease Download PDF

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US20160339018A1
US20160339018A1 US14/362,850 US201414362850A US2016339018A1 US 20160339018 A1 US20160339018 A1 US 20160339018A1 US 201414362850 A US201414362850 A US 201414362850A US 2016339018 A1 US2016339018 A1 US 2016339018A1
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compound
ocular fundus
therapeutic agent
instillation
prophylactic
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Tatsuro Ishibashi
Shintaro Nakao
Ryoichi Arita
Ken Mizuno
Akifumi Tsuchiura
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Kyushu University NUC
Kowa Co Ltd
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Kyushu University NUC
Kowa Co Ltd
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Assigned to KOWA COMPANY, LTD., KYUSHU UNIVERSITY, NATIONAL UNIVERSITY CORPORATION reassignment KOWA COMPANY, LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ARITA, RYOICHI, ISHIBASHI, TATSURO, NAKAO, Shintaro, TSUCHIURA, Akifumi, MIZUNO, KEN
Priority to US14/481,467 priority Critical patent/US20140378441A1/en
Publication of US20160339018A1 publication Critical patent/US20160339018A1/en
Priority to US15/387,113 priority patent/US10426783B2/en
Priority to US16/542,461 priority patent/US20190365776A1/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A61K31/551—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole having two nitrogen atoms, e.g. dilazep
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0046—Ear
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers

Definitions

  • the present invention relates to a drug for preventing or treating ocular fundus disease, especially diabetic retinopathy or age-related macular degeneration.
  • Diabetic retinopathy (DR, also referred to as diabetic retinal disease) is one of the three major complications of diabetes, the others being diabetic nephropathy and diabetic neuropathy, and is the second leading cause of blindness in adults after glaucoma in Japan.
  • DR Diabetic retinopathy
  • hyperglycemic microangiopathy oxygen does not reach every corner of the retina so that the retina becomes hypoxia.
  • new blood vessels neovascular vessels
  • neovascular vessels are fragile, and therefore bleeding easily occurs so that a scab-like membrane (proliferative membrane) is formed on the retina, which may be the cause of retinal detachment (proliferative diabetic retinopathy).
  • Diabetic retinopathy is broadly classified into three stages, simple diabetic retinopathy, preproliferative diabetic retinopathy, and proliferative diabetic retinopathy depending on the degree of progression. Even in the stage of simple diabetic retinopathy, maculopathy with macular edema or hard exudate (diabetic macular edema or diabetic maculopathy) may develop, which mainly results from increased vascular permeability. In either case, diagnosis based on ocular fundus findings is important.
  • Diabetic macular edema develops regardless of the disease stage of diabetic retinopathy.
  • Diabetic macular edema is a disease that is caused by accumulation of a plasma component leaked from retinal blood vessels due to increased vascular permeability in the macula and that is associated with a subjective symptom such as anorthopia or reduced visual acuity.
  • Simple diabetic retinopathy is sometimes improved by blood glucose control, but it is believed that in most cases, preproliferative diabetic retinopathy needs to be treated by retinal photocoagulation.
  • proliferative diabetic retinopathy develops so that retinal detachment or vitreous hemorrhage occurs, vitreous surgery is performed for the purpose of removing blood or grown tissue in the eye or reattaching the detached retina.
  • vitreous surgery is performed for the purpose of removing blood or grown tissue in the eye or reattaching the detached retina.
  • Age-related macular degeneration is a disease in which the macula is directly or indirectly damaged by waste products accumulated under the retinal pigment epithelium by aging. In Europe and the United States, age-related macular degeneration is the first leading cause of blindness in adults. Age-related macular degeneration is broadly classified into two types, atrophic (dry) type and exudative (wet) type. Atrophic AMD is a disease in which the retinal pigment epithelium becomes atrophic gradually so that the retina is damaged and therefore visual acuity is gradually reduced. At present, there is no known effective therapy for atrophic AMD.
  • exudative AMD is a disease in which the retina is damaged by abnormal blood vessels (choroidal neovascular vessels) that extend from the choroid into the space under the retinal pigment epithelium or the space between the retina and the retinal pigment epithelium.
  • Choroidal neovascular vessels cause accumulation of fluid (subretinal fluid) under the retina due to leakage of blood components or cause bleeding in the retina due to vascular disruption (retinal bleeding), and therefore the retina is damaged and does not properly function so that visual acuity is reduced.
