WO2004093882A1 - Remedies for diseases to be applied to eye - Google Patents

Abstract

Remedies for various diseases which are in the form of eye-drops, eye washes, etc. containing sex steroid hormones such as estrogen or progesterone, derivatives thereof, structural analogs thereof, estrogen agonists, SERM or non-feminizing estrogen (non-hormonal estrogen). Thus, remedies for diseases to be applied to the eye which have excellent therapeutic effects with relieved side effects can be provided.

Description

 Specification

Disease treatment drug applied to the eye

 The present invention relates to a therapeutic agent for a disease applied to the eye containing a sex steroid hormone, a substance having an estrogenic effect or a selective estrogen receptor modulator or a non-forming estrogen (non-formonal estrogen). The present invention relates to an agent for treating a disease applied to the eye in a form. Background art

 Hormonal replacement therapy (HRT) with estrogen alone or in combination with estrogen and progesterone can be used to treat various ocular diseases such as senile macular degeneration (AMD), macular hiatus, glaucoma, diabetic retinopathy, and senile cataract. Possible improvement in symptoms has been reported.

 However, HRT can cause serious side effects such as breast cancer, uterine cancer, gynecomastia, and impotence due to systemic administration of estrogen (or estrogen and progesterone). Therefore, HRT, despite its therapeutic potential, could not be readily used in the treatment of various diseases, especially eye diseases. Until now, glaucoma, an eye disease, was said to occur in 1 in 30 people over the age of 40. However, in a survey report of the Japan Glaucoma Society in 2002,

The result is one in seventeen, and the number of glaucoma patients is increasing. Toward an aging society, it is expected that the number of patients with glaucoma and other eye diseases will increase in the future. 'Conventional ophthalmic solutions for the treatment of eye diseases such as glaucoma reduce intraocular pressure, but have insufficient effects on ameliorating narrowing of the visual field, and may cause redness and corneal erosion of the eyes. Also, it was troublesome because I had to apply it several times a day.

 An object of the present invention is to provide a therapeutic agent for diseases caused by eye drops or the like, which overcomes the problem of side effects which is a problem of HRT.

In addition, the object of the present invention is to improve the visual field, reduce the intraocular pressure, etc., have a long duration of the therapeutic effect, and require only one instillation once or twice a day, glaucoma, cataract, etc. Eye disease The aim is to provide a radical treatment for the disease. Disclosure of the invention

 The present invention is a therapeutic agent for diseases applied to the eye, which comprises a sex steroid hormone, a substance having an estrogenic effect, a selective estrogen receptor modulator or a non-fining estrogen (non-formonal estrogen). '

 The sex steroid hormones contained in the therapeutic agent for diseases of the present invention are hormones synthesized and secreted mainly by the gonads and placenta such as the ovaries and testes, and the present invention also includes synthetic substances and derivatives having the action. It is. Sex steroid hormones are roughly divided into andogens, estrogens, and gestagens. Preferred are sex steroid hormones having 18 to 21 carbon atoms.

 Examples of androgens include testosterone, androstenedione, dehydrodrepiandrosterone, androsterone, androstanedine, methinole testosterone and its derivatives or structural analogs, and testosterone and its derivatives or structural analogs are exemplified. preferable.

 Examples of the estrogens include 17β-estradione, estrone, estriol, equilin, echirenin, ethininoleestradio, plant 'I' raw estrogen and the like, and derivatives thereof. β-estradiol and its derivatives are preferable In addition to estrogen derivatives, substances having an estrogenic effect and structural analogs of estrogen are also included, and the following compounds can be exemplified.

 Estrogen derivatives include non-fining estrogen and its derivatives. For example, the following formula described in USP 5,521,168:

(Wherein, is H, OH, OR ₂ or R ₂ , where R ₂ is CiC alkyl R ₃ is OH, = 0 or, and R ₄ is R _x> C≡CH or C = CH ₂ ), especially 2-methoxyestradiol, 2-hydroxyestradiol, Examples include methoxyestradiol, 2-methoxyestradiol 3-methyletherenole, 2-methoxyestrone, and 2-hydroxyestrone. Also, the following formula described in W002 / 40032 A2:

(Wherein, between carbons 6 and 7, 8 and 9, or 9 and 11 may have one or more double bonds conjugated to aromatic ring A, in which case one or both of R ₈ and ₁₉ Is not present, n is 1-4, R8 and R9, if present, are hydrogen or alkyl, and R13 is hydrogen, substituted or unsubstituted hydrocarbyl, halogen, amide, sulfite. R 14 is hydrogen or alkyl; R z is a substituted or unsubstituted cycloalkyl, cycloalkenyl, heterocycloalkyl or heterocycloalenyl), especially Z YC—1, 2, 4, 9, 10, 11, 12, 13, 27, and 28, and the like. Also, ZYC-5 described in Neurobiology of Disease 9, 282-293 (2002) can be mentioned.

 As gestagens, progesterone and its derivatives or structural analogues are preferred.

 Preferably, the tosei steroid honolemon is at least one selected from the group consisting of estrogen, testosterone, progesterone, androgen, and derivatives and structural analogs thereof.

 Particularly preferred as sex steroid hormones are 17β-estradiol and its derivatives and structural analogs.

17 Sex steroid hormones, such as monoestradiol, It becomes a suspension. Therefore, in the therapeutic agent for diseases of the present invention, 17) it is necessary to solubilize sex steroid hormones such as 3-estradiol, or to solubilize sex steroid hormones in water with a surfactant. From the viewpoint of the effect, it is preferable that 17 β-estradiol or a derivative thereof is water-soluble.

 1 7 β-estradiol is hydrophobic and has low solubility in water, so it forms aggregates of different sizes even when dissolved in distilled water, increases the particle size, has poor storage stability, and has the desired concentration. Setting becomes difficult. Therefore, some measures are needed to make a stable eye drop with a target concentration that can be set from a substance that is hardly soluble in water such as 17j3-estradiol. There is a need for safe and beneficial methods that ensure drug stability and do not compromise the physiochemical and biological properties of the cornea and conjunctiva. Also, it is necessary not to cause irritation or discomfort during use.

 Conventionally, there have been attempts to make eye drops from poorly soluble substances by adding additives, oils, alcohols, etc., or by increasing the viscosity of the eye drops, but the drawbacks include discomfort during eye drops was there.

 On the other hand, in the present invention, it is preferable to adopt a method in which a drug is included by using cyclodextrin, and the drug is used after being made water-soluble. Cyclodextrin is an oligosaccharide of glucose that is hydrophilic on the outside and hydrophobic on the inside (cavity), and becomes soluble and stable by making inclusions with hydrophobic drugs such as 17] 3-estradiol. It is suitable for making eye drops, because it can be used and can be used without discomfort. It is known that there are generally three types of cyclodextrins, α, β, and y.

 Of the cyclodextrins, β-cyclodextrin has problems such as hemolysis and nephrotoxicity due to poor water solubility. Therefore, it is advisable to use the following] 3-cyclodextrin derivatives. It is.

-Cyclodextrin derivatives include hydrophilic cyclodextrins and hydrophobic cyclodextrins, but hydrophilic ones are suitable for eye drops. Hydrophilic neck dextrins include methylated cyclodextrins and hydroxyalkylated cyclodextrins, and methinolated cyclodextrins include the animals and humans described below. The results of the experiment at the site showed that the results were not appropriate due to problems with eye irritation, damage to the cornea and conjunctiva, and hemolysis. In contrast, hydroxyalkylated cyclodextrins are highly safe, have no irritating effects on the eyes, have no corneal epithelial damage, and are excellent for preparing eye drops.

