JP7641566B2 - Eye drops containing ultrafine, poorly water-soluble drugs and water-soluble drugs, and methods for producing and using the same - Google Patents

Description

The present invention relates to an eye drop preparation containing a mixture of an ultrafine, poorly water-soluble drug and a water-soluble drug; a method for producing an eye drop preparation comprising mixing an ultrafine, poorly water-soluble drug and a water-soluble drug; and a method for improving the corneal permeability of a poorly water-soluble drug and a water-soluble drug using an eye drop preparation containing an ultrafine, poorly water-soluble drug and a water-soluble drug.

Eye diseases such as cataracts and glaucoma are treated with a combination of multiple drugs. In addition, when surgery is performed for eye diseases such as cataracts and retinal diseases, multiple types of eye drops are generally used for postoperative management, one for each drug, such as antibiotics and anti-inflammatory drugs.

When eye drops are used to treat and prevent eye diseases, they are applied several times a day, and the more types of eye drops used, the more frequently the patient must apply them, placing a greater burden on the patient. In particular, the majority of patients with eye diseases such as cataracts and glaucoma are elderly by nature, and the increased burden of applying eye drops can lead to a decrease in adherence to eye drops (voluntary compliance with treatment).

In order to reduce the burden on patients and the number of times that instillation is required, eye drops containing a mixture of multiple drugs have been considered. However, some drugs are water-soluble and others are poorly soluble. When poorly water-soluble drugs are used, the eye drops are in the form of a suspension, which makes it difficult to make a uniform solution, and the dispersion stability of the drug tends to be low. Methods for increasing the dispersion stability or solubilization rate of a drug in eye drops containing poorly water-soluble drugs are described in Patent Document 1 and Non-Patent Document 1.

Special Publication No. 2020-534321

Noriaki Nagai et al., Toxicology, 319 (2014), pp.53-62

However, when a poorly water-soluble drug is contained, or when a poorly water-soluble drug and a water-soluble drug are both contained, little is known about a method for making the entire solution into a homogeneous suspension, or about an eye drop that maintains the dispersion stability of the poorly water-soluble drug and the water-soluble drug.

The problem that the present invention aims to solve is to provide an eye drop that contains a poorly water-soluble drug and a water-soluble drug while maintaining a homogeneous suspension state, and a method for producing the same.

As a result of intensive research to solve the above problems, the inventors focused on the miscibility of poorly water-soluble drugs and water-soluble drugs, and came to the conclusion that ultra-fine particle size reduction of poorly water-soluble drugs is a possible way to improve the miscibility. In this case, by ultra-fine particle size reduction of poorly water-soluble drugs in the presence of an inclusion compound, it was possible to suppress aggregation between poorly water-soluble drugs and the associated precipitation. Based on this knowledge, the inventors succeeded in producing an eye drop containing ultra-fine particle size reduction of poorly water-soluble drugs and water-soluble drugs.

Moreover, it was surprisingly found that when the obtained eye drops were instilled into the eye, the corneal permeability could be improved not only for poorly water-soluble drugs but also for water-soluble drugs. Based on this knowledge, the inventors have succeeded in creating an eye drop containing ultrafinely divided poorly water-soluble drugs and water-soluble drugs, which can solve the problems of the present invention. The present invention has been completed based on these knowledge and successful examples.

That is, according to the present invention, there are provided eye drops and methods having the following aspects:
[1] An eye drop preparation comprising a nanosized poorly water-soluble drug and a water-soluble drug, and which is a homogeneous suspension after being allowed to stand for 7 days.
[2] The eye drop according to [1], wherein the nanosized poorly water-soluble drug has an average particle size of 10 nm to 400 nm.
[3] The eye drop preparation according to any one of [1] to [2], further comprising a suspending auxiliary clathrate compound.
[4] The eye drop preparation according to [3], wherein the suspending aid is at least one suspending aid selected from the group consisting of inclusion compounds, surfactants, sugars, sugar alcohols, water-soluble polymers, and amino acids.
[5] The eye drop preparation according to [4], wherein the inclusion compound is at least one cyclodextrin selected from the group consisting of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin.
[6] A step of obtaining a nano-sized poorly water-soluble drug by subjecting a poorly water-soluble drug to ultrafine processing;
and mixing the nanosized poorly water-soluble drug with a water-soluble drug to obtain an eye drop comprising the nanosized poorly water-soluble drug and the water-soluble drug.
[7] The method according to [6], wherein the ultrafine-pulverizing treatment is an ultrafine-pulverizing treatment in the presence of a suspension auxiliary.
[8] A method for improving the corneal permeability of a poorly water-soluble drug and a water-soluble drug, comprising a step of contacting the cornea with an eye drop containing a nanosized poorly water-soluble drug and a water-soluble drug, thereby improving the corneal permeability of the water-soluble drug and the water-soluble drug compared to contacting the cornea with an eye drop containing a non-nanosized poorly water-soluble drug and a water-soluble drug.

