CN103690954B - Cardiovascular protection pharmaceutical composition and application thereof - Google Patents

Abstract

The invention discloses a cardiovascular protective medicinal composition, which comprises the following ingredients: 50-200 mole parts of angiotensin converting enzyme inhibitor and 100000-600000 parts by weight of antioxidant. The invention also discloses the use of the aforementioned pharmaceutical composition and a blood substitute. The cardiovascular protection medicinal composition of the invention can effectively protect the cardiovascular system and reduce the cardiovascular side effects of hemoglobin oxygen-carrying carriers. The blood substitute of the invention has no cardiovascular side effects, overcomes the defects of the existing hemoglobin blood substitutes, and has excellent clinical application prospects.

Description

A cardiovascular protection pharmaceutical composition and its application

technical field

The invention relates to a cardiovascular protective pharmaceutical composition and its application.

Background technique

With the shortage of blood resources, the limitation of storage period and the spread of infectious diseases caused by blood transfusion, it is increasingly necessary to find substitutes that can replace blood. Currently, oxygen-carrying blood substitutes mainly include fluorocarbons and hemoglobin preparations. As an important part of blood, hemoglobin (Hb) is located in red blood cells and is the main medium for the body to transport oxygen. However, pure hemoglobin solution cannot be infused directly, because hemoglobin detached from red blood cells loses the regulation of 2,3-diphosphoglycerate (2,3-DPG), and its oxygen affinity rises sharply, which cannot effectively supply oxygen to tissues. Under the action of various enzymes, free hemoglobin will quickly depolymerize into dimers and monomers, which will be excreted by the kidneys or block the renal tubules, resulting in strong nephrotoxicity. In addition, hemoglobin lacks the function of the reductase system in red blood cells, so it will be easily oxidized into methemoglobin (MetHb), lose its oxygen-carrying capacity and generate free radicals, causing oxidative stress damage in the body. Therefore, necessary modification or treatment of hemoglobin is required, and the resulting products are collectively called hemoglobin oxygen-carrying carriers (HBOCs).

Many defects have been found in existing HBOCs products in clinical practice. HemAssist developed by Abbott is a first-generation product, which was abandoned due to the significant increase in the mortality rate of subjects found in phase III clinical trials. HemAssist and Polyheme also terminated studies due to severe cardiotoxicity in clinical trials. Hemopure (polymerized bovine hemoglobin cross-linked with glutaraldehyde) developed by Biopure in the United States is currently the only product that has been approved for clinical use, but it is only available in South Africa. Because of cardiovascular safety issues exposed in phase III clinical studies, further development was also stopped. In 2008, Natanson and his colleagues published a meta-analysis on HBOC clinical trials in the world-renowned medical journal "JAMA", pointing out that it increases the mortality of patients and has a potential risk of myocardial infarction. At the same time, "JAMA" magazine issued an editor's note for this article, pointing out that no further clinical trials should be carried out until the potential side effects of HBOC products and their mechanisms are clarified. Regarding the cardiovascular side effects after infusion of HBOCs products, the academic community generally believes that free or unpolymerized hemoglobin in HBOC, including oxygenated or non-oxygenated state, penetrates the endothelial barrier and consumes the endothelial relaxation factor nitric oxide. (NO); at the same time, the oxidation-reduction reaction of hemoglobin itself, the oxygen free radicals generated cause the body's oxidative stress response. At present, there are few drugs that can reduce the cardiovascular side effects of hemoglobin oxygen carrier.

Angiotensin converting enzyme inhibitor (Angiotensin Converting En-zyme Inhibitor, ACEI) is a skin substance isolated from the venom of snake venom, developed through a series of structure-activity relationships. According to the different chemical structures of their active parts, they can be divided into three categories: the first category is mercapto group, and the representative drug is Captopril (Captopril); the second category is phosphate group, and the representative drug is fosinopril ( Fosino Pril); the third category is carboxyl, the representative drug is Enalapril (Enala-pril). Angiotensin-converting enzyme inhibitors can dilate blood vessels, lower blood pressure, inhibit vascular hyperplasia and hypertrophy, inhibit and reverse myocardial hypertrophy, improve coronary blood supply to the heart, and reduce myocardial ischemia-reperfusion injury.

