Pyruvate formulations for asthma treatment and methods of making the same

Abstract

The present application belongs to the technical field of biological medicine, and particularly relates to a pyruvate preparation for asthma treatment and a preparation method thereof. The pyruvate preparation comprises the following components in parts by weight: 10-12 parts of pyruvate, 10-12 parts of sodium chloride, 0.5-1 part of citric acid, and 500 parts of water for injection; and the pH of the pyruvate preparation is 6-7. The pyruvate preparation is used for daily management of asthma patients, can control the increase of inflammatory cells such as eosinophilic granulocyte, reduce inflammatory reaction, and thus achieve the purpose of inhibiting asthma. The effect of the pyruvate preparation is close to that of the same kind of hormone drug dexamethasone, and the pyruvate preparation can achieve the treatment purpose while reducing the dependence of patients on hormone drugs and side effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to pyruvate preparations for the treatment of asthma and their preparation methods. Background Technology

[0002] Asthma, also known as bronchial asthma, is a chronic inflammatory disease of the airways involving various cells and cellular components, including eosinophils, mast cells, T lymphocytes, neutrophils, smooth muscle cells, and airway epithelial cells. Clinically, it mainly manifests as recurrent episodes of wheezing, shortness of breath, chest tightness, and cough. A few patients may also experience chest pain, and in severe cases, respiratory distress and sudden death can occur. Bronchial asthma is one of the most common chronic diseases worldwide, and its prevalence has been increasing in recent years. The pathogenesis of asthma is complex, and there is currently no complete cure. Existing drug treatments have limited effectiveness; therefore, the development of new drugs for treating asthma is of great significance.

[0003] Currently, the main medications used clinically to treat asthma include those 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, rapidly relieving inflammatory responses, alleviating allergy symptoms, combating shock, and regulating immune responses. They are effective for various diseases such as asthma and rheumatic diseases. However, long-term use of these hormonal drugs can reduce immune function, increase the risk of infection, affect metabolism, leading to abnormal blood sugar and blood lipids and weight gain, and have adverse effects on the digestive, nervous, and skeletal systems, such as ulcers, depression, and osteoporosis. They may also cause side effects such as skin changes, edema, and hypertension.

[0004] Given the current situation, researchers are constantly exploring new treatment approaches to reduce reliance on traditional hormonal drugs and minimize their side effects. Recently, pyruvate and its derivatives, particularly 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 asthma treatment may have certain limitations, such as degradation or deterioration during storage and use, low bioavailability, and poor stability.

[0005] Therefore, this invention proposes a pyruvate preparation for the daily management of asthma patients, which reduces dependence on and side effects of hormonal drugs by reducing airway inflammation and overreaction. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a pyruvate preparation for the treatment of asthma that can reduce airway inflammation and reduce dependence on hormone drugs.

[0007] One of the objectives of this invention is achieved through the following technical solution:

[0008] A pyruvate preparation for the treatment of asthma comprises the following components: 10-12 parts pyruvate, 10-12 parts sodium chloride, 0.5-1 part citric acid, and 500 parts water for injection; the pH of the pyruvate preparation is 6-7; the structural formula of the pyruvate is:

[0009] .

[0010] Furthermore, the preparation process of the pyruvate salt includes the following steps:

[0011]

[0012] (1) Add p-nitrobenzaldehyde, ethylenediamine, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 h under an inert atmosphere; add methanol and sodium borohydride to the reaction system and stir for 2-3 h; after the reaction is completed, the reaction solution is processed to obtain intermediate 1;

[0013] (2) Add intermediate 1 and 3,4-dihydroxybenzaldehyde to methanol and stir at 75~85°C, 5-7 bar, and in an inert gas atmosphere for 1~2 h; cool the mixture to room temperature, add palladium on carbon, replace the inert gas with hydrogen, and stir at 90~100°C, 40-45 bar for 15~30 min. After the reaction is completed, the reaction solution is treated to obtain intermediate 2.

[0014] (3) Add 3,4-dihydroxybenzaldehyde, intermediate 2, triethylamine and sodium sulfate to ethyl acetate and stir for 14-16 h under an inert atmosphere; add methanol and sodium borohydride to the reaction system and stir for 2-3 h; after the reaction is completed, the reaction solution is treated to obtain intermediate 3;

[0015] (4) Take 4-boronic acid benzaldehyde, pyruvic acid and potassium hydroxide in methanol and stir for 12-18h under an inert gas atmosphere. After the reaction is completed, the reaction solution is treated to obtain intermediate 4.