According to one aspect of the present invention, the eye drop contains an ultrafinely divided poorly water-soluble drug and a water-soluble drug, and thus the dispersion stability of the drug is high and it is possible to maintain a homogeneous suspension state. In addition, according to the present invention, the permeability to the cornea can be increased for both poorly water-soluble drugs and water-soluble drugs, which allows the pharmacological action of the drug to be effectively applied to ocular tissues, and is expected to treat and prevent eye diseases such as cataracts and glaucoma, which are the targets of the drugs.

As described in the Examples below, Figure 1A is a diagram showing the average particle size of commercially available fluorometholone bulk powder measured by a laser scattering method; Figure 1B is a diagram showing the average particle size of nanoized fluorometholone measured by a laser scattering method; Figure 1C is a diagram showing the average particle size of nanoized fluorometholone measured by a nanotracking method; and Figure 1D is an image of nanoized fluorometholone particles photographed by a scanning probe microscope. As described in the Examples below, Fig. 2A shows the results of measuring fluorometholone concentration over time; Fig. 2B shows the results of measuring rebamipide concentration over time; Fig. 2C is a photograph of a glass tube containing a test mixed ophthalmic solution; and Fig. 2D shows the number of nanosized fluorometholone particles with an average particle size of 1 μm or less in the mixed ophthalmic solution FBL-NPs 0 days and 1 month after standing. In the figures, "*1" indicates P<0.05 vs. CA-FL, and "*2" indicates P<0.05 vs. FBL-MPs. FIG. 3 is a schematic diagram of an apparatus used to evaluate the in vitro corneal permeability of a poorly water-soluble drug (fluorometholone) in a test mixed eye drop, as described in the Examples below. FIG. 4 shows the results of evaluation of in vitro corneal permeability of a poorly water-soluble drug (fluorometholone) in a test mixed eye drop, as described in the Examples below. FIG. 5 is a schematic diagram of a test system for evaluating the in vivo corneal permeability of a poorly water-soluble drug and a water-soluble drug in a test mixed eye drop, as described in the Examples below. 6A shows the results of evaluation of in vivo corneal permeability of a poorly water-soluble drug (fluorometholone) in the test mixed eye drop; FIG. 6B shows the results of evaluation of in vivo corneal permeability of a water-soluble drug (levofloxacin) in the test mixed eye drop; and FIG. 6C shows the results of evaluation of in vivo corneal permeability of a water-soluble drug (bromfenac sodium) in the test mixed eye drop, as described in the Examples described below.

Each aspect of the present invention will be described in detail below, but the present invention is not limited to the items in this section, and various aspects may be adopted as long as the object of the present invention is achieved.

Unless otherwise specified, the terms used in this specification are used in the sense commonly used by those skilled in the art who deal with pharmaceuticals, including eye drops, and should not be construed as having an unduly restrictive meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

"Content" is synonymous with concentration and means the ratio of the amount of a component to the total amount of a solution. However, the total amount of the content of the components does not exceed 100%. In this specification, unless otherwise specified, the unit of content means "mass% (wt%)". In addition, when a commercially available product is used, the content of the component is preferably the amount of the component contained in the commercially available product, but may be the amount of the commercially available product itself.
The term "and/or" means any one or any or all combinations of two or more of the associated listed items.
In a numerical range, "to" means a range that includes the numerical values before and after it, for example, "0% to 100%" means a range that is equal to or greater than 0% and equal to or less than 100%. "More than" and "less than" mean a lower limit and an upper limit, respectively, without including the numerical value before it, for example, "more than 1" means a numerical value greater than 1, and "less than 100" means a numerical value less than 100.
"Comprising" means that elements other than the elements explicitly stated as being included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the elements explicitly stated and any one or more elements, consisting of the elements explicitly stated, or consisting essentially of the elements explicitly stated. Elements include limitations such as ingredients, steps, conditions, and parameters.

The eye drop of one embodiment of the present invention contains a nano-sized poorly water-soluble drug and a water-soluble drug. The eye drop of one embodiment of the present invention is a homogeneous suspension after 7 days of standing.

Nano-sized poorly water-soluble drugs are poorly water-soluble drugs with an average particle size of less than 1 μm (nano level). Poorly water-soluble drugs are compounds that are classified as "hardly soluble (solvent volume required to dissolve 1 g or 1 mL of solute is 100 mL or more) to "hardly soluble (solvent volume required to dissolve 1 g or 1 mL of solute is 10,000 mL or more)" according to the solubility measurement method specified in the Japanese Pharmacopoeia. In contrast, water-soluble drugs are compounds that are classified as "extremely soluble (less than 1 mL) to "slightly soluble (less than 100 mL)" according to the solubility measurement method specified in the Japanese Pharmacopoeia. Note that solubility refers to the degree to which a drug dissolves within 30 minutes after powdering it, placing it in water, and shaking vigorously for 30 seconds every 5 minutes at 20±5°C, as specified in the Japanese Pharmacopoeia No. 17.

