Application of emipril in preparation of medicine for improving diseases related to choroidal neovascularization

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of emipril in preparation of a medicine for improving choroidal neovascularization related diseases. The invention aims to solve the problems of strong invasiveness and limited curative effect of the existing therapy. It is found that emipril can achieve differential regulation and control on a CD26 system in vivo, that is, the enzyme activity of CD26 in bone marrow is improved, the enzyme activity of CD26 in peripheral blood is reduced, the concentration gradient of stromal cell-derived factor-1 can be established between the bone marrow and the peripheral blood through differential regulation and control, and the concentration gradient of the stromal cell-derived factor-1 can be further improved. The bone marrow-derived cells are driven to homing to eye lesions and differentiate into repair cells, so that eye diseases are repaired or improved, meanwhile, the collection and migration of bone marrow-derived macrophages to CNV regions can be increased, and the anti-inflammatory effect is achieved. According to the application, an endogenous repair mechanism is activated through non-invasive administration, and a safer and more effective treatment method is provided for related eye diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to application of emmprizide in preparation of a drug for improving choroidal neovascularization related diseases. BACKGROUND

[0002] Neovascular age-related macular degeneration (wet AMD) is one of the main causes of severe vision loss or even blindness in the elderly population, accounting for half of the global cases of legally irreversible blindness. Wet AMD is closely related to choroidal neovascularization (CNV), and the core pathological feature is the formation of choroidal neovascularization. In the eye, the abnormal neovascular structure is unstable and prone to leakage and hemorrhage. Persistent leakage and hemorrhage of CNV can lead to local scarring and subsequent fibrosis, which damages the normal retinal structure, eventually causes photoreceptor atrophy, and leads to photoreceptor death and irreversible vision loss.

[0003] The molecular mechanism of CNV has not been fully elucidated. Studies have shown that vascular endothelial growth factor (VEGF) dysfunction plays a key role in the occurrence and development of CNV. Based on this theory, drugs that can inhibit the function of VEGF have become the main method to inhibit choroidal neovascularization (CNV). However, vascular endothelial growth factor (VEGF) is only involved in the early stage of angiogenesis, and VEGF inhibitors are only effective for less than 50% of patients with neovascular age-related macular degeneration (AMD). Therefore, there is an urgent need to develop new targets and strategies to treat CNV.

[0004] Currently, the first-line treatment for such diseases in clinical practice is to repeatedly inject anti-vascular endothelial growth factor drugs into the eyes of patients. However, this method has many shortcomings. First, its mechanism of action is mainly to inhibit the early stage of angiogenesis, and its therapeutic effect on pathological vascular structures that have already formed is limited, and it is only effective for a portion of patients and is difficult to cure. Second, repeated intraocular injection is an invasive procedure that not only brings physical pain and discomfort to patients, but also increases the risk of complications such as intraocular infection.

[0005] In order to overcome the limitations of the above method, existing technologies also propose a strategy of using exogenous mesenchymal precursor cells for transplantation therapy, hoping to repair damaged ocular tissues through transplanted cells. However, this cell therapy faces great challenges in actual application. It relies on complex in vitro cell culture, separation and purification, and invasive transplantation surgery, the whole process is complex and costly. In addition, the introduction of exogenous cells may also trigger immune rejection and involve certain ethical controversies, which all seriously limit its clinical promotion and widespread application.

[0006] Therefore, there is an urgent need in the art to develop a new therapeutic strategy with clear mechanism of action, which can be administered by non-invasive means and effectively activate the body's own repair ability, to solve the problems of limited efficacy, narrow applicability, the need for invasive administration or difficulty in implementation in the prior art for treating choroidal neovascularization. SUMMARY

[0007] Based on the above technical background, the main purpose of the present application is to provide the application of emmprizil in the preparation of drugs for improving choroidal neovascularization related diseases, in order to overcome the shortcomings in the prior art.

[0008] To achieve the aforementioned purposes, the technical solutions adopted by the present application comprise: The present application provides, in a first aspect, the application of emmprizil in the preparation of drugs for improving choroidal neovascularization related diseases.

[0009] The choroidal neovascularization related disease is neovascular age-related macular degeneration (AMD).

[0010] Emmprizil is an angiotensin converting enzyme inhibitor (ACEI) with antihypertensive activity.

[0011] It has been found through experiments that the emmprizil can differentially regulate the CD26 / DPP IV system in the bone marrow and peripheral blood of mammals, and the emmprizil can up-regulate the CD26 / DPP IV activity in the bone marrow and down-regulate the CD26 / DPP IV activity in the peripheral blood.

[0012] CD26, a type II transmembrane glycoprotein, is a dipeptidyl peptidase IV (also known as DPPIV), which is expressed on the cell surface in a membrane-bound form and released into the circulation in a soluble form.

[0013] It has been found through experiments that the emmprizil can reverse the concentration gradient of stromal cell-derived factor-1 (SDF-1) between the bone marrow and the peripheral blood by up-regulating the CD26 / DPP IV activity in the bone marrow and down-regulating the CD26 / DPP IV activity in the peripheral blood; and the concentration gradient reversal can drive the bone marrow-derived cells (BMCs) to migrate from the bone marrow to the peripheral blood and be recruited to the lesion area of the choroidal neovascularization (CNV) for the repair of choroidal neovascularization.

[0014] The hydrolysis of SDF-1 by CD26 is essential for regulating the migration of bone marrow-derived cells. It has been found that treatment with imipridone can significantly increase the enzymatic activity of CD26 in bone marrow, thereby resulting in a decrease in the level of SDF-1 in bone marrow (BM). However, the activity of CD26 in peripheral blood (PB) is decreased along with an increase in the concentration of SDF-1, thereby forming a concentration gradient of SDF-1 between the peripheral blood and the bone marrow, which promotes the migration of bone marrow cells to the peripheral blood. At the same time, the migration of bone marrow cells (BMCs) to the peripheral blood is accompanied by an increase in the concentration of SDF-1. In addition, CNV tissues also express a large amount of SDF-1.

