Application of dihydropyridine in preparation of medicine for treating choroidal neovascularization

Abstract

The invention discloses application of dihydropyridine in preparation of a medicine for treating choroidal neovascularization, and belongs to the technical field of biological medicines.The proliferation, migration and lumen formation of human choroidal endothelial cells under the hypoxia condition are inhibited through dihydropyridine, and blood vessel leakage, area and volume of laser-induced choroidal neovascularization of mice are reduced; the formation of laser-induced choroidal neovascularization of mice is relieved through dihydropyridine, and the proliferation, migration and lumen formation of hypoxia-induced human choroidal endothelial cells can be down-regulated, so that the PHD has a treatment effect on CNV.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of dihydropyridine in preparation of a drug for treating choroidal neovascularization. BACKGROUND

[0002] The late stage of age-related macular degeneration (AMD) is divided into two types, including dry AMD and wet AMD, wherein the wet AMD is mainly characterized by choroidal neovascularization (CNV), which is manifested as newly formed leaky blood vessels penetrating through Bruch's membrane and extending to the subretinal space. The leakage of these blood vessels causes tissue damage, which is replaced by fibrous tissue, resulting in subretinal fibrosis, especially macular fibrosis. At present, the main method for treating wet AMD is repeated intravitreal injection of anti-vascular endothelial growth factor drugs, which increases the medical burden of patients, and the anti-VEGF drug treatment cannot prevent the occurrence of fibrosis, and finally about half of the patients develop fibrosis after a period of treatment, resulting in reduced vision, even blindness. Therefore, it is necessary to explore a new and more effective treatment method.

[0003] Dihydropyridine (DHP) is a heterocyclic compound, which has anti-inflammatory and antioxidant effects, and has antibacterial and anti-leishmania activity, and can induce apoptosis of tumor cells. However, the intervention effect of DHP on CNV has not been determined. SUMMARY

[0004] The technical problem solved by the present application is:

[0005] The present application provides application of dihydropyridine in preparation of a drug for treating choroidal neovascularization, which can inhibit the proliferation, migration and lumen formation of human choroidal endothelial cells under hypoxic conditions, and reduce the vascular leakage, area and volume of laser-induced choroidal neovascularization in mice.

[0006] Technical scheme

[0007] In order to achieve the above-mentioned purpose, the present application is realized by the following technical scheme:

[0008] Use of a dihydropyridine in the preparation of a medicament for treating choroidal neovascularization.

[0009] Further, the medicament comprises the dihydropyridine and a pharmaceutically acceptable excipient.

[0010] Further, the excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant.

[0011] Further, the medicament is an oral or non-oral preparation.

[0012] Further, the medicament is a tablet, a capsule, a powder, a pill, a granule, a solution, a suspension, a syrup, an injection, a suppository, an adsorbent or a spray.

[0013] Advantages:

[0014] The present application provides a use of a dihydropyridine in the preparation of a medicament for treating choroidal neovascularization, which has the following advantages compared with the prior art:

[0015] 1. The present application inhibits the proliferation, migration and lumen formation of human choroidal endothelial cells under hypoxic conditions by dihydropyridine, and reduces the vascular leakage, area and volume of laser-induced choroidal neovascularization in mice;

[0016] 2. The present application relieves the formation of laser-induced choroidal neovascularization in mice by dihydropyridine, and can down-regulate the proliferation, migration and lumen formation of hypoxia-induced human choroidal endothelial cells;

[0017] 3. The present application verifies that PHD has a therapeutic effect on CNV by using dihydropyridine in the preparation of a medicament for treating choroidal neovascularization. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a representative picture of the present application DHP inhibiting hypoxia-induced HCECs proliferation EdU experiment;

[0019] Figure 2 is the ratio of the number of EdU positive cells to the number of DAPI positive cells in the present application DHP down-regulating the migration of hypoxic HCECs Transwell experiment, *** P<0.001, ### P<0.001, # P<0.05, four samples per group;

[0020] Figure 3 is a representative picture of the present application DHP inhibiting hypoxia-induced HCECs proliferation EdU experiment;

[0021] Figure 4This application is a quantitative analysis of the number of migrating cells in the migration of hypoxic HCECs regulated by DHP. *** P<0.001, ## P<0.01, # P < 0.05, three samples per group;

[0022] Figure 5 This is a representative diagram of the tube formation experiment in the in vitro tube formation experiment of this application to detect the ability of HCECs to form tubes;

[0023] Figure 6 This is a quantitative analysis of the lumen length in the in vitro lumen formation experiment of this application to detect the ability of HCECs to form lumen. *** P<0.001, ## P<0.01, # P < 0.05, five samples per group;

[0024] Figure 7 This is a representative image of the FFA assay of vascular leakage at the CNV injury site in mice. The white irregular area in the image represents the CNV vascular leakage range.

