WO2024147651A1 - Pharmaceutical composition for prevention or treatment of dry eye syndrome comprising lifitegrast and tocopherol - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating dry eye syndrome. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating dry eye syndrome comprising lifitegrast and tocopherol or a derivative thereof.

Description

PHARMACEUTICAL COMPOSITION FOR PREVENTION OR TREATMENT OF DRY EYE SYNDROME COMPRISING LIFITEGRAST AND TOCOPHEROL

The present invention relates to a pharmaceutical composition for preventing or treating dry eye syndrome. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating dry eye syndrome comprising lifitegrast and tocopherol or a derivative thereof.

Tears are composed of three layers: a mucus layer, an aqueous layer, and a lipid layer. The mucus layer is produced by goblet cells in the conjunctiva, the aqueous layer is produced by the lacrimal gland, and the outermost lipid layer is produced by the meibomian gland. Moisture in the eye is maintained by three factors: production of the tear film by secretion of tears from the lacrimal gland, loss of tears by drainage to the nose through the nasolacrimal duct, and evaporation of tears from the eye. Although the pathological mechanism of dry eye has not yet been clearly identified, several studies have reported that, if these factors are not properly balanced, dry eye syndrome occurs, which is associated with inflammatory changes in the ocular surface caused by high osmolarity of tears, and various inflammatory factors contribute not only to the pathological process of dry eye but also to ocular discomfort.

Dry eye syndrome refers to a disease condition in which abnormalities in the tear film occur due to a decrease or change in the quantity and quality of the tear film, which moisturizes the eye and maintains the eye in a smooth and comfortable state. Dry eye is known to be a major disease whose incidence is increasing and which accounts for approximately 20% of adults in Korea (data released by the Korea Centers for Disease Control and Prevention in 2011).

Recently, as the concept of dry eye has changed, dry eye has been defined as a multifactorial disease of the tear film and ocular surface, which results in symptoms of discomfort, visual disturbance, and tear film instability with potential damage to the ocular surface and is accompanied by increased osmolarity of the tear and inflammation of the ocular surface.

It has been found that Sjφgren's syndrome patients have increased expression of inflammatory cytokines such as IL-1, IL-6, IL-8, TNF-α, and IFN-γ in the tears and conjunctival epithelium, immune activation and adhesion molecules such as HLA-DR and ICAM-1 in the conjunctival epithelial cells, and cytokines related to the Th-1 or Th-17 inflammatory pathway, and that dry eye show increased concentration and activity of matrix metalloproteinase and increased apoptosis in the ocular surface epithelium, indicating that dry eye is directly associated with inflammation of the ocular surface. Therefore, for the treatment of dry eye, it is important to suppress inflammation of the ocular surface caused by high osmolarity of tears.

Currently used treatments for dry eye include environmental control, artificial tears, anti-inflammatory treatments such as steroids or cyclosporine, tear secretion stimulants, lacrimal punctal occlusion surgery, serum eye drops, therapeutic contact lenses, surgery, and the like, and are selected and used depending on the severity of the disease. However, there is still no definite treatment for dry eye.

Thereamong, combined administration of artificial tears and anti-inflammatory eye drops is the most widely used drug therapy for treating dry eye. Although artificial tears can temporarily relieve the symptoms of dry eye, they cannot fundamentally cure inflammation of the ocular surface, and among anti-inflammatory agents, steroids have the disadvantage of causing serious complications such as glaucoma or cataracts. Therefore, it is necessary to develop a new agent for treating dry eye.

Meanwhile, in 2006, Allergan Inc. (USA) developed Restasis eye drops, a dry eye therapeutic agent using the immunomodulator cyclosporine. Restasis eye drops have been reported to inhibit the production and activation of immune cells related to keratoconjunctivitis sicca and increase the amount of tear secretion (tear volume). However, Restasis eye drops have problems in that, since it exhibits medicinal efficacy through anti-inflammatory action, it has to be used repeatedly for several months to obtain a satisfactory level of efficacy, and burning sensation, a representative side effect, occurs at a high rate of about 17%.