[0015] The imipridone can promote the migration of bone marrow-derived cells (BMCs) in the bone marrow to the peripheral blood and the CNV region, and increase the proportion of bone marrow-derived cells in the peripheral blood and the CNV region. In particular, the imipridone can increase the mobilization of a subpopulation of BMCs, EPCs, to the peripheral circulation by disrupting SDF-1 / CXCR4, increase the proportion of EPCs (endothelial progenitor cells) in the peripheral blood and the CNV region, and reduce the severity of CNV.

[0016] The CD26 / DPP IV system has the ability to mobilize and expand BMCs in vivo, which provides a new BMC cell therapy strategy for the treatment of eye diseases.

[0017] BMCs include various types of stem cell and progenitor cell populations, such as endothelial progenitor cells, hematopoietic stem cells, and mesenchymal stem cells.

[0018] The imipridone can promote the migration of BMCs to the peripheral blood and the CNV region by acting on the CD26 / SDF-1 receptor, and induce the differentiation of the BMCs into cells with tissue repair or immunomodulatory functions, thereby repairing the CNV lesion.

[0019] Further, the imipridone can promote the differentiation of bone marrow-derived cells in the peripheral blood and the CNV region into cells with tissue repair or immunomodulatory functions, or recruit macrophages to migrate to the CNV region, thereby promoting the repair of the choroidal neovascularization.

[0020] Preferably, the cells with tissue repair or immunomodulatory functions include various cell subpopulations such as retinal pigment epithelial cells (RPE cells), microglial cells, etc., which can collectively promote the repair of CNV.

[0021] The microglial cells include astrocytes, oligodendrocytes, etc.

[0022] BMCs participate in the damage repair response of CNV formation, and the essence of BMCs cell therapy for CNV is to promote the repair and regeneration of tissue damage and delay the process of tissue scar healing. The repair mechanism of BMCs for CNV damage is as follows: BMCs differentiate into RPEs to play a role in regeneration and repair, BMCs activate retinal glial cells, play a secretory role, and BMCs selectively recruit bone marrow-derived macrophages to play an anti-inflammatory role.

[0023] More specifically, the mechanism of action is as follows: (1) BMCs recruit and migrate to CNV, which can differentiate into retinal pigment epithelial cells (RPEs) and have a role in damage repair; by blocking local SDF-1 / CXCR4, it can reduce the expression of endothelial cell adhesion molecules and endothelial cell lumen formation and migration. Bone marrow-derived RPEs can significantly reduce VEGF expression and have an anti-neovascularization effect. BMCs can migrate to the subretinal space and differentiate into RPEs to regenerate and repair damaged RPE cell layers. These regenerated RPEs can also protect adjacent photoreceptors.

[0024] (2) BMCs recruit and differentiate into microglial cells, activate glial cells, and play a secretory regulatory role: in laser-induced CNV, CD26 mobilizes BMCs to the CNV area, which can differentiate into microglial cells. These bone marrow-derived microglial cells aggregate in large numbers, activate glial cells, increase the expression of transforming growth factor (TGF-β) mRNA, and TGF-β can inhibit the proliferation of retinal endothelial cells. Studies have found that bone marrow-derived microglial cells differentiated from BMCs recruitment have a secretory effect, and these secretory effects have a reverse regulatory effect that can activate and increase glial cells and migration, allowing cells to adapt to changing microenvironments and have long-term protective effects. This is different from the short-term effect of anti-neovascularization therapy in the prior art, which requires repeated injections of exogenous BMCs cells, increasing the risk and cost of treatment.

[0025] (3) BMCs can recruit increased bone marrow-derived macrophage migration to the CNV area, and play an anti-inflammatory role: bone marrow-derived macrophages have an anti-inflammatory effect. Inflammation is a major mechanism in the pathogenesis of AMD, which can regulate the occurrence and development of CNV. In the laser-induced CNV area, RPE damage can induce the recruitment of bone marrow-derived macrophages. The present application further proves that the mobilization of BMCs in vivo can increase the recruitment and migration of bone marrow-derived macrophages to the CNV area when RPEs are damaged, and secrete anti-inflammatory mediators IL-10 to reduce the formation of CNV. MMP-2 / MMP-13 (matrix metalloproteinase) can degrade extracellular matrix and play a key role in promoting BMCs movement. Specifically, the recruitment and migration of BMCs to CNV and the increased expression of matrix metalloproteinase (MMP-2 / MMP-13) are related, and MMP-2 / MMP-13 is rapidly up-regulated when BMCs are recruited and migrated to the CNV damage area, and most of the MMP-2 / MMP-13 expression comes from BMCs.

[0026] Emiplimab can mobilize BMCs in vivo by regulating the enzyme activity of CD26 to provide autologous cell intervention in the CNV course and play a protective role. The experiments of the present application found that activating the CD26 peptidase system in vivo can mobilize and expand BMCs, increase the migration and homing of BMCs to the CNV damage area, and significantly inhibit the occurrence and development of CNV. Experiments have shown that the mobilization and expansion of CD26 in vivo can increase the migration of BMCs to the choroidal damage area and reduce the formation of CNV, which is similar to the effect of intraocular transplantation of BMCs.

[0027] Experiments have confirmed that activating the CD26 peptidase system can effectively mobilize and expand BMCs in vivo to intervene in the CNV course, and the mobilized and expanded BMCs exhibit endogenous damage repair effects. Specifically, the therapeutic mechanism of BMCs recruited and migrated to the intraocular lesion involves the differentiation of BMCs into RPEs to play a regenerative repair role, the activation of retinal glial cells to play a secretory protective role, and the recruitment of bone marrow-derived macrophages to play an anti-inflammatory role.