 Hydroxyalkylated cyclodextrins include hydroxyxetyl cyclodextrin, hydroxypropyl cyclodextrin, etc., especially 2-hydroxyl-hydroxypropyl-1-β-cyclodextrin, which is used for It is most preferred because of its excellent properties and low irritation and biotoxicity.

-Cyclodextrin and its derivatives can also be preferably used. _{Since α} -cyclodextrin is not so highly soluble in water by inclusion of 17β-estradiol, γ-cyclodextrin is preferred.

 Hydroxypropyl monocyclodextrin, sulfoninole β-cyclodextrin, sulfoptinoleether monocyclodextrin, and cyclodextrin can also be used.

 The water-solubilization of sex steroid hormones such as 17β-estradiol can be performed, for example, by using a sex steroid hormone as a guest compound, a host such as a cyclic compound such as a-, jS- or y-cyclodextrin or a derivative thereof. It can be carried out by inclusion in a compound. The water-soluble sex steroid hormone has good solubility in water, and effectively transfers and penetrates the drug into the intraocular tissue by instillation, so it has a high therapeutic effect and can keep the intravascular concentration low. Therefore, there are few side effects, and since it has a good feeling of use and no foreign body sensation, it is particularly suitable for eye drops. It was confirmed that the water-soluble 17 i3-estradiol migrated to the target site in the eye by instillation and activated the cells (see Examples below).

 Examples of the substance having an estrogen action or the selective estrogen receptor modulator (S ERM) contained in the therapeutic examples of the present invention include raloxifene and arzoxifene.

Estrogen structural analogs, substances with estrogenic effects, selective estrogen receptor modulators, or non-eminizing estrogens (non-formonal estrogens) were also made water-soluble by making them water-soluble. Like 17β-estradiol, it enhances translocation to intraocular target tissues, and is considered to be effective in treating diseases at the target site for treatment.

 Diseases that can be treated in the present invention are not particularly limited, and include, for example, glaucoma, glaucoma of high intraocular pressure, normotensive glaucoma, neovascular senile macular degeneration, senile macular lesion, senile macular degeneration, macular circle Foramen, cataracts, senile cataracts, retinal bleeding, central retinal arteriovenous occlusion, fundus arteriosclerosis, photopsia, diabetic retinopathy, retinochoroidal atrophy, neovascular lesions of the retina and choroid, cataract by ovariectomy , TGF-induced cataract, macular fibroplasia (epithelial membrane), retinal hiatus, retinal detachment, proliferative retinopathy, retinal pigment degeneration, corneal opacity, corneal ulcer, corneal dystrophy, epidemic keratoconjunctivitis, balsa Tumors, iritis, uveitis, chorioretinitis, iridocyclitis, eye strain, visual field narrowing due to various diseases, optic atrophy, optic neuritis, dyskinesia, abnormal color vision, presbyopia, myopia, hyperopia, astigmatism And central nervous system diseases such as Alzheimer's disease, Parkinson's disease and ALS, and mental illnesses such as fissures and depression, pituitary abnormalities and diseases caused by hormonal imbalance. Glaucoma includes Posner-Schlossmann syndrome, and corneal degeneration includes corneal endothelial cell degeneration and shedding, corneal granular degeneration (Dalenow type 1, Dalenow type 2), and corneal zonular degeneration.

 The therapeutic agent of the present invention is intended for direct application to the eye, and may be in the form of, for example, eye drops, eyewash, ointment, conjunctival injection, intraocular injection or contact lens adsorbent. .

Since the therapeutic agent for ocular diseases of the present invention directly administers a sex steroid hormone, a substance having an estrogenic effect, a selective estrogen receptor modulator or a non-eminizing estrogen (nonformonal estrogen) to the eye tissue, the treatment is carried out through the conventional circulatory system. No increase in the concentration of sex steroid hormones in the blood vessels caused by HRT, and there is no risk of causing vascular lesions or side effects, and the duration of the effect is long, so that the disease can be treated effectively with a small number of instillations. In particular, because it can reduce intraocular pressure and improve visual field stenosis, it can effectively treat not only high-tension glaucoma but also normal-tension glaucoma. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a photograph of the ophthalmic solution A of Example 1.

 FIG. 2 is a photograph of the ophthalmic solution B of Example 1.

 FIG. 3 is a graph showing the time course of the concentration of 17β-estradiol in the anterior chamber after eye drops of eye drops A to C.

 FIG. 4 is a diagram showing the visual field of the right eye of the patient of Example 6 before treatment.

 FIG. 5 is a diagram showing the visual field of the left eye of the patient of Example 6 before treatment.

 FIG. 6 is a diagram showing the visual field of the right eye of the patient of Example 6 after treatment.

 FIG. 7 is a diagram showing the visual field of the left eye of the patient of Example 6 after treatment.

 FIG. 8 is a diagram showing the visual field of the right eye of the patient of Example 7 before treatment.

 FIG. 9 is a diagram showing the visual field of the left eye of the patient of Example 7 before treatment.

 FIG. 10 is a diagram showing the visual field of the right eye of the patient of Example 7 after treatment.

 FIG. 11 is a diagram showing the visual field of the left eye of the patient of Example 7 after treatment.

 FIG. 12 is a diagram showing a change in near point before and after instillation of a patient in Example 8.

 FIG. 13 is a diagram showing the visual field of the right eye of the patient of Example 9 before treatment.

 FIG. 14 is a view showing the visual field of the left eye of the patient of Example 9 before treatment.

 FIG. 15 is a diagram showing the visual field of the right eye of the patient of Example 9 after treatment.

 FIG. 16 is a diagram showing the visual field of the left eye of the patient of Example 9 after treatment.

 FIG. 17 shows the control group of Example 10, the control group before ultraviolet irradiation (Pre 0.01 g / l and 0.1 g / 1), and the administration group after ultraviolet irradiation (Post). 1 is a photograph of an anterior eye of a mouse at 0.1 g / l and 0.1 g / l).

 FIG. 18 is a diagram illustrating a relationship between an instillation period and visual acuity in the patient of Example 11; FIG. 19 is a diagram showing the dynamic visual acuity of the patient of Example 24.

 FIG. 20 is a micrograph of the retina of a mouse to which distilled water was applied in Example 24. FIG. 21 is a micrograph of the retina of a mouse in which the water-soluble 17 β-estradiol-containing ophthalmic solution was applied in Example 24.

 FIG. 22 is an estrogen receptor antibody positive image in the retina.

 Figure 23 is an estrogen receptor antibody positive image in the lens.

 FIG. 24 is an estrogen receptor antibody positive image in the cornea.

FIG. 25 is an estrogen receptor antibody positive image in the ciliary body. FIG. 26 is an image of extraretinal granule cells when estrogen was not added and antibody was not added. —

 FIG. 27 shows a positive image of estrogen receptor antibody in extraretinal granule cells when 17] 3-estradiol was not added and an antibody was added. .

 FIG. 28 is an image of extraretinal granule cells when 17] 3-estradiol was added and no antibody was added.

 FIG. 29 shows estrogen receptor antibody-positive images in extraretinal granule cells when 17β-estradionole was added and an antibody was added.

 FIG. 30 shows the eye drops 17] 3-estradiol concentration (0 to: lOO / g / ml) and the number of estrogen receptor nuclear translocating cells.

