Ocular drug

Abstract

This ocular drug comprises: a first aqueous phase 10; a gelled oil phase 20 in which the first aqueous phase 10 is dispersed; a second aqueous phase 30 in which the oil phase 20 is dispersed; and an eye disease therapeutic agent disposed in the first aqueous phase 10.

Description

eye drops

[0001] The present invention relates to an ophthalmic drug.

[0002] Age-related macular degeneration is divided into dry and wet types, with dry age-related macular degeneration leading to blindness. Dry age-related macular degeneration is common in Japan, and is the fourth leading cause of blindness in the country, with cases increasing every year. In wet age-related macular degeneration, new blood vessels form in the choroid, damaging photoreceptor cells. The primary treatment for wet age-related macular degeneration is intravitreal injection of anti-vascular endothelial growth factor (VEGF) antibody drugs. However, intravitreal injections are highly invasive and inconvenient.

[0003] Patent No. 7005772 JP 2021-14445 A

[0004] J. Fardous,et al., "Delivery of Hydrophobic Drugs to the Posterior Ocular Region by Gel-in-Water Nanoemulsion," TVST, May 2022, Vol. 11, No. 5, Article 16

[0005] In the treatment of ocular diseases, including age-related macular degeneration, there is a need for a technology that can deliver a therapeutic agent for ocular diseases to the posterior segment of the eye with minimal invasiveness. Therefore, one object of the present invention is to provide an ophthalmic drug that can deliver a therapeutic agent for ocular diseases to the posterior segment of the eye.

[0006] An ophthalmic drug according to an embodiment of the present invention comprises a first aqueous phase, a gelled oil phase in which the first aqueous phase is dispersed, a second aqueous phase in which the oil phase is dispersed, and an eye disease therapeutic agent disposed within the first aqueous phase.

[0007] In the above-mentioned ophthalmic drug, the first aqueous phase may be gelled. In the above-mentioned ophthalmic drug, the first aqueous phase may not be gelled.

[0008] In the above eye drops, the therapeutic agent for eye diseases may be hydrophilic.

[0009] In the above eye drug, the first aqueous phase may have a diameter of 25 nm or more and 150 nm or less.

[0010] In the above eye drop, the diameter of the oil phase may be 100 nm or more and 350 nm or less.

[0011] In the above eye drops, the first aqueous phase may contain gelatin.

[0012] In the above eye drops, the gelatin may be crosslinked.

[0013] In the above eye drops, the oil phase may contain beeswax.

[0014] The eye medication may be directed to the posterior segment of the eye.

[0015] The above eye medications may be administered to a subject by eye drop administration.

[0016] The above eye medications may be administered to the subject by the eye-covering method.

[0017] A delivery agent for an eye disease therapeutic agent according to an embodiment of the present invention comprises a first aqueous phase, a gelled oil phase in which the first aqueous phase is dispersed, and a second aqueous phase in which the oil phase is dispersed.

[0018] In the delivery agent for the eye disease therapeutic agent, the eye disease therapeutic agent may be disposed in the first aqueous phase.

[0019] In the delivery agent for the therapeutic agent for eye disease, the first aqueous phase may be gelled. In the delivery agent for the therapeutic agent for eye disease, the first aqueous phase may not be gelled.

[0020] In the delivery agent for the therapeutic agent for eye diseases, the therapeutic agent for eye diseases may be hydrophilic.

[0021] In the delivery agent for a therapeutic agent for an eye disease, the first aqueous phase may have a diameter of 25 nm or more and 150 nm or less.

[0022] In the delivery agent for a therapeutic agent for an eye disease, the diameter of the oil phase may be 100 nm or more and 350 nm or less.

[0023] In the delivery agent for a therapeutic agent for an eye disease, the first aqueous phase may contain gelatin.

[0024] In the delivery agent for the therapeutic agent for eye diseases, the gelatin may be crosslinked.

[0025] In the delivery agent for the therapeutic agent for an eye disease, the oil phase may contain beeswax.

[0026] The delivery agent for the ocular disease therapeutic agent may be directed to the posterior segment of the eye.

[0027] A method for producing an ophthalmic drug according to one aspect of the present invention includes: mixing a first aqueous solution containing an ophthalmic disease therapeutic agent with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a first aqueous phase containing the ophthalmic disease therapeutic agent therein is dispersed; adding a gelling agent to the oil phase of the primary emulsion to gel the oil phase; and mixing the primary emulsion in which the oil phase has been gelled with a second aqueous solution containing a second surfactant to form a secondary emulsion containing a second aqueous phase in which the gelled oil phase is dispersed.

[0028] In the above-described method for producing an eye medication, the first aqueous solution may further contain a gelling agent, and the first aqueous solution containing the ocular disease therapeutic agent and the gelling agent may be mixed with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a gelled first aqueous phase containing the ocular disease therapeutic agent therein is dispersed.

[0029] In the method for producing the eye drop, the concentration of the first surfactant in the oil may be 125 mg / mL or more.

[0030] In the above-described method for producing an eye drop, ultrasonic waves may be applied to the mixture of the first aqueous solution and the oil when forming the primary emulsion.

[0031] In the above-mentioned method for producing an eye drop, the time for applying the ultrasonic waves may be 15 minutes or more.

[0032] In the above-described method for producing an ophthalmic drug, the concentration of the second surfactant in the second aqueous solution may be 25 mg / mL or more.

[0033] In the above-described method for producing an ophthalmic drug, when forming the secondary emulsion, ultrasonic waves may be applied to the mixture of the primary emulsion and the second aqueous solution.

[0034] In the above-mentioned method for producing an eye drop, the time for applying the ultrasonic waves may be 10 minutes or more.

[0035] A method for producing a delivery agent for an eye disease therapeutic agent according to an embodiment of the present invention includes mixing a first aqueous solution with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a first aqueous phase is dispersed; adding a gelling agent to the oil phase of the primary emulsion to gel the oil phase; and mixing the primary emulsion in which the oil phase has been gelled with a second aqueous solution containing a second surfactant to form a secondary emulsion containing a second aqueous phase in which the gelled oil phase is dispersed.

[0036] In the above-described method for producing a delivery agent for a therapeutic agent for an eye disease, the first aqueous solution may contain a gelling agent, and the first aqueous solution containing the gelling agent may be mixed with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a gelled first aqueous phase is dispersed.

[0037] In the method for producing a delivery agent for a therapeutic agent for an eye disease, the concentration of the first surfactant in the oil may be 100 mg / mL or more.

[0038] In the above-mentioned method for producing a delivery agent for a therapeutic agent for an eye disease, ultrasound may be applied to the mixture of the first aqueous solution and the oil when forming the primary emulsion.

[0039] In the method for producing a delivery agent for a therapeutic agent for an eye disease, the ultrasonic wave may be applied for 15 minutes or more.

[0040] In the method for producing a delivery agent for a therapeutic agent for an eye disease, the concentration of the second surfactant in the second aqueous solution may be 25 mg / mL or more.

[0041] In the above-mentioned method for producing a delivery agent for a therapeutic agent for an eye disease, when forming the secondary emulsion, ultrasound may be applied to the mixture of the primary emulsion and the second aqueous solution.

[0042] In the method for producing a delivery agent for a therapeutic agent for an eye disease, the time for applying ultrasound may be 10 minutes or more.

[0043] According to the present invention, an ophthalmic drug capable of delivering a therapeutic agent for an eye disease to the posterior segment of the eye can be provided.

