A kind of preparation containing pulsatilla saponin B 4 Inhalation solution and use thereof

Abstract

The present invention relates to a kind of saponin containing pulsatilla saponin B 4 Inhalation solution and use thereof. The inhalation solution comprises the active ingredient pulsatilla saponin B 4 The inhalation solution of the present invention can effectively reduce the influence of API itself and environmental factors on the preparation, and slow down the growth rate of related substances of the drug during storage. In addition, since the inhalation solution excipient of the present application is citric acid / sodium citrate, the bad taste of the raw material drug can be partially masked, and therefore, the inhalation solution has higher patient compliance. The inhalation solution of the present application uses less excipients and has good safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations and particularly relates to a pharmaceutical preparation comprising pulsatilla saponin B 4 Inhalation solution and use thereof. Background Art

[0002] Pulsatilla saponin B 4 It is a pharmacologically active pentacyclic triterpenoid saponin extracted from Pulsatilla chinensis. It has anti-inflammatory, antibacterial, immunomodulatory, anti-tumor, antioxidant, and antiviral pharmacological effects, and has the potential to be used in the treatment of asthma and chronic obstructive pulmonary disease (COPD).

[0003] Prior art CN111956655A is a saponin B 4 Made into oral and injection preparations for the treatment of COPD. 4 It is made into spray or aerosol for asthma treatment. However, the stability of this product is poor. The content of the spray decreases by 1.4%~10.0% when placed at a high temperature of 40°C for 10 days. The active ingredients of the aerosol are significantly decomposed under light conditions, and the content decreases by 2.25%~8.00% after 10 days of light exposure.

[0004] Inhalation solution is a type of inhalation preparation. The drug is dissolved in a solvent and administered through a nebulizer. It has the advantages of rapid onset of action and strong targeting.

[0005] Pulsatilla saponin B 4 Extracted from the dried root of Pulsatilla scabra, depending on the extraction process, the raw material will be mixed with a small amount of residual solvent or other impurities, making the pH value of the raw material between 4 and 8. According to research, Pulsatilla scabra saponin B 4 Unstable, the prior art CN114377027 converts Pulsatilla saponin B 4 The atomized inhalation preparation is made for the treatment of SARS-COV-2. The pH regulators used are hydrochloric acid, acetic acid, sodium hydroxide, sodium carbonate, etc. The prepared liquid medicine is tested under strong light and high temperature conditions. The pH value of the liquid medicine changes significantly and the impurities increase significantly. 4 Prepared into nanoformulations and used as TNFR2 and Foxp3 protein inhibitors for cancer treatment.

[0006] Pulsatilla saponin B 4 It has a certain bitter taste, and after being made into an inhalation solution, it has a bitter taste, which may affect the patient's compliance with the medication.

[0007] So far, there is no stable saponin B that can meet the patient's medication compliance. 4 Inhalation preparations. Summary of the invention

[0008] In order to solve the deficiencies of the prior art, the present invention provides a kind of 4 Inhalation solution and use thereof. The inhalation solution of the present invention uses citric acid / sodium citrate as a buffer stabilizer with a concentration of 1.9mM to 19mM, which can effectively reduce the impact of the API itself and environmental factors on the preparation, and slow down the growth rate of related substances during the storage of the drug. In addition, since the auxiliary material of the inhalation solution of the present application is citric acid / sodium citrate, the bad taste of the raw material can be partially masked, so the inhalation solution has higher patient compliance. The inhalation solution of the present application uses less auxiliary materials and has good safety.

[0009] The above-mentioned purpose of the present application is achieved by adopting the following technical solutions.

[0010] In one aspect, the present invention provides a method comprising: 4 Use of an inhalation solution for preparing a medicament for treating and / or preventing asthma and chronic obstructive pulmonary disease,

[0011] Wherein, the inhalation solution is composed of the active ingredient pulsatilla saponin B 4 The inhalation solution is made of citric acid and sodium citrate. The excipients in the inhalation solution include an osmotic pressure regulator and a buffer stabilizer. The buffer stabilizer is citric acid and sodium citrate.