[0028] Research has found that: (1) Emiplimab can activate the CD26 peptidase system to effectively mobilize and expand BMCs in vivo to migrate to the CNV area; (2) increasing the activity of the CD26 peptidase system can increase the recruitment and migration of BMCs to the CNV area in the eye, and the therapeutic mechanism of BMCs for CNV involves: the differentiation of BMCs into RPEs to play a regenerative repair role; the activation of retinal glial cells to play a secretory protective role; the recruitment of bone marrow-derived macrophages to play an anti-inflammatory role; (3) CD26 can mobilize BMCs in vivo to provide autologous cell intervention in the CNV course and play a protective role.

[0029] The emipliride can effectively shorten the length of choroidal neovascularization (CNV), reduce the thickening and volume of CNV, and reduce the retinal choroid permeability, thereby playing a good improvement and treatment effect on choroidal neovascularization.

[0030] The second aspect of the present application provides a drug for improving choroidal neovascularization related diseases, which comprises an active ingredient and a pharmaceutical excipient.

[0031] The active ingredient is emipliride.

[0032] The dosage of the active ingredient is 0.5-1.5 mg / kg / day.

[0033] Preferably, the dosage of the active ingredient is 1.0 mg / kg / day.

[0034] It is found through experiments that the drug plays a therapeutic effect on mammals by the following mechanisms after administration: Step one: the active ingredient emipliride in the drug differentially regulates the CD26 / DPP IV system in the bone marrow and peripheral blood of the mammal, so as to realize the up-regulation of CD26 / DPP IV activity in the bone marrow and the down-regulation of CD26 / DPP IV activity in the peripheral blood; Step two: the differential regulation in step one causes the concentration of stromal cell-derived factor-1 (SDF-1) in the bone marrow to decrease, and the concentration of stromal cell-derived factor-1 in the peripheral blood to increase, thereby forming a concentration gradient inversion of stromal cell-derived factor-1 between the bone marrow and the peripheral blood; Step three: the concentration gradient inversion of stromal cell-derived factor-1 drives the migration of bone marrow-derived cells from the bone marrow to the peripheral blood, and recruits the bone marrow-derived cells to the lesion area of the choroidal neovascularization; Step four: the bone marrow-derived cells recruited to the lesion area are induced to differentiate into one or more cells with tissue repair or immunoregulatory function, and the bone marrow-derived macrophages can also be recruited to migrate to the lesion area, thereby promoting the repair of the choroidal neovascularization, shortening the length of the choroidal neovascularization, reducing the thickening and volume of the choroidal neovascularization, and playing a good improvement and treatment effect on the choroidal neovascularization.

[0035] The drug is administered orally or subcutaneously, which avoids the complicated operation, immune rejection and ethical issues of cell transplantation, is safer and more convenient to operate.

[0036] The present application has the following beneficial effects: (1) The present application researches and finds that emmpril can mobilize endogenous bone marrow-derived cells to migrate to the lesion area of choroidal neovascularization through regulating CD26 / matrix cell-derived factor-1 (SDF-1) axis, and promote the differentiation of bone marrow-derived cells into one or more cells with tissue repair or immunoregulatory function, while increasing the recruitment and migration of bone marrow-derived macrophages to the CNV area, participating in the repair and anti-inflammatory effect of choroidal neovascularization, thereby providing a solid theoretical basis for drug application. The drug with emmpril as the active ingredient can effectively reduce the pathological severity of choroidal neovascularization by non-invasive methods such as oral administration, significantly reduce the length, thickness and volume of the lesion, and reduce vascular permeability.

[0037] (2) The present application proposes a new treatment mode of "mobilizing endogenous stem / endothelial progenitor cells for tissue repair". Compared with the invasive therapy of directly injecting exogenous stem cells, the repair method avoids the complex operation of cell transplantation, immune rejection and ethical problems, has higher safety, is more convenient to operate, and has better clinical application prospect; In addition, it is found that the in vivo mobilization of BMCs can continuously provide sufficient autologous cells to repair and regenerate the damaged and degenerative retina and choroid tissue, and the advantage of this cell therapy is that the BMCs can adapt to the surrounding changing microenvironment, and can play a long-term protective role in the eye. Unlike the current anti-angiogenic therapy, the newly formed blood vessels cannot permanently regress, and new blood vessels often occur again within a few months, and many patients need repeated injection therapy, which increases the risk and cost of treatment. The in vivo mobilization of BMC cells proposed in the present application has more practical value.

[0038] (3) The mechanism of emmpril in improving and repairing CNV disclosed by the present application not only provides a new therapy for neovascular age-related macular degeneration, but also can be applied to other eye diseases related to choroidal neovascularization, provides a new direction for developing a broad-spectrum ophthalmic drug that can activate the endogenous repair potential, and has important clinical significance and commercial value. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Show the effect of pharmacological regulation of CD26 on the proportion of CD26 cells in peripheral blood and bone marrow and enzyme activity. +

[0040] Figure 2 Show the effect of CD26 regulation on the pathological severity of CNV.

[0041] Figure 3 Show CD26 / DPP IV regulation of peripheral blood and CNV area BMC.

[0042] ​Figure 4 This demonstrates the response of SDF-1 expression to CD26 regulation in CNV mice.

[0043] Figure 5 The differentiation of BMCs in the CNV region is shown in response to enimapril treatment.

[0044] Figure 6 The expression of bone marrow-derived RPEs in CNV is shown 14 days after laser-induced CNV. Vehicle (solvent group) (Figure A) and imidapril treatment group (Figure B); GFP in both groups. + / RPE + Comparison of fluorescence area (Figure C) and CNV fluorescence area (Figure D) (*p<0.05). In the figures, GFP is green, RPE-65 is red, and Merge(GFP) is red. + / RPE-65 + The color is yellow (scale bar 50um), and DAPI, a cell nucleus-specific marker, is blue.

