A small molecule compound composition and its application

Abstract

This invention relates to a small molecule compound composition and its applications, belonging to the field of biotechnology. The small molecule compound composition of this invention includes NU7441, GW9662, and D609, or their stereoisomers. This invention is the first to discover that this small molecule compound combination can effectively inhibit oxidative damage to corneal epithelial cells caused by hyperosmolarity and significantly improve the survival rate of corneal epithelial cells. In vivo animal experiments show that, compared with the control group, the experimental group of mice injected with scopolamine-induced dry eye model mice, given eye drops containing the small molecule compound combination, showed significantly reduced corneal defects and increased tear secretion. The small molecule compound combination can effectively prevent corneal epithelial damage, has high efficacy, and no obvious drug toxicity. This small molecule compound combination may become a potent and effective drug for the clinical treatment of dry eye in the future.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to a small molecule compound composition and its application. Background Technology

[0002] Dry eye disease (DED) is a common ocular surface disorder affecting vision and quality of life. It is a multifactorial chronic disease caused by abnormalities in the quality, quantity, and dynamics of tears, leading to tear film instability or an imbalance in the ocular surface microenvironment. This can be accompanied by ocular surface inflammation, tissue damage, and neurological abnormalities, resulting in various uncomfortable ocular symptoms and visual impairment. The incidence of dry eye increases with age, with a higher incidence in women, but its exact pathogenesis remains unclear. Epidemiological surveys show a global incidence of approximately 5-30%, while the incidence in my country is approximately 21-30%, and this incidence is increasing annually with population aging and the development of internet and mobile devices. Common symptoms of dry eye include dryness, itching, pain, eye strain, and fluctuating vision. Severe cases can affect reading, entertainment, driving, and other activities, resulting in a significant decline in vision-related quality of life. Therefore, alleviating clinical symptoms, protecting visual function, and eliminating the underlying cause are the current goals of dry eye treatment.

[0003] Currently, the main drug treatments for dry eye fall into three categories: lubricating the ocular surface to promote repair, anti-inflammatory, and antibacterial. Artificial tears are currently the first-line treatment for dry eye, mimicking one or more components of the tear film to provide targeted replenishment. Studies have shown that inflammation is one of the core pathogenic factors of dry eye; anti-inflammatory treatment is used to relieve ocular inflammation, including corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), and immunosuppressants. When dry eye is associated with Demodex mites or anaerobic bacterial infections, topical antibacterial drugs such as metronidazole can be used.

[0004] Artificial tears are currently the first-line treatment for dry eye, but their main effect is to lubricate the ocular surface, essentially treating the symptoms and requiring long-term use, unable to eliminate the disease at its root. With the exploration and discovery of the pathogenesis of dry eye, ocular surface inflammation is one of the core components. Therefore, anti-inflammatory drugs such as corticosteroids are used in the treatment of dry eye. However, while controlling ocular surface inflammation, they may cause side effects such as increased intraocular pressure and lens opacity. Therefore, finding new treatments for dry eye (DED) has become particularly important. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a small molecule compound composition and its application, which has the effect of treating dry eye syndrome.

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

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

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

[0009] Through extensive combination studies and screening experiments, the inventors discovered that this combination of three small molecule compounds exhibits synergistic therapeutic effects for dry eye syndrome, requiring only a low dosage. This combination effectively antagonizes ferroptosis and functional abnormalities in corneal epithelial cells caused by oxidative stress, effectively protecting corneal epithelial cells and thus preserving the structural integrity and normal function of the ocular surface. Through extensive and in-depth research, the inventors have, for the first time, discovered that this combination of small molecule compounds effectively inhibits ferroptosis by suppressing intracellular iron ion accumulation and oxidative stress, providing protection for corneal epithelial cells under hyperosmolar conditions. It can be used for the prevention and potential treatment of ocular surface diseases caused by oxidative stress, particularly beneficial for the prevention and / or treatment of dry eye syndrome, with better efficacy than any of the three molecules individually. Furthermore, the invention experimentally verified that the combination of small molecule compounds has no significant pharmacological toxicity and can effectively control the occurrence and development of dry eye syndrome, providing new theoretical support for exploring the unknown biological activities and future clinical therapeutic effects of this combination of small molecule compounds.

[0010] In 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; and the effective dose of D609 is 1-20 μg / kg. Because NU7441, GW9662, and D609 have a certain synergistic effect, a relatively low effective dose can be used to exert their therapeutic or preventative effects on dry eye syndrome.