The poorly water-soluble drug and the water-soluble drug may be either one produced by a known method or commercially available, so long as they are used as active ingredients in eye drops. Non-limiting examples of poorly water-soluble drugs include fluorometholone, rebamipide, indomethacin, brinzolamide, triamcinolone acetonide, pirenoxine, brinzolamide, latanoprost, and isopropyl unoprostone. Non-limiting examples of water-soluble drugs include bromfenac sodium, levofloxacin, and timolol maleate. The poorly water-soluble drug and the water-soluble drug may be one of the above drugs alone or a combination of two or more of them.

If the poorly water-soluble drug manufactured or commercially available is at the nano level, it can be used as a nano-sized poorly water-soluble drug. On the other hand, typical commercially available poorly water-soluble drugs have an average particle size of 1 μm or more (micro-level). Therefore, if the poorly water-soluble drug is at the micro-level, the poorly water-soluble drug is subjected to ultrafine processing to obtain a nano-sized poorly water-soluble drug.

The ultrafine processing of poorly water-soluble drugs is not particularly limited as long as it does not significantly affect the pharmacological action of the poorly water-soluble drug, but examples include a process of physically grinding using a device such as a nano-pulverizer, and a process of repeatedly crushing using a bead mill using metal beads and centrifuging. The ultrafine processing may be either a dry grinding process in which a powder of the poorly water-soluble drug and a powder of an optional component are added to a grinding container and ground, or a wet grinding process in which such grinding is carried out in the presence of water, but a wet grinding process is preferable in order to improve the mixing of the poorly water-soluble drug with water-soluble components such as the water-soluble drug.

When performing ultrafine processing of poorly water-soluble drugs, it is preferable to add a suspension aid. By adding a suspension aid, the dispersibility of the poorly water-soluble drug in the suspension can be improved. When subjecting a poorly water-soluble drug to ultrafine processing by wet grinding, it is preferable to add the poorly water-soluble drug to an aqueous solution containing a suspension aid and then subject it to wet grinding.

The suspension aid is not particularly limited as long as it improves the dispersibility of poorly water-soluble drugs in aqueous solutions. Examples of the suspension aid include inclusion compounds such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, 2-hydroxypropyl-β-cyclodextrin, and methyl-β-cyclodextrin; surfactants such as docusate sodium, benzalkonium chloride, and sodium lauryl sulfate; water-soluble polymers such as carboxyvinyl polymers, hydroxyethyl cellulose, hydroxypropyl methylcellulose, methylcellulose, hydroxypropyl cellulose, alginic acid and its salts, polyvinyl alcohol, polyvinylpyrrolidone, macrogol, chondroitin sulfate and its salts, and hyaluronic acid and its salts; and amino acids such as glycine, leucine, and isoleucine. The suspension aid may be one produced by a known method or one that is commercially available. The suspension aid may be one of the above-mentioned ones alone or two or more of them in combination.

Among the above-mentioned suspension aids, inclusion compounds are more preferable, and 2-hydroxypropyl-β-cyclodextrin is even more preferable, because they can disperse poorly water-soluble drugs better in the eye drops. When an inclusion compound such as 2-hydroxypropyl-β-cyclodextrin is used, the nanosized poorly water-soluble drug is included in the inclusion compound, suppressing aggregation that occurs between the poorly water-soluble drug and the water-soluble drug and any components in the eye drops, as well as precipitation that accompanies the aggregation, and can improve the dispersibility of the poorly water-soluble drug while improving its miscibility with the water-soluble drug. In addition, when instilled in the eye, the corneal permeability of the water-soluble drug can be improved together with the poorly water-soluble drug.

The suspending aid is preferably added before carrying out the ultrafine processing of the poorly water-soluble drug, but may also be added after the ultrafine processing of the poorly water-soluble drug and mixed with the nano-sized poorly water-soluble drug.

Non-limiting examples of ultrafine particle size reduction of poorly water-soluble drugs are as follows.
A mixture of a powder of a poorly water-soluble drug and a powder of an optional ingredient is mixed while being ground, and then the resulting drug mixture is pulverized in a bead mill (e.g., 2,000 rpm to 4,000 rpm, 10 seconds to 5 minutes, room temperature conditions) using metal beads (e.g., zirconia beads) having a diameter of about several mm, preferably 1 mm to 3 mm, to obtain a fine drug powder. The resulting fine drug powder is then suspended in an aqueous solution containing a suspension aid to obtain a suspension containing a poorly water-soluble drug. Next, the obtained suspension containing the poorly water-soluble drug, with the poorly water-soluble drug dispersed therein, is subjected to crushing in a bead mill using metal beads (e.g., zirconia beads) having a diameter of 1 mm or less, preferably 0.05 mm to 0.5 mm (e.g., conditions of 4,000 rpm to 6,500 rpm, 10 seconds to 5 minutes, about 4°C) and centrifugation (e.g., conditions of 500 g to 1,000 g, 30 seconds to 2 minutes, about 4°C) several tens of times, preferably about 30 times, to obtain a dispersion containing a nanosized poorly water-soluble drug.

