Pharmaceutical composition for relieving vasospasm and preparation method and application thereof

Abstract

The invention provides a pharmaceutical composition for relieving vasospasm and a preparation method and application thereof.The pharmaceutical composition is prepared from main functional components including fasudil and isosorbide dinitrate, the concentration of fasudil is 10-3.5 mol / L, the concentration of isosorbide dinitrate is 10-4 mol / L, the concentration of isosorbide dinitrate is 10-15 mol / L, and the concentration of isosorbide dinitrate is 10-15 mol / L; the preparation method comprises the following steps: weighing the fasasdil hydrochloride and the isosorbide dinitrate according to the formula amount, and respectively adding the fasasdil hydrochloride and the isosorbide dinitrate into the Ringer's solution. The fasudil and the isosorbide dinitrate with specific concentration in the pharmaceutical composition have synergistic effects, so that the effects of relieving vasospasm and relaxing blood vessels can be quickly achieved, and the final relaxation degree is increased, which not only has direct significance in clinical prevention and treatment of vasospasm, but also has good clinical application prospects. And the traditional Chinese medicine composition also has a good effect on the middle and long-term patency rate of postoperative blood vessels.

Description

Technical Field

[0001] The invention relates to the field of cardiovascular diseases and peripheral vascular surgical intervention, and is a pharmaceutical composition for relieving vascular spasm, and a preparation method and application thereof. Background Art

[0002] Coronary artery disease (CAD), also known as coronary atherosclerotic heart disease, is a heart disease caused by atherosclerotic lesions in the coronary arteries that lead to narrowing or blockage of blood vessels, causing myocardial ischemia, hypoxia or necrosis. As one of the most common cardiovascular diseases worldwide, CAD is the leading cause of death and disability. The main cause of CAD is atherosclerosis, a chronic inflammatory disease associated with multiple risk factors, including hyperlipidemia, hypertension, diabetes, obesity and unhealthy lifestyles. Despite advances in drug treatment in recent years, such as the use of statins, the mortality and morbidity associated with CAD remain high.

[0003] During coronary artery bypass grafting surgery, intraoperative or postoperative vasospasm may occur due to a variety of factors, which not only affects the outcome of the surgery, especially in the early postoperative stage, but may also lead to surgical failure. Vasospasm is an important cause of death in patients, so during cardiac surgery, doctors often use a variety of vasodilators to prevent or reverse vasospasm caused by bypass grafting. Researchers around the world have invested a lot of research work in the field of anti-vasospasm treatment, which has significantly reduced the incidence of vasospasm. However, even so, vasospasm may still occur during or after surgery, so the prevention and treatment of vasospasm remains a long-standing problem in the medical field that needs to be solved.

[0004] Vasospasm is caused by abnormal contraction of vascular smooth muscle cells, which leads to vascular stenosis, reduced blood flow, ischemia of organ tissues, and in severe cases, complete occlusion of the lumen. If the spasm lasts too long, it may lead to secondary thrombosis and vascular embolism, and promote the occurrence and development of diseases such as vascular restenosis and atherosclerosis. In cardiac surgery, the use of vasodilators is an important means to prevent and treat vasospasm. For example, classic vasodilators such as papaverine hydrochloride are used to treat ischemia caused by cerebral, cardiac and peripheral vascular spasms. In addition, the treatment of vasospasm also includes interventional therapy and drug therapy. Effective prediction, prevention and treatment of vasospasm will significantly improve the survival rate and quality of life of patients. Therefore, although some progress has been made in anti-vasospasm treatment, continued research and exploration are still needed to further reduce the impact of vasospasm on patient health.

[0005] In addition, interventional surgery for coronary heart disease (such as stent implantation) is a widely used interventional method for treating coronary heart disease in clinical practice. In interventional surgery (balloon dilatation or stent implantation), the catheter access vessels, especially the radial artery and the coronary artery itself, are often prone to spasm. Clinically, antispasmodic drugs such as nitroglycerin are often injected into the catheter to relieve spasm.

[0006] Similar situations also occur in cerebrovascular interventional surgery.

