Pyruvate preparation for treating asthma and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a pyruvate preparation for treating asthma and a preparation method of the pyruvate preparation. The pyruvate preparation is prepared from the following components in parts by weight: 10 to 12 parts of pyruvate, 10 to 12 parts of sodium chloride, 0.5 to 1 part of citric acid and 500 parts of water for injection. The pH value of the pyruvate preparation is 6 to 7. The pyruvate preparation is used for daily management of asthma patients, the purpose of inhibiting asthma is achieved by controlling increase of the number of inflammatory cells such as eosinophilic granulocytes and reducing inflammatory reaction, the effect of the pyruvate preparation is close to that of similar hormone medicine dexamethasone, and the effect of the pyruvate preparation is improved while the treatment purpose is achieved. The dependence and side effects of patients on hormone drugs are reduced.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a pyruvate preparation for treating asthma and a preparation method thereof. Background Art

[0002] Asthma, also known as bronchial asthma, is a chronic inflammatory disease of the airways involving multiple cells and cell components such as eosinophils, mast cells, T lymphocytes, neutrophils, smooth muscle cells, and airway epithelial cells. Clinically, it mainly manifests as recurrent symptoms such as wheezing, shortness of breath, chest tightness, and cough. A small number of patients may also experience chest pain, and in severe cases, dyspnea and sudden death may occur. Bronchial asthma is one of the most common chronic diseases worldwide, and its prevalence has tended to increase year by year in recent years. The pathogenesis of asthma is complex, and there is currently no complete cure, and the existing drug treatments have limited effects. Therefore, the development of new drugs to treat asthma is of great significance.

[0003] At present, the drugs used in clinical treatment of asthma mainly include drugs that relieve asthma attacks, such as prednisone, prednisolone, methylprednisolone, hydrocortisone, betamethasone, and dexamethasone. These drugs have powerful anti-inflammatory, anti-allergic, anti-shock and immunosuppressive effects, can quickly relieve inflammatory reactions, reduce allergic symptoms, fight shock states, and regulate immune responses. They have significant therapeutic effects on a variety of diseases such as asthma and rheumatic diseases. However, long-term use of these hormone drugs can reduce immune function and increase the risk of infection; affect metabolism, leading to abnormal blood sugar, blood lipids and weight gain; have adverse effects on the digestive system, nervous system and skeletal system, such as ulcers, depression, osteoporosis, etc.; and may also cause side effects such as skin changes, edema and hypertension.

[0004] In view of the above situation, researchers are constantly exploring new treatment approaches in order to reduce dependence on traditional hormone drugs and reduce their side effects. Recently, pyruvic acid and its derivatives, especially sodium pyruvate, have attracted attention in the field of asthma treatment due to their potential anti-inflammatory and immunomodulatory effects. However, direct application of sodium pyruvate in the treatment of asthma may have certain limitations, such as degradation or deterioration during storage and use, low bioavailability, and poor stability.

[0005] Therefore, the present invention proposes a pyruvate preparation for daily management of asthma patients, which can reduce the dependence and side effects of hormone drugs by reducing airway inflammation and excessive response. 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 pyruvate preparation for the treatment of asthma, which can reduce airway inflammation and reduce the dependence on hormone drugs.

[0007] One of the purposes of the present invention is achieved by the following technical solution: A pyruvate preparation for treating asthma, comprising the following components: 10-12 parts of pyruvate, 10-12 parts of sodium chloride, 0.5-1 parts of citric acid, and 500 parts of water for injection; the pH of the pyruvate preparation is 6-7; the structural formula of the pyruvate is: .

