WO2010053101A1

WO2010053101A1 - Eye drop having high intraocular migration properties, fluorescent imaging agent, and methods for producing same

Info

Publication number: WO2010053101A1
Application number: PCT/JP2009/068851
Authority: WO
Inventors: 幸二西田; 耕一馬場; 均笠井; 佑治田中; 享久保田; 俊二横倉
Original assignee: 国立大学法人東北大学
Priority date: 2008-11-06
Filing date: 2009-11-04
Publication date: 2010-05-14
Also published as: JPWO2010053101A1; JP5709523B2

Abstract

Disclosed is an eye drop which can achieve high intraocular migration of a medicinal agent contained therein without the need of any carrier such as a liposome. Also disclosed is a method for producing the eye drop. The eye drop is characterized by comprising particles composed of a hydrophobic or liposoluble prodrug that can become hydrophilic or water-soluble upon hydrolysis, wherein the particles have a particle size of not smaller than 10 nm and smaller than 1 μm.

Description

Eye drops and fluorescent imaging agents with high intraocular transferability and methods for producing them

The present invention relates to an eye drop having high intraocular transferability and a method for producing the same.

Eye diseases include diseases that occur on the surface side of the eye, such as conjunctivitis, and diseases that occur in the posterior segment, such as age-related macular degeneration and (diabetic) retinopathy. A general schematic diagram of the eye structure is shown in FIG. Fig. 1 is a partially modified horizontal cross-sectional view of the right eyeball by Kenji Honse, ophthalmic solution (Nanzandou), p169, Fig. 10-1.

There are two main systems for intraocular transfer of eye drops: a system that passes through the cornea and moves into the eyeball, and a system that passes through the conjunctiva-sclera and moves into the eyeball.

The instilled drug is first mixed with tear fluid and stored in the tear fluid of the conjunctival sac. The amount of tear fluid in the conjunctival sac in a normal person is 7-8 ml, and the maximum volume of the corneal sac is 30 ml. Therefore, if the amount of normal eye drop drops, that is, 40-50 ml, which is washed away from the punctum or eyelid margin is removed, it is partially transferred into the eyeball effectively.

In addition, considering that the physiological turnover rate of tears is 8-15% per minute, the concentration of drugs in the conjunctival sac is rapidly decreasing within one hour immediately after instillation. Become. The actual rate of decrease depends on drug properties such as fat solubility, water solubility, protein binding, and pH, but a significant concentration decrease occurs within one hour.

Also, it is not uncommon for 85% of the drug to be removed by blinking that increases tear fluid excretion from the conjunctival sac. Drugs that have not migrated into the eye can be absorbed into the blood circulation from the conjunctiva and nasal mucosa and show systemic effects. For example, there is a report that 80% of the beta-blocker timolol is absorbed systemically in rabbits.

The molecular weight and polarity are important factors in drug penetration into the cornea. Drugs with a molecular weight of 100 or less have high permeability to the corneal epithelium. On the other hand, when the molecular weight of the drug is 500 or more, the permeability is extremely lowered.

Also, the more a drug is fat-soluble, the easier it is to penetrate the corneal epithelium. However, when the lipid solubility of a drug becomes high, the permeability of the hydrophilic corneal substance mainly composed of water decreases. Conversely, the more water-soluble the drug, the lower the permeability of the corneal epithelium and the easier it is to pass through the corneal stroma with water as the main component. That is, the corneal permeability of the drug is contradictory between the corneal epithelium and the substance.

Lipid solubility is an important factor for improving the corneal epithelial permeability of drugs, but for ordinary eye drops, it is necessary to dissolve the drug in an eye drop base (which is a hydrophilic liquid such as water). For this reason, the lipophilicity of drugs is set low. Therefore, by chemically modifying hydrophilic drugs in advance, the liposolubility is increased, drug distribution to the corneal epithelium is increased, and then converted to the original hydrophilic drug mainly by enzymatic reactions that occur in the corneal epithelium. There has been reported a technique for improving the corneal permeability of a drug by facilitating permeation through the corneal stroma.

An example is dipivalial epinephrine (DPE), which is a prodrug of epinephrine (see Non-Patent Document 1). This DPE was obtained by esterifying the phenolic hydroxyl group of epinephrine, which has a strong hydrophobicity, increased migration to the epithelium, and at the same time is less susceptible to oxidative degradation. Stabilization is also planned. It has been reported that this DPE undergoes hydrolysis in the corneal epithelium to become epinephrine, appears in the anterior chamber, and exhibits the same efficacy as epinephrine at doses of 1/5 or less of conventional epinephrine.

However, as in the case of DPE, a compound having a property of enhancing lipophilicity by chemically modifying a hydrophilic drug in advance and being converted into the original drug by an enzymatic reaction in aqueous humor after permeation of the corneal epithelium, When the hydrophobicity of the compound is strong, aggregates are easily formed in water and become particles. Since the particle size is usually on the order of micrometers, when used as an eye drop, penetration of the drug into the corneal epithelial layer is difficult due to the size factor of the drug particle.

In general, the drug that has passed through the cornea and reached the anterior chamber after instillation diffuses in the anterior chamber water and reaches the lens and iris. The drug penetration into the iris parenchyma is very easy and the drug has an immediate effect on the iris. In addition, the transuveal scleral flow loss system, which is one of the anterior aqueous humor flow pathways, plays an important role in reaching the ciliary body of drugs in the anterior chamber.

In the transition of eye drops to the cornea and aqueous humor, the maximum drug concentration in each tissue is, for example, the concentration of eye drops in the cornea (generally about 0.1%, 1,000 mg / L). 1/100, about 1 / 5,000 in aqueous humor. Drug concentrations in iris tissue are often considered the same as aqueous humor concentrations.

On the other hand, considering the aqueous humor flow path to the Schlemm tube, it is believed that the transfer of the drug in the anterior chamber to the posterior chamber is limited to some extent. Therefore, as described above, the eye drops move from the cornea to the anterior chamber, iris, ciliary body, and crystalline lens, and from the conjunctiva to the sclera, and partly to the retina and the surrounding tissues. However, there is a limit to the penetration of eye drops into the vitreous body located in the deep intraocular tissue of the posterior segment or the retina, which is the endocardium. Therefore, the main action site of the eye drops must be concentrated on the ocular surface and the anterior eye segment.

When local administration by instillation is ineffective for treatment, oral administration of drugs, intravenous injection, subcutaneous injection, intramuscular injection, or local administration such as vitreous injection is selected.

