A compound preparation containing pyruvate, a preparation method thereof, and applications thereof

Abstract

The present invention belongs to the field of pharmaceutical technology, and particularly relates to a compound preparation containing pyruvate, a preparation method thereof, and an application thereof. The compound preparation containing pyruvate is composed of the following components by mass percentage: Compound A: 0.02 - 0.04%, pyruvate: 0.05 - 0.1%, sodium dihydrogen phosphate: 0.2 - 2%, disodium hydrogen phosphate: 0.2 - 2%, sodium chloride: 0.7 - 0.9%, and the balance is water for injection; the structural formula of Compound A is as follows:. In the compound preparation, Compound A and pyruvate interact with each other, which can not only inhibit the pulmonary inflammatory reaction, reduce the level of inflammatory factors, but also relieve pulmonary edema.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a composite preparation containing pyruvate, and a preparation method and application thereof. Background Art

[0002] Acute respiratory distress syndrome (ARDS) is a serious respiratory disease caused by severe infection, trauma or inhalation of toxic gases, which damages the alveolar-capillary barrier and causes acute pulmonary edema, seriously affecting the patient's respiratory function. In recent years, with the continuous advancement of medical technology, the treatment of ARDS has been continuously improved, but its overall mortality rate remains high. Therefore, in-depth research on ARDS and exploration of more effective treatments are of great significance for reducing patient mortality and improving survival rates.

[0003] At present, the drugs used to relieve pulmonary edema mainly include: diuretics, vasodilators, and glucocorticoids. Diuretic drugs: Furosemide (CAS: 54-31-9), Spironolactone (CAS: 52-01-7), Hydrochlorothiazide (CAS: 58-93-5); Vasodilator drugs: Sodium nitroprussidedihydrate (CAS: 13755-38-9). Diuretics and vasodilators may have side effects of varying degrees during use, such as increased cough, increased dyspnea, palpitations, decreased blood pressure, and renal damage; these side effects may seriously affect the patient's quality of life and even endanger their lives. In addition, long-term use of certain drugs may cause patients to become dependent on the drugs, and withdrawal symptoms may occur once the drugs are stopped; some patients may develop drug resistance after long-term use of a certain drug, resulting in reduced or ineffective drug efficacy.

[0004] Vitamin C (ascorbic acid) can improve the blood flow in cells, prevent blood viscosity and blockage, and play a role in preventing arteriosclerosis; it can maintain cell vitality and improve cell quality, thereby enhancing immunity and improving bactericidal ability. At the same time, vitamin C can also promote metabolism and has a certain influence on human metabolism.

[0005] In view of the defect that current drugs for relieving pulmonary edema have poor efficacy, the present invention provides a composite preparation containing pyruvate, which can, on the one hand, reduce the inflammatory response of the lungs; on the other hand, can reduce pulmonary edema. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a composite preparation containing pyruvate. Compound A and pyruvate in the composite preparation can interact with each other, which can not only inhibit lung inflammatory response and reduce the level of inflammatory factors, but also reduce lung edema.

[0007] The second object of the present invention is to provide a method for preparing a composite preparation containing pyruvate. The method is simple and efficient, can reduce costs, and is convenient for industrial production.

[0008] A third object of the present invention is to provide an application of a composite preparation containing pyruvate.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A composite preparation containing pyruvate, which is composed of the following components by mass percentage: compound A: 0.02-0.04%, pyruvate: 0.05-0.1%, sodium dihydrogen phosphate: 0.2-2%, disodium hydrogen phosphate: 0.2-2%, sodium chloride: 0.7-0.9%, and the balance is water for injection; the structural formula of the compound A is as follows:

[0011] .

[0012] Furthermore, the preparation process of compound A comprises the following steps:

[0013]

[0014] (1) Add N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to a 3-carboxybenzaldehyde solution, stir for 1-2 hours, then add ascorbic acid, continue stirring to react, and separate and purify the reaction solution to obtain intermediate 1;

[0015] (2) Sodium pyruvate and intermediate 1 are added to a sodium hydroxide solution to obtain a mixture, which is stirred for a period of time. The reaction solution is post-treated to obtain compound A.