 FIG. 31 shows the time course of the number of estrogen receptor nuclear translocating cells after instillation of 17 β-estradiol. BEST MODE FOR CARRYING OUT THE INVENTION

 The therapeutic agent of the present invention can be produced by a conventional production method in the same manner as this kind of general drug. In the case of eye drops and eyewashes, for example, sterilized water-soluble sex steroid hormones, substances with estrogen action or ZYC-5, selective estrogen receptor modulators or non-eminizing estrogens (non-formonal estrogens) It can be produced by dissolving at a predetermined concentration in distilled water, BSS Plus, physiological saline, etc.

 In the therapeutic agent of the present invention, the sex steroid hormone, a substance having an estrogenic effect or ZYC-5, a selective estrogen receptor modulator or a non-ferminizing estrogen (non-formonal estrogen) must be contained in a therapeutically effective amount. . Therapeutically effective amounts are, for liquids, for example, 0.1 / zg to: 100 g / l, preferably 0.5 / g to lg / l, particularly preferably 1 g to 1 g / l.

Concentration of 50 Omg / 1, for solids, e.g. 0.1 μg to 100 g / kg, preferably 0.5 At g to 10 g / kg, particularly preferably 1 μg to 5 g g / kg concentration. The therapeutic agent of the present invention comprises a sex steroid hormone, a substance having an estrogenic effect or a selective estrogen receptor modulator or non-femi By changing the concentration of the Nosing Est orogen (non-formonal estrogen), the number of doses of the drug, and the dosage, or by selecting the drug administration method (for example, ophthalmic solution, eyewash, ointment) You can adjust the duration of the effect and the duration of the effect.

 For the treatment of the present invention, if necessary, conventional additives such as excipients, carriers, p

H regulators, isotonic agents, preservatives, glutathione, glucose, various salts, stabilizers, cooling agents, antioxidants, preservatives, and the like may be added.

 In addition, hydroxypropyl methylcellulose, potassium ureboxyl methylcellulose or a sodium salt thereof, polypyrrolidone, polypyrrolidone (added and heated) and the like may be added. Example

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

Example 1

 Eye drops were prepared from 17β-estradiol and clathrates thereof with various cyclodextrins as described below. For various cyclodextrins, SIGMA M0, USA was used, and for 17] 3-estradiol, Calbiochem (CALBI0CHEM), Germany was used.

Eye drops A: 17; 3-Estradiol eye drops

 17β-estradiol was dissolved in distilled water at a concentration of lmg / ml. When left undisturbed, aggregates of different sizes were formed to form a suspension. This was used as eye drop A without increasing the viscosity or adding alcohol (Fig. 1).

Ophthalmic solution B: Inclusion product of 2-hydroxypropy ^^ — j3-cyclodextrin and 17β-estradiol

13.3 mM of 2 -hydroxypropyl; 3 -cyclodextrin and 3.67 mM (1 mg / ml) of 17 / 3-estradiol are included and distilled water is added. The solution was dissolved to prepare a clear and uniform aqueous solution (Fig. 2). This aqueous solution was used as eye drop B (molar ratio (3.62: 1)). Ophthalmic solution C: a- + γ-cyclodextrin and 17] 3-estradiol

 When a-cyclodextrin and 3.67 mM (1 rag / ml) of 17β-estradiol were included and dissolved in distilled water, the suspension remained. Addition of y-cyclodextrin to this suspension resulted in a clear aqueous solution. This aqueous solution was used as eye drop C. The total amount of a— + two cyclodextrin was 24.6 mM (mol ratio 6.7: 1).

Ophthalmic solution D: Ophthalmic solution of inclusion complex of methyl-β-cyclodextrin and 17] 3-estradione

 15 mM of methyl / 3-cyclodextrin and 3.67 mM (1 mg / ml) of 17 j3

—Estradiol was included and dissolved in distilled water to prepare a clear and uniform aqueous solution. This aqueous solution was used as eye drop D.

Inclusion of 17 β-estradiol with 2-hydroxypropyl-l] -cyclodextrin, _α- + _γ -cyclodextrin or methyl-l-j3-cyclodextrin increases water solubility and makes clear and stable eye drops. It could be prepared (eye drops BD).

Example 2 Effect on keratoconjunctival tissue

 In order to see the effects of the eye drops C to D prepared in Example 1 on the eyes, ophthalmophors, mice and humans were instilled and evaluated.

 No problem occurred when eye drops B (2-hydroxypropyl-1-β-cyclodextrin inclusion) were applied to the eyes of rabbits (Dutch rabbits) four times with 50 μ1 (one drop) every 30 minutes. . In addition, no problem occurred when instilling once a day (50 // 1) for 8 months in mice and 11 months in humans.

 When eye drops C (a— + V—cyclodextrin inclusion complex) were applied to human eyes, there was no irritation.

 Eye drops D (methyl-β-cyclodextrin inclusion) were applied to eyes of rabbits (Dutch rabbits) four times with 50 Atl (one drop) every 30 minutes:

After 30 minutes, the number of corneal pyrans increased, and the blood vessels around the cornea became hyperemic;

In one hour, eye oil appeared and the area of virane expanded;

In one and a half hours, the number of pyrans has grown deeper; and At 2 hours, hyperemia became stronger, and the experiment was stopped because the corneal epithelium was completely detached in a triangular area with a base of 2 mm and a height of 3 mm in the epithelium around the cornea.

 Furthermore, when one drop (50 μ1) of eye drop D was applied to a person, there was strong irritation, tearing occurred, and the discomfort continued for about 5 hours until bedtime, but recovered the next morning. . It was found that eye drops and especially eye drops ず did not cause any particular problem even when instilled, and were found to be optimal as eye drops. On the other hand, Ophthalmic Solution D was found to be unsuitable as an ophthalmic solution because of its strong irritation, altering the physiochemical properties of the cornea and conjunctiva, and causing pathological epithelial detachment of the cornea.

Example 3 1 7—Estradiol concentration in the anterior chamber

 In order to observe the effects of the eye drops A to D prepared in Example 1, the measurement of the concentration of 17β-estradiol in the anterior chamber after instillation was performed using double rabbits (Dutch rabbit: female). Experiments were conducted in India.

 At 10 am, one drop (50 μl) of each eye drop was instilled, and 5 minutes later, another drop was instilled. At 30 minutes, 1 hour, 2 hours, and 3 hours after instillation, the cornea was punctured using a 27-gauge injection needle and an l ml injector, and 0.1 ml of aqueous humor was collected as a specimen. Specimen C

18 After column extraction, use the DPC Estradiol diantibody kit (Diagnostic

Products Corporation, USA) was used to determine the amount of 17 j8-estradiol. Using estradiol antibody and iodine modified Stora diol ¹²⁵ 1 reagent, and measuring the radioactivity in Ganmakau centers. The measured values are converted to Pg / ml. The results are shown in Figure 3. 1 7 3 —Estradiol concentration in the anterior chamber immediately before instillation is the average

It was 15 3 Pg / ml.