In the above specific example, ultrafine particle size reduction is performed using a bead mill with metal beads, but when performing this on an industrial scale, any suitable drug ultrafine particle size reduction method can be used.

The average particle size of the nano-sized poorly water-soluble drug is not particularly limited as long as it is at the nano level, i.e., less than 1 μm. For example, when the size of corneal endothelial cells is 400 nm and the permeability of the poorly water-soluble drug through the cornea is taken into consideration, the average particle size is preferably 10 nm to 400 nm, more preferably 20 nm to 300 nm, and even more preferably 50 nm to 200 nm. The average particle size of the nano-sized poorly water-soluble drug is a value measured on a number basis by a laser scattering method using a laser diffraction particle size distribution measuring device, as described in the Examples below. Note that since the water-soluble drug is dissolved in the eye drop, its average particle size is not particularly limited.

The contents of the nanosized poorly water-soluble drug, water-soluble drug, and suspending aid in the eye drop are not particularly limited, and may be appropriately set according to, for example, the desired efficacy and safety of the drug, and the solubility and average particle size of the nanosized poorly water-soluble drug.

Non-limiting preferred specific embodiments of the eye drops of the present invention include those in which the total contents of the nanosized poorly water-soluble drug, the water-soluble drug, and the suspension auxiliary are as follows, but the present invention is not limited thereto.
[Specific embodiment 1 of eye drops]
(1) Nanosized poorly water-soluble drug: 0.01% (w/v) to 5.0% (w/v)
(2) Water-soluble drugs: 0.01% (w/v) to 5.0% (w/v)
[Specific embodiment 2 of eye drops]
(1) Nanosized poorly water-soluble drug: 0.01% (w/v) to 5.0% (w/v)
(2) Water-soluble drugs: 0.01% (w/v) to 5.0% (w/v)
(3) Suspension aid: 0.1% (w/v) to 10% (w/v)
[Specific embodiment 3 of eye drops]
(1) Nanosized poorly water-soluble drug: 0.05% (w/v) to 3.0% (w/v)
(2) Water-soluble drugs: 0.1% (w/v) to 1.0% (w/v)
(3) Suspension aid: 3% (w/v) to 7% (w/v)

The eye drop of one embodiment of the present invention is a homogeneous suspension containing a nano-sized poorly water-soluble drug in a well-dispersed state. The homogeneity of the eye drop of one embodiment of the present invention is evaluated by whether it is a homogeneous suspension on the 7th day after standing. "It is a homogeneous suspension on the 7th day after standing" is evaluated by either or, preferably, both of the following: when the eye drop of one embodiment of the present invention is placed in a glass tube and left standing at room temperature in a dark place for 7 days, the homogeneous suspension is visually maintained in a uniformly dispersed state, and no obvious sedimentation or clear part of the drug is confirmed, and when the concentration of the poorly water-soluble drug is measured by HPLC after sampling at 90% of the liquid level, 80% or more of the initial concentration is measured. The eye drop of one embodiment of the present invention may be a homogeneous suspension on the 7th day after standing, but in order to maintain a homogeneous state, it is preferably a homogeneous suspension on the 14th day after standing, more preferably a homogeneous suspension on the 21st day after standing, and even more preferably a homogeneous suspension on the 28th day after standing.

The eye drop of one embodiment of the present invention may contain various other components in addition to the nano-sized poorly water-soluble drug, the water-soluble drug, and the suspension aid, so long as it does not interfere with the solution of the problems of the present invention. A typical example of the other components is water, since the eye drop of one embodiment of the present invention is a suspension. Examples of other components other than water include additives that are commonly used in the manufacture of eye drops, such as bases (such as aqueous ethanol); preservatives (such as benzalkonium chloride, sodium benzoate, ethanol, chlorobutanol, sorbic acid, potassium sorbate, sodium dehydroacetate, methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, oxyquinoline sulfate, phenethyl alcohol, benzyl alcohol, and biguanide compounds); pH adjusters (such as hydrochloric acid, boric acid, aminoethylsulfonic acid, epsilon-aminocaproic acid, citric acid, acetic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, sodium bicarbonate, sodium carbonate, borax, triethanolamine, monoethanolamine, diisopropanolamine, sulfuric acid, phosphoric acid, polyphosphoric acid, propionic acid, oxalic acid, gluconic acid, fumaric acid, lactic acid, tartaric acid, malic acid, and succinic acid, gluconolactone, ammonium acetate, etc.); isotonicity agents (sodium hydrogen sulfite, sodium sulfite, potassium chloride, calcium chloride, sodium chloride, magnesium chloride, potassium acetate, sodium acetate, sodium hydrogen carbonate, sodium carbonate, sodium thiosulfate, magnesium sulfate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, glycerin, propylene glycol, etc.); stabilizers (dibutylhydroxytoluene, trometamol, tocopherol, sodium pyrosulfite, monoethanolamine, aluminum monostearate, glycerin monostearate, etc.); fragrances (menthol, anethole, eugenol, camphor, geraniol, cineol, borneol, limonene, rhubarb, etc.); essential oils (mentha oil, cool mint oil, spearmint oil, peppermint oil, fennel oil, cinnamon oil, bergamot oil, eucalyptus oil, rose oil, etc.), but are not limited to these. Some of the above compounds also function as suspension aids, and in that case they may be added to the formulation to function as suspension aids as well.

