Disclosure of Invention
A first object of the present invention is to provide a sodium pyruvate nebulized inhalant formulation that is effective in slowing the progression of pulmonary fibrosis in patients with obstructive pulmonary disease.
The second aim of the invention is to provide a preparation method of sodium pyruvate atomized inhalation preparation.
A third object of the present invention is to provide the use of a sodium pyruvate aerosol inhalation formulation.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
A sodium pyruvate atomization inhalation preparation comprises sodium pyruvate, modified glycyrrhizic acid, sodium chloride, a pH regulator and water for injection, wherein the concentration of the sodium pyruvate is 50-120mg/mL, the concentration of the modified glycyrrhizic acid is 5-40mg/mL, the concentration of the sodium chloride is 0.85-0.95wt%, the pH of the atomization inhalation preparation is 6.5-7, the structural formula of the modified glycyrrhizic acid is shown in formula I,
。
Further, the preparation method of the modified glycyrrhizic acid comprises the following steps:
(1) Mixing glycyrrhizic acid, anhydride and pyridine, stirring for reaction to obtain an intermediate 1, mixing the intermediate 1 with thionyl chloride, and carrying out reflux reaction to obtain an intermediate 2;
(2) Adding a compound with mercapto or amino on a benzene ring into the solution of the intermediate 2 obtained in the step (1) at the temperature of 0-5 ℃, stirring and reacting for 2-3 hours, and then heating to room temperature for continuous reaction for 20-28 hours to obtain an intermediate 3;
(3) And (3) adding trimethyliodosilane into the solution of the intermediate 3 obtained in the step (3) to carry out deprotection reaction, thus obtaining the modified glycyrrhizic acid.
Further, the molar ratio of the intermediate 2, 3-amino thiophenol in the step (2) is 1:3-3.5.
Further, in the step (3), the molar ratio of the intermediate 3 to the trimethyliodosilane is 1:1.2-1.5, the temperature of the deprotection reaction is 20-30 ℃, and the removal rate of the protecting groups in the deprotection reaction is 60-70%.
Further, the dosage ratio of glycyrrhizic acid, anhydride and pyridine in the step (1) is 1g:4-6g:0.2-0.3mL, the temperature of the stirring reaction is 20-30 ℃ and the time is 24-36h.
Further, the anhydride is selected from one or two of acetic anhydride, propionic anhydride and butyric anhydride.
Further, the dosage ratio of the intermediate 1 to the thionyl chloride in the step (1) is 1g:2-3mL, the temperature of the reflux reaction is 55-65 ℃ and the time is 4-6h.
Further, the pH regulator is selected from one of disodium hydrogen phosphate-sodium dihydrogen phosphate buffer and acetic acid-sodium acetate buffer.
The preparation method of the sodium pyruvate atomization inhalation preparation comprises the following steps of dispersing sodium pyruvate, modified glycyrrhizic acid and sodium chloride in water for injection according to the formula, and regulating the pH of the solution to 6.5-7 by using a pH regulator.
The application of the sodium pyruvate aerosol inhalation preparation in preparing products for treating respiratory diseases.
Compared with the prior art, the invention has the following main beneficial effects:
The invention provides a sodium pyruvate atomization inhalation preparation, which is prepared by using modified glycyrrhizic acid and sodium pyruvate in a matching way. Wherein the modified glycyrrhizic acid is prepared by esterification reaction of glycyrrhizic acid with anhydride, nucleophilic substitution reaction of the obtained intermediate with thionyl chloride, nucleophilic addition reaction of the obtained product with 3-amino thiophenol to generate a compound with amide group, and deprotection under the action of trimethyl iodosilane.
The modified glycyrrhizic acid added by the invention has good stability, and can effectively delay the pulmonary fibrosis process of patients with obstructive pulmonary disease by synergistic action with sodium pyruvate.
The invention also provides a preparation method of the sodium pyruvate atomization inhalation preparation, which has the advantages of simple operation, easy realization and convenient mass production.