  • VEGF inhibitor vascular endothelial growth factor
  • Retinal neovascularization or choroidal neovascularization is largely responsible for the pathology of diabetic retinopathy or age-related macular degeneration, respectively, and therefore treatment intended to suppress or inhibit neovascularization has been tried.
  • Treatment of age-related macular degeneration by intravitreal injection of the above-described VEGF inhibitor has already been covered by insurance in Japan, and clinical trials of the VEGF inhibitor for diabetic retinopathy are also in progress.
  • Rho-kinase Rho-associated protein kinase: ROCK
  • fasudil or Y-27632 known as a ROCK inhibitor has the effect of inhibiting VEGF-induced neovascularization
  • fasudil When intravitreally administered to diabetic model rats as model animals with retinal microangiopathy, fasudil has the effect of protecting endothelial cells due to suppression of adhesion of neutrophils to vascular endothelium or facilitation of the synthesis of nitrogen monoxide in endothelial cells, which suggests the potential of intravitreal administration of fasudil as a new treatment strategy for early-stage diabetic retinopathy (Non-Patent Documents 4 and 5). It is also shown that Y-27632 has the effect of suppressing retinal neovascularization when intravitreally administered to model mice with hyperoxia-induced retinopathy (Non-Patent Document 6).
  • Patent Documents 1 to 3 Some patent documents disclose that novel ROCK inhibitors can be used for treatment of retinopathy, diabetic retinopathy, macular degeneration, and the like, but in these patent documents, there is no description about the specific effect of the novel ROCK inhibitors on these diseases (Patent Documents 1 to 3).
  • a drug intended to suppress chorioretinal neovascularization to treat diabetic retinopathy or age-related macular degeneration is mainly administered by intravitreal injection, but multiple injection into the human eye involves the risk of infection and imposes heavy physical, emotional, and financial burdens on patients. Therefore, development of therapy with an ophthalmic preparation has been desired.
  • a 4-fluoro-5-cycloaminosufonyl isoquinoline derivative is effective as a therapeutic agent for asthma, a substance P antagonist, a leukotriene D 4 antagonist, and a Rho-kinase inhibitor (Patent Document 3) or as a therapeutic agent for cerebrovascular disorder (Patent Document 4), but there is no report about its selective action by local administration.
  • the present invention provides a novel drug for preventing or treating ocular fundus disease, especially diabetic retinopathy or age-related macular degeneration.
  • the present invention includes the following aspects.
  • a prophylactic or therapeutic agent for ocular fundus disease comprising, as an active ingredient, (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof.
  • a pharmaceutical composition for preventing or treating ocular fundus disease comprising: (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof; and a pharmaceutically-acceptable carrier.
  • a prophylactic or therapeutic method for ocular fundus disease comprising administering an effective amount of (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof.
  • An ophthalmic preparation comprising: (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof; and a carrier acceptable to ophthalmic preparations.
  • a drug for preventing or treating ocular fundus disease especially diabetic retinopathy or age-related macular degeneration.
  • the pharmaceutical composition, especially the ophthalmic preparation, according to the present invention is very useful as a therapeutic and/or prophylactic agent for ocular fundus disease because it is not only effective even at a low dose when administered by ocular instillation but also can be administered without imposing great physical and emotional burdens on patients, can be non-invasively administered, and can be easily administered also to elderly people and children.
  • FIG. 1 shows the representative images of flat-mounted retinas of a saline instillation group (control), a 0.4% compound 1 solution instillation group, and a 0.8% compound 1 solution instillation group.
  • FIG. 2 is a graph showing the result of quantification of an ischemic retinal area (non-perfused area) in the flat-mounted retina of each of the instillation groups, wherein the vertical axis represents the relative value of the non-perfused area when the calculated proportion of non-perfused area of the saline instillation group is regarded as 100%, values are represented as mean ⁇ standard deviation, and ** indicates that p value is less than 0.01.
  • FIG. 3 is a graph showing the result of quantification of a neovascular area in the flat-mounted retina of each of the instillation groups, wherein the vertical axis represents the relative value of the neovascular area when the calculated proportion of neovascular area of the saline instillation group is regarded as 100%, values are represented as mean ⁇ standard deviation, ** indicates that p value is less than 0.01, and N.C. indicates that there is no significant difference.
  • FIG. 4 shows representative images of flat-mounted retinas of a saline instillation group (control) and a 0.4% fasudil solution instillation group.