As can be seen from Fig. 3, the concentration of 17) 3-estradiol in the anterior chamber of each ophthalmic solution was highest at the value of 30 minutes after instillation and decreased thereafter. [2-Hydroxypropyl]] The concentration of 17β-estradiol in the anterior chamber of ophthalmic solution B clathrated with 3-cyclodextrin is highest, indicating that it has penetrated the cornea and conjunctiva most strongly. From these results, it is estimated that 2-hydroxypropyl-1-j8-cyclodextrin acts as an enhancer for 17β-estradiol in permeating the cornea, that is, it facilitates permeation through the cornea. . The second highest concentration of 17 j3-estradiol in the anterior chamber is that of ophthalmic solution C containing α- + y-cyclodextrin. Was. a-Dextrin has low _melting point of 17-estradiol, and Ί-dextrin seems to work more strongly. Ophthalmic solution Α (suspension) had a low concentration in the anterior chamber and poor permeability. One of the reasons for this result is that eyedrop A had large and small agglomerates and a large particle size. M-j8-cyclodextrin inclusion ophthalmic solution D showed a high concentration of 173-estradiol in the anterior chamber (30 min after instillation: 36178 Pg / ml). This was excluded from the graph because methyl-β-cyclodextrin alters the physiochemical properties of the cornea and conjunctiva and is permeated through the pathological cornea and conjunctiva.

 From this experimental result, it was found that the ophthalmic solution of 2-hydroxypropyl-1-) 3-cyclodextrin inclusion complex was the best.

Example 4 Preparation of eye drops / eyewash

 Water-soluble inclusion complex of 17; 3-estradiol (Cyclodextrin-encapsulated 17] 3-estradiol, manufactured by Sigma-Aldrich; 2-hydroxypropyl-cyclodextrin, per gram of clathrate 17 g β-estradiol (containing 45 mg) in sterile distilled water 2.2 g / 1 (17; 8-estradiol)

The solution was dissolved at a concentration of 10 O mg / 1) to prepare eye drops / eyewash. Similarly, a water-soluble progesterone clathrate (manufactured by Sigma-Aldrich Co., Ltd.) prepared eye drops and eyewash at a concentration of 1.2 g / l (progesterone was 100 mg / l).

Example 5 Effect on Meguron pressure (animal experiment)

 For the experiment, 5 rabbits (1.8 to 2 kg in weight), 8 to 10 months old, were born.

(10 艮) Used.

 (1) Daily fluctuation of intraocular pressure

 The diurnal variation of intraocular pressure in rabbits was measured. The mean values were 14 mmHg at 9 am, 12 mraHg at 1 pm, 12 mmHg at 3 pm, and 16 mmHg at 6 pm.

 Mean intraocular pressure was 14 ± 2 mmHg.

 (2) Eye drops Z Effect of eyewash 1

Since the intraocular pressure increased in the evening as seen in the diurnal variation of intraocular pressure in rabbits, the effect was administered by administering eye drops Z eyewash at 1:00 pm. Rabbit pressure before treatment with eye drops was 14 mmHg at 1 pm. The rabbit was instilled with 2 7 1 (one drop) per eye at 1:00 pm of the 17 / 3-estradiol ophthalmic solution prepared in Example 4. The intraocular pressure of the instilled rabbit was measured at 4:30 pm three and a half hours after the instillation, and it was decreased to 1 OmmHg. This effect was maintained at least until 6:00 pm five hours after the instillation.

 Separately, a rabbit without eye drops was washed at 1:00 pm with the 17 β-estradiol eye drop prepared in Example 4 Ζ eye wash. The maximum decrease in intraocular pressure appeared 50 minutes after eyewash. The effect of lowering intraocular pressure lasted for at least 5 hours after eye washing.

 (3) Effect of eye drops / eyewash 2

 The experiment was conducted using a rabbit with only one eye having high intraocular pressure. (This data was not used to calculate the average.)

The intraocular pressure of the rabbit at 5:00 pm before instillation was 24 mmHg. It was eyewash in the afternoon at 5:00 rabbit in 1 7 β- estradiol eye drops Roh eye washes prepared in Example 4, intraocular pressure in 1 5 minutes after eyewash and they fell to 1 5 mmH _g. When the rabbit was immediately washed again with the same eye drop and eyewash, the intraocular pressure dropped to 13 mmHg 45 minutes later.

 The rabbit treated with eye drops was left untreated, and the intraocular pressure was measured at 9:00 am the next day. The value was 18 mmHg, and the effect of the eye drops on the previous day remained. When the rabbit was instilled with 17 β-estradiol eyedrop / eyewash 25 ^ 1 (1 drop) prepared in Example 4, the intraocular pressure dropped to 13 mmHg 3 hours and 15 minutes after instillation. I was At this time, 25/3 of 17 / 3-estradiol Z eyewash prepared in Example 4 was instilled again, and the intraocular pressure 3 hours and 15 minutes after reinstillation was 13 mmHg. Thereafter, when the same eye drop was added with the eyewash at 25 μΐ, the intraocular pressure was 12 raraHg 1 hour and 50 minutes later.

 On the following day (the third day), the intraocular pressure before instillation was 1 O mmHg at 9 am, at which time the 17 / 3-estradiol ophthalmic solution Z 51 prepared in Example 4 was replaced with 25 1

After one instillation, the intraocular pressure at midnight (3 hours after instillation) was 9 mraHg, the intraocular pressure at 3:15 pm was 12 mmHg, and the intraocular pressure at 6:00 pm was 1 1 It was mmHg. The effect of eye drops was found to last for a long time.

The effect remains on the next day after administration, so the estrogen receptor is involved, It may be related to the work at the DNA level. It may be due to the generation of NO. Other mechanisms are also conceivable.

 (4) Effects of eye drops 3

 The progesterone ophthalmic solution prepared in Example 4 showed an ocular hypotensive effect of an average of 4 Hg.

Example 6 Therapeutic effect of normal tension glaucoma patients

 The patient was a 75-year-old woman diagnosed with normotensive glaucoma.

 Water soluble 17β-estradiol inclusion complex (Cyclodextrin-encapsulated 173-estradiol; Sigma-Aldrich Co., Ltd.) Included with 2-hydroxypropyl-cyclodextrin. i3—estradiol

Was dissolved in sterile distilled water at a concentration of 2.2 g / l (17) 3-estradiol at a concentration of 10 Omg / 1) to prepare eye drops.

 One drop (25 1) of eye drops was applied to the above patient once daily for 10 days in the morning.

 The patient's intraocular pressure was 14 mmHg on the right and 12 mmHg on the left before instillation, but was 12 mraHg on the right and 9 mmHg on the left after instillation for 10 days.

 Visual field measurement with Octopusl—2–3 before instillation of the patient showed visual field defects (FIGS. 4 and 5), but both eyes improved very well after 10 days of instillation (FIGS. 6 and 7). ). Clinically, the treatment effect of instillation of normal tension glaucoma was confirmed.

Example 7 Therapeutic effect of open-angle glaucoma with high intraocular pressure

 The patient was a 54-year-old woman diagnosed with high-tension open-angle glaucoma.

 Water-soluble 17 / 3-estradiol clathrate (Cyclodextrin-encapsulated 17 j3-estradiol; Sigma-Aldrich; 2-t drosiki propinole-1 j3-cyclodextrin clathrate, clathrate 1 g Per 1 7; 3—estradiol

Was dissolved in sterilized distilled water at a concentration of 2.2 mg / l (17; 3-estradiol at lOOg / 1) to prepare eye drops.

 The patient received one drop of eye drops (25/1) once daily in the morning for one week.

The intraocular pressure of the patient was 23 mmHg on the right and 24 Hg on the left before instillation, Later, it was 16 脑 Hg on the right and 18 Hg on the left.