Antioxidants are a class of substances that can help capture and neutralize free radicals, thereby eliminating the damage caused by free radicals to the human body. Common antioxidants include ascorbic acid, superoxide dismutase, catalase, peroxidase, vitamin E, glutathione, sodium dithionite, N-acetylcysteine, etc. Studies have found that intake of antioxidants such as ascorbic acid and vitamin E can effectively prevent hypertension and coronary heart disease, but the therapeutic effect is not obvious.

There is no report on the combination of angiotensin-converting enzyme inhibitors and antioxidants to reduce cardiovascular side effects caused by hemoglobin oxygen carriers.

Contents of the invention

In order to overcome the defects of the prior art, the present invention provides a cardiovascular protection drug and its application, and also provides a new blood substitute.

The cardiovascular protection pharmaceutical composition of the present invention comprises the following ingredients: 50-200 mole parts of angiotensin-converting enzyme inhibitor, and 100,000-600,000 parts by weight of antioxidant.

The above molar parts: parts by mass=mol/g.

The composition includes the following ingredients: 100-200 mole parts of angiotensin-converting enzyme inhibitor and 300000-600000 parts by weight of antioxidant.

The angiotensin-converting enzyme inhibitor is N-[(S)-3-mercapto-2-methylpropionyl]-L-proline or (S)-1-[N-(1-ethoxycarbonyl -3-phenylpropyl)-L-alanyl]-L-proline.

N-[(S)-3-Mercapto-2-methylpropionyl]-L-proline, trade name Captopril, empirical formula (Hill notation) C ₉ H ₁₅ NO ₃ S, molecular weight 217.29, the structural formula is as follows:

(S)-1-[N-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl]-L-proline, trade name enalapril, empirical molecular formula (Hill Notation) C ₂₀ H ₂₈ N ₂ O ₅ , molecular weight 376.45, structural formula as follows:

The antioxidant is one or both of ascorbic acid, superoxide dismutase, catalase, peroxidase, vitamin E, glutathione, sodium dithionite, N-acetylcysteine combination of the above.

The antioxidant is a mixture of superoxide dismutase, catalase and N-acetylcysteine, and the weight ratio of the three is 1:1:1; or it is superoxide dismutase and superoxide dismutase. The mixture of catalase, the weight ratio of the two is 1:1.

The present invention also provides the use of the aforementioned composition in the preparation of medicaments for treating cardiovascular diseases.

The medicine for treating cardiovascular disease is a medicine for treating cardiovascular disease caused by hemoglobin oxygen carrier.

The drug for treating cardiovascular disease is a drug for treating vascular endothelial cell injury, hypertension, heart disease and/or coronary artery spasm.

The blood substitute of the present invention comprises hemoglobin oxygen-carrying carrier and the aforementioned composition, wherein the concentration of hemoglobin oxygen-carrying carrier is 37.5g-120g/L, and the concentration of angiotensin-converting enzyme inhibitor is 50-200μmol/L , The concentration of antioxidants is 0.1-0.6g/L.

The concentration of the hemoglobin oxygen carrier is 75-80g/L, the concentration of the angiotensin-converting enzyme inhibitor is 100-200μmol/L, and the concentration of the antioxidant is 0.3-0.6g/L.

In the hemoglobin oxygen-carrying carrier, the content of hemoglobin is 20-84%.

The hemoglobin oxygen-carrying carrier is polymerized hemoglobin, PEG-modified hemoglobin or hemoglobin encapsulated by PEG-PCL nanoparticles.

The pH of the blood substitute is 7.2-7.4.

The method for preparing the aforementioned blood substitute of the present invention comprises the following steps:

(1) According to the above ratio, take hemoglobin oxygen carrier, angiotensin converting enzyme inhibitor and antioxidant, add water, and mix well;

(2) Adjust the pH value to 7.2-7.4.

The pharmaceutical composition of the present invention can effectively protect the cardiovascular system, treat hypertension, heart disease and coronary artery spasm, and improve the cardiovascular side effects of hemoglobin oxygen-carrying carrier products. The blood substitute of the present invention overcomes the existing hemoglobin blood substitute It can effectively improve the quality of medical care and improve the prognosis of patients, and has a good prospect of clinical application.

The present invention will be further described in detail below through specific embodiments, but it is not a limitation of the present invention. According to the above-mentioned content of the present invention, according to common technical knowledge and conventional means in this field, without departing from the above-mentioned basic technical idea of the present invention. Various other modifications, substitutions, or changes may also be made.

