CN116785440A - Application of Sema4D inhibitor in preparation of medicament for preventing and/or treating choroidal neovascularization - Google Patents
Application of Sema4D inhibitor in preparation of medicament for preventing and/or treating choroidal neovascularization Download PDFInfo
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Abstract
The invention provides application of a Sema4D inhibitor in preparing medicaments for preventing and/or treating choroidal neovascularization. The Sema4D inhibitor can obviously inhibit the angiogenesis of a laser-induced choroidal neovascularization model, lighten the vascular leakage of the model, and provide a new strategy for the treatment of pathological choroidal neovascularization and leakage.
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to application of a Sema4D inhibitor in preparation of a medicine for preventing and/or treating choroidal neovascularization.
Background
Age-related macular degeneration (Age-related macular degeneration, AMD), a degenerative disease that primarily involves the macula area, is one of the most important causes of vision impairment in people over 60 years of Age. The number of people suffering from AMD rises year by year, and the estimated number of people suffering from AMD in the world reaches 2.88 hundred million by 2040 years, which causes a huge burden on society. It follows that in the future, where the aging society will become more and more advanced, AMD will become a major public health problem and have a major impact on socioeconomic and hygienic aspects.
AMD is characterized by complex and variable pathology, and depending on the pathology, AMD can be classified into dry AMD (dry-AMD is also known as non-exudative or atrophic AMD) and wet AMD (wet-AMD is also known as exudative or neovascular AMD). Dry AMD is mainly characterized by the appearance of drusen in the macular area, abnormal changes in retinal pigment epithelial cells (retinal pigment epithelium, RPE), geographic atrophy, etc. Most dry AMD progresses slowly and has a better prognosis for vision. Wet AMD is mainly characterized by choroidal neovascularization (choroidal neovascularization, CNV), an immature CNV that causes exudation and bleeding, and causes numerous complications such as retinal edema, pigment epithelial detachment, and subretinal proliferation membrane formation, leading to severe vision loss. The wet-AMD affected population, while only 10% of AMD patients, resulted in a 90% AMD related blinding rate. Therefore, the method is particularly important for early intervention and timely treatment of wet-AMD patients.
Current treatments for AMD are quite limited, with the most common treatments being laser photocoagulation, intravitreal injection of steroid hormones and anti-vascular endothelial growth factor (Vascular endothelial growth factor, VEGF) treatment. There are still many limitations to the above treatments, including the high cost of the drug, and repeated intraocular injections increase the risk of local injury, endophthalmitis, RPE atrophy and other serious complications, and more importantly, there is still a significant proportion of patients who respond poorly, or even without responding, to the treatment.
The Semaphorins family is a family of proteins containing extracellular sema domains, whose members can chemotaxis and direct nervous system development by regulating neuronal proliferation, differentiation, migration, polarity, axonal shear, synapse formation, and maturation. Sema4D (Class 4 semaphorins) is used as a star molecule in Sema family, plays an important role locally and remotely through membrane combination and extracellular dissociation, and participates in cardiovascular development and angiogenesis, maintains bone dynamic balance, regulates tumor microenvironment, immune response, maintains retinal homeostasis and other physiological or pathological processes.
The invention confirms that the Sema4D inhibitor can obviously inhibit the angiogenesis of a laser-induced choroidal neovascularization model, lighten the vascular leakage of the model and provide a new strategy for the treatment of pathological choroidal neovascularization and leakage.
Disclosure of Invention
In view of this, the present invention aims to overcome the drawbacks of the prior art and proposes the use of Sema4D inhibitors for the preparation of a medicament for the prevention and/or treatment of choroidal neovascularization.
In order to achieve the above purpose, the technical scheme of the invention is realized as follows:
in a first aspect of the invention, there is provided the use of a Sema4D inhibitor in the manufacture of a medicament for the prevention and/or treatment of choroidal neovascularisation.
Preferably, sema4D inhibitors include, but are not limited to Sema4D/PlexinB1 signaling pathway inhibitors.