[0045] Figure 7 The expression of SDF-1 and VEGF in BMCs is shown on day 14 after laser induction. Solvent control group (Figure A) and enimapril treatment group (Figure B); BMCs in both groups. + / SDF-1 + / VEGF + Comparison of fluorescence areas of the three labels (Figure C) (*p<0.05). GFP is green, VEGF is red, SDF-1 is blue, and Merge (fusion) (BMC) + / VEGF + / SDF-1 + (The color is bright white (scale bar 50um).)

[0046] Figure 8 This image shows the expression of bone marrow-derived microglia in the retinal-choroidal complex 14 days after laser-induced CNV. Immunofluorescence co-labeling was performed in the solvent group (Fig. A) and the enimapril treatment group (Fig. B); a comparison of CNV fluorescence area between the two groups (Fig. C) and bone marrow-derived microglia (GFP) expression was also observed. + / CD11b + Comparison of fluorescently labeled areas (Figure D) (*p<0.05). Microglia derived from BMCs were observed using immunofluorescence confocal microscopy. GFP-positive cells were green, CD11b-positive cells were red, and Merge (GFP-positive cells) were red. + / CD11b + ( ) is yellow, and DAPI, a cell nucleus-specific marker, is blue.

[0047] Figure 9The image shows immunofluorescence staining of retinal glial fibrillary acidic protein (GFAP) in each group 14 days after laser-induced CNV (Figure A). GFAP is labeled with red fluorescence; DIPA is used to stain cell nuclei blue (scale bar 50µm). Comparison of the fluorescence area of ​​various GFAPs (Figure B). Representative bands of GFAP (50 kDa) in the retina of each group are shown in Western blot (Figure C); Western blot grayscale analysis is shown in Figure D (*p<0.05). Expression of TGFβ-1 and IGF-1 in the retinal-choroidal complex is also shown in Figure E (*p<0.05). RT-PCR detection of TGF-β1 and IGF-1 expression levels is also presented.

[0048] Figure 10 The image shows the expression of bone marrow-derived macrophages in CNV 14 days after laser-induced CNV. Immunofluorescence co-labeling was performed in the solvent group (Fig. A) and the enimapril treatment group (Fig. B); comparison of CNV area between the two groups (Fig. C) and bone marrow-derived macrophages (GFP). + / F4 / 80 + Comparison of co-labeled fluorescent areas (Figure D) (*p<0.05). GFP positive is green, F4 / 80 positive is red, and Merge (GFP) positive is red. + / F4 / 80 + ( ) is yellow, and DAPI, a cell nucleus-specific marker, is blue.

[0049] Figure 11 This shows the number of peripheral blood bone marrow-derived macrophages (Sca-l) in each group 12 days after laser-induced CNV. + / F4 / 80 + FACS count (Figure A) and comparison (Figure B) (*p<0.05). Comparison of RPE-choroid complex IL-10 mRNA expression in each group 14 days after laser-induced CNV (Figure C) (*p<0.05). Detailed Implementation

[0050] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0051] Example The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All chemical reagents used in the embodiments of the present invention were commercially available.

[0052] Example 1: The role of the CD26 system in the pathogenesis of CNV Male GFP transgenic C57BL / 6J mice, aged 6-8 weeks, were obtained from the Department of Neurobiology, Air Force Medical University. They were housed in SPF-grade cages with free access to food and water, and the ambient temperature was maintained at 22-25°C. After one week of acclimatization, the mice were sacrificed by repeatedly flushing the bone marrow cavity to extract bone marrow from the limbs. Bone marrow cells were prepared into a suspension, and the suspension was adjusted to 1×10⁻⁶ cells before transplantation. 7 Density of cells / mL.

[0053] The bone marrow cell transplantation procedure was as follows: First, recipient wild-type C57BL / 6J mice (6-8 weeks old) were irradiated with 8 Gy of cobalt-60 to eliminate their primitive bone marrow cells. Then, a prepared bone marrow cell suspension was transplanted into the recipient wild-type mice. Thirty days after transplantation, the transfection efficiency of bone marrow cells in the recipient mice was assessed. Flow cytometry analysis showed that when the homing rate of GFP-labeled peripheral monocytes reached 80%, the bone marrow transplantation was considered successful, and the eligible mice were used for subsequent experiments.

[0054] All eligible mice were divided into five groups: a solvent group (PBS phosphate-buffered saline), an enimapril group (angiotensin-converting enzyme inhibitor; Tanabe Pharmaceutical Co., Ltd., Tianjin, Japan), a Dip-A group (dual protease inhibitor, DPP IV antagonist; Shanghai Glyco Biochemical Co., Ltd.), an enimapril + Dip-A combination therapy group, and a normal control group (mice in the normal control group did not receive laser rupture or further treatment). Except for the normal control group, the other four groups of mice were administered the corresponding drugs for 5 consecutive days before the CNV model was established. The administration methods for each group were as follows: the solvent group received 200 μL of solvent (PBS phosphate-buffered saline) via gastric tube; the enimapril group received 1 mg / kg / day via gastric tube; and the Dip-A group received 5 μmol subcutaneously twice daily. All groups of mice continued to receive the drugs for 14 days after CNV injury.

[0055] Establishment of a mouse CNV model: Recipient mice underwent laser photocoagulation after local injection of levofloxacin eye drops. Preoperatively, laser photocoagulation was performed within a 1.5 mm radius around the optic disc. A triaxial mirror was used to set the laser parameters: wavelength 532 nm, power 90–110 mW, exposure time 0.1 s, and spot diameter 75 μm. Four to six spots were created in each eye; the absence of bubbles and hemorrhage in the spots indicated effectiveness.