[0044] FIG. 1 is a schematic diagram of an eye drop according to an embodiment. G2A and 2B are graphs showing particle size distributions of W / O / W emulsions. G / O G 2D is a graph showing the particle size distribution of a W / O emulsion. G 3 is a graph showing the particle size distribution of a W / O / W emulsion. G / O / W emulsion, W / O G / W emulsion, and W G / O G 4 is a graph showing the particle size and polydispersity index (PDI) of the particle size of the W / W emulsion. GFIG. 5 is an electron microscope photograph of an aqueous / oil-in-water emulsion. FIG. 5 is a graph showing the relationship between the surfactant concentration during the preparation of a primary emulsion and the average particle size of the aqueous phase in the primary emulsion. FIG. 6 is a graph showing the relationship between the ultrasonic treatment time during the preparation of a primary emulsion and the average particle size of the aqueous phase in the primary emulsion. FIG. 7 is a graph showing the relationship between the surfactant concentration during the preparation of a secondary emulsion and the average particle size of the oil phase in the secondary emulsion. FIG. 8 is a graph showing the relationship between the volume ratio of the aqueous solution to the primary emulsion during the preparation of a secondary emulsion and the average particle size of the oil phase in the secondary emulsion. FIG. 9 is a graph showing the relationship between the ultrasonic treatment time during the preparation of a secondary emulsion and the average particle size of the oil phase in the secondary emulsion. FIG. 10 is a graph showing the change over time in the average particle size of the oil phase in the secondary emulsion. FIG. 11 is a graph showing the change over time in the polydispersity index (PDI) of the oil phase in the secondary emulsion. FIG. 12 is a graph showing the initial release amount of albumin contained in the first aqueous phase of the secondary emulsion. FIG. 13 is a graph showing the change in the release rate of a fluorescent substance contained in the first aqueous phase of the secondary emulsion over time. FIG. 14 is a graph showing the initial change in the release rate of a fluorescent substance contained in the first aqueous phase of the secondary emulsion over time. FIG. 15 is a graph showing the initial release rate of a fluorescent substance contained in the first aqueous phase of the secondary emulsion. FIG. 16 is a graph showing a calibration curve illustrating the relationship between BSA concentration and absorbance obtained by serially diluting 1% BSA without ultrasonic agitation. FIG. 17 is a graph showing the relationship between albumin concentration and absorbance for secondary emulsions containing albumin in the first aqueous phase. FIG. 18A is a fluorescence micrograph of cells cultured in a medium without fluorescein. FIG. 18B is a fluorescence micrograph of cells cultured in a medium containing a secondary emulsion containing fluorescein in the first aqueous phase. Figure 18C is a fluorescence micrograph of cells cultured in a medium supplemented with a fluorescein solution. Figure 19 is a graph showing the relationship between the concentration of the secondary emulsion and the cell viability. Figure 20 is a graph showing the relationship between the concentration of the secondary emulsion and the cell viability. Figure 21 is a graph showing the relationship between the concentration of the secondary emulsion and the cell viability. G / O G21 shows micrographs of mouse eyeballs instilled with a fluorescein solution, a W / O / W emulsion containing fluorescein in the first aqueous phase, and a W / O / W emulsion containing fluorescein in the first aqueous phase. G / O G 22 is a graph showing the fluorescence intensity in the retinal layer of the eyeball of a mouse to which each of the O / W emulsions containing coumarin-6 according to Reference Example 1 was instilled. G 23 is a micrograph of a section of the anterior segment of the eyeball of a slaughtered pig to which the O / W emulsion containing coumarin-6 according to Reference Example 1 was instilled, and a micrograph of a section of the anterior segment of the eyeball of a slaughtered pig to which the coumarin-6-containing castor oil according to Reference Example 2 was instilled. G 24 is a micrograph of a section of the posterior segment of the eyeball of a slaughtered pig to which the O / W emulsion was instilled, and a section of the posterior segment of the eyeball of a slaughtered pig to which the coumarin-6-containing castor oil of Reference Example 2 was instilled. G 25 is a micrograph of a section of the eyeball of a slaughtered pig that was given an O / W emulsion containing coumarin-6 according to Reference Example 1 by the eyeball coating method. G 26 is a graph showing the relationship between the distance from the sclera of the eyeball of a slaughtered pig that was given a coumarin-6-containing O / W emulsion according to Reference Example 1 while alive. G 27 is a micrograph of sections of the anterior and posterior segments of a pig's eyeball into which an O / W emulsion containing coumarin-6 according to Reference Example 1 was instilled by the eyeball coating method. G 28 shows micrographs of sections of the anterior and posterior segments of the eyeball of a pig that was given an O / W emulsion containing coumarin-6 according to Reference Example 1 by eye drop or eyeball coating while the pig was alive. G 29 is a graph showing the fluorescence intensity in a section of the posterior segment of the eyeball of a pig given an O / W emulsion containing coumarin-6 according to Reference Example 1. G 30 is a micrograph of a section near the sclera of an eyeball immersed in an O / W emulsion containing coumarin-6 according to Reference Example 1. G31 is a micrograph of a section near the retina of an eyeball immersed in an O / W emulsion. FIG. 32 is a micrograph for explaining the distance from the sclera to the retina in an eyeball. FIG. 33 is a micrograph of an O / W emulsion encapsulating coumarin-6 according to Reference Example 1. G 31 is a graph showing the relationship between the distance from the outside of the sclera and the fluorescence intensity in a section of an eyeball immersed in an O / W emulsion. FIG. 32 is a graph showing the relationship between the concentration of coumarin-6 and the fluorescence intensity of coumarin-6. FIG. 33 is a graph showing the relationship between the O / W emulsion containing coumarin-6 in Reference Example 1. G 35 is a graph showing the relationship between the time the eyeball was immersed in the W / W emulsion and the concentration of coumarin-6 in the retina of the eyeball. G / O G The concentration of the W emulsion and G / O G Figure 36 is a graph showing the relationship between the number of days an emulsion was stored and the average particle size. Figure 37 is a graph showing the relationship between the number of days an emulsion was stored and the polydispersity index (PDI). Figure 38 is a graph showing the stability index SI (%) of an emulsion.

[0045] The following describes embodiments of the present invention. However, it should not be understood that the following embodiments limit the present invention. From this disclosure, various alternative embodiments, examples, and operating techniques will become apparent to those skilled in the art. It should be understood that the present invention encompasses various embodiments not described herein.

[0046] As shown in FIG. 1 , the eye drop according to the embodiment includes a first aqueous phase 10, a gelled oil phase 20 in which the first aqueous phase 10 is dispersed, a second aqueous phase 30 in which the oil phase 20 is dispersed, and an eye disease therapeutic agent disposed within the first aqueous phase 10. The first aqueous phase 10 may be gelled or may not be gelled. The gelled first aqueous phase 10 and the gelled oil phase 20 are of a gelled water-in-gelled oil type (W G / O G The gelled oil phase 20 and the second aqueous phase 30 form a gelled oil-in-water type (O GThe gelled first aqueous phase 10, the gelled oil phase 20, and the second aqueous phase 30 form a gelled water-gelled oil-in-water type (W / W) emulsion. G / O G / W) complex emulsion.

[0047] The first aqueous phase 10 includes an aqueous solution. The aqueous solution includes, for example, a buffer solution. Examples of buffer solutions include, but are not limited to, 2-morpholinoethanesulfonic acid monohydrate (MES) buffer solution, acetate buffer solution, phosphate buffer solution, Tris-HCl buffer solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer solution, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer solution, and 3-morpholinopropanesulfonic acid (MOPS) buffer solution. In the present disclosure, the aqueous solution includes water. The water may be water for injection. Hereinafter, the aqueous solution constituting the first aqueous phase 10 may be referred to as the first aqueous solution.

[0048] When the first aqueous phase 10 is gelled, the gelled first aqueous phase 10 contains, for example, a gelling agent for gelling an aqueous solution. The first aqueous phase 10 is gelled by the gelling agent. Examples of gelling agents for gelling an aqueous solution include, but are not limited to, gelatin, agar, collagen, polysaccharides, sodium alginate, calcium alginate, hydroxyethyl methacrylate (HEMA), poloxamer hydrogel, and thiol-terminated poloxamer. Examples of polysaccharides include, but are not limited to, cellulose, chitin, starch, glycogen, agarose, pectin, galactomannan, hyaluronic acid, low-substituted dopamine-hyaluronic acid complex, chitosan, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. The first aqueous phase 10 may contain multiple types of gelling agents. Hereinafter, the gelling agent constituting the first aqueous phase 10 may be referred to as the "first gelling agent." The gelled first aqueous phase 10 may also contain heparin.

[0049] The gelling agent may be crosslinked with a crosslinking agent. Examples of the crosslinking agent include, but are not limited to, a combination of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide acetate (EDC) and N-hydroxysuccinimide (NHS). The gelling agent may be crosslinked with multiple types of crosslinking agents. Alternatively, the gelling agent may be self-crosslinked.

[0050] The first aqueous phase 10 is covered with a hydrophobic surfactant and forms particles dispersed in the oil phase 20. The hydrophobic surfactant is, for example, a surfactant having an HLB (Hydrophile Lipophile Balance) value range of less than 8. The hydrophobic surfactant is, for example, nonionic. Examples of hydrophobic surfactants include, but are not limited to, sucrose fatty acid esters, glycerin fatty acid esters, organic acid monoglycerides, polyglycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, lecithin, and enzyme-treated lecithin. The first aqueous phase 10 may be covered with multiple types of hydrophobic surfactants. Hereinafter, the surfactant covering the first aqueous phase 10 may be referred to as the first surfactant.

[0051] The diameter (particle size) of the first aqueous phase 10, which is coated with the hydrophobic surfactant and forms particles, is, for example, 25 nm to 150 nm, 35 nm to 90 nm, 40 nm to 80 nm, or 40 nm to 50 nm. The diameter of the first aqueous phase 10 is an average diameter measured by dynamic light scattering (DLS).

[0052] The ocular disease therapeutic agent is contained in the first aqueous phase 10 in the form of particles. An ocular disease is any disease that can affect the eye. Examples of causes of ocular diseases include, but are not limited to, proliferative disorders, infections, allergies, allergic reactions, degenerative disorders, inflammatory diseases, trauma, surgery, radiation, chemical irritation, physical irritation, and autoimmune diseases. An example of an ocular disease is a retinal disease. Examples of retinal diseases include age-related macular degeneration, retinitis pigmentosa, retinal vein occlusion, central serous chorioretinopathy, diabetic retinopathy, retinal tears, and retinal detachment.