The eye drop of one embodiment of the present invention has the property of being in the form of a suspension used for the purpose of instillation. Other properties of the eye drop of one embodiment of the present invention are not particularly limited as long as they are within the range acceptable for pharmaceutical use. For example, the pH is preferably 5.0 to 8.0, and more preferably 6.0 to 7.0; the viscosity is preferably 10 mPa·s or less, and more preferably 0.5 mPa·s to 2.0 mPa·s; and the osmotic pressure ratio is preferably 0.7 to 1.6. The viscosity is a value obtained by measurement according to the method described in the Examples below, and the pH and osmotic pressure ratio may be measured according to known methods.

The eye drops of one embodiment of the present invention are preferably instilled at one or several drops per eye once a day or in divided doses several times a day, and more preferably at one or two drops per eye once a day or in divided doses twice a day. When the eye drops of one embodiment of the present invention are instilled in divided doses several times a day, the interval between instillations is preferably one hour or more, more preferably two hours or more, and even more preferably three hours or more. The volume of one drop is not particularly limited as long as it is a commonly used amount, but is preferably about 0.01 mL to about 0.1 mL, and more preferably about 0.02 mL to about 0.05 mL, for example.

The eye drops of one embodiment of the present invention may be applied directly to the naked eye, but because the drug has good corneal permeability, they can also be applied when wearing contact lenses. The contact lenses may be either hard or soft contact lenses, and can be applied to all types of commercially available contact lenses, such as repeatedly used contact lenses, daily disposable contact lenses, weekly disposable contact lenses, biweekly disposable contact lenses, and colored contact lenses.

When the eye drop of one embodiment of the present invention is distributed on the market, it is preferably a container-packed eye drop. The container is not particularly limited as long as it is a container normally used for eye drops, and examples thereof include a multi-dose container, a single-use unit dose container, and a PFMD (Preservative Free Multi Dose) container. The material of the container is not particularly limited as long as it is a material generally used for eye drop containers, and examples thereof include resins, and specific examples thereof include resins such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polypropylene-polyethylene copolymer, polyvinyl chloride, acrylic, polystyrene, and polycyclic olefin copolymer.

Another aspect of the present invention is a method for producing the eye drop of the aspect of the present invention. The production method of the aspect of the present invention includes a step of obtaining a nano-sized poorly water-soluble drug by subjecting a poorly water-soluble drug to ultrafine processing (hereinafter also referred to as step 1), and a step of obtaining an eye drop containing a nano-sized poorly water-soluble drug and a water-soluble drug by mixing the nano-sized poorly water-soluble drug with a water-soluble drug (hereinafter also referred to as step 2).

In step 1, it is preferable to ultrafine the poorly water-soluble drug in the presence of a suspending aid. That is, as described above, it is preferable to add a mixture containing the poorly water-soluble drug to an aqueous solution containing a suspending aid, and then subject the mixture to a wet grinding process to carry out the ultrafine processing.

In step 2, the method for mixing the nanosized poorly water-soluble drug with the water-soluble drug is not particularly limited, and examples include adding the water-soluble drug to an aqueous solution containing the nanosized poorly water-soluble drug and mixing until a homogenous suspension is obtained.

In addition to steps 1 and 2, the manufacturing method of one embodiment of the present invention can include additional steps, such as a step of adjusting the pH, viscosity, osmotic pressure, etc., to within a predetermined range, and a step of heat sterilization, as necessary.

According to one embodiment of the manufacturing method of the present invention, the solubility of poorly water-soluble drugs is improved, the miscibility with water-soluble drugs is improved, and drug aggregation and precipitation can be suppressed, so that an eye drop with high uniform dispersion can be obtained, and as a result, an eye drop in which unevenness in drug concentration between drops can be eliminated.

Another aspect of the present invention is a method for improving the corneal permeability of poorly water-soluble drugs and water-soluble drugs using an eye drop according to an aspect of the present invention. The improvement method according to an aspect of the present invention includes a step of improving the corneal permeability of poorly water-soluble drugs and water-soluble drugs by contacting the cornea with an eye drop containing nanoized poorly water-soluble drugs and water-soluble drugs, compared to contacting the cornea with an eye drop containing non-nanoized poorly water-soluble drugs and water-soluble drugs.

In the method of one aspect of the present invention, the nano-sized poorly water-soluble drug is preferably a nano-sized poorly water-soluble drug obtained by subjecting the poorly water-soluble drug to ultra-fine processing in the presence of a suspending aid. In this case, the ultra-fine size of the poorly water-soluble drug and the improvement in the solubility of the poorly water-soluble drug by the suspending aid work together to increase the adhesion and retention of the poorly water-soluble drug on the cornea, which is expected to improve the efficacy of the drug in the cornea. In addition, corneal permeability due to the active transport mechanism is increased not only for poorly water-soluble drugs but also for water-soluble drugs, improving the migration of both of these drugs into the eye.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the problem of the present invention can be solved.