In 2016, Novartis (Switzerland) received FDA approval for the new drug lifitegrast which inhibits ICAM-1. Lifitegrast is evaluated as a new drug that is involved in the core stage of the inflammatory cycle and shows long-term stability, tolerability, rapid onset of efficacy, and amelioration of dry eye symptoms. Lifitegrast is marketed as the brand name of Xiidra eye drops in Korea, has the problem of being expensive, and does not improve patient convenience because one drop per administration is instilled into each eye twice a day (BID).

Korean Patent No. 10-2391198 discloses water-soluble eye drops with improved stability, obtained by adding citric acid hydrate, a stabilizer, to lifitegrast eye drops.

However, the Patent Document mentioned above aims at improving the stability of lifitegrast eye drops and does not recognize the need for eye drops comprising lifitegrast and tocopherol.

Tocopherol (vitamin E) is known as a lipid component having antioxidant effects. It is known that tocopherol replenishes lipid, which is one of the three layers (mucus layer, aqueous layer, and lipid layer), on the ocular surface, and has an antioxidant effect against reactive oxygen species that cause the pro-inflammatory cascade, and thus it can ameliorate dry eye in a conservative manner when administered orally or instilled.

Korean Patent Application Publication No. 10-2022-0092164 discloses that tocopherol is an ingredient that improves the diffusion and sustainability of eye drops.

However, the Patent Document mentioned above aims at using tocopherol together with diquafosol to improve the diffusion and sustainability of diquafosol eye drops, and does not recognize the need for eye drops comprising lifitegrast and tocopherol due to the antioxidant effect of tocopherol.

The present inventors have found that, when a mixture of lifitegrast, which has an anti-inflammatory effect, and tocopherol, which has an antioxidant effect, is instilled into an eye, it can further improve the condition of the ocular surface, and can exhibit the same or greater therapeutic effect while reducing the existing number of instillations (twice a day/one drop per administration), thereby arriving at the present invention.

[Prior Art Documents]

[Patent Documents]

(Patent Document 1) Korean Patent No. 10-2391198

(Patent Document 2) Korean Patent Application Publication No. 10-2022-0092164

An object of the present invention is to provide a novel pharmaceutical composition for preventing or treating dry eye syndrome, which is capable of more effectively ameliorating dry eye symptoms even when the number of administrations thereof is reduced compared to that of existing eye drops (a product commercially available under the brand name of Xiidra eye drops®) for dry eye treatment, which comprise lifitegrast as a main component.

The present inventors have found that, even when a mixture of lifitegrast and tocopherol is administered once a day (QD), it is capable of effectively preventing or treating dry eye syndrome by increasing tear volume, tear film break-up time, and tear film lipid layer grade and showing a significant reduction in corneal fluorescein staining score, compared to the commercially available product. This is the principle by which the object of the present invention is achieved.

In order to achieve the above object, the present invention discloses the following means.

In one aspect, the present invention achieves the above object by providing a pharmaceutical composition comprising lifitegrast as an active ingredient and tocopherol or a derivative thereof as an additive.

The pharmaceutical composition according to the present invention achieves the effect of increasing tear volume, tear film break-up time, and tear film lipid layer grade and shows a significant reduction in corneal fluorescein staining score, compared to conventional eye drops for treating dry eye syndrome comprising lifitegrast as an active ingredient.

The pharmaceutical composition according to the present invention may increase the convenience of dry eye patients by reducing the number of administrations to once a day, compared to conventional eye drops for treating dry eye comprising lifitegrast as an active ingredient.

Effects of the present invention are not limited to the effects mentioned above, and may include various effects within the scope apparent to those skilled in the art from the following description.

FIG. 1 illustrates the results of evaluating tear volume.

FIG. 2 illustrates the results of evaluating tear film break-up time.

FIG. 3 illustrates the results of evaluating corneal fluorescein staining score.

FIG. 4 illustrates the results of evaluating tear film lipid layer grade.

FIG. 5 illustrates the concentration of reactive oxygen species (ROS) in the cornea.