Description of drawings:

Fig. 1 embodiment 7 prepares the particle size distribution figure of nanoparticle

Figure 2 MTT method to determine the effect of the blood substitutes prepared in Example 8 (A) and Example 9 (B) on HUVECs cells

Figure 3 Effects of blood substitutes prepared in Example 8 (A) and Example 9 (B) on the blood pressure of Kunming mice

Effect result on canine coronary artery after the blood substitute coronary artery injection of Fig. 4 embodiment 6 preparations

Effect result on dog heart function after blood substitute coronary injection of Fig. 5 embodiment 6 preparations

detailed description:

Main instruments, reagents and animals:

Blood gas analyzer: ABL800FLEX, Radiometer Company; BC-3000PLUS, Mindray Company, China;

HEMOX ^TM analyzer: TCS Scientific, USA;

pH electrode: Mettler Toledo Company, USA;

Coronary angiography equipment: AXIOM Artis dTA system; Germany Siemens;

Cardiac ultrasound instrument: Vivid7, GE Healthcare, USA;

Captopril, enalapril and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma-Aldrich, USA;

Dimethyl sulfoxide (DMSO), acetone, dichloromethane, petroleum ether, ethyl acetate, lactic acid, and phosphate buffer (PBS, pH 7.4) were purchased from Chengdu Kelong Chemical Reagent Company;

Cell culture medium DMEM medium was purchased from Gibico BRL company;

Human umbilical vein endothelial cells (HUVECs) were obtained from the State Key Laboratory of Biotherapy, West China Hospital, Sichuan University.

Adult male Kunming mice and male Beagle dogs were purchased from Chengdu Dashuo Biotechnology Co., Ltd.

Embodiment 1 Preparation of cardiovascular protection pharmaceutical composition of the present invention

Take 434.6 μg of captopril, 4.0 mg each of superoxide dismutase, catalase, and N-acetylcysteine, add 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm. .

Embodiment 2 Preparation of cardiovascular protection pharmaceutical composition of the present invention

Take 217.3 μg of captopril and 5.0 mg of ascorbic acid, add 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a rotating speed of 600 rpm.

Embodiment 3 Preparation of cardiovascular protection pharmaceutical composition of the present invention

Take 1.5 mg of enalapril, 2.0 mg each of superoxide dismutase, catalase, and N-acetylcysteine, add 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm. .

Embodiment 4 Preparation of cardiovascular protection pharmaceutical composition of the present invention

Take 1.9 mg of enalapril, 5.0 mg each of superoxide dismutase, catalase, and N-acetylcysteine, add 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm. .

Embodiment 5 Preparation of cardiovascular protection pharmaceutical composition of the present invention

Take 108.6 μg of captopril, 0.5 mg of superoxide dismutase and 0.5 mg of catalase, add 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm.

Embodiment 6 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 0.75g of polymerized human cord blood hemoglobin freeze-dried powder (prepared according to the method on pages 7-8 of the patent ZL02133592.3 specification), in which the hemoglobin content is 80%, captopril 434.6μg, superoxidized Add 4.0 mg each of dismutase, catalase, and N-acetylcysteine to 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm. After the stirring is completed, adjust the pH value to 7.4 with a 0.1M sodium bicarbonate solution, and then adjust the volume to 20 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of polymerized human cord blood hemoglobin in the solution was 37.5g/L, the hemoglobin content was 3g/dL, the captopril content was 100μmol/L, the antioxidant content was 0.6g/L, and the oxygen affinity _P50 value was 28mmHg.

Embodiment 7 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 1.2g of PEG-modified porcine hemoglobin freeze-dried powder (prepared according to the method on pages 7-8 of the patent ZL200380109157.8 specification), in which hemoglobin content is 84%, captopril 217.3μg, ascorbic acid 5.0 mg, add 6 mL of normal saline, and stir for 5 minutes with a stirring bar at a speed of 800 rpm. After the stirring is completed, adjust the pH value to 7.4 with 0.1M sodium bicarbonate solution, and then adjust the volume to 10 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of PEG-modified porcine hemoglobin in the solution is 120g/L, the content of hemoglobin is 10g/dL, the content of captopril is 100μmol/L, the content of antioxidant is 0.5g/L, and the oxygen affinity _P50 value is 21mmHg.