In systemic administration, in order for eye drops to move into the eyeball, they must pass through the barrier of the cornea, sclera, iris ciliary body, and retinal pigment epithelial cell layer. In particular, since there is a blood-eye barrier such as the blood aqueous humor barrier existing in the iris ciliary tissue and the blood-retinal barrier existing in the retinal pigment epithelial cell layer, it is particularly difficult to reach the vitreous body and the retina.

Examples of methods for applying eye drops to the vitreous body and retina of deep intraocular tissues include local vitreous injection, retinal injection (see Patent Document 1), intra-Tenon sac injection, and vitreous implantable preparation. .

However, it is difficult to say that these methods are the optimum means when physically damaging the eyeball slightly, considering the difficulty of administration, and the burden on patients and doctors.

For the purpose of enhancing the drug's ability to move into the eye, various ideas have been made, such as direct injection into the vitreous body and retina as described above. However, it is ideal to administer a compound such as a drug in the form of eye drops from the viewpoint of the ease of administration and the burden on patients and doctors regarding administration.

Therefore, as an eye drop that can deliver a drug to the posterior eye segment, the drug is limited to a gene DNA, but an eye drop containing a specific liposome encapsulating an expression vector incorporating the gene DNA has been developed (patent) Reference 2). Furthermore, eye drops including liposomes that can deliver various drugs to the posterior eye segment as well as gene DNA have been developed (see Non-Patent Document 2).

However, eye drops using such liposomes (carriers) have a problem with the safety of the liposomes themselves. Therefore, eye drops using the above-mentioned liposomes are not yet safe enough.

JP 2006-507368 A Japanese Patent No. 3963506

In view of the above points, an object of the present invention is to provide an eye drop that does not contain a special carrier such as a liposome and has a high intraocular drug transfer property and a method for producing the same.

As a result of intensive studies to solve the above problems, the present inventors have previously made a hydrophilic or water-soluble drug (compound) into a prodrug to make it hydrophobic or fat-soluble, and this is performed by a method called a reprecipitation method. It was found that high intraocular transferability of the drug can be achieved by dispersing in the eye drop as particles having a particle size of the order of nanometer, and the present invention has been completed.

That is, the gist of the present invention is as follows.

An eye drop comprising particles comprising a prodrug that changes from hydrophobic or fat-soluble to hydrophilic or water-soluble upon hydrolysis, wherein the particle size of the particles is 10 nm or more and less than 1 μm. Eye drops.

The particle size of the particles is preferably 10 nm or more and 500 nm or less.

In the eye drop of the present invention, the prodrug is dissolved in the solvent 1 in which the prodrug dissolves, and the resulting solution A is mixed with the solvent 2 in which the prodrug does not dissolve, so that the prodrug exists as nanoparticles. And can be produced by mixing with an eye drop base.

When mixing the solution A with the solvent 2, it is preferable to supply the solution as droplets into the solvent 2.

Further, according to the present invention, a fluorescent imaging agent for observing the eye at the cellular level is provided.

The fluorescent imaging agent of the present invention comprises particles comprising a prodrug that undergoes a hydrolysis reaction to change from hydrophobic or fat-soluble to hydrophilic or water-soluble and emits fluorescence, and the particle size of the particles is It is characterized by being 10 nm or more and less than 1 μm.

Further, as an observation method using the fluorescent imaging agent of the present invention, the fluorescent imaging agent of the present invention is administered to the eyes of animals other than humans, the eyes are removed, and the extracted eyes are separated into respective parts, There is a method of observing fluorescence emitted from a site by optical means.

Furthermore, there is a method of observing fluorescence emitted by the eye by optical means without separating the extracted eye into each part.

According to the present invention, it is possible to effectively transport the compound to the intraocular tissue in the form of eye drops. In particular, effective transfer of compounds to the cornea, conjunctiva, anterior aqueous humor, ciliary body, lens, sclera, choroid, and retina is possible.

Such an operation and gain of the present invention will be clarified from embodiments for carrying out the invention described below.

FIG. 1 is a schematic diagram showing the structure of an eye. In FIG. 1, aa ′ indicates the eyeball axis, bb ′ indicates the line of sight, and cc ′ indicates the equator. FIG. 2 is a diagram showing the results of fluorescence spectrum evaluation in Example 1 and Comparative Examples 1 to 3. In FIG. 2, the curve with the whole marker is the fluorescence spectrum of fluorescein diacetate nanoparticles, the dotted curve with the triangular marker is the fluorescence spectrum of fluorescein diacetate microparticles, and the dotted line without the marker The curve is the fluorescence spectrum of sodium fluorescein, and the curve consisting of the solid line without the marker is the fluorescence spectrum of fluorescein. FIG. 3 is a diagram showing the results of time-dependent evaluation of eyeball fluorescence spectra in Examples 2 to 5 and Comparative Example 4. In FIG. 3, the curve with a round marker is a fluorescence spectrum using a rat eye as a sample 0.5 hours after administration of the eye drop, and is a dotted line with a triangular marker pointing upwards. The curve is a fluorescence spectrum in which the eye of a rat 1 hour after administration of eye drops is used as a sample, and the dotted curve with a square marker is a curve of a rat 2 hours after administration of eye drops. Fluorescence spectrum of eye sample, dotted curve with downward marker marker is a fluorescence spectrum of rat eye sample 3 hours after administration of eye drops. The dotted line that is not attached is a control. FIG. 4 is a graph showing the results of evaluation over time of fluorescence spectra of fluorescein diacetate transferred to an aqueous humor in Examples 6 to 8 and Comparative Example 5. In FIG. 4, the curve with a round marker is a fluorescence spectrum using a rat eye 10 minutes after administration of the eye drops, and the dotted curve with a triangular marker is the eye drop. It is a fluorescence spectrum using a rat eye 30 minutes after administration as a sample, and a dotted curve with a square marker is a fluorescence spectrum using a rat eye 60 minutes after administration of eye drops as a sample. The dotted curve without the marker, which is the spectrum, is the control. FIG. 5 is a diagram showing the results of evaluation of transferability of eye drops to the anterior segment / retina in Example 9 and Comparative Example 6. In FIG. 5, the curve with a round marker is the fluorescence spectrum of the anterior segment after administration of eye drops, and the dotted curve with a triangular marker is the retinal tissue after administration of eye drops. In the fluorescence spectrum, the dotted curve with a square marker is the fluorescence spectrum of the anterior segment of the control, and the dotted curve without the marker is the fluorescence spectrum of the control retinal tissue. FIG. 6 is a confocal laser microscope image of retinal tissue in Example 9 and Comparative Example 6 (the left side is Example 9 and the right side is Comparative Example 6). FIG. 7 is a graph showing the results of comparison of the fluorescence spectrum and fluorescence intensity in each tissue in Example 10. In FIG. 7, the curve with a round marker is the fluorescence spectrum of the cornea, and the dotted curve with a triangular marker is the fluorescence spectrum of the choroid and retina of the posterior eye. FIG. 8 is a graph showing the results of evaluation of changes in fluorescence intensity over time in the cornea (right side) and the retina and choroid (left side) in Examples 11 to 14. FIG. 9 is a graph showing the calibration curve used to determine the migration rate of fluorescein diacetate in Examples 11-14. FIG. 10 is a diagram showing the results of fluorescence observation of corneal tissue using a confocal laser microscope in Examples 15 to 18. In FIG. 10, the upper left is Example 15 (corneal epithelial surface cells), the upper right is Example 16 (corneal epithelial basal cells), the lower left is Example 17 (corneal parenchymal cells), and the lower right is an Example. 18 (corneal endothelial cells). FIG. 11 shows an electron micrograph of the dexamethasone derivative nanoparticles produced in Example 19. FIG. 12 shows the results of HPLC of the sample solution in Example 19 (right side) and the results of analyzing dexamethasone standards by HPLC (left side). FIG. 13 shows the evaluation results by HPLC of the eye drop of Example 19 (nanoparticles, left side) and the eye drop of Comparative Example 7 (micro particles, right side). FIG. 14 is a diagram showing the results of HPLC evaluation of intraocular transfer properties of eye drops of Example 19 and Comparative Examples 7 and 8, together with the results of controls. In FIG. 14, the leftmost bar graph (a) is the result of the eye drop of Example 19, the second bar graph (b) from the left is the result of the eye drop of Comparative Example 7, and the third bar graph from the left (C) is the result of the eye drop of Comparative Example 8, and the rightmost bar graph (d) is the result of the control. FIG. 15 shows a calibration curve used in Example 19 and Comparative Example 8 to calculate the amount of dexamethasone transferred into the eyeball by administration of eye drops.

