JP2020172440A - Eye drop for treating dry eye - Google Patents

Abstract

To provide an eye drop for treating dry eye that can achieve both of the maximized effect of dry eye therapy with diclofenac and reduced side effect.SOLUTION: This invention relates to an eye drop for treating dry eye that contains 0.01-0.05 weight/volume% of diclofenac or a pharmaceutically acceptable salt thereof.SELECTED DRAWING: None

Description

The present invention relates to eye drops for the treatment of dry eye.

Dry eye is a chronic disease of tear fluid and keratoconjunctival epithelium caused by various factors, which is accompanied by eye discomfort and visual dysfunction. Dry eye is a major eye disease that affects 10 to 20% of adults in Europe, the United States and Japan, and the number of patients is due to increased display screen usage time, air drying by air conditioning equipment, use of contact lenses, etc. Is on the rise.

Dry eye is caused by a decrease in the amount of tear fluid due to a decrease in the amount of tear fluid secreted in the lacrimal gland and an increase in the evaporation of water due to a decrease in lipids and mucin in the tear fluid. Decreased tear fluid causes chronic irritation and inflammation of the corneal and conjunctival surfaces, leading to poor quality of life for the patient. Conventionally, steroids have been used to suppress inflammation and treat dry eye, but the safety is not sufficient and side effects tend to occur. Non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac and bromfenac are known as agents that suppress inflammation in place of steroids. Patent Document 1 discloses an anti-inflammatory eye drop containing diclofenac as an active ingredient. Further, Patent Document 2 discloses that diclofenac can treat dry eye by a mechanism of action independent of anti-inflammatory that suppresses apoptosis due to high osmotic pressure of tear fluid.

Increasing the dose of the drug improves the therapeutic effect, but also causes significant side effects. Therefore, there is a need for a dosage or administration that can maximize the therapeutic effect while reducing side effects. For diclofenac, there is no known method for maximizing the therapeutic effect of dry eye and reducing side effects.

Japanese Unexamined Patent Publication No. 58-174310 International Publication No. 2015/099019

An object of the present invention is to provide an eye drop for the treatment of dry eye, which can achieve both maximization of the therapeutic effect of diclofenac and reduction of side effects.

The present inventors have found that when an eye drop containing diclofenac is administered to a dry eye patient, the therapeutic effect of dry eye can be maximized while reducing side effects under certain conditions.

That is, the present invention relates to eye drops for the treatment of dry eye containing 0.01 to 0.05% by weight / volume% of diclofenac or a pharmaceutically acceptable salt thereof.

The eye drops preferably contain 0.02 to 0.05% by weight / volume of diclofenac or a pharmaceutically acceptable salt thereof.

The eye drops preferably contain diclofenac or a pharmaceutically acceptable salt thereof of 0.01% by weight or more and less than 0.05% by weight / volume.

The eye drops preferably contain diclofenac or a pharmaceutically acceptable salt thereof of 0.02% by weight or more and less than 0.05% by weight / volume.

The eye drops preferably contain 0.02% by weight / volume of diclofenac or a pharmaceutically acceptable salt thereof.

The eye drops are preferably formulated for administration 3 to 8 times / day.

The eye drops are preferably formulated for administration 3 times / day.

It is preferred that the pharmaceutically acceptable salt of diclofenac is diclofenac sodium.

It is preferable that the dry eye is caused by apoptosis due to hyperosmotic pressure of tears.

It is preferable that the apoptosis is caused by a factor other than inflammation.

It is preferable that the dry eye is due to a decrease in mucin.

The eye drops are preferably eye drops for reducing the side effects of administration of diclofenac or a pharmaceutically acceptable salt thereof.

The side effect is preferably apoptosis of corneal surface cells.

The eye drop for treating dry eye of the present invention can maximize the therapeutic effect of dry eye while reducing side effects.

Shows the effect of eye drops on dry eye treatment. The change in the expression level of the Muc5AC gene by the eye drops is shown. The change in the expression level of the Muc6 gene by the eye drops is shown. Shows side effects of eye drops. Shows side effects of eye drops. Shows anti-inflammatory effect and side effects of eye drops.