[0053] The ocular disease therapeutic agent is, for example, hydrophilic. The ocular disease therapeutic agent includes, for example, biomolecules such as proteins, peptides, and nucleic acids. Examples of proteins include, for example, antibodies and enzymes. Examples of antibodies include, but are not limited to, anti-vascular endothelial growth factor (VEGF) antibodies and anti-angiopoietin-2 antibodies. Examples of enzymes include, but are not limited to, ranpirnase and amphikinase, which have antiviral effects. The nucleic acid may be, for example, RNA that interferes with the mRNA of a protein that causes an ocular disease. The nucleic acid may be, for example, an aptamer that binds to a protein that causes an ocular disease. Examples of proteins that cause an ocular disease include periostin, tenascin-C, (pro)renin receptor, and VEGF, which are involved in angiogenesis. Multiple types of ocular disease therapeutic agents may be contained in the first aqueous phase 10.

[0054] The gelled oil phase 20 contains an oil. Examples of the oil include, but are not limited to, fatty acid ester oils, vegetable oils, animal oils, neutral lipids (mono-substituted, di-substituted, or tri-substituted glycerides), synthetic oils and fats, liquid paraffin (mineral oil), heavy liquid isoparaffin, light liquid isoparaffin, sterol derivatives, lactate esters, polycarboxylic acid esters, 2-cetyl ethylhexanoate, silicones, and lipiodol. The oil phase 20 may contain multiple types of oils.

[0055] Examples of fatty acid ester oils include, but are not limited to, isopropyl myristate, octyldodecyl myristate, cetyl myristate, ethyl oleate, ethyl linoleate, isopropyl linoleate, isopropyl palmitate, and butyl stearate.

[0056] Examples of vegetable oils include, but are not limited to, castor oil, coconut oil, palm oil, soybean oil, cottonseed oil, rapeseed oil, sesame oil, corn oil, peanut oil, safflower oil, sunflower oil, olive oil, rapeseed oil, perilla oil, fennel oil, cacao oil, cinnamon oil, peppermint oil, and bergamot oil. Examples of animal oils include, but are not limited to, fish oil and whale oil.

[0057] Examples of neutral lipids include, but are not limited to, medium-chain triglycerides, triolein, trilinolein, tripalmitin, tristearin, trimyristin, and triarachidonin. Examples of synthetic oils and fats include, but are not limited to, azone.

[0058] Examples of sterol derivatives include, but are not limited to, cholesteryl oleate, cholesteryl linoleate, cholesteryl myristate, cholesteryl palmidate, and cholesteryl arachidate.

[0059] Examples of lactate esters include, but are not limited to, ethyl lactate and cetyl lactate. Examples of polycarboxylic acid esters include, but are not limited to, triethyl citrate, diisopropyl adipate, diethyl sebacate, and diisopropyl sebacate.

[0060] The gelled oil phase 20 contains, for example, a gelling agent for gelling oil. The gelling agent causes the oil phase 20 to be gelled. Examples of gelling agents for gelling oil include, but are not limited to, waxes, higher fatty acids, fatty acid esters, metal soaps, amino acid-based gelling agents, and hydrocarbons.

[0061] Examples of waxes include, but are not limited to, beeswax, Japan wax, hydrogenated oil, candelilla wax, carnauba wax, rice wax, montan wax, ozokerite, ceresin, paraffin wax, microcrystalline wax, petrolatum, Fischer-Tropsch wax, polyethylene wax, and ethylene-propylene copolymer.

[0062] Examples of higher fatty acids include, but are not limited to, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, behenic acid, undecylenic acid, 12-hydroxystearic acid (12-HSA), palmitoleic acid, oleic acid, linoleic acid, linolenic acid, erucic acid, docosahexaenoic acid, eicosapentaenoic acid, isohexadecanoic acid, anteisohenicosanoic acid, long-chain branched fatty acids, dimer acids, and hydrogenated dimer acids.

[0063] Examples of fatty acid esters include cholesterol fatty acid esters, sugar fatty acid esters, glycerin fatty acid esters, polyglycerin fatty acid esters, etc., and specific examples of sugar fatty acid esters include, but are not limited to, dextrin palmitate, dextrin myristate, dextrin isostearate, fructooligosaccharide stearate, inulin stearate, and dibenzylidene sorbitol.

[0064] Examples of metal soaps include, but are not limited to, calcium stearate, lithium stearate, aluminum stearate, calcium palmitate, lithium palmitate, aluminum palmitate, calcium 2-ethylhexanoate, lithium 2-ethylhexanoate, aluminum 2-ethylhexanoate, calcium 12-hydroxystearate, lithium 12-hydroxystearate, and aluminum 12-hydroxystearate.

[0065] Examples of amino acid gelling agents include, but are not limited to, N-lauroyl glutamic acid di-n-butylamide. Examples of hydrocarbons include, but are not limited to, α-olefin oligomers, polyisobutene, hydrogenated polyisobutene, polybutene, squalane, olive-derived squalane, squalene, petrolatum, and solid paraffin.

[0066] The oil phase 20 may contain multiple types of gelling agents. Hereinafter, the gelling agent constituting the oil phase 20 may be referred to as a second gelling agent.

[0067] The oil phase 20 is covered with a hydrophilic surfactant and forms particles dispersed in the second aqueous phase 30. The hydrophilic surfactant is, for example, a surfactant having an HLB (Hydrophile Lipophile Balance) value range of 8 or more. The hydrophilic surfactant is, for example, nonionic.

[0068] Examples of hydrophilic surfactants include, but are not limited to, polyoxyethylene hydrogenated castor oil (HCO-40, HCO-60, HCO-80, HCO-100, etc.), alkyl sulfates, alkyl sulfonates, alkyl sulfosuccinates, polyoxyethylene alkyl ethers, polyoxyalkylene alkyl ethers, lauryl alcohol alkoxylates, glycerin fatty acid esters, saponin, sucrose fatty acid esters, glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, polysorbates, lecithin, and polyoxyethylene-polyoxypropylene block polymers.

[0069] The oil phase 20 may be covered with multiple types of hydrophilic surfactants. Hereinafter, the surfactant covering the oil phase 20 may be referred to as the second surfactant.

[0070] The diameter (particle size) of the oil phase 20 coated with the hydrophilic surfactant and forming particles is, for example, 100 nm to 350 nm, 120 nm to 350 nm, 130 nm to 300 nm, 140 nm to 250 nm, or 160 nm to 250 nm, and does not overlap with the diameter (particle size) of the first aqueous phase 10. The diameter of the oil phase 20 is an average diameter measured by dynamic light scattering (DLS).

[0071] The second aqueous phase 30 contains an aqueous solution. Examples of the aqueous solution are the same as those contained in the first aqueous phase 10. The second aqueous phase 30 does not need to be gelled. Hereinafter, the aqueous solution constituting the second aqueous phase 30 may be referred to as the second aqueous solution.

[0072] A gelled water-in-gelled oil-in-water type (W-type) gelled oil-in-water type gelled oil contains a gelled first aqueous phase 10, a gelled oil phase 20, and a second aqueous phase 30. G / O G and a water-in-gelled oil-in-water (W / O) multiple emulsion containing a non-gelled first aqueous phase 10, a gelled oil phase 20, and a second aqueous phase 30. GThe combined emulsion (a mixture of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

[0073] The eye drops according to the embodiments may be administered to a subject by an eye drop method. The eye drop method is, for example, a method of instilling the eye drop into the conjunctival sac of a subject. The subject may be a human or a non-human animal. The subject may be a patient. The eye drops according to the embodiments may also be administered to a subject by an eye covering method. The eye covering method is a method of immersing the subject's eye in the eye drop or a solution containing the eye drop for a predetermined period of time. The eye may be immersed in the eye drop or a solution containing the eye drop by filling the cavity around the eye with the eye drop or the solution containing the eye drop. Alternatively, the eye may be immersed in the eye drop or the solution containing the eye drop by covering the eye with a container filled with the eye drop or the solution containing the eye drop. In the eyeball covering method, the time for which the eye is immersed in the eye medication or a solution containing the eye medication is not particularly limited, and may be, for example, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 15 hours, or 20 hours.

[0074] Next, a method for producing an ophthalmic drug according to an embodiment will be described. The method for producing an ophthalmic drug according to an embodiment includes mixing a first aqueous solution containing an ophthalmic disease therapeutic agent with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a first aqueous phase containing the ophthalmic disease therapeutic agent is dispersed, adding a second gelling agent to the oil phase of the primary emulsion to gel the oil phase, and mixing the primary emulsion in which the oil phase has been gelled with a second aqueous solution containing the second surfactant to form a secondary emulsion containing a second aqueous phase in which the gelled oil phase is dispersed. Note that if the first aqueous solution further contains a first gelling agent, a primary emulsion containing an oil phase in which a gelled first aqueous phase containing the ophthalmic disease therapeutic agent is dispersed is formed.

[0075] When forming the primary emulsion, the concentration of the first surfactant in the oil is, for example, 125 mg / mL or more, or 150 mg / mL or more. By setting the concentration of the first surfactant in the oil to 125 mg / mL or more, it is possible to make the particle size of the aqueous phase contained in the primary emulsion 150 nm or less, or 100 nm or less.