[0016] Furthermore, in step (1), the molar ratio of the 3-carboxybenzaldehyde, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and ascorbic acid is 1:(2-2.5):(2-2.5):(1.2-1.5); and the solvent of the 3-carboxybenzaldehyde solution is tetrahydrofuran.

[0017] Furthermore, in step (1), the stirring reaction time is 16-24 hours.

[0018] Furthermore, in step (2), the molar ratio of the intermediate 1, sodium hydroxide and sodium pyruvate is 1:(2-2.5):(2-2.5), and the solvent of the sodium hydroxide solution is a mixed solvent made of ethanol and water.

[0019] Furthermore, in step (2), the stirring reaction time is 1-2 hours.

[0020] Furthermore, in step (2), the specific steps of the post-treatment are: adjusting the reaction solution to neutrality with acid, dissolving it with water and purifying it by reverse phase high performance liquid chromatography technology, then adjusting the purified fraction to pH 8 with alkali, and finally freeze-drying it.

[0021] The preparation method of the above-mentioned composite preparation containing pyruvate comprises the following steps: compounding compound A, pyruvate, sodium dihydrogen phosphate, sodium hydrogen phosphate, sodium chloride and water for injection according to the mass percentage to obtain a composite preparation.

[0022] Furthermore, the pyruvate is sodium pyruvate.

[0023] The above-mentioned composite preparation containing pyruvate is used to prepare a drug for treating lung inflammation caused by ARDS.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention provides a composite preparation containing pyruvate, which is composed of compound A, pyruvate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water. The present invention designs and synthesizes a novel compound A, and combines it with pyruvate to form a composite preparation. The composite preparation can effectively inhibit the inflammatory response of the lungs and reduce the level of inflammatory factors; at the same time, it can also reduce the water content in the lung tissue and effectively reduce pulmonary edema.

[0026] 2. The present invention also provides a method for preparing the above-mentioned composite preparation containing pyruvate, which is simple and feasible, and provides a new method for preparing drugs for treating lung inflammation caused by ARDS. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0028] Example 1

[0029] A composite preparation containing pyruvate, which is composed of the following components by mass percentage: compound A: 0.03%, sodium pyruvate: 0.07%, sodium dihydrogen phosphate: 1%, disodium hydrogen phosphate: 2%, sodium chloride: 0.8%, and the balance is water for injection; the structural formula of the compound A is as follows:

[0030] The structural formula of the compound A is shown below:

[0031] .

[0032] The preparation process of compound A comprises the following steps:

[0033] .

[0034] (1) 3-Carboxybenzaldehyde (10 mmol) was added to 80 mL of tetrahydrofuran, and N,N'-dicyclohexylcarbodiimide (DCC, 22 mmol) and 4-dimethylaminopyridine (DMAP, 22 mmol) were added thereto under stirring. After stirring at room temperature for 2 h, ascorbic acid (14 mmol) was added and stirred at room temperature for 20 h. The reaction solution was concentrated and then passed through a column (eluent, V 二氯甲烷 :V 甲醇 =3:1) to obtain intermediate 1 after purification; 1 H NMR( C 14 H 12 O 8 , 400 MHz, d 6 -DMSO) δ 16.75 (s, 1H),10.68 (s, 1H), 9.88 (s, 1H), 8.27 (m, 1H), 8.10 (d, 1H), 7.85 (m, 1H), 7.64(t, 1H), 5.77 (s, 1H), 4.81 (m, 2H), 4.50 (m, 1H), 4.25 (m, 1H); MS (ESI) m / z= 309.05 [M+H] + .The above results confirm that the obtained product is the target product.