[0008] Furthermore, the preparation process of pyruvate comprises the following steps: (1) Add p-nitrobenzaldehyde, ethylenediamine, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 hours under an inert gas atmosphere; then add methanol and sodium borohydride to the reaction system and stir for 2-3 hours; after the reaction is completed, the reaction solution is treated to obtain intermediate 1; (2) Add intermediate 1 and 3,4-dihydroxybenzaldehyde to methanol, and stir for 1 to 2 hours at 75 to 85°C, 5 to 7 bar, and in an inert gas atmosphere; cool the mixed system to room temperature, add palladium carbon, replace the inert gas with hydrogen, and stir for 15 to 30 minutes at 90 to 100°C and 40 to 45 bar. After the reaction is completed, the reaction solution is treated to obtain intermediate 2; (3) Add 3,4-dihydroxybenzaldehyde, intermediate 2, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 hours under an inert gas atmosphere; then add methanol and sodium borohydride to the reaction system and stir for 2-3 hours; after the reaction is completed, the reaction solution is treated to obtain intermediate 3; (4) 4-boric acid benzaldehyde, pyruvic acid and potassium hydroxide were added to methanol and stirred for 12 to 18 hours under an inert gas atmosphere. After the reaction was completed, the reaction solution was treated to obtain intermediate 4; (5) Take intermediate 3, intermediate 4 and sodium bicarbonate in an ethanol aqueous solution and stir for 12 to 16 hours. After the reaction is completed, the reaction solution is treated to obtain pyruvate.

[0009] Furthermore, the molar ratio of ethylenediamine, p-nitrobenzaldehyde, triethylamine, sodium sulfate and sodium borohydride in step (1) is 1: (2-2.2): (0.5-0.8): (1-1.2): (4-6).

[0010] Furthermore, the mass ratio of 3,4-dihydroxybenzaldehyde, intermediate 1 and palladium carbon in step (2) is 3: (3.4-3.6): (0.012-0.018).

[0011] Furthermore, in step (3), the molar ratio of the intermediate 2,3,4-dihydroxybenzaldehyde, triethylamine, sodium sulfate and sodium borohydride is 1:(2-2.2):(0.5-0.8):(1-1.2):(4-6).

[0012] Furthermore, in step (4), the molar ratio of 4-boric acid benzaldehyde, pyruvic acid and potassium hydroxide is 1: (1.2-1.5): (2-2.2).

[0013] Furthermore, in step (5), the molar ratio of intermediate 3, intermediate 4 and sodium bicarbonate is 1:(4-5):(2-3).

[0014] A second object of the present invention is to provide a method for preparing a pyruvate preparation for treating asthma.

[0015] The second object of the present invention is achieved by adopting the following technical solution: The preparation method of the pyruvate preparation for asthma treatment comprises the following steps: according to the formula, pyruvate, sodium chloride and citric acid are taken, added into water for injection and stirred evenly; pH ​​buffer solution is taken and added into the above solution, and the pH value is adjusted to 6-7, so as to obtain the pyruvate preparation.

[0016] Furthermore, the pH buffer solution is one of a sodium citrate solution and a disodium hydrogen phosphate solution.

[0017] Furthermore, the preparation is in the form of an aerosol inhaler.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The preparation principle of the pyruvate of the present invention is as follows: the aldehyde group of nitrobenzaldehyde and the primary amine of ethylenediamine undergo reductive amination of the aldehyde group under alkaline conditions to obtain intermediate 1, the secondary amine of intermediate 1 undergoes reductive amination with the aldehyde group of 3,4-dihydroxybenzaldehyde under a hydrogen atmosphere, and the nitro group is reduced to an amine group to obtain intermediate 2, the amine group of intermediate 2 and the aldehyde group of 3,4-dihydroxybenzaldehyde undergo reductive amination of the aldehyde group under alkaline conditions to obtain intermediate 3; the carbonyl α-C of sodium pyruvate undergoes nucleophilic addition-elimination with the aldehyde group of 4-boronic acid benzaldehyde under alkaline conditions to obtain intermediate 4; the hydroxyl group of intermediate 3 undergoes esterification reaction with the boric acid of intermediate 4 to obtain pyruvate. The pyruvate introduces more N elements and phenylboronic acid ester bonds on the basis of retaining the basic structure of sodium pyruvate. This modified design is intended to enhance the stability and biological activity of the drug molecule, so that it can more effectively act on the key links in the pathological process of asthma, such as reducing airway inflammation.

