Eye drops for improving or preventing retinal circulatory disorders and retinal neurovascular disorders

Abstract

Eyedrops for ameliorating retinal circulatory disturbance and disorders associated with retinal nerve blood vessels, which contains fibrate-containing nanoparticles.

Description

[Technical Field]

[0001] The present invention relates to an eye drop for improving or preventing retinal circulatory disorders and retinal neurovascular disorders. This application claims priority based on Japanese Patent Application No. 2020-192736, filed on November 19, 2020, the contents of which are incorporated herein by reference. [Background technology]

[0002] Fibrates are known to have an inhibitory effect on retinal neovascularization when administered orally. For example, Patent Document 1 reports that forced oral administration of fenofibrate to mice inhibited retinal neovascularization. However, side effects (such as rhabdomyolysis) have been reported when fibrates are used in combination with statins, and fibrates are not actually used in clinical practice for the treatment of eye diseases.

[0003] As a method of administering drugs for ocular diseases, topical administration is preferable to oral administration because it has fewer systemic side effects. For example, anti-VEGF agents, which are widely used to treat retinal diseases such as age-related macular degeneration and diabetic macular edema, are mainly administered intravitreously. However, vitreous hemorrhage and lens damage have been reported, and serious ocular complications such as endophthalmitis have also been reported. From the viewpoint of safety, administration by eye drops is preferable. However, it is difficult to deliver an effective concentration of drugs to the retinal tissue located at the back of the eyeball using commonly used eye drops.

[0004] On the other hand, disorders of retinal circulation and retinal neural connections are thought to occur before obvious retinopathy symptoms are detected and are considered to be one of the causes of retinal disease. However, no eye drops effective for improving these disorders have been developed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2016-539098 Summary of the Invention [Problem to be solved by the invention]

[0006] If retinal circulatory disorders or retinal nerve-related disorders can be improved or prevented by eye drops, retinal diseases caused by these disorders can be prevented or treated safely and effectively.

[0007] Therefore, an object of the present invention is to provide an eye drop that can improve or prevent retinal circulatory disorders and retinal neurovascular coupling disorders. [Means for solving the problem]

[0008] The present invention includes the following aspects. [1] An eye drop containing nanoparticles containing fibrate for improving or preventing retinal circulatory disorders and retinal neurovascular disorders. [2] The eye drops according to [1], wherein the nanoparticles are a wet-pulverized product of fibrate. [3] The eye drop preparation according to [1] or [2], further comprising a thickener. [4] The eye drop preparation according to [3], wherein the thickener is a cellulose-based thickener. [5] The eye drop preparation according to [4], wherein the cellulose-based thickener is methylcellulose. [6] The eye drop preparation according to any one of [1] to [5], further comprising a cyclodextrin. [7] The eye drops according to [6], wherein the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin. [Effects of the Invention]

[0009] According to the present invention, there is provided an eye drop capable of improving or preventing retinal circulatory disorders and retinal neurovascular coupling disorders. [Brief explanation of the drawings]