[0035] (2) Dissolve NaOH (22 mmol) in 125 mL of mixed solvent (V 水 :V 乙醇 =1:1), sodium pyruvate (22 mmol) and intermediate 1 (10 mmol) were added, and the resulting mixture was stirred at room temperature for 1 h; the reaction solution was adjusted to neutral with 1 M hydrochloric acid, dissolved with water, purified by reverse phase high performance liquid chromatography, and the purified fraction was adjusted to pH = 8 with 1 M sodium hydroxide, and finally the fraction was lyophilized to obtain compound A.1 H NMR( C 17 H 13 NaO 10 , 400 MHz, d 6 -DMSO) δ 16.75 (s,1H), 10.68 (s, 1H), 7.88-7.80 (m, 2H), 7.68 (d, 1H), 7.40-7.32 (m, 2H), 6.67(d, 1H), 5.77 (s, 1H), 4.81 (m, 2H), 4.50 (m, 1H), 4.25 (m, 1H); MS (ESI) m / z= 378.04[M+H-Na]. The above results confirmed that the obtained product was the target product.

[0036] This embodiment also provides a method for preparing the above-mentioned composite preparation containing pyruvate, which is obtained by compounding compound A, sodium pyruvate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and water for injection according to the above-mentioned mass percentages.

[0037] Example 2

[0038] A composite preparation containing pyruvate, composed of the following components by mass percentage: compound A: 0.02%, sodium pyruvate: 0.05%, sodium dihydrogen phosphate: 0.2%, disodium hydrogen phosphate: 0.2%, sodium chloride: 0.7%, and the balance is water for injection; the structural formula of the compound A is the same as that of Example 1.

[0039] The preparation process of compound A comprises the following steps:

[0040] (1) 3-Carboxybenzaldehyde (10 mmol) was added to 80 mL of tetrahydrofuran, and N,N'-dicyclohexylcarbodiimide (DCC, 20 mmol) and 4-dimethylaminopyridine (DMAP, 20 mmol) were added thereto under stirring. After stirring at room temperature for 2 h, ascorbic acid (12 mmol) was added and stirred at room temperature for 16 h. The reaction solution was concentrated and passed through a column (eluent, V 二氯甲烷 :V 甲醇 =3:1) to obtain intermediate 1; 1 H NMR was consistent with Example 1.

[0041] (2) Dissolve NaOH (20 mmol) in 125 mL of mixed solvent (V 水 :V 乙醇=1:1), sodium pyruvate (20 mmol) and intermediate 1 (10 mmol) were added, and the resulting mixture was stirred at room temperature for 1 h; the reaction solution was adjusted to neutral with 1 M hydrochloric acid, dissolved with water, and purified by reversed-phase high performance liquid chromatography, and the purified fraction was adjusted to pH = 8 with 1 M sodium hydroxide, and finally the fraction was freeze-dried to obtain compound A. Compound A 1 H NMR was consistent with Example 1.

[0042] This example provides a method for preparing the above-mentioned composite preparation containing pyruvate, which is the same as that in Example 1.

[0043] Example 3

[0044] A composite preparation containing pyruvate, composed of the following components by mass percentage: compound A: 0.04%, sodium pyruvate: 0.1%, sodium dihydrogen phosphate: 2%, disodium hydrogen phosphate: 1.5%, sodium chloride: 0.9%, and the balance is water for injection; the structural formula of the compound A is the same as that of Example 1.

[0045] The preparation process of compound A comprises the following steps:

[0046] (1) 3-Carboxybenzaldehyde (10 mmol) was added to 80 mL of tetrahydrofuran, and N,N'-dicyclohexylcarbodiimide (DCC, 25 mmol) and 4-dimethylaminopyridine (DMAP, 25 mmol) were added thereto under stirring. After stirring at room temperature for 2 h, ascorbic acid (15 mmol) was added and stirred at room temperature for 16-24 h. The reaction solution was concentrated and passed through a column (eluent, V 二氯甲烷 :V 甲醇 =3:1) to obtain intermediate 1; 1 H NMR was consistent with Example 1.

[0047] (2) Dissolve NaOH (25 mmol) in 125 mL of mixed solvent (V 水 :V 乙醇 =1:1), sodium pyruvate (25 mmol) and intermediate 1 (10 mmol) were added, and the resulting mixture was stirred at room temperature for 1 hour; the reaction solution was adjusted to neutral with 1M hydrochloric acid, dissolved with water, and purified by reversed-phase high performance liquid chromatography technology, and the purified fraction was adjusted to pH = 8 with 1M sodium hydroxide, and finally the fraction was freeze-dried to obtain compound A. Compound A 1 H NMR was consistent with Example 1.