[0012] Preferably, the total concentration of citric acid and sodium citrate in the inhalation solution is 1.9 mM to 19 mM.

[0013] Preferably, the pH value of the inhalation solution is 5.0-7.0.

[0014] Preferably, the osmotic pressure regulator is sodium chloride.

[0015] Preferably, the inhalation solution may further include a stabilizer, and the stabilizer is disodium edetate.

[0016] Preferably, the inhalation solution may further include a pH adjuster, and the pH adjuster is hydrochloric acid or sodium hydroxide.

[0017] In another aspect, the present invention provides a method comprising pulsatilla saponin B 4 An inhalation solution comprising the active ingredient pulsatilla saponin B 4 Inhalation solution is a kind of inhalation preparation. The drug is dissolved in a solvent and administered through an atomizer. It has the advantages of rapid onset and strong targeting.

[0018] Preferably, the excipients in the inhalation solution include an osmotic pressure regulator and a buffer stabilizer.

[0019] Preferably, the buffer stabilizer is citric acid and sodium citrate. Citric acid and sodium citrate are commonly used buffer salt pairs in injections, and other commonly used buffer salts include phosphate buffer salts. The citric acid used in the present invention is an organic acid that exists naturally in large quantities in plants and can be used as a food additive and pharmaceutical excipient. Citric acid is a weak acid that will not completely dissociate in water. Citric acid, citrate and H are present in the solution at the same time. + Sodium citrate is the sodium salt of citric acid. When citric acid and sodium citrate are added to the solution at the same time, if the solution contains H + Increase, H + It will form citric acid with free citrate particles, thereby slowing down the decrease in solution pH. + decrease, citric acid will ionize to form H + , thereby slowing down the rise in solution pH.

[0020] Preferably, the total concentration of citric acid and sodium citrate in the inhalation solution is 1.9 mM to 19 mM. The inventors of the present application unexpectedly found that, completely different from other buffer systems, by using citric acid and sodium citrate, especially controlling the total concentration of citric acid and sodium citrate within 1.9 mM to 19 mM, not only can the pH value of the inhalation solution be stabilized at 5.0 to 7.0, but more importantly, it can effectively reduce impurities and environmental factors in the API (such as dissolved CO in the solvent). 2 , microbial growth, etc.) on the preparation, making the drug stable during storage and slowing down the growth rate of related substances. In addition, the buffer stabilizers citric acid and sodium citrate in the inhalation solution have a certain sour and salty taste, which can partially mask the bad taste of the raw material drug.

[0021] Preferably, the osmotic pressure regulator is selected from sodium chloride, etc. Sodium chloride is commonly used in solution dosage forms and has higher safety.

[0022] Preferably, other auxiliary materials may be added to the inhalation solution as required, including but not limited to stabilizers and pH adjusters.

[0023] Preferably, the stabilizer in the inhalation solution is disodium edetate.

[0024] Since the liquid preparation equipment, pipelines, tanks, and filling machines used in the production of liquid preparations are all made of stainless steel, metal ions may be introduced and affect the stability of the product. Disodium ethylenediaminetetraacetic acid is a commonly used chelating agent in pharmaceutical preparations, which can catalyze self-oxidation reactions and remove metal ions in preparations.

[0025] The pH regulator in the inhalation solution is selected from hydrochloric acid, sodium hydroxide, etc., which can be ionized into H + or OH -, changes the pH value of the solution and is a commonly used pH regulator for liquid preparations.

[0026] In another aspect, the present invention further provides a product comprising the above-mentioned inhalation solution, wherein the inhalation solution in the product is filled in a low-density polyethylene bottle and externally packaged in an aluminum foil bag for administration by atomization inhalation.

[0027] The inhalation solution of the present invention can be filled in a low-density polyethylene bottle or other packaging forms for atomized inhalation administration. The external packaging can be an aluminum foil bag to reduce the effect of light on the stability of the drug.

