HK1233540A1 - Ophthalmic suspension formulation - Google Patents

Description

Ophthalmic suspension formulations

Technical Field

The present invention relates generally to ophthalmic suspension formulations comprising (R) - (-) -2- (4-bromo-2-fluorobenzyl) -1,2,3, 4-tetrahydropyrrolo [1,2-a ] pyrazine-4-spiro-3 ' -pyrrolidine-1, 2',3,5' -tetraone, particularly useful for diseases of the posterior segment of the eye.

Background

Posterior segment tissues such as the vitreous, retina, choroid, and sclera are important areas of visual performance. If the area is damaged, it may often lead to severely reduced visual acuity or vision loss. Typical diseases of the posterior segment include age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, retinal vein occlusion, choroidal neovascularization, uveitis, retinitis pigmentosa, proliferative vitreoretinopathy and central serous choroidal retinopathy. In particular, age-related macular degeneration or diabetic retinopathy is a major disease causing visual loss of late-middle-aged to elderly people in developed countries such as europe, the usa and japan, which is a very problematic disease in ophthalmologic clinics as well as in the whole society (patent document 1).

In general, drug delivery through the blood to the posterior segment of the eye (e.g., the retina) is severely limited by the blood-retinal barrier (BRB) in the posterior segment of the eye. When administered in eye drops, most of the drug can be rapidly discharged from the ocular surface by the reversal of tear fluid, and then transferred to the blood circulating systemically through the nasolacrimal duct (non-patent document 1). Thus, if the amount of drug in the ophthalmic formulation is 100, the amount of delivered drug administered in the eye drops is 0.1-0.5 in the cornea where the drug is most transferred; 0.01-0.1 in the aqueous humor/iris and ciliary body; it is about 0.0001 in the lens and vitreous body, that is, it is known that the delivered amount is too low (non-patent document 2). In addition, it is generally believed that the drug is hardly delivered to the posterior segment by administration in eye drops, because the posterior segment is located further posterior to the lens or vitreous body. Also, it is generally considered that an ophthalmic suspension used in the case of a drug having low water solubility is more difficult to deliver to the posterior segment of the eye by administration of eye drops than a usual ophthalmic solution, because such a drug is not dissolved in water and is therefore generally hardly absorbed by the intraocular site (non-patent document 3). There are some ophthalmic suspensions in clinical practice, but use is limited to diseases in the anterior segment such as conjunctivitis. It is considered that means for delivering a drug to the posterior segment of the eye is only injection or surgery into the vitreous body, or systemic administration through systemic circulation blood, that is, other means is very difficult (patent document 4, non-patent document 4).

The disease of the posterior segment is an eye disease causing severe symptoms, but there are few useful drugs therefor, and furthermore, the administration is limited because the target site is the posterior segment where the drug is difficult to deliver. Therefore, treatment of diseases is now performed by injecting anti-vascular endothelial growth factor (anti-VEGF) into the vitreous, injecting steroids into the vitreous or the saccule of the eye, photodynamic therapy (PDT), operation of the vitreous, and the like. However, all of these current treatments, i.e. injection into the eye, etc., are very invasive to the patient and cause pain to the patient, and therefore development of new administration, such as eye drops, is desired.

(R) - (-) -2- (4-bromo-2-fluorobenzyl) -1,2,3, 4-tetrahydropyrrolo [1,2-a ] pyrazine-4-spiro-3 ' -pyrrolidine-1, 2',3,5' -tetraone (ranirestat) (hereinafter referred to as "compound a") has a potent inhibitory effect on aldose reductase, and the compound is also a low-toxicity compound, and therefore the compound is useful as a drug for treating diabetic complications (patent document 2, patent document 3).

Prior Art

[ patent reference ]

[ patent document 1] JP 2009-196973A

[ patent document 2] JP 2516147B

[ patent document 3] WO 1999/020276

[ patent document 4] JP 2005-097275A

[ non-patent reference ]

[ non-patent document 1] "Drug Delivery to The poster Eye section" YuukiTAKASHIMA, The achives of practical medicine, 72(2) -

[ non-patent document 2] Standard Yakugaku Series 7 "Seizaikano Science", edited by the pharmaceutical Society of Japan, issued by Tokyo Kagaku Doujin, Chapter IV "reproduction formation" 22-2 optical formation, p103-p105

[ non-patent document 3] "Tenganzai-no tekiseishiyou handbook Q & A", manufactured by the pharmaceutical Manufacturers' Association of Tokyo, Tenganzai kenkyukai, et al, JAPAN OPHTHALMOLOGISTS ASSOCIATION supervision, 9 months 2011, first edition, page 5

[ non-patent document 4] "Study to identification apparatus available catheter in inhibition of recombinant expression of" Yeast S SHIRAKAKI, gradual School of Natural Science & Technology, Doctorial thesis, Abstract of discovery and Abstract of expression output of expression, P605-P610,2007, 9 months.

Disclosure of Invention

(problem of the technology)

In order to deliver an effective concentration of drug to the posterior segment of the eye through the systemic circulating blood, because of the blood-retinal barrier (BRB), it is considered necessary to increase the concentration of drug in the systemic circulating blood by administering a very high dose. In order to avoid such a risk of high dose, the present invention aims to provide an ophthalmic preparation for posterior segment diseases and to avoid side effects due to high dose systemic exposure, and further to provide a stable ophthalmic preparation comprising compound a or a physiologically acceptable salt thereof.

(solution of problem)

The present inventors have conducted extensive studies to achieve the above object, and then have found that administration of an eye drop solution of a suspension of compound a or a physiologically acceptable salt thereof in a dispersion medium can deliver a drug to the posterior segment of the eye with high metastatic potential required for treatment while maintaining the stability of the drug and suppressing systemic exposure, and further that compound a or a physiologically acceptable salt thereof has a useful pharmacological effect on ocular diseases such as age-related macular degeneration. That is, the present invention is accomplished by suspending compound a or a physiologically acceptable salt thereof as an ophthalmic preparation.

The summary of the invention is described below.

(item 1)

An ophthalmic suspension formulation comprising (R) - (-) -2- (4-bromo-2-fluorobenzyl) -1,2,3, 4-tetrahydropyrrolo [1,2-a ] pyrazine-4-spiro-3 ' -pyrrolidine-1, 2',3,5' -tetraone (hereinafter referred to as "compound a") or a physiologically acceptable salt thereof.

(item 2)

The formulation of item 1, wherein the compound a or a physiologically acceptable salt thereof is dispersed in a dispersion medium.

(item 3)

The formulation of item 2, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the suspension is from 1nm to 20 μm.

(item 4)

The formulation of item 3, wherein the average particle size of Compound A or a physiologically acceptable salt thereof in the suspension is from 10nm to 20 μm.

(item 5)

The formulation of any one of items 2 to 4, wherein the dispersion medium is an aqueous dispersion medium.

(item 6)

The formulation of any one of items 2 to 5, wherein the dispersion medium comprises a dispersant and/or a surfactant.

(item 7)

The formulation of item 6, wherein the dispersion medium comprises a dispersant and a surfactant.

(item 8)

The formulation of any one of items 2 to 7, wherein the pH of the suspension is 3 to 9.

(item 9)

The formulation of any one of items 2 to 8, wherein the osmolality of the suspension is between 20 and 1000 mOsm.

(item 10)

The formulation of any one of items 2 to 9, comprising 1mg to 500mg of compound a or a physiologically acceptable salt thereof per 1mL of the suspension.

(item 11)

The formulation of any one of items 2 to 10, wherein the ratio of compound a or a physiologically acceptable salt thereof dissolved in the suspension to all compound a or a physiologically acceptable salt thereof in the formulation is 0.001% to 10%.

(item 12)

The formulation of any one of items 2 to 11, wherein the ratio of compound a or a physiologically acceptable salt thereof dissolved in the suspension to all compound a or a physiologically acceptable salt thereof in the formulation is 0.001% to 1%.

(item 13)

The formulation of any one of items 1 to 12 for use in treating a disease in the anterior ocular segment and/or a disease in the posterior ocular segment.

(item 14)

The formulation of item 13, wherein the disease is a VEGF-related disease.

(item 15)

The formulation of item 13 or 14, wherein the disease is age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, retinal vein occlusion, and/or cataracts.

(item 16)

A kit comprising the following combination of (1) and (2): (1) a formulation comprising compound a or a physiologically acceptable salt thereof and (2) a formulation comprising a dispersion medium.

(item 17)

The kit of item 16, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the formulation (1) is 1nm to 20 μm.

(item 18)

The kit of item 17, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the formulation (1) is from 10nm to 20 μm.

(item 19)

The kit of any one of claims 16 to 18, wherein formulation (1) or formulation (2) may comprise a dispersant and/or a surfactant.

(item 20)

The kit of any one of claims 16 to 19 for use in the treatment of a disease in the anterior ocular segment and/or a disease in the posterior ocular segment.

(item 21)

A medicament comprising compound a or a physiologically acceptable salt thereof for the treatment of a VEGF-related disease.