Embodiment 8 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 1.6g of PEG-modified bovine hemoglobin freeze-dried powder (prepared according to the method on pages 7-8 of the patent ZL200380109157.8 specification), in which the hemoglobin content is 75%, enalapril 1.5mg, superoxidized Add 2.0 mg each of dismutase, catalase, and N-acetylcysteine to 15 mL of normal saline, and stir magnetically with a stirring bar for 10 minutes at a speed of 600 rpm. After the stirring is completed, adjust the pH value to 7.2 with a 0.1M sodium bicarbonate solution, and then adjust the volume to 20 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of PEG-modified bovine blood hemoglobin in the solution was 80 g/L, the content of hemoglobin was 6 g/dL, the content of enalapril was 200 μmol/L, the content of antioxidant was 0.3 g/L, and the oxygen affinity _P50 value was 13 mmHg.

Embodiment 9 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 5.0 g of polymerized human cord blood hemoglobin freeze-dried powder (prepared according to the method on pages 7 to 8 of the patent ZL02133592.3 specification), in which the hemoglobin content is 80%, enalapril 1.9 mg, superoxidized Add 5.0 mg each of dismutase, catalase, and N-acetylcysteine to 35 mL of normal saline, and stir with a stirring bar for 30 minutes at a speed of 500 rpm. After the stirring is completed, adjust the pH value to 7.4 with a 0.1M sodium bicarbonate solution, and then adjust the volume to 50 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of polymerized human cord blood hemoglobin in the solution was 100g/L, the hemoglobin content was 8g/dL, the enalapril content was 100μmol/L, the antioxidant content was 0.3g/L, and the oxygen affinity _P50 value was 25mmHg.

Embodiment 10 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 2.0 g of polymerized human cord blood hemoglobin freeze-dried powder (prepared according to the method on pages 7-8 of the patent ZL02133592.3 specification), in which the hemoglobin content is 80%, captopril 434.6 μg, superoxidized Add 4.0 mg each of dismutase, catalase, and N-acetylcysteine to 15 mL of normal saline, and stir with a stirring bar for 15 minutes at a speed of 600 rpm. After the stirring is completed, adjust the pH value to 7.4 with a 0.1M sodium bicarbonate solution, and then adjust the volume to 20 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of polymerized human cord blood hemoglobin in the solution was 100 g/L, the hemoglobin content was 8 g/dL, the captopril content was 100 μmol/L, the antioxidant content was 0.6 g/L, and the oxygen affinity P ₅₀ value was 24 mmHg.

Embodiment 11 Preparation of blood substitute of the present invention

Under low temperature conditions of 4°C, take 0.38g of polymerized human cord blood hemoglobin freeze-dried powder (prepared according to the method on pages 7-8 of the patent ZL02133592.3 specification), in which the hemoglobin content is 80%, captopril 108.6μg, superoxidized Add 0.5 mg of dismutase and 0.5 mg of catalase, add 6 mL of normal saline, and stir with a stirring bar for 30 minutes at a speed of 500 rpm. After the stirring is completed, adjust the pH value to 7.2 with 0.1M sodium bicarbonate solution, and then adjust the volume to 10 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of polymerized human cord blood hemoglobin in the solution was 38 g/L, the hemoglobin content was 3 g/dL, the captopril content was 50 μmol/L, the antioxidant content was 0.1 g/L, and the oxygen affinity P ₅₀ value was 27 mmHg.

Embodiment 12 Preparation of blood substitute of the present invention

Put 8.57g of caprolactone (PCL) and 1.43g of polyethylene glycol monomethyl ether (mPEG5000) with a molecular weight of 5000 (mPEG5000) in a three-necked flask at a mass ratio of 6:1, add the catalyst stannous octoate and an appropriate amount of toluene, and stir in a mechanical React in an oil bath at 137° C. for 6 hours under the condition of nitrogen protection. After the temperature of the reaction system drops to room temperature, pour the polymer into cold petroleum ether to precipitate, filter the precipitate, and put the precipitate in a fume hood to volatilize the solvent until the polymer solidifies. The product is vacuum-dried, purified by dialysis, and then frozen Dry to get mPEG-PCL.