The present invention relates to eye drops for the treatment of dry eye containing 0.01-0.05% by weight / volume% diclofenac or a pharmaceutically acceptable salt thereof.

The concentration of diclophenac or a pharmaceutically acceptable salt thereof in the eye drops is 0.01 to 0.05% by weight / volume, but 0.01% by weight / volume or more and 0.05% by weight / volume. Less than, preferably 0.02 to 0.05% by weight / volume, more preferably 0.02% by weight / volume or more and less than 0.05% by weight / volume, most preferably 0.02% by weight / volume. .. Below 0.01% by weight / volume, the therapeutic effect tends to be weakened. If it exceeds 0.05% by weight / volume, side effects described later tend to appear and the therapeutic effect tends to decrease.

Pharmaceutically acceptable salts of diclofenac include diclofenac sodium and diclofenac potassium.

The pH of the eye drops is preferably 6.0 to 8.5, more preferably 7.0 to 8.0. If the pH is less than 6.0, the relief of eye irritation tends not to be obtained, and if the pH exceeds 8.5, the pH deviates from the physiological pH.

The osmotic pressure ratio of the eye drops is preferably 0.9 to 1.4. The osmotic pressure ratio referred to here means an osmotic pressure ratio when compared with a physiological saline solution.

The eye drops of the present invention may contain a buffer, an isotonic agent, a preservative, a thickener, a solubilizing agent, and a cleaning agent in addition to diclofenac or a pharmaceutically acceptable salt thereof.

Examples of the buffering agent include a combination of phosphoric acid and phosphate, a combination of boric acid and borax, and a combination of organic acid and organic acid salt. Among these, a combination of boric acid and borax is preferable. The content of the buffer in the eye drops is preferably 0.01 to 10% by weight / volume, more preferably 0.1 to 3% by weight / volume. If it is less than 0.01% by weight / volume, it tends to be difficult to sufficiently exert the effect of the present invention. Eye irritation tends to occur above 10% by weight / volume.

Examples of the tonicity agent include sugars such as glucose, sugar alcohols such as propylene glycol, glycerin, sodium chloride, potassium chloride, mannitol, sorbitol, and xylitol. Of these, sodium chloride and potassium chloride are preferable. The content of the tonicity agent in the eye drops is preferably 0.01 to 10% by weight / volume, more preferably 0.1 to 3% by weight / volume.

As preservatives, reverse soaps such as benzalkonium chloride, benzethonium chloride and chlorhexidine gluconate, parabens such as methylparaben, ethylparaben, propylparaben and butylparaben, alcohols such as chlorobutanol, phenylethyl alcohol and benzyl alcohol Can be mentioned. Of these, chlorobutanol is preferable. The content of the preservative in the eye drops is preferably 0.001 to 0.5% by weight / volume%.

Examples of the thickener include polyvinylpyrrolidone, methyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose and the like. Of these, polyvinylpyrrolidone is preferable.

Examples of the solubilizing agent include polysorbate 80 (polyoxyethylene sorbitan monooleate, trade name Tween80), polyoxyethylene oxystearate triglyceride, polyethylene glycol, α or β-cyclodextrin and the like. Of these, polysorbate 80 is preferable.

Calcium or magnesium salts may be included to relieve eye irritation. Examples of such salts include calcium salts such as calcium pantothenate, calcium chloride, calcium propionate, calcium acetate, calcium lactate, calcium gluconate, and corresponding magnesium salts. Of these, calcium pantothenate, calcium chloride, and magnesium chloride are preferable.

Among the above-mentioned combined components, polysorbate 80, borax, or polyvinylpyrrolidone is preferable.

When the eye drop of the present invention contains polysorbate 80, its concentration is preferably 0.1 to 5.0% by weight / volume, more preferably 0.3 to 3.0% by weight / volume.

When the eye drop of the present invention contains borax, its concentration is preferably 0.1 to 20.0% by weight / volume, more preferably 0.3 to 15.0% by weight / volume.

When the eye drop of the present invention contains polyvinylpyrrolidone, its concentration is preferably 1.0 to 15.0% by weight / volume, more preferably 2.0 to 10.0% by weight / volume.