[0048] This example provides a method for preparing the above-mentioned composite preparation containing pyruvate, which is the same as that in Example 1.

[0049] Comparative Example 1

[0050] The difference between this comparative example 1 and Example 1 is that compound A is omitted in the composite preparation, and the rest is the same as Example 1.

[0051] Comparative Example 2

[0052] The difference between Comparative Example 2 and Example 1 is that the compound A in the composite preparation is replaced with ascorbic acid, and the rest is the same as Example 1.

[0053] Test example

[0054] 1. Experimental Animals

[0055] Healthy BALA / c mice were selected, with a weight range of 18-22g, SPF grade, and male. During the feeding period, the room was kept ventilated, with a light / dark cycle of 12h each, a room temperature of 22-25℃, and a relative humidity of 50-55%. Before the experiment, the mice were allowed to drink water and eat freely, and were fed with ordinary feed, and were adaptively fed for 3 days.

[0056] 2. Animal grouping, modeling and drug administration

[0057] Animal grouping and modeling: The mice were fasted for 12 hours before modeling and anesthetized with 0.4% sodium pentobarbital. After anesthesia, the mouth was opened with a mouse mouth opener. A pulmonary liquid quantitative nebulizer was used to inject lipopolysaccharide (LPS) solution (at a dose of 6 mg / kg, dissolved in 0.9% sodium chloride solution) into the lungs to establish an ARDS model. The model mice were randomly divided into: positive drug group, model group, Example 1 group, Example 2 group, Example 3 group, Comparative Example 1 group, Comparative Example 2 group, and a blank control group was set up. Each mouse was given an equal amount of 0.9% sodium chloride solution, with 8 mice in each group.

[0058] Dosage regimen: Example 1-3 groups and Comparative Example 1-2 groups were respectively administered the corresponding pyruvate-containing compound preparations by atomization 2 h before modeling, 6 h after modeling, and 12 h after modeling, with a dosage of 0.4 mg / kg (calculated as sodium pyruvate content).

[0059] Positive drug group: Dexamethasone was administered at a dose of 6 mg / kg 12 h after modeling.

[0060] Blank control group: each mouse was given an equal amount of 0.9% sodium chloride solution.

[0061] 3. Index test

[0062] 3.1 Detection of inflammatory factors

[0063] After 24 hours of drug intervention, blood was collected from the orbits of the mice in the above groups, and the supernatant was obtained after centrifugation at 4°C. The inflammatory factors IL-1β and TNF-α were detected using ELISA kits. The results are shown in Table 1. Then, the thorax of the mice in the above groups was opened to expose the mouse trachea and lungs, the left main bronchus was ligated, and the left lung was flushed with 0.9% sodium chloride solution. This lavage was repeated 3 times, and the alveolar lavage fluid was collected. The above alveolar lavage fluid (BALF) was centrifuged at 4°C and the supernatant was obtained. The inflammatory factors IL-1β and TNF-α were detected using ELISA kits. The results are shown in Table 2.

[0064] 3.2 Pulmonary edema performance test

[0065] After collecting blood and BALF, the mice in each group were killed, and the right lungs of the mice were separated and dried with absorbent paper to remove water and blood. The weight was measured as wet weight, and then placed in an 80°C oven for 48 h until the weight no longer changed. After being taken out, the weight was measured as dry weight, and the wet / dry weight ratio of the lung tissue was calculated. The results are shown in Table 3.