FIG. 6 illustrates the concentration of reactive oxygen species (ROS) in the conjunctiva.

FIG. 7 illustrates the number of inflammatory immune cells in the cornea.

FIG. 8 illustrates the number of inflammatory immune cells in the conjunctiva.

Hereinafter, the present specification will be described in more detail. The terms used in the present specification are currently widely used general terms selected in consideration of their functions in the present invention, but they may change depending on the intents of those skilled in the art, precedents, or the advents of new technology. Additionally, in certain cases, there may be terms arbitrarily selected by the applicant, and in this case, their meanings are described in a corresponding description part of the present invention. Accordingly, terms used in the present invention should be defined based on the meaning of the term and the entire contents of the present invention, rather than the simple term name.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used in general and defined in dictionaries should be interpreted as having meanings identical to those specified in the context of related technology. Unless definitely defined in the present application, the terms should not be interpreted as having ideal or excessively formative meanings.

A numerical range includes numerical values defined in the range. Every maximum numerical limitation given throughout the present specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout the present specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout the present specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

Meanwhile, each description and embodiment disclosed in the present invention may also be applied to other descriptions and embodiments. That is, all combinations of various elements disclosed in the present invention fall within the scope of the present disclosure. Further, the scope of the present invention is not limited by the specific description described below.

Expressions such as "comprising" or "containing" as used in the present specification should be understood as open-ended terms that imply the possibility of including other embodiments, unless otherwise mentioned in the phrase or sentence in which the expressions are contained.

The present inventors have conducted studies to develop eye drops for treating dry eye syndrome comprising lifitegrast as an active ingredient, and as a result, have found that, when lifitegrast is mixed with tocopherol or a derivative thereof, the mixture achieves the effect of increasing tear volume, tear film break-up time, and tear film lipid layer grade and shows a significant reduction in corneal fluorescein staining score, even when the number of administrations thereof is reduced compared to that of Xiidra eye drops®, which is a commercially available product, thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

Pharmaceutical Composition for Preventing or Treating Dry Eye Syndrome

The present invention discloses a pharmaceutical composition as follows.

Specifically, the present invention discloses a pharmaceutical composition for preventing or treating dry eye syndrome comprising lifitegrast and tocopherol or a derivative thereof.

In the present invention, the term "lifitegrast" refers to an LFA-1/ICAM-1 (Leukocyte Function-association Antigen 1/InterCellular Adhesion Molecule 1) antagonist represented by the Formula below. Lifitegrast is known to inhibit the binding between LFA-1, a receptor present on the surface of T cells, and ICAM-1, present on epithelial cells, thereby preventing T cell activation (recognition of corneal/conjunctival epithelial cells as antigens) and alleviating ocular inflammation, thus normalizing tear production. Therefore, lifitegrast is known as a substance that is effective in treating dry eye.

[Formula]

In the present invention, tocopherol or a derivative thereof is meant to include tocopherols (α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, etc.), and derivatives thereof, for example, organic acid ester derivatives such as tocopherol acetate, tocopherol succinate, and tocopherol nicotinate, as well as salts thereof, without being limited thereto. Because tocopherols are easily oxidized and unstable when used alone, they are commonly used in the form of organic acid ester derivatives. In consideration of the fact that these derivatives need to be converted to active tocopherols by hydrolysis of the ester linkage portion by an enzyme such as esterase in order for the derivatives to exhibit physiological activity as tocopherols in vivo, and that various forms of tocopherol derivatives exist, the meaning of tocopherol derivatives referred to in the present invention can be clearly understood by those skilled in the art.

In the present invention, the concentration of the tocopherol or derivative thereof in the pharmaceutical composition may be 0.001 (w/v) to 0.1% (w/v) as tocopherol, preferably 0.003% (w/v) to 0.09% (w/v), without being limited thereto.

In the present invention, the concentration of lifitegrast in the pharmaceutical composition may be 0.1 to 15% (w/v) as lifitegrast, without being limited thereto.