[0028] Compared with the prior art, the present invention has at least the following beneficial technical effects:

[0029] Based on previous research, this application developed Pulsatilla saponin B 4 The inhalation solution uses citric acid / sodium citrate as a buffer stabilizer with a concentration of 1.9mM~19mM. This buffer stabilizer is different from ordinary pH adjusters. It can not only effectively reduce the impact of the API itself and environmental factors on the pH of the preparation, but also slow down the growth rate of related substances during the storage of the drug. DETAILED DESCRIPTION

[0030] Embodiments 1 to 10

[0031] Prepare Examples 1 to 10 according to the following table, add sodium chloride, citric acid / sodium citrate, disodium ethylenediaminetetraacetic acid and pulsatilla saponin B to purified water. 4 , then adjust the pH value of the prescription to 5.1~7.0 with hydrochloric acid and sodium hydroxide, and the amount of citric acid / sodium citrate added is 1.9~19mM.

[0032]

[0033] Embodiment 11 A kind of preparation containing pulsatilla saponin B 4 Inhalation solution

[0034] prescription:

[0035]

[0036] Process:

[0037] Dissolve sodium chloride, citric acid and sodium citrate in water for injection, then add pulsatilla saponin B into the container. 4 Continue stirring to dissolve, and finally fill the solution into LPDE bottles at 2 ml / tube.

[0038] The product is atomized using a jet nebulizer. According to the provisions of Item 0111 of Part IV of the 2020 edition of the "Chinese Pharmacopoeia", the product atomization rate is measured to be 1.3 mg / min and the total delivery amount is 8.4 mg.

[0039] Test Example 1: Pulsatilla saponin B 4 Effect of inhalation solution on chronic cigarette smoke inhalation + LPS-induced COPD model in rats

[0040] Test drug: Pulsatilla saponin B prepared in Example 11 4 Inhalation solution

[0041] Experimental animals: SD rats, 72

[0042] Grouping: After animal adaptive feeding, 72 male SD rats were randomly divided into 6 groups (blank group, model group, B 4 Low-dose inhalation group, B 4 Medium dose inhalation group, B 4 high-dose inhalation group, DEX group).

[0043] Model group, B 4 Low-dose inhalation group, B 4 Medium dose inhalation group, B 4 COPD model was established in high-dose inhalation group and DEX group:

[0044] (1) LPS airway insufflation: After the rats were anesthetized, LPS was non-invasively insufflated into the airways using a small animal anesthesia laryngoscope. The LPS concentration was 5 mg / mL and the dosage was 20 μL / 100 g. The first day of LPS modeling was counted as Day 0. Thereafter, airway insufflation was performed every 4 weeks. On the day of airway insufflation, the rats were not exposed to smoke due to anesthesia.

[0045] (2) Smoke exposure: Place rats in an animal smoking device, close the lid, light a cigarette, and use a vacuum pump to draw cigarette smoke into the animal smoking device and fill it up, exposing the rats to smoke. Each cigarette smoke exposure lasts for 10 minutes, followed by air exposure for 10 minutes before the next cigarette is used. Ten cigarettes are used per day for 12 consecutive weeks. The blank control group is exposed to air.

[0046] Dosage:

[0047] (1) The blank control group and the model group were administered with nebulized inhalation of normal saline.

[0048] (2) B 4 Low-dose inhalation group, B 4 Medium dose inhalation group, B 4 The high-dose inhalation group used aerosol inhalation of Pulsatilla saponin B 4For inhalation solution, the dosages were 0.47, 0.94, and 1.88 mg / kg;

[0049] (3) The DEX group model group was intraperitoneally injected with 0.05 mg / mL dexamethasone sodium phosphate, with a dosage of 1 mL / 100 g.

[0050] Each group was given medication once a day before modeling until the end of the experiment.