[0056] To verify the role of the CD26 system in the pathogenesis of CNV, the expression level and enzyme activity of the system in peripheral blood and bone marrow were measured 14 days after laser injury. CD26 activity assay: 14 days after laser photocoagulation treatment (CNV model establishment), CD26 activity in peripheral blood (PB) and bone marrow (BM) of CNV-affected mice was measured. Fasting plasma and bone marrow extracellular fluid samples (10 μL each) were collected from 6 mice in each group. Degradation efficiency was monitored using the chromogenic substrate Gly-Prop-nitroaniline (Gly-Pro-pNA; Sigma-Aldrich, St. Louis, Missouri), followed by CD26 activity measurement. After extracting fasting plasma and bone marrow extracellular fluid samples, centrifuge immediately (2000×g, 10 min) and place the samples in duplicate in 96-well plates on ice. Simultaneously, add DPP IV enzyme standard purified from porcine kidney (Sigma-Aldrich, Dieselhofen, Germany) at a concentration ranging from 0 to 10 mU / mL to the samples. Add freshly prepared Gly-Pro-pNA substrate (5 mM, dissolved in 0.04 M HEPES buffer, pH 7.4) to each well, and incubate the plate at 37°C for 60 min. Then, measure the absorbance of each well at 40 nm using a microplate reader (Bio-Rad, Hercules, California, USA). Use an equal volume of PB or BM sample with added HEPES buffer as a blank, and subtract this absorbance value from the matched measured sample value. Calculate the CD26 / DPP IV activity (U / L) according to the standard calibration curve.

[0057] Test results are as follows Figure 1 As shown, the results indicated that CD26 levels in the peripheral blood of normal control mice were significantly lower. + The cell percentage was approximately 41.3%, comparable to CNV mice injected with phosphate-buffered saline (solvent group). Meanwhile, from... Figure 1 As can be seen, the activity of CD26 in peripheral blood (PB) did not change significantly after laser damage. Treatment with enimapril in the CNV model reduced circulating CD26 activity. + The proportion of cells decreased significantly to 11.2%, accompanied by a reduction in CD26 activity. Furthermore, compared to the CNV solvent group, administration of the CD26 inhibitor Dip-A significantly reduced circulating CD26 activity. + The proportion of cells was not significantly affected, but the enzyme activity of CD26 in peripheral blood (PB) was significantly inhibited. Furthermore, compared with Dip-A alone, enimapril combined with Dip-A significantly reduced CD26 levels. + The ratio of [specific enzymes] was reduced, and it also significantly inhibited CD26 enzyme activity in peripheral blood (PB). Compared to enimapril monotherapy, Dip-A also reduced CD26 enzyme activity (e.g., [specific enzyme activity]). Figure 1(As shown in A-1C). The above results indicate that enimapril can reduce the enzyme activity and cell number of CD26 in peripheral blood in the CNV model, and Dip-A can reduce the enzyme activity of CD26 in peripheral blood.

[0058] from Figure 1 As can be seen in D-1E, congenital nasopharyngeal carcinoma lesions (CNV) do not alter CD26 in the bone marrow (BM). + The proportion of cells, and further treatment with enimapril or Dip-A did not affect CD26. + The presence and number of cells have an impact. It was also found that the activity of CD26 cells in the bone marrow did not change significantly after nasopharyngeal carcinoma injury. While enimapril treatment did not increase the number of CD26 cells in the bone marrow, it significantly stimulated an increase in the enzyme activity of CD26 cells in the bone marrow. Dip-A, on the other hand, decreased the enzyme activity of CD26 cells in the bone marrow (e.g., Figure 1 As shown in E.

[0059] Example 2 Flow Cytometry CD26-positive cells in the bone marrow and peripheral blood of the four groups of mice were detected by flow cytometry. The collected samples were incubated with FITC-labeled anti-mouse CD26 antibody (BDP harMingen) at 4°C in the dark for 30 min. Subsequently, the cells were lysed with 1× lysis buffer (BDP harMingen) at room temperature for 15 min.

[0060] The prepared samples were fixed in 4% paraformaldehyde solution in the dark for 30 min (4°C), followed by two washes. Detection was performed using an FA-CScan flow cytometer (BD Pharmaceuticals, San Jose, USA) with FlowJo vX.0.6 analysis software (FlowJo Pharmaceuticals, Ashland, USA). An isotype control antibody (BD Pharmaceuticals, Farmagan) was used as a reference. For the detection of peripheral macrophages derived from bone marrow, cell subpopulations were labeled with Lin-Sca-l-PE and F4 / 80-F fluorescently labeled antibodies, and detection was performed on the FAC Scan flow cytometer following the same procedure.

[0061] To further clarify the role of CD26 in the development of CNV lesions, retinal tissue was collected from CNV mice and stained using HE staining or immunofluorescence techniques. After discontinuation of enimapril and Dip-A, all experimental mice were anesthetized with sodium pentobarbital and subsequently euthanized by cardiac perfusion with PBS solution and 4% paraformaldehyde solution. Postoperatively, the eyeballs were enucleated and fixed.

[0062] The enucleated eyeballs were then treated with a 4% cold paraformaldehyde solution for 2 hours. After removing the anterior segment tissue and lens, the posterior segment samples were cryoprotected in a continuous sucrose solution. The prepared samples were embedded in paraffin and serially sectioned at a thickness of 3 μm. Three eyes per group (6 sections per eye) were stained with hematoxylin and eosin (HE) and observed under an optical microscope (Olympus Tokyo BX51, Japan) to detect CNV lesions. CNV thickness was defined as the vertical distance from the retinal pigment epithelium to the lesion apex, while lesion length was measured using Image-Pro Plus 6 software to determine the maximum horizontal extension of the lesion. Test results are as follows: Figure 2 As shown.

[0063] Fluorescein leakage is defined as hyperfluorescent lesions appearing during late angiography. Fluorescein penetration intensity is graded by three independent ophthalmologists according to the following criteria: 0 points represent no leakage; 1 point represents slight leakage; 2 points represent moderate leakage; and 3 points represent significant leakage.