  • FIG. 5 is a graph showing the result of quantification of an ischemic retinal area (non-perfused area) in the flat-mounted retina of each of the saline instillation group (control) and the 0.4% fasudil solution instillation group, wherein the vertical axis represents the relative value of the non-perfused area when the calculated proportion of non-perfused area of the saline instillation group is regarded as 100%, values are represented as mean ⁇ standard deviation, and N.C. indicates that there is no significant difference.
  • FIG. 6 is a graph showing the result of quantification of a neovascular area in the flat-mounted retina of each of the saline instillation group (control) and the 0.4% fasudil solution instillation group, wherein the vertical axis represents the relative value of the neovascular area when the calculated proportion of neovascular area of the saline instillation group is regarded as 100%, values are represented as mean ⁇ standard deviation, and N.C. indicates that there is no significant difference.
  • FIG. 7 shows representative fluorescein fundus angiography images of a saline instillation group (control) and a 0.8% compound 1 solution instillation group.
  • FIG. 8 is a graph showing the result of quantification of a neovascular area of each of the instillation groups by fluorescein fundus angiography, wherein the vertical axis represents the relative value of the neovascular area when the calculated proportion of neovascular area of the saline instillation group is regarded as 100%, values are represented as mean ⁇ standard error, and * indicates that p value is less than 0.05.
  • FIG. 9 is a graph showing the result of quantification of choroidal neovascularization volume of each of the instillation groups by fluorescein fundus angiography.
  • the vertical axis of the graph represents the volume of choroidal neovascularization ( ⁇ m 3 ) and values are represented as mean ⁇ standard error.
  • FIG. 10 shows representative fluorescein fundus angiography and optical coherence tomography images of a normal mouse (reference), a saline instillation group (control), and a 0.8% compound 1 solution instillation group.
  • FIG. 11 is a graph showing the maximum retinal thickness (mean ⁇ standard error, unit: ⁇ m) of each of the instillation groups measured by optical coherence tomography, wherein * indicates that p-value is less than 0.05.
  • FIG. 12 shows immunostaining images that demonstrate the effect of the compound 1 on Claudin-5 expression by VEGF stimulation or IL-6 stimulation.
  • FIG. 13 shows immunostaining images that demonstrate the effect of the compound 1 on F-Actin polymerization that is caused by VEGF stimulation or IL-6 stimulation.
  • (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine used in the present invention is a compound that is effective as a therapeutic agent for cerebrovascular disease, has antagonistic actions on substance P and leukotriene D 4 and Rho kinase inhibition activity, and can be produced by a known method such as a method described in WO 99/20620 (Patent Document 4).
  • Examples of a salt of (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine include: salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, and hydrobromic acid; and salts of organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, and camphorsulfonic acid, and hydrochlorides are particularly preferred.
  • inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, and hydrobromic acid
  • organic acids such as acetic acid, tartaric acid, lactic acid, citric acid, fum
  • (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine or a salt thereof can be present not only as an unsolvated form but also as a hydrate or a solvate.
  • a hydrate is preferable as a solvate, but in the present invention, all forms of crystalline and hydrates and solvates of compound 1 are included as (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof.
  • (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof exerts a strong neovascularization suppression effect, and is therefore useful as a prophylactic or therapeutic agent for ocular fundus disease, especially diabetic retinopathy or age-related macular degeneration.
  • the ocular fundus disease mainly refers to a lesion that develops in the retina and/or choroid.
  • ocular fundus disease examples include hypertensive or arteriosclerotic ocular fundus abnormalities, central retinal artery occlusion, retinal vein occlusion such as central retinal vein occlusion or branch retinal vein occlusion, diabetic retinopathy, diabetic macular edema, diabetic maculopathy, Eales disease, congenital retinal vascular abnormality such as Coats disease, von Hippel disease, pulseless disease, macular diseases (e.g.
  • central serous chorioretinopathy cystoid macular edema, age-related macular degeneration, macular hole, myopic macular degeneration, vitreoretinal interface maculopathy, drug-related maculopathy, and heredomacular degeneration
  • retinal detachment e.g. rhegmatogenous, tractional, and exudative retinal detachment
  • retinitis pigmetosa retinopathy of prematurity.
  • a dosage form comprising (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof can be prepared according to a known method.
  • a formulation comprising (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof can be prepared with reference to dosage form examples described in, for example, WO 00/09162 or WO 97/23222.