[0025] Figure 8 This is the statistical analysis of the degree of CNV vascular leakage measured by FFA in the CNV injury site of mice. ** P<0.01, # P < 0.05, four samples per group;

[0026] Figure 9 This is a representative image of the range of ICGA detection of CNV lesions in mice by ICGA in this application;

[0027] Figure 10 This is a statistical analysis chart of the CNV region of the CNV lesion site detected by ICGA in this application. * P<0.05, *** P < 0.001, three samples per group;

[0028] Figure 11 The present application is an immunofluorescence staining of CNV lesions in mice labeled with phalloidin, isolectin B4, and DAPI staining of mouse choroidal flat mounts;

[0029] Figure 12 This is a statistical analysis of the CNV volume of CNV lesions in mice marked by immunofluorescence staining. * P<0.05, *** P < 0.001, four samples per group. DETAILED DESCRIPTION

[0030] The application will be described in further detail below with reference to the drawings and specific examples, but should not be construed as limiting the application. Modifications or replacements of the method, steps or conditions of the application, without departing from the spirit and essence of the application, all belong to the scope of the application. The experimental methods and reagents not specified in the examples are all according to the conventional conditions in the art.

[0031] The dihydropyridine used in the application is a heterocyclic compound with antioxidant effect.

[0032] Choroidal neovascularization is a kind of choroidal abnormal angiogenesis, which can cause vision loss, even blindness.

[0033] The human choroidal endothelial cells (HCECs) in the application are purchased from Wuhan Punsai Life Science and Technology Co., Ltd.

[0034] Example 1

[0035] The application of a dihydropyridine in the preparation of a drug for treating choroidal neovascularization, the specific steps are as follows:

[0036] First, human choroidal endothelial cell culture and grouping: human choroidal endothelial cells (HCECs) are purchased from Wuhan Punsai Life Science and Technology Co., Ltd., and are grown in Dulbecco's Modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS); HCECs are divided into normal (normoxia, 95% O2 and 5% CO2 culture for 24 h), hypoxia (1% O2, 94% N2 and 5% CO2 culture for 24 h), hypoxia + low-dose DHP (10 μM), hypoxia + medium-dose DHP (20 μM) and hypoxia + high-dose DHP (40 μM) groups;

[0037] Second step, 5-ethynyl-2'-deoxyuridine (EdU) cell proliferation experiment: after 24h culture of HCVs in different groups, the chamber was taken out from the 37℃ carbon dioxide incubator, and 50μM EdU medium was added to each well according to the EdU kit instructions, and incubated at 37℃ for 2h, and the culture medium was discarded. The cells were washed with PBS for 3 times, 5min each time. Fixed with 4% PFA for 30min, and discarded the fixing solution. Add 2mg / mL glycine to neutralize the excess aldehyde group, incubate for 5min, discard the solution. Wash with PBS for 1 time, 5min; wash with PBST for 1 time, 10min; wash with PBS for 1 time, 5min. Add Apollo dye solution, incubate for 30min, discard the dye solution. Wash with PBST for 3 times, 10min each time. Add DAPI dye solution, incubate for 30min, discard the dye solution. Wash with PBS for 3 times, 10min each time. Use anti-fluorescence quenching mounting medium to mount the slide, and store at 4℃. Use fluorescence microscope to take pictures. Analyze the average proportion of EdU positive cells (red) and DAPI positive cells (blue) as an indicator of cell proliferation activity;

[0038] Third step, Transwell cell migration experiment: after 24h culture of HCVs in different groups, trypsinize the HCVs to prepare a single cell suspension, and inoculate it on the upper space of the Transwell chamber. The upper chamber is used with serum-free medium, and the lower chamber is placed with 10% FBS as the chemotactic medium, and incubated at 37℃ for 48h. Take out the chamber, fix with 4% PFA for 40min, and crystal violet staining for 30min. After washing and air-drying, randomly select 4 fields of stained cells to take pictures and count under a light microscope;