[0019] The pyruvate preparation prepared by the present invention is used for the daily management of asthma patients, and achieves the purpose of suppressing asthma by inhibiting the increase of inflammatory cells such as eosinophils and reducing inflammatory reactions. Its effect is equivalent to that of the similar hormone drug dexamethasone, and reduces the patient's dependence on hormone drugs and side effects while achieving the treatment purpose. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a schematic diagram of the process for preparing pyruvate according to the present invention; Figure 2 Schematic diagram of the hydrogen spectrum of intermediate 3 of the present invention; Figure 3 Schematic diagram of the hydrogen spectrum of pyruvate of the present invention; Figure 4 Schematic diagram of the mass spectrum of pyruvate of the present invention; Figure 5 This is a schematic diagram of the cytotoxicity test results of pyruvate of the present invention; Figure 6 Schematic diagram showing the effect of the pyruvate preparation of the present invention on IL-6 release in RAW264.7 cells; Figure 7 Schematic diagram showing the effect of the pyruvate preparation of the present invention on the release of TNF-α in RAW264.7 cells; Figure 8 Schematic diagram of the effect of the pyruvate preparation of the present invention on inflammatory cells in ovalbumin-induced asthma model mice. DETAILED DESCRIPTION

[0021] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0022] Example 1 A pyruvate preparation for treating asthma comprises the following components by weight: 10 parts of pyruvate, 10 parts of sodium chloride, 0.5 parts of citric acid, and 500 parts of water for injection; the pH of the pyruvate preparation is 7.

[0023] The preparation process of pyruvate is as follows: Figure 1 As shown, the following steps are included: (1) Add nitrobenzaldehyde (42 mmol) to 10 mL of ethyl acetate, and add ethylenediamine (20 mmol), triethylamine (10 mmol), and sodium sulfate (20 mmol) in sequence, and stir at room temperature for 14 hours; cool the reaction solution to 5°C, add 150 mL of methanol, and then add sodium borohydride (80 mmol) in batches. After the addition is completed, warm the reaction solution to room temperature and stir for 2.5 hours; filter the reaction solution and wash the filter cake with chloroform, then wash the filtrate with 20 mL of 1 M sodium hydroxide solution. After separation, dry and concentrate the organic phase, and purify it by silica gel column chromatography (eluent, n-hexane: ethyl acetate = 2:1; add triethylamine to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 1.

[0024] (2) 3 g of 3,4-dihydroxybenzaldehyde and 3.5 g of intermediate 1 were added to an autoclave containing 10 mL of methanol, and nitrogen was replaced therein to maintain an inert gas atmosphere, and the reaction was carried out at 80°C and 5 bar pressure for 1.5 h; the temperature of the mixed system was lowered to room temperature, and then 15 mg of 5% palladium carbon was added to the reactor, and the nitrogen was replaced with hydrogen and stirred at 95°C and 40 bar pressure for 20 min; the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with methanol during the filtration process. The filtrate was directly dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0025] (3) 3,4-dihydroxybenzaldehyde (12 mmol) was added to 30 mL of ethyl acetate, and intermediate 2 (5 mmol), triethylamine (6 mmol), and sodium sulfate (12 mmol) were added thereto in sequence, and stirred at room temperature for 14 hours; the reaction solution was cooled to 5°C, and 150 mL of methanol was added, and then sodium borohydride (30 mmol) was added in batches. After the addition was completed, the reaction solution was heated to room temperature and stirred for 2.5 hours; the reaction solution was filtered and the filter cake was washed with chloroform, and the filtrate was washed with 20 mL of 1 M sodium hydroxide solution. After separation, the organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 6:1; triethylamine was added to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 3.

