CN118304286B - Application of sinapine thiocyanate in preparation of medicine for treating retinal neovascular related diseases - Google Patents

Abstract

The invention discloses the application of sinapinic acid thiocyanate in the preparation of a drug for treating retinal neovascularization-related diseases, and relates to the field of biomedical technology. Sinapine thiocyanate has a significant promoting effect on the migration of human retinal vascular endothelial cells and angiogenesis in vitro; in a hypoxia-induced mouse model, it is confirmed that sinapinic acid thiocyanate has an inhibitory effect on the formation of retinal pathological neovascularization and can effectively promote the repair of avascular areas. Therefore, sinapinic acid thiocyanate has a good application prospect for the preparation of drugs for treating retinal neovascularization-related diseases.

Description

Application of sinapinic acid thiocyanate in the preparation of drugs for treating retinal neovascularization-related diseases

Technical Field

The present invention relates to the field of biomedical technology, and in particular to the use of sinapinic acid thiocyanate in the preparation of medicines for treating retinal neovascularization-related diseases.

Background Art

Sinapine thiocyanate (ST), also known as choline thiocyanate 4-hydroxy-3,5-dimethoxycinnamic acid, is an alkaloid isolated from the seeds of species of the Cruciferae family (rapeseed, radish seeds, white mustard seeds). Its chemical formula is C ₁₇ H ₂₄ N ₂ O ₅ S (UNII-A9SJB3RPE2) and its molecular weight is 368.4 g/mol. It is soluble in organic solvents such as methanol, ethanol, and DMSO, and slightly soluble in water. It has attracted much attention due to its multifaceted pharmacological effects and has shown potential application value in the fields of anti-inflammatory and anti-tumor.

Sinapine thiocyanate has a certain protective effect on vascular endothelial cells in hypertension. It protects vascular endothelial function in spontaneous hypertension by inhibiting the activation of NLRP3 inflammasome and the expression of related inflammatory mediators, and also improves angiotensin II (AngII)-induced endothelial cell damage. Sinapine thiocyanate can significantly inhibit pancreatic cancer (PC) cell proliferation and activity by targeting upregulating GADD45A.

In addition, the soluble composite microneedles composed of gelatin microspheres (GMS) loaded with mustard thiocyanate and hyaluronic acid (HA) can release continuously and efficiently in in vivo experiments, and have great potential for the treatment of allergic asthma, which also shows that the molecule has high biosafety.

Pathological neovascularization is the main cause of irreversible blindness in patients with ischemic retinopathy. Currently, there is no effective treatment for pathological neovascularization. Although anti-VEGF therapy has been widely used in the study of related diseases, many patients still do not respond to this therapy or are at risk of recurrence.

In view of this, the present invention is proposed.

Summary of the invention

The purpose of the present invention is to provide the use of sinapinic thiocyanate in the preparation of medicines for treating retinal neovascularization-related diseases.

The present invention is achieved in that:

The present invention provides an application of sinapinic thiocyanate in preparing a medicine for treating retinal neovascularization-related diseases.

The present invention has the following beneficial effects:

The present invention provides a new application of sinapinic acid thiocyanate in retinal neovascularization-related diseases. Experiments have confirmed that sinapinic acid thiocyanate has a significant promoting effect on the migration of human retinal vascular endothelial cells and angiogenesis in vitro, and has an in vitro counteracting effect on the oxidative stress caused by hydrogen peroxide; in a hypoxia-induced mouse model, it has been confirmed that sinapinic acid thiocyanate has an inhibitory effect on the formation of retinal pathological neovascularization and can effectively promote the repair of avascular areas. Therefore, sinapinic acid thiocyanate has a good application prospect for the preparation of drugs for treating retinal neovascularization-related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

Figure 1 shows the chemical structure of sinapinic acid thiocyanate (A: sinapinic acid thiocyanate 2D structure C ₁₇ H ₂₄ N ₂ O ₅ S; B: sinapinic acid thiocyanate 3D structure);

