TWI590824B - Artificial tear composition - Google Patents

Description

Artificial tear composition

The present invention relates to an artificial tear composition having moisturizing effect and at the same time having anti-inflammatory and anti-oxidation, and the composition can be used for treating ocular surface inflammation and dry eye syndrome.

The eyes are the window of human soul. In modern times, people don’t know how to maintain their eyes well. Moreover, they are looking forward to using air conditioners, using TVs and computers for a long time, and wearing contact lenses, making eye fatigue prone to some diseases, and dry eye syndrome is One of the common diseases of the eye.

Symptoms caused by dry eye syndrome are divided into three phases: mild, moderate, and severe.

The treatment of dry eye can be treated differently according to the severity of the disease. There are currently three treatment methods:

1. Mild: Artificial tears are usually used as a way to soothe dry eyes, and some use eye ointments as a treatment.

2. Moderate: often accompanied by inflammatory conditions of the ocular surface, so at this time will use anti-inflammatory eye ointment with artificial tears as a treatment, but also invasive surgery, such as: tear little obstruction surgery to reduce tear loss, to achieve Soothes the effect of dry eyes.

3. Severe: Only surgery such as sacral suture or salivary gland transplantation can be used, so that the eyes will not dry up and achieve a relaxing effect.

Furthermore, under normal conditions, the human eye is stained with fluorescent light, and the ocular surface is clear and free of debris. However, in patients with dry eye, the corneal cells are damaged due to inflammation of the ocular surface. Therefore, the dyes in the eye will enter the damaged cells under fluorescent staining, causing chaos or spots on the ocular surface.

See Table 1 for the composition of artificial tears sold in the market.

Epigallocatechin gallate (EGCG) is the most abundant component of catechins in green tea. It has been proven to have anti-inflammatory and anti-oxidant properties and can inhibit a variety of inflammatory factors (Megan). E. Cavet, et al., Molecular Vision Biology and Genetics in Vision Research , 2011; 17: 533-543).

Hyaluronic acid (HA), a natural biomaterial, an extracellular matrix, has been shown to play an important role in wound healing and inflammation (Inoue M and Katakami C, Investigative Ophthalmology & Visual Science , according to research). 1993; 34(7): 2313-2315), also commonly used to treat dry eyes, mainly because hyaluronic acid (HA) can increase the viscosity of the liquid, making the liquid stay in the ocular surface for a long time (Papa V, Aragona P, Russo S , et al. Ophthalmologica 2001; 215: 124-127, Stern ME, Beuerman RW, Fox RI, et al. Cornea 1998; 17: 584-589., Mengher LS, Pand KS, Bron AJ., et al . Br J Ophthalmol 1986; 70: 422-427); See Table 2 for the sale of artificial tears containing hyaluronic acid.

However, the artificial tears sold in the market are shown in Tables 1 and 2. Most of the salt aqueous solutions do not contain a formula for treating inflammation, and their functions are to slow down the eyes. In addition, some artificial tears are also sold in the market. Preservatives, which can also lead to deepening of symptoms of dry eye (Ankita S. Bhavsar, et al., Oman J Ophthalmol. 2011; 4(2): 50-56).

At present, doctors treat moderate dry eye syndrome by giving patients some anti-inflammatory eye ointments or syrups and artificial tears. They are used together with each other; because modern people are busy, they often have a little medicine and forget to take another medicine. It will delay the treatment effect and prolong the time course; in addition, the eye ointment will cause the patient's line of vision to be blurred when it is put into the eye, which may cause troubles in movement.

Furthermore, the artificial tears or eye drops currently sold in the market are dripped into the ocular surface for about 5 minutes, and the liquid is evaporated due to the ocular surface or the nasolacrimal duct overflows. Most of the drugs will not remain on the ocular surface, so the current treatment of the eyes When the disease doctor opens the eye drops to the patient, he or she usually squats. It takes 3 to 4 times a day. After the one is finished, it takes 5 minutes to make another one. Because of the busy life of the above modern people, two or more kinds of syrup are used at the same time. When treating eye diseases, repeated administration of the drugs may cause inconvenience to the patient, thereby affecting the effect and duration of the treatment.

For this reason, the Applicant has invented a "artificial tear composition" which has moisturizing effect and anti-inflammatory and anti-oxidation, which can effectively reduce the number of repeated administrations and effective treatment, in view of the lack of the prior art. To improve the lack of the above-mentioned practices.

