Application of MYDGF in pharmaceuticals and medical devices for the treatment of endovascular hyperplasia

Abstract

This invention discloses the application of MYDGF in pharmaceuticals and medical devices for treating vascular intimal hyperplasia, belonging to the field of pharmaceutical technology. This invention utilizes MYDGF as a target in the preparation of products for protecting vascular function, and applies MYDGF in the preparation of drugs for treating vascular intimal hyperplasia, drugs for metabolic disorders related to vascular intimal hyperplasia, and medical devices for treating vascular intimal hyperplasia. This invention provides new candidate drugs and protein therapies for the clinical treatment of intimal hyperplasia.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the pharmaceutical uses of MYDGF, particularly the application of MYDGF in pharmaceuticals and medical devices for treating endovascular hyperplasia. Background Technology

[0002] Intimal hyperplasia is a major cause of vascular stenosis, and it is common during procedures such as balloon angioplasty, bypass grafting, and stent implantation. It is characterized by abnormal dedifferentiation of vascular smooth muscle cells (VSMCs), allowing VSMCs located in the tunica media to migrate, proliferate, and secrete extracellular matrix (ECM). According to the 2021 World Health Statistics, the total number of deaths from cardiovascular disease worldwide reached 17.9 million in 2019, an increase of one-quarter since 2000. It is projected that approximately 23.6 million people will die from cardiovascular disease annually by 2030. Clinically, revascularization surgeries are commonly used to restore blood flow to the affected vessels, including balloon angioplasty, autologous implantation, and coronary artery bypass grafting. Balloon angioplasty commonly uses drug-coated balloons (DCBs) and drug-eluting stents (DESs) to prevent intimal hyperplasia. DCBs rapidly deliver anti-proliferative drugs to the vessel wall through balloon inflation. DESs release drugs into the vessel wall to prevent intimal hyperplasia. Sirolimus and paclitaxel are classic drugs widely used in DESs and DCBs. However, paclitaxel-coated balloons and stents increase the risk of lower extremity femoral-popliteal artery death after application. Therefore, researching and developing safe and effective coated balloon antiproliferative drugs is an urgent priority for preventing intimal hyperplasia. Summary of the Invention

[0003] In view of the above-mentioned deficiencies of the prior art, the present invention provides the application of MYDGF in pharmaceuticals and medical devices, providing new candidate drugs and protein therapies for the clinical treatment of endometrial hyperplasia.

[0004] The first aspect of this invention provides the application of MYDGF as a target in the preparation of products for protecting vascular function.

[0005] Furthermore, the amino acid sequence of the MYDGF is as follows:

[0006] VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL.

[0007] The second aspect of this invention provides the use of MYDGF in the preparation of medicaments for treating vascular intimal hyperplasia.

[0008] A third aspect of this invention provides the use of MYDGF in the preparation of medicaments for treating metabolic disorders related to vascular intimal hyperplasia.

[0009] The fourth aspect of this invention provides the application of MYDGF in the preparation of a medical device for treating vascular intimal hyperplasia, the medical device comprising a drug-coated ball and a drug-eluting stent.

[0010] Compared with the prior art, the present invention has the following technical effects:

[0011] 1. This invention has discovered new medical applications for the known protein MYDGF, opening up a new field of application.

[0012] 2. This invention uses MYDGF as a target for the research and development of drugs and interventions to protect vascular function.

[0013] 3. The MYDGF of the present invention can maintain the differentiation phenotype of vascular smooth muscle cells in the vascular media, effectively reduce intimal hyperplasia caused by carotid balloon injury, reduce vascular diameter stenosis, and improve the pharmacological effects of reduced peak contraction velocity, reduced average contraction velocity, increased neointimal area, increased intimal / media area, and increased collagen deposition caused by balloon injury.

[0014] 4. This invention can be applied to the clinical treatment of endometrial hyperplasia and has considerable novelty, broad application prospects and development value.