Figure 2 shows the experimental results of the effect of sinapinic thiocyanate on cell migration and in vitro angiogenesis (A: The effect of ST on HRECs migration was detected by scratch method, and the images were taken under an optical microscope at 0h, 16h, and 36h. Scale bar, 200μm; B: The experimental results of detecting the effect of ST on HRECs tube formation; HRECs were seeded into a vascular formation plate containing Matrigel, and the images were taken under an optical microscope 4h later);

Figure 3 shows the experimental results of the effect of ST on the proliferation of HRECs (A: TEER experiment was performed after adding different concentrations of drugs to HRECs, and the cell index (CI) was measured every 10 minutes. The CI value was normalized at 22 hours after the cells relatively entered the proliferation phase, and the sample size was n=3; B: Statistical analysis of the concentration required for the half-maximal effect (Effective Concentration 50, _EC50 ) of small molecule drugs in different drug groups, which was most stable in the range of 40-60 hours; C: Fitting _EC50 curve, where R2 is the statistical indicator of goodness of fit, indicating that the _EC50 fit is good; D: Under different concentrations of _H2O2 , TEER experiment was performed after adding different concentrations of drugs to _HRECs , and the CI value was normalized at 16 hours after the cells relatively entered the proliferation phase, and the sample size was n=3; E: Statistical analysis of the concentration required for the half- _maximal inhibition (Inhibitory Concentration 50, _IC50) of _H2O2 in different drug groups ), indicating the concentration required to achieve 50% effect of inhibiting cell proliferation-related biological processes, which is most stable in the range of 20-30h; F: IC ₅₀ curve of H ₂ O ₂ fitting, R ² is the statistical index of goodness of fit, indicating that the IC ₅₀ fit is good; Dunnett's multiple comparison test was used to compare the Ctrl group with the drug group and between the drug groups; *** P<0.001, **** P<0.0001);

Figure 4 shows the experimental results of the effects of different concentrations of ST on the avascular area and neovascularization of the retina (A: Schematic diagram of different concentrations of ST injected into the vitreous cavity of OIR mice; B: P13 OIR mice were intravitreally injected with PBS (control), 0.1mM, 1mM, and 10mM ST, and the mouse retinas were collected at P17, and representative images of immunofluorescence staining were selected; red is Isolectin B4, which marks retinal blood vessels; OIRSeg automatically analyzes the avascular area and neovascularization of the retina, yellow Avascular marks the avascular area, red Neovascular marks the neovascularization, and white is other retinal areas; scale, 500 μm; C: Statistical results of the weight of all OIR mice participating in the intraperitoneal injection experiment; D-E: Statistical results of the ratio of the avascular area and neovascularization area of the retina of P17 OIR mice, sample size n=6-8, all using Tukey's multiple comparison ANOVA test, ns=no significance, ** P<0.01, **** P<0.0001).

DETAILED DESCRIPTION

References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

The present invention provides application of sinapinic thiocyanate in preparing medicine for treating retinal neovascularization-related diseases.

Sinapine thiocyanate (ST), also known as choline thiocyanate 4-hydroxy-3,5-dimethoxycinnamic acid, is an alkaloid isolated from the seeds of species of the Cruciferae family (rapeseed, radish seeds, white mustard seeds). Its chemical formula is C ₁₇ H ₂₄ N ₂ O ₅ S, CAS NO. 7431-77-8, and its molecular weight is 368.4 g/mol.

