Small molecule compound composition and application thereof

Abstract

The invention relates to a small molecule compound composition and application thereof, and belongs to the technical field of biology. The small molecule compound composition provided by the invention comprises NU7441, GW9662 and D609 or stereoisomers thereof. It is found for the first time that the small molecule compound combination can effectively inhibit oxidative damage caused by hypertonic to corneal epithelial cells, and the survival rate of the corneal epithelial cells is remarkably increased. Animal in-vivo experiments show that compared with a control group, an experimental group which is subjected to injection of scopolamine dry eye model mice and is subjected to eye drop of the small molecule compound combination eye drop at the same time has the advantages that corneal defects are obviously reduced, tear secretion is increased, the small molecule compound combination can effectively prevent corneal epithelium injury, the drug effect is relatively high, and no obvious drug toxicity exists; the small molecule compound combination may become a strong and effective medicine for clinically treating dry eye in the future.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a small molecule compound composition and application thereof. Background Art

[0002] Dry eye disease (DED) is a common ocular surface disease that affects vision and quality of life. It is a multifactorial chronic disease. Abnormal tear quality, quantity and dynamics lead to unstable tear film or imbalance of ocular surface microenvironment, which may be accompanied by ocular surface inflammatory damage, tissue damage and nerve abnormalities, resulting in a variety of ocular discomfort symptoms and visual dysfunction. The incidence of dry eye increases with the age of patients, and the incidence is higher in women, but its exact pathogenesis has not yet been elucidated. Epidemiological surveys show that the global incidence of dry eye is about 5-30%, while the incidence in my country is about 21-30%. With the aging of the population and the development of terminal devices such as the Internet and mobile phones, the incidence of dry eye has increased year by year. Common symptoms of dry eye patients include dry eyes, itchy eyes, pain, visual fatigue, and visual fluctuations. In severe cases, it can affect activities such as reading, entertainment, and driving, resulting in a significant decrease in vision-related quality of life. Therefore, relieving clinical symptoms, protecting visual function, and removing the cause as much as possible are the current goals of dry eye treatment.

[0003] Currently, the main drug treatments for dry eyes are divided into three categories: lubricating the ocular surface to promote repair, anti-inflammatory, and antibacterial. Artificial tears are currently the first-line medication for the treatment of dry eyes. They can simulate one or more components of the tear film and play a directional supplementary role. Studies have shown that inflammation is one of the core causes of dry eyes. Anti-inflammatory treatments are used to relieve ocular inflammation, including glucocorticoids, nonsteroidal anti-inflammatory drugs, and immunosuppressants. When dry eyes related to Demodex or anaerobic infection occur, topical antibacterial drugs such as metronidazole can be used.

[0004] Artificial tears are currently the first-line medication for the treatment of dry eyes, but their main effect is to lubricate the ocular surface. They are essentially symptomatic treatments and require long-term use, and cannot eliminate the disease from the root. With the exploration and discovery of the pathogenesis of dry eyes, ocular surface inflammation is one of the core links in the pathogenesis of dry eyes. Therefore, anti-inflammatory drugs such as glucocorticoids are used in the treatment of dry eyes. However, while controlling ocular surface inflammation, they may cause side effects such as increased intraocular pressure and lens opacity. Therefore, it is particularly important to find new therapeutic drugs for DED. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a small molecule compound composition and application, wherein the composition has the efficacy of treating dry eye.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a small molecule compound composition, which comprises NU7441, GW9662 and D609 or stereoisomers thereof.

[0008] NU7441 is a DNA-PK inhibitor that can also inhibit mTOR and PI3K. It has a protective effect on cells by reducing the frequency of non-homologous end joining (NHEJ) and enhancing the homologous recombination repair rate after Cas9-mediated DNA splicing; GW9662 is a selective PPARγ antagonist that can reduce the increased expression of inflammatory factors IL-1B and IL-6 induced by LPS, and has a certain anti-inflammatory effect; D609 is a selective inhibitor of phosphatidylcholine-specific phospholipase (PC-PLC) that has an antagonistic effect on oxidative stress and cell ferroptosis.

