CN112716965B

CN112716965B - Application of geniposide in preparation of choroidal neovascularization therapeutic drug

Info

Publication number: CN112716965B
Application number: CN202110060217.1A
Authority: CN
Inventors: 朱琳玲; 杜姝; 黄春霞
Original assignee: Suzhou Lixiang Ophthalmic Hospital Co ltd
Current assignee: Suzhou Lixiang Ophthalmic Hospital Co ltd
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2022-12-30
Anticipated expiration: 2041-01-18
Also published as: CN112716965A

Abstract

The invention relates to the technical field of medicines, in particular to application of geniposide in preparation of a choroidal neovascularization therapeutic drug. The invention has the advantages that: the invention provides application of geniposide in choroidal neovascularization treatment, and solves the problems of repeated intraocular injection of anti-VEGF drugs and no response of part of patients in the prior art. The geniposide reduces the exudation, the damaged area and the volume of choroidal neovascularization of mice, inhibits the proliferation, the migration and the tube cavity formation of human choroidal endothelial cells under the anoxic condition, and has similar curative effect compared with the current clinical commonly used anti-vascular endothelial growth factor drug Corbescept.

Description

Application of geniposide in preparation of choroidal neovascularization therapeutic drug

Technical Field

The invention relates to the technical field of medicines, in particular to an effect of iridoid glycoside compound jasminoidin in preparing a choroid neovascularization therapeutic drug.

Background

Age-related macular degeneration (AMD) is a degenerative eye disease that occurs in people over the age of 50. AMD is classified into two broad categories, non-neovascular and neovascular, according to pathological changes. Non-neovascular AMD is characterized by a geographic atrophy of the retinal pigment epithelium and the photoreceptor cells thereon, which leads primarily to pathological Choroidal Neovascularization (CNV), characterized by abnormal blood vessel growth in the central region of the retina, i.e., under the macula, which breaks through the Bruch's membrane and penetrates into the subretinal pigment epithelium region, resulting in exudation, hemorrhage, retinal edema, pigment epithelial detachment, and fibrotic scarring, resulting in visual impairment. AMD is the leading cause of blindness in the elderly in developed countries, reducing the quality of life of patients with AMD. With the trend of aging population and increasing life expectancy of China, the incidence rate of AMD of China is also increasing year by year. While neovascular AMD accounts for only 10% of all AMD patients, neovascular AMD-induced blindness accounts for 90% of AMD patients, and neovascular AMD is therefore of greater interest to researchers.

The current major treatments for wet AMD are anti-Vascular Endothelial Growth Factor (VEGF) drugs such as Conbeccept (CON) and Ranibizumab (RAN), but these are not effective in all AMD patients, some patients do not respond (about 10%) to treatment and after a period of treatment (about two years) the patients begin to develop resistance to drugs, furthermore, anti-VEGF drugs require repeated intravitreal injections, increase the economic burden and difficulty of seeing a doctor on patients, and cause various intraocular and systemic side effects such as endophthalmitis, intraocular hemorrhage and stroke. Therefore, new effective treatment strategies for CNV are under way.

Geniposide (GEN) is an iridoid glycoside compound extracted from dried mature fruit of fructus Gardeniae. Previous researches find that the geniposide has various pharmacological effects of anti-angiogenesis, anti-inflammation and neuroprotection. In addition, geniposide is an agonist of glucagon-like peptide-1receptor (GLP-1R) (Li N, zhou H, wu Q, duan M, deng W, tang Q. Sting-IRF3 receptors to lipopolysaccharide-induced cardiac dysfunction, inflammation, apoptosis and apoptosis by activating NLR 3 Redox Biol 2019, 101215.Song P, shen DF, meng YY, kong CY, zhang X, yuan YP, yan L, tang QZ, ma ZG. Geniposide precursors detection section 186-96), whereas GLP-1R is widely expressed in various tissues of the human body, including retina (Hebsgaard JB, pyC, yildiam E, knudsen LB, heegard S, kvist pH. Glucose-receptor Diipt-expression in 2018: 2304-8). Recent studies have shown that functional GLP-1R is expressed in Retinal Pigment Epithelium (RPE) cells, promoting apoptosis of RPE cells and progression of Diabetic Retinopathy (DR) (Puddu a, sanguineti R, montecuco F, viaani gl. Reliable pigment cells expresson functional receptor for glucose-polypeptide-1 (GLP-1), mediators Inflamm 2013 975032.Kim di, park mj, choice jh, lim sk, choice hj, park sh. Hyperbaric-induced GLP-1R lateral suspensions cells RPE cells, cell biology J59. However, the role and mechanism of geniposide for CNV development have not been clear so far.