Preferably, sema4D inhibitors include, but are not limited to, sema4D neutralizing antibodies.
Preferably, the choroidal neovascularization disease is pathological choroidal neovascularization and leakage.
More preferably, the choroidal neovascularization disease is pathological choroidal neovascularization and leakage caused by argon ion laser damage.
In a second aspect of the invention there is provided a medicament for the prophylaxis and/or treatment of choroidal neovascularisation, said medicament comprising a Sema4D inhibitor.
Preferably, the medicament further comprises anti-VEGF (anti-vascular endothelial growth factor).
More preferably, the Sema4D inhibitor is a Sema4D neutralizing antibody.
Compared with the prior art, the invention has the following advantages:
the inventor finds that the Sema4D inhibitor can obviously inhibit the angiogenesis of a laser-induced choroidal neovascularization model in the research process, lightens the vascular leakage of the model, and provides a new strategy for the treatment of pathological choroidal neovascularization and leakage. In addition, the inventors have found that Sema4D/PlexinB1 signaling pathway inhibitors are effective in inhibiting choroidal angiogenesis and leakage and have synergistic effects with anti-VEGF treatment; meanwhile, the effect of inhibiting Sema4D/plexinB1 signal pathway on pathologic choroidal angiogenesis and leakage is proved in-vivo gene knockout mice and in-vitro angiogenesis models.
Drawings
Fig. 1 is a graph showing the effect of various groups of medicines injected into a vitreous cavity on OCT tomography of the fundus and fundus photography and thickness variation statistics (A. OCT schematic diagrams before and after various groups of medicines are injected into the vitreous cavity show that 4 administration modes cannot damage the structure of retina; B. Statistical graphs of the thickness of the whole retina after various groups of medicines are injected into the vitreous cavity have no obvious difference; C. Eyeball slices after various groups of medicines are injected into the vitreous cavity have no obvious Tunel staining positive areas);
FIG. 2 is a graph showing statistics of the leakage area and volume of choroidal neovascularization by intravitreal injection of each group of drugs (A. Choroidal neovascularization modeling and 7 days after intravitreal injection of each group of drugs administration, FFA and CD31 staining are typical; B. Statistics of FFA leakage area between groups show that the Sema4D neutralizing antibody group (anti-Sema 4D) can significantly reduce leakage area as well as the Conbasic group (combeppt), and that both can have synergistic effects when used simultaneously; C. CD31 volume statistics show the same trend as FFA);
FIG. 3 is a graph of contrast and choroidal flatmount staining results and associated statistics (therapeutic effect) of Sema4 d-/-mice in a laser induced CNV model (A. 7 days after modeling of choroidal neovascularization, typical graphs of Sema4 d-/-and Sema4d+/+ mice FFA and CD31 staining; B/C. FFA leak area and CD31 volume statistics for two groups, it can be seen that the Sema4 d-/-group is significantly lower than the Sema4d+/+ group);
FIG. 4 shows the choroidal budding experiments (A. Typical bright field and phalloidin staining patterns for exogenous administration of recombinant SEMA4D protein in the choroidal budding model; B. Statistical results for vessel length (left) and vessel number (right) in FIG. A. It can be seen that the addition of recombinant SEMA4D significantly increases the vessel length (left) and vessel number (right) of the explants, C. Sema 4D-/-and Sema4d+/+ mice, typical bright field and phalloidin staining patterns in the choroidal budding model; D. Statistical results for vessel length (left) and vessel number (right) in panel C, it can be seen that Sema 4D-/-mice have lower vessel length (left) and vessel number (right) than Sema4d+/+ mice).
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the inventive concepts pertain. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The present invention will be described in detail with reference to examples.
The following examples are provided in the following sections:
SPF grade male wild type C57BL/6J mice (Beijing velutini Hua Co.) and Sema4 d-/-mice (Jiangsu JieXueyaokang) at 6-8 weeks of age.