In the present invention, pharmaceutically acceptable additives may, if necessary, be added to the pharmaceutical composition according to conventional methods. For example, buffering agents such as sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate anhydrous, sodium acetate, sodium thiosulfate pentahydrate, and epsilon-aminocaproic acid, etc.; tonicifying agent s such as sodium chloride, potassium chloride, and concentrated glycerin, etc.; pH adjusters such as hydrochloric acid, sodium hydroxide, and potassium hydroxide, etc.; surfactants such as polyoxyethylene sorbitan monooleate (polysorbate), polyoxyl 40 stearate, and polyoxyethylene hydrogenated castor oil, etc. may be selected and added as needed. The pH of the pharmaceutical composition according to the present invention is preferably in the range of 4 to 8, which is a range generally acceptable for ophthalmic preparations.

The pharmaceutical composition of the present invention may be subjected to filtration sterilization or other sterilization treatment processes, and these sterilized eye drops are also included within the scope of the present invention.

In the present invention, the pharmaceutical composition may be used so that one drop per eye is instilled once a day, without being limited thereto.

In the present invention, instilling "one drop" of the pharmaceutical composition of the present invention generally means instilling 10 μL to 60 μL of the eye drop of the present invention.

In the present invention, "instilling the pharmaceutical composition once a day" means instilling the eye drop of the present invention once within 24 hours.

Described below are the results of tests for measurement of tear volume, tear film break-up time, corneal fluorescein staining score, tear film lipid layer grade, measurement of the number of immune cells by flow cytometry, measurement of reactive oxygen species by DCF-DA, and measurement of inflammatory cytokines by Luminex immunobead assay, which are efficacy tests. However, these experimental examples are for better understanding of the present invention and are not to be construed as limiting the scope of the present invention.

Experimental Examples

Design of Experiment

1. Experimental Dry Eye (EDE) Model

An experimental dry eye model was induced in 8-week-old C57BL/6 mice by subcutaneously injecting 0.5 mg/0.2 mL scopolamine hydrobromide three times a day in a state in which the mice were exposed to a ventilator for 18 hours a day for a total of 5 days. At this time, the humidity was maintained at 30% and the temperature was maintained at 25°C.

2. Treatment Group Setting

Mice were divided into the following groups according to the type of eye drops to be administered (see Table 1 below).

(1) Untreated control (hereinafter referred to as "UT group")

(2) Experimental dry eye (hereinafter, referred to as "EDE group")

(3) EDE + 0.05% cyclosporine eye drops (instilled once a day (QD); dose per administration: 2 μL; hereinafter referred to as "0.05% CsA group")

(4) EDE + 5% lifitegrast drops (instilled twice a day (BID), dose per administration: 2 μL; hereinafter referred to as "5% LF group")

(5) EDE + 10% lifitegrast drops (instilled once a day (QD), dose per administration: 2 μL; hereinafter referred to as "10% LF group")

(6) EDE + 5% lifitegrast + DL-α-tocopherol acetate drops (instilled once a day (QD), dose per administration: 2 μL; hereinafter referred to as "5% LF+TO group")

(7) EDE + 10% lifitegrast + DL-α-tocopherol acetate drops (instilled once a day (QD), dose per administration: 2 μL; hereinafter referred to as "10% LF+TO group")

Group setting

Untreated control (UT group)

Experimental dry eye (EDE group)

EDE + 0.05% cyclosporine eye drops (0.05% CsA group)

EDE + 5% lifitegrast drops (5% LF group)

EDE + 10% lifitegrast drops (10% LF group)

EDE + 5% lifitegrast + DL-α-tocopherol acetate drops (5% LF+TO group)

EDE + 10% lifitegrast + DL-α-tocopherol acetate drops (10% LF+TO group)

Here, the 5% LF group was instilled twice within 24 hours, and specifically, it was administered to both eyes regularly at 9:00 and 18:00 at 9-hour intervals. In addition, the 0.05% CsA group, the 10% LF group, the 10% LF+TO group, and the 5% LF+TO group were each administered to both eyes regularly at 9:00.