[0010] [Figure 1]1 shows the results of particle size distribution analysis by laser diffraction / scattering method for fibrate nanoparticles prepared in the examples. [Figure 2] 1 shows atomic force microscope images of fibrate nanoparticles prepared in the examples. [Figure 3A] This shows the change in body weight over time in diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: Administered eye drops containing fibrate nanoparticles (n = 6). Control group: Administered eye drops containing vehicle nanoparticles (n = 6). NS: No significant difference. [Figure 3B] This shows the time course of casual blood glucose levels in diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: Administered eye drops containing fibrate nanoparticles (n = 6). Control group: Administered vehicle nanoparticle eye drops (n = 6). NS: No significant difference. [Figure 3C] This shows the change over time in intraocular pressure (left eye) of diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: Administered eye drops containing fibrate nanoparticles (n = 6). Control group: Administered eye drops containing vehicle nanoparticles (n = 6). NS: No significant difference. [Figure 4A] This shows the time course of mean blood pressure in diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: Administered eye drops containing fibrate nanoparticles (n = 6). Control group: Administered vehicle nanoparticle eye drops (n = 6). NS: No significant difference. [Figure 4B] This shows the time course of ocular perfusion pressure in diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: administration of eye drops containing fibrate nanoparticles (n = 6). Control group: administration of vehicle nanoparticle eye drops (n = 6). NS: no significant difference. [Figure 5] This shows the time course of baseline optic nerve head blood flow in diabetic model mice subjected to an eye drop administration test. Mean ± SEM. Treatment group: administration of eye drops containing fibrate nanoparticles (n = 6). Control group: administration of vehicle nanoparticle eye drops (n = 6). NS: no significant difference. [Figure 6]This figure shows the changes in ocular blood flow after flicker stimulation in diabetic model mice at 8, 10, 14, and 12 weeks of age, which were subjected to an eye drop administration test. Treatment group: fibrate-containing eye drops (n=6). Control group: vehicle eye drops (n=6). [Figure 7] This shows the change in total blood flow after flicker stimulation at each age in diabetic model mice subjected to an eye drop administration test. Treatment group: administration of eye drops containing fibrate nanoparticles (n=6). Control group: administration of vehicle nanoparticle eye drops (n=6). [Figure 8] These figures show changes in ocular blood flow after hyperoxia stimulation in diabetic model mice at 8, 10, 14, and 12 weeks of age, which were subjected to an eye drop administration test. Treatment group: fibrate-containing eye drops (n=6). Control group: vehicle eye drops (n=6). [Figure 9] This shows the change in total blood flow after hyperoxia stimulation at each age in diabetic model mice subjected to an eye drop administration test. Treatment group: administration of eye drops containing fibrate nanoparticles (n=6). Control group: administration of vehicle nanoparticle eye drops (n=6). [Figure 10] This shows the results of evaluating the a-wave implicit time and b-wave implicit time in the electroretinogram of diabetic model mice subjected to an eye drop administration test. Treatment group: Fibrate nanoparticle-containing eye drops administered (n=6). Control group: Vehicle nanoparticle eye drops administered (n=6). DETAILED DESCRIPTION OF THE INVENTION

[0011] [Eye drops] In one embodiment, the present invention provides an eye drop for improving retinal circulatory disorders and retinal neurovascular coupling disorders, which contains nanoparticles containing a fibrate.

[0012] "Retinal circulation" refers to blood flow in the retina. "Retinal circulation disorder" refers to impaired blood flow in the retina. "Retinal neurovascular coupling" refers to changes in blood flow in the retina in response to neural activity. "Retinal neurovascular coupling disorder" refers to impaired response of blood flow changes in the retina in response to neural activity. When retinal blood vessels are blocked or narrowed by a thrombus or embolus, retinal circulation is impaired, preventing the supply of oxygen and nutrients to the optic nerve. In addition, the retinal tissue falls into a state of relative hypoxia due to the prevention of an increase in retinal blood flow that corresponds to the increased oxygen demand associated with neural activity, resulting in retinal neurovascular dysfunction.

[0013] The onset of retinal circulatory disorders and retinal neurovascular coupling disorders can be quantitatively and noninvasively confirmed by measuring changes in retinal blood flow due to flicker stimulation. In a normal retina without any damage, retinal blood flow usually increases with flicker stimulation. However, if retinal circulatory disorders and retinal neurovascular coupling disorders occur, the increase in retinal blood flow due to flicker stimulation is inhibited. Therefore, if retinal blood flow does not increase with flicker stimulation, it can be evaluated that retinal circulatory disorders and retinal neurovascular coupling disorders have occurred. Furthermore, if an increase in retinal blood flow due to flicker stimulation can be confirmed after a state in which retinal blood flow did not increase with flicker stimulation, it can be evaluated that retinal circulatory disorders and retinal neurovascular coupling disorders have improved.