Under conditions of ischemia and hypoxia, retinal tissue can promote angiogenesis by secreting vascular endothelial growth factor (VEGF) and other cytokines that stimulate angiogenesis. We named this type of regenerated blood vessel neovascularization. Different from the physiological angiogenesis during development, the wall structure of the neovascularization is weak and the permeability is high, which can easily lead to changes such as tissue leakage, bleeding, and edema, which ultimately seriously affect vision and may cause visual field loss or blindness. Currently, the main method for treating retinal neovascularization is to inhibit the function of VEGF through high-frequency intraocular injection of VEGF inhibitors, such as VEGF monoclonal antibodies or VEGFR competitive binding proteins. Although this method can inhibit the formation of new blood vessels, it requires repeated administration and cannot solve problems such as retinal ischemia and hypoxia. It is a temporary solution and does not address the root cause.

The inventors found that sinapine thiocyanate has a significant promoting effect on the migration of human retinal vascular endothelial cells and angiogenesis in vitro. In in vitro experiments, sinapine thiocyanate has a counteracting effect on the oxidative stress caused by hydrogen peroxide; in a hypoxia-induced mouse model, sinapine thiocyanate has an inhibitory effect on the formation of retinal pathological neovascularization and can effectively promote the repair of avascular areas. Therefore, sinapine thiocyanate has a good application prospect for the preparation of drugs for treating retinal neovascularization-related diseases.

The invention is expected to provide a new treatment option for patients who do not respond to or have relapsed from anti-VEGF therapy, and also help provide a new solution for the treatment of retinal neovascularization-related diseases.

In a preferred embodiment of the present invention, the retinal neovascularization-related disease is selected from at least one of ischemic retinopathy, neovascular glaucoma, ischemic choroidopathy and secondary retinopathy.

In a preferred embodiment of the present invention, ischemic retinopathy includes but is not limited to at least one of diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, central retinal artery occlusion, branch retinal artery occlusion (BRAO), ciliary retinal artery occlusion, distal retinopathy, Purtscher-like retinopathy, retinal periphlebitis, ocular ischemic syndrome, fundus lesions of Takayasu arteritis, radiation retinopathy, familial exudative vitreoretinopathy and age-related macular degeneration.

In a preferred embodiment of the present invention, ischemic choroidal lesions include but are not limited to at least one of polypoidal choroidal vasculopathy, high myopic choroidal neovascularization and idiopathic choroidal neovascularization.

Secondary retinal diseases include but are not limited to: proliferative vitreoretinopathy, vitreous hemorrhage, fibrous proliferation and tractional retinal detachment.

In a preferred embodiment of the present invention, the structural formula of sinapinic acid thiocyanate is as follows:

.

In a preferred embodiment of the present invention, sinapinic thiocyanate is used to treat retinal neovascularization-related diseases in at least one of the following ways:

(1) Promote the migration of retinal vascular endothelial cells;

(2) Promote angiogenesis of retinal endothelial cells in vitro;

(3) Inhibit and/or counteract the oxidative stress produced by retinal vascular endothelial cells;

(4) Prolong the cell proliferation period of retinal vascular endothelial cells;

(5) Reduce the area of avascular zone and promote the repair of avascular zone;

(6) Inhibit the formation of pathological neovascularization.

The cell proliferation phase refers to the period when cells divide (including mitosis and meiosis) under certain conditions to produce new cells to replace aging and dead cells.

In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable additive.

Other pharmaceutically acceptable additives are selected from one or more of starch, pregelatinized starch, microcrystalline cellulose, lactose, lactose starch complex, lactose cellulose complex, mannitol, mannitol starch complex, sorbitol, povidone, copovidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked polyvidone, magnesium stearate, sodium stearyl fumarate, talc, and stearic acid.

In an optional embodiment, the above-mentioned medicine is a liquid pharmaceutical preparation (such as a kind of injection), such as a solution, a suspension and a gel usually containing a liquid carrier, such as water and/or a pharmaceutically acceptable organic solvent. In addition, such liquid preparations may also include a pH adjusting agent, an emulsifier or a dispersant, a buffer, a preservative, a wetting agent, a gelling agent (such as methylcellulose), such as defined above. The medicine may be isotonic, that is, they may have the same osmotic pressure as blood. The isotonicity of the medicine may be adjusted by using sodium chloride and other pharmaceutically acceptable agents, such as glucose, maltose, boric acid, sodium tartrate, propylene glycol and other inorganic or organic soluble substances. The viscosity of the liquid composition may be adjusted by a pharmaceutically acceptable thickener such as methylcellulose. Other suitable thickeners include, for example, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, carbomer, etc. The preferred concentration of the thickener depends on the selected agent.