In this example, the "%" indicating the content of a component indicates "mass volume percent concentration (w/v%)". Statistical analysis was performed using unpaired Student's t-test for comparison between two groups, and Dunnett's multiple comparison for comparison between multiple groups. A significant difference was determined when p<0.05.

Fluorometholone and rebamipide were used as poorly water-soluble drugs. Levofloxacin, bromfenac sodium, and timolol maleate were used as water-soluble drugs. 2-hydroxypropyl-β-cyclodextrin was used as cyclodextrin. Using these, mixed eye drops containing two types of drugs, a finely divided poorly water-soluble drug and a water-soluble drug, were prepared, and the degree of dispersion and dispersion stability of the poorly water-soluble drug in the mixed eye drops, as well as the corneal permeability, were evaluated.

[Preparation of test mixed eye drops]
1. Suspension containing nanosized fluorometholone, bromfenac sodium, and levofloxacin (FBL-NPs)
0.2 g of fluorometholone (FL) bulk powder (average particle size: 4 μm to 5 μm), 0.0004 g of benzalcohol chloride (BAC), 0.04 g of D-mannitol, and 0.2 g of methylcellulose (MC) were ground in an agate mortar for about 1 hour to obtain a drug mixture. The resulting drug mixture was then placed in a 2.0 mL tube containing 2.0 g of zirconia beads with a diameter of 2.0 mm, and milled using a bead mill (3,000 rpm, 30 seconds, with cooling; manufactured by Wakenyaku Co., Ltd.) to obtain a fine drug powder. The resulting fine drug powder was suspended in a 5% aqueous solution of 2-hydroxypropyl-β-cyclodextrin (HPβCD) to prepare a 0.5% fluorometholone-containing suspension.

The obtained 0.5% fluorometholone-containing suspension was dispersed in a tabletop ultrasonic cleaner to disperse the components in the liquid, and then poured into a 2.0 mL tube containing 2 g of zirconia beads with a diameter of 0.1 mm until the tube was filled to about 80%. The suspension was crushed in a bead mill (5,500 rpm, 30 seconds, 4°C) and centrifuged (800 g, 60 seconds, 4°C) 30 times to prepare a fluorometholone nanoparticle-containing dispersion (FL-NPs).

Bromfenac sodium (BF) and levofloxacin (LV) were further dissolved in the obtained FL-NPs to prepare a suspension (FBL-NPs) containing final concentrations of 0.1% FL, 0.001% BAC, 0.1% D-mannitol, 5% HPβCD, 0.5% MC, 0.1% BF, and 0.5% LV.

The pH of FBL-NPs was measured at room temperature using a pH meter (main body: Seven Compact S220, electrode: InLab MicroPro-ISM) (manufactured by Mettler Toledo) and was found to be approximately 6.5.

The viscosity of FBL-NPs was measured using a spindle rotor method with an RB80 type viscometer (manufactured by Toki Sangyo Co., Ltd.) by adjusting 20 mL of sample to 20°C, immersing a BL adapter (code No. 10) in the sample, and measuring at 60 rpm for 3 minutes. The viscosity was 1.2 mPa·s to 1.7 mPa·s.

2. Suspension containing nanosized rebamipide and timolol maleate (TM/REB-NPs)
In the same manner as in the preparation of FBL-NPs, a suspension containing 2% REB, 0.5% TM, 0.001% BAC, 0.1% D-mannitol, 5% HPβCD and 0.5% MC as final concentrations (TM/REB-NPs) was prepared using bulk rebamipide (REB) powder (average particle size: 0.05 μm to 0.2 μm) as the poorly water-soluble drug and timolol maleate (TM) as the water-soluble drug.

3. Suspension containing fluorometholone, bromfenac sodium and levofloxacin (FBL-MPs)
0.2 g of FL bulk powder, 0.0004 g of BAC, 0.04 g of D-mannitol, and 0.2 g of MC were ground in an agate mortar for about 1 hour to obtain a drug mixture. The obtained drug mixture was then placed in a 2.0 mL tube containing 2.0 g of zirconia beads with a diameter of 2.0 mm, and milled using a bead mill (3,000 rpm, 30 seconds, with cooling; manufactured by Wakenyaku Co., Ltd.) to obtain a fine drug powder. The obtained fine drug powder was suspended in a 5% HPβCD aqueous solution to prepare a 0.5% fluorometholone-containing suspension.

The components of the resulting 0.5% fluorometholone-containing suspension were dispersed using a tabletop ultrasonic cleaner, and BF and LV were further dissolved to prepare a suspension (FBL-MPs) in which the final concentration of each component was the same as that of FBL-NPs and in which FL was not nanosized.