[0014] Retinal circulatory disorders and retinal neurovascular correlation disorders contribute to various vitreous retinal diseases. Examples of such vitreous retinal diseases include, but are not limited to, diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, age-related macular degeneration, and central serous chorioretinopathy. The eye drops of the present embodiment can improve retinal circulatory disorders and retinal neurovascular correlation disorders, and can therefore be used to treat or prevent such vitreous retinal diseases.

[0015] <Fibrate-containing nanoparticles> The eye drop of the present embodiment contains nanoparticles containing fibrate (hereinafter also referred to as "fibrate nanoparticles"). The fibrate nanoparticles are solid nanoparticles, preferably fibrate nanoparticles.

[0016] "Fibrate" is a general term for compounds that act as lipid-lowering drugs among amphipathic carboxylic acid derivatives. Examples of fibrates include, but are not limited to, gemfibrozil, fenofibrate, bezafibrate, clofibrate, ciprofibrate, beclofibrate, binifibrate, ciprofibrate, clinofibrate, etofibrate, nicofibrate, pirifibrate, lonifibrate, simfibrate, theofibrate, pemafibrate, fibric acid derivatives, and pharmaceutically acceptable salts or esters of the fibric acid derivatives. Among these, fenofibrate is preferred as the fibrate. One fibrate may be used alone, or two or more may be used in combination.

[0017] The content of fibrate in the eye drops of this embodiment is not particularly limited, but may be, for example, 0.1 to 10% (w / v) based on the total amount of the eye drops. The content of fibrate is preferably 0.5 to 5% (w / v), more preferably 1 to 4% (w / v), and even more preferably 1.5 to 3% (w / v), based on the total amount of the eye drops.

[0018] "Nanoparticles" are particles having a particle diameter of 1 nm or more and less than 1000 nm. From the viewpoint of cellular uptake efficiency, the fibrate nanoparticles contained in the eye drop solution of this embodiment preferably have a particle diameter in the range of 10 to 300 nm, more preferably in the range of 30 to 150 nm. Examples of fibrate nanoparticles include those having a particle size distribution with an average diameter of 50 to 120 nm as measured by laser diffraction / scattering method or dynamic light scattering method.

[0019] Fibrate nanoparticles can be obtained by wet-milling fibrate. Wet-milling of fibrate can be carried out using a bead mill, a ball mill, or the like. Dry-milling may be carried out before wet-milling. By carrying out dry-milling before wet-milling, fibrate can be efficiently nano-sized. It can be said that fibrate nanoparticles are a wet-milled product of fibrate, a wet bead milled product, or a wet ball milled product. The wet-milled product of fibrate may be a wet-milled product of a dry-milled product of fibrate, a wet bead milled product of a dry-milled product of fibrate, or a wet ball milled product of a dry-milled product of fibrate.

[0020] <Water> The eye drops of the present embodiment contain water as a dispersion medium. The water may be of a grade suitable for use in eye drops, such as purified water, sterilized purified water, water for injection, or distilled water for injection.

[0021] <Optional ingredients> The eye drop of the present embodiment may contain optional ingredients in addition to fibrate nanoparticles, such as thickeners, preservatives, tonicity agents, cyclodextrins, buffers, and pH adjusters.

[0022] (thickener) The eye drops of the present embodiment preferably contain a thickener, which prevents the fibrate nanoparticles from becoming meringue-like or creamy during preparation, thereby enabling the preparation of a good fibrate nanoparticle dispersion. Examples of thickeners include, but are not limited to, cellulose-based thickeners such as methyl cellulose, hydroxyethyl cellulose, and hydroxypropyl methyl cellulose; vinyl-based thickeners such as carboxyvinyl polymer, polyvinyl alcohol (fully or partially saponified), and polyvinylpyrrolidone; sodium alginate, sodium chondroitin sulfate, and macrogol. Among these, the thickener is preferably a cellulose-based thickener, and more preferably methylcellulose, because it improves the retention of fibrate nanoparticles and enhances their intraocular penetration.