(item 22)

The medicament of item 21, wherein the disease is age-related macular degeneration and/or diabetic retinopathy.

(item 23)

The formulation of any one of items 2 to 4, wherein the suspension comprises a dispersant and/or a surfactant.

(item 24)

The formulation of item 23, wherein the suspension comprises a dispersant and a surfactant.

(Effect of the invention)

The present invention can provide a stable ophthalmic preparation comprising compound a or a physiologically acceptable salt thereof, which has a therapeutic effect not only on diseases of the anterior segment but also on diseases of the posterior segment and can avoid side effects due to systemic exposure to high doses. The solubility of compound a in water is low, and therefore if an aqueous solution preparation containing compound a is prepared, its concentration should be low. However, by preparing a formulation comprising compound a as a suspension, the formulation will become a highly concentrated formulation which is expected to deliver a sufficient amount of drug to the affected site at the actual dosing frequency (e.g., 1 to 6 times per day). In addition, compound a has another problem that the stability in a solution state is very poor, the compound in the solution can be decomposed within several hours, but a suspension formulation containing compound a can provide an ophthalmic formulation having the ability to withstand decomposition and also having good stability for practical storage. The ophthalmic preparation of the present invention can provide a breakthrough treatment for diseases in the posterior segment of the eye, which are mainly treated by an invasive method, with less burden on patients.

Drawings

Figure 1 shows the results regarding the storage stability of the content and particle size of compound a in ophthalmic suspension formulations. The ordinate of the upper graph represents the content (%) of compound a in the suspension formulation, and the abscissa represents the time (hr). The vertical axis of the lower graph represents the mean particle size (nm) of compound a in the suspension formulation, and the horizontal axis represents time (days).

FIG. 2 shows the evaluation of transferability to the posterior segment of the eye in rats (I-1). In the upper three panels of the four panels, the vertical axis represents the concentration of compound a in each tissue (the first, second and third panels show ophthalmic formulation a1, ophthalmic formulation Z and the results of oral administration, respectively). The unit of the vertical axis is μ g/g for the concentration in retina and cornea and μ g/mL for the concentration in plasma. The horizontal axis represents each tissue (from the left, retina, cornea and plasma). In the lowest graph of the four graphs, the vertical axis represents the retina/plasma ratio, and the horizontal axis represents the formulations administered (from the left, oral administration, ophthalmic formulation Z, ophthalmic formulation a 1). The retina/plasma ratio indicates "concentration of compound a in retina (μ g/g)/concentration of compound a in plasma (μ g/mL)".

FIG. 3 shows the evaluation of transferability to the posterior segment of the eye in rats (I-2). In each figure, the vertical axis represents the concentration of compound a in each tissue (from the upper side, retina, cornea, and plasma). The unit of the vertical axis is μ g/g for the concentration in retina and cornea and μ g/mL for the concentration in plasma. The horizontal axis represents each administered formulation (from the left, ophthalmic formulation Z and ophthalmic formulation a 1).

FIG. 4 shows the evaluation of transferability to the posterior segment of the eye in rats (II). In each figure, the vertical axis represents the concentration of compound a in each tissue (from the upper side, retina, cornea, and plasma). The horizontal axis represents the number of doses of the eye drops (from the left, 1 dose/eye, 3 doses/eye, 5 doses/eye).

FIG. 5 shows the evaluation of the transferability to the posterior segment of the eye in rats (III). In the upper three of the four figures, the vertical axis represents the concentration of compound a in each tissue (the first, second and third figures show the results for retina, cornea and plasma, respectively). The horizontal axis represents each administered suspension formulation, and each mean particle size (nm) of compound a in each suspension formulation. In the lowest of the four figures, the vertical axis represents the retinal/plasma ratio and the horizontal axis represents each administered suspension formulation, and each mean particle size (nm) of compound a in each suspension formulation. The retina/plasma ratio indicates "concentration of compound a in the retina (μ g/g)/concentration of compound a in the plasma (μ g/mL)".

FIG. 6 shows the evaluation of the transferability to the posterior segment of the eye in rats (IV). In each figure, the vertical axis represents the concentration of compound a in each tissue (from the upper side, retina, cornea, and plasma). The unit of the vertical axis is μ g/g for the concentration in retina and cornea and μ g/mL for the concentration in plasma. The horizontal axis represents the pH of each suspension formulation administered and each suspension formulation.

FIG. 7 shows the evaluation of transferability to the posterior segment of the eye in rats (V). In the figure, the vertical axis represents each concentration of compound a in the retina (μ g/g). The horizontal axis represents each average particle size (nm) of compound A, B or C in each suspension formulation administered and each suspension formulation.

Figure 8 shows the effect of compound a on anti-VEGF action. In the figure, the vertical axis represents distance migration of HRECs, which is the distance migration (%) of each wound width shortly after the wound. The horizontal axis represents the respective test conditions [ DMSO (%), drugs (aldose reductase inhibitor or Lucentis: (common name: Rakizumab)) and their dosages, VEGF concentration (ng/mL) ].

FIG. 9 shows the evaluation of transferability to the posterior segment of the eye in rats (III-2). In each figure, the vertical axis represents the concentration of compound a in each tissue (from the upper side, retina, cornea, and plasma). The horizontal axis represents the concentration of each suspension formulation administered.

The "mean ± se" at the upper right of fig. 2,3,4 to 9 means that each indicated value represents the mean value in each group, and each error range represents se (standard error). "n" represents the number of samples per group.

Detailed Description

The formulation of the present invention is an ophthalmic formulation characterized in that compound a or a physiologically acceptable salt thereof (hereinafter, it may be referred to as "the present drug" as a whole) is suspended in a dispersion medium. The formulation of the present invention includes an ophthalmic suspension comprising compound a or a physiologically acceptable salt thereof and a dispersion medium, and a kit for preparing a suspension for use by dispersing compound a or a physiologically acceptable salt thereof in a dispersion medium.

The active ingredient, compound a, in the formulations of the invention may be in free form or in the form of a salt with a physiologically acceptable, i.e. pharmaceutically acceptable, inorganic or organic base. Inorganic and organic bases include, for example, alkali metals such as sodium and potassium, ammonium hydroxide, isopropylamine, diethylamine, ethanolamine, piperidine and lysine. Also, compound a of the present invention or a physiologically acceptable salt thereof may also be in the form of a hydrate or a solvate, and thus compound a or a physiologically acceptable salt thereof in the present invention includes such hydrates and solvates. The details thereof are described in patent document 2. Compound a or a physiologically acceptable salt thereof can be prepared, for example, according to patent document 2.

The term "suspension" as used herein refers to a state in which compound a or a physiologically acceptable salt thereof is dispersed as a solid in a dispersion medium, and also includes a suspension in which the drug of the present invention is partially dissolved in a dispersion medium. If the drug of the present invention precipitates or aggregates in suspension due to storage, the suspension may be shaken loosely before use to return to a conventional suspension state, which is also included in the suspension of the present invention. However, the present invention does not include formulations in which the drug is dispersed, emulsified or encapsulated in oil and fat droplets, such as liposome and emulsion formulations. In particular, the drug particles in the formulations of the invention do not need to be coated with a fat or oil ingredient, and the invention does not include, for example, suspension formulations in which the drug of the invention is encapsulated in liposomes, or oil-in-water emulsion formulations in which oil and fat droplets comprising the drug of the invention are dispersed in water.

The proportion of compound a or a physiologically acceptable salt thereof dissolved in the suspension to all compound a or a physiologically acceptable salt thereof in the formulation is usually 0.001% to 10%, preferably 0.001% to 5%, further preferably 0.001% to 2%, more preferably 0.001% to 1%, even more preferably 0.001% to 0.5%, and particularly preferably 0.001 to 0.1% from the viewpoints of transferability to the retina in the posterior segment of the eye, chemical stability, physical stability of particle size, and the like. Preferably, the formulation of the present invention does not include a component having a solubilizing action to enhance the solubility of the drug of the present invention as an additive, but may include such a component having a solubilizing action unless the amount of the component may affect the solubility of the drug of the present invention. Such solubilizing ingredients include, for example, cyclodextrins.

The "dispersion" herein refers to a state in which compound a or a physiologically acceptable salt thereof is uniformly suspended in a dispersion medium, and also includes temporary suspensions and partially aggregated suspensions unless they are problematic for use as ophthalmic preparations.

The average particle size of the solid compound a or a physiologically acceptable salt thereof suspended in the suspension formulation should not be limited, but is preferably 20 μm or less, more preferably 2 μm or less, even more preferably 700nm or less, even more preferably 650nm or less, even more preferably 460nm or less, even more preferably 300nm or less, even more preferably 230nm or less, particularly preferably 200nm or less, from the viewpoint of manufacturing handleability and transferability to the posterior segment.