[0039] Fourth step, tube formation test: after 24h culture of HCVs in different groups, trypsinize the HCVs to prepare a single cell suspension. Add 200μL Matrigel to each well of the 24-well plate, and place it in a 37℃ carbon dioxide incubator for 30min to allow it to solidify. Add the collected HCVs suspension on the Matrigel, and observe whether the cells form a closed tube network under phase contrast microscope after 6h, and measure the total length of the tube structure;

[0040] Step 5: Establishment of CNV model in mice: CNV model was established in male C57BL / 6 mice (8 weeks old) by krypton laser. The mice were anesthetized by intraperitoneal injection of 10 mg / kg of xylazine and 80 mg / kg of chloral hydrate, and their pupils were dilated with compound tropicamide. The mice were then irradiated with a handheld light source while maintaining their body temperature. The experimental eye of the mouse was fixed in front of a slit lamp, and a 5.4 mm handheld contact lens was placed in front of the cornea with the assistance of 1% methylcellulose. A krypton ion laser machine was used with a laser wavelength of 647.1 nm, a power of 300 mw, a spot diameter of 50 μm, and an exposure time of 0.05 seconds. Four points were coagulated around the optic disc and at a distance of 2 papillary diameters from the optic disc, located at 3, 6, 9, and 12 o'clock, respectively. The presence of bubbles was used as an indication that Bruch's membrane had been broken, and the absence of vitreous hemorrhage was used as a success criterion for coagulation.

[0041] Step 6: Intravitreal injection of DHP or Conbercept: Immediately after laser induction, the CNV group of C57BL / 6 mice was weighed, anesthetized, and their pupils were dilated with compound tropicamide. The eye surface was moistened with physiological saline, and the mice were made to lie on their sides on the operating table. Under a dissecting microscope, the eyelids were probed to expose the corneal limbus. A 10-0 needle was used to make an incision 1 mm behind the corneal limbus, and a 33G syringe was used to inject 1 μl of CON into the vitreous cavity. Postoperative erythromycin eye ointment was applied to the eye to prevent infection. The C57BL / 6 mice were randomly divided into normal (without any treatment), CNV 7 days (without any injection), low-dose DHP (OT53030000, SimSon Pharma Limited, USA; 5 μM, 1 μl) intravitreal injection, medium-dose DHP (10 μM, 1 μl) intravitreal injection, high-dose DHP (20 μM, 1 μl) intravitreal injection, and CON intravitreal injection groups. DHP was dissolved in 0.1% DMSO;

[0042] Step 7: Fundus angiography of mice: Mice were randomly divided into normal, 7-day CNV, 7-day CNV + 0.1% DMSO, low-dose DHP intravitreal injection, medium-dose DHP intravitreal injection, high-dose DHP intravitreal injection, and RAN intravitreal injection groups. Mice were anesthetized and sacrificed, and the pupils were dilated with tropicamide. 10% sodium fluorescein diluted with water for injection to 2% sodium fluorescein or indocyanine green (ICGA) was injected intraperitoneally (0.3 ml). Fundus fluorescein angiography (FFA) or indocyanine green angiography (ICGA), respectively, was performed. Confocal laser retinal tomography was used to record the images starting 100-140 seconds after injection. Recording time was 30 minutes. CNV grading: Grade 0 (no exudate), Grade 1 (mild exudate), Grade 2a (moderate exudate), and Grade 2b (severe exudate). Changes in choroidal exudate area were analyzed using Image J software. The researchers used a freehand drawing tool to draw the edge of the ICGA extravasation area and calculated the pixel area using the region of interest (ROI) manager of ImageJ software. The pixel area was then converted to microns squared (microns) based on the ratio between pixels and microns (the ratio we used was 0.5249). 2 , mm 2 ) to obtain the final CNV area;

