Preparation method of sinapultide phospholipid surfactant liposome

Abstract

The invention relates to the technical field of pharmaceutical preparations, and discloses a preparation method of sinapultide phospholipid surfactant lipidosome. According to the preparation method of the sinapultide phospholipid surfactant lipidosome, nitrogen is introduced in the film evaporation process, the efficiency of the film evaporation process is improved, meanwhile, the process parameters of the film evaporation process are optimized, the stability of the product is remarkably improved, and the yield of the sinapultide phospholipid surfactant lipidosome is improved. And the quality indexes such as biological activity, content, related substances and the like of the product all accord with related regulations in the industry.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and specifically relates to a method for preparing sinapeptide phospholipid surfactant liposomes. Background Art

[0002] Liposome is a bilayer lipid vesicle made of lipid materials that can be used as a drug carrier. When amphiphilic lipid molecules are dispersed in the aqueous phase, the hydrophobic tails of the molecules tend to gather together to avoid the aqueous phase, while the hydrophilic heads of the molecules are exposed to the aqueous phase, spontaneously forming closed vesicles with a lipid bilayer structure. The preparation methods of liposomes mainly include solvent injection method and thin film dispersion method.

[0003] The solvent injection method is to quickly inject the organic phase (such as ethanol, ether, etc.) containing liposome membrane materials into a large amount of aqueous phase containing drugs, and then prepare drug-loaded liposomes by ultrasound and other methods. A small amount of organic phase will be quickly diluted after being injected into a large amount of aqueous phase, and the liposome membrane material in the organic phase will quickly form drug-loaded SUV liposomes. The film dispersion method is to dissolve the liposome membrane material in a suitable solvent, remove the solvent to form a lipid film, and then hydrate it to form liposomes. In the drug encapsulation process, fat-soluble drugs can be added together with the film material, and water-soluble drugs can be added together with the hydration solution, and finally drug-loaded liposomes are prepared.

[0004] Neonatal respiratory distress syndrome refers to the symptom of breathing difficulties in newborns, which can cause respiratory failure and even lead to death of the child. Neonatal respiratory distress syndrome mainly occurs in premature infants, but can also occur in full-term infants. Its cause is not only related to abnormal lung development, but also closely related to the lack of alveolar surfactant. Sinapultide phospholipid surfactant injection (FDA-approved trade name: Surfaxin, this product is the world's first synthetic lung surfactant), is composed of synthetic peptide Sinapultide (KL4), phospholipid DPPC, POPG sodium salt and palmitic acid PA. It is a product designed based on the characteristics of natural human lung surfactant on the basis of 21 amino acid peptide KL4, simulating human lung surfactant protein SP-B, and is used to prevent neonatal respiratory distress syndrome (NRDS).

[0005] Sinaputide phospholipid surfactant injection acts as a pulmonary surfactant, promoting the formation of a lipid monolayer at the air / liquid interface inside the alveoli. The main lipid component of the monolayer is DPPC, and the DPPC layer is stabilized by the electrostatic interaction between the positively charged lysine residues of KL4 and the negatively charged polar head groups of phospholipids, thereby maintaining the order stability of the phospholipid layer. PA helps stabilize the order of phospholipids and helps the monolayer reduce surface tension by improving the fluidity, membrane properties and phase behavior of phospholipids. POPG, Na can enhance fluidity and promote the transformation of liposomes into monolayers.

[0006] The main components of Sinaputide Phospholipid Surfactant Injection include dipalmitoylphosphatidylcholine (DPPC), palmitoleoylglycerophosphoglycerol sodium salt (POPG, Na), palmitic acid (PA) and Sinaputide (KL4), which are insoluble in water but soluble in ethanol. Sinaputide Phospholipid Surfactant Injection is a complex liposome injection with poor stability. Summary of the invention

[0007] The preparation process of Sinaputepide phospholipid surfactant liposomes mainly includes Tris buffer preparation, API ethanol solution preparation, liposome solution formation, thin film evaporation (ethanol removal), weighing, filling, and capping. The inventors of the present application have found that liposome solution preparation and thin film evaporation (TFE) are the most critical process control steps in the production process of Sinaputepide phospholipid surfactant liposomes. Among them, the liposome solution formation step, the stability of the formed liposomes is greatly affected by factors such as the design of the stirring paddle and the stirring speed, while the thin film evaporation (TFE) step, the ability to remove ethanol is greatly affected by the design of the thin film evaporator and process parameters.

[0008] The purpose of the present application is to provide a method for preparing Sinaputepide phospholipid surfactant liposomes to solve the problem of poor stability of Sinaputepide phospholipid surfactant liposomes.