Disclosure of Invention

The invention aims to provide a treatment method of Choroidal Neovascularization (CNV), which aims to solve the problems of side effects caused by repeated intraocular injection of anti-VEGF drugs and partial non-response of patients in the prior art.

In a first aspect of the present invention, there is provided the use of Geniposide (GEN) in the manufacture of a medicament for the treatment of choroidal neovascularization.

Further, the application is the application in preparing the medicine for reducing the exudation of choroidal neovascularization.

Further, the application is the application in preparing the medicine for reducing the damaged area of the choroidal neovascularization.

Further, the application is the application in preparing the medicine for reducing the volume of choroidal neovascularization.

Further, the application is the application in preparing the medicine for inhibiting proliferation, migration or lumen formation of Human Choroid Endothelial Cells (HCECs) under the hypoxic condition.

In a second aspect of the present invention, there is provided the use of geniposide in the preparation of a choroidal neovascularization inhibitor.

In a fourth aspect, the invention provides the use of geniposide in the preparation of a medicament for inhibiting proliferation, migration or luminal formation of human choroidal endothelial cells under hypoxic conditions.

In a third aspect of the present invention, there is provided a therapeutic agent for choroidal neovascularization, said agent comprising geniposide as an active ingredient.

The invention has the advantages that:

1. the invention provides application of geniposide in choroidal neovascularization treatment, and solves the problems of repeated intraocular injection of anti-VEGF drugs and no response of part of patients in the prior art.

2. The geniposide reduces the exudation, the damaged area and the volume of choroidal neovascularization of mice, inhibits the proliferation, the migration and the tube cavity formation of human choroidal endothelial cells under the condition of oxygen deficiency, and has similar curative effect compared with the Conbai (CON) which is a medicament for resisting Vascular Endothelial Growth Factor (VEGF) and is commonly used in clinic at present.

3. The geniposide is administered by intravenous injection, and avoids intraocular multiple injections and possible side effects such as endophthalmitis.

Drawings

FIG. 1 geniposide tail vein injection improves CNV exudation in mice. Graph A shows the experimental design, the first day of the experiment, the establishment of mouse CNV model, and the subsequent experiment after gardenoside tail vein injection on day 2 to day 6 and killing the mouse on day 7. Fig. B is a representative view of Fundus Fluorography (FFA). Panel C is an analysis of changes in choroidal exudate area using Image J software.

FIG. 2. Geniposide tail vein injection reduces CNV injured area in mice. Panel A is a representative image of indocyanine green angiography (ICGA). The B picture is the statistics of CNV regions, which shows that the damaged regions of CNV of three doses of geniposide and CON groups are reduced, and the action of geniposide is dose-dependent, compared with CNV 7-day group. ^* P<0.05， ^** P<0.01, compared to the CNV 7 day group.

Figure 3 tail vein injection of geniposide reduced the volume of CNV lesions in mice. Panel A shows the choroidal patch, which was fluorescently labeled with Collagen IV (Collagen IV, red), isolectin-B4 (isolectin-B4, green) and DAPI (blue). The B picture is the statistics of CNV volume, showing that the CNV volume of three doses of geniposide tail vein injection and CON intravitreal injection groups is reduced, and the effect of 30mg/kg/d is similar to that of 60mg/kg/d geniposide. ^* P<0.05， ^** P<0.01, compared to the CNV 7 day group.

FIG. 4 geniposide inhibits proliferation of human choroidal endothelial cells under hypoxic conditions. Panel A shows the EdU incorporation assay for detecting proliferation of Human Choroidal Endothelial Cells (HCECs). ^** P<0.01, compared to the normal group. ^# P<0.05, compared to the CNV 7 day group. Panel B is a statistical analysis of the ratio of EdU-positive cells to DAPI-positive cells.

FIG. 5 jasminoidin inhibits migration of human choroidal endothelial cells under hypoxic conditions. Panel A is Transwell experiment detection HMigration of CECs. Panel B is a statistical analysis of the number of migrating cells. ^** P<0.01, compared to the normal group. ^# P<0.05, compared to the hypoxic group.