[0007] In the above cases, the use of appropriate vascular antispasmodics is an important measure to reduce complications and mortality. However, which antispasmodics to use is a question that clinicians and scientists have been discussing. Summary of the invention

[0008] The technical problem to be solved by the present invention is to provide a pharmaceutical composition for relieving vasospasm.

[0009] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned pharmaceutical composition for relieving vasospasm.

[0010] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned pharmaceutical composition for relieving vasospasm.

[0011] In order to solve the above technical problems, the technical solution of the present invention is:

[0012] A pharmaceutical composition for relieving vasospasm, named FI solution, the main active ingredient of which is a mixed solution of Fasudil and Isosorbide dinitrate (ISDN), wherein the concentration of Fasudil is 10 -3.5 mol / L, isosorbide dinitrate concentration is 10 -4 mol / L.

[0013] Preferably, the pharmaceutical composition for relieving vasospasm is composed of Ringer's solution, fasudil, isosorbide dinitrate, heparin sodium injection and sodium bicarbonate, wherein the final concentration of fasudil in each 500 ml of Ringer's solution is 10 - 3.5 mol / L; the final concentration of isosorbide dinitrate was 10 -4 mol / L; the final concentration of heparin sodium injection is 1700U / L; the final concentration of sodium bicarbonate is 0.1064g / L.

[0014] In the above-mentioned pharmaceutical composition, Ringer's solution is a solution, and fasudil and isosorbide dinitrate are main solutes. The fasudil, molecular formula is C 14 H 17N3O2S, molecular weight 327.83; the isosorbide dinitrate, molecular formula C6H8N2O8, molecular weight 236.14; the heparin sodium solution, molecular formula ca. (C 12 H 16 NS2Na3) 20 ; The sodium bicarbonate has a molecular formula of NaHCO3 and a molecular weight of 84.01.

[0015] Preferably, the pharmaceutical composition for relieving vasospasm contains the following in every 150 ml of the Ringer's solution: 15.5 mg of fasudil hydrochloride, 3.5 mg of isosorbide dinitrate, 250 U of sodium heparin, 190 μl of 8.4% sodium bicarbonate, with a pH value of 7.4.

[0016] The preparation method of the above-mentioned pharmaceutical composition for relieving vasospasm comprises the following specific steps (taking the preparation of 150mI as an example):

[0017] (1) Weigh 150 ml of Ringer's solution;

[0018] (2) Weigh 15.5 mg of farsudil hydrochloride and 3.5 mg of isosorbide dinitrate and add them separately into 150 ml of Ringer's solution.

[0019] Preferably, the preparation method of the above-mentioned pharmaceutical composition for relieving vasospasm comprises the following specific steps (taking the preparation of 150 ml as an example):

[0020] (1) Weigh 150 ml of Ringer's solution;

[0021] (2) Weigh 15.5 mg of farsudil hydrochloride, 3.5 mg of isosorbide dinitrate, and 40 μl of heparin sodium injection and add them into 150 ml of Ringer's solution;

[0022] (3) Weigh 0.84 g of sodium bicarbonate and add 10 ml of distilled water to obtain an 8.4% solution. Take 190 μl and add it to 150 ml of Ringer's solution.

[0023] The application of the above-mentioned pharmaceutical composition in preparing a drug for relieving vasospasm.

[0024] Preferably, in the application of the above-mentioned pharmaceutical composition, fasudil and isosorbide dinitrate act synergistically, and the prepared drug for relieving vasospasm is a drug for relieving vasospasm during surgery or interventional treatment of coronary heart disease and peripheral vascular disease.

[0025] Preferably, in the application of the above-mentioned pharmaceutical composition, the coronary heart disease and peripheral vascular disease surgeries include coronary artery bypass grafting surgery, percutaneous coronary intervention surgery, and other peripheral vascular intervention surgeries (such as cerebrovascular intervention surgery).