Then, 30 mg of free human cord blood hemoglobin freeze-dried powder (hemoglobin content 85%) was dissolved in 2 mL of PBS (pH 7.4), and 5 mL of dichloromethane containing 100 mg of mPEG-PCL was dropped into it under ultrasonic vibration at a speed of 1 mL per minute. solution, after the dropwise addition, quickly pour the prepared colostrum into 20ml of 0.1% PVA solution, emulsify on the emulsification disperser for 5 minutes, then add 100ml of 0.05% PVA solution to dilute and stir for 30 minutes, and then evaporate The organic solvent was evaporated on the instrument, washed with PBS solution and centrifuged at 12000g. Collect the nanovesicles in the lower layer, dissolve them in 5ml of phosphate buffer, purify them through gel chromatography, collect the eluate with characteristic absorption of hemoglobin in the region of 400-600nm, concentrate by ultrafiltration and freeze-dry to obtain the free human umbilical cord PEG-PCL nanoparticles freeze-dried powder of hemoglobin. The particle size of the nanoparticles is about 224nm (as shown in Figure 1), and the hemoglobin drug loading rate is 20%.

Take 5 batches of 600 mg of lyophilized nanoparticle powder, 108.6 μg of captopril, 0.5 mg of superoxide dismutase and 0.5 mg of catalase, add 6 mL of normal saline, and stir magnetically with a stirring bar for 30 minutes at a speed of 500 rpm. Adjust the pH value to 7.2 with a 0.1M sodium bicarbonate solution, and then adjust the volume to 10 mL to obtain the non-cardiovascular active hemoglobin oxygen-carrying carrier solution of the present invention, which is stored at 4°C for use.

The content of nanoparticle freeze-dried powder in the solution is 60g/L, the content of hemoglobin is 1g/dL, the content of captopril is 50 μmol/L, the content of antioxidant is 0.1g/L, and the oxygen affinity _P50 value is 6mmHg.

The beneficial effect of the present invention is illustrated in the mode of experimental example below:

Preliminary experiments have found that if the antioxidants used in Examples 1-5 and hemoglobin oxygen-carrying carriers (HBOCs) are used alone at the same time, there is almost no ability to alleviate the cardiovascular side effects caused by HBOCs.

Since the inhibitor angiotensin-converting enzyme inhibitor (ACEI) has a certain relieving effect on the cardiovascular side effects caused by HBOCs, the inventors set it as a positive control, and compared the composition of the present invention with the negative control group (alone HBOCs) and the positive control group (HBOCs+ACEI) for comparative tests, the specific test examples are as follows;

Experimental Example 1 Effect of the composition of the present invention on vascular endothelial cells

1. Experimental grouping

Negative control group 1 (HBOCs group 1): PEG-modified bovine hemoglobin (hemoglobin concentration 6g/dL; prepared according to the method on pages 7-8 of the patent ZL200380109157.8 specification);

Control group 1 (HBOCs+ACEI group 1): use the above PEG to modify bovine hemoglobin and add enalapril (hemoglobin concentration 6g/dL, enalapril concentration 200μmol/L);

Experiment 1 group (composition group 1 of the present invention: HBOCs+ACEI+antioxidant group): use the final product prepared in Example 8.

Negative control group 2 (HBOCs group 2): Use polymerized human cord blood hemoglobin (hemoglobin concentration 8g/dL, prepared according to the method on pages 7-8 of the patent ZL02133592.3 specification);

Control group 2 (HBOCs+ACEI group 2): use the above polymerized human cord blood hemoglobin and add enalapril (hemoglobin concentration 8g/dL, enalapril concentration 100μmol/L);

Experiment 2 group (composition group 2 of the present invention: HBOCs+ACEI+antioxidant group): use the final product prepared in Example 9.

The sample was heated to 37°C in a water bath just before use.

2. Experimental method

Experimental operation: Human umbilical vein endothelial cell lines (HUVECs) were selected and placed in a 96-well plate containing 100 μL of DMEM medium at a cell density of 3×10 ³ . After being cultured at 37°C for 24 hours in a cell culture incubator, 100 μL of the above samples were added to treat for 2 hours. . The blank control group was treated with 100 μL of DMEM medium for 2 h. Then the cell viability was detected by MTT assay.