[0009] The inventors have found through a large number of combination studies and screening tests that these three small molecule compound combinations have the effect of synergistic treatment of dry eye, and the dosage is low. These three small molecule compound combinations can effectively antagonize the cell iron death and dysfunction caused by the oxidative stress suffered by corneal epithelial cells, and it can effectively protect corneal epithelial cells and then protect the structural integrity and normal function of the ocular surface. After extensive and in-depth research, the inventors first found that the small molecule compound combination can effectively inhibit the occurrence of cell iron death by inhibiting the accumulation of intracellular iron ions and oxidative stress, and has a protective effect on corneal epithelial cells under hypertonic conditions, which can be used for the prevention and potential treatment of ocular surface diseases caused by oxidative stress, especially beneficial to the prevention and / or treatment of dry eye, and the effect is better than the three molecules alone. In addition, the present invention has verified through experiments that the small molecule compound combination has no obvious pharmacological toxicity, and can effectively control the occurrence and development of dry eye, providing new theoretical support for the development of unknown biological activity of such small molecule compound combinations and future clinical therapeutic effects.

[0010] As a preferred embodiment of the first aspect, the effective dose of NU7441 in the small molecule compound composition is 1-20 μg / kg; the effective dose of GW9662 is 1-20 μg / kg; the effective dose of D609 is 1-20 μg / kg. Since NU7441, GW9662 and D609 have certain synergistic effects, a lower effective dose can be used to exert their effect of treating or preventing dry eye.

[0011] As a preferred embodiment of the first aspect, the effective dose of NU7441 can also be 1 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg or any numerical range therein; the effective dose of GW9662 can also be 1 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg or any numerical range therein; the effective dose of D609 can also be 1 μg / kg, 5 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg or any numerical range therein.

[0012] As a preferred embodiment of the first aspect, in the small molecule compound combination, the effective dose of NU7441 is 5-15 μg / kg; the effective dose of GW9662 is 5-15 μg / kg; and the effective dose of D609 is 5-15 μg / kg.

[0013] As a preferred embodiment of the first aspect, the effective dose of NU7441 can also be 5μg / kg, 8μg / kg, 10μg / kg, 12μg / kg, 15μg / kg or any numerical range therein; the effective dose of GW9662 can also be 5μg / kg, 8μg / kg, 10μg / kg, 12μg / kg, 15μg / kg or any numerical range therein; the effective dose of D609 can also be 5μg / kg, 8μg / kg, 10μg / kg, 12μg / kg, 15μg / kg or any numerical range therein.

[0014] In a second aspect, the present invention provides a pharmaceutical composition for preventing and / or treating dry eye, the pharmaceutical composition comprising the small molecule compound composition described in the first aspect, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0015] As a preferred embodiment of the second aspect, the pharmaceutical composition further comprises an anti-dry eye damage protection drug.

[0016] As a preferred embodiment of the second aspect, the preparation form of the pharmaceutical composition includes one or more of eye drops, eye ointment or gel.

[0017] In a third aspect, the present invention provides use of the small molecule compound composition described in the first aspect or the pharmaceutical composition described in the second aspect in the preparation of a drug for preventing and / or treating dry eye.

[0018] Through the cell survival rate experiment detection, it can be known that the small molecule compound combination of the present invention can effectively inhibit the oxidative damage of corneal epithelial cells induced by hypertonic stress and significantly improve the survival rate of corneal epithelial cells. Although the three components in the compound combination all show a certain cell protective effect when treated alone, the effect of combined use is the best. This result reflects the powerful effect of the small molecule compound combination in antagonizing ferroptosis and protecting cells.