  • a preparation comprising (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof can also be prepared according to a known method.
  • an ophthalmic preparation can be prepared by using, if necessary, a tonicity agent, a buffering agent, a surfactant, a preservative, and the like.
  • the pH of the ophthalmic preparation shall be within a range acceptable to ophthalmologic preparations, and is preferably in the range of 4 to 8.
  • the preparation according to the present invention is preferably used as an ophthalmologic preparation, especially as a preparation for ocular instillation.
  • an ophthalmic preparation may be any one of an aqueous ophthalmic preparation, a non-aqueous ophthalmic preparation, an ophthalmic suspension, an ophthalmic emulsion, and an ophthalmic ointment.
  • Such a dosage form suitable for administration can be produced by a (preparation) method known to those skilled in the art.
  • pharmaceutically-acceptable carriers especially carriers acceptable to ophthalmic preparations, such as a tonicity agent, a chelating agent, a stabilizer, a pH adjuster, a preservative, an antioxidant, a solubilizing agent, a thickening agent, and the like can be added to the formulation.
  • An ophthalmic preparation according to the present invention comprises (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof that is the active ingredient used in the present invention and a carrier acceptable to ophthalmic preparations.
  • the ophthalmic preparation can be prepared by, for example, dissolving or suspending desired ingredients such as the above-described ingredients in an aqueous solvent such as sterile purified water or saline or a non-aqueous solvent such as vegetable oil (e.g., cottonseed oil, soybean oil, sesame oil, or peanut oil) so that the osmotic pressure of the solution or suspension is adjusted to a predetermined value and then subjecting the solution or suspension to sterilization such as filtration sterilization.
  • an ointment base can be added in addition to the above-described various ingredients.
  • Preferred examples of the ointment base include, but are not limited to: oily bases such as petrolatum, liquid paraffin, and polyethylene; emulsion bases obtained by emulsifying an oil phase and an aqueous phase with a surfactant; and water-soluble bases such as hydroxypropyl methylcellulose, carboxymethylcellulose, and polyethylene glycol.
  • oily bases such as petrolatum, liquid paraffin, and polyethylene
  • emulsion bases obtained by emulsifying an oil phase and an aqueous phase with a surfactant
  • water-soluble bases such as hydroxypropyl methylcellulose, carboxymethylcellulose, and polyethylene glycol.
  • the adult dose of (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof is 0.025 to 10000 ⁇ g, preferably 0.025 to 2000 ⁇ g, more preferably 0.1 to 2000 ⁇ g per day. Further, the other preferable adult dose is 0.025 to 200 ⁇ g, or 0.025 to 100 ⁇ g per day.
  • the frequency of administration is not particularly limited, but the above daily dose is preferably administered once or in divided doses.
  • one to several drops of the preparation shall be instilled into the eye at a time.
  • a fasudil solution having a desired concentration was prepared in the same manner as in the preparation of the above-described compound 1 solution except that a predetermined amount of fasudil dihydrochloride (LC Laboratories) was used instead of (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine monohydrochloride dihydrate.
  • Fasudil solution 0.4% solution (instillation volume: 20 ⁇ L)
  • Test animals C57BL/6JJcl mice (sex: male, 8 to 14 mice per group)
  • Test groups were a saline instillation group (control), a 0.4% compound 1 solution instillation group, a 0.8% compound 1 solution instillation group, and a 0.4% fasudil solution instillation group, and saline, the compound 1 solution, or the fasudil solution was administered by ocular instillation three times a day.
  • ischemic retinal areas (non-perfused areas) and neovascular areas were quantified for evaluation by flat-mounting or fluorescein fundus angiography. More specifically, 1 g/kg of pentobarbital (Nembutal) was administered intraperitoneally for euthanasia under deep anesthesia, and then both eyeballs were excised and fixed with 4% paraformaldehyde (PFA) at 37° C. for 1 hour, and the corneal limbus was circumferentially incised to remove the cornea and iris from the eyeball. Then, after fixation with 4% PFA at 37° C.
  • pentobarbital Naembutal
  • PFA paraformaldehyde
  • the lens, sclera, and choroid were removed, and the eye cup including the retina was isolated. Further, the retina was fixed with 4% PFA at 37° C. for 3 hours, washed with PBS three times (15 min per wash, 37° C.), dehydrated (methanol 50% ⁇ 100%, 10 min per methanol treatment, 37° C.), washed with PBS three times (15 min per wash, 37° C.), and blocked with Blocking Buffer (1% BSA, 0.5% Triton-X in PBS) for 60 minutes (37° C.).