[0064] Figure 2 As shown in Figure A, the CNV length and thickness of mice in the solvent group (PBS treatment group) were 221.1±13.4 μm and 52.3±6.2 μm, respectively. After enimapril treatment, the CNV length and thickness of mice significantly decreased to 140.3±12.1 μm and 27.5±5.2 μm, respectively, which was observed in the enimapril treatment group (P<0.05). Compared with the solvent group (PBS treatment group), Dip-A treatment had no significant effect on CNV length and thickness. It can also be seen that after enimapril treatment, the CNV volume of mice was significantly reduced. Compared with the solvent group, Dip-A treatment had no significant effect on the CNV volume of mice. The combination of enimapril and Dip-A can reduce CNV volume, but the reduction in CNV volume by the combination is less than that by enimapril alone.

[0065] Figure 2 Figures B and C in the image show the immunofluorescence staining results, which demonstrate that enimapril inhibits damage to choroidal neovascularization (CNV) and reduces CNV volume. However, the inhibitory effect of enimapril on CNV damage is partially offset by treatment with enimapril plus Dip-A. In contrast, Dip-A alone failed to change CNV volume.

[0066] In addition, observation of the permeability of the lesion area using FFA (fluorescein imaging) technology revealed that the normal control group showed moderate or significant fluorescein leakage, with a median FFA score of 2; after treatment with enimapril, the score dropped to a median of 1.2.

[0067] The above results indicate that enimapril treatment can significantly reduce the length, thickness, and volume of CNVs, demonstrating its inhibitory effect on CNV damage. Furthermore, enimapril can also reduce fluorescein leakage in the lesion area.

[0068] Example 3 Histopathological Analysis To investigate whether BMC cells are involved in CD26-mediated CNV pathogenesis, the proportion of BMC cells in peripheral blood and their distribution in CNV lesion areas were measured. The test results are as follows: Figure 3 As shown.

[0069] BMC cells in PB were double-labeled using Sca-1 and c-kit antibodies, and BMC cells were observed in CNV lesion areas using fluorescence microscopy. Figure 3 A), such as Figure 3 As shown in Figure A. Figures A and B show that CNV damage did not affect the proportion of BMC (bone marrow-derived cells) in PB (peripheral blood) or their presence in the lesion area. However, enimapril treatment significantly increased the proportion of BMC cells in PB, indicating that enimapril can promote the increase of BMC cells in PB.

[0070] Figure 3 Figures C and D show that in PB and CNV lesions, the use of Dip-A alone to inhibit CD26 enzyme activity did not change the proportion of BMC cells in PB and CNV. Enimapril can increase the proportion of BMCs in CNV; when Dip-A is used to inhibit CD26 enzyme activity, the promoting effect of enimapril on BMCs is partially offset, as shown in Figures C and D. Figure 3 B-3D.

[0071] The above results indicate that enimapril can promote the proportion of BMCs in PB and CNV lesions, but after using Dip-A, Dip-A reduces the promoting effect of enimapril on BMCs by inhibiting the enzyme activity of CD26.

[0072] Example 4 Immunofluorescence staining After refixation of the excised orbital specimen, it was vertically frozen sectioned to a thickness of 8 μm. Following 10 min of permeation and 30 min of blocking, the sections were incubated overnight at 4°C with a primary antibody targeting the target protein. BMCs were observed to be stained with labeled GFP using fluorescence microscopy. RPE (retinal pigment epithelial cells) differentiated from BMCs were identified by double-positive staining for RPE 65 and GFP. Furthermore, macrophages and glial cells in the CNV region were identified using F4 / 80 and CD11b markers, respectively. The resulting sections were then co-incubated with a matched secondary antibody for 1 h, stained with DAPI, and finally observed using a confocal microscope. Figure 4 As shown.

[0073] Given that SDF-1 has been confirmed as a substrate of CD26 / DPP IV, further verification was conducted to determine whether truncated CD26 affects the pathogenic mechanism of CNV. The concentrations of SDF-1 in peripheral blood and bone marrow were measured before laser injury and at 3, 7, and 14 days post-surgery. The procedure was as follows: Mice in the four groups underwent laser photocoagulation on days 0, 3, 7, and 14, and centrifuged peripheral plasma and bone marrow extracellular fluid samples were prepared (five samples per time point, five replicates per group (n=5)). Bone marrow extracellular fluid samples were collected by rinsing bilateral femoral contents into 400 μL of sample buffer and then centrifuged. The concentration of soluble SDF-1 in each sample was detected using an ELISA kit containing anti-mouse SDF-1 antibody (Shanghai Yandao Instrument Co., Ltd.). The absorbance of each sample was measured at 450 nm using a microplate reader, and the SDF-1 concentration was calculated based on the absorbance curve of recombinant mouse SDF-1 standard (Shanghai Yandao Instrument Co., Ltd.). Statistical analysis: All data are expressed as mean ± SD and analyzed using SPSS 22.0 software. Test results are as follows: Figure 4 As shown.

[0074] The test results found that ( Figure 4(Figures A and B in the image): Compared to the normal control group, laser damage did not significantly alter SDF-1 levels in peripheral blood or bone marrow within 14 days of CNV onset. However, when CNV mice were treated with enimapril, peripheral blood SDF-1 concentrations significantly increased to 1252.4 ± 206.3 pg / mL on day 3, peaked at 1689.8 ± 174.3 pg / mL on day 7, and eventually decreased to 912.4 ± 179.5 pg / mL. Compared to baseline levels, bone marrow SDF-1 significantly decreased on day 3 after enimapril treatment (baseline value 923.7 ± 95.3 pg / mL vs. 1690.7 ± 187.3 pg / mL). On days 7 and 14, SDF-1 expression in the enimapril-treated mice further decreased (9 picograms / mL). Meanwhile, when CD26 enzyme activity was inhibited using Dip-A, the effect of enimapril on SDF-1 in peripheral blood (PB) and bone marrow (BM) was eliminated at all time points. Furthermore, in both PB and BM, treatment with the CD26 antagonist Dip-A (dual protein A) alone did not significantly alter SDF-1 levels. Figure 4 A-4B).