[Eye drops of the present invention]
The eye drop of the present invention is an eye drop containing particles comprising a prodrug that undergoes a hydrolysis reaction and changes from hydrophobic or fat-soluble to hydrophilic or water-soluble, and the particle size of the particles is 10 nm or more and 1 μm. It is characterized by being less than. As will be described later, the prodrug contained in the eye drop and the fluorescent imaging agent of the present invention is excellent in intraocular transferability because the particle size of the particle composed of the prodrug is on the order of nanometers. First, a method for producing nanoparticles having such a particle size on the order of nanometers will be described.

<Nanoparticle production method>
Nanoparticles composed of prodrugs can be produced by a method called a reprecipitation method. A prodrug is a compound that undergoes a hydrolysis reaction to change from hydrophobic or fat-soluble to hydrophilic or water-soluble as described above. That is, the prodrug is hydrophobic or fat-soluble. Specific examples of the prodrug will be described later.

In the present specification, “hydrophobic or fat-soluble” refers to a property that the solubility in water at 20 ° C. is less than 0.01 g / L. “Hydrophilic or water-soluble” refers to the property that the solubility in water at 20 ° C. is 0.01 g / L or more.

In the reprecipitation method, the prodrug is dissolved in the solvent 1 in which the prodrug dissolves, and the obtained solution A is mixed with the solvent 2 in which the prodrug does not dissolve. Thereby, a dispersion liquid in which nanoparticles composed of the prodrug and the solvent 1 are dispersed in the solvent 2 is obtained. The manufacturing method of the eye drop of the present invention is characterized by including a step of obtaining such a dispersion.

The above “dissolve” means that 1 mg or more of prodrug dissolved in 100 g of solvent 1 at 20 ° C., and “does not dissolve” means prodrug that dissolves in 100 g of solvent 2 at 20 ° C. Say less than 1 mg.

As described later, in producing the eye drop of the present invention, the dispersion is mixed with the eye drop base. Therefore, the solvent 2 needs to be not harmful to the living body at the same time that the prodrug is not dissolved. Therefore, the most preferable as the solvent 2 is water which is the liquid most contained in the living body. In addition, examples of the solvent 2 include hydrophilic liquids such as an ethanol aqueous solution (ethanol concentration is 0.08 mL / mL or less). The amount of the solvent 2 used is usually 50 to 1,000,000 parts by weight, preferably 1000 to 100,000 parts by weight per 100 parts by weight of the solution A.

Examples of the solvent 1 include alcohols such as ethanol, and organic solvents such as acetone, dimethyl sulfoxide (DMSO), 1-methyl-2-pyrrolidone, and tetrahydrofuran. The amount of solvent 1 used is usually 100 to 100,000 parts by weight, preferably 1000 to 50000 parts by weight, per 100 parts by weight of the prodrug.

As described above, in the reprecipitation method, the prodrug is dissolved in the solvent 1, and the obtained solution A is mixed with the solvent 2. In this case, the solution A is utilized using a tubular object such as a syringe needle. Is preferably supplied as droplets in the solvent 2 and the solution A and the solvent 2 are mixed. When the solution A is supplied as droplets in this way, the solvent 1 and the solvent 2 can be diluted infinitely, so that the droplet of the solution A quickly diffuses and disappears in the solvent 2. At this time, since the prodrug dissolved in the solution A does not dissolve in the solvent 2, the solubility rapidly decreases, and the prodrug is precipitated. In this way, nanoparticles having a particle size of the order of nanometers can be easily produced in a state of being uniformly dispersed in the solvent 2.

In the eye drop of the present invention, the particle size of the prodrug-containing particles is 10 nm or more and less than 1 μm, preferably 10 nm or more and 500 nm or less, more preferably 10 nm or more and 250 nm or less. If the particle size is not in the range of such a small value, the prodrug cannot be transferred to the posterior eye part, and high intraocular transferability cannot be achieved for the eye drop.

In the present specification, the particle size refers to the particle diameter of a particle measured by a scanning electron microscope and a dynamic light scattering method regardless of the shape of the particle.

The particle size is adjusted by the diameter of the tubular object such as the syringe needle, the stirring method when mixing the solution A and the solvent 2, and the temperature of the solution A and the solvent 2 when mixing the solution A and the solvent 2 can do. Moreover, the fine dispersion degree in the solvent 2 of particle | grains (solution A) can also be raised by using surfactant, a suspending agent, etc. An example of a surfactant is polysorbate 80, and an example of a suspending agent is polyvinylpyrrolidone K30. Furthermore, the particle size varies depending on the types of the solvent 1 and the solvent 2 and the concentration of the prodrug in the solution A.