Also, the average particle size of solid compound a or a physiologically acceptable salt thereof suspended in the suspension formulation of the present invention is preferably 1nm or more, more preferably 5nm or more, and even more preferably 10nm or more. The average particle size preferably ranges from 10nm to 20 μm or from 1nm to 20 μm, more preferably from 10nm to 2 μm or from 1nm to 2 μm, more preferably from 10nm to 700nm or from 1nm to 700nm, even more preferably from 1nm to 650nm, further preferably from 1nm to 460nm, even more preferably from 1nm to 300nm, even more preferably from 10nm to 300nm or from 5nm to 300nm, even more preferably from 1nm to 230nm, even more preferably from 5nm to 200nm, particularly preferably from 10nm to 200nm or from 10nm to 230 nm.

As used herein, mean particle size refers to the mean particle size of compound a or a physiologically acceptable salt thereof, micronized compound a or a physiologically acceptable salt thereof, or solid compound a or a physiologically acceptable salt thereof suspended in a suspension formulation. The average particle size as used herein refers to the average particle size obtained by the apparatus and method described below. When measuring the average particle size in a suspension formulation, the concentration of the suspension can be diluted to its measurable concentration.

The above-mentioned compound a or a physiologically acceptable salt thereof having an acceptable particle size can be prepared by wet milling or dry milling.

Wet milling preparation can be carried out by stirring or dispersing the compound in a suitable solvent (for milling) with a stirrer, homogenizer, or the like. Alternatively, the milled compound may be prepared by milling the compound in a suitable solvent (for milling) using a wet jet mill such as Star Burst, and a ball mill, bead mill, homomixer, homogenizer, etc. For example, compound a or a physiologically acceptable salt thereof in a solvent for milling may be milled using a planetary ball mill (LP-4/2, imomanufacturing co., LTD.) with a compound content of 1-500mg/mL at a speed of 30-370 rpm.

The dry milling preparation can be carried out with the following apparatus: air-flow type pulverizers such as a screw Jet Mill, Jet-O-Mill, a counter-current Mill and a Jet Mill; shear type crushers such as hammer mills, sifters and sample mills; rolling ball mills, such as ball mills, bead mills, and the like.

Further, in addition to the break-down method of making particles smaller by dispersion, grinding, or the like, the ground compound a used herein or a physiologically acceptable salt thereof can also be prepared by the build-up (built-up) method using spray drying, crystallization, or freeze drying.

Ophthalmic formulations can be classified in the present invention if the formulation is/becomes in suspension for eye drops in the last step. For example, in the case of preparing the formulation of the present invention by dry milling, compound a or a physiologically acceptable salt thereof may be milled with the above-mentioned milling apparatus so as to bring the particle size to a desired, specifically, each of the above-mentioned average particle sizes, and then suspended in a dispersion medium to obtain a formulation. The formulation of the present invention also includes an embodiment (i.e., a kit) in which a suspension is prepared in use, that is, the compound a or a physiologically acceptable salt thereof and a dispersion medium are separately prepared, and the compound a or a physiologically acceptable salt thereof is suspended in the dispersion medium at the time of use.

In the case of preparing the preparation of the present invention by wet milling, a mixture of compound a or a physiologically acceptable salt thereof and a solvent for milling may be milled with the above-mentioned milling apparatus, the solvent for milling may be removed by freeze-drying or other means, and then the lyophilized product is suspended in a dispersion medium to obtain the preparation. The formulation of the present invention also includes an embodiment (i.e., a kit) in which the lyophilized product and the dispersion medium are separately prepared and the lyophilized product is suspended in the dispersion medium at the time of use. That is, the embodiment of the present invention also includes a kit comprising a combination of the following (1) and (2): (1) a lyophilized composition comprising the drug of the present invention and (2) a dispersion medium. As shown below, when compound a or a physiologically acceptable salt thereof is ground together with a solvent for grinding containing a surfactant, a dispersant, and the like, and the solvent for grinding is removed by freeze-drying or other means, the lyophilized product of compound a or a physiologically acceptable salt thereof may contain the surfactant, the dispersant, and the like, and also contain a part of the solvent for grinding.

In the above case, the dispersion medium may be used as a solvent for milling, and the milled suspension may also be provided as a formulation of the present invention by dilution optionally without freeze-drying.

The formulations of the present invention also include those that are used up in one injection or one week or other period, as well as those that prepare suspensions at the time of use, the use of which is limited to one week, one month or other period after the suspension is prepared.

The dispersion medium used herein refers to a biocompatible solvent that can disperse compound a or a physiologically acceptable salt thereof in a liquid formulation, and may include single-component solvents and multi-component solvents, as long as the solubility of compound a in the dispersion medium is preferably 0.4mg/mL or less, more preferably 0.1mg/mL or less. Specifically, the dispersion medium includes an aqueous solvent as well as an oily solvent such as castor oil, polyoxyethylene hydrogenated castor oil, and liquid paraffin. And, it may comprise a mixture of two or more solvents.

A preferred dispersion medium for use herein is an aqueous dispersion medium. The aqueous dispersion medium means an aqueous solvent containing 90w/w% or more of water, preferably 95w/w% or more of water, more preferably 99w/w% or more of water, relative to the entire dispersion medium solvent. A particularly preferred solvent for the dispersion medium is water.

Solvents in aqueous dispersion media other than water include ethanol, glycerol, propylene glycol, anise oil, phenethyl alcohol, monoethanolamine, acetic acid, glacial acetic acid, hydrochloric acid, benzyl alcohol, and polyethylene glycol. However, as mentioned above, the dispersion medium as used herein does not include a dispersion medium for an oil-in-water emulsion formulation in which oil and fat droplets comprising the drug of the present invention are dispersed in water.

The dispersion medium used herein may contain additives such as dispersing agents, surfactants, wetting agents, tonicity agents, buffering agents, preservatives, pH adjusting agents. Preferably, the dispersion medium comprises a surfactant and/or a dispersant.

Preferred dispersion media for use herein include water, more preferred dispersion media are water containing a surfactant or dispersant, even more preferred are water containing both a surfactant and a dispersant.

Further, preferred dispersion media used herein also include aqueous solvents containing a surfactant or a dispersant, and aqueous solvents containing both a surfactant and a dispersant.

The pH of the dispersion medium used herein is usually 3 to 9, preferably 3 to 8, more preferably 4 to 7, particularly preferably 4 to 6. The pH of the dispersion medium can be adjusted with the pH adjusting agent described below.

The solvent for milling used herein means a solvent used for wet milling of compound a or a physiologically acceptable salt thereof, wherein the solubility of compound a is preferably 0.4mg/mL or less. Specifically, solvents for milling used herein include water, polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), heptane, and hexane. And, it may comprise a mixture of two or more solvents. The preferred mixed solvent is an aqueous solvent containing 90w/w% or more of water with respect to the whole solvent, and optionally including the above-mentioned polyhydric alcohol, more preferably 95w/w% or more of water with respect to the whole solvent, and particularly preferably 99w/w% or more of water. The preferred solvent for milling used herein is water, which may contain additives such as surfactants, dispersants and salts, as appropriate, to aid in milling compound a or a physiologically acceptable salt thereof. The above-mentioned dispersion medium can be used as a solvent for milling.

The formulations of the invention may be provided after sterilization, which may be carried out, for example, by filtration, irradiation or autoclaving a suspension of compound a or a physiologically acceptable salt thereof in a dispersion medium. Where appropriate, compound a or a physiologically acceptable salt thereof, the lyophilized suspension, the dispersion medium and optionally added additives may be sterilized separately. Also, the whole process or a part of the process for preparing the ophthalmic preparation of the present invention may be performed in a sterile environment.

The particle size in the present invention is measured, for example, in the manner explained below, in consideration of the state of particles, the size of particles, and the like, but is not limited thereto.

For solid compound a or a physiologically acceptable salt thereof dispersed in a suspension formulation, its particle size is 1nm to 5 μm, preferably 10nm to 5 μm; the particle size measurement was carried out by diluting the suspension formulation with a dispersion medium to adjust the content of compound a or a physiologically acceptable salt thereof in the suspension formulation to about 200 μ g/mL, and then measuring the diluted sample with a measuring instrument Zeta Sizer nanoS (Malvern Instruments Ltd, Malvern UK). The measurement/calculation of particle size was performed by dynamic light scattering, where the material RI and the dispersant RI were 1.33, and the average of the calculated Z-means of particle size was shown as the measured particle size.

A particle size of 5 μm or more for solid compound a or a physiologically acceptable salt thereof dispersed in the suspension formulation; the content of compound a in the suspension formulation was adjusted to about 10 to 50 μ g/mL by diluting the suspension formulation with a dispersion medium, dispersing the diluted suspension with ultrasonic waves (15 seconds) and a stirrer (speed: 1200 rpm), and analyzing with a laser diffraction particle size analyzer: HEROS/BR-multi and moisture dispersing device: CUVETTE (Sympatec GmbH) [ Range R3, using a 50mL cell, trigger conditions (time base: 1000.00ms, determination: 10s actual time) ] and the measurement of the particle size was carried out by calculating the X50 value of the particle size by means of the calculation mode HRLD. The X50 values are shown as measured values.