[0075] Further, the expression of SDF-1 in the neovascularization region of the retina of mice treated with enimapril was observed by immunofluorescence staining. Figure 4 (Figures C and D) showed that imipreli significantly increased SDF-1 expression in this region.

[0076] The above results indicate that enimapril can increase the expression of SDF-1 in peripheral blood (PB) and the neovascularization region of the mouse retina, while enimapril can decrease the expression of SDF-1 in bone marrow (BM). Example 5: Fluorescein angiography The severity of CNV lesions was assessed using the sodium fluorescein assay. Four groups of mice were injected intraperitoneally with 0.15 mL of 2% sodium fluorescein solution, and retinal leakage was recorded.

[0077] Observation was performed using a confocal scanning laser ophthalmoscope (Heidelberg Engineering GmbH, Germany) from 2 minutes to the late stage (6-8 minutes) after injection.

[0078] Based on the theoretical hypothesis that mobilized retinal pigment epithelial cells (BMCs) partially clear CNV lesions by differentiating into various constituent cells, this study further investigated the role of CD26 in BMC differentiation. Using RPE-65 and GFP co-localization techniques, retinal pigment epithelial (RPE) cells differentiated from BMCs were identified. Figure 5 As shown.

[0079] from Figure 5As can be seen, after treatment with enimapril, the CNV area was significantly reduced, and the proportion of PRE cells differentiated from BMCs was significantly increased. Figure 5 A). In addition, macrophages and microglia differentiated from bone marrow stromal cells (BMCs) were also labeled with F4 / 80 and CD11 b antibodies, respectively. In the CNV group, the median percentage of F4 / 80-positive BMCs was 27.3%, compared to 3% in the solvent control group. After enimapril treatment, this percentage significantly increased to 41.5%. Figure 5 B). Importantly, following injection of imipreliable, CD11b-labeled microglia significantly aggregated in the retinochoroidal complex region, reaching a median ratio of 39.7% relative to total GFP+-BMCs (e.g., ...). Figure 5 C).

[0080] The above results indicate that enimapril can significantly reduce the area of ​​CNV, promote the differentiation of bone marrow stromal cells into retinal pigment epithelial (RPE) cells and microglia, and recruit macrophages to migrate to the CNV region. At the same time, enimapril can promote the aggregation of microglia in the retinochoroidal complex region.

[0081] Example 6: Detection of BMC migration and differentiation from CNV to retinal pigment epithelial cells (RPEs) (1) Determining CNV expression of bone marrow-derived RPEs: Immunofluorescence-labeled laser confocal microscopy was used to observe and compare the expression differences of bone marrow-derived RPEs in the RPE-choroid complex between the control and treatment groups (GFP+BMCs and RPEs-specifically labeled RPE-65 co-labeled bone marrow-derived RPEs). The test results are as follows: Figure 6 As shown; (2) Expression changes of BMCs, SDF-1 and VEGF in the RPE-choroid complex: Immunofluorescence-labeled laser confocal microscopy was used to observe and compare the expression differences between the control group and the treatment group (SDF-1, VEGF and GFP+BMCs were co-labeled), and the test results are shown in the figure. Figure 7 As shown.

[0082] from Figure 6 It can be seen that in the CNV model 14 days after laser induction, CD26-mobilized and expanded BMCs were recruited and migrated to the CNV damage area. The expression of bone marrow-derived RPEs was significantly increased in the imipreliate group compared with the solvent group (Figure D), and the area of ​​CNV damage area was reduced (Figure C).

[0083] from Figure 7 It can be seen that the recruitment of bone marrow-derived RPEs in the CNV injury area of ​​the enimapril group significantly reduced the expression of stromal cell-derived factor SDF-1 and vascular endothelial growth factor VEGF compared with the solvent group.

[0084] The above results indicate that under laser-induced CNV or hypoxic injury conditions, damaged retinal vasculature (RPEs) can increase the expression of SDF-1 and receptor CXCR4. SDF-1 can chemotactically induce basal cell membranes (BMCs) to homing to the RPE injury site, regenerating and repairing the damaged RPEs. This invention mobilizes endogenous BMCs; the functionally activated cells may migrate to the damaged areas of the retina and choroid, differentiating into RPEs. Their effect may be similar to the treatment of intraocular BMC transplantation. The endogenous damage repair pathway used in this invention differs from previous invasive intraocular cell injection routes, offering higher safety and clinical feasibility.

[0085] Experiment 7: Detection of BMCs recruiting and differentiating glial cells and their secretory regulation: (1) Determining the expression of microglia derived from bone marrow in the retinal nerve fiber layer and RPE-choroid complex: The expression difference between the control group and the enimapril treatment group was observed and compared using immunofluorescence-labeled laser confocal microscopy (GFP). + BMCs and CD11b + Co-labeled microglia derived from BMCs; test results as follows Figure 8 As shown. (2) Detection of retinal glial cell activation: Immunohistochemistry was used to detect the expression of glial fibrillary acidic protein (GFAP) positive marker areas in the nerve fiber layer, representing glial activation. Western blotting was used to detect the expression of GFAP protein in the retina; the test results are shown in the figure. Figure 9 (Figures A and B) are shown; (3) RT-PCR was used to detect the mRNA expression levels of retinal neuroprotective cytokines (TGF-β1 and IGF-1), and the test results are shown in Figures A and B. Figure 9 As shown in Figures C, D, and E.

[0086] from Figure 8 It can be seen that in the laser-induced CNV region, CD26-mobilized and expanded BMCs recruited to the CNV can be expressed as microglia; compared with the solvent group, the enimapril treatment group can significantly reduce the area of ​​the CNV region and increase the proportion of bone marrow-derived microglia.

[0087] from Figure 9 It can be seen that enimapril can activate the response of glial cells, activate glial levels, increase the expression of transforming growth factor (TGF-β) mRNA, and has no effect on the expression of insulin-like growth factor (IGF-1) mRNA. CD26 inhibitors can block the regulatory effect of BMCs on glial activation.