The eye drops of the present invention are used for the treatment of dry eye. Dry eye is a chronic disease of tear fluid and keratoconjunctival epithelium caused by various factors, accompanied by eye discomfort and visual dysfunction. Apoptosis of corneal tissue is one of the causes of the onset of dry eye, and this apoptosis is further classified into apoptosis due to hyperosmotic pressure of tear fluid and apoptosis due to inflammation.

The mechanism of apoptosis due to hyperosmotic pressure of tear fluid is as follows. When the amount of tears decreases due to a decrease in the amount of tears secreted or the air dries, the osmotic pressure of the tears increases. As a result, water is drained from the cells and the cells are exposed to osmotic stress of shrinking. On the other hand, the cell takes in external sodium ions and the like into the cell, the intracellular ionic strength increases, and apoptosis occurs. Eye tissues in which apoptosis occurs include the cornea, conjunctiva, and lacrimal gland. More specifically, cells in which apoptosis occurs include corneal epithelial cells, conjunctival epithelial cells, and lacrimal gland cells.

Diclofenac suppresses apoptosis even under hyperosmolar conditions of tear fluid by promoting the expression and nuclear translocation of the NFAT5 (Nuclear Factor of Activated T-cells 5) protein. The NFAT5 protein activates the BGT-1 (betaine / GABA transporter-1) gene and the like. As a result, cells can respond to osmotic stress by incorporating organic osmoregulators that do not affect ionic strength into the cells. Since the increase in intracellular ionic strength can be avoided, it is considered that apoptosis is suppressed even under hyperosmotic conditions of tear fluid. Patent Document 2 discloses that diclofenac suppresses apoptosis due to high osmotic pressure in an in vitro system using corneal epithelial cells, which does not contain inflammatory cells. As described above, the eye drops of the present invention can be used for the treatment of dry eye caused by apoptosis due to hyperosmotic pressure of tears. The eye drops of the present invention can be suitably prescribed to dry eye patients who exhibit apoptosis due to hyperosmolarity of tear fluid but do not exhibit apoptosis due to inflammation.

The eye drops of the present invention can suppress apoptosis in the cornea, conjunctiva, and lacrimal gland due to hyperosmosis of tear fluid. More specifically, it can suppress apoptosis of corneal epithelial cells, conjunctival epithelial cells, and lacrimal gland cells.

The present inventors have also found that eye drops containing diclofenac may have side effects. Examples of this side effect include corneal injury in ocular tissue, particularly induction of apoptosis, and specific examples thereof include apoptosis of corneal epithelial cells and conjunctival epithelial cells. In the eye drops for treating dry eye of the present invention, side effects are reduced by setting the concentration of diclofenac or a pharmaceutically acceptable salt thereof to 0.01 to 0.05% by weight / volume.

In addition, the eye drops of the present invention increase mucin in tears by increasing the expression level of the core protein of mucin. One of the causes of dry eye is the promotion of evaporation of water content due to the decrease of mucin in tears, and the eye drops of the present invention can increase mucin. It is the first effect found by the present inventors that an eye drop containing diclofenac has an effect of increasing mucin. Therefore, the eye drops of the present invention are effective for dry eye caused by a decrease in mucin.

When using the eye drops of the present invention, the amount of eye drops for one eye at a time is preferably 1 to 3 drops, and more preferably 1 to 2 drops. In addition, the amount of instillation for one eye at one time is preferably 10 to 300 μL, more preferably 20 to 200 μL, and even more preferably 30 to 100 μL.

The eye drops of the present invention are preferably formulated for administration of 3 to 8 times / day, and more preferably for administration of 3 times / day. In particular, when the concentration of diclofenac or a pharmaceutically acceptable salt thereof in the eye drops is 0.01% by weight / volume% or more and less than 0.05% by weight / volume%, 0.02% by weight / volume% or more 0.05. When less than weight / volume% or 0.02 weight / volume%, it is preferably formulated for 3 doses / day.

In the examples, the present invention will be specifically described, but the present invention is not limited to these.