[0076] When forming the primary emulsion, ultrasound or pressure may be applied to the mixture of the first aqueous solution and oil to agitate the mixture of the first aqueous solution and oil. The time for applying ultrasound to the mixture of the first aqueous solution and oil is, for example, 3 minutes or more, 10 minutes or more, 15 minutes or more, or 20 minutes or more. By applying ultrasound to the mixture of the first aqueous solution and oil for 3 minutes or more, it is possible to reduce the particle size of the aqueous phase contained in the primary emulsion to 150 nm or less, or 100 nm or less. By applying ultrasound to the mixture of the first aqueous solution and oil for 15 minutes or more, it is possible to reduce the particle size of the aqueous phase contained in the primary emulsion to 50 nm or less.

[0077] When forming the secondary emulsion, the concentration of the second surfactant in the second aqueous solution is, for example, 25 mg / mL or more, or 35 mg / mL or more. By making the concentration of the second surfactant in the second aqueous solution 25 mg / mL or more, it is possible to make the particle size of the oil phase contained in the secondary emulsion 350 nm or less, or 250 nm or less.

[0078] When forming the secondary emulsion, ultrasonic waves or pressure may be applied to the mixture of the primary emulsion and the second aqueous solution to agitate the mixture of the primary emulsion and the second aqueous solution. The time for applying ultrasonic waves to the mixture of the primary emulsion and the second aqueous solution is, for example, 10 minutes or more, or 15 minutes or more. By applying ultrasonic waves to the mixture of the primary emulsion and the second aqueous solution for 10 minutes or more, it is possible to reduce the particle size of the oil phase contained in the secondary emulsion to 350 nm or less, or 200 nm or less.

[0079] Example 1 A crosslinker-containing aqueous solution was prepared by adding heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) to a post-addition concentration of 10 mg / mL, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide acetate (EDC, Peptide Institute, Inc.) to a post-addition concentration of 10 mg / mL, and N-hydroxysuccinimide (NHS, Fujifilm Wako Pure Chemical Industries, Ltd.) to a post-addition concentration of 6 mg / mL, to 50 mmol / L 2-morpholinoethanesulfonic acid monohydrate (MES) buffer solution (pH 5.6, Dojindo Laboratories, Ltd.), and the crosslinker-containing aqueous solution was allowed to stand in a refrigerator for 30 minutes.

[0080] A surfactant-containing oil was prepared by dissolving a nonionic hydrophobic surfactant (ER-290, Ryoto Sugar Ester, registered trademark, Mitsubishi Chemical Foods Corporation) in isopropyl myristate (IPM, Fujifilm Wako Pure Chemical Industries, Ltd.) at 50 mg / mL by shaking at room temperature.

[0081] A gelatin-containing aqueous solution was prepared by dissolving gelatin powder (pig skin gelatin type A, Sigma-Aldrich) in phosphate-buffered saline (PBS) in an incubator so that the final concentration would be 5 wt% when the crosslinker was added. 100 μL of a crosslinker-containing aqueous solution was added to 900 μL of the gelatin-containing aqueous solution, and the resulting mixture was thoroughly pipetted. The mixture and the surfactant-containing oil were mixed in a 1:2 ratio to obtain an oil-water mixture.

[0082] Immediately after mixing the aqueous mixture and the surfactant-containing oil, the container containing the oil-water mixture was placed on ice and stirred for 3 minutes with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds and 70% output amplitude. The ultrasonically treated oil-water mixture was left standing in a refrigerator overnight to obtain a primary emulsion (W emulsion) in which the gelled first aqueous phase was dispersed in the oil. G A 100% emulsion (a 100% emulsion in water / oil) was prepared.

[0083] Example 2 A primary emulsion (W / O emulsion) in which an ungelled first aqueous phase was dispersed in oil was prepared by replacing 900 μL of the gelatin-containing aqueous solution in Example 1 with 900 μL of water.

[0084] (Example 3) A surfactant-containing aqueous solution was prepared by dissolving a nonionic hydrophilic surfactant (polyethylene hydrogenated castor oil-60, NIKKOL HCO-60, Nippon Surfactant Kogyo Co., Ltd.) at 50 mg / mL in PBS in a water bath at 75°C. The surfactant-containing aqueous solution and the primary emulsion (W) prepared in Example 1 were mixed. G The oil and water emulsions were mixed at a ratio of 5:1 to obtain an oil-water mixture.

[0085] The container containing the oil-water mixture was placed on ice and stirred with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds for 3 minutes at 70% output amplitude. The ultrasonically treated oil-water mixture was left to stand in a refrigerator overnight to obtain a secondary emulsion (W emulsion) containing a non-gelled oil phase in which the gelled first aqueous phase was dispersed, and a second aqueous phase in which the non-gelled oil phase was dispersed. G An oil-in-water emulsion was prepared.

[0086] Example 4 The same surfactant-containing aqueous solution as in Example 3 and the primary emulsion (W / O emulsion) prepared in Example 2 were mixed in a ratio of 5:1 and stirred under the same conditions as in Example 3 to prepare a secondary emulsion (W / O / W emulsion) containing an ungelled oil phase in which an ungelled first aqueous phase was dispersed, and a second aqueous phase in which the ungelled oil phase was dispersed.

[0087] (Example 5) The same surfactant-containing aqueous solution as in Example 3 was prepared. In addition, the same primary emulsion (W G A 100% beeswax / o emulsion was prepared. The primary emulsion was centrifuged at 3,500 rpm for 10 minutes using a centrifuge (KUBOTA 5100, Kubota Manufacturing Co., Ltd.), and 1 mL of supernatant was collected. Beeswax was added to the collected 1 mL of supernatant to a final concentration of 2.5 wt% in the oil phase, and the resulting mixture was returned to the centrifuged primary emulsion. The primary emulsion was then placed in a 75°C water bath for 15 seconds, and stirred with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / second for 10 seconds, with stirring at 70% output amplitude, to re-disperse the gelled first aqueous phase in the oil.

[0088] The surfactant-containing aqueous solution and the primary emulsion were mixed at a ratio of 5:1 to prepare an oil-water mixture. The container containing the oil-water mixture was placed on ice and stirred with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds for 3 minutes at 70% output amplitude. The ultrasonically treated oil-water mixture was left to stand in a refrigerator overnight to obtain a secondary emulsion (W) containing a gelled oil phase in which the gelled first aqueous phase was dispersed, and a second aqueous phase in which the gelled oil phase was dispersed. G / O G A water-in-water emulsion was prepared.

[0089] (Example 6) The same primary emulsion (W G A secondary emulsion (W / O emulsion) containing a gelled oil phase in which a non-gelled first aqueous phase is dispersed and a second aqueous phase in which the gelled oil phase is dispersed was prepared in the same manner as in Example 5, except that the same primary emulsion (W / O emulsion) as in Example 2 was used instead of the non-gelled first aqueous phase. G A water-in-water emulsion was prepared.

[0090] (Example 7) The secondary emulsions prepared in each of Examples 3 to 6 were diluted 600 times with PBS. The average diameter and polydispersity index (PDI) of the particles contained in the diluted secondary emulsions were measured using a particle size measurement system (nanoSAQLA, Otsuka Electronics) that uses dynamic light scattering (DLS). As a result, as shown in Figures 2 and 3, the particle diameter in each secondary emulsion was approximately 200 nm, and the W G / O G The particle size of the W / W emulsion was the smallest. In addition, the polydispersity index (PDI) of each secondary emulsion was 0.3 or less. G / O G The polydispersity index (PDI) of the secondary emulsion was the smallest, indicating that the particle size distribution was narrow. Although the particles contained in the secondary emulsion have a multilayer structure, the average diameter of the particles measured by dynamic light scattering (DLS) is the average diameter of the outermost part of the particles.

[0091] (Example 8) W produced in Example 3 GThe oil-in-water emulsion was diluted 100 times, and 10 μL of the diluted emulsion was added to a carbon-coated copper grid (Glassy Film GLF-C10, STEM Cu100P grid, Oken Shoji Co., Ltd.). The emulsion was then dried in a vacuum for 30 minutes, and the particles were observed using a transmission electron microscope (JEOL, JEM-2100HC) at an accelerating voltage of 200 kV and uniaxial tilt. As a result, as shown in Figure 4, it was confirmed that gelled aqueous phase particles were contained within oil phase particles of approximately 200 nm. Therefore, W G It was confirmed that the particles contained in the / O / W emulsion had a multilayer structure.

[0092] Example 9 A crosslinker-containing aqueous solution was prepared by adding heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) to a post-addition concentration of 10 mg / mL, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide acetate (EDC, Peptide Institute, Inc.) to a post-addition concentration of 10 mg / mL, and N-hydroxysuccinimide (NHS, Fujifilm Wako Pure Chemical Industries, Ltd.) to a post-addition concentration of 6 mg / mL, to 50 mmol / L 2-morpholinoethanesulfonic acid monohydrate (MES) buffer solution (pH 5.6, Dojindo Laboratories, Ltd.), and the crosslinker-containing aqueous solution was allowed to stand in a refrigerator for 30 minutes.