[0023] The thickener may be used alone or in combination of two or more kinds. The content of the thickener in the eye drops of the present embodiment is not particularly limited, but is, for example, preferably 0.01 to 3% (w / v) of the total amount of the eye drops, more preferably 0.05 to 2% (w / v), and even more preferably 0.1 to 1% (w / v).

[0024] (preservative) The eye drops of the present embodiment may contain a preservative. By including a preservative, the shelf life of the eye drops is improved. Examples of preservatives include, but are not limited to, cationic soaps such as benzalkonium chloride, benzethonium chloride, and chlorhexidine gluconate; parabens such as methylparaben, ethylparaben, propylparaben, and butylparaben; and alcohols such as chlorobutanol, phenylethyl alcohol, and benzyl alcohol. Among these, the cationic surfactants such as invert soaps are preferred as preservatives because they are widely used in eye drops and have a high preservative effect, and benzalkonium chloride is more preferred.

[0025] The preservatives may be used alone or in combination of two or more. The content of the preservative in the eye drops of the present embodiment is not particularly limited, but is, for example, preferably 0.0005 to 0.5% (w / v) of the total amount of the eye drops, more preferably 0.001 to 0.1% (w / v), and even more preferably 0.001 to 0.01% (w / v).

[0026] (Tonicity agent) The eye drops of the present embodiment may contain an isotonicity agent, which can reduce irritation to the eyes. Examples of isotonic agents include, but are not limited to, sugars such as glucose; polyhydric alcohols such as propylene glycol, glycerin, mannitol, sorbitol, and xylitol; and inorganic salts such as sodium chloride and potassium chloride. Among these, polyhydric alcohols are preferred as the isotonic agent, and mannitol is more preferred, since they can protect the cornea from irritation caused by protective agents and the like.

[0027] The isotonicity agent may be used alone or in combination of two or more kinds. The content of the isotonic agent in the eye drops of the present embodiment is not particularly limited, but is, for example, preferably 0.01 to 3% (w / v), more preferably 0.05 to 2% (w / v), and even more preferably 0.1 to 1% (w / v), of the total amount of the eye drops.

[0028] (cyclodextrins) The eye drops of the present embodiment preferably contain cyclodextrins, which can suppress aggregation of fibrate nanoparticles and disperse the fibrate nanoparticles uniformly. "Cyclodextrins" refers to cyclodextrin or cyclodextrin derivatives. Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and derivatives thereof. Examples of cyclodextrin derivatives include alkyl derivatives, hydroxyalkyl derivatives, sulfoalkyl ether derivatives, and sugar-bound derivatives. Among these, as cyclodextrins, hydroxyalkyl derivatives are preferred, and 2-hydroxypropyl-β-cyclodextrin is more preferred, because they have excellent dispersibility of fibrate nanoparticles.

[0029] The cyclodextrins may be used alone or in combination of two or more. The content of cyclodextrins in the eye drops of the present embodiment is not particularly limited, but is, for example, preferably 0.1 to 15% (w / v), more preferably 1 to 10% (w / v), and even more preferably 3 to 8% (w / v), of the total amount of the eye drops.

[0030] (pH adjuster) The eye drops of the present embodiment may contain a pH adjuster, such as dilute hydrochloric acid or sodium hydroxide.

[0031] (buffering agent) The eye drops of the present embodiment may contain a buffering agent, such as sodium citrate hydrate, sodium acetate hydrate, sodium bicarbonate, trometamol, boric acid, borax, sodium hydrogen phosphate hydrate, and sodium dihydrogen phosphate.

[0032] Examples of the eye drops of this embodiment include eye drops containing fibrate nanoparticles, water, and a thickener; eye drops containing fibrate nanoparticles, water, a thickener, and cyclodextrins; eye drops containing fibrate nanoparticles, water, a thickener, cyclodextrins, and a preservative; and eye drops containing fibrate nanoparticles, water, a thickener, cyclodextrins, a preservative, and a tonicity agent.