FIG. 6 geniposide inhibits lumenal formation of human choroidal endothelial cells under hypoxic conditions. Panel A is an in vitro lumen formation assay to detect the lumen formation of HCECs. Panel B is a statistical analysis of lumen length. ^** P<0.01, compared to the normal group. ^# P<0.05, compared to the hypoxic group.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example (b):

1. experimental methods

(1) Establishment and grouping of mouse CNV model

Krypton laser-induced CNV mice were anesthetized with intraperitoneal injection of 0.5% pentobarbital, compound tropicamide mydriasis, hand-held light irradiation, and mice were kept at body temperature. The mouse experimental eye was fixed in front of a slit lamp, a 5.4mm hand-held contact lens was placed in front of the cornea with the aid of 1% methylcellulose, and krypton ion laser was used with a laser wavelength of 647.1nm, a power of 300mw, a spot diameter of 50 μm, and an exposure time of 0.05 sec. Photocoagulation is performed around the optic disc at equal distances at 2 nipple diameter positions of the optic disc, 4 points are counted, and the 4 points are respectively positioned at 3, 6, 9 and 12 points, so that bubbles are generated to indicate that the Bruch membrane is broken and no vitreous hemorrhage is taken as a success standard of photocoagulation. C57BL/6 mice were randomly divided into seven groups: a normal group (without any treatment), a CNV 7-day group (without any injection), a Phosphate Buffer Solution (PBS) tail vein injection group, a geniposide (15 mg/kg/d) tail vein injection group, a geniposide (30 mg/kg/d) tail vein injection group, a geniposide (60 mg/kg/d) tail vein injection group and a CON vitreous intracavity injection group. Wherein the geniposide group is injected into tail vein once a day, and the injection lasts from 2 days to 6 days after laser molding. The PBS group was injected with equal volumes of sterile PBS.

(2) Intravitreal injection of Corbina Xipu (CON) into mouse

CNV group C57BL/6 mice are weighed, 0.5% pentobarbital is used for intraperitoneal injection and anesthesia, compound tropicamide powder is used for enlarging pupils on two sides, normal saline is used for moistening the ocular surface, levofloxacin is used for dropping, the mice are laid on the side on an operating table, the eyelids are opened under a dissecting microscope, and the corneoscleral margin is exposed. A10-0 needle was used to make an incision 1mm behind the corneal scleral edge, a 33G syringe was inserted into the vitreous cavity, and 1. Mu.l of CON (trade name Lamu, chengdu Konghong Biotech Co., ltd., 10 mg/ml) was injected. The postoperative erythromycin eye ointment is applied to eyes to prevent infection.

(3) Mouse fundus angiography

C57BL/6 mice were randomly divided into seven groups: a normal group, a CNV 7-day group, a PBS tail vein injection group, a geniposide (15 mg/kg/d) tail vein injection group, a geniposide (30 mg/kg/d) tail vein injection group, a geniposide (60 mg/kg/d) tail vein injection group and a CON vitreous body cavity injection group. Mice were sacrificed by intraperitoneal injection of 0.5% pentobarbital, compound tropicamide mydriasis. 10% fluorescein sodium was diluted with water for injection to 2% fluorescein sodium or indocyanine green reagent, and 0.3ml was intraperitoneally injected, and Fundus Fluorography (FFA) or indocyanine green angiography (ICGA) was performed, respectively. Recording of the contrast process with a confocal laser retinal tomography scanner was started 100-140 seconds after injection. The recording time was 30min. CNV classification: grade 0 is no bleed, grade 1 is light bleed, grade 2a is moderate bleed, and grade 2b is heavy bleed. Changes in choroidal exudative area were analyzed using Image J software. Researchers draw an edge in the ICGA-oozed region by a freehand drawing tool, and apply a region of interest (ROI) manager of ImageJ software to calculate the pixel region. The pixel area is then converted to micron squared (microns) according to the ratio between pixel and micron (we use a ratio of 0.5249) ² ，mm ² ) And obtaining the final CNV area.