For the tubular object such as the syringe needle, generally, the droplet size of the solution A supplied into the solvent 2 is reduced by reducing the diameter of the tube, so that the particle size of the obtained nanoparticles is reduced. With respect to the stirring method, the particle size decreases as the stirring speed is increased, and the particle size increases as the stirring speed is decreased. The influence of the temperature of the solution A and the solvent 2 on the particle size depends on the prodrug used in the production of the eye drop of the present invention. Empirically, a prodrug that dissolves in a concentration range of 0.01 μg or more and less than 1 mg in 100 g of solvent 2 at 20 ° C. increases in particle size with increasing temperature and dissolves in solvent 2 at 20 ° C. and 100 g. In a prodrug with an amount of less than 0.01 μg, the particle size decreases as the temperature of Solution A and Solvent 2 increases.

The temperature range for forming the nanoparticles is generally 0 to 100 ° C. at 1 atm in the solution A and the solvent 2.

The dispersion of nanoparticles obtained by mixing the solution A and the solvent 2 is composed of a prodrug, a solvent 1 such as an organic solvent, and a solvent 2 such as water. Organic solvents are harmful to living organisms and should be removed by dialysis. This removal may be performed after the dispersion is in the form of eye drops described later.

<Eye drops>
The eye drop of the present invention is obtained by mixing a dispersion liquid in which nanoparticles composed of the above prodrug (including solvent 1 if not removed by dialysis or the like) in solvent 2 are mixed with an eye drop base. Can be obtained. This operation is performed aseptically. Alternatively, the eye drops may be sterilized using a filter sterilization filter having a pore size of 0.22 μm and 0.45 μm. The eye drop base is a liquid containing water and various additives. Further, when the nanoparticles contain the solvent 1, the solvent 1 is removed by dialysis or the like.

In addition, the said dispersion liquid can be freeze-dried, and this can also be mixed and used for an eye drop base at the time of use.

Examples of the additive include isotonic agents such as sodium chloride, concentrated glycerin, sorbitol, glucose;
Buffering agents such as sodium hydrogen phosphate, sodium acetate, boric acid, citric acid;
Surfactants such as polysorbate 80, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 60;
Stabilizers such as sodium citrate, sodium edetate;
Preservatives such as benzalkonium chloride, benzethonium chloride, methyl paraoxybenzoate, chlorhexidine gluconate;
Thickeners such as polyvinylpyrrolidone K30, hydroxyethylcellulose, propylene glycol, methylcellulose, macrogol 4000;
Suspending agents such as carboxyvinyl polymer, macrogol 6000, polyvinylpyrrolidone K25, polyvinylpyrrolidone K30;
PH adjusters such as hydrochloric acid, glacial acetic acid, sodium hydroxide;
Solubilizing agents such as magnesium chloride, ethanol, propylene glycol;
Chelating agents can be mentioned.

The concentration of the prodrug in the eye drop of the present invention containing the above-described additives as appropriate is appropriately determined depending on the type of the prodrug, but is usually in the range of 0.001 wt% to 10 wt%. .

Prodrugs are compounds that change from hydrophobic or lipophilic to hydrophilic or water-soluble upon hydrolysis, and usually become active as active ingredients in eye drops upon hydrolysis. However, there is no problem even if it is active before undergoing the hydrolysis reaction.

Examples of such prodrugs include esterification, acetalization, amidation, thioesterification, or ketalization of a hydrophilic drug, which undergoes a hydrolysis reaction by a hydrolase in the corneal epithelium, and the ester group. And prodrugs in which the substituent is removed.

As an example, a prodrug obtained by esterifying indomethacin is shown below. By this esterification, the solubility of indomethacin in water at 20 ° C. is changed from 0.01 g / L or more (hydrophilic or water-soluble) to less than 0.01 g / L (hydrophobic or fat-soluble).

The pH of the eye drop of the present invention is usually 5-9. The pH of the eye drop can be adjusted with the above pH adjuster.

The osmotic pressure ratio (ratio to the osmotic pressure of 0.9% by weight physiological saline) of the eye drop of the present invention is usually 0.6 to 1.6. The osmotic pressure ratio of the eye drop can be adjusted with the above-mentioned tonicity agent.

Such an eye drop of the present invention is an eye drop containing particles composed of a prodrug that undergoes a hydrolysis reaction and changes from hydrophobic or fat-soluble to hydrophilic or water-soluble, and the prodrug is as described above. Since it has properties and the particle size of the particles is in a specific range, it is excellent in intraocular transferability. In particular, prodrugs are effectively transferred to the cornea, conjunctiva, anterior aqueous humor, ciliary body, lens, choroid, sclera, and retina.

Specifically, since the prodrug constituting the particle is hydrophobic or lipophilic, it has excellent affinity with the corneal epithelium, and when the eye drop of the present invention is instilled, the prodrug penetrates into the corneal epithelium. . A hydrolase such as an esterase enzyme group exists in the corneal epithelium, and the prodrug is hydrolyzed and changed to hydrophilic or water-soluble by the action of the hydrolase. The next site to break through the corneal epithelium for the prodrug to enter the eye is the corneal stroma, which is hydrophilic. Therefore, since the hydrolyzed prodrug (hereinafter referred to as prodrug B) is excellent in affinity with the corneal stroma, the prodrug B penetrates into the corneal stroma. After breaking through the corneal stroma, prodrug B moves into the eyeball and moves not only to the anterior segment but also to the posterior segment, ie, the anterior aqueous humor, ciliary body, crystalline lens, choroid, and retina. it is conceivable that. In addition, the prodrug is considered to be delivered to the posterior segment by the route from the conjunctiva to the sclera after instillation.

(Use / Dose and Applicable Diseases of Eye Drops)
The usage and dosage of the eye drop of the present invention are appropriately determined depending on the type of prodrug.

Since the eye drop of the present invention is excellent in intraocular transferability, neovascular macular disease (age-related macular degeneration, etc.), diabetic retinopathy, retinal artery occlusion, retinal vein occlusion, hypertensive retinopathy, central serous choroid Retinopathy, retinitis pigmentosa, acute retinitis pigmentosa, multiple disappearing white spot syndrome, retinitis pigmentosa, proliferative vitreoretinopathy, secondary retinal detachment, cancer-related retinopathy, glaucoma, metastatic choroidal tumor It can be suitably applied to diseases in which a drug needs to be administered to the posterior eye segment such as the retina for treatment.