[0026] The beneficial effects of the present invention are:

[0027] The pharmacological effects of the above-mentioned pharmaceutical composition (FI solution) for relieving vasospasm on transplanted blood vessels used in coronary heart disease and peripheral vascular disease surgery have been preliminarily studied, and it has been found that the synergistic effect of fasudil and isosorbide dinitrate at a specific concentration can more quickly relieve vasospasm and dilate blood vessels, and increase the final degree of dilation. This is not only of direct significance in the clinical prevention and treatment of vasospasm, but also has a good effect on the medium- and long-term patency of blood vessels after surgery. This formula shows great application potential in the preparation of therapeutic drugs for cardiovascular diseases and peripheral vascular lesions, and in the relief of vasospasm and improvement of medium- and long-term patency during surgery and interventional treatment. It can also lay a scientific foundation for the study of the mechanism of anti-vasospasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the vasodilation curve when potassium chloride is a vasoconstrictor.

[0029] Figure 2 This is the vasodilation curve when U46619 is a vasoconstrictor.

[0030] Figure 3 This is the vasoconstriction curve when potassium chloride is used as a vasoconstrictor.

[0031] Figure 4 This is the vasoconstriction curve when U46619 is a vasoconstrictor.

[0032] Figure 5 This is the release expression diagram of ROCK2 protein after incubation with FI solution.

[0033] Figure 6 It is the expression graph of cGMP release after incubation with FI solution. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0035] In the following pharmaceutical composition, Ringer's solution is the main solvent, the specification is 500ml, purchased from Guangdong Otsuka Pharmaceutical Co., Ltd.; Fasudil, the molecular formula is C 14 H 17 N3O2S, molecular weight 327.83, specification 100 mg, purchased from Dalian Meilun Biotechnology Co., Ltd.; isosorbide dinitrate, molecular formula C6H8N2O8, specification 50 mg, purchased from MCE (MedChemExpress); heparin sodium solution, molecular formula ca. (C 12 H 16NS2Na3) 20 , specification is 2ml: 12500 units, purchased from Jiangsu Wanbang Biochemical Pharmaceutical Group Co., Ltd.; sodium bicarbonate, molecular formula is NaHC03, molecular weight is 84.01.

[0036] Example 1

[0037] A pharmaceutical composition (FI solution) for relieving vasospasm, comprising Ringer's solution (main solvent), fasudil, isosorbide dinitrate, heparin sodium solution, and sodium bicarbonate, wherein:

[0038] Green's solution: 150ml

[0039] Fasudil: 15.5 mg

[0040] Isosorbide dinitrate: 3.5 mg

[0041] Heparin sodium: 250U

[0042] 8.4% Sodium bicarbonate: 190 μl

[0043] PH value: 7.4.

[0044] The preparation method of the above-mentioned pharmaceutical composition (FI solution) for relieving vasospasm comprises the following specific steps:

[0045] (1) Weigh 150 ml of Ringer's solution;

[0046] (2) Weigh 15.5 mg of fasudil, 3.5 mg of isosorbide dinitrate, and 40 μl of heparin sodium injection and add them into 150 ml of Ringer's solution;

[0047] (3) Weigh 0.84 g of sodium bicarbonate and add 10 ml of distilled water to obtain an 8.4% solution. Take 190 μl and add it to 150 ml of Ringer's solution.

[0048] Example 2

[0049] (1) Collection and pretreatment of internal mammary artery:

[0050] The distal end of the grafted blood vessel (internal mammary artery) discarded during coronary artery bypass grafting surgery was placed in 6 mL of Krebs buffer at 4°C filled with a mixed gas of 95% CO2 and 5% O2, and immediately transferred to the laboratory. The peripheral tissue was carefully separated in the buffer with dissecting scissors to expose the blood vessels, and the blood vessels were segmented into 3 mm segments. Each segment of the blood vessel ring was suspended in the bath of a microvascular tension measuring instrument (Model 620, DMT Company, Denmark) for at least 45 minutes.

[0051] (2) Vascular function experiments:

[0052] Vascular status recovery:

[0053] 6 ml of Krebs buffer was added to the bath of the microvascular tension measuring instrument, the temperature was set at 37°C, and a 95% CO2, 5% O2 mixed gas was continuously introduced until the end of the experiment. The separated segmented blood vessels were placed on the hanging needle used to measure the blood vessel tension in the bath, and the "length-tension" curve of the blood vessel was completed by using specific software, so that the tension of the blood vessel was set at the physiological pressure and maintained in this state for 1-1.5 hours, so that the following experiments were carried out under the physiological pressure of the blood vessel.