  • Blocking Buffer 1% BSA, 0.5% Triton-X in PBS
  • the retina was treated with a primary antibody (0.7% FITC-conjugates Anti-lectin Ab in PBS, 4° C., overnight) and washed with PBS three times (15 min per wash, 37° C.). Then, 4 to 6 cuts were radially made in the eye cup, and the flat-mounted retina was covered with Crystal Mount. Then, the image of the flat-mounted retina was taken with a fluorescence microscope (BZ-9000, KEYENCE Corp, Osaka, Japan), and the proportion of the non-perfused area to the entire retina and the proportion of the neovascular area to the entire retina were calculated by the following formulas in NIH image J software. The thus obtained values were converted to relative values by regarding the obtained values of the saline instillation group (control) as 100%, and statistical analysis was performed by Wilcoxon test.
  • a primary antibody (0.7% FITC-conjugates Anti-lectin Ab in PBS, 4° C., overnight
  • PBS three times (15 min per wash, 37°
  • Non-perfused area area of avascular region/area of entire retina
  • Neovascular area area of neovascular region/area of entire retina
  • each of the animals was first given tropicamide by ocular instillation to cause mydriasis, anesthetized by intraperitoneal administration of 100 mg/kg of Ketalar and 10 mg/kg of Selactar, given 12 ⁇ L/g of a contrast agent for fluorescein fundus angiography by intraperitoneal injection, and subjected to imaging using Optos200TX (OPTOS PLC) to take a fluorescein fundus angiography image to calculate the proportion of a neovascular area to the entire retina by the above formula in NIH image J software.
  • OPS PLC Optos200TX
  • FIGS. 1 to 3 The results of ocular instillation of the compound 1 obtained by flat mounting are shown in FIGS. 1 to 3 , and the results of ocular instillation of fasudil obtained by flat mounting are shown in FIGS. 4 to 6 .
  • FIG. 1 shows the representative images of flat-mounted retinas of the saline instillation group (control), the 0.4% compound 1 solution instillation group, and the 0.8% compound 1 solution instillation group. As can be seen from FIG.
  • FIGS. 2 and 3 show the results of quantification of the ischemic retinal area (non-perfused area) and the neovascular area. As can be seen from FIG.
  • FIG. 4 shows the representative images of flat-mounted retinas of the saline instillation group (control) and the 0.4% fasudil solution instillation group.
  • the ischemic retinal area (non-perfused area) and the neovascular area are conspicuously observed in both the groups.
  • FIGS. 5 and 6 show the results of quantification of the ischemic retinal area (non-perfused area) and the neovascular area. As can be seen from FIG.
  • Non-Patent Documents 5 discloses that fasudil is effective when injected into the vitreous body, but the effect of ocular instillation of fasudil could not be confirmed in the present invention.
  • FIGS. 7 and 8 show the results of fluorescein fundus angiography.
  • FIG. 7 shows the representative fluorescein fundus angiography images of the saline instillation group (control) and the 0.8% compound 1 solution instillation group.
  • FIG. 7 shows the neovascular region is conspicuously observed in the saline instillation group, whereas suppression of the neovascular area is observed in the 0.8% compound 1 solution instillation group.
  • FIG. 8 shows the results of quantification of the neovascular area. As can be seen from FIG.
  • FIGS. 1 to 3 , FIG. 7 , and FIG. 8 indicate that the development of the ischemic area and the neovascular area in the OIR model is conspicuously suppressed by ocular instillation of the compound 1. Further, the results shown in FIGS. 4 to 6 indicate that the effect obtained by the compound 1 cannot be obtained by fasudil.
  • mice CNV model known as a model of age-related macular degeneration or the like was examined in the following manner.
  • Test animals C57BL/6JJcl mice (6- to 10-week old, sex: male, 11 to 12 mice per group)
  • mice The preparation of a CNV model and evaluation were performed with reference to a literature (e.g., J. Leukoc. Biol. 2003; 74: 25-32, or Am. J. Pathol. 1998; 153: 1641-1646). More specifically, each of the mice was given tropicamide by ocular instillation to cause mydriasis, anesthetized by intraperitoneal administration of 100 mg/kg of Ketalar and 10 mg/kg of Selactar, and subjected to photocoagulation at 4 spots per eye. The photocoagulation was performed by krypton laser irradiation (75- ⁇ m spot size, 0.1 seconds duration, 200 mW) with a slit-lamp delivery system using a cover glass as a contact lens.