Detailed Description
The technical scheme of the invention is further described below with reference to the specific embodiments. It will be understood by those skilled in the art that the following examples are illustrative of the present invention and are not to be construed as limiting the invention. The specific conditions not specified in the examples were carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used, unless otherwise specified, are all conventional products obtained from commercial sources.
Example (one)
Example 1
The sodium pyruvate atomization inhalation preparation comprises sodium pyruvate, modified glycyrrhizic acid, sodium chloride, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and water for injection, wherein the concentration of the sodium pyruvate is 80mg/mL, the concentration of the modified glycyrrhizic acid is 25mg/mL, the concentration of the sodium chloride is 0.85wt%, and the pH value of the atomization inhalation preparation is 6.7.
The preparation process of the modified glycyrrhizic acid comprises the following steps:
(1) Mixing glycyrrhizic acid and propionic anhydride according to the dosage ratio of 1g to 5g to 0.25mL of glycyrrhizic acid, propionic anhydride and pyridine at 25 ℃, adding pyridine, stirring and reacting for 30h, purifying by a column to obtain an intermediate 1, slowly dripping thionyl chloride into the intermediate 1 according to the dosage ratio of 1g to 2.5mL of the intermediate 1 and thionyl chloride at 0 ℃, carrying out reflux reaction at 60 ℃ for 5h, and carrying out reduced pressure distillation on a reaction solution after the reaction is finished to obtain an intermediate 2;
the nuclear magnetic resonance of intermediate 1 is as follows (shown in fig. 1):
1H-NMR(400MHz,DMSO)δ=0.90-0.95(m,13H),1.13(m,3H),1.24-1.26(d,8H),1.32(m,2H),1.57(m,6H),1.64(m,2H),1.71-1.75(m,4H),1.91-1.92(m,4H),2.02(s,8H),3.05(t,1H),3.61(t,1H),4.49(t,2H),4.51(t,H),4.78(s,H),5.07-5.10(m,3H),5.37-5.41(m,2H),5.61-5.69(m,2H),5.78(s,H),12.09(s,H),12.87(s,2H). The above results confirm that the obtained product is the target product.
The nuclear magnetic resonance of intermediate 2 is as follows (shown in fig. 2):
1H-NMR(400MHz,DMSO)δ=0.90-0.95(m,13H),1.01-1.13(m,5H),1.22-1.26(d,7H),1.32-1.39(m,4H),1.46(m,1H),1.52-1.57(m,4H),1.64-1.68(m,2H),1.71-1.76(m,2H),1.91-2.02(m,12H),3.05(t,1H),3.61(t,1H),4.50-4.52(t,3H),4.78(s,2H),5.00-5.06(m,3H),5.37-5.41(m,2H),5.59-5.61(m,2H),5.78(s,1H), The above results confirm that the obtained product is the target product.
(2) Adding 3-aminophenylthiophenol into tetrahydrofuran solution of the intermediate 2 at the temperature of 4 ℃ according to the mol ratio of the intermediate 2 to the 3-aminophenylthiophenol of 1:3.2, stirring for reaction for 2.5 hours, then heating to room temperature for continuous reaction for 24 hours, and separating and purifying the reaction liquid by column chromatography after the reaction is finished to obtain an intermediate 3;
The nuclear magnetic resonance of intermediate 3 is as follows (shown in fig. 3):
1H-NMR(400MHz,DMSO)δ=0.90-0.95(m,13H),1.01-1.13(m,5H), 1.25-1.30(m,7H),1.32-1.39(m,4H),1.46(m,1H),1.57(m,3H),1.61-1.64(m,2H),1.71-1.77(m,2H),1.85(,m1H),1.91(m,1H),2.01-2.03(s,10H),3.05(t,1H),3.41(s,3H),3.61(t,1H),4.50-4.52(t,3H),4.78(s,1H),5.09(t,1H),5.30-5.37(m,3H),5.41(m,1H),5.61-5.67(m,2H),5.78(s,1H),7.03(t,3H),7.41(t,12H),7.77(t,3H),8.22(d,3H),9.30(s,1H),10.03(s,2H), The above results confirm that the obtained product is the target product.