[0093] A surfactant-containing oil was prepared by dissolving a nonionic hydrophobic surfactant (ER-290, Ryoto Sugar Ester, registered trademark, Mitsubishi Chemical Foods Corporation) in isopropyl myristate (IPM, Fujifilm Wako Pure Chemical Industries, Ltd.) at the concentration shown in Table 1 by shaking at room temperature.

[0094] A gelatin-containing aqueous solution was prepared by dissolving gelatin powder (pig skin gelatin type A, Sigma-Aldrich) in phosphate-buffered saline (PBS) in an incubator so that the final concentration would be 5 wt% when the crosslinker was added. 100 μL of a crosslinker-containing aqueous solution was added to 900 μL of the gelatin-containing aqueous solution, and the resulting mixture was thoroughly pipetted. The mixture and the surfactant-containing oil were mixed in a 1:2 ratio to obtain an oil-water mixture.

[0095] Immediately after mixing the aqueous mixture and the surfactant-containing oil, the container containing the oil-water mixture was placed on ice and stirred with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds and 70% output amplitude for the time listed in Table 2. The ultrasonically treated oil-water mixture was left to stand in a refrigerator overnight to obtain a primary emulsion (W emulsion) in which a gelled first aqueous phase was dispersed in the oil. G A 100% aqueous emulsion (a 100% aqueous emulsion) was formed.

[0096] The 600-fold diluted primary emulsion (W) was measured at 25°C using a particle size measurement system (nanoSAQLA, Otsuka Electronics) using dynamic light scattering (DLS). G The average diameter of particles contained in the emulsion was measured, and the polydispersity index (PDI) was calculated. As a result, as shown in Figure 5, the particle size tended to decrease as the concentration of the hydrophobic surfactant increased, i.e., 100 mg / mL or more. Furthermore, as shown in Figure 6, the particle size tended to decrease as the ultrasonic treatment time increased, i.e., 15 min or more.

[0097] Example 10 A surfactant-containing aqueous solution was prepared by dissolving a nonionic hydrophilic surfactant (polyethylene hydrogenated castor oil-60, NIKKOL HCO-60, Nippon Surfactant Industry Co., Ltd.) in PBS in a water bath at 75°C to a concentration shown in Table 3.

[0098] The same primary emulsion as in Example 1 was prepared using a nonionic hydrophobic surfactant concentration of 50 mg / mL and an ultrasonic treatment time of 3 minutes. The primary emulsion was centrifuged at 3,500 rpm for 10 minutes using a centrifuge (KUBOTA 5100, Kubota Manufacturing Co., Ltd.), and 1 mL of supernatant was collected. Beeswax was added to the collected 1 mL of supernatant to a final concentration of 2.5 wt% in the oil phase, and the mixture was returned to the centrifuged primary emulsion. The primary emulsion was then stirred in a 75°C water bath using an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / second and 70% output amplitude for 10 seconds to re-disperse the gelled first aqueous phase in the oil.

[0099] The surfactant-containing aqueous solution and the primary emulsion were mixed in the ratios shown in Table 4 to obtain oil-water mixtures.

[0100] The container containing the oil-water mixture was placed on ice and stirred using an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds and 70% output amplitude for the time shown in Table 5. The ultrasonically treated oil-water mixture was left to stand in a refrigerator overnight to obtain a secondary emulsion (W emulsion) containing a gelled oil phase in which the gelled first aqueous phase was dispersed, and a second aqueous phase in which the gelled oil phase was dispersed. G / O G A water-in-water emulsion was formed.

[0101] The secondary emulsion was diluted 600 times with PBS. Using a particle size measurement system (nanoSAQLA, Otsuka Electronics) using dynamic light scattering (DLS), the average diameter of the particles contained in the secondary emulsion was measured, and the polydispersity index (PDI) was calculated. As a result, as shown in FIG. 7, the particle size tended to be smaller when the concentration of the hydrophilic surfactant was 25 mg / mL or higher. Also, as shown in FIG. 8, the particle size tended to be smaller when the ratio of the surfactant-containing aqueous solution to the primary emulsion was higher. Also, as shown in FIG. 9, the particle size tended to be smaller when the ultrasonic treatment time was 10 minutes or longer.

[0102] (Example 11) The secondary emulsions prepared in each of Examples 3 to 6 were stored at 4°C, and the average diameter and polydispersity index (PDI) of the particles contained in the emulsions were measured on days 0, 1, 3, and 7 after preparation in the same manner as in Example 7. The rate of increase in the average particle diameter relative to day 0 is shown in Figure 10, and the polydispersity index (PDI) is shown in Figure 11. G / O G W / O emulsion and W / O G The W / W emulsion showed a low rate of increase in the average particle diameter over time and was stable. G / O G W / O emulsion and W / O G The / W emulsion maintained a low polydispersity index (PDI) over time.

[0103] W in which the oil phase is not gelled G In the W / O / W emulsion and the W / O / W emulsion, the average particle diameter increased over time. This is thought to be due to the particles agglomerating over time and then fusing together.

[0104] Example 12: The same W as in Example 3 was used except that 200 ng / mL of rat albumin (Sigma-Aldrich) was dissolved in the first aqueous phase. G a W / O / W emulsion identical to that of Example 4 except that 200 ng / mL of rat albumin (Sigma-Aldrich) was dissolved in the first aqueous phase; and a W / O / W emulsion identical to that of Example 5 except that 200 ng / mL of rat albumin (Sigma-Aldrich) was dissolved in the first aqueous phase. G / O G A W / O / W emulsion was prepared and allowed to stand at 4°C for 30 minutes. The amount of rat albumin leaked into the second aqueous phase of the emulsion was then measured using the ELISA method. The amount of albumin measured indicated the amount of albumin that could not be retained in the first aqueous phase 30 minutes after the emulsion was prepared. As a result, as shown in Figure 12, the W / O / W emulsion contained more albumin than the W / O / W emulsion. G The O / W emulsion has less leakage, G W / O / W emulsion G / O G These results indicate that gelling the first aqueous and oil phases can suppress leakage of the therapeutic agent into the second aqueous phase in the early stages after emulsion preparation.

[0105] (Example 13) A W / O / W emulsion was prepared in the same manner as in Example 4, except that 0.28 mg / mL of fluorescein (Novartis Pharma) was dissolved in the first aqueous phase and the concentration of the nonionic hydrophobic surfactant was 150 mg / mL. A W / O / W emulsion was prepared in the same manner as in Example 5, except that 0.28 mg / mL of fluorescein (Novartis Pharma) was dissolved in the first aqueous phase and the concentration of the nonionic hydrophobic surfactant was 150 mg / mL. G / O GA fluorescein solution was prepared by dissolving fluorescein in phosphate buffer (PBS, pH 7.4) to a concentration of 0.28 mg / mL.

[0106] W / O / W emulsion, W G / O G The emulsion in water and the fluorescein solution were each placed in a 3.5 kDa dialysis tube (Spectra / Por, registered trademark, Pre-wetted RC Tubing, REPLIGEN, Rancho Dominguez) to reduce the surface area of ​​the dialysis tube to 16 mm 2 I did.

[0107] The dialysis tubes were placed in a 100-mm dish containing 60 mL of phosphate buffer solution (PBS, pH 7.4) as receptor solution. The dish was shaken at 37°C and 150 rpm. 1 mL of receptor solution was collected at 10, 20, 30, 45, 60, 120, 180, 240, 300, and 360 minutes after the start of shaking, and 1 mL of fresh phosphate buffer solution (PBS, pH 7.4) was added to the dish.

[0108] Using a spectrofluorometer (microplate reader, Gemini EM, registered trademark, Molecular Devices Japan), the recovered receptor solution was irradiated with excitation light at a wavelength of 494 nm, and the fluorescence intensity at a wavelength of 521 nm was measured. A calibration curve was prepared by serially diluting the fluorescein solution, and the fluorescence intensity was converted to fluorescein concentration, creating the four-parameter logistic curve shown in Figure 13. Figure 14 is an enlarged graph of the first 10 minutes of the graph in Figure 13. Figure 15 also shows the initial release rate of fluorescein calculated from the graph in Figure 14.

[0109] W G / O G The W / O / W emulsion released less fluorescein over time than the W / O / W emulsion. The W / O / W emulsion had a 16.4% decrease in initial release rate compared to the fluorescein solution, and the W / O / W emulsion released less fluorescein over time than the W / O / W emulsion. G / O GThe initial release rate of the fluorescein / water emulsion was 35.8% lower than that of the fluorescein solution. G / O G The initial release rate of the W / O / W emulsion was 23.1% lower than that of the W / O / W emulsion. G / O G This indicates that the drug / water emulsion has sustained release properties.