[0088] The above results indicate that in laser-induced CNV, recruited bone marrow-derived microglia can differentiate into microglia. These microglia, accumulating in large numbers, not only increase glial activation in the retinal neurofibrillary layer but also secrete regulatory TGF-β, which inhibits retinal endothelial cell proliferation. TGF-β derived from retinal endothelial cells (RPEs) can induce microglia to produce an anti-inflammatory phenotype. These experiments verify that recruited and differentiated bone marrow-derived microglia from BMCs can activate glial cells and exert secretory regulatory effects. These secretory effects may also have a reverse regulatory effect, increasing glial cell migration and activation, enabling cells to adapt to the constantly changing microenvironment, thus providing long-term protection. This differs from the short-term effects of current anti-angiogenic therapies. The mechanism of action discovered in this invention eliminates the need for repeated injections to improve and treat CNV, reducing treatment risks and costs.

[0089] Experiment Example 8: Recruitment of bone marrow-derived macrophages and detection of their secretion of anti-inflammatory mediators (1) Determining the expression of peripheral blood bone marrow-derived macrophages: FACS counting (Sca-l) was used. + / F4 / 80 + (1) Labeling peripheral blood bone marrow-derived macrophages); (2) Determining the expression of CNV bone marrow-derived macrophages: Immunofluorescence-labeled laser confocal microscopy was used to observe CNV-recruited bone marrow-derived macrophages (GFP). + BMCs and macrophage-specific marker F4 / 80 (co-labeled bone marrow-derived macrophages) were used to compare the expression differences between the control and treatment groups. Figure 10 and 11 As shown.

[0090] from Figure 10 and 11 It can be seen that in laser-induced CNV, the number of peripheral circulating bone marrow-derived macrophages mobilized by CD26 in the enimapril group was significantly increased compared with that in the solvent group. Enimapril can increase the recruitment and expression of bone marrow-derived macrophages in the CNV damage area, upregulate the expression level of IL-10 mRNA, and alleviate CNV formation.

[0091] These results indicate that bone marrow-derived macrophages have an anti-inflammatory effect in the repair of CNVs. Inflammation is a major mechanism in the pathogenesis of AMD and can regulate the occurrence and development of CNVs. Studies have confirmed that in laser-induced CNVs, RPE damage can induce the recruitment of bone marrow-derived macrophages.

[0092] These results further confirm that in vivo mobilization of BMCs during RPE injury can increase the recruitment and migration of bone marrow-derived macrophages to the CNV and secrete the anti-inflammatory mediator IL-10, thereby alleviating CNV formation. Therefore, increasing the recruitment of in vivo mobilized BMCs to the CNV injury area may be related to both increased autologous cell number and cell motility.

[0093] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. Application of enimapril in the preparation of drugs to improve choroidal neovascularization-related diseases.

2. The application according to claim 1, characterized in that, Enimapril can differentially regulate CD26 / DPP IV in bone marrow and peripheral blood. Enimapril can increase the activity of CD26 / DPP IV in bone marrow and decrease the activity of CD26 / DPP IV in peripheral blood.

3. The application according to claim 2, characterized in that, Enimapril reverses the concentration gradient of stromal cell-derived factor-1 between bone marrow and peripheral blood by upregulating the activity of CD26 / DPP IV in bone marrow and downregulating the activity of CD26 / DPP IV in peripheral blood.

4. The application according to claim 3, characterized in that, By reversing the concentration gradient of the stromal cell-derived factor-1, bone marrow-derived cells can be driven to migrate from the bone marrow to the peripheral blood and be recruited to the lesion area of ​​the choroidal neovascularization for the repair of choroidal neovascularization.

5. The application according to claim 1, characterized in that, The imipreli, by regulating CD26 activity, can promote the migration of bone marrow-derived cells from the bone marrow to lesions in the peripheral blood and choroidal neovascularization, thereby increasing the proportion of bone marrow-derived cells in these lesions.

6. The application according to claim 5, characterized in that, Bone marrow-derived cells that migrate to the choroidal neovascularization lesion area can differentiate into cells with tissue repair or immune regulation functions, and can recruit macrophages to migrate to the choroidal neovascularization lesion area, thereby promoting the repair of choroidal neovascularization.

7. The application according to claim 6, characterized in that, Bone marrow-derived cells that migrate to the choroidal neovascularization lesion area can differentiate into retinal pigment epithelial cells to play a regenerative and repair role. Bone marrow-derived cells that migrate to the choroidal neovascularization lesion area can activate retinal glial cells to play a secretory role. Bone marrow-derived cells selectively recruit bone marrow-derived macrophages to play an anti-inflammatory role.

8. The application according to claim 7, characterized in that, Retinal pigment epithelial cells differentiated from bone marrow-derived cells can significantly reduce VEGF expression and have an anti-angiogenic effect. Bone marrow-derived cells can migrate to the subretinal space and differentiate into retinal pigment epithelial cells, regenerating and repairing the damaged retinal pigment epithelial cell layer. The regenerated retinal pigment epithelial cells can also protect adjacent photoreceptors.

9. The application according to claim 7, characterized in that, CD26-recruited bone marrow-derived cells to the choroidal neovascularization lesion area can differentiate into microglia, increase glial activation in the retinal neurofibrillary layer, and secrete regulatory TGF-β. TGF-β secreted by microglia can inhibit the proliferation of retinal endothelial cells and induce microglia to produce an anti-inflammatory phenotype. Bone marrow-derived cells recruited and differentiated into bone marrow-derived microglia have secretory functions. Bone marrow-derived cells can increase the recruitment and migration of bone marrow-derived macrophages to the choroidal neovascularization lesion area and secrete the anti-inflammatory mediator IL-10, exerting an anti-inflammatory effect and reducing the formation of choroidal neovascularization lesions.

10. A drug that can improve choroidal neovascularization-related diseases, characterized in that, The drug includes an active ingredient and pharmaceutical excipients, wherein the active ingredient is enimapril.

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