That is, as prodrugs, drugs that are hydrolyzed to treat these diseases (eg, synthetic steroids, fibrinolytics, anticoagulants, platelet aggregation inhibitors, prostaglandin-related drugs, sympathetic blockers, carbonic acid Dehydrase inhibitor, sympathomimetic, parasympathomimetic, hyperosmotic, neuroprotective, vasodilator, anti-inflammatory enzyme, vitamin preparation, diuretic, calcium antagonist, anti-inflammatory, cytostatic It is preferable to select a compound obtained by esterifying, acetalizing, amidating, thioesterifying or ketalizing a drug such as a non-steroidal anti-inflammatory agent.

In addition, even if the eye drop of the present invention is applied to diseases in which a drug needs to be administered to the anterior segment such as keratitis and conjunctivitis, a sufficient therapeutic effect can be obtained.

The eye drop of the present invention can be used not only for humans but also for the treatment of eye diseases such as monkeys, cows, horses, dogs, cats and other mammals.

[Fluorescent imaging agent]
Since the eye drop of the present invention is excellent in intraocular mobility, the prodrug migrates to the posterior eye region and penetrates the entire eye.

Therefore, if the prodrug is hydrolyzed to generate fluorescence, the fluorescence from the eye or each part of the eye (cornea, conjunctiva, anterior chamber, ciliary body, lens, vitreous body, choroid, retina, Can be observed (observed).

The fluorescent imaging agent of the present invention that makes it possible to observe such particles is a particle formed of a prodrug that undergoes a hydrolysis reaction to change from hydrophobic or fat-soluble to hydrophilic or water-soluble and emits fluorescence. And the particle size of the particles is from 10 nm to less than 1 μm, preferably from 10 nm to 500 nm, and more preferably from 10 nm to 250 nm.

Prodrugs satisfying the above conditions include diacetate fluorescein, 3′-O-acetyl-2 ′, 7′-bis (carboxyethyl) -4 or 5-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM) and 5- or 6- (N-succinimidyloxycarbonyl) -fluorescein 3 ', 6' -diacetate (CFSE) and the like.

Specifically, as an observation method using the fluorescent imaging agent of the present invention, the fluorescent imaging agent is administered to the eyes of animals other than humans, the eyes are removed, the extracted eyes are separated into each part, and the part And a method of observing fluorescence emitted from the optical means. The extracted eye may be observed without being separated into each part. Information obtained by observation is digitized, for example, and the digitized information is output by the output means.

Examples of the optical means include a spectrofluorometer.

Further, as the output means, means for displaying the digitized information on a display two-dimensionally or three-dimensionally, means for electrically outputting the digitized information, means for printing the digitized information, Means for recording digitized information on a recording medium can be mentioned.

Observing may be one, plural or all of the separated parts.

Alternatively, the fluorescent imaging agent of the present invention may be administered to the eyes of animals other than humans, the eyes may be removed, the removed eyes may be separated into each part, and the part may be observed with a confocal laser microscope. Of course, one, a plurality or all of the separated parts may be observed. Further, the eyes may be observed directly without being separated into each part. At that time, the eyes may be observed without being removed or removed.

As in the case of the eye drop of the present invention, the fluorescent imaging agent of the present invention dissolves the prodrug by dialysis or the like from a dispersion in which the above-mentioned prodrug nanoparticles are dispersed in the solvent 2 in which the prodrug is not dissolved. Is obtained by removing the solvent 1 Alternatively, the dispersion may be obtained by mixing with an eye drop base. Even in this case, the solvent 1 is removed.

Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

<Creation of nanoparticles>
Fluorescein diacetate (solubility is 2.9X10 ^-3 g / L, calculated by ACD / Solubility DB (manufactured by Advanced Chemistry Development)) 25 mg is dissolved in 1 g of acetone, and 200 mg of the resulting solution is syringe needle with a caliber of 0.41 mm Then, it was poured into 10 g of water containing polyvinylpyrrolidone K30 (PVP) while stirring with a magnetic stirrer (stirring speed: 1200 rpm) to obtain a nanoparticle dispersion.

Then, this nanoparticle dispersion was dialyzed using a dialysis tube to remove acetone.

When the particle size of the nanoparticles was measured by a scanning electron microscope and a dynamic light scattering method, the individual particle size (particle diameter) was 10 nm or more and 500 nm or less.

<Preparation of eye drops>
The obtained nanoparticle dispersion was mixed with water containing salt, and the final concentration of each component was 0.05% by weight of fluorescein diacetate, 0.9% by weight of salt, and 2% by weight of PVP. Thus, an eye drop was obtained. The pH of the eye drop was 7.

<Eye fluorescence spectrum evaluation>
[Example 1]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained above. After 0.5 hour, the rats were killed by anesthesia, and the eyes instilled with eye drops were sufficiently washed with physiological saline and then removed and further washed with physiological saline.

The washed eyes were mixed with 5 ml of a mixed solution (volume ratio 1: 1) of a sodium hydroxide aqueous solution (100 mM) and a DMSO aqueous solution. In this mixture, fluorescein diacetate was present in the state of fluorescein after hydrolysis, and the eyeball was dissolved.

The fluorescence spectrum of the obtained sample solution was evaluated. Specifically, the fluorescence intensity when the sample solution was excited with light having a wavelength of 488 nm was measured. The results are shown in FIG. The fluorescence spectrum was evaluated twice (conditions 1 and 2) while changing the measurement conditions. The measurement result in the case of the condition 1 is a diagram on the left side of FIG. 2, and the measurement result in the case of the condition 2 is a diagram on the right side of FIG. Condition 2 differs from condition 1 in the slit width of the excitation side slit and the fluorescence side slit of the spectrofluorophotometer. Specifically, in condition 1, the excitation side slit and the fluorescence side slit are each 5.0 nm, and in condition 2, the excitation side slit and the fluorescence side slit are each 2.5 nm.

[Comparative Example 1]
Fluorescein diacetate was recrystallized from an acetone solution in which fluorescein diacetate was dissolved to obtain a powder of fluorescein diacetate. This powder was refined in an agate bowl to obtain microparticles. The microparticles were mixed with water containing sodium chloride and polyvinylpyrrolidone K30 (PVP) to obtain an eye drop. In this eye drop, the final concentration of each component was 0.05% by weight of fluorescein diacetate, 0.9% by weight of sodium chloride, and 2% by weight of PVP. The pH of the eye drop was 7.