[0054] Establishment of vascular resting force state:

[0055] After the blood vessels have fully rested, rotate the micrometer to dilate the blood vessels. The dilation process is carried out in steps. Generally, the blood vessel wall pressure reaches 100mmHg (13.3kPa) after 3-4 dilations. At this time, the software simulates and calculates the inner diameter of the blood vessel L100. Adjust the micrometer by 0.9*L100 to relax the blood vessels. When the blood vessels are balanced, the blood vessel Resting Force state is established. At this time, the dilation state of the blood vessels in vitro is closest to the physiological state in vivo. Record the transmural pressure and inner diameter D100 in this state. After the blood vessels reach the Resting Force state, in order to restore the tension changes caused by the stretching process of the blood vessels, this state should be maintained for 1 hour to fully restore the vascular function.

[0056] In order to study the effects of different treatment factors on vasodilation and anti-contraction functions, the experiment was divided into 4 parts:

[0057] Part 1: Vasodilation by FI solution after vasoconstriction by KCl

[0058] The same internal mammary artery was cut into three sections. After the internal mammary artery was constricted with potassium chloride (30 mM) in advance, 18 μl of 10 -1 mol / L fasudil (final concentration was 10 -3.5 mol / L), 6μl concentration is 10 -1 mol / L isosorbide dinitrate (final concentration was 10 -4 mol / L), Fasudil + isosorbide dinitrate mixed solution (10 -3.5 mol / L+10 -4 mol / L). The three groups of solutions were compared in terms of their effects on vasodilation of the internal mammary artery (n=8, 8 biological replicates). Figure 1 The maximum relaxation degrees were 91.1%, 71.1% and 100% respectively, and after relaxation with FI solution, the relaxation speed was fast and lasting.

[0059] Part 2: Relaxation of vasoconstricted vessels by U46619 followed by FI solution

[0060] The same internal mammary artery was cut into three segments and then treated with U46619 (10 -8 mol / L) to pre-constrict the internal mammary artery, and then 18 μl of 10 -1 mol / L fasudil (final concentration was 10 -3.5 mol / L), 6μl concentration is 10 -1 mol / L isosorbide dinitrate (final concentration was 10 -4 mol / L), Fasudil + isosorbide dinitrate mixed solution (10 -3.5 mol / L+10 -4 mol / L). The three groups of solutions were compared in terms of their effects on vasodilation of the internal mammary artery (n=8, 8 biological replicates). Figure 2 The maximum relaxation degrees were 94.3%, 87.8% and 100%, respectively. After relaxation with FI solution, the relaxation speed was fast and lasting.

[0061] Part 3: Stepwise contraction of KCl solution after pretreatment of blood vessels with FI solution

[0062] The same internal mammary artery was cut into three sections. The internal mammary artery was first constricted with potassium chloride (100 mM) to obtain a contraction value and then washed away. Then 18 μl of 10 -1 mol / L fasudil (final concentration was 10 -3.5 mol / L), 6μl concentration is 10 -1 mol / L isosorbide dinitrate (final concentration was 10 -4 mol / L), Fasudil + isosorbide dinitrate mixed solution (10 -3.5 mol / L+10 -4 After the internal mammary artery was pretreated with 100 mol / L of potassium chloride, the internal mammary artery was contracted in a stepwise manner with 5-120 mM potassium chloride. The anti-contractile effects of the three groups of solutions after pretreatment of the internal mammary artery were compared (n=8, 8 biological replicates). Figure 3 The percentages of the maximum contraction values ​​after pretreatment with potassium chloride (100 mM) were 63.2%, 147.9% and 49.1%, respectively. It was found that after pretreatment with FI solution, the anti-contraction effect was strong and lasting.