For dry milling compound a or a physiologically acceptable salt thereof, a laser diffraction particle size analyzer was used: HEROS/BR-multi and dry dispersion apparatus (Sympatec GmbH) [ range R3, trigger conditions (start: ch.25. gtoreq.0.5%, stop: ch.25. ltoreq.0.5%, duration 2 seconds or 10 seconds as actual time), dispersion pressure 2.0 bar ] were measured for their particle size and the X50 value calculated from the calculation mode HRLD was shown as measured value.

The surfactant used herein is a material having a hydrophilic group and a hydrophobic group (lipophilic group) in its molecule; when the concentration is higher than a certain concentration, micelle, vesicle or lamellar structure can be formed; polar substances and non-polar substances can be uniformly mixed; has the function of reducing surface tension; and has a molecular weight of 6,000 or less; and it is an additive that helps to wet the nanoparticles of compound a or a physiologically acceptable salt thereof. Specific surfactants for use herein include polysorbate 80, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, alkyldiaminoethylglycine hydrochloride, polyethylene glycol 40 stearate, glycerin, propylene glycol, sodium chondroitin sulfate, aluminum monostearate, alkylallyl polyether alcohol, cholesterol, sucrose fatty acid ester, sorbitan sesquioleate, squalane, stearyl alcohol, cetyl alcohol, cetomacrogol 1000, diethyl sebacate, sodium dodecylbenzenesulfonate, sorbitan trioleate, nonylphenoxy polyoxyethylene ethanesulfonate ammonium, polyoxyethylene oleylamine, polyoxyethylene stearyl ether, polyoxyethylene cetyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan polyether beeswax, polyoxyethylene nonylphenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan polyether beeswax, polyoxyethylene nonylphenyl ether, polyoxyethylene polyoxypropylene glycol, polyoxyethylene sorbitan, Polyoxyethylene polyoxypropylene cetyl ether, polyethylene glycol 35 castor oil, polysorbate 20, polysorbate 60, polyethylene glycol 400, polyethylene glycol 4000, polyethylene glycol 6000, sorbitan monooleate, glycerol monostearate, sorbitan monostearate, lauryl dimethyl amine oxide solution, sodium lauryl sulfate, lauric acid diethanolamide, sodium lauroyl sarcosinate, lauromacrogol (lauromarogol), sodium polyoxyethylene lauryl ether phosphate and polyoxyethylene oleyl ether phosphate.

Preferably, it includes polysorbate 80, polyoxyethylene hydrogenated castor oil, polyoxyethylene castor oil, alkyldiaminoethylglycine hydrochloride, polyethylene glycol 40 stearate, glycerin, propylene glycol, sodium chondroitin sulfate, aluminum monostearate, polyethylene glycol 4000 and polyethylene glycol 6000; more preferred are polysorbate 80, alkyldiaminoethylglycine hydrochloride and polyoxyethylene hydrogenated castor oil. Also, two or more surfactants, preferably 2 to 3 surfactants, may be used. The amount of surfactant is preferably 0.001 to 5% by weight relative to the total amount of suspension.

The dispersant used herein is an additive that is a polymer having a molecular weight of 6,000 or more, and can enter the space between nanoparticles to help prevent aggregation. Specific dispersants for use herein include carboxyvinyl polymers, polyvinylpyrrolidone (povidone), methylcellulose, hydroxypropylmethylcellulose (hypromellose), hydroxyethylcellulose, hydroxypropylcellulose, polyvinyl alcohol, carboxymethylcellulose sodium (sodium carboxymethylcellulose), tyloxapol, gum ghatti, gum arabic, acacia powder, karaya gum, xanthan gum, aminoalkyl methacrylate copolymer RS, propylene glycol alginate, sodium carboxymethyl starch, agar powder, dioctyl sodium sulfosuccinate, and dextrin.

Preferably, it comprises carboxyvinyl polymer, hydroxyethylcellulose, polyvinylpyrrolidone (povidone), methylcellulose, hydroxypropylmethylcellulose (hypromellose), polyvinyl alcohol, sodium carboxymethylcellulose (sodium carboxymethylcellulose), and tyloxapol; more preferred are carboxyvinyl polymers, hydroxyethylcellulose, polyvinylpyrrolidone (povidone), methylcellulose, hydroxypropylmethylcellulose (hypromellose), and polyvinyl alcohol. Also, two or more dispersants may be used.

The amount of dispersant is preferably 0.001 to 5% by weight relative to the total amount of suspension.

Wetting agents include ethanol, oleic acid, magnesium silicate, light anhydrous silicic acid, and phosphorylcholine.

Tonicity agents include sodium chloride, potassium chloride, sorbitol, dextrose, sucrose, D-mannitol, ethanol, oleic acid, magnesium silicate, light anhydrous silicic acid, and choline phosphate, with sodium chloride being preferred.

The buffering agent includes sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium acetate, citric acid, sodium citrate, sodium hydrogen carbonate and tromethamine, preferably disodium hydrogen phosphate and citric acid.

Preservatives include quaternary ammonium salts such as benzalkonium chloride, benzethonium chloride and cetylpyridinium chloride; parabens such as methyl paraben, ethyl paraben, propyl paraben and butyl paraben; benzyl alcohol; phenyl ethyl alcohol; sorbic acid and sorbate salts; chlorhexidine gluconate solution.

The pH regulator includes hydrochloric acid, citric acid, glacial acetic acid, phosphoric acid, sodium dihydrogen phosphate, sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, and disodium hydrogen phosphate hydrate.

The ophthalmic suspension formulation of the present invention optionally containing the above-mentioned additives as appropriate may be prepared with compound a or a physiologically acceptable salt thereof, wherein the amount of compound a or a physiologically acceptable salt thereof is usually 1 to 500mg, preferably 5 to 300mg, more preferably 10 to 200mg, particularly preferably 20 to 300mg, 25 to 300mg, 10 to 150mg or 25 to 230mg per 1mL of the dispersion medium, but the present invention should not be limited to the above-mentioned amount.

The pH of the suspension formulation of the present invention is generally 3 to 9, preferably 3 to 8, more preferably 4 to 7, and particularly preferably 4 to 6. The pH of the suspension may be adjusted with the above-mentioned pH adjusting agents.

The osmotic pressure of the suspension preparation of the present invention is usually 20-1000 mOsm, preferably 100-700 mOsm, more preferably 180-500 mOsm, and particularly preferably 200-360 mOsm. The osmotic pressure of the suspension can be adjusted with the tonicity agents described above.

The osmotic pressure of the above suspension can be measured, for example, with a supernatant solution obtained by centrifuging the suspension. For example, the measurement may be performed using an osmolarity measuring device "OSMOSTAT OM-6040" (ARKRAY, Inc.).

The formulations of the present invention may contain other active ingredients without inhibiting the pharmacological effects of the present invention.

According to the present invention, it has been found that compound a or a physiologically acceptable salt thereof can inhibit the promotion of cell migration by VEGF stimulation, as shown in the test examples below. Therefore, compound a or a physiologically acceptable salt thereof in the preparation of the present invention is expected to have a therapeutic effect on various ophthalmic diseases, because the compound has an inhibitory action on aldose reductase, an inhibitory action on VEGF production, and an inhibitory action on the promotion of cell migration by VEGF stimulation. Furthermore, the suspension formulation of the present invention containing compound a or a physiologically acceptable salt thereof particularly has good transferability to the posterior segment, and therefore the suspension formulation of the present invention is also expected to be therapeutically suitable for diseases of the posterior segment requiring administration to the posterior segment, such as age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, retinal vein occlusion, choroidal neovascularization, uveitis, retinitis pigmentosa, proliferative vitreoretinopathy and central serous chorioretinopathy, but the disease of interest of the present invention should not be limited to these diseases.

In particular, the formulations of the invention are expected to have therapeutic applications in diseases associated with VEGF, diseases caused by VEGF intervention, or diseases following diseases that occur as a result of VEGF intervention.

In addition, the preparation of the present invention is also expected to have a significant therapeutic effect when it is therapeutically applied to diseases of the anterior segment requiring administration to the anterior segment, such as keratitis, conjunctivitis, neovascular glaucoma, dry eye and cataract, or diseases in which a drug needs to be transported across the blood-aqueous barrier (BAB) or cornea.

The dosage and administration of the ophthalmic suspension formulation of the present invention should be appropriately determined based on the efficacy of the drug, the administration route, symptoms, age, body weight, and the like. The preferred dosage and administration in the present invention is, for example, administration of a suspension formulation containing 1 to 500mg/mL of Compound A or a physiologically acceptable salt thereof in eye drops in an amount of 1 to 2 drops per eye for a total of 1 to about 6 times a day. Generally, one drop in the eye drops is in an amount of 20 to 80. mu.L, preferably 30 to 50. mu.L. The period of administration in the present invention should be determined according to the severity of symptoms and the like, and includes, for example, 1 or more weeks, preferably about 1 week to about 4 weeks, more preferably about 4 or more weeks.

The formulations of the present invention may also be used for ophthalmic diseases in mammals other than humans, such as monkeys, cows, horses, dogs, and cats.