With respect to the obtained eye drop (the particle size (particle diameter) of each particle in the eye drop is 1 μm or more and 100 μm or less), the fluorescence spectrum was evaluated in the same manner as in Condition 1 of Example 1. The results are shown in FIG.

[Comparative Example 2]
25 mg of fluorescein was dissolved in 1 g of dimethyl sulfoxide, and 200 mg of the resulting solution was stirred with a magnetic stirrer (stirring speed: 1200 rpm) with a syringe needle having a diameter of 0.41 mm in 10 g of water containing polyvinylpyrrolidone K30 (PVP). To obtain a dispersion.

Then, this dispersion was subjected to dialysis using a dialysis tube to remove dimethyl sulfoxide. The resulting dispersion is mixed with water containing salt so that the final concentration of each component is 0.05% by weight for fluorescein, 0.9% by weight for salt and 2% by weight for PVP. To obtain eye drops. The pH of the eye drop was 7. The obtained eye drop was evaluated for the fluorescence spectrum in the same manner as in Condition 2 of Example 1. The results are shown in FIG.

[Comparative Example 3]
A dispersion was obtained by dissolving 5 mg of sodium fluorescein in 10 g of water containing polyvinylpyrrolidone K30 (PVP).

The obtained dispersion is mixed with water containing salt so that the final concentration of each component is 0.05% by weight for sodium fluorescein, 0.9% by weight for salt and 2% by weight for PVP. Thus, an eye drop was obtained. The pH of the eye drop was 7. The obtained eye drop was evaluated for the fluorescence spectrum in the same manner as in Condition 1 of Example 1. The results are shown in FIG.

In FIG. 2, “fluorescein diacetate nanoparticles” refers to the eye drop of Example 1, “fluorescein diacetate microparticles” refers to the eye drop of Comparative Example 1, and “fluorescein” refers to the eye drop of Comparative Example 2. "Fluorescein sodium" indicates the eye drop of Comparative Example 3.

2 that the amount of the eye drop of the present invention migrates into the eye is larger than that of the eye drop of the comparative example.

<Evaluation of time dependence of eyeball fluorescence spectrum>
[Examples 2 to 5]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 0.5 (Example 2), 1 (Example 3), 2 (Example 4), and 3 (Example 5), the rats were killed by anesthesia, and the eye instilled with eye drops was sufficiently saline. After washing, it was removed and further washed with physiological saline.

The fluorescence spectrum of the obtained sample solution was evaluated. Specifically, the fluorescence intensity when all sample solutions were excited with light having a wavelength of 488 nm was measured. The results are shown in FIG. 3 (the left side of FIG. 3 is the evaluation result of the fluorescence spectrum, and the right side of FIG. 3 is the measurement result of the fluorescence intensity at a wavelength of 525 nm when excited with light having a wavelength of 488 nm).

[Comparative Example 4]
An ophthalmic solution containing no fluorescein diacetate and 0.9% by weight of sodium chloride and 2% by weight of PVP was used as a control. The pH of the eye drop was 7.

For the obtained eye drop, the fluorescence spectrum was evaluated in the same manner as in Example 2. The results are shown in FIG. Results are shown as “control”.

As shown in FIG. 3, in the eye drops of Examples 2 to 5, fluorescein diacetate was transferred into the eye, and the transferred fluorescein diacetate (hydrolyzed to fluorescein when transferred) was at least applied to the eye drops. Then, after 0.5 hours, the eye drops move with time, and in the eye drop of Comparative Example 4, since the eye drop does not contain fluorescein diacetate, fluorescence due to autofluorescence of cells and the like It can be seen that it was detected slightly. Therefore, it can be seen that the fluorescence detected by using the eye drops of Examples 2 to 5 is not autofluorescence of cells or the like.

<Evaluation over time of fluorescence spectrum of fluorescein diacetate transferred to anterior aqueous humor>
[Examples 6 to 8]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 10 (Example 6), 30 (Example 7), and 60 (Example 8) minutes, the rats were killed by anesthesia, and the eye instilled with an eye drop was thoroughly washed with physiological saline and then removed and further physiologically removed. Washed with brine.

2 μl of anterior aqueous humor was collected and mixed with 3 ml of a mixed solution of aqueous sodium hydroxide (100 mM) and aqueous DMSO (volume ratio 1: 1). In this mixture, fluorescein diacetate was present in the state of fluorescein after hydrolysis.

Fluorescence spectrum evaluation was performed on the obtained sample solution. Specifically, the fluorescence intensity when all sample solutions were excited with light having a wavelength of 488 nm was measured. The results are shown in FIG.

[Comparative Example 5]
An ophthalmic solution containing no fluorescein diacetate and 0.9% by weight of sodium chloride and 2% by weight of PVP was used as a control. The pH of the eye drop was 7. The obtained eye drop was evaluated for the fluorescence spectrum in the same manner as in Example 8. The results are shown in FIG. Results are shown as “control”.

As shown in FIG. 4, in the eye drops of Examples 6 to 8, fluorescein diacetate was transferred to the anterior aqueous humor, and the transferred fluorescein diacetate (which was hydrolyzed into fluorescein when transferred) contained at least the eye drops. 30 minutes after instillation, it moves to the outside of the anterior aqueous humor over time, and in the eye drop of Comparative Example 5, the eye drop does not contain fluorescein diacetate. It turns out that the fluorescence by fluorescence was detected slightly. Therefore, it can be seen that the fluorescence detected by using the eye drops of Examples 6 to 8 is not autofluorescence such as anterior aqueous humor.

<Evaluation of transferability of eye drops to the anterior segment and retina>
[Example 9]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. One hour later, the rats were killed by anesthesia, and the eyes instilled with eye drops were sufficiently washed with physiological saline and then removed and further washed with physiological saline.

The washed eye is cut and separated into the anterior segment and the posterior segment, the retinal tissue is removed from the posterior segment, and each sample of the anterior segment and retinal tissue is washed with an aqueous solution of sodium hydroxide (100 mM) and DMSO. The mixture was mixed with 5 ml of a mixture (volume ratio 1: 1). In this mixed solution, fluorescein diacetate was present in the state of fluorescein after hydrolysis, and each sample was dissolved.

Fluorescence spectrum evaluation was performed on the obtained sample solution. Specifically, the fluorescence intensity when all sample solutions were excited with light having a wavelength of 488 nm was measured. The results are shown in FIG. The retinal tissue was also observed with a confocal laser microscope. The result is shown in FIG.