[0009] In order to achieve the above invention purpose, the technical solution adopted in this application is:

[0010] The present application provides a method for preparing sinapeptide phospholipid surfactant liposomes, comprising the following steps:

[0011] S1. Add the heated API ethanol solution to Tris buffer and mix well to obtain a mixed drug solution; wherein the API ethanol solution contains dipalmitoylphosphatidylcholine, palmitoleoylglycerophosphoglycerol sodium salt, palmitic acid and sinapeptide;

[0012] S2, transferring the mixed liquid to a thin film evaporator, and performing thin film evaporation under nitrogen flow to remove ethanol; wherein,

[0013] The conditions for the thin film evaporation include: the feeding rate of the mixed liquid is 80 to 150 mL / min, the jacket temperature of the thin film evaporator is 60 to 80°C, the vacuum degree is -0.85 to -1.00 bar, the rotation speed of the thin film evaporator is 500 to 2000 rpm, the nitrogen flow rate is 5 to 15 L / min, and the stirring speed of the receiving tank is 65 to 75 rpm.

[0014] In an optional embodiment, the heating jacket of the thin film evaporator used in step S2 has a honeycomb structure;

[0015] And / or, the thin film evaporator has an inclination angle with the ground of 45° to 60°.

[0016] The thin film evaporator is a kind of high-efficiency evaporation equipment that uses a high-speed rotating scraper to disperse the liquid into a uniform film to concentrate the material. The heating jacket used in the thin film evaporator is a honeycomb jacket structure, which can enhance the stability of the cylinder inside the thin film evaporator, improve the load-bearing capacity, and have a good heat transfer effect. The inclination angle of the thin film evaporator to the ground is 45°~60°, which can effectively promote the uniform flow of the liquid medicine in the evaporation tube, improve the evaporation efficiency, and make the liquid medicine after evaporation more uniform.

[0017] In an optional embodiment, in step S2, before the thin film evaporation, the temperature of the mixed liquid is controlled to be 28-32° C. Before the thin film evaporation, the temperature of the mixed liquid is controlled within the range of 28-32° C., which can improve the fluidity and uniformity of the liposome mixed liquid while ensuring that the product quality is not affected by the temperature.

[0018] In an optional embodiment, in step S1, the temperature of the heated API ethanol solution is 43-53°C;

[0019] And / or, before adding the heated API ethanol solution into the Tris buffer, the method further comprises heating the Tris buffer to 43-53°C.

[0020] In an optional embodiment, in step S1, the mixing operation is performed in a liposome forming tank, and the liposome forming tank includes at least 3 layers of stirring paddles with holes from bottom to top. The design of such stirring paddles is conducive to the mixing of products, promotes the formation of a uniform and stable liposome liquid, and can also avoid the generation of a large number of bubbles that affect the subsequent thin film evaporation feed rate.

[0021] And / or, the mixing conditions include: a stirring speed of 65 to 75 rpm and a stirring time of 20 to 40 min.

[0022] In an optional embodiment, before adding the heated API ethanol solution to the Tris buffer, the method further comprises filtering the Tris buffer and the API ethanol solution;

[0023] Optionally, the Tris buffer is filtered at least twice, and the pore size of the filter is 0.2 μm; the API ethanol solution is filtered at least twice, and the pore size of the filter is 0.22 μm.

[0024] In an optional embodiment, in the API ethanol solution, the concentration of dipalmitoylphosphatidylcholine is 21.8-22.2%, the concentration of palmitoleoylglycerophosphoglycerol sodium salt is 7.14-7.54%, the concentration of palmitic acid is 3.8-4.2%, and the concentration of sinapeptide is 0.7-0.9%, calculated in percentage by weight.

[0025] In an optional embodiment, after the thin film evaporation is completed, the preparation method further comprises the step of transferring the drug solution after the thin film evaporation to a receiving tank and adding water for injection to determine the weight;

[0026] The weight determination conditions include: the liquid temperature is 38-42° C., and the stirring speed is 45-55 rpm.

[0027] In an optional embodiment, the pH value of the Tris buffer is 7.55-7.65, and it contains tromethamine and sodium chloride, the concentration of tromethamine is 2.4-2.6 mg / mL, and the concentration of sodium chloride is 7.5-7.8 mg / mL.

[0028] Based on the above technical solution, this application has at least the following beneficial effects:

[0029] (1) The preparation method of sinapeptide phospholipid surfactant liposomes provided in the present application introduces nitrogen during the thin film evaporation process, thereby improving the efficiency of the thin film evaporation process. At the same time, the process parameters of the thin film evaporation process are optimized, which significantly improves the stability of the product, so that the product's biological activity, content, related substances and other quality indicators all meet the relevant industry regulations.

[0030] Specifically, the Sinaputide phospholipid surfactant has poor stability under high temperature conditions, while the thin film evaporation temperature is relatively high. In order to prevent the thin film evaporation process from affecting the product stability, the thin film evaporation parameters and evaporation efficiency are controlled to ensure that the thin film evaporation time is shortened under the condition of complete evaporation of ethanol, thereby ensuring qualified product quality.