4. Suspension containing rebamipide and timolol maleate (TM/REB-MPs)
In a manner similar to that for preparing FBL-MPs, REB was used as the poorly water-soluble drug and TM was used as the water-soluble drug to prepare a suspension (TM/REB-MPs) containing final concentrations of 2% REB, 0.5% TM, 0.001% BAC, 0.1% D-mannitol, 5% HPβCD, and 0.5% MC.

5. Commercially available eye drops (CA-FL, CA-BF, CA-LV, CA-REB)
As a commercially available FL eye drop, "Flumetholone eye drop 0.1%" (0.1% FL; manufactured by Santen Pharmaceutical Co., Ltd.) was used (CA-FL), as a commercially available BF eye drop, "Blonac eye drop 0.1%" (0.1% BF; manufactured by Senju Pharmaceutical Co., Ltd.) was used (CA-BF), as a commercially available LV eye drop, "Cravit eye drop" (0.5% LV; manufactured by Santen Pharmaceutical Co., Ltd.) was used (CA-LV), and as a commercially available REB eye drop, "Mucosta eye drop UD 2%" (2% REB; manufactured by Otsuka Pharmaceutical Co., Ltd.) was used (CA-REB).

[Measurement of average particle size of poorly water-soluble drugs in test mixed ophthalmic solutions]
The average particle size of fluorometholone in FBL-MPs and FBL-NPs was measured on a number basis by a laser scattering method using a laser diffraction particle size distribution analyzer "SALD-7100" (manufactured by Shimadzu Corporation). The measurement results for bulk fluorometholone in FBL-MPs are shown in Figure 1A, and the measurement results for nanoized fluorometholone in FBL-NPs are shown in Figure 1B. As these results show, the average particle size of bulk fluorometholone was 3.98 μm, while the average particle size of nanoized fluorometholone was 74 nm, confirming that it was sufficiently finely divided.

The nanosized fluorometholone was also measured by number using a nanotracking particle size measurement device "NANOSIGHT LM10" (Malvern). The results are shown in Figure 1C. As shown in Figure 1C, the average particle size of the nanosized fluorometholone was 119 nm, and it was found to be nano-sized by the nanotracking method as well.

Furthermore, the results of photographing nano-sized fluorometholone using a scanning probe microscope "SPM-9700" (Shimadzu Corporation) are shown in Figure 1D. As shown in Figure 1D, the image also shows that the nano-sized fluorometholone is nano-sized.

[Measurement of the degree of dispersion of poorly water-soluble drugs in test mixed ophthalmic solutions]
10 mL of FBL-NPs and 10 mL of FBL-MPs were each taken and centrifuged at a relative centrifugal force of 100,000 g for 60 minutes using a centrifuge "Optima MAX-MP" (manufactured by BECKMAN COULTER) to collect the supernatant. The concentration of fluorometholone in the obtained supernatant was measured by high performance liquid chromatography according to a conventional method.

The measurement results confirmed that FBL-NPs containing nanosized fluorometholone had a higher concentration of fluorometholone in the supernatant and a higher degree of dispersion in the mixed eye drops than FBL-MPs containing bulk fluorometholone.

[Measurement of dispersion stability of poorly water-soluble drugs in test mixed eye drops]
20 mL of FBL-NPs, 20 mL of FBL-MPs and 20 mL of CA-FL were each dispensed into a glass tube and allowed to stand in a dark place at room temperature. At this time, the height of the liquid surface in the glass tube was 4 cm from the bottom. At the start of the standing (0 d) and on the 1st, 4th, 7th, 14th, 21st and 28th days (1M) after the standing, 50 μL of samples were taken from each glass tube at 0.4 cm (90% part) from the liquid surface, and the fluorometholone concentration was measured using high performance liquid chromatography. In the same manner, the rebamipide concentration was measured for TM/REB-NPs, TM/REB-MPs and CA-REB.

The measurement results of fluorometholone concentration and rebamipide concentration over time are shown in Figure 2A and Figure 2B, respectively.

Furthermore, photographs of the 0d and 1M glass tubes are shown in Figure 2C. Furthermore, these were visually inspected and the dispersibility of fluorometholone was evaluated according to the following criteria, and the results are shown in Table 1.
<Evaluation of Dispersibility>
○: Uniformly dispersed state is maintained. △: Some drug has settled and clear areas are observed in the solution. ×: The drug has completely settled and the solution is clear.

As shown in Figures 2A and 2B, the mixed eye drops containing nanosized fluorometholone and nanosized rebamipide had fluorometholone and rebamipide concentrations of approximately 90% on the first day after the start of incubation, whereas the mixed eye drops containing microsized fluorometholone and rebamipide had concentrations of approximately 25% and 10%, respectively, and only a few percent in the commercially available eye drop suspension.

As shown in Figure 2C, even when visually inspected, the mixed eye drops containing nanosized fluorometholone maintained a dispersed state, whereas the mixed eye drops containing microsized fluorometholone and the commercially available eye drop suspension were confirmed to have aggregated and/or precipitated fluorometholone when left to stand.