Examples

The present invention will be described in detail below with reference to examples, reference examples, comparative examples, tests, and the like, but the present invention is not limited thereto.

Reference example 1: preparation of Compound A

Compound A is according toT, Negoro et al J. Med. chem. 1998, 41, 4118-4129The process as described in (1). To the crude product thereof (10 g) were added activated carbon (50% humidity, 0.8 g) and 2-propanol (101 g), and the reaction solution was heated to its reflux temperature (about 84 ℃ C.) and allowed to stand at the same temperature for 30 minutes. The reaction solution was filtered at the same temperature and washed with 2-propanol (13.8 g). The resulting filtrate was heated to 75 ℃ or higher, cooled to 60 ℃, held at 60 ℃ for 1 hour, and then cooled to 0 ℃. The precipitated solid was collected on the filter and dried in vacuo to give compound a as white crystals (9.3 g).

XRD; 2θ = 11.5, 15.4, 15.7, 16.3, 16.9, 18.2, 19.3, 20.1, 20.9, 21.6,22.2, 23.3, 24.0, 24.7, 25.1, 26.4, 27.5, 28.4, 28.8, 29.6, 29.9, 30.9, 31.9,32.4。

Differential Scanning Calorimetry (DSC) showed an endothermic peak with an extrapolated onset of melting of 186.7 ℃.

The measurement of the above powder X-ray diffraction was carried out using powder X-ray diffraction system XRD-6100 (Shimadzu Corp.), under the following conditions: an X-ray tube: CuK α (wavelength 1.54 angstroms), tube voltage: 30.0kV, tube current: 20.0mA, drive shaft: θ -2 θ, measurement range: 5-40 degrees, step width: 0.020 degree, speed: 2.00 (degree/minute), count time: 0.60 second. The above Differential Scanning Calorimetry (DSC) measurements were performed using Thermo Plus 2 (Rigaku Corporation) under flowing air between 25 ℃ and 250 ℃ with samples weighed at about 10mg in an aluminum container and a ramp rate of 5 ℃/min.

The compound a obtained above was granulated using a jet mill, and grinding conditions were changed to obtain three compounds a having different average particle sizes. The average particle sizes of the three compounds A obtained were 1.43. mu.m, 6.29. mu.m and 21.98. mu.m.

The average particle size of the compound a obtained by granulation with a jet mill (dry milling) was measured using the above laser diffraction particle size analyzer, and is represented by X50 value of the particle size calculated in the calculation mode LD.

Reference example 2: preparation of Dispersion Medium (pH 5.0)

To a 0.9% (w/v) aqueous solution of sodium chloride (a) of 0.02mol/L aqueous disodium hydrogenphosphate was added a 0.01mol/L aqueous solution of citric acid (b) of 0.9% (w/v) sodium chloride to adjust the pH of the solution (a) to 5.0 (addition ratio (a): (b) = about 1: 1), yielding a citrate-phosphate buffer solution having a pH of 5.0. 20g of hydroxypropylmethylcellulose was dissolved in 380g of pure water to prepare 400g of a 5% hydroxypropylmethylcellulose aqueous solution, to which 1600g of a citrate-phosphate buffer solution having a pH of 5.0 was added to prepare 2000g of a 1% hydroxypropylmethylcellulose aqueous solution. 500mg of 27-33% aqueous solution of alkyldiaminoethylglycine hydrochloride was dissolved in 400g of 1% aqueous hydroxypropyl methylcellulose, 2.5g of polysorbate 80 was further added thereto, and the mixture was dissolved. The weight of the solution was adjusted to 500g by adding a 1% hydroxypropylmethylcellulose aqueous solution, and the pH of the solution was adjusted to 5.0 with a 1mol/L hydrochloric acid aqueous solution to obtain a dispersion medium (pH 5.0). The composition of the prepared dispersion medium (pH 5.0) is shown below.

[ Table 1]

Composition (I)	Measurement of
		Citric acid	0.76 mg/g
Disodium hydrogen phosphate	1.13 mg/g
		Sodium chloride	7.2 mg/g
Hydroxypropyl methylcellulose	9.9 mg/g
		27-33% aqueous solution of alkyldiaminoethylglycine hydrochloride	1 mg/g
Polysorbate 80	5 mg/g
		Hydrochloric acid	Small amount of

Example 1: preparation of ophthalmic suspension formulations

4g of Compound A (average particle size: 1.43 μm) granulated with a jet mill and 12mL of the dispersion medium (pH 5.0) prepared in reference example 2 were put in a screw bottle, and the mixture was stirred with a stirrer for 30 minutes. The entire sample mixture was then transferred to a grind tank. The screw-top bottle was washed with 4mL of dispersion medium and the washing solution was also placed in the milling jar. To each of the prepared milling pots, 50g of zirconia beads having respective diameter sizes defined below were added. The milling jar was placed in a planetary ball mill (LP-4/2, ITO manual co., LTD.) and the contents thereof were milled at 300rpm for 2 hours. Each of the ground solutions was filtered through a sieve to remove beads, and then defoamed for 30 seconds using a blender/defoamer, "THINKY MIXER AR-250" (THINKY CORPORATION) to give suspension formulations A1, A2, A3 and B-D.

Suspension formulations a1, a2 and A3: the beads used had a diameter size of 0.5 mm.

Suspension formulation B: the beads used had a diameter size of 2.0 mm.

Suspension formulation C: the beads used had a diameter size of 3.0 mm.

Suspension formulation D: the beads used had a diameter size of 5.0 mm.

2.2G of jet-milled Compound A (average particle size: 1.43 μm, 6.29 μm or 21.98 μm) and 10mL of a dispersion medium (pH 5.0) were added to a screw bottle and stirred with a stirrer for 10 minutes to give a suspension formulation E, F or G.

Each average particle size of compound a suspended in suspension formulations a1 to A3 and B to G was measured by the method described above. The obtained average particle sizes are shown below.

Suspension formulation A1: 209.0 nm

Suspension formulation A2: 195.2 nm

Suspension formulation A3: 227.0 nm

Suspension formulation B451.2 nm

Suspension preparation C: 609.9 nm

Suspension formulation D, 801.2 nm

Suspension formulation E: 1944 nm

Suspension formulation F9560 nm

Suspension preparation G at 20430 nm

Reference example 3: quantitative analysis of drug in suspension formulations for each eye

To 100 μ L of each suspension formulation A1 to A3 and B to G was added 800 μ L of 1% HPMC and 100 μ L of acetonitrile and each mixture was vortexed. 100 μ L of each shaking mixture was put into a 10mL volumetric flask, to which 1% HPMC/acetonitrile (1: 1) was added and the mixture was completely dissolved. The total volume of the solution was adjusted to 10mL with 1% HPMC/acetonitrile (1: 1) accurately, giving each of its analytical samples. The suspension preparations a1 to A3 and B to G were analyzed for the content of each compound a (which is the combined content of the dissolved compound a and the suspended compound a) using ultra high performance liquid chromatography (SHIMADZU) using a column YMC-Pack Pro C185 μm 150 × 4.6 mm. The analysis results are shown below.

Suspension formulation A1: 200.1 mg/mL

Suspension formulation A2: 216.2 mg/mL

Suspension formulation A3: 207.1 mg/mL

Suspension formulation B211.0 mg/mL

Suspension formulation C195.6 mg/mL

Suspension formulation D218.8 mg/mL

Suspension formulation E135.4 mg/mL

Suspension formulation F201.8 mg/mL

Suspension formulation G224.5 mg/mL.

Reference example 4: preparation of the Dispersion Medium (pH 3.0)

To an aqueous solution (c) of 0.9% (w/v) sodium chloride in 0.02mol/L disodium hydrogenphosphate aqueous solution was added an aqueous solution (d) of 0.01mol/L aqueous citric acid in 0.9% (w/v) sodium chloride to adjust the pH of the solution (c) to 3.0 (addition ratio (c): (d) = about 2: 8), yielding a citrate-phosphate buffer solution having a pH of 3.0. The resulting citrate-phosphate buffer solution of pH3.0 was treated in a similar manner to the case of the above-described citrate-phosphate buffer solution of pH5.0 to obtain a dispersion medium (pH 3.0). The composition of the prepared dispersion medium (pH 3.0) is shown below.

[ Table 2]

Composition (I)	Measurement of
		Citric acid	1.22 mg/g
Disodium hydrogen phosphate	0.45 mg/g
		Sodium chloride	7.2 mg/g
Hydroxypropyl methylcellulose	9.9 mg/g
		27-33% aqueous solution of alkyldiaminoethylglycine hydrochloride	1 mg/g
Polysorbate 80	5 mg/g
		Hydrochloric acid	Small amount of

Reference example 5: preparation of the Dispersion Medium (pH 8.0)

To an aqueous solution (e) of 0.9% (w/v) sodium chloride in 0.02mol/L disodium hydrogenphosphate aqueous solution was added an aqueous solution (f) of 0.9% (w/v) sodium chloride in 0.02mol/L sodium dihydrogenphosphate aqueous solution to adjust the pH of the solution (e) to 8.0 (addition ratio (e): (f) = about 19: 1), resulting in a phosphate buffer solution of pH 8.0. The resulting phosphate buffer solution of pH 8.0 was treated in a similar manner to that in the case of the above citrate-phosphate buffer solution of pH5.0 (provided that a 1mol/L aqueous sodium hydroxide solution was used instead of a 1mol/L aqueous hydrochloric acid solution), to obtain a dispersion medium (pH 8.0). The composition of the prepared dispersion medium (pH 8.0) is shown below.