[0110] Example 14 As in Example 12, a W / O / W emulsion was prepared by dissolving 200 ng / mL rat albumin (Sigma-Aldrich) in the first aqueous phase, and the emulsion was then subjected to ultrasonic treatment at 75° C. for 15 seconds and allowed to stand at 4° C. for 30 minutes. A 10% BSA solution (Sera Care) was also diluted to prepare a 1% BSA solution, which was then stirred with ultrasonic waves under the same conditions as used to prepare the W / O / W emulsion and allowed to stand at 4° C. for 30 minutes. The rat albumin concentrations in the second aqueous phase of the W / O / W emulsion and the 1% BSA solution were then measured by ELISA.

[0111] Figure 16 shows the calibration curve obtained by serially diluting 1% BSA without ultrasonic agitation. Figure 17 also shows the calibration curve obtained by serially diluting 1% BSA with ultrasonic agitation. Without ultrasonic agitation, the calibration curve showed that the absorbance increased with increasing albumin concentration. However, with ultrasonic agitation, the calibration curve showed that the increase in absorbance was suppressed even when the albumin concentration before agitation increased, indicating that albumin was denatured by agitation. On the other hand, sufficient absorbance was measured in the second aqueous phase of the W / O / W emulsion, indicating that ultrasonic agitation did not significantly denaturate albumin. These results indicate that significant protein denaturation was suppressed by adding the protein to the first aqueous phase of the emulsion.

[0112] Example 15 Hepatocytes were isolated from male Sprague-Dawley rats (Japan SLC) aged 6 to 8 weeks and weighing 160 to 230 g. A two-stage collagenase perfusion method was used to isolate hepatocytes. The viability of hepatocytes, as determined by trypan blue exclusion, was approximately 90%.

[0113] Serum-free Dulbecco's modified Eagle's medium (DMEM, Funakoshi) supplemented with 0.05 mg / L epidermal growth factor (Funakoshi), 10 mg / L bovine pancreatic insulin (Sigma-Aldrich), 7.5 mg / L hydrocortisone (Sigma-Aldrich), and 60 mg / L L-proline (Sigma-Aldrich) was prepared, and the hepatocytes were suspended in the medium.

[0114] The same W as in Example 3 was used except that 2.52 mg / mL of a hydrophilic fluorescent dye (fluorescein, Novartis Pharma) was dissolved in the first aqueous phase. G An O / W emulsion and a fluorescein solution prepared by dissolving fluorescein in phosphate buffer solution (PBS, pH 7.4) were prepared.

[0115] Hepatocytes were plated on a collagen-coated 35 mm dish at 2.5 × 10 4 cells / cm 2 The cells were cultured at 37°C in 5% CO2, and the medium was changed 4 hours after seeding. Next, the first aqueous phase was diluted 100-fold with W-100 containing fluorescein. G 2 mL each of the / O / W emulsion and 100-fold diluted fluorescein solution was placed in a dish.

[0116] The cells were then cultured at 37°C for 1 hour, the medium was removed from the dish, and the cells were washed several times with PBS. Next, the cells were fixed with 10% neutral formalin buffer (Fujifilm Wako Pure Chemical Industries, Ltd.) and stained with Rhodamine B / Hoechst 33258 (Thermo Fisher Scientific) for 20 minutes, and then washed several times with PBS.

[0117] The cells were observed using a confocal laser microscope (TCS SP8, Leica). As a result, as shown in Figure 18, W containing fluorescein in the aqueous phase GIt was confirmed that cells cultured in a medium supplemented with the / O / W emulsion incorporated fluorescein.

[0118] (Example 16) The primary emulsion (W) prepared in Example 1 was G The primary emulsion (a 10 ...

[0119] A surfactant-containing aqueous solution was prepared by dissolving a nonionic hydrophilic surfactant (polyethylene hydrogenated castor oil-60, NIKKOL HCO-60, Nippon Surfactant Kogyo Co., Ltd.) at 50 mg / mL in PBS in a water bath at 75°C. G The oil and water emulsions were mixed at a ratio of 5:1 to obtain an oil-water mixture.

[0120] The container containing the oil-water mixture was placed on ice and stirred for 3 minutes and 3 seconds at 1 pulse / 0.7 seconds using an ultrasonic homogenizer (Sonifier 250, Branson) at an output amplitude of 70%. The ultrasonically treated oil-water mixture was left to stand in a refrigerator overnight to obtain a secondary emulsion (W emulsion) containing an ungelled oil phase in which the gelled first aqueous phase was dispersed, and a second aqueous phase in which the ungelled oil phase was dispersed. G An oil-in-water emulsion was prepared.

[0121] Human umbilical vein endothelial cells (HUVEC, RIKEN BioResource Research Center) were cultured at 3.4 × 10 4 cells / cm 2 The cells were seeded in a 96-well plate at a density of 1000 x g / ml and cultured in endothelial cell growth medium 2 (EGM-2, Lonza) at 37°C under 5% CO2. The medium was changed 24 hours after seeding, and after another 24 hours, the cells were incubated with a secondary emulsion (W GA secondary emulsion (a 0.1% / o / w emulsion) was added to the medium at a concentration of 0%, 0.1%, 1%, or 10%. 24 hours after the addition of the secondary emulsion, the cell viability was measured using a cell counting kit (CCK-8, Dojindo Laboratories). As a result, as shown in Figure 19, it was confirmed that the cells were viable. These results indicate that the secondary emulsion is not cytotoxic.

[0122] Example 17 The same W solution as in Example 5 was used except that 2.52 mg / mL of a hydrophilic fluorescent dye (fluorescein, Novartis Pharma) was dissolved in the first aqueous phase and the concentration of the nonionic hydrophobic surfactant was 150 mg / mL. G / O G A W / O / W emulsion and a W / O / W emulsion identical to those in Example 4 were prepared, except that 2.52 mg / mL of a hydrophilic fluorescent dye was dissolved in the first aqueous phase. In addition, a 2.52 mg / mL fluorescein solution was prepared by dissolving fluorescein in phosphate buffer (PBS, pH 7.4).

[0123] Six-week-old ICR male mice (Slc: ICR, Japan SLC) were treated with W G / O G Five microliters of each of the W / O / W emulsion, W / O / W emulsion, and fluorescein solution was carefully instilled into the eyes of the mice, and the mice were asked to blink 10 times after instillation. This procedure was repeated three times. The mice were then instilled twice daily for three consecutive days. On the third day, the mice were sacrificed and their eyes were removed.

[0124] The eyeballs were washed with PBS and embedded in OCT compound (Sakura Finetech Japan). The embedded material was frozen at -80°C and 20 μm sections were prepared using a microtome (CM 1860 UV, Leica Microsystems). The sections were placed on slides and observed under a fluorescence microscope (TCS SP8, Leica Microsystems). As a result, as shown in Figure 20, W G / O G Intense fluorescence was observed in the retinal layer of the eyeball of the mouse to which the / W emulsion was instilled.

[0125] The fluorescence intensity in the retinal layer was analyzed using ImageJ software. The fluorescence intensity in the retinal layer of the control mouse that did not receive eye drops was set to 1, and the relative fluorescence intensity in the retinal layer of the mouse that received eye drops was calculated. As a result, as shown in Figure 21, G / O G The fluorescence intensity of the retinal layer of the mice instilled with the W / O / W emulsion was significantly stronger than that of the mice instilled with the W / O / W emulsion or fluorescein solution. G / O G This indicates that by incorporating a hydrophilic drug into the first aqueous phase of a / W emulsion, the hydrophilic drug can be efficiently delivered to the posterior segment of the eye, such as the retina.

[0126] Reference Example 1 A nonionic hydrophilic surfactant (polyethylene hydrogenated castor oil-60, NIKKOL HCO-60, Nippon Surfactant Industries Co., Ltd.) was added to a phosphate buffer solution (PBS, pH 7.4) to a concentration of 50 mg / mL, and dissolved at 75°C in a thermostatic bath (AS ONE, THERMAX TM-1A) to form an aqueous phase.

[0127] Hydrophobic fluorescent dye coumarin-6 (Tokyo Chemical Industry Co., Ltd.) was added to castor oil (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 3 mg / mL. Furthermore, beeswax was added to the castor oil to a concentration of 5 wt %, and the mixture was dissolved at 75°C in a thermostatic bath (AS ONE, THERMAX TM-1A) to form an oil phase.

[0128] An ultrasonic homogenizer (Sonifier 250, Branson) was used in a thermostatic bath (AS ONE, THERMAX TM-1A) to stir the water phase and oil phase (water phase:oil phase = 7:1) at 75°C for 3 minutes at 1 pulse / second and 70% output amplitude to form an O / W emulsion encapsulating coumarin-6 in the oil phase. The O / W emulsion was allowed to stand at room temperature to gel the oil phase, resulting in an O / W emulsion encapsulating coumarin-6 in the gelled oil phase. G A water-in-water emulsion was formed.

[0129] (Reference Example 2) A hydrophobic fluorescent dye, coumarin-6 (Tokyo Chemical Industry Co., Ltd.), was added to castor oil (Fujifilm Wako Pure Chemical Industries, Ltd.) to a concentration of 3 mg / mL to prepare coumarin-6-added castor oil. Ｇ The coumarin-6-added castor oil was diluted 8 times with castor oil so that the concentration of coumarin-6 in the / W emulsion was the same as that in the coumarin-6-added castor oil.