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

[0012] In a preferred embodiment of the first aspect, the effective dose of NU7441 in the combination of small molecule compounds 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 may also be 5 μg / kg, 8 μg / kg, 10 μg / kg, 12 μg / kg, 15 μg / kg or any value range thereof; the effective dose of GW9662 may also be 5 μg / kg, 8 μg / kg, 10 μg / kg, 12 μg / kg, 15 μg / kg or any value range thereof; the effective dose of D609 may also be 5 μg / kg, 8 μg / kg, 10 μg / kg, 12 μg / kg, 15 μg / kg or any value range thereof.

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

[0015] As a preferred embodiment of the second aspect, the pharmaceutical composition further includes a protective agent against dry eye damage.

[0016] As a preferred embodiment of the second aspect, the pharmaceutical composition is formulated in one or more of the following forms: eye drops, eye ointment, or gel.

[0017] Thirdly, the present invention provides the 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 medicament for the prevention and / or treatment of dry eye syndrome.

[0018] Cell survival rate experiments showed that the combination of small molecule compounds in this invention can effectively inhibit the oxidative damage of corneal epithelial cells to hyperosmolar stress and significantly improve the survival rate of corneal epithelial cells. Although the three components in the compound combination all showed certain cell protection effects when used individually, the combined use had the best effect. This result reflects the powerful antagonistic effect of the small molecule compound combination on ferroptosis and cell protection.

[0019] Animal experiments have shown that, compared with the control group, the small molecule compound combination eye drops of the present invention effectively protect corneal epithelial cells from oxidative damage caused by dryness stress and greatly reduce corneal epithelial cell ferroptosis, keeping corneal epithelial cells intact and approaching normal levels as much as possible. The results demonstrate the strong antioxidant and antiferroptosis effects of the small molecule compound combination in vivo, as well as its protective effect on the ocular surface; moreover, the small molecule compound combination has no obvious drug toxicity and has high safety.

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

[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 beneficial effects of the present invention are as follows:

[0023] This invention achieves a synergistic effect by specifically combining small molecule compounds NU7441, GW9662, and D609, enabling the treatment or prevention of dry eye at lower doses. This invention is the first to discover that this combination of small molecule compounds can effectively inhibit oxidative damage to corneal epithelial cells caused by hyperosmolarity, significantly improving corneal epithelial cell survival. Furthermore, in vivo animal experiments show that, compared to the control group, mice injected with scopolamine-induced dry eye showed significantly reduced corneal defects and increased tear secretion in the experimental group. The small molecule compound combination effectively prevents corneal epithelial damage, exhibits high efficacy, and has no significant drug toxicity. This combination of small molecule compounds may become a potent and effective drug for the clinical treatment of dry eye in the future. Attached Figure Description

[0024] Figure 1 A schematic diagram showing the effect of small molecule compound combinations on the protective effect of HCE-2 cells;

[0025] Figure 2 A schematic diagram showing the experimental results of the survival rate of HCE-2 cells for each component and combination of small molecule compounds.

[0026] Figure 3 A schematic diagram showing the results of intracellular iron staining.

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

[0028] Figure 5 This is a schematic diagram of the results of corneal TUNEL staining in animal experiments. Detailed Implementation

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

[0030] The information on the small molecule compounds in this embodiment 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] I. Methods

[0036] 1. Experimental Materials and Procedures

[0037] 1.1 Cell culture medium preparation: DMEM / F12 + 10% fetal bovine serum + 1% P / S antibiotics (penicillin + streptomycin)

[0038] 1.2 Preparation of small molecule compound combinations:

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

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

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

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

[0043] 1.3 Preparation of hypertonic solution: Sodium chloride was dissolved in ultrapure water to a final concentration of 0.94M, and then diluted with culture medium to a final concentration of 94mM (osmotic pressure 500mOSM).

[0044] 1.4 Cell treatment:

[0045] HCE-2 corneal epithelial cell line purchased from ATCC was cultured in DMEM / F12 medium containing 10% fetal bovine serum and 1% P / S antibiotics. Cells were divided into a negative control group, a model control group (hyperosmolarity), and an experimental group (hyperosmolarity + small molecule compound co-treatment group). Cells were cultured at 37°C in a 5% CO2 incubator, and cell changes were observed, followed by corresponding molecular biological assays.