In a preferred embodiment of the present invention, the dosage form of the drug is selected from any one of tablets, pills, powders, suspensions, gels, emulsions, granules, capsules, injections and sprays.

In a preferred embodiment of the present invention, the drug is used for intravitreal injection.

In a preferred embodiment of the present invention, the dosage of the drug injected into the vitreous cavity is 0.0001-0.01 μmol.

The dosage is, for example, 0.0001-0.001 μmol or 0.0005-0.01 μmol.

In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

Example 1

In this example, the effect of ST on human retinal endothelial cells (HRECs) was investigated in vitro.

(1) Scratch test

To explore whether ST (sinapine thiocyanate, TargetMol Chemicals In, product number T3392, chemical structure shown in Figure 1) affects the migration ability of HRECs, the migration ability of HRECs was evaluated by scratch test (Figure 2, Panel A). The specific operation is as follows: HRECs were seeded in a 12-well plate at a cell density of 5 × 10 ⁵ /mL. When the confluence of HRECs reached about 100%, a sterilized 200μL pipette tip was used to scratch the monolayer of confluent cells in the center of the culture well to form a scratch, and then the cells were washed with Ultra-saline A buffer. The drug was configured in the culture medium according to the experimental concentration, returned to the incubator and continued to be cultured for a specific period of time, and the cell migration was observed under a microscope every 24 hours, and photos were taken for subsequent statistical analysis. The results showed that ST at concentrations of 16 nM, 400 nM, 2 μM, 10 μM and 50 μM could promote the recovery rate of HRECs 36 hours after scratching, and 16 nM had the best recovery effect, indicating that ST can accelerate the migration of HRECs.

(2) Detection of the effect of ST on angiogenesis of HRECs in vitro:

Considering the important role of HRECs in angiogenesis, a Matrigel-based in vitro angiogenesis assay was performed to test whether ST promotes in vitro angiogenesis of HRECs. The specific steps are as follows: (1) Place Matrigel, sterile enzyme-free 10 µL pipette tips, and μ-slide angiogenesis plates in a 4°C refrigerator overnight. (2) Plate: Add 10 µL of 4°C pre-cooled Matrigel to the wells of the μ-slide angiogenesis plate, taking care not to generate bubbles, and then place it in a 37°C cell culture incubator for 10 minutes. (3) Inoculation: Add HRECs at a concentration of 1 × 10 ⁴ /50 µL to the wells of the angiogenesis plate containing Matrigel, and place it in a cell culture incubator for about 4-8 h. Observe the angiogenesis under a microscope every 2 h, and take photos for subsequent statistical analysis.

In this example, PBS was added to the Ctrl group as a negative control, and observation 4 hours after inoculation revealed that both 16 nM and 2 μM ST could promote angiogenesis of HRECs in vitro (as shown in Figure 2 B).

The above results indicate that ST has a significant promoting effect on the migration and in vitro angiogenesis of human retinal endothelial cells.

Example 2

This example is based on the trans-endothelial electrical resistance assay (TEER) experiment of the xCELLigence RTCA MP real-time cell analyzer to observe the effect of ST on HRECs activity under pathological and physiological conditions. The specific operations are as follows: (1) Plating: HRECs cells are digested and centrifuged, and the cells are counted under a microscope. (2) Real-time monitoring: Trans-endothelial electrical resistance assay (TEER), first add culture medium to the xCELLigence RTCA MP analyzer to measure the baseline, and then inoculate into the E-Plate 96-well culture dish at a volume of about 5,000 cells per well. (3) Treatment conditions: When the cells are cultured to the logarithmic phase of cell proliferation, different concentrations of ST and H ₂ O ₂ are given, and the changes in cell growth are recorded in real time. The cell index (CI) is collected every 10 minutes, and the machine automatically normalizes.