[0063] Part 4: Stepwise contraction of blood vessels after pretreatment with FI solution followed by U46619 solution

[0064] The same internal mammary artery was cut into three sections. The internal mammary artery was first constricted with potassium chloride (100 mM) to obtain a contraction value and then washed off. Then, 18 μl of 10 mol / L fasudil (final concentration 10 -3.5 mol / L), 6μl concentration is 10 -1 mol / L isosorbide dinitrate (final concentration was 10 -4 mol / L), Fasudil + isosorbide dinitrate mixed solution (10 -3.5 mol / L+10 -4 mol / L) was used to pre-treat the internal mammary artery, and then the -10 -10 -4.5 log M concentration of U46619 stepwise contraction. Comparison of the anti-contractile effects of the three groups of solutions after pretreatment of the internal mammary artery (n = 8, 8 biological replicates). Figure 4 , the maximum shrinkage was the percentage of the shrinkage value after pretreatment with potassium chloride (100 mM) was 60.7%, 154.5% and 37.4%, respectively. It was found that after pretreatment with FI solution, the anti-contraction effect was strong and lasting.

[0065] Example 3

[0066] Internal mammary artery rings from the same patient were divided into two groups for culture. Both groups of blood vessels were treated in advance and immersed in 6 ml Krebs buffer. In addition, FI solution (Fasudil 10 -3.5 log M and isosorbide dinitrate 10 - 4 log M), and the control group was added with Krebs solution of equal volume to FI solution. The blood vessels were placed in a CO2 incubator (95% O2, 5% CO2), and after 24 hours of incubation, they were transferred to -80°C for storage until analysis. Due to the low protein content of individual patient samples, each group of experimental internal mammary artery rings came from 1-3 patients, involving a total of 15 patients, divided into five experiments.

[0067] To study the effects of different molecules on vasodilation, the molecular experiment was divided into 4 parts:

[0068] Part 1: ROCK2 protein expression after 24 h incubation with FI solution

[0069] Western Blot (WB) analysis process of ROCK protein expression

[0070] 1. Sample preparation and lysis:

[0071] The internal mammary artery rings of the experimental group and the control group were collected and quickly frozen in liquid nitrogen. Before analysis, the vascular tissue was washed with ice-cold PBS buffer to remove surface blood and impurities. Then 150 μl of lysis buffer (containing protease inhibitors) was added, and the cells were lysed using an ultrasonic disruptor to obtain a homogenous tissue lysate. After lysis, the cells were centrifuged at 12,000 g for 10 minutes, and the supernatant was collected for subsequent analysis.

[0072] 2. Protein concentration determination:

[0073] The protein concentration in the lysate was determined using the BCA method (protein quantification kit).

[0074] 3. SDS-PAGE gel electrophoresis:

[0075] Mix 50 μg of protein sample with SDS-PAGE loading buffer and boil for 8 minutes to denature the protein. Then, perform electrophoresis on SDS-PAGE gel at a predetermined concentration (10%-15%). Run the gel at 60V until the sample enters the separation gel, then increase to 100V and continue running until it reaches the appropriate position.

[0076] 4. Transfer membrane:

[0077] After electrophoresis, the separated proteins were transferred to a PVDF membrane under the conditions of 90 min and a constant voltage of 120 V to ensure uniform transfer on the membrane.

[0078] 5. Blocking and antibody incubation:

[0079] The membrane was blocked in 5% skim milk powder solution for 1 hour with shaking at room temperature. After blocking, the membrane was incubated with anti-ROCK2 primary antibody (1:1000 dilution) at 4°C overnight. The next day, the membrane was washed 3 times for 10 minutes each time using TBST buffer.

[0080] 6. Secondary antibody incubation and development:

[0081] The membrane was incubated with HRP-labeled secondary antibody (1:2000 dilution) and shaken at room temperature for 1 hour. Afterwards, the membrane was washed again 3 times. Finally, chemiluminescence (ECL) was used for color development and photos were taken for preservation.

[0082] 7. Result analysis:

[0083] Western Blot images were analyzed using Gel Quant Net software to measure the band intensity of ROCK protein and calculate the relative expression of ROCK protein based on the internal reference (β-actin).

[0084] 8. Experimental Repetition and Statistical Analysis:

[0085] To ensure the reliability of the results, four independent experiments were performed in each group. The data were statistically analyzed using GraphPad software, and the differences between the two groups were tested using t-test. Figure 5 The expression of ROCK2 in the internal mammary artery after treatment with FI solution decreased significantly, indicating that FI solution inhibited the expression of ROCK2, thereby playing a vasodilation role.

