Use of a fumaric acid supplement and pharmaceutical compositions

Abstract

The present application relates to the field of biological medicine, and particularly relates to application of fumarate supplement and pharmaceutical composition; the application is used for preparing one or more than two kinds of drugs for promoting collateral circulation establishment, promoting blood vessel growth and promoting vascular endothelial cell proliferation. The fumarate supplement can promote the proliferation of Willis circle and retinal vascular endothelium, can promote the increase of the number of new collaterals, improve the cardiac perfusion, realize the angiogenesis and collateral circulation formation in the adult rat myocardial infarction model, can help the patients with ischemic cardiovascular and cerebrovascular diseases to form blood revascularization, reduce ischemic injury and reduce the mortality of patients. Compared with the traditional operation and cytokine treatment, the application has a wider application range and a lower treatment cost, and has a significant clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of a fumaric acid supplement and a pharmaceutical composition. Background Technology

[0002] Ischemic cardiovascular and cerebrovascular diseases refer to a series of syndromes caused by insufficient blood supply to the heart and brain tissue due to narrowing or blockage of blood vessels. Timely and effective revascularization can effectively alleviate symptoms and salvage damaged tissue cells. Currently, revascularization mainly relies on interventional surgery, thrombolysis, and bypass surgery. However, these treatment strategies have strict time windows after onset, require highly skilled operators, and face postoperative complications such as reembolization. Therefore, the search for new revascularization methods still has significant research value and broad clinical application prospects.

[0003] Collateral circulation refers to the vascular bypass that forms between the main arteries when the feeding arteries are severely narrowed or blocked. Clinical evidence shows that good collateral circulation can reduce ischemic injury and lower patient mortality by forming effective revascularization. Therefore, the status of collateral circulation is an important factor affecting the prognosis of ischemic diseases, and promoting the establishment of collateral circulation is a feasible treatment approach. However, there are currently no drugs or methods to directly promote collateral circulation. Current clinical research mainly focuses on whether a series of angiogenesis-promoting cytokines, such as VEGF, HIF-1α, hepatocyte growth factor (HGF), and fibroblast growth factor (FGF), can promote the formation of collateral circulation in patients with ischemic diseases and improve the prognosis of ischemic diseases. However, all clinical studies on collateral circulation in phase II and III have failed to obtain positive results. Moreover, cytokine therapy is expensive and difficult to popularize widely. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the above-mentioned defects of the prior art and provide an application of fumaric acid supplement and a pharmaceutical composition that can promote the establishment of collateral circulation, promote angiogenesis, and promote the proliferation of vascular endothelial cells.

[0005] The technical solution adopted by the present invention to solve its technical problem is as follows: the application of a fumaric acid supplement for the preparation of one or more drugs that promote the establishment of collateral circulation, promote angiogenesis, and promote the proliferation of vascular endothelial cells.

[0006] Preferably, the fumaric acid supplement comprises one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumarate hydratase inhibitors, and pharmaceutically acceptable derivatives of fumarate hydratase inhibitors.

[0007] Preferably, it is a drug for preparing revascularization therapy.

[0008] Preferably, it is used to prepare a drug for the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases.

[0009] Preferably, the fumaric acid supplement is further salted and / or chemically modified.

[0010] More preferably, the chemical modification includes at least one of acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, ubiquitination, biotin labeling, and fluorescent protein labeling.

[0011] Based on the same inventive concept, the present invention also provides a pharmaceutical composition for promoting the establishment of collateral circulation; the pharmaceutical composition includes a fumaric acid supplement.

[0012] Preferably, the fumaric acid supplement in the pharmaceutical composition comprises one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumarate hydratase inhibitors, and pharmaceutically acceptable derivatives of fumarate hydratase inhibitors.

[0013] Preferably, the dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion, or liniment.

[0014] Preferably, the dosage of the pharmaceutical composition is 0.1~100 mg / kg.