[0051] Test results:

[0052] The results of the long-term cigarette smoke inhalation + LPS-induced COPD model in rats showed that: 4 The (0.47, 0.94, 1.88 mg / kg, ih) groups could significantly reduce the increase in total white blood cell count, with inhibition rates of 35.48%, 35.49%, and 46.79%, respectively (P<0.001); the classification count observation significantly reduced the increase in neutrophils in BALF, with inhibition rates of 52.98%, 63.58%, and 69.92%, respectively; and significantly reduced the increase in macrophages, with inhibition rates of 51.46%, 64.35%, and 68.35%, respectively. The results of H&E staining of pathological sections showed that B 4 The three dose groups and the DEX group significantly reduced the infiltration of macrophages and neutrophils into lung tissue. The expressions of IL-1β, TGF-β1, and MUC5B mRNA in the lung tissue of rats in the model group were significantly increased (P<0.001); 4 The three dose groups and the DEX group could significantly inhibit the expression of IL-1β mRNA, MUC5B and MUC5AC mRNA; there was an inhibitory trend on the expression of TGF-β1 mRNA, but there was no statistical difference. The protein levels of IL-1β, IL-8, IL-17, MMP-12, MMP-9, TGF-β1, TNF-α, MUC5B and MUC5AC in the lung tissue of rats in the model group were significantly increased (P<0.001). 4 The three dose groups and the DEX group could significantly reduce the increase in IL-1β, IL-17, MUC5B and MUC5AC protein levels, but had no significant effect on the increase in IL-8, MMP-12, MMP-9, TGF-β1 and TNF-α protein levels. 4 Masson staining results of pathological sections in the medium-dose group showed that collagen deposition in lung tissue was significantly reduced (P<0.05), while other groups including the DEX group had no significant effect. 4 Both DEX and β-catenin can significantly reduce the increase of cell apoptosis. The results of immunohistochemical staining of pathological sections showed that B 4Both XTEN and DEX can significantly reduce the expression of TWIST protein in the lung tissue of rat COPD model and increase the expression of E-cadherin protein in the lung tissue.

[0053] That is, in the COPD model of rats induced by long-term cigarette smoke combined with LPS, B 4 (0.47, 0.94, 1.88 mg / kg) nebulized inhalation administration can significantly reduce the expression of IL-1β, IL-17, TNF-α, MUC5AC, MUC5B proteins and IL-1β, MUC5AC, MUC5B mRNA in lung tissue, inhibit the increase of inflammatory cells in BALF, reduce inflammatory cell infiltration in lung tissue, inhibit the apoptosis of lung tissue cells, reduce Twist expression in lung tissue and increase E-cadherin protein expression, reduce airway resistance and improve lung function in model rats, and play a role in alleviating COPD symptoms.

[0054] Comparative Examples 1 to 3

[0055] Comparative Examples 1 to 3 were prepared according to the following table. Sodium chloride and Pulsatilla saponin B were added to purified water. 4 , without adding citrate, and then adjust the formulation pH to 5.2-6.6 with hydrochloric acid and sodium hydroxide.

[0056]

[0057] Comparative Examples 4-5

[0058] Adding Pulsatilla saponin B with different purification processes into purified water 4 , no citrate and no sodium chloride are added.

[0059]

[0060] Comparative Example 6

[0061] Comparative Example 6 was prepared according to the following table. Sodium chloride, citric acid / sodium citrate and pulsatilla saponin B were added to purified water. 4 Then, the pH value of the formulation was adjusted to 6.0 with hydrochloric acid and sodium hydroxide, and the amount of citric acid / sodium citrate added was 40 mM.

[0062]

[0063] Comparative Examples 7-8

[0064] Comparative Example 7 was prepared according to the following table. Sodium chloride, disodium hydrogen phosphate / sodium phosphate and pulsatilla saponin B were added to purified water. 4, and then adjust the pH value of the prescription to 6.0 with hydrochloric acid and sodium hydroxide, the amount of disodium hydrogen phosphate / sodium phosphate added is 3.8mM, and the amount of acetic acid / sodium acetate added is 13.6mM.