[0016] (5) Take intermediate 3, intermediate 4 and sodium bicarbonate and stir in an aqueous solution of ethanol for 12-16 hours. After the reaction is completed, the reaction solution is treated to obtain pyruvate.

[0017] Further, 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).

[0018] Further, in step (2), the mass ratio of 3,4-dihydroxybenzaldehyde, intermediate 1, and palladium on carbon is 3:(3.4~3.6):(0.012~0.018).

[0019] Further, in step (3), the molar ratio of intermediates 2,3,4-dihydroxybenzaldehyde, triethylamine, sodium sulfate, and sodium borohydride is 1:(2~2.2):(0.5~0.8):(1~1.2):(4~6).

[0020] Further, in step (4), the molar ratio of 4-boronic acid benzaldehyde, pyruvic acid, and potassium hydroxide is 1:(1.2~1.5):(2~2.2).

[0021] Furthermore, the molar ratio of intermediate 3, intermediate 4, and sodium bicarbonate in step (5) is 1:(4-5):(2~3).

[0022] The second objective of this invention is to provide a method for preparing a pyruvate preparation for the treatment of asthma.

[0023] The second objective of this invention is achieved by the following technical solution:

[0024] The preparation method of the above-mentioned pyruvate preparation for the treatment of asthma includes the following steps: according to the formula, take pyruvate, sodium chloride and citric acid, add them to water for injection and stir evenly; separately take pH buffer and add it to the above solution to adjust the pH value to 6-7, and the pyruvate preparation is obtained.

[0025] Furthermore, the pH buffer solution is one of sodium citrate solution and disodium hydrogen phosphate solution.

[0026] Furthermore, the dosage form of the preparation is a nebulized inhaler.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The preparation principle of the pyruvate salt of this invention is as follows: The aldehyde group of nitrobenzaldehyde undergoes reductive amination with the primary amine of ethylenediamine 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, simultaneously reducing the nitro group to an amino group to obtain intermediate 2. The amino group of intermediate 2 undergoes reductive amination with the aldehyde group of 3,4-dihydroxybenzaldehyde 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 with the boric acid of intermediate 4 to obtain the pyruvate salt. This pyruvate salt, while retaining the basic structure of sodium pyruvate, introduces a greater amount of nitrogen and phenylboronic acid ester bonds. This modification design aims to enhance the stability and bioactivity of the drug molecule, enabling it to act more effectively on key aspects of the asthma pathogenesis process, such as reducing airway inflammation.

[0029] The pyruvate preparation prepared in this invention is used for the daily management of asthma patients. It reduces the inflammatory response by inhibiting the increase of inflammatory cells such as eosinophils, thereby achieving the purpose of suppressing asthma. Moreover, its effect is comparable to that of dexamethasone, a similar hormone drug, which reduces the patient's dependence on hormone drugs and side effects while achieving the therapeutic goal. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of the process for preparing pyruvate according to the present invention;

[0031] Figure 2 This is a schematic diagram of the proton spectrum of intermediate 3 of the present invention;

[0032] Figure 3 This is a schematic diagram of the proton NMR spectrum of the pyruvate of this invention;

[0033] Figure 4 This is a schematic diagram of the mass spectrometry of the pyruvate salt of the present invention;

[0034] Figure 5 This is a schematic diagram of the cytotoxicity test results of pyruvate in this invention;

[0035] Figure 6 This is a schematic diagram illustrating the effect of the pyruvate formulation of the present invention on IL-6 release in RAW264.7 cells;

[0036] Figure 7 This is a schematic diagram illustrating the effect of the pyruvate formulation of the present invention on TNF-α release in RAW264.7 cells;

[0037] Figure 8 This is a schematic diagram of the inflammatory cells in an ovalbumin-induced asthma model mouse induced by the pyruvate preparation of the present invention. Detailed Implementation

[0038] The present invention will now be further described with reference to the accompanying drawings and specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, all reagents or instruments used are conventional products obtained through commercial channels.

[0039] Example 1

[0040] A pyruvate preparation for the treatment of asthma comprises, by weight, the following components: 10 parts pyruvate, 10 parts sodium chloride, 0.5 parts citric acid, and 500 parts water for injection; the pH of the pyruvate preparation is 7.