[0015] The present invention has the following beneficial effects: In in vitro experiments, fumaric acid supplementation increased the proliferation level of human umbilical vein endothelial cells, and similar results were obtained in in vivo experiments. It promoted the proliferation of the Willis Circle (a first-order collateral circulation network) in the brain and retinal vascular endothelium. In an adult rat model of myocardial infarction, it increased the number of newly formed collateral branches, improved cardiac blood perfusion, and facilitated angiogenesis and collateral circulation formation. It can help patients with ischemic cardiovascular and cerebrovascular diseases achieve revascularization, reduce ischemic damage, and lower patient mortality. Compared with traditional surgery and cytokine therapy, it has a wider range of applications and lower treatment costs, demonstrating significant clinical application value.

[0016] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the accompanying drawings. Attached Figure Description

[0017] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the changes in cell proliferation levels in human brain microvascular endothelial cells after administration of MMF in an in vitro experiment: Figure 1 -A is a fluorescence microscope image. Figure 1 -B represents the statistical results; Figure 2 This is a graph showing the proliferation of Willis Circle and retinal endothelial cells in mice after administration of MMF and FH-IN: Figure 2 -A is an immunofluorescence staining image of Willis Circle in mice on day 4 after they were given MMF; Figure 2 -B represents the statistical results of Willis Circle proliferation in mice on day 4 after administration of MMF; Figure 2 -C is an immunofluorescence staining image of Willis Circle on day 4 after mice were administered FH-IN; Figure 2 -D represents the statistical results of Willis Circle proliferation in mice on day 4 after administration of FH-IN; Figure 2 -E is an immunofluorescence staining image of the retina of mice on day 6 after they were given MMF; Figure 2 -F represents the statistical results of retinal proliferation in mice on day 6 after administration of MMF; Figure 3 This is a statistical graph showing the changes in the proliferation level of Willis Circle endothelial cells in mice after administration of MMF in Example 3 of this invention: Figure 3 -A is an immunofluorescence staining image. Figure 3 -B represents the statistical results; Figure 4 This is a diagram of the vascular condition in a mouse myocardial infarction model treated with MMF according to Example 4 of this invention: Figure 4 -A is a statistical chart of the diameter and number of newly formed blood vessels; Figure 4 -B is the angiography image of the control group. Figure 4 -C is an angiographic image of the MMF group. Detailed Implementation

[0018] To make the objectives, solutions, and beneficial technologies of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. It should be noted that the embodiments described in this specification are merely illustrative of the invention and are not intended to limit the invention.

[0019] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0020] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, "multiple" in "one or more" means two or more, and "more than" in "one or more" means two or more.

[0021] An embodiment of the present invention provides an application of fumaric acid supplement for the preparation of one or more drugs that promote the establishment of collateral circulation, promote angiogenesis, and promote the proliferation of vascular endothelial cells.

[0022] Fumaric acid supplements can increase fumaric acid levels.

[0023] In some embodiments of the present invention, the fumaric acid supplement comprises one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumaric acid hydratase inhibitors, and pharmaceutically acceptable derivatives of fumaric acid hydratase inhibitors.

[0024] Fumaric acid is a key member of the tricarboxylic acid cycle, participating in oxidative phosphorylation in cells and driving the formation of energy and biosynthetic precursors. Currently, dimethyl fumarate (DMF) is the primary fumarate supplement used clinically, as it can be metabolized into fumarate within cells. DMF is mainly used to treat multiple sclerosis, acting by regulating the immune system and reducing inflammation in the central nervous system. Monomethyl fumarate (MMF) is the active metabolite of DMF in vivo, more readily permeating cell membranes than fumarate and metabolizing into fumarate within cells. Compared to cytokines and other drugs, fumarate and its derivatives, as natural components, are relatively easier to obtain and less expensive. Fumarate hydratase inhibitors (FH-IN) can increase intracellular fumarate levels and can also be used as fumarate supplements.

[0025] In some embodiments of the present invention, a medicament is prepared for revascularization therapy.

[0026] In some embodiments of the present invention, drugs are used to prepare for the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases.