[0065]

[0066] Comparative Example 9

[0067] Comparative Example 9 was prepared according to the following table. Sodium chloride, disodium edetate and pulsatilla saponin B were added to purified water. 4 , and then adjust the pH value of the prescription to 6.1 with hydrochloric acid and sodium hydroxide without adding citrate.

[0068]

[0069] Test Example 1

[0070] Take 20 mL of the solution of Examples 6, 7, 8 and Comparative Examples 2, 4, and 5, add 0.1 mol / L NaOH or 0.1 mol / L HCl dropwise until the pH value is greater than 9 or less than 4, and record the amount of NaOH solution or HCl solution used.

[0071]

[0072] Test Example 2

[0073] Examples 1 to 10 and Comparative Examples 1 to 3, 6 to 9 were placed at a high temperature of 60° C. to examine the pH value and related stability of each formulation.

[0074]

[0075] Test Example 3

[0076] Example 11 was placed in an environment of 40°C and RH75% for 3 months, and the changes in pH, content and related substances were evaluated.

[0077]

[0078] Test Example 4

[0079] The solutions of Examples 6 to 9 and Comparative Example 5 and saline were taken and the bitterness was evaluated.

[0080]

[0081] Test Example 5

[0082] Examples 10 and 11 were packed in LDPE bottles, one part was placed directly under strong light, another part was sealed in an aluminum bag and placed under strong light, and another part was placed for 10 days to evaluate the changes in pH value and related substances.

[0083]

[0084] During production and storage, the inhalation solution of the present application is susceptible to acid and alkali, causing changes in the solution, which may affect the stability of the drug and further affect the safety of the drug. Test Example 1 studies the buffering capacity of Examples 6 to 8 and Comparative Examples 2, 4, and 5 for acids and alkalis. The results show that the buffering capacity for acids and alkalis when only hydrochloric acid and sodium hydroxide are used as pH regulators (Comparative Example 2) or when no pH regulator is added (Comparative Examples 4 and 5) is significantly lower than that of Examples 6 to 8, and the buffering capacity of citric acid / sodium citrate decreases with decreasing concentration. Test Example 2 studies the stability of Examples 1 to 10, Comparative Examples 1 to 3, and Comparative Examples 6 to 9 under high temperature conditions of 60°C. The results show that the pH of Examples 1 to 10 is stable at 60°C for 10 days, and the difference with 0 days is within 0.1, and the total impurity growth of related substances does not exceed 0.43%. Comparative Examples 1 to 3 only use hydrochloric acid and sodium hydroxide as pH regulators, and no citric acid / sodium citrate is added. The difference between the pH at 60°C for 10 days and the pH at 0 days is 0.1-1.0, and the pH is easily affected by the environment and is not stable enough; the total impurity growth of related substances at high temperature for 10 days is 0.52%, 0.54%, and 0.61%, which is slightly worse than Comparative Example 6 with a citric acid / sodium citrate concentration of 40 mM (the total impurity growth at high temperature for 10 days is 0.48%), Comparative Example 7 using phosphate buffer (the total impurity growth at high temperature for 10 days is 0.45%), and Comparative Example 8 using acetic acid / sodium acetate buffer (the total impurity growth at high temperature for 10 days is 0.54%), and is significantly worse than Examples 1, 3, and 4 with similar pH (the total impurity growth at high temperature for 10 days is 0.33%, 0.27%, and 0.33%, respectively).

[0085] Both Example 10 and Comparative Example 9 added disodium ethylenediaminetetraacetate as a stabilizer. The growth of related substances in Example 10 in which citrate was added as a pH regulator was consistent with that in Example 3 in which disodium ethylenediaminetetraacetate was not added as a stabilizer (the total impurities increased by 0.27% after 10 days at high temperature), and was better than that in Comparative Example 9 in which citrate was not added as a pH regulator (the total impurities increased by 0.41% after 10 days at high temperature).