 Although the visual field had a scotoma, a slight defect in the right eye (Fig. 8) recovered almost normally (Fig. 1Q) and a progressive defect in the left eye (Fig. 9) improved slightly (Fig. 11). ).

 When the instillation was further continued, the patient's intraocular pressure increased to 2 OmraHg on the right and 19 mmHg on the left 3 weeks before the instillation. The 17-estradiol concentration of 100 g / 1 is considered to be close to the lower limit of the concentration that is effective. Effective treatment is possible by increasing the concentration or increasing the number of drops per day. Clinically, the treatment effect of open-angle glaucoma with high intraocular pressure by instillation was confirmed. Example 8 Therapeutic effect of presbyopia

 The patient had presbyopia with mild myopia of 2.5 diopters and astigmatism of 1. O D

59-year-old man.

 When the near point of the patient was measured without correcting the left eye with a lens, it was found to be 29 cm.

 Water-soluble 17 j8—Estradiol clathrate (Cyclodextrin-encapsulated 17β-estradiol; Sigma-Aldrich; 2-Hydrochaflohi / Ray) Inclusion with 3-cyclodextrin, 1 g per clathrate 7 β-estradiol (containing 45 mg) was dissolved in sterile distilled water at a concentration of 2.2 g / l (17 j3-estradiol at a concentration of 10 Omg / 1) to prepare eye drops.

 The patient received one drop of eye drops (25 l) at 9 am in the morning. The near point of the person who was 29 cm before instillation became 27 cm 1 hour after instillation, and 24 cm after 3 hours, with a maximum refraction improvement of 5 cm (Fig. 12). The distant point averaged 41 ± 2 cm.

 Clinically, the treatment effect of presbyopia by instillation has been demonstrated.

Example 9 Treatment of Retinitis Pigmentosa

The patient is an 86-year-old woman who has already undergone cataract surgery and has an artificial lens. Visual field narrowing is caused by retinitis pigmentosa. One drop (501) of ophthalmic solution containing 17β-estradiol prepared in Example 4 at a concentration of 100 mg / l was administered to the patient once daily (at 8:00 in the morning). The field of view gradually improved. It was confirmed that both visual acuity and visual field had improved 77 days after the start of instillation. The visual acuity before instillation is Vd = 0.4 XI OL (corrected visual acuity 0.6)

 V s = 0.8 XOL (corrected visual acuity 0.8) but after 77 days

 Vd = 0.4 X I OL (corrected vision 0.8)

 V s = 0.5 X I OL (corrected visual acuity 1.0) and improvement in visual acuity were confirmed.

In addition, it was confirmed that the visual field measured with the OCTOPUS 1-2-3 static perimeter improved before and 77 days after the start of instillation. (See Figures 13-16)

Example # 0 Cataract treatment (animal experiment)

 As shown below, see the therapeutic effect of cataract by 2-hydroxypropyl-1] 3-cyclodextrin inclusion complex 17j3-estradiol ophthalmic solution (17 ^ -estradiol concentrations of 0.1 lg / 1 and 0.0 lg / 1) Animal experiments were performed with double blinds.

 Control group (control): UV-A + UV-B (0.36 J: 100 1 hour each) was applied to mouse eyes (four eyes) on day 1, day 3 and day 5

Irradiation was performed three times, once each from 22:00 to 23:00. However, one drop of PBS 1 minute before and 1 minute after each UV irradiation (50 drops, 2nd, 4th, 6th, 7th, 8th, 9th and 10th One drop of PBS was instilled once each day.

 UV irradiation pre-administration group (Pre 0.0 lg / 1 and Pre 0.0 lg / 1): Ultraviolet UV-A + UV-B (0.36 J each: Ι Ο Ο μΨΧ Ι hours) ) Was irradiated once on each of the first, third, and fifth days, from 22:00 to 23:00, for a total of three irradiations. One minute before each UV irradiation, apply 1 drop (50 // 1) of 17-estradiol concentration of 0.1 lg / 1 (4 eyes) or 0.01 g / l (4 eyes) and apply ultraviolet light. One minute after the irradiation, one drop of PBS was instilled, and once on each of the second, fourth, sixth, seventh, eighth, ninth, and tenth days, the 17-estradiol concentration was 0. One drop of lg / 1 or 0.0 lg / 1 eye drops was instilled.

UV-irradiated administration groups (P ost 0.lg / 1 and P ost 0.0 lg / 1): Ultraviolet rays UV-A + UV-B (0.36 J each: 100 / zWX 1 hour) To Irradiation was performed three times, once each on the first day, the third day, and the fifth day at 22 to 23 o'clock. However, one drop of PBS was instilled one minute before each UV irradiation, and one minute after the UV irradiation, 17 j3-estradiol concentration 0.1 lg / 1 (4 eyes) or 0.0 1 g / l (4 eyes) One drop of the ophthalmic solution is instilled once a day on the second, fourth, sixth, seventh, eighth, ninth, and tenth day, and once on each of the tenth day. One drop of eye drops of lg / 1 or 0.01 g / 1 was instilled.

 At 22 o'clock on the 10th, a photograph of the anterior segment was taken (Fig. 17).

 As can be seen from Fig. 17, when the mouse eyes were exposed to UV-A + UV-B for 3 days, cataracts appeared on the 10th day in the control group to which only PBS was administered. On the other hand, it was confirmed that cataract hardly occurred in the pre-irradiation group and the post-irradiation group of the 17-estradiol-containing eye drops. Cataract appeared less frequently and the pharmacological effect was higher in the group receiving the high-concentration 17 β-estradiol eye drops than in the group receiving the low-concentration eye drops. Cataracts appeared less in the group before UV irradiation than in the group after irradiation, and the pharmacological effects were stronger. It was confirmed that 2-hydroxypropyl-1-j3-cyclodextrin clathrate 17 estradiol had both preventive and therapeutic effects on cataract by eye drops.

 2-Hydroxypropynolone β-cyclodextrin inclusion in the mouse eyes 17 β-estradiol (17 β-estradiol concentration 1 Omg / 1) Eye drops are applied to the eye and nuclei in the lens cortex are estrogen receptor antibodies As described later, perinuclear and intranuclear staining were performed, and it was confirmed that 17 / 3-estradiol had reached the lens cortical cells.

Example 1 1 Cataract treatment (Clinical therapeutic effect)

The patient's cataract, by dissolving 1 7 J3- estradiol inclusion in water-soluble as used in Example 4 in sterile distilled water, 1 7] 3- estradiol lm _g / l of the concentration of the eye drops were prepared, When one drop (501) was instilled once daily at 8:00 in the morning, visual acuity was improved (see Figure 18). In addition, it was observed that opacity of the cataract was reduced even with a microscopic microscope for ophthalmic examination.

Example 1 2 Treatment of Chalazionoma 8 talented woman children right) chalazia, Example 1 1 1 7 one estradiol lmg / 1 concentration of eye drops 1 drop prepared in (50 _w l) eye drops were at once in the morning, masses after 1 2 days vanishing Had lost.

Example 13 Myopia treatment

 1.13-year-old boy [sight: Vd = 0.7 (1.5 X-0.5D)

 V s = 0.6 (1.5 X—0.5 D)]

One drop (501) of ophthalmic solution having a concentration of 100 Atg / 1 of 17 β-estradiol prepared by dissolving the water-soluble 17β-estradiol clathrate used in Example 4 in distilled water was used. I applied it once a day (at 8:00 in the morning)

 Vd = 1.0 (1.5 X-0.25 D)

 V s = 1.0 (1.5 X-0.25 D)

Improved.