[0041] The preparation process of pyruvate is as follows: Figure 1 As shown, it includes the following steps:

[0042] (1) Nitrobenzaldehyde (42 mmol) was added to 10 mL of ethyl acetate, followed by ethylenediamine (20 mmol), triethylamine (10 mmol), and sodium sulfate (20 mmol). The mixture was stirred at room temperature for 14 hours. The reaction solution was cooled to 5°C, and 150 mL of methanol was added. Then, sodium borohydride (80 mmol) was added in portions. After the addition was complete, the reaction solution was heated to room temperature and stirred for 2.5 h. The reaction solution was filtered and the filter cake was washed with chloroform. The filtrate was then washed with 20 mL of 1 M sodium hydroxide solution. After separation, the organic phase was dried and concentrated. The intermediate 1 was purified by silica gel column chromatography (eluent: hexane: ethyl acetate = 2:1; triethylamine was added to the eluent at 1% of the eluent volume).

[0043] (2) Add 3g of 3,4-dihydroxybenzaldehyde and 3.5g of intermediate 1 to a high-pressure vessel containing 10mL of methanol, replace the nitrogen gas in the vessel, maintain an inert gas atmosphere, and react at 80°C and 5bar for 1.5h. Cool the temperature of the mixture to room temperature, then add 15mg of 5% palladium on carbon to the reactor, replace the nitrogen gas with hydrogen gas, and stir at 95°C and 40bar for 20min. After cooling the reaction solution to room temperature, filter it. During the filtration process, wash the filter cake with methanol. Dry and concentrate the filtrate directly, and purify it by silica gel column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0044] (3) Add 3,4-dihydroxybenzaldehyde (12 mmol) to 30 mL of ethyl acetate, then add intermediate 2 (5 mmol), triethylamine (6 mmol), and sodium sulfate (12 mmol) sequentially, and stir at room temperature for 14 hours; lower the reaction temperature to 5°C, add 150 mL of methanol, and then add sodium borohydride (30 mmol) in batches. After the addition is complete, raise the temperature of the reaction solution to room temperature and stir for 2.5 h; 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: dichloromethane: methanol = 6:1; add triethylamine to the eluent, the amount added is 1% of the eluent volume) to obtain intermediate 3.

[0045] The above intermediate 3 1 H NMR image as follows 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).

[0046] (4) Add 10 mmol of benzaldehyde 4-borate and 13 mmol of sodium pyruvate to 10 mL of methanol. Under a nitrogen atmosphere, add 21 mmol of potassium hydroxide at 0°C. 甲醇 V 水 =1:1 mixed solution was prepared as 0.2M potassium hydroxide solution. After the addition was completed, the reaction system was heated to room temperature and stirred for 16 hours. After the reaction was completed, the reaction solution was filtered to obtain filter cake. The filter cake was washed with cold ethanol, and then dissolved with ethyl acetate. The pH was adjusted to 5 with 1M HCl to separate the ethyl acetate phase. After drying and concentration, intermediate 4 was obtained.

[0047] (5) Add intermediate 3 (10 mmol), intermediate 4 (50 mmol), and sodium bicarbonate (46 mmol) to a 150 mL mixture of ethanol and water (1:1) and stir for 14 h. The reaction solution is concentrated and purified by column chromatography (eluent: dichloromethane: methanol = 1:1) to obtain pyruvate.

[0048] Pyruvate 1 The schematic diagram of the H NMR results is shown below. Figure 3 MS results diagram as follows Figure 4 As shown, the specific results are as follows:

[0049] 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.

[0050] This embodiment also provides a method for preparing a pyruvate preparation, the specific process of which is as follows:

[0051] Take 10g of pyruvate, 10g of sodium chloride, and 0.5g of citric acid, add 50mL of water for injection and stir well; separately add 0.5mol / L sodium citrate solution to the above solution, adjust the pH to 7, and add water for injection to 500mL to obtain the pyruvate preparation.

[0052] Example 2

[0053] A pyruvate preparation for the treatment of asthma comprises, by weight, the following components: 11 parts pyruvate, 12 parts sodium chloride, 0.8 parts citric acid, and 500 parts water for injection; the pH of the pyruvate preparation is 6.5.