Statistical analysis: Cell viability was analyzed by one-way analysis of variance, and LSD test was used to compare the differences between pairs. P<0.05 was considered to have a significant difference. Data are presented as mean ± standard deviation, n=5. *P<0.05 and **P<0.01 vs. negative control group.

3. Experimental results

The experimental results are shown in Figure 2. Compared with the blank control group, the endothelial cells in HBOCs group 1 and HBOCs group 2 were significantly damaged and the cell viability was significantly reduced (P<0.001 and P<0.001vs. blank control group). Compared with HBOCs group 1, although the cell viability of HBOCs+ACEI group 1 added with enalapril increased to a certain extent, it did not reach a statistical difference (P>0.05). The cell activity of the composition group of the present invention was significantly higher than that of the control group (P<0.01) (Fig. 2A). Compared with HBOCs group 2, the control HBOCs+ACEI group 2 added with enalapril significantly increased cell viability (P<0.05), while the pharmaceutical composition of the present invention after adding enalapril and antioxidants, cell viability increased The effect was stronger (P<0.01) (Fig. 2B)

Conclusion: The experimental results show that the single hemoglobin oxygen carrier can damage the vascular endothelial cells, and the angiotensin-converting enzyme inhibitor can reduce the damage to the vascular endothelial cells, but the effect is not obvious. However, the use of the composition of the present invention, that is, the combination of angiotensin-converting enzyme inhibitors and antioxidants can significantly reduce the damage of hemoglobin oxygen-carrying carriers to vascular endothelial cells (P<0.01), indicating that the composition of the present invention can be used to reduce hemoglobin Cardiovascular side effects of oxygen carrier-like.

Experimental Example 2 Vasoactive detection of the composition of the present invention

1. Experimental grouping

Negative control group 1 (HBOCs group 1): PEG-modified bovine hemoglobin (hemoglobin concentration 6g/dL; prepared according to the method on pages 7-8 of the patent ZL200380109157.8 specification);

Control group 1 (HBOCs+ACEI group 1): use the above PEG to modify bovine hemoglobin and add enalapril (hemoglobin concentration 6g/dL, enalapril concentration 200μmol/L);

Experiment 1 group (composition group 1 of the present invention: HBOCs+ACEI+antioxidant group): use the final product prepared in Example 8.

Negative control group 2 (HBOCs group 2): Use polymerized human cord blood hemoglobin (hemoglobin concentration 8g/dL, prepared according to the method on pages 7-8 of the patent ZL02133592.3 specification);

Control group 2 (HBOCs+ACEI group 2): use the above polymerized human cord blood hemoglobin and add enalapril (hemoglobin concentration 8g/dL, enalapril concentration 100μmol/L);

Experiment 2 group (composition group 2 of the present invention: HBOCs+ACEI+antioxidant group): use the final product prepared in Example 9. The sample was heated to 37°C in a water bath just before use.

2. Experimental method

Experimental operation: 12 male Kunming mice, weighing 22-25 g, were randomly entered into the above 4 groups, with 3 mice in each group. After the conscious animals were weighed, they were placed in the BP-2000 non-invasive blood pressure detection system, the tail pressure was measured, and the basic blood pressure was measured, and then 16% blood volume was injected from the tail vein according to the groups (theoretical maximum tolerance, about 0.3ml/25g mouse) for the above samples. Another 3 animals were injected with the same dose of normal saline through tail vein as blank control group. Then, detect the change of blood pressure 10 minutes and 30 minutes after the bolus injection in each group.

Statistical analysis: One-way analysis of variance was used, and LSD test was used to compare the differences between pairs. P<0.05 was considered to have a significant difference. The results are expressed as mean ± standard deviation, *P<0.05 and **P<0.01 vs. control group 1 or control group 2; vs. Control 1 or Control 2.

3. Experimental results

Compared with the basic blood pressure, there was no significant difference in the blank control group, but 10 minutes after injecting the sample, the blood pressure of HBOCs group 1 and HBOCs group 2 increased significantly. The comparison shows that the blood pressure of the HBOCs+ACEI group 1 added with enalapril has no significant difference from the blood pressure of the HBOCs group 1, while the composition of the present invention group 1 added with enalapril and antioxidants at the same time, the blood pressure is significantly lower than that of the HBOCs Group 1 (P<0.01), meanwhile, was also significantly lower than HBOCs+ACEI group 1 (P<0.05) (Fig. 3A). Similarly, the blood pressure of the composition group 1 of the present invention added with enalapril and antioxidants was significantly lower than that of the HBOCs group 2 (P<0.01) and only the HBOCs+ACEI group 2 (P<0.05) (Fig. 3B).