[0019] Animal experiments have shown that compared with the control group, the embodiment group of the small molecule compound combination eye drops of the present invention effectively protects corneal epithelial cells from oxidative damage caused by dryness stress, and greatly reduces corneal epithelial cell ferroptosis, so that the corneal epithelial cells remain intact and approach normal levels to the greatest extent. The results reflect the powerful antioxidant and anti-ferroptosis of the small molecule compound combination in vivo and its protective effect on the ocular surface; and the small molecule compound combination has no obvious drug toxicity and is highly safe.

[0020] As a preferred embodiment of the third aspect, when administered to animals, the effective dose of NU7441 in the small molecule compound composition is 1-20 μg / kg; the effective dose of GW9662 is 1-20 μg / kg; the effective dose of D609 is 1-20 μg / kg. Since NU7441, GW9662 and D609 have certain synergistic effects, a lower effective dose can be used to exert their effect of treating or preventing dry eye.

[0021] As a preferred embodiment of the third aspect, when administered to animals, the effective dose of NU7441 in the small molecule compound composition is 5-15 μg / kg; the effective dose of GW9662 is 5-15 μg / kg; and the effective dose of D609 is 5-15 μg / kg.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention achieves a synergistic technical effect by specifically combining the small molecule compounds NU7441, GW9662 and D609, and can use a lower dose to treat or prevent dry eye. The present invention discovered for the first time that the small molecule compound combination can effectively inhibit the oxidative damage to corneal epithelial cells caused by hypertonicity and significantly improve the survival rate of corneal epithelial cells. In addition, in vivo animal experiments showed that compared with the control group, the experimental group that was given small molecule compound combination eye drops while injecting scopolamine dry eye model mice had significantly reduced corneal defects and increased tear secretion. The small molecule compound combination can effectively prevent damage to the corneal epithelium, has a high efficacy, and has no obvious drug toxicity. The small molecule compound combination may become a strong and effective drug for the clinical treatment of dry eye in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the effect of small molecule compound combination on the protective effect of HCE-2 cells;

[0025] Figure 2 It is a schematic diagram of the experimental results of the survival rate of HCE-2 cells by various components and combinations of small molecule compounds;

[0026] Figure 3 This is a schematic diagram of the intracellular iron ion staining results;

[0027] Figure 4 This is a schematic diagram of the results of animal experiments - corneal fluorescein staining and tear secretion measurement;

[0028] Figure 5 Schematic diagram of the results of animal experiment - corneal TUNEL staining. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0030] The information of the small molecule compounds in this example is as follows:

[0031] NU7441: CAS No.: 503468-95-9, Molecular formula: C 25 H 19 NO3S; molecular weight: 413.49;

[0032] GW9662: CAS No.: 22978-25-2, Molecular formula: C 13 H9ClN2O3; molecular weight: 276.68;

[0033] D609: CAS No.: 83373-60-8, Molecular formula: C 11 H 15 KOS2, molecular weight: 266.46.

[0034] Example 1 Bright field photography

[0035] 1. Methods

[0036] 1. Experimental Materials and Procedures

[0037] 1.1 Preparation of cell culture medium: DMEM / F12 + 10% fetal bovine serum + 1% P / S double antibody (penicillin + streptomycin)

[0038] 1.2 Preparation of small molecule compound combinations:

[0039] NU7441: Dissolve NU7441 powder in ultrapure water to a final concentration of 30 μg / mL;

[0040] GW9662: Dissolve GW9662 powder in ultrapure water to a final concentration of 30 μg / mL;

[0041] D609: Dissolve D609 powder in ultrapure water to a final concentration of 30 μg / mL;

[0042] The above small molecule compound mother solutions were mixed in equal volumes to obtain a small molecule compound composition. The concentrations of the components after mixing were: NU7441: 10 μg / mL, GW9662: 10 μg / mL, and D609: 10 μg / mL.

[0043] 1.3 Preparation of hypertonic solution: Dissolve sodium chloride with ultrapure water to a final concentration of 0.94 M, then dilute with culture medium to a final concentration of 94 mM (osmotic pressure 500 mOSM).