(3) Adding trimethyliodosilane into methylene dichloride solution of the intermediate 3 at 25 ℃ according to the mol ratio of the intermediate 3 to the trimethyliodosilane of 1:1.3, carrying out deprotection reaction, washing reaction liquid with water after the reaction is finished, collecting an organic phase, drying, and separating and purifying the organic phase by column chromatography to obtain the modified glycyrrhizic acid (the structural formula is shown as the formula I).
The nuclear magnetic resonance result of the modified glycyrrhizic acid is (shown in fig. 4):
1H-NMR(400MHz,DMSO)δ=0.90-0.95(m,13H),1.01-1.13(m,5H) 1.25-1.26(m,4H),1.30-1.39(m,8H),1.46(m,1H),1.57-1.64(m,5H),1.71-1.76(m,2H),1.85(m,1H),1.91-1.93(m,2H),2.01-2.03(m,5H),3.05(t,1H),3.41(s,3H),3.61(t,1H),3.91t(,1H),4.01(t,1H),4.50-4.52(m,2H),4.60(m,1H),4.71-4.78(m,6H),5.41(d,1H),5.61(d,1H),5.78(s,1H),7.03(d,3H),7.41(t,3H),7.77(m,3H),8.22(d,3H),9.30(s,1H),10.03(s,2H), The above results confirm that the obtained product is the target product.
The preparation method of the sodium pyruvate aerosol inhalation preparation comprises the following specific steps:
According to the formula, sodium pyruvate, modified glycyrrhizic acid and sodium chloride are dispersed in water for injection, the pH value of the solution is regulated to 6.7 by using disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and the solution is filled by adopting a sterile filling process.
Example 2
The sodium pyruvate atomization inhalation preparation comprises sodium pyruvate, modified glycyrrhizic acid, sodium chloride, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution and water for injection, wherein the concentration of the sodium pyruvate is 120mg/mL, the concentration of the modified glycyrrhizic acid is 40mg/mL, the concentration of the sodium chloride is 0.95wt%, and the pH value of the atomization inhalation preparation is 6.5.
The preparation process of the modified glycyrrhizic acid comprises the following steps:
(1) Mixing glycyrrhizic acid and butyric anhydride according to the dosage ratio of 1g to 4g to 0.2mL of glycyrrhizic acid, butyric anhydride and pyridine at 20 ℃, adding pyridine, stirring and reacting for 24h, purifying by a column to obtain an intermediate 1, slowly dripping thionyl chloride into the intermediate 1 according to the dosage ratio of 1g to 3mL of the intermediate 1 at 0 ℃, carrying out reflux reaction at 55 ℃ for 6h, and carrying out reduced pressure distillation on the reaction solution after the reaction is finished to obtain an intermediate 2;
(2) Adding 3-aminophenylthiophenol into tetrahydrofuran solution of the intermediate 2 at 0 ℃ according to the mol ratio of the intermediate 2 to the 3-aminophenylthiophenol of 1:3.5, stirring for reaction for 3 hours, then heating to room temperature for continuous reaction for 20 hours, and separating and purifying the reaction liquid by column chromatography after the reaction is finished to obtain an intermediate 3;
(3) Adding trimethyliodosilane into methylene dichloride solution of the intermediate 3 at a temperature of 30 ℃ according to a molar ratio of 1:1 of the intermediate 3 to trimethyliodosilane for deprotection reaction, washing reaction liquid with water after the reaction is finished, collecting an organic phase, drying, and separating and purifying the organic phase by column chromatography to obtain the modified glycyrrhizic acid (the structural formula is shown as formula I).
The preparation method of the sodium pyruvate aerosol inhalation preparation comprises the following specific steps:
According to the formula, sodium pyruvate, modified glycyrrhizic acid and sodium chloride are dispersed in water for injection, the pH value of the solution is regulated to 6.5 by using disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and the solution is filled by adopting a sterile filling process.