The results showed that under physiological conditions, with DMSO and cell culture medium as negative control groups, 80nM, 400nM, 2μM, 10μM and 50μM ST all had a certain promoting effect on the proliferation of HRECs, among which 400nM had the most obvious effect and significantly prolonged the proliferation plateau (Figure 3A). ST was added at 40h, and the EC ₅₀ curve of ST in HRECs proliferation was fitted by concentration gradient. R ² was a statistical indicator of goodness of fit. The closer to 1, the better the fitting effect. EC ₅₀ was 172.53nM (Figure 3B and Figure C).

Furthermore, in this example, hydrogen peroxide (H ₂ O ₂ ) was used to induce cellular oxidative stress. Different concentrations of H ₂ O ₂ (50 μM, 100 μM, and 300 μM) were added to the cell culture medium to simulate the HREC model under pathological conditions, and TEER was used to monitor cell resistance in real time.

Freshly prepared 50μM, 100μM, 300μM H ₂ O ₂ and 400nM ST were added to the cell culture medium according to different groups, and the cell status was observed by real-time detection of CI values. The results showed that 300μM H ₂ O ₂ could cause direct cell death and ST could not save it; 100μM H ₂ O ₂ partially inhibited cell proliferation, and the promoting effect of ST could partially offset the inhibitory effect of 100μM H ₂ O ₂ and significantly prolong its proliferation platform; HRECs were less inhibited under 50μM H ₂ O ₂ conditions, and the supporting effect of ST was mainly reflected in prolonging the proliferation time (Figure 3, Figure D).

The IC ₅₀ curve of HRECs proliferation was fitted by H ₂ O ₂ concentration gradient. R2 is a statistical indicator of goodness of fit. The closer to 1, the better the fit. The IC ₅₀ is 118.45 μM (E and F in Figure 3). The IC ₅₀ is most stable in the range of 20-30 h.

Example 3

This example uses a mouse hypoxia-induced retinal pathological neovascularization model to explore the therapeutic effect of ST on retinal pathological neovascularization. The specific operation is as follows: Newborn mice (C57BL/6J) were purchased from Jicui Yaokang Biotechnology Co., Ltd. and raised in the animal room of Sichuan Provincial People's Hospital. When the mice were born on the seventh day (P7), they were placed in a culture chamber with an oxygen concentration of 75% together with the mother mice. The temperature and humidity were suitable, and the feed and water sources were sufficient. After 5 days of feeding in the warehouse, that is, at P12, the mice were taken out of the high oxygen culture chamber and placed in a normal environment to continue to be raised until P13, P17, and P21, respectively, to complete the construction of the hypoxia-induced OIR mouse model. OIR mice are the abbreviation of Oxygen-induced retinopathy, which is a hypoxia-induced model. When mice are fed with high oxygen, the blood vessels in the center of the retina will degenerate to form an avascular area. When they are taken out of high oxygen, the retina will form a relatively hypoxic state, which will induce the repair of the avascular area, but the repair process will be accompanied by the formation of pathological neovascularization.

OIR mice with normal body weight and no significant differences between groups were selected. On P13 (day 13), 1 uL of different concentrations of ST (0.1 mM, 1 mM or 10 mM) or control PBS was injected into the vitreous cavity of the eyes. Retinal flat mounts were made on P17, and immunofluorescence staining was performed and imaging was performed using a laser confocal microscope to explore the therapeutic effect of the drug on pathological neovascularization (Figure 4A).