[0054] (1) Nitrobenzaldehyde (44 mmol) was added to 10 mL of ethyl acetate, followed by ethylenediamine (20 mmol), triethylamine (16 mmol), and sodium sulfate (24 mmol) in sequence. The mixture was stirred at room temperature for 16 hours. The reaction solution was cooled to 5°C, and 150 mL of methanol was added. Then, sodium borohydride (120 mmol) was added in batches. After the addition was completed, the reaction solution was heated to room temperature and stirred for 2 hours. The reaction solution was filtered. During the filtration process, 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. The intermediate 1 was purified by silica gel column chromatography (eluent: hexane: ethyl acetate = 2:1; triethylamine was added to the eluent at 1% of the eluent volume).

[0055] (2) Add 3g of 3,4-dihydroxybenzaldehyde and 3.6g of intermediate 1 to a high-pressure vessel containing 10mL of methanol, replace the nitrogen gas in the vessel, maintain an inert gas atmosphere, and react at 75°C and 5bar for 1h. Cool the temperature of the mixture to room temperature, then add 18mg of 5% palladium on carbon to the reactor, replace the nitrogen gas with hydrogen gas, and stir at 100°C and 40bar for 15min. Cool the reaction solution to room temperature and filter it. During the filtration process, wash the filter cake with methanol. Dry and concentrate the filtrate directly, and purify it by silica gel column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0056] (3) Add 3,4-dihydroxybenzaldehyde (10 mmol) to 30 mL of ethyl acetate, then add intermediate 2 (5 mmol), triethylamine (5 mmol), and sodium sulfate (10 mmol) to it in sequence, and stir at room temperature for 14 hours; lower the reaction solution to 5°C, add 150 mL of methanol, and then add sodium borohydride (20 mmol) in batches. After the addition is complete, raise the temperature of the reaction solution to room temperature and stir for 2.5 h; 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: dichloromethane: methanol = 6:1; add triethylamine to the eluent, the amount added is 1% of the eluent volume) to obtain intermediate 3.

[0057] (4) Add 10 mmol of benzaldehyde 4-borate and 15 mmol of sodium pyruvate to 10 mL of methanol. Under a nitrogen atmosphere, add 22 mmol of potassium hydroxide at 0°C. (Add 21 mmol of potassium hydroxide V) 甲醇 V 水 =1:1 mixed solution was prepared as 0.2M potassium hydroxide solution. After the addition was completed, the reaction system was heated to room temperature and stirred for 12 hours. After the reaction was completed, the reaction solution was filtered to obtain filter cake. The filter cake was washed with cold ethanol, and then dissolved with ethyl acetate. The pH was adjusted to 5 with 1M HCl to separate the ethyl acetate phase. After drying and concentration, intermediate 4 was obtained.

[0058] (5) Add intermediate 3 (10 mmol), intermediate 4 (60 mmol), and sodium bicarbonate (60 mmol) to a 150 mL mixture of ethanol and water in a 1:1 ratio and stir for 12 h. The reaction solution is concentrated and purified by column chromatography (eluent: dichloromethane: methanol = 1:1) to obtain pyruvate. 1 The H NMR and MS results are consistent with the structure determined in Example 1.

[0059] The preparation process of 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; separately add 0.5mol / L disodium hydrogen phosphate solution to the above solution, adjust the pH value to 6.5, and add water for injection to 500ml to obtain pyruvate preparation.

[0060] Example 3

[0061] A pyruvate preparation for the treatment of asthma comprises, by weight, the following components: 12 parts pyruvate, 11 parts sodium chloride, 1 part citric acid, and 500 parts water for injection; the pH of the pyruvate preparation is 6.

[0062] The preparation process of pyruvate is as follows:

[0063] (1) Add benzaldehyde (40 mmol) to 10 mL of ethyl acetate, and add ethylenediamine (20 mmol), triethylamine (15 mmol), and sodium sulfate (22 mmol) to it in sequence. Stir at room temperature for 15 hours. Cool the reaction solution to 5°C, add 150 mL of methanol, and then add sodium borohydride (100 mmol) in batches. After the addition is complete, heat the reaction solution to room temperature and stir for 3 hours. Filter the reaction solution. During the filtration process, wash the filter cake with chloroform and 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: hexane: ethyl acetate = 2:1; add triethylamine to the eluent, the amount added is 1% of the eluent volume) to obtain intermediate 1.