[0086] Part 2: Release of cGMP molecules after 24 h incubation with FI solution

[0087] ELISA process for cGMP expression

[0088] 1. Sample preparation:

[0089] Rinse the tissue with pre-cooled PBS (0.01M, pH=7.4) to remove residual blood, weigh and mince the tissue. Add the minced tissue and the corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9mL of PBS, the specific volume can be adjusted appropriately according to experimental needs, and keep records. Add protease inhibitors to PBS) into a glass homogenizer and grind thoroughly on ice. Finally, centrifuge the homogenate at 4°C, 5000×g for 8 minutes, and take the supernatant for detection.

[0090] 2. ELISA kit preparation:

[0091] Use a cGMP ELISA kit (cGMP ELISA kit purchased from Elabscience). Prepare the ELISA kit according to the manufacturer's instructions, ensure that all reagents are equilibrated at room temperature, and prepare standard and sample diluents. Take the kit out of the refrigerator 20 minutes in advance and equilibrate to room temperature (18-25°C).

[0092] 3. Preparation before testing:

[0093] 1. Take out the test kit from the refrigerator 20 minutes in advance and equilibrate to room temperature (18-25°C).

[0094] 2. Washing solution: dilute the concentrated washing solution with double distilled water (1:24).

[0095] 3. Standard working solution: Centrifuge the standard at 10000×g for 1 minute, add 1mL of standard & sample diluent to the freeze-dried standard, tighten the tube cap, let it stand for 10 minutes, turn it upside down several times, and after it is fully dissolved, gently mix it to avoid foaming, and prepare a 50pmol / mL standard working solution. Then dilute it in multiples as needed. Prepare the following concentrations: 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0pmol / mL. Multiple dilution method: Take 7 EP tubes, add 500μl of standard & sample diluent to each tube, draw 500μl from the 50pmol / mL standard working solution into the first EP tube and mix it to make a 25pmol / mL standard working solution, and follow this step to draw and mix in sequence.

[0096] 4. Biotinylated antibody working solution: Calculate the required amount for the experiment before the experiment (calculated as 50μl / well). 15 minutes before use, centrifuge the concentrated biotinylated antibody at 800×g for 1 minute, and dilute the 100× concentrated biotinylated antibody to 1× working concentration with biotinylated antibody diluent (for example: 10μl concentrated solution + 990μl diluent). Prepare and use immediately.

[0097] 5. HRP enzyme conjugate working solution: HRP enzyme conjugate is HRP enzyme conjugate with avidin. Calculate the amount required for the experiment before the experiment (calculated as 100μl / well). 15 minutes before use, centrifuge the concentrated HRP enzyme conjugate at 800×g for 1 minute, and dilute the 100× concentrated HRP enzyme conjugate to 1× working concentration with enzyme conjugate diluent (for example: 10μl concentrated solution + 990μl diluent). Prepare and use immediately.

[0098] 4. Operation steps:

[0099] 1. Set up standard wells, blank wells and sample wells respectively. Add 50μl of diluted standard to the standard wells, 50μl of standard & sample diluent to the blank wells, and 50μl of the sample to be tested to the remaining wells (all samples and standards to be tested are set up in duplicate wells during the test). Immediately add 50μl of the prepared biotinylated antibody working solution to each well. Cover the ELISA plate with a film and incubate at 37℃ for 45 minutes. Tip: When adding the sample, add the sample to the bottom of the ELISA plate, try not to touch the wall of the well, and gently shake to mix and avoid bubbles. The sample addition time should be controlled within 10 minutes.

[0100] 2. Shake off the liquid in the wells and pat dry on clean absorbent paper. Add 350μl of washing solution to each well, soak for 1 minute, absorb or shake off the liquid in the ELISA plate, and pat dry. Repeat this washing step 3 times. After washing, please proceed to the next step immediately and do not let the microplate dry.

[0101] 3. Add 100 μl of HRP enzyme conjugate working solution to each well, cover the ELISA plate with a film, and incubate at 37°C for 30 minutes.