[0130] (Reference Example 3) Slaughtered pig heads were purchased from Fukuoka Meat Sales Co., Ltd. Generally, the size of a pig's eyeball is about seven times the size of a mouse's eyeball and is approximately the same size as a human eyeball. 50 μL of the O2 solution containing coumarin-6 according to Reference Example 1 was poured into the eye of the first pig head. G The coumarin-6-added castor oil of Reference Example 2 was instilled into the eye of a second pig head at 10-minute intervals for a total of 6 times (for a total of 1 hour).

[0131] Immediately after administration, the eyeballs were removed from the heads of the first and second pigs, embedded in OCT compound (Sakura Finetech Japan), and frozen at -80°C. 20 μm sections of the eyeballs were prepared using a microtome (CM 1860 UV, Leica Microsystems). The sections were placed on slides and observed under a fluorescence microscope (TCS SP8, Leica Microsystems).

[0132] As shown in FIG. 22, the O containing coumarin-6 according to Reference Example 1 G In both the eyeballs to which the O / W emulsion and the eyeballs to which the coumarin-6-containing castor oil of Reference Example 2 were instilled, penetration of coumarin-6 into the corneal epithelial layer was confirmed in the anterior segment of the eye. However, as shown in FIG. G In both the eyeballs to which the / W emulsion was instilled and the eyeballs to which the coumarin-6-added castor oil of Reference Example 2 was instilled, penetration of coumarin-6 into the corneal epithelial layer was not confirmed in the posterior segment of the eye.

[0133] (Reference Example 4) A slaughtered pig's head was purchased from Fukuoka Meat Sales Co., Ltd. The flesh around the pig's eyes was pulled and fixed with tape to keep the eyes open. 2 mL of the O-ring containing coumarin-6 according to Reference Example 1 was injected into each of the four open eyes. G The pigs were given a 1:1 ratio (0.01:1) / (0.01:1) emulsion in water, with their open eyes kept immersed in the emulsion. 0, 1, 3, and 20 hours after the emulsion was given, the eyeballs were removed from the pig's head, and sections of the eyeballs were observed in the same manner as in Reference Example 3.

[0134] As shown in Figure 24, it was confirmed that coumarin-6 reached the posterior segment of the eye over time. Next, the fluorescence intensity from the sclera to the retina in the fluorescence images was analyzed using image software (ImageJ). The distance from the sclera to the retina was 1500 μm. As shown in Figure 25, it was confirmed that coumarin-6 penetrated from the sclera toward the retina over time, but coumarin-6 did not reach the retina.

[0135] Reference Example 5 At Rakuno Gakuen Educational Corporation and Rakuno Gakuen University, a surviving pig was anesthetized and subjected to muscle relaxation. Under anesthesia, the pig was placed on its side. 100 μL of the O2 solution containing coumarin-6 according to Reference Example 1 was added. G The pigs were euthanized immediately after the procedure, and the eyeballs were removed immediately after euthanasia and after being left to stand for 1 hour after euthanasia. Sections of the eyeballs were examined in the same manner as in Reference Example 3.

[0136] As shown in Figure 26, it was confirmed that coumarin-6 was delivered to the outer sclera of the posterior segment of the eye in both the eyeballs removed immediately after euthanasia and the eyeballs removed one hour after euthanasia. This result indicates that in living animals, the emulsion moves rapidly to the posterior segment of the eye after instillation due to the production of tears and the circulation of aqueous humor.

[0137] (Reference Example 6) At Rakuno Gakuen Educational Corporation and Rakuno Gakuen University, a surviving pig was anesthetized and subjected to muscle relaxation. Under anesthesia, the pig was placed on its side. The flesh around the eyes was pulled and fixed with tape to keep the eyes open. 600 μL of the O2 solution containing coumarin-6 according to Reference Example 1 was added. G The pigs were euthanized one hour after the administration of the emulsion, and the eyeballs were removed from the heads of the pigs immediately after euthanasia and one hour after euthanasia, and sections of the eyeballs were examined in the same manner as in Reference Example 3.

[0138] As shown in Figure 27, it was confirmed that coumarin-6 was delivered to the outer sclera of the posterior segment of the eye in both the eyeballs removed immediately after euthanasia and the eyeballs removed one hour after euthanasia. This result indicates that in living animals, when the eye is immersed in the emulsion, the emulsion rapidly moves to the posterior segment of the eye.

[0139] (Reference Example 7) The fluorescence intensity of the images of the posterior segment of the eye obtained in Reference Examples 5 and 6 was analyzed using image software (ImageJ), and the relative fluorescence intensity RI of each sample was calculated using the following formula: RI = (SI - CI) / CI. In the above formula, SI is the measured fluorescence intensity of each sample, and CI is the fluorescence intensity measured in the image of the control that was not given the emulsion. A graph of the calculated relative fluorescence intensity is shown in Figure 28. Between the eye drop method and the eye-covering method, the emulsion penetrated more deeply into the posterior segment of the eye with the eye-covering method. Furthermore, compared to immediately after eye drop or eye-covering, the emulsion penetrated more deeply into the posterior segment of the eye one hour after eye drop or eye-covering.

[0140] (Reference Example 8) Slaughtered pig heads were purchased from Fukuoka Meat Sales Co., Ltd. The pig eyeballs were removed and placed in a 50 mL screw-cap tube (SARSTEDT), and 6 mL of the O solution containing coumarin-6 according to Reference Example 1 was added thereto. G A corneal in-water emulsion was added to the eyeballs, and the eyeballs were immersed in the emulsion. The eyeballs were removed 0, 3, 6, 12, 18, and 24 hours after the start of immersion, and sections of the eyeballs were observed in the same manner as in Reference Example 3.

[0141] As shown in Figure 29, it was confirmed that coumarin-6 penetrated near the sclera toward the retina as time passed. Furthermore, as shown in Figure 30, it was confirmed that coumarin-6 penetrated near the retina as time passed. Using image software (ImageJ), the fluorescence intensity from the sclera to the retina in the fluorescence image was analyzed as shown in Figure 31. As a result, it was confirmed that the fluorescence intensity on the retina side increased as time passed, as shown in Figure 32.

[0142] As shown in Figure 33, the concentration of coumarin-6 and the fluorescence intensity of coumarin-6 are proportional to each other. The concentration of coumarin-6 in the retina was calculated from the fluorescence image shown in Figure 30, and as shown in Figure 34, the concentration of coumarin-6 in the retina reached approximately 5 μg / mL 18 hours after the eyeball was immersed in the emulsion. For the following reasons, if the coumarin-6 encapsulated in the oil phase of the emulsion is replaced with a therapeutic agent for eye disease, the therapeutic agent for eye disease is thought to reach the retina earlier than coumarin-6.

[0143] An example of an ophthalmic therapeutic agent is aflibercept. Assuming that the weight of a pig's retina is 0.14 g and the volume of the pig's retina is 1.37 mL, the effective concentration of aflibercept is 4.25 μg / mL. Here, coumarin-6 is hydrophobic, whereas aflibercept is hydrophilic. Hydrophilic substances penetrate the sclera more easily than hydrophobic substances. Therefore, the ophthalmic therapeutic agent reaches the retina in a shorter time than coumarin-6, and it is believed that the ophthalmic therapeutic agent reaches an effective concentration in the retina in less than 18 hours.

[0144] The emulsions used in Examples 1 and 3 to 8 contained two phases. Ｇ It is a water-in-water emulsion, but it contains three phases. G / O G / W emulsion or W / O G The outermost layer and diameter of the W emulsion are the same. G / O G / W emulsion or W / O GThe dynamics of the O / W emulsion Ｇ It is considered to be similar to a water-in-water emulsion.

[0145] Example 18 A surfactant-containing oil was prepared by dissolving a nonionic hydrophobic surfactant (ER-290, Ryoto Sugar Ester, registered trademark, Mitsubishi Chemical Foods Corporation) in isopropyl myristate (IPM, Fujifilm Wako Pure Chemical Industries, Ltd.) at 150 mg / mL by shaking at room temperature.

[0146] A crosslinker-containing aqueous solution was prepared by adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, Peptide Institute) to a post-addition concentration of 10 mg / mL and N-hydroxysuccinimide (NHS, Fujifilm Wako Pure Chemical Industries) to a post-addition concentration of 6 mg / mL to 2-morpholinoethanesulfonic acid monohydrate (MES) buffer solution (pH 5.6, Dojindo Laboratories).

[0147] A gelatin-containing aqueous solution was prepared by dissolving gelatin powder (pig skin gelatin type A, Sigma-Aldrich) in phosphate buffered saline (PBS) in an incubator so that the final concentration would be 5 wt% when the crosslinker was added. The crosslinker-containing aqueous solution and heparin sodium (Fujifilm Wako Pure Chemical Industries, Ltd.) with a post-addition concentration of 10 mg / mL were added to the gelatin-containing aqueous solution to obtain a crosslinker- and gelatin-containing aqueous solution.