[0046] 2. Taking photos in open space

[0047] 2.1 Selection of photographic time points: After processing the cells according to the above method, photographs of the cells in the six-well plates were taken using a microscope to observe and record changes in cell number and morphology. The time points after treatment where changes between cell groups were more obvious were selected for recording.

[0048] 2.2 Imaging instrument: Leica DMIL inverted biological microscope for imaging.

[0049] II. Results

[0050] The results are as follows Figure 1 As shown, the treatment of corneal epithelial cells with a hypertonic solution prepared with sodium chloride is a well-established dry eye model and has been widely used in experiments. Figure 1 In the study, compared with the negative control group, the number of HCE-2 cells in the hyperosmolar group was significantly reduced after 24 hours of drug administration, indicating that hyperosmolarity can induce HCE-2 cell damage, and the model was successfully established. However, compared with the hyperosmolar group, the number of HCE-2 cells in the experimental group (hyperosmolar + small molecule compound composition) was significantly increased, indicating that the small molecule compound composition can effectively protect against oxidative damage to corneal epithelial cells caused by hyperosmolarity and improve the survival rate of corneal epithelial cells.

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

[0052] I. Methods

[0053] 1. Experimental Materials and Procedures

[0054] 1.1 Cell culture medium preparation: DMEM / F12 + 10% fetal bovine serum + 1% P / S double antibiotics (penicillin + streptomycin);

[0055] 1.2 Preparation of small molecule compound combinations:

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

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

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

[0059] Mixing equal volumes of the mother liquors of the three small molecule compounds yields a small molecule compound combination, with the final concentration of each component of the small molecule compound being 10 μg / mL.

[0060] 1.3 Preparation of hypertonic solution: Sodium chloride was dissolved in ultrapure water to a final concentration of 0.94M, and then diluted with culture medium to a final concentration of 94mM (osmotic pressure 500mOSM).

[0061] 1.4 Cell Preparation: HCE-2 corneal epithelial cell line purchased from ATCC was seeded into 96-well plates at a density of 10,000 cells per well and cultured for 24 hours. The cells were divided into a negative control group, a model control group (hyperosmolarity), experimental group 1 (hyperosmolarity + NU7441), experimental group 2 (hyperosmolarity + GW9662), experimental group 3 (hyperosmolarity + D609), and experimental group 4 (hyperosmolarity + small molecule compound combination). Cell changes were observed, and subsequent experiments were conducted.

[0062] 1.5 Preparation of CCK8 staining solution: The cell culture medium and CCK8 staining stock solution are prepared at a ratio of 10:1.

[0063] 1.6 After the cells were treated with the drug to a specific time point, the supernatant was removed, 100 μL of staining solution was added to each well, and the cells were incubated at 37°C for 1 hour.

[0064] 1.7 The absorbance of each well at 450 nm was measured using an ELISA reader for comparison.

[0065] Survival rate calculation formula:

[0066] Cell viability % = [A(drug-treated) - A(blank)] / [A(negative control) - A(blank)]

[0067] II. Results

[0068] The results are as follows Figure 2 As shown in Example 1, the composition of NU7441, GW9662, and D609 effectively protects against oxidative damage to corneal epithelial cells caused by hyperosmolarity and improves their survival rate. Compared to hyperosmolar treatment, although each small molecule combined with hyperosmolarity alone can increase the cell CCK8 level to some extent, the combination of hyperosmolarity and small molecule compounds most effectively increased the cell CCK8 level. This indicates that the combination of small molecule compounds can effectively inhibit oxidative damage to HCE-2 cells caused by hyperosmolarity and significantly improve the survival rate of HCE-2 cells.

[0069] Example 3 Intracellular iron staining

[0070] I. Methods

[0071] 1. Experimental Materials and Procedures

[0072] 1.1 Cell culture medium preparation: DMEM / F12 + 10% fetal bovine serum + 1% P / S antibiotics

[0073] 1.2 Preparation of small molecule compound combinations:

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

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

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

[0077] Mixing equal volumes of the mother liquors of three small molecule compounds yields a combination of small molecule compounds.

[0078] 1.3 Preparation of hypertonic solution: Sodium chloride was dissolved in ultrapure water to a final concentration of 0.94M, and then diluted with culture medium to a final concentration of 94mM (osmotic pressure 500mOSM).

[0079] 1.4 Cell Preparation: HCE-2 corneal epithelial cell line purchased from ATCC was seeded into 12-well plates at a density of 50,000 cells per well and cultured for 24 hours. The cells were divided into a negative control group, a model control group (hyperosmolarity), and an experimental group (hyperosmolarity + small molecule compound combination). After 24 hours of incubation, cell changes were observed, and subsequent experiments were conducted.