[0033] More specifically, examples include eye drops containing fibrate nanoparticles, water, and a cellulose-based thickener; eye drops containing fibrate nanoparticles, water, a cellulose-based thickener, and cyclodextrins; eye drops containing fibrate nanoparticles, water, a cellulose-based thickener, cyclodextrins, and benzalkonium chloride; and eye drops containing fibrate nanoparticles, water, a cellulose-based thickener, cyclodextrins, benzalkonium chloride, and mannitol.

[0034] More specifically, examples include eye drops containing fibrate nanoparticles, water, and methylcellulose; eye drops containing fibrate nanoparticles, water, methylcellulose, and 2-hydroxypropyl-β-cyclodextrin; eye drops containing fibrate nanoparticles, water, methylcellulose, 2-hydroxypropyl-β-cyclodextrin, and benzalkonium chloride; and eye drops containing fibrate nanoparticles, water, methylcellulose, 2-hydroxypropyl-β-cyclodextrin, benzalkonium chloride, and mannitol.

[0035] <Manufacturing method> The eye drops of this embodiment can be produced by adding optional ingredients such as a thickener to a fibrate, mixing the mixture, dispersing the mixture in an aqueous dispersion medium preferably containing cyclodextrins, and wet-pulverizing the mixture. Dry pulverization may be performed before wet pulverization. "Dry pulverization" refers to pulverization in a gas. "Wet pulverization" refers to pulverization in a liquid. Hereinafter, specific examples of the method for producing the eye drop of the present embodiment will be described, but the present invention is not limited to these.

[0036] A thickener or the like is added to the fibrate as needed, and the mixture is then mixed and pulverized. In this case, components to be mixed with the fibrate include components other than cyclodextrins and water. These components may be added after dry pulverization and before wet pulverization, but it is preferable to mix the thickener and isotonic agent with the fibrate before pulverization. Mixing and pulverization may be performed first in an agate mortar or the like, followed by dry pulverization using a bead mill. Mixing and pulverization are preferably performed under low-temperature conditions (e.g., 4°C). For dry pulverization using a bead mill, zirconia beads with a diameter of about 2 mm can be used.

[0037] The finely pulverized material obtained by dry milling is dispersed in an aqueous solution containing cyclodextrins. Optional ingredients such as preservatives may be added at this time. Next, wet milling is performed using a bead mill or similar. Zirconia beads with a diameter of approximately 0.1 mm can be used for wet milling using a bead mill. Wet milling is preferably performed under low-temperature conditions (e.g., 4°C). If bubbles are generated during wet milling or the mixture becomes meringue or creamy, it can be returned to a suspension state by adding a small amount of dispersion medium and performing centrifugation or similar.

[0038] After wet pulverization, the beads are removed and the mixture is sterilized by filtration using a 0.2 μm filter. This allows the eye drop preparation of the present embodiment to be obtained. When using the obtained eye drop preparation, the concentration of the fibrate nanoparticles may be adjusted appropriately using a diluent containing components other than the fibrate nanoparticles.

[0039] <How to use> The eye drops of this embodiment can be used to improve retinal circulatory disorders and retinal neurovascular coupling disorders. For example, the eye drops of this embodiment can be administered to patients who have or are at risk of developing vitreoretinal diseases such as diabetic retinopathy, retinal vein occlusion, retinal artery occlusion, age-related macular degeneration, and central serous chorioretinopathy due to retinal circulatory disorders and retinal neurovascular coupling disorders. The eye drops of this embodiment are expected to improve retinal circulatory disorders and retinal neurovascular coupling disorders, thereby curing, alleviating, or preventing the onset of these vitreoretinal diseases.