[0015] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0016] Figure 1 This is an experimental result showing the biological correlation between membrane proliferation and MYDGF in a specific embodiment of the present invention;

[0017] Figure 2 This is a specific embodiment of the present invention, showing the effect of MYDGF on endometrial hyperplasia;

[0018] Figure 3 This is a specific embodiment of the present invention, showing the effect of MYDGF on carotid intimal hyperplasia after balloon injury;

[0019] Figure 4 This is a specific embodiment of the present invention, showing the effect of MYDGF on the phenotypic transformation of vascular media smooth muscle cells;

[0020] Figure 5 This is a schematic diagram of the MYDGF three-dimensional structure according to a specific embodiment of the present invention;

[0021] Figure caption: Values ​​are expressed as mean ± standard error. Detailed Implementation

[0022] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0023] It should be noted that the illustrations provided in the following embodiments are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0024] Some exemplary embodiments of the invention have been described for illustrative purposes. It should be understood that the invention may be implemented in other ways not specifically shown in the accompanying drawings.

[0025] Unless otherwise specified, all materials, instruments and reagents used in this invention are commercially available or self-made.

[0026] 1. Preparation of MYDGF drugs

[0027] Preparation of topical medication: Weigh 20 mg of Pluronic F-127 into 100 μL of 50 μg / mL MYDGF solution, dissolve in a 40°C water bath for 30 min until it forms a gel, and prepare fresh before use.

[0028] In-situ drug preparation: 50ug / ml MYDGF solution, prepared fresh before use.

[0029] Myeloid-derived growth factor (MYDGF) is a paracrine protein secreted by bone marrow-derived monocytes and macrophages. MYDGF, also known as C19orf10, belongs to the MYDGF family. The amino acid sequence of MYDGF is as follows:

[0030] VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDH QHFTCTIWRPQGKSYLYFTQFKAAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAV AHRPGAFKAELSKLVIVAKASRTEL with Figure 5 The structure shown.

[0031] 2. Experimental Methods

[0032] 2.1 Construction of a rat carotid artery balloon injury model

[0033] SPF-grade SD rats (300-350g) were acclimatized to a standard maintenance diet for 3-5 days, weighed, and randomly divided into three groups: the Sham group, the Model group, and the MYDGF treatment group. The specific procedures were as follows: SD rats were fasted for 12 hours before surgery, but allowed free access to water. They were anesthetized by intraperitoneal injection of 3% pentobarbital at 0.15ml / 100g; successful anesthesia was achieved when no corneal reflex was observed. After shaving the neck, the rats were placed in a supine position and fixed on the operating table. The surgical area was disinfected with iodine. A midline incision was made in the neck. The left common carotid artery was bluntly dissected beside the left trachea. The internal carotid artery and external carotid artery were then dissected upwards. At the origin of the external carotid artery, the distal end and small branch arteries were permanently ligated with 4.0 suture. The proximal end of the common carotid artery and the origin of the internal carotid artery were temporarily clamped with hemostatic clips. Make a V-shaped incision at the distal end of the external carotid artery using ophthalmic scissors, and insert a 2F Fogarty balloon catheter approximately 3-4 cm into it. Inflate the balloon with 2 mL of air using a syringe connected to the balloon. Pull the balloon back until a friction sensation is felt, yet the balloon can still be pulled; this is the appropriate pressure. Repeat this five times, then remove the balloon catheter. Ligate the external carotid artery at the incision site and release the hemostatic clips from the common carotid and internal carotid arteries. Observe the common carotid artery; a clear pulsation should be visible, indicating unobstructed blood flow.

[0034] In the MYDGF treatment group, MYDGF solution was administered in situ to the injured vessel for 1 minute before suturing the external carotid artery incision, and a medicated gel was applied to the periphery of the injured vessel. Three days postoperatively, penicillin (200,000 U / animal) was injected intraperitoneally to prevent infection.

[0035] 2.2 Determination of plasma MYDGF levels

[0036] After anesthetizing each group of animals with intraperitoneal injection of sodium pentobarbital solution, approximately 500 μL of whole blood was collected from the inner canthus into an anticoagulant tube containing EDTA. The plasma was collected within 30 minutes by centrifugation at 1000g for 15 minutes at 4°C. The supernatant was then collected as plasma, and the MYDGF content in the plasma was detected using a rat enzyme-linked immunosorbent assay (ELISA) kit.

[0037] 2.3 Vascular Doppler Ultrasound

[0038] All animals were anesthetized with isoflurane aerosol, and their abdomens were shaved and skin prepared. Vascular changes in each group of animals were detected using a Doppler ultrasound imaging system (Fineo, Suzhou, China).