Mice were kept in SPF-grade animal houses at 25℃in Tianjin medical university, cycled for 12h of light, and given sufficient food and water. All studies concerning animal use were approved by the institutional animal care and use committee of the university of Tianjin medical science and followed the ARVO statement made by the American society of visual and ophthalmology research. All experimental procedures were in accordance with national institutes of health laboratory animal care and use guidelines.
CD31 anti-body (MAB 1398Z) (Millipore Co.); ophthalmic surgical microscope (motic company); microinjectors (Hamilton, usa); LSM 900 laser confocal microscope (Zeiss); confocal laser fundus angiography System (Heidelberg Co.), sema4D neutralizing antibody (invitrogen, 14-1001-82), sema4D recombinant protein (acrobiosystems, CD 0-H5257).
The method of the examples is as follows:
example 1 modeling method of laser-induced CNV model
This example includes therapeutic experiments and Sema4d gene knockout experiments. Wherein the therapeutic experiment is 20 male 6-8 week old C57BL/6J mice, and the Sema4d gene knockout experiment is 5 male 6-8 week old Sema4d+/+ mice and 5 Sema4 d-/-mice. Two times after mydriasis of compound topiramate eye drops, 1.6g/ml tribromoethanol (0.2 ml/10g body weight) is adopted to anesthetize mice, photocoagulation is carried out at 3, 6, 9 and 12 o' clock positions around a video disc, and laser parameters are set: 100 μm in diameter, 150ms in exposure time, and 200mW in power, based on bubble generation, suggesting that the Bruch film was broken.
EXAMPLE 2 intravitreal injection
This example included a safety trial and a therapeutic trial, the safety trial aimed at 20C 57BL/6J male mice for 6-8 week adults, with the mice randomly divided into 4 groups of 5. The administration was according to the following dosing system: the first group was 1.5ul (0.75ul IgG+0.75ul normal saline), the second group was 1.5ul (0.75 ul of kangbuji+0.75 ul normal saline), the third group was 1.5ul (0.75 ul of sema4d neutralizing antibody+0.75 ul normal saline), and the fourth group was 1.5ul (0.75 ul of sema4d neutralizing antibody+0.75 ul of kangbuji).
In the therapeutic experiments, 20C 57BL/6J mice in example 1 were randomly divided into 4 groups of 5 mice each, and immediately after successful molding of the CNV model, intravitreal injections were given. The grouping and the drug delivery system are consistent with the safety experiment.
All of the above intravitreal injections used a 33-gauge Hamilton microinjector. Wherein, the concentration of the Sema4D neutralizing antibody is 1ug/ul, and the concentration of the Kangbai cilp is 10mg/ml.
EXAMPLE 3 choroidal flatmount immunofluorescent staining
CO 2 After anesthetizing mice, eye samples were collected, fixed with 4% paraformaldehyde for 1 hour, and the choroidal-RPE-scleral complex was isolated under a microscope using scissors forceps, permeabilized with 1% Triton in PBS for 12 hours, and then incubated in blocking buffer (PBS containing 0.3% Triton and 2% BSA) at 4 ℃ for 12 hours. Subsequently, the samples were incubated at 4℃for 2 days with primary antibody (diluted 1:150 in blocking buffer). Followed by three washes in PBS containing 0.1% Triton and incubation at room temperature for 2 hours in a suitable concentration of secondary antibody (dilution in blocking buffer, 1:300), followed by three washes in PBS containing 0.1% Triton followed by overnight washes and then blocking with neutral resinAnd (3) a sheet. The images were taken with a zeiss LSM 900 confocal microscope. Quantitative analysis was performed using Image J and imaris software.
The statistical graph of the effect of the drug on OCT (optical coherence tomography) and fundus photography of the fundus and thickness variation of the fundus is shown in FIG. 1, and the statistics of the leakage area of the choroidal neovascularization and the volume of the neovascularization are shown in FIG. 2. As can be seen from fig. 1, the 4 administration modes do not damage the general structure of retina, the thickness of the whole retina after the intravitreal injection of each group of medicine has no obvious difference, and each group has no obvious Tunel staining positive area; as can be seen from fig. 2, the Sema4D neutralizing antibody group (third group) reduced the leakage area as significantly as the cobra and western medicine group (second group), and both Sema4D neutralizing antibody and cobra and western medicine used simultaneously can have a synergistic effect.