[0026] The above intermediate 3 1 H NMR images Figure 2 As shown, 1 H NMR (C 44 H 46 N4O8, 400MHz, DMSO-d6): δ 9.50 (s, 8H), 7.36 (s, 2H), 7.18 (d, 4H), 6.66-6.52 (m, 16H), 4.32 (s, 4H), 3.61 (s, 4H), 3.49 (s, 4H), 2.37 (s, 4H).

[0027] (4) Add 4-boronic acid benzaldehyde (10 mmol) and sodium pyruvate (13 mmol) to 10 mL of methanol, and add potassium hydroxide (21 mmol) at 0°C under a nitrogen atmosphere (21 mmol potassium hydroxide V 甲醇 :V 水 =1:1 mixed solution is prepared as 0.2M potassium hydroxide solution), after the dropwise addition is completed, the reaction system is heated to room temperature and stirred for 16 hours; after the reaction is completed, the reaction solution is filtered to obtain a filter cake, the filter cake is washed with cold ethanol, and then the filter cake is dissolved with ethyl acetate, the pH is adjusted to 5 with 1M HCl, the ethyl acetate phase is separated, and the intermediate 4 is obtained after drying and concentration.

[0028] (5) Intermediate 3 (10 mmol) and intermediate 4 (50 mmol) and sodium bicarbonate (46 mmol) were added to a mixed solution of 150 mL of ethanol and water in a ratio of 1:1, and the mixture was stirred for 14 h. The reaction solution was concentrated and purified by column chromatography (eluent, dichloromethane:methanol = 1:1) to obtain pyruvate.

[0029] Pyruvate 1 Schematic diagram of H NMR results is shown in Figure 3 , MS result diagram as shown Figure 4 As shown, the specific results are: 1 H NMR (C 84 H 62 B4N4Na4O 20 , 400MHz, DMSO-d6): δ [M+H] + m / z: 1583.39, ESI-MS (m / z): 1582.40.

[0030] This embodiment also provides a method for preparing a pyruvate preparation, and the specific process is as follows: Take 10 g of pyruvate, 10 g of sodium chloride, and 0.5 g of citric acid, add 50 mL of water for injection and stir evenly; take another 0.5 mol / L sodium citrate solution and add it to the above solution, adjust the pH value to 7, and add water for injection to 500 mL to obtain the pyruvate preparation.

[0031] Example 2 A pyruvate preparation for treating asthma comprises the following components by weight: 11 parts of pyruvate, 12 parts of sodium chloride, 0.8 parts of citric acid, and 500 parts of water for injection; the pH of the pyruvate preparation is 6.5.

[0032] (1) Add nitrobenzaldehyde (44 mmol) to 10 mL of ethyl acetate, and add ethylenediamine (20 mmol), triethylamine (16 mmol), and sodium sulfate (24 mmol) in sequence, and stir at room temperature for 16 hours; cool the reaction solution to 5°C, add 150 mL of methanol, and then add sodium borohydride (120 mmol) in batches. After the addition is completed, warm the reaction solution to room temperature and stir for 2 hours; filter the reaction solution, wash the filter cake with chloroform during the filtration process, and then wash the filtrate with 20 mL of 1M sodium hydroxide solution. After separation, dry and concentrate the organic phase, and purify it by silica gel column chromatography (eluent, n-hexane: ethyl acetate = 2:1; add triethylamine to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 1.