[0040] The eye drops of this embodiment can be used as an eye drop for treating or preventing vitreous retinal diseases. The vitreous retinal diseases to be treated with the eye drops of this embodiment may be mild, moderate, or severe. When the vitreous retinal disease is mild, administration of the eye drops of this embodiment can be expected to cure the vitreous retinal disease. When the vitreous retinal disease is moderate or severe, the eye drops of this embodiment may be used in combination with existing medical treatments (anti-vascular endothelial growth factor (VEGF) inhibitors or steroid drugs) or surgical treatments (retinal photocoagulation, vitreous surgery, etc.).

[0041] The eye drops of the present embodiment can be used to prevent retinal circulatory disorders and retinal neurovascular coupling disorders. By prophylactically administering the eye drops of the present embodiment to a subject at high risk of developing retinal circulatory disorders and retinal neurovascular coupling disorders, the occurrence of retinal circulatory disorders and retinal neurovascular coupling disorders can be prevented.

[0042] The administration interval of the eye drops of this embodiment can be appropriately determined depending on the patient's symptoms, weight, age, sex, etc. The administration interval can be, for example, 2 to 3 times a day.

[0043] [Other embodiments] In one embodiment, the present invention provides a method for improving or preventing retinal circulatory disorders and retinal neurovascular disorders, comprising administering the eye drop preparation of the above embodiment to a subject. In one embodiment, the present invention provides use of nanoparticles containing a fibrate in the manufacture of an eye drop for improving or preventing retinal circulatory disorders and retinal neurovascular coupling disorders. In one embodiment, the present invention provides the eye drops of the above embodiment for use in improving or preventing retinal circulatory disorders and retinal neurovascular coupling disorders. In one embodiment, the present invention provides use of the eye drops of the above embodiment for improving or preventing retinal circulatory disorders and retinal neurovascular coupling disorders. In one embodiment, the present invention provides a method for treating or preventing a vitreoretinal disease, comprising administering the eye drop of the embodiment to a subject. In one embodiment, the present invention provides the use of nanoparticles comprising a fibrate in the manufacture of an eye drop for treating or preventing a vitreoretinal disease. In one embodiment, the present invention provides the eye drops of the above embodiment for use in treating or preventing a vitreoretinal disease. In one embodiment, the present invention provides use of the eye drops of the above embodiments for treating or preventing vitreoretinal diseases. [Example]

[0044] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0045] [Preparation of eye drops] <Preparation of eye drops containing fibrate nanoparticles> Fibrate-containing eye drops were prepared according to the following procedure. 1) 450 mg of fenofibrate (Takeda Teva), 112.5 mg of methylcellulose, and 112.5 mg of D-mannitol were mixed in an agate mortar and finely ground (at low temperature (4°C) for 1 hour). 2) The finely pulverized material from 1) was collected and dry-pulverized (at low temperature (4°C)) using a bead-type cell disrupter (Micro Smash MS-100R, Tomy Seiko Co., Ltd.) (2 times with 2 mm zirconia beads, 3,000 rpm for 30 seconds each time) or a multi-sample cell disrupter (Shake Master Neo, Biomedical Science Co., Ltd.) (500 rpm with zirconia beads for 10 minutes). 3) 450 mg of the dry-ground material obtained in 2) was weighed out and diluted to 15 mL with 5% (w / v) 2-hydroxypropyl-β-cyclodextrin aqueous solution. Next, 75 μL of 1% (w / v) benzalkonium chloride (BAC) aqueous solution was added. 4) The mixture obtained in 3) was subjected to 30 wet grinding cycles using a bead cell disrupter (Micro Smash MS-100R, Tomy Seiko Co., Ltd.) (30 cycles with 0.1 mm zirconia beads, 15,000 rpm, 30 seconds per cycle, at a low temperature (4°C)). If bubbles were generated during wet grinding or the mixture became meringue-like, the mixture was centrifuged (800 × g, 60 seconds, 4°C) as needed to return it to a suspension state. 5) The wet-pulverized material obtained in 4) was wet-pulverized using a multi-sample cell disrupter (Shake Master Neo, Biomedical Science Co., Ltd.) at 1,500 rpm with 0.1 mm zirconia beads for 1 hour at low temperature (4°C). 6) Remove the zirconia beads from the super-crushed product obtained in 5), filter and sterilize with a 0.2 μm filter, and use it as an eye drop containing fibrate. If necessary, adjust the concentration appropriately with the following vehicle (dispersion medium) before use. Composition of Vehicle Benzalkonium chloride 0.005% (w / v) Mannitol 0.5% (w / v) Methylcellulose 0.5% (w / v) 2-Hydroxypropyl-β-cyclodextrin 5% (w / v)