[0102] 4. Shake off all liquid in the wells and wash the plate 5 times using the same method as step 2.

[0103] 5. Add 90 μl of substrate solution (TMB) to each well, cover the ELISA plate with a film, and incubate at 37°C in the dark for about 15 minutes.

[0104] 6. Add 50 μl of stop solution to each well to terminate the reaction. Tip: The order of adding the stop solution should be as consistent as possible with the order of adding the substrate solution.

[0105] 7. Immediately measure the optical density (OD value) of each well at a wavelength of 450 nm using an enzyme reader.

[0106] 5. Result judgment:

[0107] 1. Calculate the average OD value of the standard and sample duplicate wells, and fit the standard curve of the fourth-order polynomial function with concentration as the horizontal axis and OD value as the vertical axis.

[0108] 2. If the sample OD value is lower than the lower limit of the standard curve, it should be appropriately diluted and re-measured and multiplied by the corresponding dilution factor when calculating the sample concentration.

[0109] 6. Statistical analysis:

[0110] The cGMP concentration data of different experimental groups were statistically analyzed using GraphPad software, and the differences between the two groups were analyzed using t-test. Figure 6 The expression of cGMP in the internal mammary artery after treatment with FI solution increased significantly, indicating that FI solution increased the expression of cGMP, thereby playing a vasodilation role.

[0111] The above-mentioned detailed description of the pharmaceutical composition for relieving vasospasm, its preparation method and application with reference to the specific embodiments is illustrative rather than restrictive, and several embodiments can be listed according to the limited scope. Therefore, changes and modifications without departing from the overall concept of the present invention should fall within the scope of protection of the present invention.

Claims

1. A pharmaceutical composition for relieving vasospasm, characterized in that: The main active ingredients are fasudil and isosorbide dinitrate, among which the concentration of fasudil is 10 -3.5 mol / L, isosorbide dinitrate concentration is 10 -4 mol / L.

2. The pharmaceutical composition for relieving vasospasm according to claim 1, characterized in that: It is composed of Ringer's solution, fasudil, isosorbide dinitrate, heparin sodium injection and sodium bicarbonate, wherein the final concentration of fasudil in each 500ml Ringer's solution is 10 -3.5 mol / L; the final concentration of isosorbide dinitrate was 10 -4 mol / L; the final concentration of heparin sodium injection is 1700U / L; the final concentration of sodium bicarbonate is 0.1064g / L.

3. The pharmaceutical composition for relieving vasospasm according to claim 1, characterized in that: Every 150 ml of the Ringer's solution contains: 15.5 mg of fasudil hydrochloride, 3.5 mg of isosorbide dinitrate, 250 U of heparin sodium, 190 μl of 8.4% sodium bicarbonate, and the pH value is 7.

4.

4. The method for preparing the pharmaceutical composition for relieving vasospasm according to claim 1, characterized in that: Weigh fadil hydrochloride and isosorbide dinitrate according to the formula amount and add them into Ringer's solution respectively.

5. The method for preparing the pharmaceutical composition for relieving vasospasm according to claim 4, characterized in that: The specific steps are: (1) Weigh 15.5 mg of fasudil hydrochloride, 3.5 mg of isosorbide dinitrate, and 40 μl of heparin sodium injection and add them to 150 ml of Ringer's solution; (2) Prepare 8.4% sodium bicarbonate solution and add 190 μl of it into 150 ml of Ringer's solution.

6. Use of the pharmaceutical composition according to any one of claims 1 to 3 in the preparation of a drug for relieving vasospasm.

7. The use of the pharmaceutical composition according to claim 6, characterized in that: The prepared medicine for relieving vascular spasm is a medicine for relieving vascular spasm during surgery or interventional treatment of coronary heart disease and peripheral vascular disease.

8. The use of the pharmaceutical composition according to claim 7, characterized in that: The coronary heart disease and peripheral vascular disease surgeries include coronary artery bypass grafting, percutaneous coronary intervention, and other peripheral vascular intervention surgeries.

9. The use of the pharmaceutical composition according to claim 8, characterized in that: The coronary heart disease and peripheral vascular disease surgery is a cerebrovascular interventional surgery.

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