[0033] (2) 3 g of 3,4-dihydroxybenzaldehyde and 3.6 g of intermediate 1 were added to an autoclave containing 10 mL of methanol, and nitrogen was replaced therein, maintaining an inert gas atmosphere, and reacting at 75°C and 5 bar pressure for 1 h; the temperature of the mixed system was lowered to room temperature, and then 18 mg of 5% palladium carbon was added to the reactor, and nitrogen was replaced with hydrogen, and then stirred at 100°C and 40 bar pressure for 15 min; the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with methanol during the filtration process. The filtrate was directly dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0034] (3) 3,4-dihydroxybenzaldehyde (10 mmol) was added to 30 mL of ethyl acetate, and intermediate 2 (5 mmol), triethylamine (5 mmol), and sodium sulfate (10 mmol) were added thereto in sequence, and stirred at room temperature for 14 hours; the reaction solution was cooled to 5°C, and 150 mL of methanol was added, and then sodium borohydride (20 mmol) was added in batches. After the addition was completed, the reaction solution was heated to room temperature and stirred for 2.5 hours; the reaction solution was filtered and the filter cake was washed with chloroform, and the filtrate was washed with 20 mL of 1 M sodium hydroxide solution. After separation, the organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 6:1; triethylamine was added to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 3.

[0035] (4) 4-Borate benzaldehyde (10 mmol) and sodium pyruvate (15 mmol) were added to 10 mL of methanol. Potassium hydroxide (22 mmol) was added at 0°C under a nitrogen atmosphere (21 mmol potassium hydroxide V 甲醇 :V 水=1:1 mixed solution is prepared as 0.2M potassium hydroxide solution), after the dropwise addition is completed, the reaction system is heated to room temperature and stirred for 12 hours; after the reaction is completed, the reaction liquid is filtered to obtain a filter cake, the filter cake is washed with cold ethanol, and then the filter cake is dissolved with ethyl acetate, the pH is adjusted to 5 with 1M HCl, the ethyl acetate phase is separated, and the intermediate 4 is obtained after drying and concentration.

[0036] (5) Intermediate 3 (10 mmol) and intermediate 4 (60 mmol) and sodium bicarbonate (60 mmol) were added to a mixed solution of 150 mL of ethanol and water (1:1), and the mixture was stirred for 12 h. The reaction solution was concentrated and purified by column chromatography (eluent, dichloromethane:methanol = 1:1) to obtain pyruvate. 1 The results of H NMR and MS were consistent with the structure determined in Example 1.

[0037] The preparation process of the pyruvate preparation is as follows: take 11g of pyruvate, 12g of sodium chloride, and 0.8g of citric acid, add 50ml of water for injection and stir evenly; take another 0.5mol / L disodium hydrogen phosphate solution and add it to the above solution, adjust the pH value to 6.5, and add water for injection to 500ml to obtain the pyruvate preparation.

[0038] Example 3 A pyruvate preparation for treating asthma comprises the following components by weight: 12 parts of pyruvate, 11 parts of sodium chloride, 1 part of citric acid, and 500 parts of water for injection; the pH of the pyruvate preparation is 6.

[0039] The preparation process of pyruvate is as follows: (1) Benzaldehyde (40 mmol) was added to 10 mL of ethyl acetate, and ethylenediamine (20 mmol), triethylamine (15 mmol), and sodium sulfate (22 mmol) were added thereto in sequence, and stirred at room temperature for 15 hours; the reaction solution was cooled to 5°C, and 150 mL of methanol was added, and then sodium borohydride (100 mmol) was added in batches. After the addition was completed, the reaction solution was heated to room temperature and stirred for 3 hours; the reaction solution was filtered, and the filter cake was washed with chloroform during the filtration process, and then the filtrate was washed with 20 mL of 1M sodium hydroxide solution. After separation, the organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent, n-hexane: ethyl acetate = 2:1; triethylamine was added to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 1.