[0044] 1.4 Treat cells:

[0045] Corneal epithelial cell line HCE-2 cells purchased from ATCC were cultured in DMEM / F12+10% fetal bovine serum+1% P / S dual antibody culture medium and divided into negative control group, model control group (hypertonic), experimental group: hypertonic + small molecule compound combination co-treatment group. The cells were cultured in a 37°C, 5% CO2 incubator to observe cell changes and perform corresponding molecular biological tests.

[0046] 2. Bright field photography

[0047] 2.1 Selection of photography time point: After the cells are treated according to the above method, use a microscope to take pictures of the cells in the six-well plate to observe and record the changes in cell number and morphology. Select the time point after treatment when the changes between cell groups are more obvious as the recording time point.

[0048] 2.2 Photographic instrument: Leica DMIL inverted biological microscope was used for photography.

[0049] 2. Results

[0050] The results are as follows Figure 1 The treatment of corneal epithelial cells with a hypertonic solution of sodium chloride is a mature dry eye model and is widely used in experiments. Figure 1 Compared with the negative control group, the number of HCE-2 cells in the hyperosmotic group after 24h administration was significantly reduced, indicating that hyperosmotic pressure can induce HCE-2 cell damage and the model was successfully established in this experiment. Compared with the hyperosmotic group, the number of HCE-2 cells in the experimental group (hyperosmotic pressure + small molecule compound combination) increased significantly, indicating that the small molecule compound combination can effectively protect the oxidative damage of corneal epithelial cells caused by hyperosmotic pressure and improve the survival rate of corneal epithelial cells.

[0051] Example 2 Cell viability experiment (CCK8 staining)

[0052] 1. Methods

[0053] 1. Experimental Materials and Procedures

[0054] 1.1 Preparation of cell culture medium: DMEM / F12 + 10% fetal bovine serum + 1% P / S double antibody (penicillin + streptomycin);

[0055] 1.2 Preparation of small molecule compound combinations:

[0056] NU7441: Dissolve NU7441 powder in ultrapure water to a final concentration of 30 μg / mL.

[0057] GW9662: Dissolve GW9662 powder in ultrapure water to a final concentration of 30 μg / mL.

[0058] D609: Dissolve D609 powder in ultrapure water to a final concentration of 30 μg / mL.

[0059] The three small molecule compound stock solutions were mixed in equal volumes to form a small molecule compound combination, and the final concentration of each component of the small molecule compound was 10 μg / mL.

[0060] 1.3 Preparation of hypertonic solution: Dissolve sodium chloride with ultrapure water to a final concentration of 0.94 M, then dilute with culture medium to a final concentration of 94 mM (osmotic pressure 500 mOSM).

[0061] 1.4 Cell preparation: HCE-2 cells, a corneal epithelial cell line purchased from ATCC, were inoculated into 96-well plates, with 10,000 cells per well, and cultured for 24 hours. The cells were divided into a negative control group, a model control group (hypertonic), experimental group 1: hypertonic + NU7441, experimental group 2: hypertonic + GW9662, experimental group 3: hypertonic + D609, and experimental group 4: hypertonic + small molecule compound combination. The cells were incubated for 24 hours, and the cell changes were observed and the following experiments were carried out.

[0062] 1.5 Prepare CCK8 staining solution: Cell culture medium and CCK8 staining stock solution are prepared in a ratio of 10:1.

[0063] 1.6 After the cells were treated with drugs for a specific time point, the supernatant was removed, 100 μL of dye solution was added to each well, and the cells were placed in a 37°C incubator for 1 hour.

[0064] 1.7 Use an ELISA reader to measure the absorbance of each well at 450 nm for comparison.