 2.13-year-old boy [sight: Vd = 0.4 (1.5 X-0.75 D)

 V s = 0.3 (1.5 X-1.25 D))

One drop (50 μΐ) of ophthalmic solution having a concentration of 17 β-estradiol lmg / 1 prepared in Example 7 was instilled once every morning, and the following visual acuity improvement was observed 45 days after instillation. .

 Vd = 1.0 (1.5 X-0.5D)

 V s = 0.5 (1.5 X- 1.OD)

Example 14 Presbyopia treatment

 Correction near vision before treatment (Near corrected vision)

 V d = 0.6 / 3 OcmXN J B

 V s = 0.4 / 3 OcmXN J B

A 49-year-old man with presbyopia was given 1 drop (501) of 17 / 3-estradiol at a concentration of 1 mg / 1 prepared in Example 11 in the morning once a day for 14 days. After instillation, near corrected vision improved as follows.

 Vd = 1.0 / 3 OcmXN J B

 V s = 0.7 / 3 OcmXN J B

Example 15 Eye fundus bleeding treatment To a 80-year-old man with retinal hemorrhage in the right eye, 1 drop of 50 mg of 1 7/1 β-estradiol prepared in Example 11 (50 1) once a day At 8 o'clock, she was given adona 3 Omg three times a day. After 45 days, the bleeding had disappeared.

Example 16 Corneal ulcer treatment

 A 76-year-old female student who had corneas and ulcers that alternately recurred in her eyes. One drop (50 // 1) of ophthalmic solution at a concentration of / 1 was applied twice a day at 8:00 and 17:00 in the morning, and it was relieved in 3 weeks. It is considered possible to treat autoimmune diseases.

Example 17 Treatment of corneal degeneration

 1. To the left eye of a 63-year-old woman diagnosed with granular corneal degeneration, 1 drop of 1 mg / 1 17 j3-estradiol eye drop prepared in Example 11 (50 1) Two times a day (at 8 o'clock and 15 o'clock), my eyesight improved in two weeks.

 V s = 0.6 (nc) → V s = 0.4 (0.8)

2. One drop of the same ophthalmic solution for a 68-year-old woman diagnosed with granular corneal degeneration

(50 1) In the morning, once in the right eye, in a week

 Vd = 0.3 (0.9) → Vd = 0.3 (1.0)

 Two months later, Vd was significantly improved to 0.5 (1.2). () Indicates corrected vision.

Example 18 Treatment of iridocyclitis

 8 A 50-year-old woman with iridocyclitis in her right eye was treated with 1 μl of 17 j3-estradiol at a concentration of 1 mg / 1 in 50 μl drops of the eye drops prepared in Example 11. When she was instilled at 8:00 in the morning, she recovered one week later (the inflammatory cells in the anterior chamber disappeared).

Example 19 Treatment of Postner-Schlossmann Syndrome

 36 A 6-year-old woman with iritis and increased intraocular pressure (T os = 36 mmHg) in her left eye and diagnosed with Posner-Schlossmann syndrome was prepared in Example 11 1 One drop of 1 mg / l of superdiol in a dose of 5 µmΐ was instilled for 1 week at 8:00 in the morning. Irisitis disappeared and intraocular pressure was normalized (T os = l 7 mmHg).

Example 20 Central nervous system disease treatment For an 80-year-old woman diagnosed with schizophrenia (schizophrenia), the aqueous 1 "raw 17β-estradiol inclusion complex used in Example 4 was dissolved in sterile distilled water to obtain a solution. An ophthalmic solution with a concentration of β-estradiol lmg / 1 was prepared, and this solution was instilled once a day (50 μl) once a day. As a result, visual acuity and mental condition were improved as follows. Was.

Starting V d = 0 1 X I OL (0.3)

 V s = 0 09 X I OL (0.1)

 I lmg / 1 1 drop per morning (50 / l)

 4 days later V d = o 1 X I OL (0.4)

 V s = 0 1 X I OL (0.1)

 He said that he had seen it better.

1 lmg / 1 drop per morning once (50 _W l)

 42 days later V d = 0, 1 5 X I OL (0.4)

 V s = 0, 09 X I OL (0.1)

 He said he was getting better.

 1 lmg / 1 morning (50 μ （)

 7 7 days later V d = 0 3 X I OL (0.6)

 V s = 0 1 X I OL (0.15)

 1 1 mg / 1 twice (50 1)

 15 days later Vd = 0 2 X I OL (0.4)

 V s = 0 0 9 X I O L (0.2)

 () Indicates corrected vision.

 It has been confirmed that it is effective for treating schizophrenia patients. It is also considered to be effective for the treatment of other central nervous system diseases such as Alzheimer's disease, Parkinson's disease, ALS, psychosis, schizophrenia, schizophrenia, and depression.

Example 21 Treatment of Choroidal Atrophy

 When the right eye of a 65-year-old man was administered 5 drops once a day with 1 713-estradiol lmg / 1 prepared in Example 11, 0.08 (n'c) showed corrected visual acuity.

When the above eye drops were administered to the left eye of a 70-year-old woman at a dose of 50 1 once a day, the corrected visual acuity improved to 0.1. Example 22 Treatment of diabetic retinopathy

 To a 74-year-old female patient with diabetic retinopathy, 50 μl of the 17 β-estradiol 1 mg / 1 eye drop prepared in Example 11 was instilled once daily daily. Approximately two months after the start of administration, ecchymosis of the fundus was observed with a fundus photograph, and it was slightly improved. About 100 days after the start of administration, ecchymosis of the fundus was cured. Example 2 3 Treatment of macular hole

 In the right eye of a 57-year-old woman with a macular hole and a visual acuity of 0.7, an ophthalmic solution containing 1-β-estradiol was added to 2-hydroxypropyl-1-i3-cyclodextrin. When one drop of 1) was continuously instilled twice a day for one month, the macular hole was healed and visual acuity was restored to 1.2.

Example 24 Effect of improving dynamic visual acuity

 Includes 2-hydroxypropyl-1-β-cyclodextrin in the left eye of a 60-year-old man with corrected vision V s = 0.1 (2. OX—2.75—0.50 DAX 1 15 °) 1 7; 8-Estradiol ophthalmic solution (10 μg / ml ί7β-estradiol concentration) 50 μ 1 (1 drop) was instilled once, and the measurement of dynamic visual acuity was performed using a two-deck dynamic visual acuity meter Κ V- This was performed using 100. Dynamic visual acuity improved rapidly between 30 minutes and 1 hour and 15 minutes. (Fig. 19)

Example 2 5 Regeneration protection of corneal endothelial cells

In a 30-year-old female patient with decreased endothelial cells due to wearing of contact lenses, one drop of 17 j3-estradiol (1 _mg / l) eyedrop prepared in Example 11 (50 μm)

1) was improved 10 days after instillation (once a day).