[0043] Step 8, choroidal flattening and immunofluorescence staining: Mice were randomly divided into CNV 7 days, CNV 7 days + 0.1% DMSO, low-dose DHP intravitreal injection, medium-dose DHP intravitreal injection, high-dose DHP intravitreal injection and RAN intravitreal injection groups. The mice were killed by intraperitoneal injection of 20 mg / kg xylazine and 80 mg / kg ketamine, and the eyeballs were immediately removed and quickly fixed in 4% paraformaldehyde (PFA) for 1 hour. Under a dissecting microscope, the sclera was cut open along the equator, the anterior segment of the eye was removed, and the retinal neuroepithelium was carefully separated to obtain the retina-retinal pigment epithelium (RPE)-choroid complex eye cup, which was repeatedly rinsed with cold ICC buffer. Under dim light, 1 μg / μl Alexa 594 phalloidin and Alexa 488 isolectin-B4 antibody was added to ICC buffer at a dilution of 1:150. After thorough mixing, the cells were transferred to EP tubes containing eye cups for staining and placed in a dark place at 4 ℃The retinas were incubated overnight in the refrigerator. After rewarming for 2 h at room temperature, the eyecups were washed thoroughly with ICC buffer. The retinas-RPE-choroids complex was flat-mounted on glass slides, and four radial cuts were made along the optic disc direction. The slides were observed under a fluorescence microscope. The three-dimensional images of CNV were generated using ZEN software (Zeiss, Germany), and the volume of CNV was measured using Imaris software (Switzerland);

[0044] Step 9, Statistical analysis: Each data was expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments. The comparison between two groups of data used the two-tailed Student's t-test, while the comparison between multiple groups of data used one-way ANOVA and Bonferroni post-test. P < 0.05 was considered statistically significant.

[0045] Example 2

[0046] Use of a dihydropyridine in the preparation of a medicament for treating choroidal neovascularization, DHP down-regulates HCECs proliferation induced by hypoxia:

[0047] Compared with the normal group, the proportion of EdU positive cells of HCECs in the hypoxia group increased, and 10 μM, 20 μM and 40 μM DHP reduced the proportion of EdU positive cells induced by hypoxia, and the higher the concentration of DHP, the stronger the effect of reducing the proportion of EdU positive cells of HCECs. As shown in Figure 1 and Figure 2 These results show that DHP inhibits HCECs proliferation induced by hypoxia.

[0048] HCECs were randomly divided into normal, hypoxia, hypoxia + DHP 10 μM (treated for 24 hours), hypoxia + DHP 20 μM and hypoxia + DHP 40 μM groups. Figure 1 is a representative picture of the EdU experiment of DHP inhibiting HCECs proliferation induced by hypoxia in the present application, Figure 2 is the ratio of the number of EdU positive cells to the number of DAPI positive cells of HCECs proliferation induced by hypoxia in the present application. *** P < 0.001, ### P < 0.001, # P < 0.05. Four samples in each group.

[0049] Example 3

[0050] Use of a dihydropyridine in the preparation of a medicament for treating choroidal neovascularization, DHP hinders HCECs migration induced by hypoxia:

[0051] Compared with the normal group, the number of migrated cells of HCECs increased after hypoxia treatment, and 10 μM, 20 μM and 40 μM DHP could reduce the number of migrated cells of HCECs in the hypoxia group, as shown in Figure 3 and Figure 4 The data show that DHP has a down-regulating effect on the migration of HCECs treated by hypoxia.

[0052] HCECs were randomly divided into normal, hypoxia, hypoxia+DHP 10 μM, hypoxia+DHP 20 μM and hypoxia+DHP 40 μM groups. The migration ability of HCECs was detected by Transwell experiment. Figure 3 is a representative picture of the Transwell experiment of DHP down-regulating the migration of HCECs treated by hypoxia in the present application; Figure 4 is a quantitative analysis of the number of migrated cells in the migration of HCECs treated by hypoxia and down-regulated by DHP in the present application, *** P<0.001, ## P<0.01, # P<0.05. Each group has three samples.

[0053] Example 4

[0054] The use of a dihydropyridine in the preparation of a medicament for treating choroidal neovascularization, DHP reduces the tube formation of HCECs under hypoxia conditions:

[0055] Hypoxia treatment increases the tube length of HCECs, and DHP reduces the tube length of HCECs under hypoxia conditions from 10 μM, and 40 μM still plays a role in inhibiting the tube formation of HCECs, and 40 μM DHP has a stronger inhibitory effect on tube formation than 10 μM and 20 μM DHP, indicating that the effect of DHP on inhibiting tube formation is dose-dependent.

[0056] DHP reduces the tube formation of HCECs under hypoxia, HCECs are randomly divided into normal, hypoxia, hypoxia+DHP 10 μM, hypoxia+DHP 20 μM and hypoxia+DHP 40 μM groups. The ability of HCECs to form tubes was detected by in vitro tube formation experiment. Figure 5 is a representative picture of the tube formation experiment in the detection of the ability of HCECs to form tubes by in vitro tube formation experiment in the present application, Figure 6 is a quantitative analysis of the tube length in the detection of the ability of HCECs to form tubes by in vitro tube formation experiment in the present application, *** P<0.001, ## P<0.01, # P<0.05, each group has five samples.