[0040] (2) 3 g of 3,4-dihydroxybenzaldehyde and 3.4 g of intermediate 1 were added to an autoclave containing 10 mL of methanol, and nitrogen was replaced therein to maintain an inert gas atmosphere, and the reaction was carried out at 85°C and 5 bar pressure for 2 h; the temperature of the mixed system was lowered to room temperature, and then 12 mg of 5% palladium carbon was added to the reactor, and the nitrogen was replaced with hydrogen, and the mixture was stirred at 90°C and 40 bar pressure for 15 min; the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with methanol during the filtration process. The filtrate was directly dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0041] (3) 3,4-dihydroxybenzaldehyde (11 mmol) was added to 30 mL of ethyl acetate, and intermediate 2 (5 mmol), triethylamine (5.5 mmol), and sodium sulfate (11 mmol) were added thereto in sequence, and stirred at room temperature for 14 hours; the reaction solution was cooled to 5°C, and 150 mL of methanol was added, and then sodium borohydride (25 mmol) was added in batches. After the addition was completed, the reaction solution was heated to room temperature and stirred for 2.5 hours; the reaction solution was filtered and the filter cake was washed with chloroform, and the filtrate was washed with 20 mL of 1 M sodium hydroxide solution. After separation, the organic phase was dried and concentrated, and purified by silica gel column chromatography (eluent, dichloromethane: methanol = 6:1; triethylamine was added to the eluent in an amount of 1% of the volume of the eluent) to obtain intermediate 3.

[0042] (4) 4-Borate benzaldehyde (10 mmol) and sodium pyruvate (12 mmol) were added to 10 mL of methanol. Potassium hydroxide (20 mmol) was added at 0°C under a nitrogen atmosphere (21 mmol potassium hydroxide V 甲醇 :V 水 =1:1 mixed solution is prepared as 0.2M potassium hydroxide solution), after the dropwise addition is completed, the reaction system is heated to room temperature and stirred for 18 hours; after the reaction is completed, the reaction liquid is filtered to obtain a filter cake, the filter cake is washed with cold ethanol, and then the filter cake is dissolved with ethyl acetate, the pH is adjusted to 5 with 1M HCl, the ethyl acetate phase is separated, and the intermediate 4 is obtained after drying and concentration.

[0043] (5) Intermediate 3 (10 mmol) and intermediate 4 (40 mmol) and sodium bicarbonate (40 mmol) were added to a mixed solution of 120 mL of ethanol and water (1:1), and the mixture was stirred for 16 h. The reaction solution was concentrated and purified by column chromatography (eluent, dichloromethane:methanol = 1:1) to obtain pyruvate. 1 The results of H NMR and MS were consistent with the structure determined in Example 1.

[0044] The preparation process of the pyruvate preparation is as follows: take 12g of pyruvate, 11g of sodium chloride, and 1g of citric acid, add 50ml of water for injection and stir evenly; take another 0.5mol / L disodium hydrogen phosphate solution and add it to the above solution, adjust the pH value to 6, and add water for injection to 500ml to obtain the pyruvate preparation.

[0045] Test Example 1 Pyruvate cytotoxicity assay In the medium of DMEM + 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin double antibody, the concentration of mouse T lymphoma EL-4 cells was adjusted to 5×10 4 / mL, take 100μL and inoculate into a 96-well plate; Example 1-3 groups: add different concentrations (120μg / mL, 60μg / mL, 30μg / mL, 15μg / mL, 7.5μg / mL) of pyruvate solution to each well (the pyruvate solution prepared in Example 1 is dissolved in a culture medium containing 0.2% DMSO to prepare a 120μg / mL pyruvate stock solution, and then the pyruvate stock solution is diluted with the culture medium to the required pyruvate solution of different concentrations); blank control group: no pyruvate is added. The same concentration of pyruvate solution is set up in 3 replicate wells and placed in a 37°C, 5% CO2 incubator for culture; after 48h of incubation, 30μL of 5mg / mL MTT solution is added, and the reaction is terminated after continuing to incubate for 4h; the absorbance (OD value) of each well is measured at 570nm using an enzyme reader to calculate the cell survival rate. Cell survival rate (%) = OD value of the experimental group / OD value of the negative control group × 100%, the specific results are as follows Figure 5 shown.

[0046] Depend on Figure 5 It can be seen that the survival rate of cells is greater than 80% when the concentration of pyruvate is below 120 μg / mL. Therefore, it can be concluded that the pyruvate prepared by the present invention has no cytotoxicity below 120 μg / mL.