Number of right eye endothelial cells (per mm ² ) 28 0 1 → 34 1 2

Number of left eye endothelial cells (per mm ² ) 2 9 6 → 34 9 6

The measuring instrument used NONCON ROBO (KONAN INC) Suzukiura microscope. Example 26 Improvement of photophobia

For a 2-year-old woman who complained of photophobia, 2-hydroxypropyl-1-β-cyclodextrin inclusion complex 17 β-estradiol eye drops (17 j8—estradiol concentration 1 Omg / 1 (10 g / ml) ) Was instilled once a day with one drop (501), and the symptoms improved. Example 27 Treatment of Epidemic Keratoconjunctivitis

 26-year-old woman with epidemic keratoconjunctivitis was given 2-hydroxypropyl-cyclodextrin clathrate 17 beta-estradiol ophthalmic solution (17 beta-estradiol lmg / 1) daily After a single administration, the amount of eye oil increased, but the vilanic superficial keratitis was cured on the fourth day.

Example 28 Treatment of Eye Strain

 A 30-year-old man who complained of eye fatigue by computer input was given 2-hydroxypropyl-1-β-cyclodextrin inclusion complex 17 3-estradiol eye drops (17 β-estradiol concentration 1 Omg / 1). When administered once a day (5 // 1), eye strain improved within 3 weeks.

Example 29 Transfer of 9 Estrogen Receptor into the Nucleus of Extraretinal Granulosa Cells (Animal Concentration of 17 j3-estradiol prepared in Example 4 in 3 month old male mice (body weight: about 20 g) 100 Omg / 1 ophthalmic solution 50 was instilled once in the morning on the first day, twice in the morning and evening in the second, and once in the morning on the third day. The cells of the condylar granule layer (photoreceptor cells) were used as a sample, and a sample from a mouse similarly instilled with distilled water was used as a control.

 The sample was immunostained using the anti-estrogen receptor clone AER311 (mouse monoclonal IgG2aΚ) (200 ^ g / 200 Ail) and examined under a microscope (X100). Observed. The results are shown in FIGS. 19 and 20.

 In the control, it was confirmed that the transfer of estrogens to the nuclei of cells (photoreceptor cells) in the outer nuclear layer of the mouse retina was small (see Fig. 20), but the eye drops containing water-soluble 17i3-estradiol were instilled. In the cells of the outer granular layer of the mouse retina (visual cells), it was confirmed that the receptor was translocated into the cell nucleus more than in the control (see Fig. 21). It was confirmed that 17β-estradiol migrated deep into the eye (retina) with this water-soluble ophthalmic solution, which proved that the desired cells were activated.

Example 30 Molecular Biology Experiment (Animal Experiment)

By inclusion with 2-hydroxypropyl-j3-cyclodextrin, Since it was found that 17 β-estradione was sufficiently transferred into the eye by instillation, a more detailed molecular biology experiment was performed.

 (Experimental materials and methods)

Experimental materials>

 The mice used were C57B LZ6 (purchased from Saitama experimental animals). Ether was used for anesthesia during dissection.

 The eye drops were prepared from 2-hydroxypropyl 1-cyclodextrin inclusion complex 17-j3 estradiol (SIGMA M0, USA), and the 17-β estradiol concentration 0.01--: L 0 ig / Used in ml. ,

 AER311, an anti-estrogen receptor monoclonal antibody clone, was obtained from Upstate (NY,

USA). Reagents necessary for immunostaining, including the secondary antibody, were purchased from VENTANA (AZ, USA).

<Frozen section preparation method>

After a predetermined time after instillation, anesthesia was performed with ether. After euthanasia, the eyeball partially containing the optic nerve was excised, and fixed for 1 hour while cooling with PBS containing 2% paraformaldehyde on ice. Then, it was embedded using TISSU MOUNT (Shiraimatsu Kikai) on isopentane cooled with dry ice. Slicing is used Kurioshiyutatto (LEICA CM1 ⁹ 00, GERMANY), soaked in 5 minutes HE fixative for post-fixed (7-9% ethanol, 2 0% formalin, 1% acetic acid). After washing with PBS, they were subjected to staining scan.

<Detection of estrogen receptor by immunostaining>

 Thin sections were immunostained using VENTANA HX system. For the staining protocol, select the DAB Non-Paraffin protocol, perform antigen activation treatment with the conditioner CC1, and use an anti-estrogen receptor antibody diluted to a concentration of 4 / g / ml with an antibody diluent as the primary antibody. Reacted for minutes. Color development with secondary antibody and diaminobenzidine (DAB) should be performed using Ventana Amplification Kit ¾ ■. Also, post-counterstain

BLUING REAGENT was used. After completion of staining, the cells were transparentized using ethanol and xylene, and mounted using a soft mount (Wako Pure Chemical Industries).

<Observation of stained section>

Use an upright microscope to observe the immunostained sections, and use a digital camera (Nikon CoolPix 900), displayed on a personal computer with image processing software Photoshop, and analyzed.

 (Result)

 <Ocular distribution of estrogen receptor>

 It is known that estrogen has a clear mechanism of action: activation of intracellular estrogen receptor by binding to estrogen, translocation to the nucleus, and increase in transcriptional activity of specific genes by binding to enhancer. ing. Therefore, we examined where in the eye the receptor required for estrogen to act was located. As a result, anti-estrogen receptor antibody-positive images were obtained in the retina containing the outer nuclear layer (Fig. 22), a part of the lens (Fig. 23), and the cornea (Fig. 24) as observed by DAB coloration. Was. The ciliary body had a strong background and was difficult to distinguish. (Figure 25).

 It was confirmed whether 17-estradiol migrated deep into the eye by instillation and was effective in treating various retinal diseases and optic nerve diseases such as glaucoma.

<Translocation of estrogen receptor to the nucleus by instillation of 17j3-estradiol> As described above, the presence of the estrogen receptor was observed in the retina. But the figure

Its localization is cytoplasmic, as shown in 22, and this state is inactive. Numerous translocations to the nucleus have been reported in cultured cells and the like when estrogen binds. However, it is completely unclear whether estrogen induces nuclear translocation of this receptor in vivo and this can be confirmed by immunostaining of tissue sections. First, it was examined whether estrogen instillation could cause nuclear translocation of the retinal receptor.

 (1) Nuclear translocation of estrogen receptor in the retina

 One drop of eye drops of estrogen (2-hydroxypropinole j3-cyclodextrin inclusion complex 17/3-estradiole 10 μg / ml) was given to the mice (501). Four instillations were performed on the morning and evening of the second day, and on the morning of the third day. One hour after the last instillation, the eyes were enucleated, frozen, sectioned, and stained with an anti-estrogen receptor antibody. As a result, in the extraretinal granule cells, a certain percentage of the cells transferred to the nucleus could be confirmed (Figures 26 to 29). Based on this result, it was decided to angularly analyze the change in the concentration of ophthalmic 17β-estradiol and the change with time after addition.

(2) Instillation 1 7] Change due to 3-estradiol concentration In order to determine the concentration of ophthalmone 17 / 3-estradiol capable of translocating the estrogen receptor of extraretinal granule cells to the nucleus, as in (1), the concentration of 2-hydroxypropyl-1-β-cyclotide was determined in the same manner as in (1). Dextrin clathrate 1 7 β-estradio is applied 4 times a day, morning and evening on the first day, morning and evening on the third day, and after 1 hour after the last instillation, the eyeball is extracted, frozen, and sliced. Stained with anti-estrogen receptor antibody. Figure 30 shows the results. 17—Estradiol concentration of 0.1 // g / ml or more in the case of eye drops, significantly reduced receptor nuclei compared to PBS-only eye drops (17 i8—estradiol 0 ig / ml) Migration was observed. Nuclear translocation increased in a dose-dependent manner up to 17] 3-estradiol concentration of 100 ^ g / ml.