[ Table 3]

Composition (I)	Measurement of
		Sodium dihydrogen phosphate	0.10 mg/g
Disodium hydrogen phosphate	2.15 mg/g
		Sodium chloride	7.2 mg/g
Hydroxypropyl methylcellulose	9.9 mg/g
		27-33% aqueous solution of alkyldiaminoethylglycine hydrochloride	1 mg/g
Polysorbate 80	5 mg/g
		Sodium hydroxide	Small amount of

Example 2: preparation of ophthalmic suspension formulations

Suspension formulations H, I or J were prepared in a similar manner to example 1, using 1.0mm diameter beads and a dispersion medium (pH 3.0 of reference example 4, pH5.0 of reference example 2, or pH 8.0 of reference example 5). The respective pH of the resulting suspension formulation is shown below.

Suspension formulation H (pH 3): 3.09

Suspension formulation I (pH5): 5.07

Suspension formulation J (pH 7): 7.10

Each average particle size of compound a suspended in suspension formulations H, I and J was measured by the method described above. The obtained average particle sizes are shown below.

Suspension formulation H (pH 3) 352.1 nm

Suspension formulation I (pH5) 263.2 nm

Suspension formulation J (pH 7) 256.0 nm

The content of each compound a in suspension formulations H, I and J was analyzed in the above manner, and the analysis results are shown below.

Suspension formulation H (pH 3): 217.9 mg/mL

Suspension formulation I (pH5) 220.0 mg/mL

Suspension formulation J (pH 7) 222.4 mg/mL

Comparative example 1: preparation of ophthalmic solution formulations comprising Compound A

To a solution of 0.9% (w/v) sodium chloride in 0.1mol/L sodium dihydrogenphosphate aqueous solution was added a solution of 0.9% (w/v) sodium chloride in 0.1mol/L disodium hydrogenphosphate solution to adjust the pH of the solution to 8.0, and the solution was diluted 1.25 times with pure water (hereinafter referred to as "pH 8.0 solution"). To 1mL of the obtained solution, 400. mu.g of jet-milled Compound A and 0.08mL of ethanol were added to dissolve Compound A, to obtain solution preparation Z.

Comparative example 2: preparation of ophthalmic suspension formulations comprising [5- [ (1Z,2E) -2-methyl-3-phenylpropenylidene ] -4-oxo-2-thioxothiazolidin-3-yl ] acetic acid or (2S,4S) -6-fluoro-2 ',5' -dioxospiro [ chromane-4, 4' -imidazolidine ] -2-carboxamide

2g of [5- [ (1Z,2E) -2-methyl-3-phenylallylidene ] -4-oxo-2-thioxothiazolidin-3-yl ] acetic acid (hereinafter referred to as "Compound B") and 6mL of the dispersion medium (pH 5.0) prepared in reference example 2 were charged into a screw bottle, and the mixture was stirred with a stirrer for 30 minutes. The entire sample mixture was then transferred to a grind tank. The screw-top bottle was washed with 2mL of dispersion medium and the washing solution was also placed in the milling jar. 50g of beads having a diameter of 1.0mm are added to the milling jar. The milling jar was placed in a planetary ball mill (LP-4/2, ITO manual co., LTD.) and the contents thereof were milled at 300rpm for 6 hours. The milled solution was filtered through a sieve to remove beads, and then defoamed for 30 seconds using a blender/defoamer "THINKY MIXERAR-250" (THINKY CORPORATION) to give suspension formulation X1. Further, 1.84g of (2S,4S) -6-fluoro-2 ',5' -dioxospiro [ chromane-4, 4' -imidazolidine ] -2-carboxamide (hereinafter referred to as "Compound C") and 6mL of the dispersion medium (pH 5.0) prepared in reference example 2 were charged into a screw bottle, and the mixture was stirred with a stirrer for 30 minutes. The resulting sample was treated in a similar manner to compound B above to give suspension formulation Y1.

1.1g of Compound B and 5mL of the dispersion medium (pH 5.0) prepared in reference example 2 were placed in a screw bottle, and the mixture was stirred for 10 minutes with a stirrer, to give a suspension formulation X2. In a similar manner, suspension formulation Y2 was also prepared from compound C.

The respective average particle sizes of compound B and compound C suspended in suspension formulations X1, Y1, X2 and Y2 were measured in a similar manner to the measurement of the average particle size of compound a. The obtained average particle sizes are shown below.

Suspension preparation X1: 555.7 nm

Suspension preparation Y1: 290.8 nm

Suspension formulation X2: 8970 nm

Suspension preparation Y2: 5430 nm

Each content of compound B and compound C in suspension formulations X1, Y1, X2, and Y2 was analyzed in the above-described manner, and the analysis results are shown below.

Suspension formulation X1: 215.4 mg/mL

Suspension formulation Y1: 214.1 mg/mL

Suspension formulation X2: 187.7 mg/mL

Suspension formulation Y2: 196.8 mg/mL

The results of the examples and reference examples herein are summarized as follows.

[ Table 4]

Test 1: evaluation of storage stability of ophthalmic suspension formulations comprising Compound A

With respect to the ophthalmic suspension formulation, the change in the content of compound a stored at 37 ℃ and the change in the particle size of compound a stored at 25 ℃ were evaluated according to the following procedures.

Ophthalmic suspension formulations were prepared according to the procedure described in example 1, using a dispersion medium (ph 5.0) and beads having a diameter of 0.5 mm. The resulting ophthalmic suspension formulations were analyzed for compound a content and for the average particle size of the suspended compound a by the methods described above. The analysis results were 220.95mg/mL and 170.2nm, respectively. The prepared ophthalmic suspension formulation was stored at 37 ℃ for a prescribed time and then subjected to ultrasonic treatment to homogenize it. To 100 μ L of the ophthalmic suspension formulation, 800 μ L of 1% HPMC and 100 μ L acetonitrile were added and the mixture was vortexed. 100 μ L of the shaking mixture was put into a 10mL volumetric flask, to which 1% HPMC/acetonitrile (1: 1) was added to adjust the total volume of the solution to exactly 10 mL. The resulting solution was used as an analytical sample of compound a. Each sample was analyzed by ultra high performance liquid chromatography (SHIMADZU) for each storage time using column YMC-Pack Pro C185 μm 150X 4.6 mm. The analysis results are shown in FIG. 1.

Subsequently, an ophthalmic suspension formulation was prepared according to the procedure described in example 1, using a dispersion medium (ph 5.0). In the milling, beads with a diameter of 0.5mm were used for 2 hours, then the beads were changed to beads with a diameter of 0.02mm and the mixture was milled for 2 hours. The resulting ophthalmic suspension formulations were analyzed for compound a content and for the average particle size of the suspended compound a by the methods described above. The analysis results were 168.5mg/mL and 180.5nm, respectively. The prepared ophthalmic suspension formulations were stored at 25 ℃ and the average particle size of the stored samples was analyzed by the method described above for each time period. The analysis results are shown in FIG. 1.

Figure 1 shows that the amount of compound a in the ophthalmic suspension formulations of the present invention did not decrease much even after 72 hours of storage. Also, even after 14 days of storage, there was no change in the average particle size. The results indicate that the ophthalmic formulation of the present invention is chemically and physically stable and does not require storage in cold places, i.e. can be stored at ambient temperature.

Test 2: evaluation of transferability to the posterior segment of the eye in rats (I-1) and (I-2)

For both eyes of the diabetic rat model, an ophthalmic suspension formulation a1 in which compound a was suspended as a suspension formulation (200 mg/mL) at a generally tolerated dose, or an ophthalmic solution formulation Z in which compound a was dissolved (400 μ g/mL), was administered to one eye (which is the maximum tolerated dose of rat eye drops) at 5-minute intervals as eye drops in an amount of 5 μ L for a total of 5 times. Each concentration of compound a in the cornea, retina and plasma was measured 60 minutes after the administration.

Eye drops have a limit on the volume that can be tolerated once, unlike other dosage forms. Therefore, the amount of drug delivered to the target tissue obtained from the eye drops of the maximum tolerated dose is important. The tolerated volume varies according to the animal species, and the maximum volume in rats is 5 μ Ι _ for one eye.

The solubility of compound a in water is very low, and therefore in the preparation of its ophthalmic solution formulation, an additive is added to the solution to make the solubility of compound a higher than the original solubility, thereby preparing ophthalmic solution formulation Z (400 μ g/mL).