It is an object of the present invention to provide an artificial tear composition having a moisturizing effect and having both anti-inflammatory and anti-oxidant functions for treating ocular surface inflammation and moderate dry eye.

In order to achieve the above object, the technical means of the present invention consists in combining the following ingredients into an artificial tear composition; the composition comprises an anti-inflammatory and anti-oxidant substance, for example: gallocatechin gallate (EGCG) ), a substance that increases the viscosity of the liquid, such as hyaluronic acid (HA) and an artificial tear.

Another object of the present invention is to provide an artificial tear composition which can prolong the retention of ocular surface time, thereby reducing the step of repeated administration, and effectively shortening the course of treatment for ocular surface inflammation and moderate dry eye.

In order to achieve the above object, the technical means of the present invention is to add a substance which can increase the viscosity of the liquid, for example, hyaluronic acid (HA) in artificial tears, so that the liquid stays in the ocular surface for a long time, thereby increasing the retention of the medicament in the eye. The table time, in turn, promotes the effect of the present invention in the treatment of moderate dry eye.

1‧‧‧Artificial Tear Treatment Group

2‧‧‧Artificial tears added to hyaluronic acid (HA) treatment group

3‧‧‧Artificial tears added to the gallocatechin gallate (EGCG) treatment group

4‧‧‧The artificial tear composition treatment group of the invention

A‧‧‧ artificial tears only with the gallogalate gallate (EGCG) group

B‧‧‧Inventive tear composition of the invention (ie containing hyaluronic acid (HA))

Figure 1A is a graph showing the effect of the artificial tear composition of the present invention on cell viability.

Figure 1B is a graph showing the effect of the artificial tear composition of the present invention on cytotoxicity.

Figure 2 is a graph showing the effect of the artificial tear composition of the present invention on inflammatory factors.

Figure 3 is a graph showing the results of a Schirmer test after treatment of moderate dry eye syndrome in animals using the artificial tear composition of the present invention.

Figure 4 is a graph showing changes in the inflammatory factors following treatment of moderate dry eye in animals using the artificial tear composition of the present invention.

Figure 5 is a diagram showing the use of the artificial tear composition of the present invention in the treatment of animals. The result of fluorescent staining of the ocular surface after dry eye syndrome, this figure needs to be represented by a color pattern.

Figure 6 is a graph showing changes in corneal epithelial cells and corneal thickness after treatment of moderate dry eye in animals using the artificial tear composition of the present invention.

Figure 7 is a comparison of the results of the artificial tear composition added with hyaluronic acid (HA) applied to the time of retention of the ocular surface of the animal. This figure is represented by a color pattern.

In order to facilitate the reviewer's understanding and understanding of the artificial tear composition of this case, the following examples are combined with the drawings, which are described in detail below.

Example 1

This embodiment provides a composition comprising a combination of an anti-inflammatory and anti-oxidant substance and a substance which increases the viscosity of the liquid to determine whether the composition will be human corneal epithelial cells (Human). Comeal Epithelial Cells (HCEC) have adverse effects on activity, whether they cause damage to human corneal epithelial cells (HCEC), and whether they can reduce the inflammatory response of human corneal epithelial cells (HCEC).

In the present embodiment, the anti-inflammatory and anti-oxidant substance is a gallocatechin gallate (EGCG), and the substance which increases the viscosity of the liquid is a hyaluronic acid (HA).

The gallocatechin gallate (EGCG) is used at a concentration of between about 1 μg/ml and 200 μg/ml; the hyaluronic acid (HA) is used in a volume percent concentration of between about 0.01% and 0.3%.

Cell activity assay

In this experiment, human corneal epithelial cells (HCEC) were cultured for 24 hours, and then the human corneal epithelial cells (HCEC) were induced by lipopolysaccharide (LPS) at a concentration of 500 ng/ml for 3 hours to produce the human corneal epithelial cells (HCEC). Inflammatory reaction, then add the aforementioned The composition of gallocatechin gallate (EGCG) and the hyaluronic acid (HA) was cultured for 3 days, and the cell activity experiment was performed for 5 days; the cells on the first day and the third day were collected, using conventional techniques. WST-8, with an enzyme-linked immunosorbent Assay (ELISA), measured the relative absorbance at a wavelength of 450 nm to measure cell viability.

The results are shown in Fig. 1A, in which the control group is the human corneal epithelial cell (HCEC) not induced by lipopolysaccharide (LPS), and the experimental group is added with the gallocatechin gallate ( EGCG) and the composition of hyaluronic acid (HA); as shown in Fig. 1A, there is no statistically significant difference in the relative absorbance values of the experimental group on the first day compared with the control group, and the experimental group and the control group on the third day There is also no statistically significant difference in relative absorbance values, so the composition does not adversely affect cell viability.