[0031] (2) The preparation method of the sinapeptide phospholipid surfactant liposomes provided in the present application uses a thin film evaporator with a honeycomb jacket structure, in conjunction with specific thin film evaporation conditions, which can further improve the stability of the product.

[0032] (3) The key technologies of the preparation method of Sinaputepitide phospholipid surfactant liposomes provided in the present application mainly include the formation of liposome solution and the design of thin film evaporator as well as the optimization setting of process parameters. Under the process parameters provided in the present application, the stability, biological activity, content, related substances and other quality indicators of the prepared Sinaputepitide phospholipid surfactant liposomes meet the requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1 is a schematic diagram of the structure of a thin film evaporator provided in an embodiment of the present application;

[0035] Figure 2 Schematic diagram of the structure of the liposome formation tank provided in the examples of the present application. DETAILED DESCRIPTION

[0036] In order to further explain the technical means and results taken by the present application to achieve the predetermined invention purpose, the specific implementation methods, technical solutions and features of the present application are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0037] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0038] The present application is further described in detail below in conjunction with specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in the present application.

[0039] Example 1

[0040] The Sinaputide phospholipid surfactant liposomes were prepared as follows:

[0041] (1) Preparation of Tris buffer

[0042] Add 80% of the prescribed amount of water for injection at a temperature of 15 to 30° C. into the preparation tank, start stirring, add the prescribed amount of tromethamine (33.07 g) and sodium chloride (102.6 g), adjust the pH of the solution to 7.55 to 7.65 with glacial acetic acid, then add water for injection to the full amount (13.5 L), continue stirring, and stop stirring after complete dissolution to obtain Tris buffer;

[0043] (2) Preparation of API ethanol solution

[0044] 51.6 g palmitic acid, 285.4 g DPPC, 94.3 g POPG, Na and 10.9 g KL4 were added to 1.3 kg ethanol in sequence, the temperature was set to 48°C ± 5°C for dissolution, and visual inspection was performed until each API was completely dissolved to obtain an API ethanol solution;

[0045] (3) Liposome formation

[0046] The Tris buffer prepared in step (1) was passed through two consecutive 0.2 μm filters, and the filtrate was placed in Figure 2 The liposome preparation tank (including 3 layers of stirring paddles with holes from bottom to top) shown in the figure is placed in the liposome preparation tank, and the obtained solution is heated to 48±5°C; the API ethanol solution heated to 48±5°C is passed through two consecutive 0.22μm filters in sequence, and the filtrate is introduced into the liposome preparation tank containing Tris buffer, and the stirring speed is set to 70rpm, stirred for 30 minutes, and mixed to obtain a mixed drug solution;

[0047] (4) Thin film evaporation

[0048] Before thin film evaporation, the temperature of the mixed solution was controlled to 30±2℃, and then the mixed solution was transferred to Figure 1 In the thin film evaporator (TFE, the heating jacket is a honeycomb structure, and the angle between the heating jacket and the ground is 45°), thin film evaporation is performed to remove ethanol while nitrogen is introduced, and the liquid medicine after thin film evaporation is collected in a receiving tank; wherein the key process parameters of thin film evaporation are shown in Table 1;

[0049] Table 1 Key process parameters of thin film evaporation in Example 1

[0050] Process parameters scope Feeding rate 115mL / min TFE jacket temperature 70℃ Vacuum degree -0.9bar TFE speed 1000rpm Nitrogen flow rate 10L / min Receiving tank stirring speed 70rpm

[0051] (5) Fixed weight

[0052] The drug solution in the receiving tank was heated to 40° C. and stirred at a speed of 50 rpm. 10 kg of water for injection filtered through a 0.22 μm filter was added and mixed to obtain the sinapeptide phospholipid surfactant liposomes of this example.

[0053] Example 2

[0054] Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (3) of the present example, the stirring speed in the liposome preparation tank was 65 rpm and the stirring time was 40 minutes.

[0055] Example 3

[0056] Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (3) of the present example, the stirring speed in the liposome preparation tank was 75 rpm and the stirring time was 20 minutes.

[0057] Example 4

[0058] The Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (4) of the present embodiment, the key process parameters of the thin film evaporation are shown in Table 2:

[0059] Table 2 Key process parameters of thin film evaporation in Example 4

[0060] Process parameters scope Feeding rate 80mL / min TFE jacket temperature 80℃ Vacuum degree -0.85bar TFE speed 2000rpm Nitrogen flow rate 5L / min Receiving tank stirring speed 75rpm

[0061] Example 5

[0062] The Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (4) of the present embodiment, the key process parameters of the thin film evaporation are shown in Table 3:

[0063] Table 3 Key process parameters of thin film evaporation in Example 5

[0064] Process parameters scope Feeding rate 150mL / min TFE jacket temperature 60℃ Vacuum degree -1.00bar TFE speed 500rpm Nitrogen flow rate 15L / min Receiving tank stirring speed 65rpm

[0065] Comparative Example 1

[0066] The sinapeptide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (3) of the present example, Figure 2 The liposome preparation tank shown is replaced with a liposome preparation tank commonly used in the field, the manufacturer is: Jiangsu Cen Tu Engineering Technology Co., Ltd., model: CDYD-100.