[0027] In some embodiments of the present invention, the fumaric acid supplement is further salted and / or the fumaric acid supplement is chemically modified.

[0028] In some embodiments of the present invention, the chemical modification includes at least one of acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, ubiquitination, biotin labeling, and fluorescent protein labeling.

[0029] The pharmaceutical composition of this invention is used to promote the establishment of collateral circulation; the pharmaceutical composition includes a fumaric acid supplement.

[0030] In some embodiments of the present invention, the fumaric acid supplement in the pharmaceutical composition comprises one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumarate hydratase inhibitors, and pharmaceutically acceptable derivatives of fumarate hydratase inhibitors.

[0031] In some embodiments of the present invention, the dosage form of the pharmaceutical composition is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion, or liniment. Multiple dosage forms meet different therapeutic needs, improve patient compliance, optimize drug efficacy and safety, and can satisfy different clinical needs and patient preferences, as well as adapt to different routes of administration.

[0032] In some embodiments of the present invention, the dosage of the pharmaceutical composition is 0.1~100 mg / kg.

[0033] Example The following examples describe the disclosure of this invention in more detail. These examples are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of this disclosure. Unless otherwise stated, all parts, percentages, and ratios reported in the following examples are based on weight. Unless otherwise stated, all reagents used in the examples (such as DMF, MMF, FH-IN, etc.) are available commercially or synthesized using conventional methods and are ready for use without further processing. Unless otherwise stated, the instruments used in the examples are commercially available.

[0034] Example 1: In vitro experiment of human brain microvascular endothelial cells (HBMEC) An in vitro experiment was conducted on human brain microvascular endothelial cells to administer MMF, and the cell proliferation level was detected. If the cell proliferation level increased, it indicates that MMF has the potential to promote the formation of collateral circulation.

[0035] Experimental methods: (1) Seeding cells into plates: Seed 10,000 HBMEC cells per well into an eight-well plate and incubate for 16 hours in a cell culture incubator until the cells adhere to the plate. (2) Cell treatment: DMEM low-glucose medium containing 1 mg / mL glucose was used for preparation. The medium formula was DMEM + 0.2% FBS + 1% P / S + 1% ECGS. The eight-well plates were replaced with this medium. After 1 hour, MMF was added to a final concentration of 10 μM, 25 μM or 50 μM. (3) EdU labeling: Prepare a 2 mM 5-ethynyl-2'-deoxyuridine (EdU) working solution with sterile water; add 2 μL of the working solution to each well of an eight-well plate with a final EdU concentration of 2 μM. Continue incubation for 2 h; (4) Cell fixation and washing: After EdU labeling of cells, remove the culture medium and add 4% paraformaldehyde (PFA) fixative, fix at room temperature for 10 min. Remove the fixative and wash three times with PBS for 5 min each time; (5) EdU staining: EdU staining was performed using the EdU cell proliferation imaging kit (ThermoFisher C10340); the washing buffer was removed, and the cells were incubated with permeabilization buffer (PBS containing 5% donkey serum + 0.3% Triton X-100) at room temperature for 30 minutes; the reaction solution was prepared according to the instructions, and 100 μL of the reaction solution was added to each well. The culture plate was gently shaken to ensure that the reaction mixture could evenly cover the sample; the cells were incubated at room temperature in the dark for 30 minutes; the reaction solution was removed, and the cells were washed 3 times with washing buffer for 5 minutes each time. 100 μL of mounting medium containing DAPI was added to each well to stain the cell nuclei; the maximum excitation wavelength of Azide 647 is 650 nm, and the maximum emission wavelength is 671 nm. High-resolution images were taken using a fluorescence microscope.

[0036] Cell proliferation level is expressed as the proportion of EDU(+)DAPI(+) double-positive cells to DAPI(+) cells.