[0064] (2) Add 3g of 3,4-dihydroxybenzaldehyde and 3.4g of intermediate 1 to a high-pressure reactor containing 10mL of methanol, replace the nitrogen gas in the reactor, maintain an inert gas atmosphere, and react at 85°C and 5bar for 2h. Cool the temperature of the mixture to room temperature, then add 12mg of 5% palladium on carbon to the reactor, replace the nitrogen gas with hydrogen gas, and stir at 90°C and 40bar for 15min. Cool the reaction solution to room temperature and filter it. Wash the filter cake with methanol during the filtration process. Dry and concentrate the filtrate directly, and purify it by silica gel column chromatography (eluent: dichloromethane: methanol = 10:1) to obtain intermediate 2.

[0065] (3) Add 3,4-dihydroxybenzaldehyde (11 mmol) to 30 mL of ethyl acetate, then add intermediate 2 (5 mmol), triethylamine (5.5 mmol), and sodium sulfate (11 mmol) sequentially, and stir at room temperature for 14 hours; lower the reaction temperature to 5°C, add 150 mL of methanol, and then add sodium borohydride (25 mmol) in batches. After the addition is complete, raise the temperature of the reaction solution to room temperature and stir for 2.5 h; 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: dichloromethane: methanol = 6:1; add triethylamine to the eluent, the amount added is 1% of the eluent volume) to obtain intermediate 3.

[0066] (4) Add 10 mmol of benzaldehyde 4-borate and 12 mmol of sodium pyruvate to 10 mL of methanol. Under a nitrogen atmosphere, add 20 mmol of potassium hydroxide at 0°C. (Add 21 mmol of potassium hydroxide V) 甲醇 V 水 =1:1 mixed solution was prepared as 0.2M potassium hydroxide solution. After the addition was completed, the reaction system was heated to room temperature and stirred for 18 hours. After the reaction was completed, the reaction solution was filtered to obtain filter cake. The filter cake was washed with cold ethanol, and then dissolved with ethyl acetate. The pH was adjusted to 5 with 1M HCl to separate the ethyl acetate phase. After drying and concentration, intermediate 4 was obtained.

[0067] (5) Add intermediate 3 (10 mmol), intermediate 4 (40 mmol), and sodium bicarbonate (40 mmol) to a 120 mL mixture of ethanol and water in a 1:1 ratio and stir for 16 h. The reaction solution is concentrated and purified by column chromatography (eluent: dichloromethane: methanol = 1:1) to obtain pyruvate. 1 The H NMR and MS results are consistent with the structure determined in Example 1.

[0068] The preparation process of 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; add 0.5mol / L disodium hydrogen phosphate solution to the above solution, adjust the pH value to 6, and add water for injection to 500ml to obtain pyruvate preparation.

[0069] Experimental Example 1

[0070] Cytotoxicity assay of pyruvate

[0071] The concentration of EL-4 mouse T-lymphoma cells was adjusted to 5 × 10⁻⁶ cells in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. 4Cells / mL, 100 μL was seeded into 96-well plates; Examples 1-3: different concentrations (120 μg / mL, 60 μg / mL, 30 μg / mL, 15 μg / mL, 7.5 μg / mL) of pyruvate solution were added to each well (the pyruvate solution prepared in Example 1 was dissolved in medium containing 0.2% DMSO to prepare a 120 μg / mL pyruvate stock solution, and then diluted with medium to the required concentrations of pyruvate solution); Blank control group: no pyruvate was added. Pyruvate solution of the same concentration was used in triplicate, and the plates were incubated at 37°C in a 5% CO2 incubator; after 48 h of incubation, 30 μL of 5 mg / mL MTT solution was added, and the reaction was terminated after another 4 h of incubation; the absorbance (OD value) of each well was measured at 570 nm using a microplate reader, and the cell viability was calculated. Cell viability (%) = OD value of experimental group / OD value of negative control group × 100%, specific results are shown below. Figure 5 As shown.

[0072] Depend on Figure 5 It is known that the cell survival rate is greater than 80% at pyruvate concentrations below 120 μg / mL. Therefore, it can be concluded that the pyruvate prepared in this invention has no cytotoxicity at concentrations below 120 μg / mL.