[0039] 2.4 Vascular pathology examination

[0040] Left carotid arteries from each group were embedded in OCT scans and frozen at -20°C. Vascular sections with a thickness of 10 μm were prepared using a cryostat (Leica, Germany). Frozen sections of carotid artery tissue from each group were stained using an HE staining kit (Solepro, Beijing, China) and a Masson staining kit (Solepro, Beijing, China).

[0041] 2.5 Immunofluorescence

[0042] 10 μm frozen sections of blood vessels were incubated at room temperature for at least 30 min before immunofluorescence experiments. The sections were gently rinsed three times with PBS, blocked with 2% BSA at room temperature or 37°C for 1 h, and then incubated with MYDGF antibody (1:500, RDsystems, USA), α-SMA antibody (1:500, Bio-Science, China), and OPN antibody (1:150, Proteintech, USA) at room temperature for 2 h or overnight at 4°C. After gentle rinsing with PBS, the sections were stained with DAPI (Beyotime, China) for 30 s, rinsed with PBS, and mounted with an anti-quenching mounting medium (Beyotime, China). Images were taken using a confocal microscope (Olympus, Japan).

[0043] 2.6 Data Statistics

[0044] Experimental data are expressed as mean ± standard error. T-tests were used for statistical analysis, with P < 0.05 indicating statistical significance. All experimental results were statistically analyzed and plotted using Graphpad Prism 6.0.

[0045] 3. Experimental Results

[0046] 3.1 Biological correlation between endometrial hyperplasia and MYDGF

[0047] Changes in MYDGF levels in plasma and left carotid artery of SD rats after balloon injury are as follows: Figure 1 As shown, Figure 1 A shows the curve of MYDGF content in plasma of SD rats after balloon injury over time; Figure 1 B shows representative images of MYDGF immunofluorescence staining in frozen sections of the left carotid artery of each group of animals 28 days after balloon injury; 1C shows the statistical results of MYDGF immunofluorescence staining in frozen sections of the left carotid artery of each group of animals. (***P<0.001, n=6-7)

[0048] In existing technologies, MYDGF has various cardiovascular protective effects, including promoting cardiomyocyte proliferation and neonatal cardiac regeneration, protecting against heart failure caused by stress overload, protecting against podocyte damage and proteinuria in diabetic nephropathy, regulating neutrophil interstitial activity and inflammatory response, addressing tissue damage, and reducing endothelial damage and atherosclerosis. Enzyme-linked immunosorbent assay (ELISA) results showed that MYDGF levels in the plasma of rats after balloon injury were significantly lower than in the control group (**P<0.01, n=8-10). Figure 1 A). Immunofluorescence staining was performed on the expression of MYDGF in the left common carotid artery of each group. The results showed that MYDGF was highly expressed in the normal control group. Balloon injury induced neointimal formation, and the expression of MYDGF was significantly reduced compared with the control group (***P<0.01, n=6-7). Figure 1 B, C) Experimental results show that MYDGF is biologically related to intimal hyperplasia. Based on this characteristic, MYDGF can be used as a target in the research and development of vascular function protection drugs and interventions.

[0049] 3.2 Effects of MYDGF on carotid artery diameter and blood flow velocity after balloon injury

[0050] The results of Doppler ultrasound imaging and blood perfusion observation of the left carotid artery in each group of animals are as follows: Figure 2 As shown, where Figure 2 A is a representative image of the left carotid artery Doppler ultrasound results of animals in each group 28 days after balloon injury surgery; Figure 2 B is a statistical chart of the carotid artery diameter; Figure 2 C is a statistical chart of PSV for each group; Figure 2 D is the EDV statistics chart for each group; Figure 2 E is a representative graph showing the measurement results of left carotid artery blood flow in each group of animals; Figure 2 F is a statistical chart of average PSV; Figure 2 G is the average EDV statistical chart; Figure 2 H mean blood flow statistics. (*P<0.05, ***P<0.001, # P<0.05, ## P<0.01, n=5-7)

[0051] Depend on Figure 2 Experimental results showed that quantitative analysis of representative Doppler ultrasound images revealed a significant reduction in both the diameter and blood flow velocity of the left carotid artery in the model group compared to the normal control group. Furthermore, MYDGF treatment partially offset the carotid artery stenosis caused by balloon mechanical injury. Blood flow measurement results (…) Figure 2D, 2E) indicate that MYDGF treatment reduced the peak systolic velocity (PSV) and end-diastolic velocity (EDV) (*P<0.05, ***P<0.001). # P<0.05, ## P<0.01, n=6-7)( Figure 2 A-2D). (***P<0.001, # P<0.05, ## P<0.01, n=5-7).