On days 7 and 14 after inducing EDE, tear volume and tear film break-up time were measured in both eyes of all the mice, and corneal fluorescein staining score was examined. After measuring the tear volume, tear film break-up time, and corneal fluorescein staining score on day 14 after inducing EDE, the mice were sacrificed and then subjected to flow cytometry and the like. Each group consisted of 7 animals.

Experimental Methods

1. Measurement of Tear Volume

The amount of tear secretion (tear volume) was measured by placing phenol-red impregnated cotton threads in contact with the conjunctival sac on the lateral canthus side of the mouse eye for 20 seconds, measuring the length thereof using SMZ microscopy, and then converting the value into the volume by substituting the length into a prescribed formula.

2. Measurement of Tear Film Break-Up Time (TBUT)

For the examination of tear film break-up time to determine tear film stability, which is the most important clinical factor in dry eye, 1 μL of 1% fluorescent dye (fluorescein) was instilled into the lower conjunctival sac of the mouse, and then the mouse was allowed to blink their eyes about three times. Next, the eyes were gently opened, and then the time (seconds) until first damage occurred in the stained tear film was measured using a slit lamp microscope equipped with a cobalt blue filter. The measurement was repeated three times to obtain an average value.

3. Measurement of Corneal Fluorescent Staining Score

1 μL of 1% fluorescent staining solution was instilled into the eyes of each mouse, followed by washing with saline. Next, the cornea was observed under a slit-lamp microscope, and the degree of epithelial damage (the degree of fluorescence staining) was evaluated by scoring.　To this end, the cornea was divided into four zones, and then each zone was evaluated from 0 to 4 points as follows and the points were summed up (a total of 0 to 16 points).

① 0: Absent

② 1: Slightly punctuate staining < 30 spots

③ 2: Punctate staining > 30 spots, but not diffuse

④ 3: Severe diffuse staining but no positive plaque

⑤ 4: Severe diffuse staining with positive fluorescein plaque

4. Measurement of Tear Film Lipid Layer Grade

A whiteboard was fixed at a designated location and light was reflected on the lower half of the cornea. Trinocular stereo zoom microscopy (SZM45TR-STL2, SOPTOP, Zhejiang, China) was used to evaluate the lipid layer interference pattern of the corneal background after blinking. The tear film lipid layer was classified into 6 grades (grades 1 to 6) as follows.

(1) Grade 1: grey appearance of low reflectivity and a faintly visible meshwork pattern

(2) Grade 2: more compact meshwork pattern with grey appearance of average reflectivity

(3) Grade 3: vertical and horizontal grey waves of good visibility

(4) Grade 4: yellow-grey and yellow spread lipid layer interference fringes superimposed on a background

(5) Grade 5: brown-yellow and brown spread lipid layer interference fringes superimposed on a background

(6) Grade 6: blue and brown spread lipid layer interference fringes superimposed on a background

5. Measurement of Number of Immune Cells by Flow Cytometry and Reactive Oxygen Species by DCF-DA Assay

The conjunctiva was excised from 8 eyes of 4 animals in each group, which was the amount required for tissue analysis, split with scissors, and shaken with 0.5 mg/ml collagenase type D at 37°C for 60 minutes. The tissue was ground, passed through a cell strainer, and then centrifuged. Flow cytometry was performed using a fluorescein-conjugated anti-CD4 antibody and a phycoerythrin-conjugated anti-IFN-γ antibody to calculate the number of CD4-IFN-γ+ T cells, and the number of DCF-DA cells was calculated using the CM-H2DCFDA kit.

6. Measurement of Inflammatory Cytokines by Luminex Immunobead Assay

Conjunctival tissue was excised and collected from 6 eyes of 3 animals in each group, immersed in lysis buffer containing a protease inhibitor for 30 minutes, centrifuged, and then stored at -80°C. Thereafter, the concentrations of IFN-γ, IL-1β, and TNF-α in the conjunctiva were analyzed by Luminex multiplex immunobead assay.