[0047] Test Example 2 Effects of pyruvate preparations on the release of IL-6 and TNF-α in RAW264.7 cells RAW264.7 cells were cultured in a sterile DMEM high-glucose medium containing DMEM+10% fetal bovine serum in a 5% CO2, 37°C cell culture incubator for 24 h. RAW264.7 cells growing in the logarithmic period were taken and adjusted to 2×10 5Cell suspension was prepared at 1 mL / well and inoculated into a 6-well plate; after culturing for 24 hours at 37°C in a 5% CO2 cell culture incubator, the culture medium was replaced. For the blank control group, the culture medium was replaced with a new one in the wells; for the Example 1-3 groups, the culture medium containing 1 μg / mL lipopolysaccharide and 20 μg / mL pyruvate preparations in the Example 1-3 groups was replaced in the wells; for the lipopolysaccharide model group, the culture medium containing 1 μg / mL lipopolysaccharide was replaced in the wells; 3 replicate wells were set for each group; the culture medium was collected in the wells, centrifuged at 4000 r / min for 15 minutes, and the supernatant was taken. The ELISA method was used to detect the levels of IL-6 and TNF-α in the cell culture medium. The specific results are as follows: Figure 6-7 shown.

[0048] Depend on Figure 6-7 It can be seen that pyruvate can reduce the levels of TNF-α and IL-6 induced by LPS. It can be concluded that pyruvate can inhibit LPS-induced inflammation in RAW264.7 cells.

[0049] Test Example 3 Effects of pyruvate preparations on inflammatory cells in ovalbumin-induced asthma model mice Animal grouping: Female BALB / c mice, weighing 23±2g, aged 6-8 weeks, were randomly divided into 6 groups: blank control group, ovalbumin model group, Example 1-3 group, positive control group, 6 mice in each group. The mice were placed in an environment of 23±2°C and 12h / day lighting time, and the experiment was started after the animals were adapted to the environment for one week.

[0050] Animal modeling: Starting from the first day of the experiment, except for the blank control group, each group of animals was intraperitoneally injected with 200 μL of PBS suspension containing 20 μg of ovalbumin and 2 mg of aluminum hydroxide, once every 14 days, and continued to be administered twice. The blank control group was replaced with an equal volume of PBS. Starting from the 28th day, except for the normal control group, the mice in each group were placed in a transparent sealed container and atomized with a provocation solution (PBS solution containing 1% ovalbumin) for 20 minutes, once a day for 3 days, and the blank control group was replaced with PBS instead of the provocation solution. Each group was gavaged 1 hour before each provocation step.

[0051] Each group was given medication: the positive control group was given dexamethasone (30 mg / kg), the pyruvate preparation group of Example 1-3 was given pyruvate preparation (20 mg / kg based on the mass of pyruvate), and the blank control group and the ovalbumin model group were given an equal amount of normal saline for 3 days.

[0052] Test method: Mice were killed 48 hours after the last challenge, and bronchoalveolar lavage fluid (BALF) of mice in each group was collected and centrifuged at 4°C for 10 min at a speed of 1000 r / min. The precipitated cells were resuspended in 0.5 mL PBS and counted using a hematology analyzer. The specific results are as follows: Figure 8 shown.

[0053] Depend on Figure 8 It can be seen that, with the blank control group mice as the benchmark, the number of eosinophils in the bronchoalveolar lavage fluid of the asthmatic mice in the model group increased dramatically, indicating that a large number of inflammatory cells, namely eosinophils, infiltrated the airways of the mice, and the modeling was successful. Compared with the model group, the number of inflammatory cells such as eosinophils and macrophages in the positive control group mice was significantly reduced, and the number of inflammatory cells in the bronchoalveolar lavage fluid of the asthmatic mice treated with the pyruvate preparations of Examples 1 to 3 decreased to varying degrees with the change of dose, and the effect was more obvious, and close to the effect of the hormone drug dexamethasone in the positive control group.