[0065] Survival rate calculation formula:

[0066] Cell survival rate % = [A (drug added) - A (blank)] / [A (negative control) - A (blank)]

[0067] 2. Results

[0068] The results are as follows Figure 2 As shown. From the results of Example 1 above, it can be seen that the composition composed of NU7441, GW9662 and D609 can effectively protect the oxidative damage of corneal epithelial cells caused by hypertonicity and improve the survival rate of corneal epithelial cells. Compared with hypertonic treatment, although each small molecule + hypertonicity can increase the cell CCK8 level to a certain extent, the hypertonicity + small molecule compound combination co-treatment group most effectively increases the cell CCK8 level. It shows that the combination of small molecule compounds can effectively inhibit the oxidative damage of HCE-2 cells caused by hypertonicity and significantly improve the survival rate of HCE-2 cells.

[0069] Example 3 Intracellular iron staining

[0070] 1. Methods

[0071] 1. Experimental Materials and Procedures

[0072] 1.1 Preparation of cell culture medium: DMEM / F12+10% fetal bovine serum+1% P / S double antibody

[0073] 1.2 Preparation of small molecule compound combinations:

[0074] NU7441: Dissolve NU7441 powder in ultrapure water to a final concentration of 30 μg / mL.

[0075] GW9662: Dissolve GW9662 powder in ultrapure water to a final concentration of 30 μg / mL.

[0076] D609: Dissolve D609 powder in ultrapure water to a final concentration of 30 μg / mL.

[0077] The small molecule compound combination is obtained by mixing equal volumes of three small molecule compound mother solutions.

[0078] 1.3 Preparation of hypertonic solution: Dissolve sodium chloride with ultrapure water to a final concentration of 0.94 M, then dilute with culture medium to a final concentration of 94 mM (osmotic pressure 500 mOSM).

[0079] 1.4 Prepare cells: HCE-2 cells, a corneal epithelial cell line purchased from ATCC, were inoculated into 12-well plates, with 50,000 cells per well, and cultured for 24 hours. The cells were divided into a negative control group, a model control group (hypertonicity), and an experimental group: hypertonicity + small molecule compound combination. After incubation for 24 hours, the cell changes were observed and the following experiments were performed.

[0080] 1.5 Prepare intracellular iron ion staining solution: Cell culture medium and iron ion staining stock solution are prepared in a ratio of 4000:1.

[0081] 1.6 After the cells were treated with drugs for a specific time point, the supernatant was removed, 500 μL of dye solution was added to each well, and the cells were placed in a 37°C incubator for 30 minutes.

[0082] 1.7 Direct observation under microscope.

[0083] (2) Results

[0084] The results are as follows Figure 3 Compared with the hypertonic treatment, the small molecule compound combination can effectively reduce the intracellular iron ion level. This shows that the small molecule compound combination can effectively inhibit the ferroptosis of HCE-2 cells caused by hypertonicity and significantly improve the survival rate of HCE-2 cells.

[0085] Example 4 Animal Experiment - Corneal Fluorescein Staining and Tear Secretion Measurement

[0086] (1) Methods

[0087] 1. Animal Injection

[0088] 1.1 The injected animals were 6-8 week old male C57 / BL6 mice. The experiment was divided into: negative control group, model control group (scopolamine), experimental group (scopolamine + small molecule compound combination co-treatment group (NU7441 concentration was 8 μg / kg; GW9662 concentration was 8 μg / kg; D609 concentration was 12.5 μg / kg)).

[0089] 1.2 Scopolamine injection was performed by subcutaneous injection: Scopolamine was dissolved in PBS, and the injection dose for each animal was 1.5 mg / 0.3 ml, 3 times a day for 5 consecutive days.

[0090] 1.3 The small molecule compound combination group was administered with eye drops: each small molecule was dissolved in PBS to prepare a mother solution and then diluted to equal volume. The final concentration for each animal was calculated based on the animal weight. The concentration of NU7441 was 8 μg / kg; the concentration of GW9662 was 8 μg / kg; and the concentration of D609 was 12.5 μg / kg.

[0091] 1.4 After injection, the mice were subjected to corneal sodium fluorescein staining on the designated date, and the corneal epithelial staining was observed under a slit lamp microscope.