Experimental Results

1. Tear Volume

The results of the experiment conducted on both eyes of 7 animals per group are shown in FIG. 1 and Table 2 below. Referring to FIG. 1 and Table 2, the tear volume on day 7 after inducing dry eye was 0.046±0.006 μL in the UT group, 0.019±0.006 μL in the EDE group, 0.023±0.005 μL in the 0.05% CsA group, 0.023±0.006 μL in the 5% LF group, 0.024±0.006 μL in the 5% LF+TO group, 0.022±0.006 μL in the 10% LF group, and 0.027±0.007 μL in the 10% LF+TO group. Also, the tear volume on day 14 after inducing dry eye was 0.041±0.005 μL in the UT group, 0.015±0.003 μL in the EDE group, 0.024±0.005 μL in the 0.05% CsA group, 0.025±0.006 μL in the 5% LF group, 0.029±0.009 μL in the 5% LF+TO group, 0.025±0.006 μL in the 10% LF group, and 0.029±0.007 μL in the 10% LF+TO group. It was found that the tear volume on day 14 of the experiment significantly increased in all the 0.05% CsA, 5% LF, 5% LF+TO, 10% LF, and 10% LF+TO groups compared to the EDE group.

Group	Day 7 (μL)	Day 14 (μL)
UT group	0.046±0.006	0.041±0.005
EDE group	0.019±0.006	0.015±0.003
0.05% CsA group	0.023±0.005	0.024±0.005
5% LF group	0.023±0.006	0.025±0.006
10% LF group	0.022±0.006	0.025±0.006
5% LF+TO group	0.024±0.006	0.029±0.009
10% LF+TO group	0.027±0.007	0.029±0.007

2. Tear Film Break-Up Time

The results of the experiment conducted on both eyes of 7 animals per group are shown in FIG. 2 and Table 3 below. Referring to FIG. 2 and Table 3, the tear film break-up time on day 7 after inducing dry eye was 2.52±0.8 sec in the UT group, 1.2±0.3 sec in the EDE group, 1.25±0.4 sec in the 0.05% CsA group, 1.22±0.3 sec in the 5% LF group, 1.28±0.4 sec in the 5% LF+TO group, 1.22±0.4 sec in the 10% LF group, and 1.3±0.4 sec in the 10% LF+TO group. Also, the tear film break-up time on day 14 after inducing dry eye was 2.76±0.6 sec in the UT group, 0.84±0.3 sec in the EDE group, 1.39±0.4 sec in the 0.05% CsA group, 1.26±0.4 sec in the 5% LF group, 1.51±0.5 sec in the 5% LF+TO group, and 1.27±0.6 sec in the 10% LF group, and 1.4±0.5 sec in the 10% LF+TO group. It was found that the tear film break-up time on day 14 significantly of the experiment increased in the 5% LF+TO group compared to the EDE group.

Group	Day 7 (sec)	Day 14 (sec)
UT group	2.52±0.8	2.76±0.6
EDE group	1.2±0.3	0.84±0.3
0.05% CsA group	1.25±0.4	1.39±0.4
5% LF group	1.22±0.3	1.26±0.4
10% LF group	1.22±0.4	1.27±0.6
5% LF+TO group	1.28±0.4	1.51±0.5
10% LF+TO group	1.3±0.4	1.4±0.5

3. Corneal Fluorescein Staining Score

The results of the experiment conducted on both eyes of 7 animals per group are shown in FIG. 3 and Table 4 below. Referring to FIG. 3 and Table 4 below, it was shown that the corneal fluorescein staining score on day 7 after inducing dry eye was 3.3±1.7 points in the UT group, 12.0±1.5 points in the EDE group, 10.7±1.9 points in the 0.05% CsA group, 11.4±2.0 points in the 5% LF group, 9.9±2.7 points in the 5% LF+TO group, 10.9±1.7 points in the 10% LF group, and 10.2±1.6 in the 10% LF+TO group. Also, it was shown that the corneal fluorescein staining score on day 14 after inducing dry eye was 4.5±1.5 points in the UT group, 13.8±1.5 points in the EDE group, 9.9±1.5 in the 0.05% CsA group, 10.8±1.6 points in the 5% LF group, 8.7±1.2 points in the 5% LF+TO group, 10.0±0.9 points in the 10% LF group, and 9.2±1.1 points in the 10% LF+TO group. It was found that the corneal fluorescein staining score on day 14 of the experiment significantly decreased in all the 0.05% CsA group, the 5% LF group, the 5% LF+TO group, the 10% LF group, and the 10% LF+TO group compared to the EDE group. In particular, it was found that the corneal fluorescein staining score on day 14 of the experiment significantly decreased in the 5% LF+TO group compared to the 5% LF group.