In addition to this, the present invention is,Diabetic Retinopathy, INTECH, Phapter 15 "Prophylactic Medical Treatment of Diabetic Retinopathy", Akihiro Kakehashiet al disclose an experiment of orally repeating administration of compound a to SDT rats, wherein the dose of compound a that weakens retinal capillaries and inhibits VEGF production in the retina is 1.0 mg/kg. In this test, the dose of compound A (1.0 mg/kg) was administered orally repeatedly once a day for 21 days. Each concentration of compound a in the cornea, retina and plasma was measured 60 minutes after the final administration. The results are shown in FIGS. 2 and 3.

Fig. 2 shows the delivered amount (concentration) of compound a to plasma, cornea and retina by eye-drop administration of ophthalmic suspension formulation a1, eye-drop administration of ophthalmic solution formulation Z and oral repeated administration. The respective delivery ratios of compound a to plasma, cornea and retina are shown below.

The concentration ratio of compound a in each tissue of ophthalmic suspension preparation a1 administered by eye drop was

Plasma: cornea: retina = about 1: 101: 33.5.

the concentration ratio of Compound A in each tissue of ophthalmic solution preparation Z administered by eye drop was

Plasma: cornea: retina = about 1: 1689: 1.

and, by repeated oral administration, the concentration ratio of Compound A in each tissue is

Plasma: cornea: retina = about 1:1: 1.

In addition, each retina/plasma ratio [ retina/plasma ratio = (concentration of compound a in retina (μ g/g))/(concentration of compound a in plasma (μ g/mL)) ] of each administration group is shown below.

The retina/plasma ratio of the group administered with ophthalmic suspension formulation a1 = 33.5.

The retina/plasma ratio of the administration group of ophthalmic solution formulation Z = 1.1.

The retina/plasma ratio of the oral repeat dosing group = 1.0.

In rats to which ophthalmic solution preparation Z was administered in eye drops, the retina/plasma ratio was 1.1, which was almost the same as the retina/plasma ratio (1.0) by oral administration. The results indicate that most ophthalmic solution formulation Z enters the systemic circulating blood through the nasolacrimal duct and then reaches the retina.

On the other hand, in rats to which the ophthalmic suspension formulation a1 of the present invention was administered, the retinal/plasma ratio was 33.5, which is more than 30 times the retinal/plasma ratio (1.0) obtained by orally administering an effective dose of compound a to SDT rats for diabetic retinopathy or the retinal/plasma ratio (1.1) obtained by administering the ophthalmic solution formulation Z. This indicates that the ophthalmic suspension formulation can be delivered to the retina by a direct delivery route.

The respective concentrations of compound a in the respective tissues to which the rat ophthalmic suspension formulation a1 and ophthalmic solution formulation Z were administered in test 2 are shown in fig. 3. In rats to which ophthalmic solution formulation Z was administered, although the concentration of compound a was higher than the original solubility (400 μ g/mL), the delivery rate to the retina was low (0.0473 μ g/g), i.e., this test was not considered to be predictive of therapeutic effect. On the other hand, in rats given ophthalmic suspension formulation a1, the delivery rate to the retina was high enough (342 μ g/g) to be expected to be a therapeutic effect, where the concentration of compound a in the formulation was the generally tolerated dose as a suspension (200 mg/mL).

The concentration of compound a in the suspension formulation administered in the eye drops is 500 times that of the solution formulation [ (200 mg/mL)/(400 μ g/mL) ], but its amount in the retina derived from the suspension formulation is much higher than that of the solution formulation (7230 times [ (342 μ g/g)/(0.0473 μ g/g) ].on the other hand, for delivery to the cornea of the anterior segment and delivery to the plasma, its respective amounts in the cornea and plasma from the suspension formulation are only 14 times and 232 times that of the solution formulation, respectively, although the amount of compound a in the suspension formulation administered in the eye drops is 500 times that of the solution formulation.

It has been found that ophthalmic suspension formulations containing compound a can be delivered in a more adequate concentration of compound a than oral administration through systemic circulation or via eye drops of ophthalmic solution formulations. Accordingly, ophthalmic suspension formulations containing compound a or a physiologically acceptable salt thereof are believed to have sufficient separation between its effects and its side effects, and thus may allow diseases in the posterior segment of the eye, including the retina, to be safely treated.

Test 3: evaluation of the transferability to the posterior segment of the eye in rats (II)

For both eyes of SD rats, ophthalmic preparation a2 was administered as eye drops once, three times or five times at 5-minute intervals in an amount of 5 μ L for one eye. Each concentration of compound a in the cornea, retina and plasma was measured 60 minutes after the administration. The results are shown in FIG. 4.

As shown in fig. 4, ophthalmic suspension formulations resulted in an increase in plasma concentration of compound a with dosing frequency. However, the concentration in the retina is not affected by the frequency of dosing, i.e. the required amount of compound a is delivered to the retina in one dose. From this result, it has been found that an ophthalmic suspension formulation comprising compound a or a physiologically acceptable salt thereof can suppress an increase in circulating levels by such a low administration frequency and also allow the formulation to be sufficiently delivered into the retina.

Test 4: evaluation of the transferability to the posterior segment of the eye in rats (III)

To both eyes of SD rats, ophthalmic preparation A3 or ophthalmic preparations B to G were administered once in the form of eye drops in an amount of 5. mu.L to one eye. Each concentration of compound a in the cornea, retina and plasma was measured 60 minutes after the administration. The results are shown in FIG. 5.

In addition, the ophthalmic preparation a3 was diluted with a dispersion medium to prepare ophthalmic suspension preparations having different concentrations of compound a (20 mg/mL), and then the diluted preparations were administered to SD rats. At 60 minutes after the administration, each concentration of compound A in the cornea, retina and plasma (III-2) was measured. The results are shown in FIG. 9.

As shown in fig. 5, the smaller the average particle size, the higher the concentration of compound a in the retina. However, there was no large difference in the concentration of compound a in cornea and plasma, as long as the average particle size was less than 9560 nm. Ocular administration using the suspension formulation showed higher retinal/plasma ratios (about 4-12) than oral administration or ocular administration with solution (oral administration: 1.0, ocular administration with solution: 1.1), even for all ophthalmic suspension formulations with various mean particle sizes. Also, ophthalmic suspension formulations containing compound a having an average particle size of 700nm or less have a large difference between the concentration in the retina and the concentration in the plasma. In view of this result, it is believed that ophthalmic suspension formulations can exhibit high efficacy and also reduce side effects.

As shown in fig. 9, the higher the concentration of compound a in the suspension formulation, the higher the concentration of compound a in the retina. It is generally believed that drug delivery to the posterior segment depends on the amount of drug dissolved in water. However, in this test, it is surprising that the amount of compound a delivered to the retina increases with the concentration of compound a in suspension, regardless of the amount of compound a dissolved. Also, ocular administration with the suspension formulation showed a higher retinal/plasma ratio (about 12) than oral administration or ocular administration with solution (oral administration: 1.0, ocular administration with solution: 1.1), regardless of the suspension concentration.

Test 5: evaluation of the transferability to the posterior segment of the eye in rats (IV)

Ophthalmic preparation H (pH 3), ophthalmic preparation I (pH5), or ophthalmic preparation J (pH 7) was administered once in eye drops in an amount of 5 μ L for one eye to both eyes of SD rats. Each concentration of compound a in the cornea, retina and plasma was measured 60 minutes after the administration. The results are shown in FIG. 6.

As shown in fig. 6, ophthalmic formulation I (pH5), ophthalmic formulation H (pH 3), and ophthalmic formulation J (pH 7) all showed delivery levels to the retina sufficient to be able to treat diseases of the posterior segment of the eye. In particular, ophthalmic formulation I (pH5) showed very high delivery to the retina. However, delivery to the cornea and plasma is not affected by this pH.

Test 6: evaluation of the transferability to the posterior segment of the eye in rats (V)

Ophthalmic preparation I, ophthalmic preparation F, ophthalmic preparation G, ophthalmic preparation X1, ophthalmic preparation X2, ophthalmic preparation Y1, or ophthalmic preparation Y2 was administered once as eye drops to both eyes of SD rats in an amount of 5 μ L to one eye. Each concentration of compound a in the retina was measured 60 minutes after the administration. The results are shown in FIG. 7.

As shown in fig. 7, the ophthalmic suspension formulations containing compound a delivered significantly more to the retina than the ophthalmic suspension formulations containing compound B or C. Even when compound a in the ophthalmic suspension formulation has an average particle size of 9560nm, the required amount of compound a is delivered to the retina. However, in the case of compound B or compound C, compounds with any average particle size were hardly delivered to the retina.