[0054] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A pyruvate preparation for the treatment of asthma, characterized in that The composition comprises the following components by weight: 10-12 parts of pyruvate, 10-12 parts of sodium chloride, 0.5-1 parts of citric acid, and 500 parts of water for injection; the pH of the pyruvate preparation is 6-7; the structural formula of the pyruvate is: 。 2. The pyruvate preparation for treating asthma according to claim 1, characterized in that: The preparation process of the pyruvate comprises the following steps: (1) Add p-nitrobenzaldehyde, ethylenediamine, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 hours under an inert gas atmosphere; then add methanol and sodium borohydride to the reaction system and stir for 2-3 hours; after the reaction is completed, the reaction solution is treated to obtain intermediate 1; (2) Add intermediate 1 and 3,4-dihydroxybenzaldehyde to methanol, and stir for 1 to 2 hours at 75 to 85°C, 5 to 7 bar, and in an inert gas atmosphere; cool the mixed system to room temperature, add palladium carbon, replace the inert gas with hydrogen, and stir for 15 to 30 minutes at 90 to 100°C and 40 to 45 bar. After the reaction is completed, the reaction solution is treated to obtain intermediate 2; (3) Add 3,4-dihydroxybenzaldehyde, intermediate 2, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 hours under an inert gas atmosphere; then add methanol and sodium borohydride to the reaction system and stir for 2-3 hours; after the reaction is completed, the reaction solution is treated to obtain intermediate 3; (4) 4-boric acid benzaldehyde, pyruvic acid and potassium hydroxide were added to methanol and stirred for 12 to 18 hours under an inert gas atmosphere. After the reaction was completed, the reaction solution was treated to obtain intermediate 4; (5) Take intermediate 3, intermediate 4 and sodium bicarbonate in an aqueous solution of ethanol and stir for 12 to 16 hours. After the reaction is completed, the reaction solution is treated to obtain pyruvate.

3. The pyruvate preparation for treating asthma according to claim 2, characterized in that: The molar ratio of ethylenediamine, p-nitrobenzaldehyde, triethylamine, sodium sulfate and sodium borohydride in step (1) is 1: (2-2.2): (0.5-0.8): (1-1.2): (4-6).

4. The pyruvate preparation for treating asthma according to claim 2, characterized in that: The mass ratio of 3,4-dihydroxybenzaldehyde, intermediate 1 and palladium carbon in step (2) is 3: (3.4-3.6): (0.012-0.018).

5. The pyruvate preparation for treating asthma according to claim 2, characterized in that: The molar ratio of the intermediate 2,3,4-dihydroxybenzaldehyde, triethylamine, sodium sulfate and sodium borohydride in step (3) is 1:(2-2.4):(1-1.2):(2-2.4):(4-6).

6. The pyruvate preparation for treating asthma according to claim 2, characterized in that: The molar ratio of 4-boric acid benzaldehyde, pyruvic acid and potassium hydroxide in step (4) is 1: (1.2-1.5): (2-2.2).

7. The pyruvate preparation for treating asthma according to claim 2, characterized in that: The molar ratio of intermediate 3, intermediate 4 and sodium bicarbonate in step (5) is 1:(4-5):(2-3).

8. The method for preparing a pyruvate preparation for treating asthma according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: taking pyruvate, sodium chloride and citric acid according to the weight parts, adding them into water for injection and stirring evenly to obtain a mixed solution; and taking a pH buffer solution to adjust the pH value of the mixed solution to 6-7, so as to obtain a pyruvate preparation.

9. The method for preparing a pyruvate preparation for treating asthma according to claim 8, characterized in that: The pH buffer solution is one of a sodium citrate solution and a disodium hydrogen phosphate solution.

10. The method for preparing a pyruvate preparation for treating asthma according to claim 1, characterized in that: The dosage form of the preparation is a nebulizer inhalation.

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