Group	Day 7 (points)	Day 14 (points)
UT group	3.3±1.7	4.5±1.5
EDE group	12.0±1.5	13.8±1.5
0.05% CsA group	10.7±1.9	9.9±1.5
5% LF group	11.4±2.0	10.8±1.6
10% LF group	10.9±1.7	10.0±0.9
5% LF+TO group	9.9±2.7	8.7±1.2
10% LF+TO group	10.2±1.6	9.2±1.1

4. Tear Film Lipid Layer Grade

The results of the experiment conducted on both eyes of 7 animals per group are shown in FIG. 4 and Table 5 below. Referring to FIG. 4 and Table 5, it was shown that the tear film lipid layer grade on day 7 after inducing dry eye was 5.4±0.6 points in the UT group, 2.6±1.1 points in the EDE group, 3.0±1.1 points in the 0.05% CsA group, 3.1±1.0 points in the 5% LF group, 3.2±1.0 points in the 5% LF+TO group, 3.2±1.0 points in the 10% LF group, and 3.7±1.2 points in the 10% LF+TO group. Also, it was shown that the tear film lipid layer grade on day 14 after inducing dry eye was 5.0±0.8 in the UT group, 1.9±0.7 points in the EDE group, 3.4±1.0 points in the 0.05% CsA group, 3.7±1.1 points in the 5% LF group, 4.0±1.4 points in the 5% LF+TO group, 3.8±1.1 points in the 10% LF group, and 4.2±0.9 points in the 10% LF+TO group. It was found that the tear film lipid layer grade on day 14 of the experiment significantly decreased in all the 0.05% CsA group, the 5% LF group, the 5% LF+TO group, the 10% LF group, and the 10% LF+TO group compared to the EDE group.

Group	Day 7 (points)	Day 14 (points)
UT group	5.4±0.6	5.0±0.8
EDE group	2.6±1.1	1.9±0.7
0.05% CsA group	3.0±1.1	3.4±1.0
5% LF group	3.1±1.0	3.7±1.1
10% LF group	3.2±1.0	3.8±1.1
5% LF+TO group	3.2±1.0	4.0±1.4
10% LF+TO group	3.7±1.2	4.2±0.9

5. Measurement of Reactive Oxygen Species by DCF-DA Assay

Because the eye tissues of mice are very small, the tissues of 7 mice in each group were collected and expressed as one value (N=1). Specifically, the concentration of reactive oxygen species in the cornea was 18.3% in the UT group, 58.99% in the EDE group, 50.45% in the 0.05% CsA group, 46.71% in the 5% LF group, 33.08% in the 5% LF+TO group, 44.17% in the 10% LF group, and 38.19% in the 10% LF+TO group (see FIG. 5 and Table 6 below). The concentration of reactive oxygen species in the conjunctiva was 21.02% in the UT group, 59.71% in the EDE group, 52.85% in the 0.05% CsA group, 48.03% in the 5% LF group, 32.53% in the 5% LF+TO group, 47.96% in the 10% LF group, and 44.36% in the 10% LF+TO group (see FIG. 6 and Table 6 below). It was found that the concentration of reactive oxygen species in the cornea and conjunctiva decreased in the 5% LF+TO group and the 10% LF+TO group, suggesting that inflammation of the cornea and conjunctiva could be reduced in the 5% LF+TO group and the 10% LF+TO group compared to the EDE group.