(1) Suppression test of corneal epithelial cell damage (Example 1, Comparative Examples 1 to 3)
Diclofenac sodium was added to water at a concentration of 0.05% by weight / volume% (Example 1) or 0.1% by weight / volume% (Comparative Example 3) to prepare eye drops.

The lacrimal glands of both eyes of the rat were removed to create a dry eye model. From 1 week after the removal of the lacrimal gland, the above eye drops or vehicle eye drops (Comparative Example 2) were administered to both eyes three times a day for 4 weeks. Five weeks after the removal of the lacrimal gland, a section of eye tissue was prepared and TUNEL-stained to evaluate the apoptosis of corneal cells, and the number of TUNEL-positive cells was calculated. The result is shown in FIG. The measurement results in rats in which the lacrimal glands of both eyes have not been removed are shown as Sham (Comparative Example 1).

In FIG. 1, in Example 1, the apoptosis of corneal cells could be suppressed to the same level as in Comparative Example 3, although the concentration was half that of Comparative Example 3.

(2) Measurement of mucin expression in the keratoconjunctiva (Examples 2 to 3, Comparative Examples 4 to 7)
Eye drops containing diclofenac sodium at a concentration of 0.02% by weight / volume% (Examples 2 to 3) or 0.1% by weight / volume% (Comparative Examples 5 and 7) were applied to both eyes of healthy rats every hour. Was administered by eye drops three times. In Comparative Examples 4 and 6, rats (Control) that had not been treated with eye drops were used.

The conjunctiva and cornea were collected 1 hour after the final instillation. Using the collected conjunctiva and cornea, RNA was extracted by the following method, and the expression of the Muc5AC gene and the Muc6 gene in the corneal conjunctiva was examined.

RNA extraction: RNA contained in the keratoconjunctiva was extracted by TRIzol RNA Isolation Reagents (Thermo Fisher Scientific Inc.).

Reverse transcription reaction: Reverse transcription reaction was carried out using RiverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., LTD.) To obtain cDNA.

Real-time PCR: Quantitative PCR was performed on 10-fold diluted cDNA by ViiA7 real-time PCR system (Thermo Fisher Scientific Inc.) using KOD SYBR qPCR Mix (TOYOBO Co., LTD.). Each primer was added so as to have a final concentration of 0.2 μM per reaction. Primers with the following sequences were used for quantitative PCR.

Muc5AC
forward 5'-ACTCTGCCCACCACAAGC-3' (SEQ ID NO: 5)
reverse 5'-TGCCATCTATCCAAATCAGTCCAAT-3' (SEQ ID NO: 6)

Muc6
forward 5'-TGCTGTCTCCAGCACAAAAC-3' (SEQ ID NO: 7)
reverse 5'-TCAGAAGTCTGCCGTCACTGC-3' (SEQ ID NO: 8)

β-actin
forward 5'-AGACCTTCAACACCCCAG-3'(SEQ ID NO: 3)
reverse 5'-CACGATTTCCTCTCAGC-3' (SEQ ID NO: 4)

The expression levels of the Muc5AC gene and the Muc6 gene obtained by quantitative PCR were corrected by the expression level of β-actin, which is an endogenous control. The corrected expression level of Muc5AC mRNA in the keratoconjunctiva is shown in FIG. 2, and the expression level of Muc6 mRNA in the keratoconjunctiva is shown in FIG.

As shown in FIGS. 2 to 3, 0.02% by weight / volume% diclofenac eye drops increased the expression levels of the Mu5AC gene (Example 2) and the Muc6 gene (Example 3) in the keratoconjunctiva. It was confirmed that the amount of mucin was increased. On the other hand, 0.1% diclofenac eye drops did not have the effect of significantly increasing the amount of mucin in the keratoconjunctiva, and sometimes decreased it (Comparative Examples 5 and 7).

(3) Side effect suppression test 1 (Example 4, Comparative Examples 8 to 9)
Both healthy rats were treated with eye drops containing diclofenac sodium at a concentration of 0.05% by weight / volume% (Example 4) or 0.1% by weight / volume% (Comparative Example 9), or vehicle eye drops (Comparative Example 8). The eye was instilled 8 times every hour.