[0037] Figure 1 -A is the obtained fluorescence microscope image. Figure 1 -B represents the statistical results; Figure 1 In -A, MERGE represents the co-localization image of DAPI and EDU; the group without MMF is abbreviated as CTRL or NC; the group with 10μM MMF is abbreviated as MMF 10uM; the group with 25μM MMF is abbreviated as MMF 25uM; and the group with 50μM MMF is abbreviated as MMF 50uM. The results are as follows... Figure 1 As shown, after treatment with 10 μM MMF, 25 μM MMF and 50 μM MMF for 48 h, the proliferation level of HBMEC was significantly increased, indicating that MMF has the potential to promote the formation of collateral circulation.

[0038] Example 2: Willis Circle and retinal vascular endothelial proliferation experiment in developing rats In suckling rats, MMF and FH-IN (fumarate hydratase inhibitor) were administered during development, and the proliferation of Willis Circle and retinal endothelial cells was detected. Increased cell proliferation levels indicated that MMF and FH-IN could promote collateral circulation formation. FH-IN can increase intracellular fumarate levels; if FH-IN can promote collateral circulation formation, it indirectly suggests that increasing intracellular fumarate levels can promote collateral circulation formation.

[0039] Experimental animals: C57BL6 / J mice were selected as experimental subjects and divided into experimental group and control group.

[0040] Drug preparation: MMF was dissolved in ddH2O to a final concentration of 10 mg / mL, aliquoted, and stored at -20°C protected from light; FH-IN was dissolved in 10% DMSO + 90% corn oil to a final concentration of 5 mg / mL, aliquoted, and stored at -20°C protected from light.

[0041] Eight litters of suckling mice were randomly divided into four groups, with approximately 16 mice from two litters in each group. These groups were designated as the MMF administration group, the FH-IN administration group, and their respective control groups (collectively referred to as the CTRL groups). From day 1 to day 4 after birth, the experimental groups received daily injections of either MMF or FH-IN insulin, while the control groups received daily injections of an equal volume of ddH2O or 10% DMSO + 90% corn oil. On day 4, Willis Circle specimens were collected: EDU was injected intraperitoneally, and the whole brain was harvested 4 hours later, fixed in 4% PFA for 16 hours, thoroughly washed, and the Willis Circle specimen was removed. On day 6, the retina was collected: EDU was injected intraperitoneally, and the eyeball was harvested 4 hours later, fixed at room temperature for 15 minutes, and thoroughly washed. Willis Circle and retinal specimens were co-stained with EDU and ERG (endothelial cell markers) to prepare fully embedded specimens. Images were captured using a confocal microscope for statistical analysis. Cell proliferation level was expressed as the proportion of EDU(+) and ERG(+) double-positive cells to ERG(+) cells.

[0042] The specific operating method is as follows: Intraperitoneal injection: Fix the suckling mice, draw an appropriate amount of drug solution using an insulin needle, insert the needle parallel to the skin, avoid the internal organs, and enter the peritoneal cavity at a 45° angle; slowly inject 12μL of drug solution per mouse; the control group of MMF was injected with ddH2O, and the control group of FH-IN was injected with 10% DMSO + 90% corn oil. Sampling: Willis Circle was collected on day 4 after birth; 5-ethynyl-2'-deoxyuridine (EdU) was prepared as a 5 mg / mL working solution using PBS; 12 μL of EdU was injected intraperitoneally into each suckling mouse 4 hours before sampling; the mice were sacrificed, the whole brain was immediately removed, fixed in 4% PFA for 24 hours, thoroughly washed, and the Willis Circle vessels were removed; Retinal tissue was collected on day 6 after birth; 5-ethynyl-2'-deoxyuridine (EdU) was prepared into a working solution of 5 mg / mL using PBS; 12 μL of EdU was injected intraperitoneally into each suckling mouse 4 hours before collection; the mice were euthanized, and the eyeballs were immediately removed, fixed in 4% PFA at room temperature for 15 min, thoroughly washed, and the retina was dissected.