Cytotoxicity test

In this experiment, human corneal epithelial cells (HCEC) were cultured for 24 hours, and then the human corneal epithelial cells (HCEC) were induced by lipopolysaccharide (LPS) at a concentration of 500 ng/ml for 3 hours to produce the human corneal epithelial cells (HCEC). The inflammatory reaction was further carried out by adding the above-mentioned composition of gallocatechin gallate (EGCG) and the hyaluronic acid (HA) for 3 days, and the cytotoxicity test was carried out for 5 days; the first day and the third day were collected. The cells were assayed for cytotoxicity by measuring the relative absorbance at a wavelength of 490 nm using the conventional technique LDH in conjunction with the enzyme-linked immunosorbent assay (ELISA).

The results are shown in Fig. 1B, in which the cell lysis group is a negative control group, and the control group is a human corneal epithelial cell (HCEC) not induced by lipopolysaccharide (LPS). The composition is a combination of the gallocatechin gallate (EGCG) and the hyaluronic acid (HA); as shown in the first panel, the first day of the experimental group and the control group There was no statistically significant difference in relative absorbance values. There was no statistically significant difference in the relative absorbance values between the experimental group and the control group on day 3, so the composition did not cause damage to the cells.

Inflammatory factor gene expression experiment

In this experiment, human corneal epithelial cells (HCEC) were cultured for 48 hours, and then the human corneal epithelial cells (HCEC) were induced by lipopolysaccharide (LPS) at a concentration of 500 ng/ml for 3 hours to produce the human corneal epithelial cells (HCEC). The inflammatory reaction is further carried out by adding the above-mentioned composition of gallocatechin gallate (EGCG) and the hyaluronic acid (HA) for 2 hours, collecting the cells, and first extracting the ribonucleic acid of the cells with a phenol reagent (Trizol reagent). RNA), followed by reverse transcription polymerization (RT-PCR) using the High-Capacity cDNA Reverse Transcription Kits manufactured by the manufacturer (Applied Biosystems, ABI) to generate complementary deoxyribonucleic acid (cDNA), and then using the TagMan produced by the manufacturer (ABI). ® Fast Universal Master Mix (2X) for the detection of inflammatory factors by Quantitative Real-Time PCR (Q-PCR) for inflammatory factors (IL-1β, IL-6, IL-8 and TNF-α) Gene expression of (IL-1β, IL-6, IL-8, and TNF-α).

The results are shown in Fig. 2. The control group was the human corneal epithelial cells (HCEC) not induced by lipopolysaccharide (LPS), and only the hyaluronic acid (HA) was added to the hyaluronic acid (HA). In order to add the composition of the gallocatechin gallate (EGCG) and the hyaluronic acid (HA); as shown in Fig. 2, the relative transcription rate of the four inflammatory factors in the experimental group (relation) The transcription ratio was lower than the other two groups, indicating a decrease in the expression of inflammatory factors, and the relative transcription rates of the four inflammatory factors added with the hyaluronic acid (HA) were higher than those of the other two groups, and the hyaluronic acid (HA) reduced inflammation. The factor has no effect, thus confirming that the gallocatechin gallate (EGCG) has an effect of reducing the inflammatory response.

The WST-8, LDH, RT-PCR and Q-PCR in the foregoing examples are experimental kits and are conventional techniques, and the ribonucleic acid (RNA) extracted from cells by using a phenol reagent (Trizol reagent) is also a conventional technique. Therefore, it will not be repeated in this manual.

Example 2

This experiment is aimed at whether the mixed gallocatechin gallate (EGCG) and the hyaluronic acid (HA) composition are the same as the physiological characteristics of the human ocular surface, such as pH, osmotic pressure and viscosity.

In this experiment, the pH meter was used separately, and the brand was EUTECH INSTRUMENTS model pH510; the micro osmometer was used as the Advanced Instruments model 3320; the program rheometer device (detecting viscosity) was adopted. The label is a microcomputer program rheometer device of the Brookfield model DV III.

The results are shown in Table 3 below. The composition of the gallocatechin gallate (EGCG) and the hyaluronic acid (HA) has physiological properties similar to those of normal human tear fluid.

The pH meter, the micro osmometer and the program rheometer device in the foregoing embodiments are experimental instruments and are conventional techniques, and therefore, they are not described in the specification.