One hour after the final instillation, a section of eye tissue was prepared, and TUNEL staining was performed by the following method to evaluate the apoptosis of corneal cells, and the number of TUNEL-positive cells was calculated. The result is shown in FIG.

For TUNEL staining, eyeballs were collected from test animals after administration of eye drops. It was immersed in 4% paraformaldehyde and then embedded in paraffin. A section on the surface of the eye was prepared so as to have a thickness of about 4 μm, TUNEL staining (fluorescence) was performed, and a fluorescence-stained image was taken.

As shown in FIG. 4, since healthy rats were used in this test, apoptosis of corneal epithelial cells was hardly observed in Comparative Example 8. In Comparative Example 9, apoptosis of corneal epithelial cells was observed by administration of eye drops containing diclofenac. On the other hand, in Example 4, apoptosis of corneal epithelial cells could be suppressed by reducing the concentration of diclofenac.

(4) Side effect suppression test 2 (Example 5, Comparative Examples 10 to 11)
Eye drops containing diclofenac sodium at a concentration of 0.02% by weight / volume% (Example 5) or 0.1% by weight / volume% (Comparative Example 11) were applied to both eyes of healthy rats once every hour. A total of 3 eye drops were administered. In Comparative Example 10, a rat (Control) that had not been treated with eye drops was used.

Approximately 10 minutes after the final instillation, the cornea was observed by fluorescein staining. Both eyes of awake animals were instilled with 2 μL of 0.5% fluorescein solution to stain the corneal epithelium. Approximately 7 minutes after instillation of fluorescein solution, induction of anesthesia with isoflurane (3-4%) was started, and approximately 10 minutes after instillation of fluorescein solution, fluorescence with a GFP2-ET filter attached under anesthesia (maintenance 1-3%). Animal corneum was photographed using a stereoscopic microscope (MZ10F, Leica) and a digital camera (DFC450C, Leica). The cornea was divided into nine parts, left, right, upper and lower, and scored from 0 to 3 points according to the following criteria, the degree of corneal epithelial damage was evaluated on a scale of 27 points, and the average value of the obtained points was calculated. The result is shown in FIG.
0 points: Almost unstained 1 point: Partially stained (mild disorder)
2 points: About 2/3 staining (moderate disability)
3 points: Staining on almost the entire surface (severe disability)

In FIG. 5, in Comparative Example 11, corneal epithelial damage was observed by administration of eye drops containing diclofenac. On the other hand, in Example 5, corneal epithelial damage could be suppressed by reducing the concentration of diclofenac. The results show that when the concentration of diclofenac is 0.1% by weight / volume, corneal epithelial damage due to diclofenac appears, whereas when the concentration of diclofenac is 0.02% by weight / volume, the corneal conjunctival damage is slight (Figs. 4 to 5). ) Therefore, the anti-inflammatory effect and the corneal conjunctival protective effect became apparent, and it is considered that the corneal epithelial damage was reduced as compared with Comparative Example 10.

(5) Side effect suppression test 3 (Example 6, Comparative Examples 12 to 13)
Eye drops containing diclofenac sodium at a concentration of 0.02% by weight / volume% (Example 6) or 0.1% by weight / volume% (Comparative Example 13) were applied to both eyes of healthy rats three times every hour. After a total of 3 instillations, the keratoconjunctiva was collected. In Comparative Example 12, rats (Control) that had not been treated with eye drops were used. Using the collected keratoconjunctiva, RNA was extracted by the following method, and the expression of the MMP-2 gene in keratoconjunctival cells was examined.

RNA extraction: RNA extraction was performed using TRIzol RNA Isolation Reagents (Thermo Fisher Scientific Inc.) according to the method described in the attached instruction.

Reverse transcription reaction: Using RiverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO Co., LTD.), The reverse transcription reaction was carried out according to the method of the attached instruction manual to obtain cDNA.