  • a literature e.g., J. Leukoc. Biol. 2003; 74: 25-32, or Am. J. Pathol. 1998; 153: 1641-1646. More specifically, each of the mice was given tropicamide by o
  • Test groups were a saline instillation group (control), a 0.4% compound 1 solution instillation group, and a 0.8% compound 1 solution instillation group.
  • Saline or the compound 1 solution was administered three times a day by ocular instillation. The day when photocoagulation treatment was performed was defined as day 0.
  • Saline or the compound 1 solution was administered by ocular instillation from day 0 to day 7, a flat-mount was prepared on day 7, and blood vessels were stained with FITC-lectin for evaluation. More specifically, a spot with bleeding or tissue destruction was excluded, the CNV volume of each spot adopted was calculated by NIS-Elements AR Version 4.13, and then an average value ( ⁇ m 3 ) per eye was calculated.
  • FIG. 9 The results of the test are shown in FIG. 9 .
  • the CNV volume of the control group (Control) was 109177 ⁇ 26399 ⁇ m 3
  • the CNV volume of the 0.4% compound 1 solution instillation group was 56408 ⁇ 9007 ⁇ m 3
  • the CNV volume of the 0.8% compound 1 solution instillation group was 88387 ⁇ 33678 ⁇ m 3 , that is, choroidal neovascularization was suppressed.
  • mice transgenic for VEGF gene which develop retinal neovascularization
  • Test animals Kimba mice (available from Lions Eye Institute Ltd., 6 mice per group)
  • each of the mice was first given tropicamide by ocular instillation to cause mydriasis and then anesthetized by intraperitoneal administration of 100 mg/kg of Ketalar and 10 mg/kg of Selactar, given 6 ⁇ L/g of a contrast agent for fluorescein fundus angiography by intraperitoneal injection, and subjected to fluorescein fundus angiography using Heidelberg Retina Angiograph (HRA, Heidelberg, Germany).
  • mice were given tropicamide by ocular instillation to cause mydriasis, anesthetized by intraperitoneal administration of 100 mg/kg of Ketalar and 10 mg/kg of Selactar, and subjected to imaging with The Cirrus HD-OCT (Carl Zeiss Meditec, Dublin, Calif.) by 5-line scan (scan length: 6 mm) in X and Y axis directions to measure an average maximum retinal thickness (statistical analysis was performed by Student's t test).
  • Cirrus HD-OCT Carl Zeiss Meditec, Dublin, Calif.
  • FIGS. 10 and 11 The results of the test are shown in FIGS. 10 and 11 .
  • the upper images are representative fluorescein fundus angiography images of the test groups and the lower images are representative optical coherence tomography images of the test groups.
  • fluorescein fundus angiography and optical coherence tomography images of a normal mouse are also shown as a reference example (left side, maximum retinal thickness of this example: 276 ⁇ m).
  • FIG. 10 in the case of saline instillation, edema is observed and retinal thickening is also observed, whereas in the case of 0.8% compound 1 solution instillation, suppression of edema is observed and the retina is comparable in thickness to that of the normal mouse.
  • FIG. 11 is a graph showing the values of retinal thickness. As can be seen from FIG. 11 , the average maximum retinal thickness of the saline instillation group (control) was 361.8 ⁇ 36.1 ⁇ m, whereas the average maximum retinal thickness of the 0.8% compound 1 solution instillation group was 256.7 ⁇ 35.7 ⁇ m, that is, retinal thickening was significantly suppressed.
  • b-END3 (bEND.3: ATCC CRL-2299TM) cells, which are mouse brain microvascular endothelial cells, were subcultured in 3.5 cm dishes (9 ⁇ 10 5 cells/dish), and a test was started from day 6 after subculture.
  • the cells were subjected to drug treatment under the following five or six conditions: no treatment (shown in FIGS. 12 and 13 as “control”); VEGF stimulation (25 ng/mL, 24 hours) (shown in FIGS. 12 and 13 as “VEGF (25 ng/mL 24 hours) stimulation”); pretreatment with the compound 1 (3 ⁇ M or 30 ⁇ M, 3 hours) followed by VEGF stimulation (25 ng/mL, 24 hours) (shown in FIGS.