	Reactive oxygen species (ROS)
	Cornea (%)	Conjunctiva (%)
UT group	18.3	21.02
EDE group	58.99	59.71
0.05% CsA group	50.45	52.85
5% LF group	46.71	48.03
10% LF group	44.17	47.96
5% LF+TO group	33.08	32.53
10% LF+TO group	38.19	44.36

6. Measurement of Number of Inflammatory Immune Cells by Flow Cytometry

Because the eye tissues of mice are very small, the tissues of 7 mice in each group were collected and expressed as one value (N=1). Specifically, the proportion of inflammatory T cells in the cornea was 5.48% in the UT group, 40.04% in the EDE group, 28.32% in the 0.05% CsA group, 38.86% in the 5% LF group, 14.92% in the 5% LF+TO group, 25.15% in the 10% LF group, and 17.51% in the 10% LF+TO group (see FIG. 7 and Table 7 below). Also, the proportion of inflammatory T cells in the conjunctiva was 3.45% in the UT group, 37.57% in the EDE group, 26.92% in the 0.05% CsA group, 30.32% in the 5% LF group, 12.49% in the 5% LF+TO group, 23.61% in the 10% LF group, and 15.51% in the 10% LF+TO group (see FIG. 8 and Table 7 below). It was found that the proportion of inflammatory T cells in the cornea and conjunctiva tended to decrease in the 0.05% CsA group, the 5% LF+TO group, the 10% LF group, and the 10% LF+TO group, in particular, the 5% LF+TO group and the 10% LF group, compared to the EDE group.

	CD4+IFN-γ+T cells
	Cornea (%)	Conjunctiva (%)
UT group	5.48	3.45
EDE group	40.04	37.57
0.05% CsA group	28.32	26.92
5% LF group	38.86	30.32
10% LF group	25.15	23.61
5% LF+TO group	14.92	12.49
10% LF+TO group	17.51	15.51

7. Measurement of Inflammatory Cytokines by Luminex Immunobead Assay

It was found that IL-1β and TNF-α in the conjunctiva tended to decrease in all the 0.05% CsA group, the 5% LF+TO group, the 10% LF group, and the 10% LF+TO group compared to the EDE group (see Table 8 below).

Conjunctiva	IFN-γ (pg/ml)	IL-10 (pg/ml)	IL-17 (pg/ml)	IL-1β (pg/ml)	IL-6 (pg/ml)	TNF-α (pg/ml)
UT group	49.8	11.5	1.1	34.9	54.6	< 0
EDE group	64.0	22.0	3.0	113.5	72.3	3.3
0.05% CsA group	68.0	16.8	1.5	83.5	72.8	1.3
5% LF group	49.8	10.8	2.0	82.1	62.3	0.3
10% LF group	63.4	14.2	4.8	75.7	60.7	0.3
5% LF+TO group	30.6	3.5	0.3	67.2	55.0	< 0
10% LF+TO group	63.4	10.1	0.7	80.6	73.8	< 0

8. Conclusion

In the dry eye mice, the eye drops comprising the mixture of lifitegrast and tocopherol tended to increase tear volume, ameliorate corneal epithelial damage, increase tear film lipid layer grade, and decrease immune cells. In the 5% LF+TO group and the 10% LF+TO group, reactive oxygen species in the cornea and conjunctiva tended to decrease, and immune cells tended to decrease compared to those in the other treatment groups. In particular, it was found that the corneal fluorescein staining score in the 5% LF+TO group significantly decreased compared to that in the 5% LF group.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only of a preferred embodiment thereof, and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereto.

Claims (5)

1. A pharmaceutical composition for preventing or treating dry eye syndrome comprising lifitegrast and tocopherol or a derivative thereof.
2. The pharmaceutical composition according to claim 1, wherein a concentration of the tocopherol or derivative thereof in the pharmaceutical composition is 0.001 to 0.1% (w/v) as tocopherol.
3. The pharmaceutical composition according to claim 1, wherein a concentration of the lifitegrast in the pharmaceutical composition is 0.1 to 15% (w/v) as lifitegrast.
4. The pharmaceutical composition according to claim 1, wherein the tocopherol derivative is tocopherol acetate.
5. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is used so that one drop per eye is instilled once a day.

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