Example 3
The sodium pyruvate atomization inhalation preparation comprises the following components of sodium pyruvate, modified glycyrrhizic acid, sodium chloride, acetic acid-sodium acetate buffer and water for injection, wherein the concentration of the sodium pyruvate is 50mg/mL, the concentration of the modified glycyrrhizic acid is 5mg/mL, the concentration of the sodium chloride is 0.85wt%, and the pH of the atomization inhalation preparation is 7.
The preparation process of the modified glycyrrhizic acid comprises the following steps:
(1) Mixing glycyrrhizic acid, acetic anhydride and butyric anhydride according to the dosage ratio of 1g to 6g to 0.3mL of glycyrrhizic acid, acetic anhydride and butyric anhydride at 30 ℃, adding pyridine, stirring and reacting for 36h, purifying by a column to obtain an intermediate 1, slowly dripping thionyl chloride into the intermediate 1 according to the dosage ratio of 1g to 2mL of the intermediate 1 at 0 ℃, then carrying out reflux reaction for 4h at 65 ℃, and carrying out reduced pressure distillation on the reaction liquid after the reaction is finished to obtain an intermediate 2;
(2) Adding 3-aminophenol into the tetrahydrofuran solution of the intermediate 2 at the temperature of 5 ℃ according to the mol ratio of the intermediate 2 to the 3-aminophenol of 1:3.5, stirring for reaction for 2 hours, then heating to room temperature for continuous reaction for 28 hours, and separating and purifying the reaction liquid by column chromatography after the reaction is finished to obtain an intermediate 3;
(3) Adding trimethyliodosilane into methylene dichloride solution of the intermediate 3 at 20 ℃ according to the mol ratio of the intermediate 3 to the trimethyliodosilane of 1:1.5, carrying out deprotection reaction, washing reaction liquid with water after the reaction is finished, collecting an organic phase, drying, and separating and purifying the organic phase by column chromatography to obtain the modified glycyrrhizic acid (the structural formula is shown as the formula I).
The preparation method of the sodium pyruvate aerosol inhalation preparation comprises the following specific steps:
According to the formula, sodium pyruvate, modified glycyrrhizic acid and sodium chloride are dispersed in water for injection, the pH value of the solution is regulated to 7 by using acetic acid-sodium acetate buffer solution, and the solution is filled by adopting a sterile filling process, so that the injection is obtained.
(II) comparative example
Comparative example 1
Comparative example 1 glycyrrhizic acid was used instead of modified glycyrrhizic acid, the remainder being as in example 1.
Comparative example 2
Comparative example 2 monoammonium glycyrrhizinate was used instead of modified glycyrrhizic acid, and the rest was the same as in example 1.
Comparative example 3
Comparative example 3 was conducted in the same manner as in example 1 except that no modified glycyrrhizic acid was added.
The performance of the sodium pyruvate aerosol inhalation formulation prepared by the present invention is further described below.
1. Biological Activity of nebulized inhalant formulations
The biological activities of the aerosol inhalation formulations of examples 1 to 3 and comparative examples 1 to 3 before and after atomization were examined by cytopathic method (L929) using a vibrating screen type atomizer in combination with the aerosol inhalation formulations of examples 1 to 3 and comparative examples 1 to 3, and the results are shown in Table 1.
TABLE 1
From an examination of Table 1, the nebulized inhalation formulations of examples 1-3 showed no significant differences in biological activity between before and after nebulization. The aerosol inhalation formulation of comparative example 3 showed a significant difference in biological activity before and after atomization, and the aerosol inhalation formulations of comparative examples 1 and 2 showed a slight difference in biological activity before and after atomization. As a result of analysis, comparative example 1 was conducted by using glycyrrhizic acid instead of modified glycyrrhizic acid, comparative example 2 was conducted by using monoammonium oxalate instead of modified glycyrrhizic acid, and comparative example 3 was conducted without adding modified glycyrrhizic acid. From the above, the modified glycyrrhizic acid provided by the invention can improve the stability of sodium pyruvate.