  • VEGF+Compound 1 3 ⁇ M 3 hours pretreatment” or “VEGF+Compound 1 (30 ⁇ M 3 hours) pretreatment”
  • IL-6 stimulation 10 ng/mL, 24 hours
  • IL-6 stimulation 10 ng/mL 24 hours
  • IL-6+Compound 1 30 ⁇ M 3 hours
  • the cells after the test were subjected to immunostaining according to the following procedure to evaluate the expression of Claudin-5 or F-Actin. More specifically, the cells after the test were treated with 100% methanol at ordinary temperature for 5 minutes, further treated with 50% methanol for 5 minutes, and washed with PBS twice (5 min per wash). Then, a cover glass-sized cell sample was prepared by trimming with a cotton swab, enclosed with a Dako pen, blocked with 10% normal goat serum (10% normal goat serum ready-to-use (Invitrogen)) (30 min, ordinary temperature), and allowed to stand at 4° C. overnight.
  • normal goat serum (10% normal goat serum ready-to-use (Invitrogen)
  • the cell sample was subjected to primary antibody treatment by dropping 50 to 70 ⁇ L of a 25-fold dilution of rabbit anti-claudin-5 antibody (Rabbit anti-Claudin-5 (Invitrogen 34-1600)) or rabbit anti-F-actin antibody (Rabbit anti-F-actin (Biossusa bs-1571R)) and then washing with PBS three times (10 min per wash).
  • rabbit anti-claudin-5 antibody Rabbit anti-Claudin-5 (Invitrogen 34-1600)
  • rabbit anti-F-actin antibody Rassusa bs-1571R
  • the cell sample was allowed to stand at ordinary temperature for 60 minutes under dark conditions, and was then subjected to secondary antibody treatment with a 200-fold dilution of anti-rabbit IgG FITC labeled with Alexa Fluo488TM (Alexa Fluo488 anti-Rabbit IgG FITC) and washed with PBS three times (10 min per wash). Then, the cell sample was subjected to nuclear staining with DAPI and covered with Crystal Mount as a cover glass, and then its image was taken with a microscope (400 ⁇ magnification). The results for Claudin-5 are shown in FIG. 12 and the results for F-actin are shown in FIG. 13 .
  • FIG. 12 The results of immunostaining for Claudin-5 are shown in FIG. 12 and the results of immunostaining for F-Actin are shown in FIG. 13 .
  • FIG. 12 improvement in Claudin-5 expression, which is decreased by VEGF stimulation or IL-6 stimulation, by pretreatment with the compound 1 is observed.
  • FIG. 13 suppression of F-Actin polymerization, which is caused by VEGF stimulation or IL-6 stimulation, by pretreatment with the compound 1 is observed.
  • (S)-( ⁇ )-1-(4-fluoro-5-isoquinolinesulfonyl)-2-methyl-1,4-homopiperazine, a salt thereof, or a solvate thereof used in the present invention has an excellent neovascularization suppression effect, and is therefore useful as a drug for preventing or treating ocular fundus disease, especially diabetic retinopathy or age-related macular degeneration.

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10376523B2 (en) 2014-09-25 2019-08-13 Kowa Company, Ltd. Aqueous composition containing ripasudil, or a salt, or a solvate thereof

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2015229671A (ja) * 2014-06-06 2015-12-21 株式会社デ・ウエスタン・セラピテクス研究所 網膜色素上皮細胞保護剤
CN106604730B (zh) 2014-09-24 2019-12-20 兴和株式会社 角膜厚度调节剂
JP6244038B2 (ja) * 2014-09-25 2017-12-06 興和株式会社 医薬製剤
JP2016135780A (ja) * 2015-01-16 2016-07-28 興和株式会社 水性組成物
KR20160108121A (ko) 2015-03-06 2016-09-19 코와 가부시키가이샤 수성 조성물
US20180296474A1 (en) * 2015-10-13 2018-10-18 INSERM (Institut National de la Santé et de la Recherche Médicale Methods and pharmaceutical compositions for the treatment of retinal capillary non-perfusion