[0148] A surfactant-containing oil and an aqueous solution containing a crosslinker and gelatin were mixed at a ratio of 2:1 to obtain an oil-water mixture. The container containing the oil-water mixture was placed on ice and stirred for 15 minutes with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds and 70% output amplitude. The ultrasonically treated oil-water mixture was left standing in a refrigerator overnight to obtain a primary emulsion (W emulsion) in which a gelled first aqueous phase was dispersed in the oil. G A 100% emulsion (a 100% emulsion in water / oil) was prepared.

[0149] A nonionic hydrophilic surfactant (polyethylene hydrogenated castor oil-60, NIKKOL HCO-60, Nippon Surfactant Kogyo Co., Ltd.) was dissolved in PBS in a water bath at 75°C to a concentration of 50 mg / mL to prepare an aqueous solution containing castor oil.

[0150] The primary emulsion was centrifuged at 3,500 rpm for 10 minutes using a centrifuge (KUBOTA 5100, Kubota Manufacturing Co., Ltd.), and the supernatant was collected. Beeswax was added to the collected supernatant to a final concentration of 2.5 wt% in the oil phase, and the resulting mixture was returned to the centrifuged primary emulsion. The primary emulsion was then stirred in a 75°C water bath with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / second for 10 seconds and at 70% output amplitude, yielding a primary emulsion in which the gelled first aqueous phase was re-dispersed in the oil.

[0151] The castor oil-containing aqueous solution and the primary emulsion were mixed at a ratio of 5:1 to prepare an oil-water mixture, which was then stirred with an ultrasonic homogenizer (Sonifier 250, Branson) at 1 pulse / 0.7 seconds for 3 minutes at 70% output amplitude. The ultrasonically treated oil-water mixture was left standing in a refrigerator overnight to obtain a secondary emulsion (W) containing a gelled oil phase in which the gelled first aqueous phase was dispersed, and a second aqueous phase in which the gelled oil phase was dispersed. G / O G A water-in-water emulsion was prepared.

[0152] Human retinal pigment epithelial cells (ARPE-19, American Type Culture Collection) were cultured at 3.1 × 10 ５ cells / cm 2 The cells were seeded onto a 96-well plate at a density of 100 μg / well and cultured in DMEM / Ham's F-12 medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS) and penicillin / streptomycin (Nacalai Tesque) at 37°C under 5% CO2. The medium was changed 24 hours after seeding, and after another 24 hours, the cells were incubated with the secondary emulsion (W G / O G Secondary emulsion (W emulsion) was added to the medium at a concentration of 0%, 0.1%, or 1%. 24 hours after the addition of the secondary emulsion, the cell viability was measured using a cell counting kit (CCK-8, Dojindo Laboratories). As a result, as shown in Figure 35, it was confirmed that the cells survived regardless of the emulsion concentration. These results indicate that the secondary emulsion (W emulsion) G / O GThese results indicate that secondary emulsions (in-water emulsions) do not have cytotoxicity. Generally, the concentration of secondary emulsion administered to a subject is less than 1%.

[0153] (Example 19) A secondary emulsion (W) containing a non-gelled oil phase in which a gelled first aqueous phase is dispersed, and a second aqueous phase in which the non-gelled oil phase is dispersed, was prepared in the same manner as in Example 3. G A first emulsion (a W / O / W emulsion) containing an ungelled oil phase in which a first ungelled aqueous phase is dispersed, and a second emulsion (a W / O / W emulsion) containing a second aqueous phase in which the ungelled oil phase is dispersed, both prepared in the same manner as in Example 4, were prepared.

[0154] A secondary emulsion (W) containing a gelled oil phase in which a gelled first aqueous phase is dispersed, and a second aqueous phase in which the gelled oil phase is dispersed, was prepared in the same manner as in Example 5. G / O G and a secondary emulsion (W / O emulsion) containing a gelled oil phase in which a non-gelled first aqueous phase is dispersed, and a second aqueous phase in which the gelled oil phase is dispersed, both prepared in the same manner as in Example 6. G A water-in-water emulsion was prepared.

[0155] The four types of secondary emulsions prepared were stored at 4°C, and on days 0, 1, 3, 7, 14, 30, 60, 90, and 270, samples were diluted 600-fold with PBS, and the average diameter and polydispersity index (PDI) of the particles contained in the diluted secondary emulsions were measured using a particle size measurement system (nanoSAQLA, Otsuka Electronics) that uses dynamic light scattering (DLS).

[0156] As a result, as shown in FIG. 36, the W / O / W emulsion and W G It was shown that the particle size of the O / W emulsion increased over time, and the particles aggregated and coalesced. G / O G W / O emulsion and W / O GIn the W / O / W emulsion, the particle size increase was moderate, and it was shown that the aggregation and coalescence of particles was suppressed. G Compared with the O / W emulsion, G / O G W / O emulsion and W / O G The polydispersity index (PDI) of the / W emulsion was small, indicating that the particle size distribution was narrow.

[0157] The four types of secondary emulsions stored in the tubes for 270 days were photographed from the side, and the depth of the entire solution in the tube and the depth of the emulsified layer in the tube were determined using image analysis software (ImageJ). Furthermore, the volume of the entire solution, V Ｅ The volume of the emulsified layer V is calculated from the depth of the emulsified layer. Ｃ Next, the stabilization index SI (%) of each of the four secondary emulsions was calculated using the following formula: SI = V Ｃ / V Ｅ The closer the stabilization index SI is to 100%, the more uniformly the emulsion is dispersed. Also, when the stabilization index SI is lower than 100%, it indicates that oil-water separation occurs due to a decrease in creaming, in which emulsified particles float to the surface. As shown in Figure 38, G / O G The SI value of the W / W emulsion was the highest, and the SI value of the W / O emulsion was the highest. G The SI value of the / W emulsion was the next highest, and G The SI value of the W / O / W emulsion was the next highest, and the SI value of the W / O / W emulsion was the lowest.

[0158] 10: First aqueous phase, 20: Oil phase, 30: Second aqueous phase

Claims

1. An eye medication comprising: a first aqueous phase; a gelled oil phase in which the first aqueous phase is dispersed; a second aqueous phase in which the oil phase is dispersed; and an eye disease therapeutic agent disposed within the first aqueous phase.

2. The eye drop of claim 1, wherein the first aqueous phase is gelled or the first aqueous phase is non-gelled.

3. The eye drop according to claim 1, wherein the eye disease treatment agent is hydrophilic.

4. The eye drop according to claim 1, wherein the first aqueous phase has a diameter of 25 nm or more and 150 nm or less.

5. The ophthalmic solution according to claim 1, wherein the diameter of the oil phase is 100 nm or more and 350 nm or less.

6. The ophthalmic solution of claim 1, wherein the first aqueous phase comprises gelatin.

7. The eye drop of claim 6, wherein the gelatin is crosslinked.

8. The eye drop of claim 1, wherein the oil phase comprises beeswax.

9. The eye medication of claim 1, which is directed to the posterior segment of the eye.

10. The eye medication of claim 1, which is administered to a subject by eye drop administration.

11. The eye medication of claim 1, which is administered to a subject by an eye-covering method.

12. A delivery agent for an eye disease therapeutic agent comprising: a first aqueous phase; a gelled oil phase in which the first aqueous phase is dispersed; and a second aqueous phase in which the oil phase is dispersed.

13. A method for producing an eye medication, comprising: mixing a first aqueous solution containing an eye disease therapeutic agent with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a first aqueous phase containing the eye disease therapeutic agent therein is dispersed; adding a gelling agent to the oil phase of the primary emulsion to gel the oil phase; and mixing the primary emulsion in which the oil phase has been gelled with a second aqueous solution containing a second surfactant to form a secondary emulsion containing a second aqueous phase in which the gelled oil phase is dispersed.

14. The method of claim 13, wherein the concentration of the first surfactant in the oil is 125 mg / mL or more.

15. The method of claim 13, wherein ultrasonic waves are applied to the mixture of the first aqueous solution and the oil when forming the primary emulsion.

16. The method for producing an ophthalmic drug according to claim 15, wherein the ultrasonic wave is applied for 15 minutes or more.

17. The method for producing an eye medication according to claim 13, wherein the concentration of the second surfactant in the second aqueous solution is 25 mg / mL or more.

18. The method of claim 13, further comprising applying ultrasound to the mixture of the primary emulsion and the second aqueous solution when forming the secondary emulsion.

19. The method for producing an eye drug according to claim 18, wherein the ultrasonic wave is applied for 10 minutes or more.

20. A method for producing a delivery agent for an eye disease therapeutic agent, comprising: mixing a first aqueous solution with an oil containing a first surfactant to form a primary emulsion containing an oil phase in which a first aqueous phase is dispersed; adding a gelling agent to the oil phase of the primary emulsion to gel the oil phase; and mixing the primary emulsion in which the oil phase has been gelled with a second aqueous solution containing a second surfactant to form a secondary emulsion containing a second aqueous phase in which the gelled oil phase is dispersed.

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