[0043] Preparation of Willis Circle fully embedded specimens: 5% donkey serum + 1% Triton X-PBS was used as blocking and antibody diluents. Willis Circle vessels were placed in EP tubes, and 50 μL of blocking solution was added per vessel. The tubes were incubated overnight at 4°C with shaking. EdU 488 staining solution was prepared according to the EdU cell proliferation imaging kit (ThermoFisher C10337). After staining for 12 h, the tubes were thoroughly washed with 0.3% Triton X-PBS. Primary antibody preparation: ERG (endothelial cell marker) antibody was diluted 1:200. 50 μL of primary antibody diluent was added per vessel. The tubes were incubated at 4°C with shaking for 48 h, and then thoroughly washed with 0.3% Triton X-PBS. Secondary antibody preparation: The corresponding secondary antibody was diluted 1:200, and sma-cy3 antibody (labeling vascular smooth muscle) was diluted 1:500. 50 μL of secondary antibody diluent was added per vessel. The tubes were incubated at 4°C with shaking for 16 h, and then washed with 0.3% Triton X-PBS. Wash thoroughly with X-PBS; mount using mounting medium containing DAPI.

[0044] Preparation of fully embedded retinal specimens: 5% donkey serum + 0.5% Triton X-PBS were used as blocking and antibody diluents. Retinas were placed in 96-well plates, and 200 μL of blocking solution was added per retina. The plates were blocked on a horizontal shaker for 1 h. Primary antibodies were prepared: ERG (endothelial cell nuclear marker) antibody and CD31 (endothelial cell membrane marker) antibody were diluted 1:200. 50 μL of primary antibody diluent was added per vessel. The plates were incubated at 4°C for 16 h on a shaker, followed by thorough washing with 0.5% Triton X-PBS. Secondary antibodies were prepared: the corresponding secondary antibody was diluted 1:200. 50 μL of secondary antibody diluent was added per vessel. The plates were incubated on a horizontal shaker at room temperature for 2 h, followed by thorough washing with 0.5% Triton X-PBS. EdU 488 staining solution was prepared according to the EdU cell proliferation imaging kit (ThermoFisher C10337). After staining for 30 min, 0.3% Triton X-PBS was added. Wash thoroughly with X-PBS; mount using mounting medium containing DAPI.

[0045] The proliferation of Willis Circle and retinal endothelial cells in mice after administration of MMF and FH-IN is shown in the figure. Figure 2 ;in Figure 2 -A is an immunofluorescence staining image of Willis Circle in mice on day 4 after they were given MMF; Figure 2 -B represents the statistical results of Willis Circle proliferation in mice on day 4 after administration of MMF; Figure 2 -C is an immunofluorescence staining image of Willis Circle on day 4 after mice were administered FH-IN; Figure 2-D represents the statistical results of Willis Circle proliferation in mice on day 4 after administration of FH-IN; Figure 2 -E is an immunofluorescence staining image of the retina of mice on day 6 after they were given MMF; Figure 2 -F represents the statistical results of retinal proliferation in mice on day 6 after MMF administration. α-SMA is a vascular smooth muscle marker used to indicate the vascular wall; ERG labels the endothelial cell nuclei, and EDU labels S-phase proliferating cells. The EDU-ERG MERGE, a co-localization image of EDU and ERG, is used to quantitatively analyze the proportion of endothelial cell proliferation. The results showed that after administration of MMF or FH-IN, the proliferation proportion of Willis Circle and retinal endothelial cells in mice significantly increased. This indicates that MMF and FH-IN can promote collateral circulation formation.

[0046] Example 3: Effects of MMF on vascular endothelial proliferation in an adult rat model of cerebral infarction (Willis Circle) The effect of MMF on Willis Circle vascular endothelium was tested using an adult rat cerebral infarction model; if an increase in cell proliferation level was observed, it indicates that MMF can promote the formation of collateral circulation.

[0047] Experimental animals: Eight-week-old male C57BL / 6J mice with normal development were selected as experimental subjects and divided into experimental group and control group.

[0048] Drug preparation: Dissolve MMF in ddH2O to a final concentration of 10 mg / mL, dispense into containers, and store at -20°C protected from light.