[0046] The final concentrations of each drug in the eye drops prepared as above are shown below. Fenofibrate 2% (w / v) Benzalkonium chloride 0.005% (w / v) Mannitol 0.5% (w / v) Methylcellulose 0.5% (w / v) 2-Hydroxypropyl-β-cyclodextrin 5% (w / v)

[0047] (Particle size distribution analysis by laser diffraction / scattering method) For the eye drops containing fibrate prepared as above, particle size distribution analysis was performed using a laser diffraction type particle size distribution measuring device (SALD-7100, Shimadzu Corporation). The results are shown in Fig. 1 and Table 1. The fibrate nanoparticles had a particle diameter in the range of 34 to 244 nm. Fig. B shows an atomic force microscope image of the fibrate nanoparticles.

[0048] [Table 1]

[0049] [Preparation of Vehicle eye drops] Prepare the Vehicle described in <Preparation of eye drops containing fibrate>, and use this as the Vehicle eye drops. '

[0050] [Eye drop administration test] Using 6-week-old female diabetic model mice (C57BL / 6J db / db), an eye drop administration test was carried out as shown in Table 2. Eye drops were administered twice a day, in the morning and evening.

[0051] [Ocular blood flow measurement test] Ocular blood flow measurements were performed under isoflurane inhalation anesthesia (induction 3.5-4.0% / min, maintenance 1.5-2.5% / min). The mice were kept at a body temperature of 36-37°C using a heating pad during the experiment. Measurements of each evaluation item began at 8 weeks of age. Measurements were performed every other week.

[0052] [Table 2]

[0053] <Measurement of whole body parameters> Systemic parameters, including body weight, casual blood glucose level, intraocular pressure (left eye), mean blood pressure, and intraocular perfusion pressure, were measured every other week from 8 weeks of age. Between-group analysis between the treatment and control groups was performed using two-way repeated-measures ANOVA with Sidak's multiple test. Between-age analysis was performed using the Johckheere-Terpstra test.

[0054] The results are shown in Figures 3A to 4B (mean ± SEM). Body weight tended to increase with age in both the treatment and control groups, but there was no significant difference in body weight gain between the treatment and control groups (Figure 3A). Random blood glucose levels were maintained at high levels in both the treatment and control groups. There were no significant differences in random blood glucose levels between groups of 10 weeks of age (Fig. 3B). There were no significant differences in intraocular pressure (Fig. 3C), mean blood pressure (Fig. 4A), and ocular perfusion pressure (Fig. 4B) between the treatment and control groups. There were also no significant differences between the age groups in either group.

[0055] <Baseline optic disc blood flow measurement> Baseline optic nerve head (ONH) blood flow measurements were performed biweekly from 8 weeks of age using a laser speckle blood flowmeter (LSFG-Micro, Softcare). Measurements were performed on the left eye. Between-group analysis between treatment and control groups was performed using two-way repeated-measures ANOVA. Between-age analysis was performed using the Johckheere-Terpstra test.

[0056] The results are shown in Figure 5 (mean ± SEM). There was no significant difference in resting blood flow before stimulation between the treatment and control groups. There was also no significant difference between the age groups in either group.