[0080] 1.5 Preparation of intracellular iron staining solution: The cell culture medium and the iron staining stock solution were prepared at a ratio of 4000:1.

[0081] 1.6 After the cells were treated with the drug to a specific time point, the supernatant was removed, 500 μL of staining solution was added to each well, and the cells were incubated at 37°C for 30 minutes.

[0082] 1.7 Direct observation under a microscope.

[0083] (2) Results

[0084] The results are as follows Figure 3 As shown, compared with hyperosmolar treatment, the small molecule compound combination can effectively reduce the intracellular iron ion level. This indicates that the small molecule compound combination can effectively inhibit hyperosmolar-induced ferroptosis in HCE-2 cells and significantly improve the survival rate of HCE-2 cells.

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

[0086] (1) Method

[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), and experimental group (scopolamine + small molecule compound co-treatment group (NU7441 concentration of 8 μg / kg; GW9662 concentration of 8 μg / kg; D609 concentration of 12.5 μg / kg)).

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

[0090] 1.3 The small molecule compound combination group was administered via eye drops: Each small molecule was dissolved in PBS to prepare a stock solution, which was then diluted with an equal volume. The final concentrations for each animal were calculated based on the animal's body weight: NU7441 concentration was 8 μg / kg; GW9662 concentration was 8 μg / kg; and D609 concentration was 12.5 μg / kg.

[0091] 1.4 After injection, the mice underwent corneal fluorescein staining on a specified date, and the corneal epithelial staining was observed under a slit-lamp microscope.

[0092] 1.5 After injection, mice underwent a phenol red cotton thread test to measure tear secretion on a specified date. A phenol red cotton thread was held with micro-forceps and placed at the outer canthus of both eyes of the mouse for 15 seconds. The length of the wet and reddened part of the phenol red cotton thread was measured as an estimate of the tear volume (in millimeters).

[0093] (2) Results

[0094] like Figure 4 As shown, subcutaneous injection of scopolamine, a dry eye model mouse, significantly increased corneal epithelial loss and reduced tear secretion. Animal experiments demonstrated that, compared to the control group, the small molecule compound administered as eye drops effectively prevented corneal epithelial cell damage, restored tear secretion, and showed no significant drug toxicity in the experimental group of dry eye model mice.

[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), and experimental group (scopolamine + small molecule compound co-treatment group (NU7441 concentration of 8 μg / kg; GW9662 concentration of 8 μg / kg; D609 concentration of 12.5 μg / kg)).

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

[0099] 1.3 Small molecule compound combinations were administered via eye drops: The small molecules were dissolved in PBS to prepare a stock solution, which was then diluted with an equal volume. The final concentrations for each animal were calculated based on the animal's body weight: NU7441 was 8 μg / kg; GW9662 was 8 μg / kg; and D609 was 12.5 μg / kg.

[0100] 1.4 Mice injected with the drug were euthanized by cervical dislocation at the designated date. After dissecting the eyeballs, corneal tissue paraffin sections were prepared and then subjected to TUNEL immunofluorescence staining.

[0101] (2) Results

[0102] like Figure 5 As shown, subcutaneous injection of scopolamine, a dry eye model mouse, significantly increased corneal epithelial cell death. Animal experiments demonstrated that, compared to the model control group, the small molecule compound effectively reduced corneal epithelial cell death in the experimental group of dry eye model mice treated with eye drops.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A small molecule compound composition, characterized in that, The small molecule compound composition consists of NU7441, GW9662 and D609; the CAS number of NU7441 is 503468-95-9, the CAS number of GW9662 is 22978-25-2, and the CAS number of D609 is 83373-60-8.

2. The small molecule compound composition according to claim 1, characterized in that, The effective dose of NU7441 in the combination of small molecule compounds 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 the prevention and / or treatment of dry eye syndrome, characterized in that, The pharmaceutical composition comprises a small molecule compound composition as described in any one of claims 1-3, or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier, and a protective drug against dry eye damage.

5. The pharmaceutical composition according to claim 4, characterized in that, The pharmaceutical composition is formulated as any one of eye drops, eye ointment, or gel.

6. The use of the small molecule compound composition according to any one of claims 1-3 or the pharmaceutical composition according to any one of claims 4-5 in the preparation of a medicament for the prevention and / or treatment of dry eye syndrome.

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