[0049] Sixteen adult mice were randomly divided into two groups of eight each: the MMF administration group and the control group (CTRL group). Seven days after administration, middle cerebral artery occlusion (MCAO) surgery was performed. After another seven days of administration, samples were collected. EdU was injected 24 hours before sample collection. Willis Circle specimens were co-stained with EDU and ERG (endothelial cell markers) to prepare fully embedded specimens. Images were taken using a confocal microscope and statistically analyzed. Cell proliferation level was expressed as the proportion of EDU(+)ERG(+) double-positive cells to ERG(+) cells.

[0050] The specific operating method is as follows: Administration: Hold a 1mL syringe connected to a gavage needle; fix the mouse, draw MMF drug solution with a 1mL syringe, insert the gavage needle into the corner of the mouse's mouth, slide it into the throat, and after confirming that it has entered the stomach, slowly inject it; the MMF drug dose is 200μL / mouse, and the control group is given an equal amount of ddH2O.

[0051] Construction of the MCAO model: During the operation, mice were deeply anesthetized by injection of chloral hydrate (5mg / mL; 0.2mL / 20g), the hair on the top of the skull was removed, the skin was cut and the skull was opened, and the left middle cerebral artery was ligated; the temporalis muscle and skin were sutured, and the mice were placed in an incubator to wait for them to wake up naturally; the condition of the mice should be closely monitored for the first three days after the operation, and the surviving mice were harvested one week later.

[0052] Sample collection: 5-ethynyl-2'-deoxyuridine (EdU) was prepared into a working solution of 5 mg / mL using PBS; 24 hours before sample collection, each adult mouse was injected intraperitoneally with 100 μL of EdU using an insulin needle; 7 days after MCAO, the mice were euthanized by cervical dislocation, and the whole brain was immediately removed, fixed in 4% PFA for 24 hours, thoroughly washed, and the Willis Circle vessels were removed.

[0053] Preparation of Willis Circle fully embedded specimens: 5% donkey serum + 1% Triton X-PBS was used as blocking and antibody diluents. Willis Circle vessels were placed in EP tubes, and 75 μL of blocking solution was added per vessel. The tubes were incubated overnight at 4°C with shaking. EdU 488 staining solution was prepared according to the EdU cell proliferation imaging kit (ThermoFisher C10337). After staining for 12 h, the tubes were thoroughly washed with 0.3% Triton X-PBS. Primary antibody preparation: ERG (endothelial cell marker) antibody was diluted 1:200. 75 μL of primary antibody diluent was added per vessel. The tubes were incubated at 4°C with shaking for 48 h, and then thoroughly washed with 0.3% Triton X-PBS. Secondary antibody preparation: The corresponding secondary antibody was diluted 1:200, and sma-cy3 antibody (labeling vascular smooth muscle) was diluted 1:500. 75 μL of secondary antibody diluent was added per vessel. The tubes were incubated at 4°C with shaking for 16 h, and then washed with 0.3% Triton X-PBS. Wash thoroughly with X-PBS; mount using mounting medium containing DAPI.

[0054] The proliferation level of Willis Circle endothelial cells in mice after administration of MMF is shown in the figure. Figure 3 : Figure 3 -A is an immunofluorescence staining image. Figure 3 -B represents statistical results; α-SMA is a vascular smooth muscle marker used to indicate the vascular wall; ERG labels the endothelial cell nucleus, EDU labels S-phase proliferating cells, and EDU-ERG MERGE, a co-localization image of EDU and ERG, is used to quantitatively analyze the endothelial cell proliferation rate. The results showed that MMF administration significantly increased the proliferation rate of Willis Circle cells in mice, and compared with the control group, the proliferation rate of Willis Circle endothelial cells was also increased. This indicates that MMF can promote collateral circulation formation.

[0055] Example 4: Effects of MMF on the diameter and number of newly formed collateral vessels in an adult rat model of myocardial infarction By examining the effects of MMF on the diameter and number of newly formed collateral vessels in an adult rat model of myocardial infarction, the ability of MMF to promote collateral circulation can be directly evaluated.