[Comparative Example 6]
An ophthalmic solution containing no fluorescein diacetate and 0.9% by weight of sodium chloride and 2% by weight of PVP was used as a control. The pH of the eye drop was 7. The obtained eye drop was evaluated for the fluorescence spectrum in the same manner as in Example 9. The results are shown in FIG. Results are shown as “control”. The retinal tissue was also observed with a confocal laser microscope. The result is shown in FIG.

5 and 6, in the eye drop of Example 9, fluorescein diacetate migrates to both the anterior segment and retinal tissue (when it migrates, it is hydrolyzed to fluorescein), and transition to the anterior segment It can be seen that the amount of the ophthalmic solution of Comparative Example 6 is large, and since the ophthalmic solution does not contain fluorescein diacetate, fluorescence due to autofluorescence of cells and the like was slightly detected. Therefore, it turns out that the fluorescence detected by using the eye drop of Example 9 is not autofluorescence of cells or the like.

<Comparison of fluorescence spectrum and fluorescence intensity in each tissue>
[Example 10]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 5 minutes, the rats were killed by anesthesia, and the eyes instilled with eye drops were sufficiently washed with physiological saline and then removed and further washed with physiological saline.

The washed eye is quickly frozen and solidified, and then the eye is cut and the cornea, anterior aqueous humor, conjunctiva, sclera (posterior eye part), ciliary body, lens, vitreous body (anterior eye part), vitreous body (Rear eye part) and retina and choroid (rear eye part) were separated, and each was mixed with 5 ml of a mixed solution of sodium hydroxide aqueous solution (100 mM) and DMSO aqueous solution (volume ratio 1: 1). In this mixed solution, fluorescein diacetate was present in the state of fluorescein after hydrolysis, and each sample was dissolved.

The obtained sample solution cornea, the retina of the posterior eye part and the choroid were evaluated for fluorescence spectrum. The results are shown in FIG. 7 (left side). Moreover, the fluorescence intensity in wavelength 525nm was measured about the sample solution of each structure | tissue. The results are shown in FIG. 7 (right side).

From FIG. 7 (left side), in the eye drop of Example 10, fluorescein diacetate migrates to both the cornea and the retina and choroid of the posterior eye (when it migrates, it is hydrolyzed into fluorescein). It can be seen that the amount is greater in the cornea. In addition, FIG. 7 (right side) shows that when the eye drop of the present invention is instilled, fluorescein diacetate migrates to the entire eye including the posterior segment, although there is a difference in the migration amount.

<Evaluation of temporal change and transition rate of fluorescence intensity in retina and choroid of cornea and posterior eye>
[Examples 11 to 14]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 5 (Example 11), 30 (Example 12), 60 (Example 13), and 120 (Example 14), the rats were killed by anesthesia, and the eye instilled with eye drops was thoroughly washed with physiological saline. Then, it was extracted and further washed with physiological saline.

The washed eye is quickly frozen and solidified, and then the eye is cut to separate the cornea and the posterior eye retina and choroid, each of which is mixed with a sodium hydroxide aqueous solution (100 mM) and a DMSO aqueous solution (volume ratio). 1: 1) Mixed with 5 ml. In this mixed solution, fluorescein diacetate was present in the state of fluorescein after hydrolysis, and each sample was dissolved.

Fluorescence intensity at a wavelength of 525 nm was measured for the obtained sample solution. The results are shown in FIG.

From FIG. 8, it can be seen that when eye drops are administered, fluorescein diacetate is transferred to the cornea (left of FIG. 8) and the retina and choroid (right of FIG. 8) in 5 minutes. Fluorescein).

Next, based on a calibration curve representing the relationship between the concentration of fluorescein diacetate and the fluorescence intensity (FIG. 9), the retina and choroid of the entire eyeball, cornea, posterior eye segment in Examples 1 and 10 and Comparative Examples 1 to 3 The concentration of fluorescein acetate was determined. From the concentration and the concentration of fluorescein diacetate in the eye drops, the rate of transfer of fluorescein diacetate to the eyeball 30 minutes after instillation and to the cornea, retina and choroid at 5 minutes after instillation It was. The results are shown in Table 1 below. The transition rate of Comparative Examples 1 and 3 is not displayed, and the transition rate of the cornea of Comparative Example 2 and the transition rate to the retina and choroid of the posterior segment are not displayed. This is because sufficient fluorescence intensity could not be obtained.

<Fluorescence observation of corneal tissue using confocal laser microscope>
[Examples 15 to 18]
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 0.5 hour, the rats were killed by anesthesia, and the eyes instilled with eye drops were sufficiently washed with physiological saline and then removed and further washed with physiological saline. Confocal laser for the corneal site of the eye (corneal epithelial surface cells (Example 15), corneal epithelial basal cells (Example 16), corneal parenchymal cells (Example 17), and corneal endothelial cells (Example 18)) The fluorescence was observed with a microscope. An argon laser having a wavelength of 488 nm was used as an excitation light source. The results are shown in FIG.

FIG. 10 shows that by using the fluorescent imaging agent of the present invention, the cornea structure can be observed in a tomographic fluorescence while maintaining the shape of the eyeball of the eye.

<Quantitative evaluation of intraocular transfer of eye drops containing dexamethasone prodrug>
[Example 19]
<Creation of drug nanoparticles>
Dexamethasone sorbate (refer to the following formula, also referred to as “dexamethasone derivative” hereinafter. Solubility is 1.2 × 10 ⁻³ g / L, calculated by ACD / Solubility DB (manufactured by Advanced chemistry development)) 20 mg is dissolved in 1 g of ethanol. Then, 100 mg of the obtained solution was injected into 10 g of water being stirred with a magnetic stirrer (stirring speed: 1200 rpm) with a syringe needle having a diameter of 0.41 mm to obtain a nanoparticle dispersion. Dexamethasone (the solubility is 0.035 g / L, calculated by ACD / Solubility DB (manufactured by Advanced chemistry development)) is generated after hydrolysis from the dexamethasone sorbate derivative.

When the particle size of the nanoparticles was measured by a scanning electron microscope and a dynamic light scattering method, each particle size (particle diameter) was 10 nm or more and 500 nm or less.
An electron micrograph of the dexamethasone derivative nanoparticles is shown in FIG.