(4) Choroidal patch and immunofluorescence staining

C57BL/6 mice were randomly divided into six groups: CNV 7-day group, PBS tail vein injection group, geniposide (15 mg/kg/d) tail vein injection group, geniposide (30 mg/kg/d) tail vein injection group, geniposide (60 mg/kg/d) tail vein injection group and CON vitreous intracavity injection group. Mice were sacrificed by intraperitoneal injection with 0.5% excess pentobarbital, the eyeballs were immediately enucleated, and placed in 4% paraformaldehyde for rapid fixation for 1h. Under a dissecting microscope, the sclera is cut along the equator circuit, the anterior segment of the eye is removed, the retinal nerve epithelial layer is carefully separated, the Retinal Pigment Epithelium (RPE) -choroidal complex eye cup is obtained, and the eye cup is repeatedly washed with cold ICC buffer. Under dark light, 1. Mu.g/. Mu.l of Collagen IV (Collagen IV) and isolectin-B4 (isolectin-B4) antibody were added to ICC buffer at a dilution of 1. After mixing well, the mixture was transferred to an EP tube equipped with an eye cup for staining, and placed in a refrigerator at 4 ℃ in the dark overnight. Rewarming for 2h at normal temperature, and then fully washing the eye cup with ICC buffer solution. The retina-RPE-choroid complex was spread on a glass slide, 4 radial incisions were made in the direction of the optic disc, and the mounting was observed with a fluorescence microscope. Three-dimensional images of CNVs were generated with ZEN software (Zeiss, germany) while measuring the volume of CNVs.

(5) 5-ethynyl-2'-deoxyuridine (5-ethynyl-2' -deoxyuridine, edU) incorporation experiment for detecting human vascular endothelial cell proliferation

Culturing Human Choroidal Endothelial Cells (HCECs) and randomly dividing the HCECs into normal groups (20% O) ₂ ，5％CO ₂ Culture for 24 h), hypoxic group (1% ₂ ，94％N ₂ ，5％CO ₂ Cultured for 24 h) and hypoxia + gardenoside group (treated for 24h at 50. Mu.g/ml), proliferation of HCECs was detected by EdU incorporation assay, observed and photographed by fluorescence microscope.

(6) Transwell experiment for detecting migration of HCVEC

HCECs were randomly divided into normal group, hypoxic group and hypoxic + jasminoidin group, migration of HCVEC was detected by Transwell experiment, and 5 areas were randomly selected under phase contrast microscope (200X) for counting.

(7) Transwell chamber co-culture lumen forming model detection of lumen formation of HCVEC

The HCECs were randomly divided into normal, hypoxic and hypoxic + geniposide groups, the Transwell cell co-culture lumen shaping model detected the lumen formation of HCVEC, and the lumen length was measured using Image-Pro Plus 6.0 software.

(8) Statistical analysis

Data are expressed as mean ± standard error of the mean (SEM) of at least three independent experiments. Two sets of data were compared using the two-sided Student-t test, while multiple sets of data were compared using one-way anova and Bonferroni post hoc testing. P <0.05 is statistically different.

2. Results of the experiment

To test the therapeutic effect of geniposide in choroidal neovascularization, we established a laser-induced mouse CNV model with three doses (15 mg/kg/d, 30mg/kg/d, and 60 mg/kg/d) of geniposide injected caudal vein and with the currently widely used anti-VEGF drug Corbescept intravitreal injection as a positive control.

(1) Geniposide tail vein injection for improving CNV exudation of mice

As shown in fig. 1, fundus fluorography showed the change of the exudation area of choroid in geniposide group; wherein, the A picture is the experimental design, the first day of the experiment, the mouse CNV model is established, the geniposide tail vein injection is carried out on the 2 nd day to the 6 th day, the mouse is sacrificed on the 7 th day, and the subsequent experiment is carried out. Fig. B is a representative diagram of Fundus Fluorography (FFA). Panel C is an analysis of changes in choroidal exudate area using Image J software. The geniposide tail vein injection and CON intravitreal injection groups had increased grade 0 and grade 1 exudation, while grade 2a and grade 2b exudation were reduced compared to the CNV 7 day group. The results show that the exudation of the three doses of geniposide and CNV injured areas of the CON group is reduced, and the action of the geniposide is dose-dependent. ^** P<0.01, compared to the CNV 7 day group.

FFA of mouse fundus fluorography shows that 30mg/kg/d and 60mg/kg/d geniposide tail vein injection significantly reduce the exudation of CNV region, and the effect is similar to that of combaici cypress (FIGS. 1B and 1C).