The results showed that compared with the control group, ST at concentrations of 0.1 mM, 1 mM, and 10 mM could significantly reduce the area of avascular zone (Figure 4B), suggesting the key role of drugs in regulating vascular repair. In addition, ST at concentrations of 0.1 mM and 1 mM could significantly inhibit the formation of pathological neovascularization (Figure 4B), confirming that ST could inhibit the formation of pathological neovascularization to a certain extent. Compared with the control group, intraperitoneal injection of ST at concentrations of 0.1 mM, 1 mM, and 10 mM had no significant effect on the body weight of OIR mice (Figure 4C). Compared with the control, intraperitoneal injection of ST at concentrations of 0.1 mM, 1 mM, and 10 mM could significantly reduce the proportion of avascular zone in the retina of P17 OIR mice (Figure 4D). Compared with the control, intraperitoneal injection of ST at concentrations of 0.1 mM, 1 mM, and 10 mM could significantly reduce the proportion of neovascular area in the retina of P17 OIR mice (Figure 4E).

The results showed that intravitreal injection of 1 uL 1mM ST could inhibit the formation of pathological neovascularization in OIR mice to the greatest extent and promote the repair process of avascular areas.

In summary, sinapinic acid thiocyanate significantly promotes the migration of human retinal vascular endothelial cells and angiogenesis in vitro, and has an in vitro counteracting effect on the oxidative stress caused by hydrogen peroxide; in a hypoxia-induced mouse model, it was confirmed that sinapinic acid thiocyanate has an inhibitory effect on the formation of retinal pathological neovascularization and can effectively promote the repair of avascular areas. Therefore, sinapinic acid thiocyanate has a good application prospect for the preparation of drugs for the treatment of retinal neovascularization-related diseases.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims (9)

1. Use of sinapine thiocyanate in the preparation of a drug for treating retinal neovascularization-related diseases, characterized in that the structural formula of the sinapine thiocyanate is as follows: . 2. The use according to claim 1, characterized in that the retinal neovascularization-related disease is selected from at least one of ischemic retinopathy, neovascular glaucoma, ischemic choroidopathy and secondary retinopathy. 3. The use according to claim 2, characterized in that the ischemic retinopathy is selected from at least one of diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, central retinal artery occlusion, branch retinal artery occlusion, ciliary retinal artery occlusion, distal retinopathy, Purtscher-like retinopathy, retinal periphlebitis, ocular ischemic syndrome, fundus lesions of large arteritis, radiation retinopathy, familial exudative vitreoretinopathy and age-related macular degeneration. 4. The use according to claim 2, characterized in that the ischemic choroidopathy is selected from at least one of polypoidal choroidal vasculopathy, high myopic choroidal neovascularization and idiopathic choroidal neovascularization; The secondary retinal lesions are selected from proliferative vitreoretinopathy, vitreous hemorrhage, fibrous proliferation, and tractional retinal detachment. 5. The use according to claim 1, characterized in that the sinapinic acid thiocyanate is used to treat retinal neovascularization-related diseases in at least one of the following ways: (1) Promote the migration of retinal vascular endothelial cells; (2) Promote angiogenesis of retinal endothelial cells in vitro; (3) Inhibit and/or counteract the oxidative stress produced by retinal vascular endothelial cells; (4) Prolong the cell proliferation period of retinal vascular endothelial cells; (5) Reduce the area of avascular zone and promote the repair of avascular zone; (6) Inhibit the formation of pathological neovascularization. 6. The use according to claim 1, characterized in that the medicine further comprises a pharmaceutically acceptable additive. 7. The use according to claim 1, characterized in that the dosage form of the drug is selected from any one of tablets, pills, powders, suspensions, gels, emulsions, granules, capsules, injections and sprays. 8. The use according to claim 1, characterized in that the drug is used for vitreous cavity injection. 9. The use according to claim 8, characterized in that the dosage of the drug injected into the vitreous cavity is 0.0001-0.01 μmol.