[0073] Experimental Example 2

[0074] Effects of pyruvate preparations on IL-6 and TNF-α release in RAW264.7 cells

[0075] RAW264.7 cells were cultured for 24 h in sterile DMEM high-glucose medium containing DMEM and 10% fetal bovine serum in a 5% CO2, 37°C cell culture incubator; RAW264.7 cells in logarithmic growth phase were harvested and adjusted to 2 × 10⁻⁶ cells / cells. 5 Cell suspension was seeded at 1 mL / well in 6-well plates. After culturing at 37°C in a 5% CO2 incubator for 24 h, the culture medium was replaced. For the blank control group, fresh culture medium was used in each well. For Examples 1-3, fresh culture medium containing 1 μg / mL lipopolysaccharide and 20 μg / mL pyruvate preparation from Examples 1-3 was used in each well. For the lipopolysaccharide model group, fresh culture medium containing 1 μg / mL lipopolysaccharide was used in each well. Each group was divided into 3 replicates. After culturing for another 24 h, the culture medium was collected, centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The levels of IL-6 and TNF-α in the cell culture medium were detected using ELISA. Specific results are shown below. Figure 6-7 As shown.

[0076] Depend on Figure 6-7It is known that pyruvate can reduce LPS-induced TNF-α and IL-6 levels, leading to the conclusion that pyruvate can inhibit LPS-induced inflammation in RAW264.7 cells.

[0077] Experimental Example 3

[0078] Effects of pyruvate preparations on inflammatory cells in ovalbumin-induced asthma model mice

[0079] Animal grouping: Female BALB / c mice, weighing 23±2g, 6-8 weeks old, were randomly divided into 6 groups: blank control group, ovalbumin model group, Example 1-3 groups, and positive control group, with 6 mice in each group. They were placed in an environment of 23±2℃ with a lighting time of 12h / day. The experiment began after the animals had adapted to the environment for one week.

[0080] Animal modeling: Starting on day 1, except for the blank control group, all animals in each group were intraperitoneally injected with 200 μL of PBS suspension containing 20 μg ovalbumin and 2 mg aluminum hydroxide, once every 14 days, for two consecutive days. The blank control group was replaced with an equal volume of PBS. Starting on day 28, except for the normal control group, all mice in each group were placed in a transparent, sealed container and nebulized with a stimulation solution (PBS solution containing 1% ovalbumin) for 20 minutes, once a day for 3 days. The blank control group was replaced with PBS. One hour before each stimulation step, all groups were administered the drug by gavage.

[0081] Dosage in each group: The positive control group was given dexamethasone (30 mg / kg), the pyruvate preparation group in Examples 1-3 was given pyruvate preparation (20 mg / kg based on pyruvate mass), and the blank control group and ovalbumin model group were given an equal volume of physiological saline for 3 days.

[0082] Test method: Mice were sacrificed 48 hours after the last challenge, and bronchoalveolar lavage fluid (BALF) was collected from each group of mice. The BALF was centrifuged at 4°C for 10 min at 1000 rpm. The pelleted cells were resuspended in 0.5 mL of PBS, and cell counts were performed using a hematology analyzer. Specific results are as follows: Figure 8 As shown.

[0083] Depend on Figure 8 It can be seen that, compared with the blank control group mice, the number of eosinophils in the bronchoalveolar lavage fluid of the model group asthmatic mice increased dramatically, indicating that a large number of inflammatory cells, namely eosinophil infiltration, appeared in 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. The number of inflammatory cells in the bronchoalveolar lavage fluid of asthmatic mice treated with pyruvate preparations in Examples 1-3 decreased to varying degrees with changes in dosage, and the effect was more obvious and close to the effect of the hormone drug dexamethasone in the positive control group.

[0084] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

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

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

5. The pyruvate preparation for the treatment of asthma according to claim 2, characterized in that, The molar ratio of intermediates 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 the treatment of asthma according to claim 2, characterized in that, The molar ratio of 4-boronic acid benzaldehyde, pyruvic acid and potassium hydroxide in step (4) is 1:(1.2~1.5):(2~2.2).

7. The pyruvate preparation for the treatment of 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 the treatment of asthma according to any one of claims 1 to 7, characterized in that, Includes the following steps: According to the stated weight proportions, take pyruvate, sodium chloride, and citric acid, add them to water for injection, and stir until homogeneous; separately, add pH buffer to the above solution and adjust the pH value to 6-7 to obtain the pyruvate preparation.

9. The method for preparing the pyruvate preparation for asthma treatment according to claim 8, characterized in that, The pH buffer solution is either sodium citrate solution or disodium hydrogen phosphate solution.

10. The method for preparing the pyruvate preparation for asthma treatment according to claim 8, characterized in that, The formulation is a nebulized inhaler.

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