2. Pharmacological Properties of the nebulized inhalant formulation
Rats were anesthetized with pentobarbital sodium (1%, 40 mg/kg) on days 1, 14 of the experiment, then injected with 0.2mL of lipopolysaccharide physiological saline solution (200 μg/μl), and then rotated vertically and laterally to uniformly distribute lipopolysaccharide in both lungs. Groups of rats on days 2-28 (except day 14) were placed in self-made closed mold boxes, and all experimental rats were given a treatment of smoking 30min each day at morning, 5 cigarettes each time, to construct a model of Chronic Obstructive Pulmonary Disease (COPD).
The grouping and administration method comprises randomly dividing 50 rats with successful mould construction into 5 groups, immediately performing lung function test on the rats with successful mould construction, collecting alveolar lavage fluid of the rats, detecting IL-6 and IL-1β concentration by enzyme-linked immunosorbent assay, performing experimental group including lavage physiological saline 0.6mg/kg, and performing experimental group including 0.6g/kg of each of the preparations of examples 1-3 and comparative examples 1-3 (i.e. 0.6mg/kg sodium pyruvate in the aerosol inhalation preparation) in an aerosol manner, and performing blank control group including 10 healthy rats and 35mg/kg of lavage physiological saline. The negative control group was a lung function test performed on rats after the first day of successful modeling.
Continuously administering or adding water 14d, 15d anesthetizing each group of rats, cutting neck skin, blunt separating subcutaneous tissue to expose trachea, circular cutting a small incision between tracheal cartilage, inserting tracheal cannula and fastening, supine, head-low position, and placing in sealed plethysmograph box of small animal pulmonary function tester. One end of the tracheal tube was connected to an animal ventilator and a period of calm breath was traced to detect Forced Vital Capacity (FVC), forced expiratory volume for 0.3s (FEV 0.3), FEV0.3/FVC, maximum expiratory peak flow rate (PEFR), and the results are shown in table 2.
The following treatment or water 14d, 15d collection of each group of rat alveolar lavage fluid, enzyme-linked immunosorbent assay detection of IL-6, IL-1β concentration, results are shown in Table 3.
TABLE 2
TABLE 3 Table 3
From an examination of Table 2, the lung function parameters FVC, FEV0.3/FVC, and PEFR were significantly reduced in rats compared to the blank control group, indicating that the rats had airflow obstruction, indicating successful molding. The experimental data in table 2 shows that the maximum peak expiratory flow rate of rats was close to the negative control group after 14 days of treatment with the sodium pyruvate nebulized inhalation formulation of examples 1-3 of the present invention. And the peak expiratory flow maximum expiratory peak flow rate of rats was lower than that of the negative control group after 14 days of treatment with the sodium pyruvate nebulized inhaled formulation of comparative examples 1-3. From the above, the sodium pyruvate and the modified glycyrrhizic acid in the sodium pyruvate aerosol inhalation preparation of the invention act synergistically, and can effectively delay the pulmonary fibrosis progress of patients with obstructive pulmonary diseases.
From an examination of Table 3, it is understood that the sodium pyruvate aerosol inhalation formulation of the present invention can effectively inhibit the expression of pulmonary inflammatory factor, compared to the model group. The aerosol inhalation formulations of comparative examples 1-3 have a reduced ability to inhibit inflammatory factor expression compared to examples 1-3. Further analysis shows that comparative examples 1-3 differ from examples 1-3 in the use of different glycyrrhizic acid. In conclusion, the sodium pyruvate atomized inhalation preparation prepared from the modified glycyrrhizic acid provided by the invention has excellent anti-inflammatory effect.
Finally, the above embodiments are only for illustrating the technical solution of the present invention, and are not limited thereto. While the basic principles and main features of the present invention have been described above with specific embodiments, modifications or substitutions may be made thereto without departing from the spirit of the invention as claimed.