[0057] <Measurement of ocular blood flow changes due to flicker stimulation> Changes in ocular blood flow induced by flicker stimulation were measured every two weeks from 8 weeks of age. Flicker stimulation was performed for 3 minutes at a frequency of 12 Hz, and ocular blood flow measurements were taken every 20 seconds using an LSFG-Micro. Measurements were performed on the left eye. Between-group analysis between the treatment and control groups was performed using two-way repeated-measures ANOVA with Sidak's multiple test.

[0058] The results are shown in Figures 6 and 7 (mean ± SEM). Each graph in Figure 6 shows the changes in blood flow after flicker stimulation at 8, 10, 14, and 12 weeks of age. Figure 7 is a graph summarizing the total blood flow changes at each age. "Base" on the horizontal axis of Figure 6 indicates the baseline optic disc blood flow before stress. In the control group, impaired blood flow response after flicker stimulation was confirmed. On the other hand, in the treatment group, the increased blood flow response after flicker stimulation was not impaired and was maintained. From 10 weeks of age onwards, there was a significant difference in the change in blood flow after flicker stimulation between the treatment and control groups. These results confirmed that administration of eye drops containing fibrate nanoparticles can prevent impairment of retinal neurovascular connections.

[0059] <Measurement of changes in ocular blood flow due to hyperoxia stimulation> Changes in ocular blood flow due to hyperoxia were measured biweekly from 8 weeks of age. Flicker stimulation was performed at a frequency of 12 Hz for 3 minutes, and measurements were taken every 20 seconds using an LSFG-Micro. Measurements were taken in the left eye. Between-group analysis between the treatment and control groups was performed using two-way repeated-measures ANOVA with Sidak's multiple test.

[0060] The results are shown in Figures 8 and 9 (mean ± SEM). Each graph in Figure 8 shows the changes in blood flow after hyperoxia stimulation at 8, 10, 14, and 12 weeks of age. Figure 9 is a graph summarizing the changes in total blood flow at each age. "Base" on the horizontal axis in Figure 8 indicates the baseline optic nerve head blood flow before stimulation. In the control group, impaired hypoxia after hyperoxia stimulation was confirmed. On the other hand, in the treatment group, hypoxia after hyperoxia stimulation was not impaired and was maintained. These results confirmed that administration of fibrate nanoparticle-containing eye drops can prevent damage to glial cells and vascular endothelial cells.

[0061] <Electroretinogram (ERG)> Electroretinograms were performed biweekly from 8 weeks of age on the left eye. Intergroup analysis between the treatment and control groups was performed using two-way repeated measures ANOVA with Sidak's multiple test.

[0062] The results are shown in Figure 10 (mean ± SEM). The prolongation of b-wave latency observed in the control group was improved in the treatment group. From 12 weeks of age onwards, the b-wave latency in the treatment group was significantly shortened compared to the control group. These results confirmed that administration of eye drops containing fibrate nanoparticles can suppress retinal dysfunction. [Industrial Applicability]

[0063] According to the present invention, there is provided an eye drop capable of improving or preventing retinal circulatory disorders and retinal neurovascular association disorders.

[0064] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the scope of the appended claims.

Claims

1. A pharmaceutical composition comprising nanoparticles containing fenofibrate, the nanoparticles are wet-milled fenofibrate; The nanoparticles have a particle size in the range of 10 to 300 nm. An eye drop for improving or preventing retinal circulatory disorders and retinal neurovascular disorders.

2. The eye drop of claim 1 further comprising a thickening agent.

3. The eye drop according to claim 2, wherein the thickener is a cellulose-based thickener.

4. The eye drop according to claim 3, wherein the cellulose-based thickener is methylcellulose.

5. The eye drop preparation according to any one of claims 1 to 4, further comprising a cyclodextrin.

6. 6. The eye drop preparation according to claim 5, wherein the cyclodextrin is 2-hydroxypropyl-β-cyclodextrin.

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