Furthermore, the number of fluorometholone particles in a mixed eye drop containing nanoized fluorometholone with an average particle size of less than 1 μm was measured immediately after preparation (day 0) and on the 28th day, and the results are shown in Figure 2D. As Figure 2D shows, it was confirmed that the concentrations of both were almost constant.

These results show that nanosizing poorly water-soluble drugs improves their dispersibility during preparation and their long-term dispersion stability in mixed eye drops containing poorly water-soluble drugs and water-soluble drugs.

[Measurement of corneal permeability (in vitro) of poorly water-soluble drugs in test mixed eye drops]
As shown in Figure 3, a device was prepared in which a donor chamber and a reservoir chamber were separated by a rabbit cornea. The rabbit cornea had a total thickness of 520 μm (epithelial layer 50 μm; stroma layer 450 μm; endothelial layer 20 μm).

3.0 mL of the test mixed eye drop or a commercially available eye drop suspension was added to the donor chamber, and 3.0 mL of glucose-containing HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid) buffer solution was added to the reservoir chamber.

The device was incubated in a water bath set at 35°C, and 50 μL of sample was taken from the reservoir chamber 30, 60, 120, 240, 300, and 360 minutes after the start of the measurement, and the concentration of fluorometholone in the sample was measured using high performance liquid chromatography. The results are shown in Figure 4.

With the commercially available eye drop suspension (CA-FL), the fluorometholone concentration on the reservoir side did not increase, indicating that drug permeation was hindered by the rabbit cornea. In contrast, with the mixed eye drop containing nanoized fluorometholone (FBL-NPs), the fluorometholone concentration in samples taken from the reservoir chamber increased over time, indicating that the nanoized drug has excellent corneal permeability.

[Measurement of corneal permeability (in vivo) of each drug in the test mixed eye drop]
The corneal permeability of each drug was evaluated for the test mixed eye drop FBL-NPs, and three commercially available eye drops containing fluorometholone, bromfenac sodium, and levofloxacin alone (CA-FL, CA-LV, and CA-BF).

Figure 5 shows a schematic overview of the test. Male Japanese white rabbits were anesthetized with inhaled influenza, and then a microsyringe equipped with a silicone tube (φ0.5 mm) with an insulin injection needle (29 G) at the tip was used to insert the needle directly under the cornea. 5 mL of the test eye drops or 5 mL of a commercially available eye drop was dripped into the rabbit's eye in this state. After dripping, 5 μL of aqueous humor from within the rabbit's eye was collected into the microsyringe every 10 minutes for up to 90 minutes. The drug concentration in the collected aqueous humor was measured using high performance liquid chromatography.

Figure 6 shows the results of measuring the concentrations of fluorometholone, bromfenac sodium, and levofloxacin over time in the collected aqueous humor when FBL-NPs, or CA-FL, CA-LV, and CA-BF were instilled.

As shown in Figure 6A, for the poorly water-soluble drug fluorometholone, the fluorometholone concentration was higher at all measurement times when the mixed eye drops containing nanoized fluorometholone were instilled compared to when the commercially available eye drops were instilled. This result demonstrated that the corneal permeability of the poorly water-soluble drug fluorometholone is improved by nanoizing it.

As shown in Figures 6B and 6C, it is surprising that when the poorly water-soluble drug fluorometholone is nanosized, the concentrations of not only fluorometholone but also the coexisting water-soluble drugs bromfenac sodium and levofloxacin are higher at all measurement times when a mixed eye drop containing nanoized fluorometholone is instilled, compared to when a commercially available eye drop is instilled. This result indicates that nanoizing the poorly water-soluble drug fluorometholone synergistically improves the corneal permeability of water-soluble drugs that coexist in a mixture with nanoized fluorometholone.

The eye drops and method of one embodiment of the present invention have high dispersion stability of the drug and are capable of maintaining a homogeneous suspension state. Furthermore, due to excellent permeability of the drug through the cornea, the pharmacological action of the drug can be effectively applied to ocular tissues, and can contribute to the health and welfare of individuals seeking treatment and prevention of eye diseases such as cataracts and glaucoma.

Claims (4)

An eye drop comprising a nanosized poorly water-soluble drug and a water-soluble drug, and being a homogeneous suspension after 7 days of standing,
Further containing 2-hydroxypropyl-β-cyclodextrin as a suspending aid.
The eye drop . The eye drop according to claim 1, wherein the nanosized poorly water-soluble drug has an average particle size of 10 nm to 400 nm. 3. The eye drop according to claim 1, further comprising at least one suspending aid selected from the group consisting of surfactants, sugars, sugar alcohols, water-soluble polymers and amino acids . A step of obtaining a nano-sized poorly water-soluble drug by subjecting the poorly water-soluble drug to ultrafine processing;
and mixing the nanosized poorly water-soluble drug with a water-soluble drug to obtain an eye drop comprising the nanosized poorly water-soluble drug and the water-soluble drug ,
The ultrafine-graining treatment is performed in the presence of 2-hydroxypropyl-β-cyclodextrin as a suspension aid.
The manufacturing method .