(2) Geniposide tail intravenous injection reduced the mouse CNV injury area.

Referring to fig. 2, indocyanine green angiography (ICGA) of mouse fundus oculi showed that geniposide tail vein injection reduced areas of CNV damage in mice. Wherein Panel A is indocyanine green angiography (ICGA)Represents a figure. The B picture is the statistics of CNV regions, which shows that the damaged regions of CNV of three doses of geniposide and CON groups are reduced, and the action of geniposide is dose-dependent, compared with CNV 7-day group. ^* P<0.05， ^** P<0.01, compared to CNV 7 day group.

ICGA (fluorescence angiography) of mouse fundus shows that 30mg/kg/d and 60mg/kg/d geniposide tail vein injection obviously reduces the area of CNV (common cnidium) injury area, and the effect is similar to that of combaxipu (figures 2A and 2B).

(3) Gardenoside tail intravenous injection reduced the volume of CNV lesions in mice.

As shown in FIG. 3, fluorescence labeling of Collagen IV (Collagen IV; vascular endothelial cell marker) and isolectin (isolectin-4; vascular endothelial cell marker) showed that geniposide tail vein injection reduced the volume of CNV injury in mice. Wherein, the A picture is fluorescence labeling of Collagen IV (Collagen IV, red), isolectin-B4 (isolectin-B4, green) and DAPI (blue) after choroidal plating. The B picture is the statistics of CNV volume, showing that the CNV volume of three doses of geniposide tail vein injection and CON intravitreal injection groups is reduced, and the effect of 30mg/kg/d is similar to that of 60mg/kg/d geniposide. ^* P<0.05， ^** P<0.01, compared to the CNV 7 day group.

Fluorescence labeling of collagen IV and isolectin showed that tail vein injection of jasminoidin at 30mg/kg/d and 60mg/kg/d significantly reduced the volume of mouse CNV, and the effect was similar to that of combaici (FIGS. 3A and 3B).

(4) Geniposide inhibits proliferation of human choroidal endothelial cells under hypoxic conditions

See FIG. 4, experiments with 5-ethynyl-2' -deoxyuridine (EdU) incorporation to detect proliferation of Human Choroidal Endothelial Cells (HCECs). Wherein, A is the EdU incorporation experiment for detecting the proliferation of endothelial cells of human vascular membrane. ^** P<0.01, compared to the normal group. ^# P<0.05, compared to CNV 7 day group. Panel B is a statistical analysis of the ratio of EdU-positive cells to DAPI-positive cells. The hypoxic group had an increased proportion of EdU-positive cells compared to the normal group, while geniposide was able to down-regulate the proportion of EdU-positive cells under hypoxic conditions (fig. 4A and 4B).

(5) Geniposide inhibits migration of human choroidal endothelial cells under hypoxic conditions

See FIG. 5, the transwell experiment detects the migration of HCVEC. Wherein, A is the migration of HCECs detected by Transwell experiment. Statistical analysis of number of migrating cells in B-panels. ^** P<0.01, compared to the normal group. ^# P<0.05, compared to the hypoxic group. The hypoxic group migrated HCECs more than the normal group, while jasminoidin was able to inhibit migration of HCECs under hypoxic conditions (fig. 5A and 5B).

(6) Geniposide inhibits human choroidal endothelial cell luminal formation under hypoxic conditions

See FIG. 6, the transwell chamber coculture lumen profiling model detects the lumen formation of HCVECs. In which panel A is an in vitro tube lumen formation assay to detect tube lumen formation of HCECs. Panel B is a statistical analysis of lumen length. ^** P<0.01, compared to the normal group. ^# P<0.05, compared to the hypoxic group. The luminal formation of HCECs was increased in the hypoxic group compared to the normal group, whereas geniposide was able to inhibit luminal formation of HCECs under hypoxic conditions (FIGS. 6A and 6B).

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.

Claims

1. The application of geniposide in preparing choroidal neovascularization intravenous injection therapeutic medicine; the application is the application in preparing the medicine for reducing the exudation of choroidal neovascularization, reducing the injury area of the choroidal neovascularization, reducing the volume of the choroidal neovascularization and inhibiting the proliferation, migration or lumen formation of human choroidal endothelial cells under the hypoxic condition.