Example 4 mouse fluorescein fundus angiography
For all mice in example 1, on day 7 after CNV molding, after mydriasis of compound topiramate eye drops, 1.6g/ml of tribromoethanol (0.2 ml/10g body weight) was injected intraperitoneally to anesthetize the mice, 10% fluorescein sodium (0.1 ml/kg) was injected intraperitoneally, the heads of the mice were fixed with the lenses aligned to the pupil positions, and a mouse fundus fluorescence angiography was acquired using a hadburg confocal laser fundus scanning system (Heidelberg Retina Angiography, HRA) and quantitatively analyzed using Image J software.
EXAMPLE 5 choroidal explant vascular sprouting experiment
This example is divided into exogenous SEMA4D budding experiments and SEMA4D knockout budding experiments. The exogenous SEMA4D budding experiment selects 6 male 6-8 week old C57BL/6J mice, and randomly divides the mice into 3 control groups and 3 SEMA4D groups. Sema4d knockout budding experiments 6 male 6-8 week old mice were selected, of which Sema4 d-/-group 3, sema4d+/+ group 3. The choroidal in vitro vascular sprouting model was constructed by culturing mouse choroidal grafts in matrigel. Fresh eyeballs are taken from 2 month old mice, extrabulbar fascia and muscles are separated, choroidal scleral complex is obtained by separation, the complex is sheared into 1 x 1mm size plant pieces, and the plant pieces are transferred into a culture medium for incubation for 5 hours (37 ℃,5% CO) 2 ). The matrigel is sucked into 10ul and dripped into the center of a 48-well plate at 37 DEG CIncubating for 5min until solidification, transferring the separated implant to the surface of matrigel, sucking the culture medium, dripping 10ul matrigel on the surface of the implant, and incubating for 5min again until solidification. Adding a culture medium (wherein the SEMA4D group of the exogenous SEMA4D sprouting experiment is added with SEMA4D recombinant protein (the concentration is 1.6 ug/ml)), photographing by an inverted microscope in a bright field after 4 days, and observing the sprouting condition of blood vessels. The cytoskeleton was labeled with the dye phalloidin and photographed by a zeiss LSM 900 confocal microscope. Quantitative analysis was performed using Image J software and vessel length and vessel count were quantified.
As a result, as shown in FIG. 4, the addition of recombinant SEMA4D in the growth of C57BL/6J mouse choroidal explants significantly increased the vascular length and vascular fraction of the explants (A, B). Meanwhile, by comparing Sema4d+/+ mice with Sema4 d-/-mice, the latter choroidal explants had lower vessel lengths and vessel branches.
The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
- Use of a sema4d inhibitor for the preparation of a medicament for the prevention and/or treatment of choroidal neovascularization.
- 2. The use according to claim 1, characterized in that: sema4D inhibitors include, but are not limited to, sema4D/plexinB1 signaling pathway inhibitors.
- 3. The use according to claim 1, characterized in that: sema4D inhibitors include, but are not limited to, sema4D neutralizing antibodies.
- 4. The use according to claim 1, characterized in that: the choroidal neovascularization disease is pathologic choroidal neovascularization and leakage.
- 5. The use according to claim 1, characterized in that: the choroidal neovascularization disease is pathologic choroidal neovascularization and leakage caused by argon ion laser injury.
- 6. A medicament for preventing and/or treating choroidal neovascularization, characterized by: the medicament comprises a Sema4D inhibitor.
- 7. A medicament according to claim 3, characterized in that: the medicament also comprises anti-VEGF.
- 8. A medicament according to claim 3, characterized in that: sema4D inhibitors include, but are not limited to, sema4D neutralizing antibodies.
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