Real-time PCR: Quantitative PCR was performed on 10-fold diluted cDNA using KOD SYBR qPCR Mix (TOYOBO Co., LTD.) And StepOne Plus (Thermo Fisher Scientific Inc.) according to the method described in the attached manual. .. Each primer was added so as to have a final concentration of 0.4 μM per reaction. As a primer, a primer having the following base sequence was used.

Mmp-2
forward 5'-TGGGGGGAGAATTCTCACTTG-3'(SEQ ID NO: 1)
reverse 5'-CCATCAGCGTTCCATACTT-3'(SEQ ID NO: 2)

β-actin
forward 5'-AGACCTTCAACACCCCAG-3' (SEQ ID NO: 3)
reverse 5'-CACGATTTCCTCTCAGC-3' (SEQ ID NO: 4)

The expression level of the Mmp-2 gene obtained by quantitative PCR was corrected by the expression level of the β-actin gene, which is an endogenous control. The result is shown in FIG. In Comparative Example 13, the expression of the Mmp-2 gene was observed by administration of an eye drop containing diclofenac, but in Example 6, the expression of the Mmp-2 gene could be suppressed by reducing the concentration of diclofenac. The Mmp (Matrix metalloproteinase) -2 gene is constitutively expressed in the cornea, and its expression is induced during inflammation, causing keratoconjunctival damage. Suppression of the expression of the Mmp-2 gene means suppression of corneal surface damage. This result shows that when the concentration of diclofenac is 0.1% by weight / volume, the anti-inflammatory effect of diclofenac (Fig. 1) and the inflammation due to damage to the keratoconjunctiva (Figs. 4 to 5) antagonize each other. It is considered that the anti-inflammatory effect was manifested because the keratoconjunctival disorder was slight at 01 to 0.05% by weight / volume (FIGS. 4 to 5).

Based on the above, eye drops containing diclofenac at a concentration of 0.01 to 0.05% by weight / volume% have a stronger protective effect on the keratoconjunctiva than eye drops containing a higher concentration, and are themselves. It was shown that the keratoconjunctival disorder was reduced. In addition, eye drops containing diclofenac at a concentration of 0.01 to 0.05% by weight / volume increase mucin in the keratoconjunctiva as compared with eye drops containing diclofenac at a higher concentration or control eye drops. It was confirmed that the action was strong. Therefore, eye drops containing diclofenac at a concentration of 0.01 to 0.05% by weight / volume% have a synergistic effect of anti-inflammatory effect, mucin increasing effect, and reduction of keratoconjunctival damage, resulting in dry eye. Treatment can be optimized.

Claims (13)

Eye drops for the treatment of dry eye containing 0.01-0.05% by weight / volume% diclofenac or a pharmaceutically acceptable salt thereof. The eye drop according to claim 1, which comprises 0.02 to 0.05% by weight / volume% of diclofenac or a pharmaceutically acceptable salt thereof. The eye drop according to claim 1, which comprises diclofenac or a pharmaceutically acceptable salt thereof in an amount of 0.01% by weight or more and less than 0.05% by weight / volume. The eye drop according to claim 3, which comprises diclofenac or a pharmaceutically acceptable salt thereof in an amount of 0.02% by weight or more and less than 0.05% by weight / volume. The eye drop according to any one of claims 1 to 4, which comprises 0.02% by weight / volume% of diclofenac or a pharmaceutically acceptable salt thereof. The eye drop according to any one of claims 1 to 5, which is formulated for administration of 3 to 8 times / day. The eye drop according to any one of claims 3 to 5, which is formulated for administration 3 times / day. The eye drop according to any one of claims 1 to 7, wherein the pharmaceutically acceptable salt of diclofenac is diclofenac sodium. The eye drop according to any one of claims 1 to 8, wherein the dry eye is caused by apoptosis due to hyperosmotic pressure of tears. The eye drop according to claim 9, wherein the apoptosis is apoptosis caused by a factor other than inflammation. The eye drop according to any one of claims 1 to 8, wherein the dry eye is caused by a decrease in mucin. To reduce the side effects of administration of diclofenac or its pharmaceutically acceptable salt,
The eye drop according to any one of claims 1 to 11. The eye drop according to claim 12, wherein the side effect is apoptosis of corneal surface cells.

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