 (3) 1 7) 8_Change of estradiol after instillation

 In order to determine how long the translocation of estrogen receptor into the nucleus by 17-β-estradiol is continued, 17-estradiol at a concentration of 10 β-g / ml was added for the purpose of examining the long-term translocation of estrogen receptor into the nucleus. Eye drops were instilled three times every 10 minutes, and after 2, 8, 24, and 48 hours from the first instillation, the eyes were excised, frozen, sectioned, and stained with anti-estrogen receptor antibody. Figure 31 shows the results. As can be seen from the figure, nuclear translocation was observed in 36% of extraretinal granule cells 2 hours after instillation, but reached 22% and 19% after 24 hours and 48 hours. Control with addition of 7β-estradiol at 0 μg / ml Control 18 The number of transferred cells decreased to slightly more than 18%.

 Instillation of 17β-estradiol confirmed that its receptor translocated to the nucleus in extraretinal granule cells in a dose-dependent manner. This result clearly shows that 17 β- and estradiol act directly on the deep layer of the eye by instillation. The effect decreases after 1-2 days. After nuclear translocation, the estrogen receptor is proteolytically degraded.

 (Summary)

2-Hydroxypropyl-1] 3-cyclodextrin clathrate The effect of 17 β-estradiol on the eyeball by eye drops could be confirmed by molecular biology using a model animal. This suggests that in clinical applications, this method would allow estrogen such as 17j3-estradiol to act on the eye more easily. It is shown as data.

 When 0.01 μg / ml or more of 17-estradiol was instilled, the translocation of estrogen receptor of extraretinal granule cells into the nucleus was significantly recognized as compared with the control of PBS instillation. Nuclear translocation is dose-dependent at concentrations from 0.1 to 100 wg / ml, with concentrations of 0.1 μg / ml to 100 g / ml or higher being 17 β -It was found that the concentration of ostradiol-containing ophthalmic solution was suitable.

 In addition, since the effect lasts for a long time (24 hours or more) from the time course of translocation into the nucleus, it is sufficient to administer once or twice a day or twice a day, and the frequency of administration of eye drops is small. . From this experiment, it is assumed that eye drops can be used to treat various retinal diseases and optic nerve diseases such as glaucoma.The ophthalmic solution containing 2-hydroxypropyl-1-cyclodextrin 17 β-estradiol is expected to be effective in treating various diseases. It can be said that it is an excellent ophthalmic treatment.

Industrial applicability

 ADVANTAGE OF THE INVENTION According to this invention, the problem of the side effect which is a problem of HRT can be overcome, and it becomes possible to provide a therapeutic agent for eye diseases such as glaucoma and cataract, central nervous disease or mental illness, which has a high therapeutic effect.

 Further, according to the present invention, it is possible to provide a therapeutic agent for eye diseases such as glaucoma, cataract, and fundus disease, which has a long duration of effect and requires only one instillation once a day or once every two to three days. . In particular, since it can reduce intraocular pressure and improve visual field narrowing, it can effectively treat not only high-tension glaucoma but also normal-tension glaucoma.

Claims

The scope of the claims

1. A therapeutic agent for diseases to be applied to the eye, comprising a sex steroid hormone, a substance having an estrogenic effect, a selective estrogen receptor modulator or a non-feminizing estrogen.

 2. The therapeutic agent according to claim 1, wherein the raw steroid honolemon is selected from the group consisting of estrogen, testosterone, progesterone, androgen and derivatives thereof.

 3. The therapeutic agent according to claim 1, wherein the sex steroid hormone is 17β-estradiol or a derivative thereof.

 4. The therapeutic agent according to any one of claims 1 to 3, wherein the sex steroid hormone is a water-soluble sex steroid hormone.

 5. The therapeutic agent according to any one of claims 1 to 4, wherein the sex steroid hormone is a sex steroid hormone included by cyclic dextrin.

6. Sex steroid hormones are hydroxypropyl] 3-cyclodextrin, hydroxyxetinolate β-cyclodextrin, snorehoptinoleatene beta-sic mouth dextrin, sulfonyl-beta-cyclodextrin, γ-cyclodextrin The therapeutic agent according to any one of claims 1 to 5, which is a sex steroid hormone encapsulated by δ-cyclodextrin or hydroxypropyl-γ-cyclodextrin.

 7. The therapeutic agent according to claim 1, wherein the sex steroid hormone is a sex steroid hormone encapsulated by 2-hydroxypropyl-3-cyclodextrin.

 8. The therapeutic agent according to claim 1, wherein the substance having an estrogenic effect or a selective estrogen receptor modulator or a non-feminizing estrogen is water-solubilized.

 9. The therapeutic agent according to any one of claims 1 to 8, which is in the form of eye drops, eyewashes, ointments, conjunctival injections, intraocular injections, or contact lens adsorbents.

10. The disease is glaucoma, high tension glaucoma, normal tension glaucoma, senile macular degeneration, macular Hole, cataract, senile cataract, retinal bleeding, central retinal arteriovenous obstruction, fundus arteriosclerosis, photoopia, diabetic retinopathy, retinochoroidal atrophy, retinochoroidal neovascular lesion, cataract by ovariectomy, TGF jS Cataract, macular fibroplasia, epiretinal membrane, retinal tear, retinal detachment, proliferative retinopathy, retinal pigment degeneration, corneal opacity, corneal piran, corneal ulcer, corneal endothelial cell degeneration, corneal degeneration, epidemic Keratoconjunctivitis sicca, chalazion, iritis, uveitis, chorioretinitis, iridocyclitis, eye strain, visual field narrowing due to various diseases, optic nerve atrophy, optic neuritis, dynamic visual acuity, abnormal color vision, presbyopia The therapeutic agent according to any one of claims 1 to 9, wherein the therapeutic agent is selected from the group consisting of myopia, hyperopia, astigmatism and central nervous system diseases, mental illness, pituitary abnormalities, and diseases due to hormonal imbalance.

11. Use of a sex steroid hormone, a substance having an estrogenic effect, a selective estrogen receptor modulator or a non-ferminizing estrogen as an active ingredient in the manufacture of a therapeutic agent for diseases applied to the eye.

 12. The use according to claim 11, wherein the 'I raw steroid honolemon is selected from the group consisting of estrogen, testosterone, progesterone, androgen and derivatives thereof.

 13. The use according to claim 11 or 12, wherein the sex steroid hormone is 17β-estradiol or a derivative thereof.

 14. The use according to any one of claims 11 to 13, wherein the sex steroid hormone is a water-solubilized sex steroid hormone.

 15. Use according to any one of claims 11 to 14, wherein the sex steroid hormone is a sex steroid hormone encapsulated by a cyclic dextrin.

 1 6. Sex steroid hormones are hydroxypropyl-cyclodextrin, hydroxyxethyl-j3-cyclodextrin, sulfobutylether-3] cyclodextrin, sulfonyl-cyclodextrin, cyclodextrin, delta-cyclodextrin The use according to any one of claims 11 to 15, which is a sex steroid hormone encapsulated by hydroxypropyl-γ-cyclodextrin.

17. The sex steroid hormone according to any one of claims 11 to 16, wherein the sex steroid hormone is a sex steroid hormone encapsulated by 2-hydroxypropyl-1] 3-cyclodextrin. Use according to clause 1.

 18. The use according to claim 11, wherein the substance having an estrogenic effect, the selective estrogen receptor modulator or the non-fining estrogen is water-solubilized.