Conclusion: the experimental results show that the hemoglobin oxygen carrier can cause blood pressure to increase significantly, and the combination of angiotensin converting enzyme inhibitors can reduce its side effects, but the effect is not obvious. The combined use of inhibitors and antioxidants can significantly reduce the hypertensive response caused by hemoglobin oxygen carriers (P<0.05), indicating that the composition of the present invention can be used to reduce cardiovascular side effects of hemoglobin oxygen carriers.

Experimental Example 3 Effect of the composition of the present invention on canine coronary artery

1. Experimental grouping

HBOCs group: According to the method on pages 7-8 of the patent ZL02133592.3 specification, prepare a polymerized human umbilical cord blood hemoglobin solution (hemoglobin concentration 3g/dL) for the negative control group;

HBOCs+ACEI group: take the above polymerized human cord blood hemoglobin solution and add captopril (hemoglobin concentration 3g/dL, captopril concentration 100μmol/L) for the control group (no antioxidant);

Composition group of the present invention: the final product prepared in Example 6 was used in the experimental group (containing antioxidant).

The solution was oxygenated with 95% oxygen + 5% carbon dioxide bubbles for 10 minutes before use, and then the temperature was raised to 37°C in a water bath.

2. Experimental method

Experimental operation: Select 6 adult male Beagle dogs with normal heart function, weighing 8-10 kg, and divide them into negative control group (2 dogs), control group (no antioxidant, 2 dogs) and experimental group (with antioxidant , 2 only). Beagle dogs in each group were intramuscularly injected with morphine before operation, induced anesthesia with fentanyl citrate, midazolam, propofol, and vecuronium bromide, connected to a ventilator after tracheal intubation, and anesthetized with Liyuexime and propofol , fentanyl citrate, and vecuronium bromide, and intravenous injection as needed until the end of the operation. The left femoral artery was punctured to monitor blood pressure, arterial blood gas and serum electrolyte levels, and the femoral vein was punctured to connect a three-way extension tube to the level of the inferior vena cava to establish an infusion channel. The coronary catheter was punctured percutaneously into the femoral artery of the lower extremity, retrograde along the descending aorta to the root of the ascending aorta, and then passed through the coronary artery, the catheter was fixed at the left and right coronary artery ostium, and the non-ionic contrast agent iohexol (onepaque ) 10mL, developed after X-ray irradiation, as the basic value. After 10 minutes of stabilization, the negative control group, the control group (without antioxidant) and the experimental group (with antioxidant) were injected with 10 mL of polymerized human cord blood hemoglobin, polymerized human cord blood hemoglobin + captopril and Example 1 Prepare the final product, and then immediately inject 10mL of the above-mentioned contrast agent for development.

Detection indicators: (1) Cardiac function indicators: Cardiac ejection fraction was collected by PHILIPS IE33 color ultrasound diagnostic system, and the collection time was immediately after each coronary angiography. (2) The change rate of coronary artery was obtained by analyzing the change of the lumen diameter of the left anterior descending coronary artery displayed on the angiography images before and after the injection. Negative values indicated vasoconstriction. The analysis software was QuantCor QCA from Siemens, Germany. (3) Femoral artery was collected, and blood gas and electrolyte indicators were monitored in real time with ABL800FLEX blood gas analyzer from Radiometer Company, including oxygen partial pressure (PO ₂ ), oxygen saturation (SO ₂ ), carbon dioxide partial pressure (PCO ₂ ), pH value, Hemoglobin concentration, Ca ²⁺ concentration, K ⁺ concentration, Mg ²⁺ concentration, etc.

Statistical analysis: One-way analysis of variance was used, and LSD test was used to compare the differences between pairs. P<0.05 was considered to have a significant difference. Data are presented as mean ± standard deviation. *P<0.05 and ***P<0.001 vs. basal state; and vs. negative control group.