EP4088719A1 (fr) * 2015-10-13 2022-11-16 Institut National de la Santé et de la Recherche Médicale (INSERM) Procédés et compositions pharmaceutiques pour le traitement de non-perfusion capillaire rétinienne
JP6928479B2 (ja) 2017-05-12 2021-09-01 学校法人順天堂 拒絶反応抑制剤

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0349482A (ja) 1989-07-18 1991-03-04 Matsushita Electric Ind Co Ltd ファインダー表示装置
KR100452715B1 (ko) * 1995-12-21 2004-12-17 알콘 래보레이토리스, 인코퍼레이티드 녹내장및안구국소빈혈을치료하기위한특정이소퀴놀린술포닐화합물의용도
EP0956865B2 (fr) 1996-08-12 2010-08-18 Mitsubishi Tanabe Pharma Corporation MEDICAMENTS COMPRENANT UN INHIBITEUR DE LA Rho KINASE
AU9646198A (en) 1997-10-22 1999-05-10 Nippon Shinyaku Co. Ltd. Isoquinoline derivative and drug
JP4212149B2 (ja) * 1998-06-11 2009-01-21 株式会社デ・ウエスタン・セラピテクス研究所 医薬
EP1459742B9 (fr) 1998-08-17 2012-05-02 Senju Pharmaceutical Co., Ltd. Agent pour prévenir et traiter l'asthénopie et la pseudomyopie
US7109208B2 (en) 2001-04-11 2006-09-19 Senju Pharmaceutical Co., Ltd. Visual function disorder improving agents
US20050014783A1 (en) * 2003-05-29 2005-01-20 Schering Aktiengesellschaft Use of Rho-kinase inhibitors in the treatment of aneurysm and cardiac hypertrophy
GB0425026D0 (en) 2004-11-12 2004-12-15 Biofocus Discovery Ltd Compounds which bind to the active site of protein kinase enzymes
CA2585490A1 (fr) * 2004-11-12 2006-05-18 Galapagos Nv Composes heteroaromatiques de l'azote qui se lient au site actif d'enzymes de type proteines kinases
MY139797A (en) * 2004-11-29 2009-10-30 Kowa Co (s)-(-)-1-(4-fluoroisoquinolin-5-yl)sulfonyl-2-methyl-1, 4-homopiperazine hydrochloride dihydrate
TWI367098B (en) * 2004-12-23 2012-07-01 Kowa Co Treating agent for glaucoma
US20060142270A1 (en) * 2004-12-23 2006-06-29 Kowa Co., Ltd. Preventing or treating agent for glaucoma
WO2006090783A1 (fr) * 2005-02-25 2006-08-31 Kowa Co., Ltd. Procédé de fabrication d’halogénure de 4-fluoroisoquinoléine-5-sulfonyle ou d’un sel de celui-ci
JP2006290827A (ja) * 2005-04-13 2006-10-26 Kowa Co 緑内障予防又は治療剤
WO2006115244A1 (fr) * 2005-04-25 2006-11-02 D. Western Therapeutics Institute, Inc. Dérivé de 4-bromoisoquinoléine et préparation pharmaceutique comprenant celui-ci
JP4800720B2 (ja) * 2005-09-21 2011-10-26 興和株式会社 点眼用組成物
JP4168071B2 (ja) * 2005-12-08 2008-10-22 興和株式会社 点眼用組成物
TWI527798B (zh) * 2009-06-19 2016-04-01 Western Therapeutics Inst Inc D Substituted isoquinoline derivatives
AU2010332010A1 (en) * 2009-12-14 2012-08-02 Inspire Pharmaceuticals, Inc. Bridged bicyclic Rho kinase inhibitor compounds, composition and use
JP2012006918A (ja) * 2010-05-27 2012-01-12 Santen Pharmaceut Co Ltd イソキノリンスルホニル誘導体を有効成分として含有する網脈絡膜変性疾患の予防または治療剤
CA2805242A1 (fr) * 2010-07-19 2012-01-26 Inspire Pharmaceuticals, Inc. Composes inhibiteurs de rho-kinases bifonctionnels, composition et utilisation
AU2011282887A1 (en) * 2010-07-27 2013-02-07 Inspire Pharmaceuticals, Inc. Method for treating ophthalmic diseases using kinase inhibitor compounds in prodrug forms
JP2013129604A (ja) * 2011-12-20 2013-07-04 Kowa Co 緑内障治療薬を含有する徐放性フィルム製剤
CN106604730B (zh) * 2014-09-24 2019-12-20 兴和株式会社 角膜厚度调节剂

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10376523B2 (en) 2014-09-25 2019-08-13 Kowa Company, Ltd. Aqueous composition containing ripasudil, or a salt, or a solvate thereof

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US20190365776A1 (en) 2019-12-05
EP2990040A4 (fr) 2017-07-05

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