[0092] 1.5 After injection, the mice were subjected to the phenol red cotton thread test to measure the tear secretion on the designated date. The phenol red cotton thread was clamped with micro forceps and placed on the outer canthus of both eyes of the mice. After 15 seconds, the length of the moistened and reddened part of the phenol red cotton thread was measured as the estimated value of the tear volume (in millimeters).

[0093] (2) Results

[0094] like Figure 4 As shown, subcutaneous injection of scopolamine in dry eye model mice can significantly increase corneal epithelial defects and reduce tear secretion. Animal experiments in vivo showed that compared with the model control group, the small molecule compound can effectively prevent corneal epithelial cell damage and restore tear secretion in the experimental group of dry eye model mice, without obvious drug toxicity.

[0095] Example 5 Animal experiment - corneal TUNEL staining

[0096] 1. Animal Injection

[0097] 1.1 The injected animals were 6-8 week old male C57 / BL6 mice. The experiment was divided into: negative control group, model control group (scopolamine), experimental group (scopolamine + small molecule compound combination co-treatment group (NU7441 concentration was 8 μg / kg; GW9662 concentration was 8 μg / kg; D609 concentration was 12.5 μg / kg)).

[0098] 1.2 Scopolamine injection was performed by subcutaneous injection: Scopolamine was dissolved in PBS, and the injection dose for each animal was 1.5 mg / 0.3 ml, 3 times a day for 5 consecutive days.

[0099] 1.3 The small molecule compound combination was administered by eye drops: the small molecules were dissolved in PBS to prepare a mother solution and then diluted to equal volumes. The final concentrations for each animal were calculated based on the animal weight: NU7441 concentration was 8 μg / kg; GW9662 concentration was 8 μg / kg; and D609 concentration was 12.5 μg / kg.

[0100] 1.4 The injected mice were killed by cervical dislocation on the designated date, and the eyeballs were dissected to prepare corneal tissue paraffin sections, followed by TUNEL immunofluorescence staining.

[0101] (2) Results

[0102] like Figure 5 As shown, subcutaneous injection of scopolamine can significantly increase corneal epithelial cell death in dry eye model mice. In vivo animal experiments have shown that small molecule compounds can effectively reduce corneal epithelial cell death in the experimental group of dry eye model mice compared with the model control group.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A small molecule compound composition, characterized in that: The small molecule compound composition includes NU7441, GW9662 and D609 or stereoisomers thereof.

2. The small molecule compound composition according to claim 1, characterized in that: The effective dose of NU7441 in the small molecule compound combination is 1-20 μg / kg; the effective dose of GW9662 is 1-20 μg / kg; and the effective dose of D609 is 1-20 μg / kg.

3. The small molecule compound composition according to claim 2, characterized in that: The effective dose of NU7441 in the small molecule compound combination is 5-15 μg / kg; the effective dose of GW9662 is 5-15 μg / kg; and the effective dose of D609 is 5-15 μg / kg.

4. A pharmaceutical composition for preventing and / or treating dry eye, characterized in that: The pharmaceutical composition comprises the small molecule compound composition according to any one of claims 1 to 3, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition also includes an anti-dry eye damage protection drug.

6. The pharmaceutical composition according to claim 4 or 5, characterized in that The pharmaceutical composition may be in the form of one or more of eye drops, eye ointments or gels.

7. Use of the small molecule compound composition according to any one of claims 1 to 3 or the pharmaceutical composition according to any one of claims 4 to 6 in the preparation of a drug for preventing and / or treating dry eye.

8. The use according to claim 7, characterized in that When administered to animals, the effective dose of NU7441 in the small molecule compound composition is 1-20 μg / kg; the effective dose of GW9662 is 1-20 μg / kg; and the effective dose of D609 is 1-20 μg / kg.

9. The use according to claim 8, characterized in that When administered to animals, the effective dose of NU7441 in the small molecule compound composition is 5-15 μg / kg; the effective dose of GW9662 is 5-15 μg / kg; and the effective dose of D609 is 5-15 μg / kg.

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