[0066] Table 1 Serum IL-1β and TNF-α levels

[0067]

[0068] Table 2 IL-1β and TNF-α levels in bronchoalveolar lavage fluid

[0069]

[0070] As shown in the test results of Table 1-2, compared with the blank control group, IL-1β and TNF-α in the serum and alveolar lavage fluid of the model group were significantly increased, indicating that the established ADRS model can cause severe inflammatory reactions. Compared with the model group, the levels of IL-1β and TNF-α in the serum and alveolar lavage fluid of Example 1-3 groups were significantly reduced, and were comparable to the levels of IL-1β and TNF-α in the positive drug group. The addition of compound A was omitted in the comparative example 1 group, and the comparative example 2 group replaced compound A with an equal amount of ascorbic acid. The decrease in IL-1β and TNF-α levels was relatively small, indicating that compound A is superior to unmodified ascorbic acid in reducing the level of inflammatory factors. In addition, compound A and sodium pyruvate can synergistically inhibit inflammatory reactions in lung tissue and significantly reduce the level of inflammatory factors.

[0071] Table 3 Ratio of wet weight to dry weight of mouse lung tissue

[0072]

[0073] The ratio of wet weight to dry weight of lung tissue can be used to evaluate the degree of pulmonary edema. It can be seen from the experimental data in Table 3 that compared with the blank control group, the ratio of wet weight to dry weight of lung tissue of mice in the model group increased significantly, indicating that the water content of the mouse lungs increased significantly; compared with the model group, the ratio of wet weight to dry weight of mice in Example 1-3 groups decreased significantly, which was equivalent to the effect of the positive drug group. Compared with Example 1-3 groups, the effect of Comparative Example 1-2 group was poor. The above results show that compound A and sodium pyruvate in the composite preparation of the present invention can synergistically reduce lung water content, thereby effectively improving pulmonary edema.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. On the basis of the present invention, some modifications or replacements may be made thereto, but these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of protection claimed by the present invention.

Claims

1. A composite preparation containing pyruvate, characterized in that: The following components are included by mass percentage Composition: Compound A: 0.02-0.04%, sodium pyruvate: 0.05-0.1%, sodium dihydrogen phosphate: 0.2-2%, disodium hydrogen phosphate: 0.2-2%, sodium chloride: 0.7-0.9%, and the balance is water for injection; the structural formula of the compound A is as follows: 。 2. A composite formulation containing pyruvate according to claim 1, characterized in that: The preparation process of compound A comprises the following steps: (1) Add N,N'-dicyclohexylcarbodiimide and 4-dimethylaminopyridine to a 3-carboxybenzaldehyde solution, stir for 1-2 hours, then add ascorbic acid, continue stirring to react, and separate and purify the reaction solution to obtain intermediate 1; (2) Sodium pyruvate and intermediate 1 are added to a sodium hydroxide solution to obtain a mixture, which is stirred for a period of time. The reaction solution is post-treated to obtain compound A.

3. A composite formulation containing pyruvate according to claim 2, characterized in that: In step (1), the molar ratio of the 3-carboxybenzaldehyde, N,N'-dicyclohexylcarbodiimide, 4-dimethylaminopyridine and ascorbic acid is 1:(2-2.5):(2-2.5):(1.2-1.5); the solvent of the 3-carboxybenzaldehyde solution is tetrahydrofuran.

4. A composite formulation containing pyruvate according to claim 2, characterized in that: In step (1), the stirring reaction time is 16-24 hours.

5. A composite formulation containing pyruvate according to claim 2, characterized in that: In step (2), the molar ratio of the intermediate 1, sodium hydroxide and sodium pyruvate is 1:(2-2.5):(2-2.5); the solvent of the sodium hydroxide solution is a mixed solvent made of ethanol and water.

6. A composite formulation containing pyruvate according to claim 2, characterized in that: In step (2), the stirring reaction time is 1-2 hours.

7. A composite formulation containing pyruvate according to claim 2, characterized in that: In step (2), the specific steps of the post-treatment are: adjusting the reaction solution to neutrality with acid, dissolving it with water and purifying it by reverse phase high performance liquid chromatography technology, then adjusting the purified fraction to pH 8 with alkali, and finally freeze-drying it.

8. A method for preparing a composite formulation containing pyruvate according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: compounding compound A, sodium pyruvate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride and water for injection according to the mass percentage to obtain the compound.

9. The use of the composite formulation containing pyruvate according to any one of claims 1 to 7, characterized in that: Used to prepare drugs for treating lung inflammation caused by acute respiratory distress syndrome.

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