[0056] Experimental animals: Eight-week-old male C57BL / 6J mice with normal development were selected as experimental subjects and divided into experimental group and control group.

[0057] Drug preparation: Dissolve MMF in ddH2O to a final concentration of 10 mg / mL, dispense into containers, and store at -20°C protected from light.

[0058] Sixteen adult mice were randomly divided into two groups of eight each: the MMF administration group and the control group (hereinafter referred to as the CTRL group). Seven days after administration, a myocardial infarction model was constructed. Seven days after the operation, Microfil angiography was performed to count the diameter and number of newly formed blood vessels.

[0059] The specific operating method is as follows: Administration: Hold a 1mL syringe connected to a gavage needle; fix the mouse, draw MMF drug solution with a 1mL syringe, insert the gavage needle into the corner of the mouse's mouth, slide it into the throat, and after confirming that it has entered the stomach, slowly inject it; the MMF drug dose is 200μL / mouse, and the control group is given an equal amount of ddH2O.

[0060] Establishment of a mouse myocardial infarction model: Mice were deeply anesthetized by intraperitoneal injection of anesthetic (5g tribromoethanol + 10mL tert-amyl alcohol + 400mL PBS, 0.3mL / 30g), followed by endotracheal intubation; thoracotomy: a transverse incision of approximately 1-1.5cm was made in the 4th-5th intercostal space in the precordial region; the pectoral muscles were bluntly dissected, followed by the longitudinal muscles below, exposing the intercostal spaces; a small hole was made with forceps, and the intercostal spaces were then opened with forceps; a thoracotomy instrument was inserted; ligation: the pericardium was first torn open to expose the heart; 10-0... Ligate the left anterior descending branch with sutures at a location 3.5 mm from the lowest point of the left atrial appendage, with a ligation width of approximately 1.5-2 mm and a depth of approximately 0.5-1 mm until a 10-0 suture needle is visible penetrating the myocardial layer. Gently cross the sutures on both sides; observe that the area below the ligation line turns pale before ligating again. Suture the neck and chest. After weaning from the ventilator, observe the mouse's respiratory status and place it on a 35-37℃ warming pad. If the breathing improves significantly, remove the endotracheal tube; if the breathing is weak, continue ventilator support.

[0061] Microfil angiography: Mice were deeply anesthetized by intraperitoneal injection of anesthetic (5g tribromoethanol + 10mL tert-amyl alcohol + 400mL PBS, 0.3mL / 30g). After intravenous injection of anticoagulant (0.1g heparin + 10mL saline) into the penis, the mice were euthanized by isoflurane gas anesthesia. The tissue below the diaphragm was cut open, and needles were fixed below the diaphragm on both sides. The diaphragm was then cut open to expose the thoracic cavity. The ribs on both sides were cut open, turned upwards, and fixed. The area was washed with PBS to expose the field of view. Angiography was performed from the thoracic aorta... Insert a 6-0 suture under the aorta, tie a slipknot, connect the indwelling needle, and connect the fluid to the end of the vasodilator. Prepare adhesive tape. Insert the indwelling needle into the thoracic aorta, secure the tape after successful insertion, then ligate the sutures and continuously infuse the vasodilator at a rate of 6 mL / min. Cut open the white thymus. Ligate the blood vessel and infuse the perfusion fluid to one end of the PFA. Infuse for about 2-3 minutes, observing the heart turning white. Take 600 μL of the perfusion fluid, add 60 μL of white fixative, and quickly mix with a 1 mL syringe. Draw up the perfusion fluid. Inject the solution, then remove air bubbles; close the PFA perfusion switch, connect the perfusion gel, and remove air bubbles; connect a 1mL syringe to the microinfusion pump, and inject approximately 200μL of perfusion gel at a rate of 200μL / min. After the yellow perfusion gel enters the thoracic aorta, slow the injection rate to 10μL / min, and slowly inject 10μL at a time; observe under a microscope when the yellow perfusion gel passes through the aortic arch to the ascending aorta, and record the remaining perfusion gel volume (<10μL). Add a second 10μL perfusion volume; observe the heart under a microscope. The perfusion should be complete when the small arteries are fully filled with perfusion gel, generally 10-15μL; then close the indwelling needle switch and place the mouse vertically in the tray; rinse the heart with physiological saline or PBS every 2-3 minutes to prevent the yellow perfusion gel from adhering to the heart surface; generally, the perfusion gel will be fixed in 30 minutes to 1 hour, at which point the heart can be removed, placed in the PFA, and fixed overnight at 4°C. Replace the PBS after 24 hours.