3. Experimental results

1. The results of coronary angiography image analysis are shown in Figure 4. The coronary arteries in the HBOCs group showed significant contraction or spasm, and the degree of coronary arterial contraction in the HBOCs+ACEI group was alleviated (P<0.05vs.HBOCs group), while the combination of the present invention In the HBOCs group, there was no vasoconstriction or spasm in the coronary arteries before and after the injection, and the state was the best (P<0.01vs.HBOCs group).

2. Ejection fraction As shown in Figure 5, there was no significant difference in the ejection fraction of the hearts of the three groups of animals at the base, but the ejection fraction of the hearts of the HBOCs+ACEI group was significantly higher than that of the HBOCs group after injecting the sample (P<0.05 ), but compared with the basic state, there is still a significant decrease (P<0.05); and the ejection fraction of the composition group of the present invention is significantly higher than that of the HBOCs group (P<0.05), and there is no significant difference compared with the basic state (P >0.05).

3. The blood gas and electrolyte indexes of animals in each group were maintained at normal levels during the experiment.

Conclusion: the experimental results show that the hemoglobin oxygen carrier can cause coronary artery constriction or spasm, resulting in reduced cardiac function, angiotensin converting enzyme inhibitors can reduce its side effects, but it is difficult to make the body return to normal levels, and the combination of the present invention Combination of angiotensin-converting enzyme inhibitors and antioxidants can significantly reduce the side effects of hemoglobin oxygen carriers and restore the body to normal levels.

For the side effects caused by hemoglobin oxygen-carrying carriers, such as vascular endothelial cell damage, high blood pressure, and coronary artery spasm, the inventor's previous experimental research found that the use of antioxidants alone could hardly alleviate them. At the same time, as shown in the above experimental examples 1-3 , using angiotensin-converting enzyme inhibitors alone has a certain relief effect, but the effect is still not obvious. However, when the present invention uses antioxidants and angiotensin-converting enzymes in combination, it can very effectively reduce the aforementioned side effects (p <0.05), indicating that in the composition of the present invention, the antioxidant and angiotensin-converting enzyme play a synergistic effect.

In summary, the composition of the present invention can prevent damage to vascular endothelial cells, reduce hypertensive reactions, prevent coronary artery spasm, maintain heart function, and effectively reduce cardiovascular side effects of hemoglobin oxygen-carrying carriers. Some blood substitutes have the disadvantage of cardiovascular side effects, and the clinical application prospect is good.

Claims (6)

1. A pharmaceutical composition for treating cardiovascular diseases caused by hemoglobin oxygen-carrying carriers, characterized in that: its active ingredients are composed of the following ingredients: 200 molar parts of angiotensin-converting enzyme inhibitors, 300,000 to 300,000 parts of antioxidants 600000 parts by mass; The angiotensin-converting enzyme inhibitor is N-[(S)-3-mercapto-2-methylpropionyl]-L-proline or (S)-1-[N-(1-ethoxycarbonyl -3-phenylpropyl)-L-alanyl]-L-proline; The antioxidant is a mixture of superoxide dismutase, catalase and N-acetylcysteine, and the weight ratio of the three is 1:1:1; The molar parts: parts by mass are mol/g. 2. The use of the composition according to claim 1 in the preparation of medicines for treating cardiovascular diseases caused by hemoglobin oxygen carriers. 3. A blood substitute, characterized in that: its active ingredients are composed of hemoglobin oxygen-carrying carrier and the composition according to claim 1, wherein the concentration of hemoglobin oxygen-carrying carrier is 37.5g～120g/L, blood vessel The concentration of the tensin-converting enzyme inhibitor is 200 μmol/L, and the concentration of the antioxidant is 0.3-0.6 g/L. 4. The blood substitute according to claim 3, characterized in that: the concentration of the hemoglobin oxygen-carrying carrier is 75-80 g/L. 5. The blood substitute according to claim 3 or 4, characterized in that: In the hemoglobin oxygen-carrying carrier, the hemoglobin content is 20-84% (w/w); The hemoglobin oxygen-carrying carrier is polymerized hemoglobin, PEG-modified hemoglobin or hemoglobin encapsulated by PEG-PCL nanoparticles. 6. A method for preparing the blood substitute according to any one of claims 3 to 5, characterized in that it comprises the following steps: (1) According to the proportioning described in any one of claims 3 to 5, get hemoglobin oxygen carrier, angiotensin-converting enzyme inhibitor and antioxidant, add water, and mix; (2) Adjust the pH value to 7.2-7.4.