Test 7: inhibition of the stimulation of cell migration by VEGF stimulation by Compound A

By passingJ Diabetes Complications. 2012 ;26(5):369-77The experimental method described in (cell migration assay) evaluates the anti-VEGF effect of compound a. In the experiments, normal human retinal capillary endothelial cells (HREC) obtained from Cell System coThe Cell culture was CS-C medium (Cell systemco., LTD.). Compound a, compound B, or compound C was dissolved in dimethyl sulfoxide (DMSO), and each solution was diluted with a cell culture to adjust the concentration in DMSO to 0.1%, and each 0.1% solution was used for the experiment. For Lucentis, its formulation stock was diluted with cell culture at a dilution ratio of 50 μ L of formulation stock per 4mL of cell culture, and the diluted solution was used for experiments. HREC were seeded in 6-well plates and incubated until confluence was 80-90%. The cell culture was changed to a cell culture containing 0.1% fetal bovine serum 20-24 hours before measuring cell migration stimulated by VEGF. Then, the cultured cell monolayer was injured at one point per well with a 200 μ L pipette tip, and the width of the wound was measured with a microscope. After injury, cell cultures were changed to cell cultures containing VEGF, each aldose reductase inhibitor (compound a, compound B, compound C) or Lucentis, depending on each assay condition. Approximately 18 hours after changing the cell culture, each width of the wound was measured microscopically and the anti-VEGF effect was evaluated by comparing the width to the width of the just-injured wound. The results are shown in FIG. 8.

As shown in fig. 8, the results indicate that VEGF stimulation promotes HREC migration, whereas Lucentis and aldose reductase inhibitors inhibit migration. The effect of inhibiting HREC migration with 1nM ((0.42 ng/mL) L) Compound A was the same as the level of 100nM ((28 ng/mL)/L) Compound C and more potent than 1000nM ((320 ng/mL)/L) Compound B. In addition, the migration inhibition of compound a was more effective than that of other aldose reductase inhibitors, as shown by the action ratio shown in the following table. And the migration inhibition of compound a at 1nM was at the same level as the anti-VEGF antibody Lucentis.

[ Table 5]

AR inhibitors	AR inhibition, Ki (nM)
		Compound A	0.23 (X 1.0)
Compound C	1.9 (X 8.3)
		Compound B	62 (X 270)

Diabetic Retinopathy, INTECH, Phapter 15, "Prophylactic Medical Treatment of Diabetic Retinopathy", Akihiro KakehashiEt al show that repeated oral administration of compound a to SDT rats in a non-obese type 2 diabetes model can attenuate retinal capillaries and inhibit VEGF production in the retina, and this finding indicates that prophylactic administration of compound a for VEGF-related diseases before promoting VEGF production is expected to progress to a certain level. However, this document does not clearly show that compound a is effective in therapy after the onset of the disease by reducing the effects of VEGF that have already been produced. In addition, non-patent document 1 shows only data of oral administration, but does not show the possibility of suppressing progress by eye drop administration.

This experiment has clearly shown that compound a or a physiologically acceptable salt thereof can inhibit the promoting effect of VEGF stimulation on cell migration, and also shows an anti-VEGF effect on VEGF that has already been produced. This finding suggests that the present invention may treat VEGF-related diseases such as age-related macular degeneration and diabetic retinopathy after onset of disease. Also, the anti-VEGF effect of compound a was more potent than other aldose reductase inhibitors, and it was at the same level as Lucentis as an anti-VEGF antibody preparation.

Reference example 6: preparation of suspension formulation samples (1) to (4)

Preparation of sample (1): an ophthalmic suspension formulation containing compound a (250 mg/mL) was prepared according to the procedure of example 1, using a dispersion medium (ph 5.0) and beads having a diameter of 0.5 mm.

Preparation of sample (2): according to reference example 2, a dispersion medium (ph 5.0) containing 0.3% polyoxyethylene hydrogenated castor oil was prepared by using polyoxyethylene hydrogenated castor oil instead of polysorbate 80 in the procedure of reference example 2. 15 mL of the dispersion medium and 5g of Compound A were placed in a screw bottle, and the mixture was subjected to sonication for 5 minutes. The entire contents were transferred to syringe Star Burst Mini (Sugino Machine Limited) for milling and the screw-threaded vial was washed with 5mL of dispersion medium and the wash solution was also placed in the syringe. The contents of the syringe were triturated at a trituration pressure of 245MPa for 30 minutes to prepare an ophthalmic suspension formulation comprising compound a (250 mg/mL).

Preparation of sample (3): 1g of jet-milled Compound A was added to 10g of glycerin, and the mixture was stirred with a stirrer for 1 hour to prepare a suspension formulation containing Compound A.

Preparation of sample (4): 1g of jet-milled compound a was added to 10g of water, and the mixture was stirred with a stirrer for 1 hour to prepare a suspension formulation containing compound a.

Test 8: evaluation of solubility of Compound A

Samples (1) to (4) were each centrifuged at 500. mu.L (150,000 rpm, 10 minutes, 5 ℃) using a centrifuge HITACH-GX (Hitachi Koki Co., Ltd.). To 100. mu.L of the resulting supernatant, 800. mu.L of 1% HPMC and 100. mu.L of acetonitrile or water were added, and the mixture was shaken by vortexing to obtain each of the analysis samples. The amount of each compound A dissolved in each sample was analyzed by ultra high performance liquid chromatography (SHIMADZU) using a column YMC-Pack Pro C185 μm 150X 4.6 mm. The analysis results are shown in table 6. Table 6 shows that the percentage of compound a dissolved in the aqueous suspension is very small.

[ Table 6]

The respective average particle sizes of compound a suspended in samples (1) and (2) were analyzed in the above manner. The results were 244.2nm and 276.7nm, respectively. The particle sizes in the samples (2) stored at 25 ℃ for one month and two months were 277.3nm and 256.9nm, respectively.

INDUSTRIAL APPLICABILITY

In view of these examples, reference examples, experiments, and the like, the ophthalmic preparation of the present invention has good transferability to the posterior segment of the eye, and can be suitably used for treating ophthalmic diseases such as diseases of the posterior segment of the eye.

Claims (24)

1. An ophthalmic suspension formulation comprising (R) - (-) -2- (4-bromo-2-fluorobenzyl) -1,2,3, 4-tetrahydropyrrolo [1,2-a ] pyrazine-4-spiro-3 ' -pyrrolidine-1, 2',3,5' -tetraone (hereinafter referred to as "compound a") or a physiologically acceptable salt thereof.

2. The formulation of claim 1, wherein Compound A or a physiologically acceptable salt thereof is dispersed in the dispersion medium.

3. The formulation of claim 2, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the suspension is from 1nm to 20 μm.

4. The formulation of claim 3, wherein the average particle size of Compound A or a physiologically acceptable salt thereof in the suspension is from 10nm to 20 μm.

5. The formulation of any one of claims 2 to 4, wherein the dispersion medium is an aqueous dispersion medium.

6. A formulation according to any one of claims 2 to 5, wherein the dispersing medium comprises a dispersant and/or a surfactant.

7. The formulation of claim 6, wherein the dispersion medium comprises a dispersant and a surfactant.

8. The formulation of any one of claims 2 to 7, wherein the pH of the suspension is from 3 to 9.

9. The formulation of any one of claims 2 to 8, wherein the osmolality of the suspension is from 20 mOsm to 1000 mOsm.

10. The formulation of any one of claims 2 to 9, comprising 1mg to 500mg of compound a or a physiologically acceptable salt thereof per 1mL of the suspension.

11. The formulation as claimed in any one of claims 2 to 10, wherein the proportion of compound a or a physiologically acceptable salt thereof dissolved in the suspension to all compound a or a physiologically acceptable salt thereof in the formulation is from 0.001% to 10%.

12. The formulation as claimed in any one of claims 2 to 11, wherein the proportion of compound a or a physiologically acceptable salt thereof dissolved in the suspension to all compound a or a physiologically acceptable salt thereof in the formulation is from 0.001% to 1%.

13. The formulation of any one of claims 1 to 12 for use in the treatment of a disease of the anterior segment and/or a disease of the posterior segment.

14. The formulation of claim 13, wherein the disease is a VEGF-related disease.

15. The formulation of claim 13 or 14, wherein the disease is age-related macular degeneration, diabetic retinopathy, diabetic macular edema, myopic choroidal neovascularization, retinal vein occlusion, and/or cataracts.

16. A kit comprising the following combination of (1) and (2): (1) a formulation comprising compound a or a physiologically acceptable salt thereof and (2) a formulation comprising a dispersion medium.

17. The kit of claim 16, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the formulation (1) is from 1nm to 20 μm.

18. The kit of claim 17, wherein the average particle size of compound a or a physiologically acceptable salt thereof in the formulation (1) is from 10nm to 20 μm.

19. The kit of any one of claims 16 to 18, wherein formulation (1) or formulation (2) may comprise a dispersant and/or a surfactant.

20. The kit of any one of claims 16 to 19 for use in the treatment of a disease of the anterior segment and/or a disease of the posterior segment.

21. A medicament comprising compound a or a physiologically acceptable salt thereof for the treatment of a disease associated with VEGF.

22. The medicament of claim 21, wherein the disease is age-related macular degeneration and/or diabetic retinopathy.

23. A formulation according to any one of claims 2 to 4, wherein the suspension comprises a dispersant and/or a surfactant.

24. The formulation of claim 23, wherein the suspension comprises a dispersant and a surfactant.