[0057] Example 5

[0058] A dihydropyridine is used in the preparation of a drug for treating choroidal neovascularization. DHP reduces laser-induced CNV vascular leakage in mice:

[0059] The leakage degree of CNV in the 7-day CNV group of mice was mainly 2a, the leakage degree after intravitreal injection of 5μM and 10μM DHP was mainly 1, and the leakage degree after treatment with 20μM DHP was mainly 0. Moreover, the reduction in leakage degree of the three concentrations of DHP compared with the 7-day CNV group was statistically significant, as shown in Figure 2. Figure 7 and Figure 8 These results suggest that DHP has the effect of downregulating vascular leakage in CNV.

[0060] DHP attenuated laser-induced CNV vascular leakage in mice. The mice were randomly divided into normal, CNV 7 days, CNV 7 days + DHP 5 μM (injected once into the vitreous after laser injury, volume 1 μl), CNV 7 days + DHP 10 μM, and CNV 7 days + DHP 20 μM groups. Figure 7 This is a representative image of the FFA assay of vascular leakage at the CNV injury site in mice. The white irregular area in the image represents the CNV vascular leakage range. Figure 8 This is the statistical analysis of the degree of CNV vascular leakage measured by FFA in the CNV injury site of mice. ** P<0.01, # P < 0.05, four samples in each group.

[0061] Example 6

[0062] The use of a dihydropyridine in the preparation of a drug for treating choroidal neovascularization. DHP reduces the area of ​​laser-induced CNV in mice:

[0063] After intravitreal injection of DHP, the area of ​​laser-induced CNV in mice was significantly reduced. Figure 9 and Figure 10 As shown, DHP reduced the CNV area in mice.

[0064] DHP reduces the area of ​​laser-induced CNV in mice. The mice were randomly divided into normal, CNV 7 days, CNV 7 days + DHP 5 μM, CNV 7 days + DHP 10 μM, and CNV 7 days + DHP 20 μM groups. Figure 9 This is a representative picture of the ICGA range of CNV lesion sites detected by ICGA in this application. Figure 10 This is a statistical analysis chart of the CNV region of the CNV lesion site detected by ICGA in this application. * P<0.05, *** P < 0.001, three samples per group.

[0065] Example 7

[0066] Use of a dihydropyridine in the manufacture of a medicament for treating choroidal neovascularization, DHP reduces laser-induced CNV volume in mice:

[0067] The results of immunofluorescent staining showed that DHP significantly reduced the lesion volume of CNV 7 days as shown in Figure 11 and Figure 12 .

[0068] DHP reduces laser-induced CNV volume in mice, mice were randomly divided into normal, CNV 7 days, CNV 7 days + DHP 5 μM, CNV 7 days + DHP 10 μM and CNV 7 days + DHP 20 μM groups. Figure 11 To immunofluorescently stain the CNV lesion of mice, phalloidin (cytoskeleton marker, red), isolectin B4 (vascular endothelial cell marker, green) and DAPI (nucleus marker, blue) were used to stain the mouse choroid smears, Figure 12 is a statistical analysis of the CNV volume of the CNV lesion of mice immunofluorescently stained in the present application, * P<0.05, *** P<0.001, four samples in each group.

[0069] It should be noted that the above examples are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that the technical solutions of the present application can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. Use of a dihydropyridine in the manufacture of a medicament for treating choroidal neovascularization.

2. Use of a dihydropyridine according to claim 1 for the preparation of a medicament for the treatment of choroidal neovascularization, characterized in that: The medicament comprises a dihydropyridine and a pharmaceutically acceptable excipient.

3. Use of a dihydropyridine according to claim 2 for the preparation of a medicament for the treatment of choroidal neovascularization, characterized in that: The excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant.

4. Use of a dihydropyridine according to claim 2 for the preparation of a medicament for the treatment of choroidal neovascularization, characterized in that: The medicament is an oral or non-oral dosage form.

5. Use of a dihydropyridine according to claim 2 for the preparation of a medicament for the treatment of choroidal neovascularization, characterized in that: The medicament is a tablet, a capsule, a powder, a pill, a granule, a solution, a suspension, a syrup, an injection, a suppository, an adsorbent, or a spray.

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