Example 3

The present invention provides a composition which is composed of the anti-inflammatory and anti-oxidant substance, the substance which increases the viscosity of the liquid, and an artificial tear liquid. The artificial tear composition of the invention is tested for efficacy test for treating dry eye with the artificial tear composition of the present invention.

In the present experiment, the anti-inflammatory and anti-oxidant substance is the gallocatechin gallate (EGCG), and the substance which increases the viscosity of the liquid is the hyaluronic acid (HA).

The gallic acid gallate (EGCG) is used at a concentration of about 1 μg/ml to 200 μg/ml; the hyaluronic acid (HA) is used in a concentration of hyaluronic acid contained in commercially available artificial tears. The percentage concentration is about 0.01% to 0.3%; the artificial tears used in this embodiment do not contain the hyaluronic acid (HA) and the preservative, and the composition thereof comprises a sodium chloride, potassium chloride, calcium chloride and Sodium dihydrogen phosphate, etc.

Lacrimal gland secretion test (Schirmer test)

This experiment first used Benzalkonium Chloride (BAC) to instill into the animal's eyes (in this experiment, the animal is a large white rabbit) 3 times a day for 3 weeks, causing the white rabbit eyes to become inflamed, resulting in Symptoms of moderate dry eye, followed by artificial tears, hyaluronic acid (HA) and gallocatechin gallate (EGCG) and other different compositions for treatment, the course of treatment is 2 times a day, a total of 2 week.

The lacrimal gland secretion test is a method for measuring the amount of tears in clinically tested dry eye patients, and is to place a test paper on the lower eyelid to observe the amount of tears by capillary phenomenon.

The results are shown in Fig. 3, which are respectively a control group, which is a group with no induced dry eye and a dry eye group. The dry eye group is a moderate dry eye induced by the hydroxychloroaniline (BAC). The artificial tear treatment group 1 was used, and the hyaluronic acid (HA) treatment group was added with the artificial tears. 2. The artificial tear was added to the gallocatechin gallate (EGCG) treatment. Group 3 and a treatment group 4 using the artificial tear composition of the present invention; as shown in Figure 3, After treatment of the artificial tear composition of the present invention, the artificial tear composition was used to treat group 4, and the tear content was restored to a normal value.

Inflammatory factor gene expression experiment

This experiment first used the monohydroxychloroaniline (BAC) to drip into the eyes of the white rabbit three times a day for 3 weeks, induced the white rabbit eyes to become inflamed, causing symptoms of moderate dry eye syndrome, and then using artificial tears, hyaluronic acid Different compositions such as (HA) and gallocatechin gallate (EGCG) were treated for 2 days, for 2 weeks; then the protein of the cornea of the rabbit was extracted for enzyme-linked immunology. The adsorption method (ELISA) is to test the gene expression of inflammatory factors (IL-1β, IL-6, IL-8 and TNF-α), and to observe whether the inflammatory factors are artificial tears or hyaluronic acid added by the above groups. The treatment with different compositions such as (HA) and gallocatechin gallate (EGCG) decreased.

The results are shown in Fig. 4, which are respectively a control group, which is a group without induced dry eye and a dry eye group. The dry eye group is a moderate dry eye induced by the hydroxychloroaniline (BAC). In the treatment group 1, the artificial tears were added to the hyaluronic acid (HA) treatment group 2, and the artificial tear was added to the gallate catechin gallate (EGCG) treatment group 3 And treating the group 4 with the artificial tear composition; as shown in the fourth figure, the artificial tear composition of the present invention is treated with the group 4 and the artificial tear is added to the gallocatechin gallate. (EGCG) treatment group 3, after treatment, each of the inflammatory factors (IL-1β, IL-6, IL-8 and TNF-α) decreased significantly and was similar to the concentration of the control group, and the artificial tear treatment group 1 and The artificial tears were added to the hyaluronic acid (HA) treatment group 2, and the concentration of each of the inflammatory factors (IL-1β, IL-6, IL-8, and TNF-α) was not significantly decreased after treatment, indicating that the gallic catechin Gallic acid ester (EGCG) has the effect of reducing the expression of each of these inflammatory factors (IL-1β, IL-6, IL-8 and TNF-α).

Eyeball fluorescent staining experiment (Fluorescein Staining)

This experiment firstly used monohydroxychloroaniline (BAC) to drip into the eyes of a white rabbit three times a day for 3 weeks, inducing inflammation of the white rabbit's eyes, causing symptoms of moderate dry eye, and then using the present invention. The artificial tear composition was treated twice a day for 2 weeks; then, the white rabbit eyes were subjected to a fluorescent staining experiment.