<Preparation of eye drops>
A 2 wt% PVP aqueous solution (1 g) was added to the obtained nanoparticle dispersion (10 g), and then freeze-dried to remove ethanol to obtain a nanoparticle powder. Subsequently, the nanoparticle powder obtained by freeze-drying was mixed with 1 g of water containing sodium chloride, and the nanoparticle powder was redispersed in water. An eye drop was obtained by adjusting the final concentration of each component to 0.2% by weight of the dexamethasone derivative, 0.9% by weight of sodium chloride, and 2% by weight of PVP.

<Evaluation of intraocular transfer of dexamethasone derivative nanoparticle eye drops>
One eye of a 4-week-old male rat was instilled with 10 μl of the eye drop obtained in the above <Preparation of eye drop>. After 5 minutes, the rats were killed by anesthesia, and the eyes instilled with eye drops were sufficiently washed with physiological saline and then removed and further washed with physiological saline.

The washed eyes were mixed with 500 μl of DMSO, and this solution was sonicated (60 minutes). Subsequently, this solution was passed through a syringe filter having a pore size of 0.2 μm.

The obtained sample solution was evaluated by high performance liquid chromatography (High Performance Liquid Chromatography: HPLC). FIG. 12 (left) shows a chromatogram derived from a dexamethasone standard product. The result (chromatogram) of Example 19 is shown in FIG. 12 (right). Also in the sample solution containing the eye instilled with the eye drop of Example 19, a peak derived from dexamethasone similar to that of the dexamethasone standard product was detected, indicating that dexamethasone was transferred into the eyeball.

As a control, an ophthalmic solution containing no dexamethasone was instilled into rats, and HPLC evaluation was performed in the same manner, and a slight peak was detected (see FIG. 14). This is presumed that a small amount of some compound derived from the eyeball having the same retention time as dexamethasone was detected.

[Comparative Example 7]
The powder of dexamethasone was mixed with an aqueous solution containing salt and containing 2% by weight of PVP, and an aqueous microparticle dispersion was obtained by ultrasonic treatment. In this dispersion, the final concentration of each component was 0.2% by weight of dexamethasone, 0.9% by weight of sodium chloride, and 2% by weight of PVP to obtain an eye drop. About the obtained eye drop (particle size (particle diameter) of each particle in the eye drop is 1 μm or more and 100 μm or less), rats were instilled in the same manner as in Example 19 to evaluate HPLC.

FIG. 13 and FIG. 14 show the results of comparing nanoparticle eye drops (Example 19) and microparticle eye drops (this comparative example) with respect to dexamethasone content in the eyeball after instillation. In FIG. 14, the leftmost bar graph “a” shows the result of Example 19, and the second bar graph “b” from the left shows the result of this comparative example. The dexamethasone content was expressed as the area of the spectrum obtained by HPLC measurement.

13 and 14, it can be seen that nanoparticle eye drops have higher intraocular mobility than microparticle eye drops. From these examples and comparative examples, it was shown that the eye drop of the present invention having enhanced intraocular transferability was effective.

[Comparative Example 8]
The dexamethasone derivative was mixed with an aqueous solution containing sodium chloride and containing 2% by weight of PVP, and an aqueous microparticle dispersion was obtained by ultrasonic treatment. In this dispersion, the final concentration of each component was 0.2% by weight of the dexamethasone derivative, 0.9% by weight of sodium chloride, and 2% by weight of PVP to obtain an eye drop. The obtained eye drop (the particle size (particle diameter) of each particle in the eye drop is 1 μm or more and 100 μm or less) was instilled into a rat in the same manner as in Example 19 and evaluated by HPLC.

The dexamethasone content in the eyeball after instillation was estimated from the area of the spectrum obtained by HPLC measurement using a calibration curve (see FIG. 15). However, in the case of the dexamethasone derivative microparticle eye drop (this comparative example), a sufficient amount of dexamethasone that can be calibrated was not transferred into the eye. On the other hand, when the same calculation was made for the results of the eye drops of Example 19 (dexamethasone derivative nanoparticle eye drops), intraocular transferability was observed, and the amount was 1.06 ± 0.34 μg / g tissue. .

The above results are summarized in Table 2 below.

Intraocular migration is not observed with the dexamethasone derivative microparticle eyedrops, and the HPLC peak area obtained from the dexamethasone derivative microparticle eyedrops (the third c bar from the left in FIG. 14) shows that the eyedrops do not contain dexamethasone. (Control: 0.9% by weight of salt and 2% by weight of PVP only; d on the rightmost side in FIG. 14) can be confirmed from the fact that the peak area has almost the same area value.

Furthermore, a (leftmost bar graph, HPLC evaluation result of eye drop of Example 19) and c (third bar graph from the left, HPLC evaluation result of eye drop of Comparative Example 8) in FIG. From the comparison, it can be seen that, in order to improve the intraocular transfer of dexamethasone, not only the drug prodrug but also the drug nanoparticles are important.

11 cornea 12 iris 13 ciliary body 14 sclera 15 choroid 16 retina 17 fovea 18 optic nerve 19 disc recess 20 vitreous 21 lens 22 eyeball conjunctiva 23 posterior chamber 24 anterior chamber

Claims

An eye drop comprising particles comprising a prodrug that undergoes a hydrolysis reaction and changes from hydrophobic or fat-soluble to hydrophilic or water-soluble,
An eye drop wherein the particle size of the particles is 10 nm or more and less than 1 μm.
The eye drop according to claim 1, wherein the particle size of the particles is 10 nm or more and 500 nm or less.
Dissolving the prodrug in solvent 1 in which the prodrug dissolves;
The method for producing an eye drop according to claim 1 or 2, comprising a step of mixing the obtained solution A with a solvent 2 in which the prodrug does not dissolve.
The method for producing eye drops according to claim 3, wherein the solution A is supplied as droplets into the solvent 2 and mixed.
A fluorescence imaging agent comprising particles comprising a prodrug that undergoes a hydrolysis reaction to change from hydrophobic or fat-soluble to hydrophilic or water-soluble and emits fluorescence,
The fluorescent imaging agent, wherein a particle size of the particles is 10 nm or more and less than 1 μm.
6. The fluorescent imaging agent according to claim 5, wherein a particle size of the particles is 10 nm or more and 500 nm or less.
The fluorescent imaging agent according to claim 5 or 6 is administered to an eye of an animal other than a human, the eye is removed, the removed eye is separated into each part, and fluorescence emitted from the part is observed by optical means Fluorescence observation method characterized by doing.
A fluorescence imaging method comprising: administering the fluorescent imaging agent according to claim 5 or 6 to an eye of an animal other than a human; removing the eye; and observing fluorescence emitted from the removed eye by optical means. .