[0086] In summary, citric acid / sodium citrate at a concentration of 1.9 mM to 19 mM reduced the effect of the environment on the solution pH and had an important effect on the activity of pulsatilla saponin B. 4It has a certain stabilizing effect. When the citrate concentration is too high, the stronger buffering capacity is of little significance. On the contrary, the stability of the active ingredient will be reduced due to the high salt concentration. The solution stability when using phosphate buffer is slightly worse than that of the prescription using citrate buffer.

[0087] Test Example 3 studied the stability of Example 11 at 40°C. The results showed that after adding citric acid / sodium citrate to Example 11 and observing it at 40°C for 3 months, the pH and content of the product did not change significantly, the impurities increased less, the product properties were stable, and it could withstand a high temperature of 40°C.

[0088] Test Example 4 Study on the effect of citric acid / sodium citrate on Pulsatilla saponin B 4 The results show that after adding citric acid / sodium citrate to Examples 6 to 9, the taste of citric acid / sodium citrate masks the taste of Pulsatilla saponin B to a certain extent. 4 The unpleasant smell is eliminated, and the bitterness is reduced compared to Comparative Example 5 in which citric acid / sodium citrate is not added.

[0089] Test Example 5 studies the light stability of the product. The results show that the light stability of the product with 0.01% disodium ethylenediaminetetraacetate added (Example 10) is worse than that of the product without disodium ethylenediaminetetraacetate added (Example 11), and the stability of the two products is significantly improved after being packaged in aluminum bags.

Claims

1. Use of an inhalation solution containing pulsatilla saponin B4 in the preparation of a medicament for treating and / or preventing asthma and chronic obstructive pulmonary disease, in, The inhalation solution is prepared from the active ingredient pulsatilla saponin B4 and excipients. The excipients in the inhalation solution include an osmotic pressure regulator and a buffer stabilizer. The buffer stabilizer is citric acid and sodium citrate. Wherein, the concentration of pulsatilla saponin B4 is 1.00 w / w %; The concentration of the osmotic pressure regulator is 0.87w / w%; The total concentration of citric acid and sodium citrate in the inhalation solution is 1.9 mM to 19 mM; The osmotic pressure regulator is sodium chloride.

2. The use according to claim 1, characterized in that The pH value of the inhalation solution is 5.0-7.

0.

3. The use according to claim 1 or 2, characterized in that: The auxiliary materials of the inhalation solution also include a stabilizer, and the stabilizer is disodium ethylenediaminetetraacetate.

4. The use according to claim 1 or 2, characterized in that: The auxiliary materials of the inhalation solution also include a pH regulator, and the pH regulator is hydrochloric acid or sodium hydroxide.

5. An inhalation solution comprising pulsatilla saponin B4, characterized in that The inhalation solution is prepared from the active ingredient pulsatilla saponin B4 and excipients. The excipients in the inhalation solution include an osmotic pressure regulator and a buffer stabilizer. The buffer stabilizer is citric acid and sodium citrate. Wherein, the concentration of pulsatilla saponin B4 is 1.00 w / w %; The concentration of the osmotic pressure regulator is 0.87w / w%; The total concentration of citric acid and sodium citrate in the inhalation solution is 1.9 mM to 19 mM; The osmotic pressure regulator is sodium chloride.

6. The inhalation solution according to claim 5, characterized in that The pH value of the inhalation solution is 5.0-7.

0.

7. The inhalation solution according to claim 5 or 6, characterized in that The auxiliary materials of the inhalation solution also include a stabilizer, and the stabilizer is disodium ethylenediaminetetraacetate.

8. The inhalation solution according to claim 5 or 6, characterized in that The auxiliary materials of the inhalation solution also include a pH regulator, and the pH regulator is hydrochloric acid or sodium hydroxide.

9. An inhalation solution comprising pulsatilla saponin B4, characterized in that The prescription of the inhalation solution is as follows: 。 10. A product comprising the inhalation solution according to any one of claims 5 to 9, wherein the inhalation solution in the product is filled in a low-density polyethylene bottle and externally packaged in an aluminum foil bag for administration by aerosol inhalation.

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