The results are shown in Fig. 5, in which the control group was no induced dry eye syndrome, the dry eye group was induced by the hydroxychloroaniline (BAC), and the treatment group was the artificial tear combination using the present invention. Treatment of the substance; as shown in Fig. 5, in the treatment group using the artificial tear composition of the present invention, the eyes recovered after treatment without chaos or spots.

Eye corneal epithelial cell section staining experiment

This experiment firstly used monohydroxychloroaniline (BAC) to drip into the eyes of a white rabbit three times a day for 3 weeks, inducing inflammation of the white rabbit's eyes, causing symptoms of moderate dry eye, and then using the present invention. The artificial tear composition was treated twice a day for 2 weeks; the corneal epithelial cells of the animal eye were first sectioned, followed by hematoxylin-eosin staining (ie, HE staining), and observed with a microscope at 10 times eyepiece.

In rabbits with moderate dry eye, the corneal epithelial cells are inflamed. The thickness of the epithelial cell layer of the original 3-5 layers will become thinner and become 1-3 layers, and the overall thickness of the cornea will become thinner. See page 6 for results. As shown in the figure, the control group is non-induced dry eye syndrome, the dry eye group is induced by the use of the hydroxychloroaniline (BAC), and the treatment group is treated with the artificial tear composition of the present invention; As can be seen from Fig. 6, in the treatment group of the artificial tear composition of the present invention, the thickness of the epithelial cell arrangement after treatment was restored to the normal thickness, and the overall corneal thickness also returned to normal.

Artificial tear ocular surface retention time experiment

In this experiment, an artificial tear containing only the gallocatechin gallate (EGCG) and the artificial tear composition of the present invention were simultaneously dropped into the eye of the animal (in this experiment, the animal is a mouse) After 15 minutes, a non-invasive 3D in vivo molecular imaging system (labeled Xenoge) was used to observe the retention time of artificial tears on the ocular surface of the mouse.

The results are shown in Fig. 7, wherein the artificial tears are only added with gallocatechin gallate (EGCG) group A and one is the artificial tear composition using the present invention (ie, contains Hyaluronic acid (HA)) group B; as shown in Fig. 7, the artificial tear composition of the present invention (i.e., containing hyaluronic acid (HA)) group B remains after 15 minutes of instillation into the ocular surface, and In the artificial tears, only the gallocatechin gallate (EGCG) group A was added, and the phenomenon of retention of the medicated water could not be observed. Therefore, the artificial tear composition of the present invention (that is, containing hyaluronic acid (HA)) was not observed. Group B has the addition of the hyaluronic acid (HA), which can increase the viscosity of the liquid (that is, the artificial tear composition of the present invention), so that the liquid stays in the ocular surface for a long time, thereby effectively reducing the number of repeated administrations, and It can shorten the course of treatment for ocular surface inflammation and dry eye syndrome.

In the foregoing embodiments, the lacrimal gland secretion test, ELISA, eyeball fluorescence staining, and hematoxylin-eosin staining are experimental kits and are conventional techniques, and the non-invasive 3D living molecular imaging system is a conventional instrument, and therefore, no longer This description has been added.

The detailed description of the preferred embodiments of the present invention is not intended to limit the scope of the present invention, and the equivalent implementations or modifications of the present invention should be included in the present invention. In the scope of patents.

Claims (6)

An artificial tear composition mainly consisting of a combination of hyaluronic acid gallate (EGCG) having an anti-inflammatory substance, hyaluronic acid (HA) which can increase the viscosity of a liquid, and an artificial tear liquid The artificial tear composition has a pH ranging from pH 7 to pH 8.5. The artificial tear composition according to claim 1, wherein the artificial tear component comprises sodium chloride, potassium chloride, calcium monochloride and sodium dihydrogen phosphate. The artificial tear composition of claim 1, wherein the gallocatechin gallate (EGCG) concentration is in the range of 1 μg/ml to 200 μg/ml. The artificial tear composition according to claim 1, wherein the gallocatechin gallate (EGCG) is a catechin component in green tea. The artificial tear composition of claim 1, wherein the hyaluronic acid (HA) concentration is in the range of 0.01% to 0.3% by volume. The artificial tear composition according to claim 1, wherein the composition can treat moderate dry eye, relieve inflammation of the ocular surface of the patient, restore normal tear value, and repair the cornea.