[0062] Vascular features in a mouse model of myocardial infarction treated with MMF are shown below. Figure 4 ; Figure 4 -A statistical analysis of the diameter and number of newly formed blood vessels showed that, compared with the control group, the diameter and number of newly formed collateral blood vessels after myocardial infarction increased after MMF administration. Figure 4 -B represents the angiography images of the control group. Figure 4 -C shows the angiographic images of the MMF group. The images clearly show a significant increase in red collateral vessels after drug administration. This indicates that in an adult rat myocardial infarction model, MMF administration increases the number of newly formed collateral vessels and improves cardiac blood perfusion, demonstrating that MMF promotes collateral circulation formation.

[0063] The above examples demonstrate that in in vitro experiments, HBMEC showed increased cell proliferation after administration of MMF; similar results were obtained in in vivo experiments: administration of MMF or FH-IN promoted the proliferation of Willis circle and retinal vascular endothelium in developing rats; in an adult rat cerebral infarction model, administration of MMF promoted the proliferation of Willis circle vascular endothelium; in an adult rat myocardial infarction model, administration of MMF increased the number of newly formed collaterals and improved cardiac blood perfusion; therefore, it can be inferred that the mechanism of action of fumaric acid includes promoting the proliferation of vascular endothelial cells, thereby achieving angiogenesis and collateral circulation formation.

Claims

1. An application of a fumaric acid supplement, characterized in that, This is used to prepare one or more drugs that promote the establishment of collateral circulation, promote angiogenesis, and promote the proliferation of vascular endothelial cells.

2. The application of the fumaric acid supplement according to claim 1, characterized in that, The fumaric acid supplement includes one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumaric acid hydratase inhibitors, and pharmaceutically acceptable derivatives of fumaric acid hydratase inhibitors.

3. The application of the fumaric acid supplement according to claim 1 or 2, characterized in that, Drugs used to prepare for revascularization therapy.

4. The application of the fumaric acid supplement according to claim 1 or 2, characterized in that, Used to prepare drugs for the prevention and treatment of ischemic cardiovascular and cerebrovascular diseases.

5. The application of the fumaric acid supplement according to claim 1 or 2, characterized in that, The fumaric acid supplement is also salted and / or chemically modified.

6. The application of the fumaric acid supplement according to claim 5, characterized in that, The chemical modification includes at least one of acetylation, amination, methylation, phosphorylation, glycosylation, lipidation, ubiquitination, biotin labeling, and fluorescent protein labeling.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition is used to promote the establishment of collateral circulation; the pharmaceutical composition includes fumaric acid supplements.

8. The pharmaceutical composition according to claim 7, characterized in that, The fumaric acid supplement includes one or more of the following: fumaric acid, pharmaceutically acceptable derivatives of fumaric acid, monomethyl fumarate, pharmaceutically acceptable derivatives of monomethyl fumarate, dimethyl fumarate, pharmaceutically acceptable derivatives of dimethyl fumarate, fumaric acid hydratase inhibitors, and pharmaceutically acceptable derivatives of fumaric acid hydratase inhibitors.

9. The pharmaceutical composition according to claim 7 or 8, characterized in that, The dosage form is any one of aerosol, spray, powder, pill, tablet, film, ointment, suppository, paste, solution, injection, mixture, lotion or liniment.

10. The pharmaceutical composition according to claim 7 or 8, characterized in that, The dosage is 0.1~100mg / kg.

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