[0052] 3.3 Effect of MYDGF on carotid intimal hyperplasia after balloon injury

[0053] Representative results of HE staining and Masson staining of frozen sections of the left carotid artery of each group of animals are as follows: Figure 3 As shown. Figure 3 A is a representative image showing the HE staining results of the left carotid artery of each group of animals 28 days after balloon injury surgery. Figure 3 B is a statistical chart showing the measurement results of the left carotid artery inner diameter of each group of animals; Figure 3 C is a statistical graph showing the measurement results of the intimal hyperplasia area of ​​the left carotid artery in each group of animals; Figure 3 Figure D is a statistical graph showing the measurement results of the medial area of ​​the left carotid artery in each group of animals; Figure E is a representative graph showing the measurement results of the circumference of the left carotid artery in each group of animals; Figure F is a representative graph showing the Masson staining results of the left carotid artery in each group of animals; Figure G is a statistical graph showing the Masson staining results; (***P<0.001, # P<0.05, n=7-12)

[0054] HE staining results showed that, compared with the model group, MYDGF administration 28 days after balloon injury significantly reduced the neointimal area and intima / media ratio, and collagen deposition (*P<0.05, n=6). Figure 3 (A, B, C, F, G), while the area of ​​the middle membrane and the perimeter of the outer elastic plate were not affected. (n=6)( Figure 2 (D, E). The experimental results demonstrate that MYDGF has an inhibitory effect on the formation of new endometrium after mechanical damage.

[0055] 3.4 Effects of MYDGF on Phenotypic Transformation of Vascular Media Smooth Muscle Cells

[0056] Immunofluorescence staining results of the left carotid artery of each group of animals are as follows: Figure 4 . Figure 4 A shows representative OPN staining images of frozen sections of the left carotid artery of each group of animals 28 days after balloon injury surgery. Figure 4 B is a representative image of α-SMA staining of frozen sections of the left carotid artery of each group of animals; Figure 4 C is a statistical graph of OPN staining results; Figure 4D is a statistical graph of α-SMA staining results.

[0057] Immunofluorescence results of frozen sections of vascular tissue showed that the expression of the contractile marker α-SMA in the vascular tissue of the balloon injury model group was significantly lower than that in the normal control group, while the expression of the synthetic marker OPN was higher than that in the normal control group. The experimental results indicate that MYDGF treatment can upregulate the expression of α-SMA and inhibit the expression of OPN (*P<0.05). # P<0.05, ## P<0.01, n=4-6)( Figure 4 MYDGF (Advanced Dihydrotestosterone) can inhibit neointimal formation by maintaining the differentiation phenotype of vascular media VSMCs.

[0058] The above experiments demonstrate that MYDGF can maintain the differentiation phenotype of vascular smooth muscle cells (VSMCs) in the vascular media, effectively inhibit intimal hyperplasia caused by balloon injury in rat carotid arteries, and reduce vascular stenosis. It exerts pharmacological effects by reducing peak contraction velocity, reducing average contraction velocity, increasing neointimal area, increasing intima / media area, increasing collagen deposition, and inhibiting the phenotype transformation of vascular smooth muscle cells in the vascular media caused by balloon injury. This indicates that MYDGF, as the sole active ingredient, can effectively treat intimal hyperplasia and related metabolic disorders. It can also be formulated into drug preparations with different excipients for the treatment of proliferative vascular diseases. MYDGF can also be incorporated into various clinical medical devices for the preparation of drug-coated balloons and drug-eluting stents.

[0059] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. Application of MYDGF in the preparation of drugs for treating vascular intimal hyperplasia caused by mechanical injury.

2. Application of MYDGF in the preparation of drugs for treating metabolic disorders related to vascular intimal hyperplasia caused by mechanical injury.

3. The application of MYDGF in the preparation of medical devices for treating vascular intimal hyperplasia caused by mechanical injury, characterized in that, The medical device includes a drug-coated ball and a drug-eluting stent.

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