[0067] Comparative Example 2

[0068] The sinapeptide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (4) of the present example, Figure 1 The thin film evaporator shown is replaced with a thin film evaporator commonly used in the field, the manufacturer is: Jiangsu Cen Tu Engineering Technology Co., Ltd., model: TCT-0.3.

[0069] Comparative Example 3

[0070] The Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (4) of the present embodiment, the key process parameters of the thin film evaporation are shown in Table 4:

[0071] Table 4 Key process parameters of thin film evaporation in comparative example 3

[0072]

[0073]

[0074] Comparative Example 4

[0075] The Sinaputide phospholipid surfactant liposomes were prepared according to the method of Example 1, except that in step (4) of the present embodiment, the key process parameters of the thin film evaporation are shown in Table 5:

[0076] Table 5 Key process parameters of thin film evaporation in comparative example 4

[0077] Process parameters scope Feeding rate 160mL / min TFE jacket temperature 90℃ Vacuum degree -0.8bar TFE speed 2500rpm Nitrogen flow rate 17L / min Receiving tank stirring speed 80rpm

[0078] Experimental Example 1

[0079] The key indexes of the products prepared in Examples 1-5 and Comparative Examples 1-4 were tested respectively, and the test results are shown in Table 6.

[0080] Table 6 Key index test results of the products prepared in Examples 1-5 and Comparative Examples 1-4

[0081]

[0082]

[0083] Table 6 (Continued) Key index test results of the products prepared in Examples 1-5 and Comparative Examples 1-4

[0084]

[0085] Table 6 (Continued) Key index test results of the products prepared in Examples 1-5 and Comparative Examples 1-4

[0086]

[0087]

[0088] Experimental Example 2

[0089] The products prepared in Example 1 and Comparative Examples 1-4 were respectively subjected to stability tests (temperature of 5±3° C., humidity of 25%), and key indicators were tested. The test results are shown in Table 7.

[0090] Table 7 Stability test results of products prepared in Example 1 and Comparative Examples 1-4

[0091]

[0092]

[0093] Table 7 (Continued) Stability test results of products prepared in Example 1 and Comparative Examples 1-4

[0094]

[0095] Table 7 (Continued) Stability test results of products prepared in Example 1 and Comparative Examples 1-4

[0096]

[0097]

[0098] Table 7 (Continued) Stability test results of products prepared in Example 1 and Comparative Examples 1-4

[0099]

[0100]

[0101] Table 7 (Continued) Stability test results of products prepared in Example 1 and Comparative Examples 1-4

[0102]

[0103] The above is only a preferred specific implementation of the present application; however, the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solution and its improved ideas of the present application within the technical scope disclosed in the present application, which should be included in the protection scope of the present application.

Claims

1. A method for preparing sinaputide phospholipid surfactant liposomes, characterized in that: The steps include: S1. Add the heated API ethanol solution to Tris buffer and mix well to obtain a mixed drug solution; wherein the API ethanol solution contains dipalmitoylphosphatidylcholine, palmitoleoylglycerophosphoglycerol sodium salt, palmitic acid and sinapeptide; S2, transferring the mixed liquid to a thin film evaporator, and performing thin film evaporation under nitrogen flow to remove ethanol; wherein, The conditions for the thin film evaporation include: the feeding rate of the mixed liquid is 80 to 150 mL / min, the jacket temperature of the thin film evaporator is 60 to 80°C, the vacuum degree is -0.85 to -1.00 bar, the rotation speed of the thin film evaporator is 500 to 2000 rpm, the nitrogen flow rate is 5 to 15 L / min, and the stirring speed of the receiving tank is 65 to 75 rpm.

2. The preparation method according to claim 1, characterized in that: The heating jacket of the thin film evaporator used in step S2 has a honeycomb structure; And / or, the thin film evaporator has an inclination angle with the ground of 45° to 60°.

3. The preparation method according to claim 1, characterized in that: In step S2, before the thin film evaporation, the temperature of the mixed liquid is controlled to be 28-32°C.

4. The preparation method according to claim 1, characterized in that: In step S1, the temperature of the heated API ethanol solution is 43-53°C; And / or, before adding the heated API ethanol solution into the Tris buffer, the method further comprises heating the Tris buffer to 43-53°C.

5. The preparation method according to claim 1, characterized in that: In step S1, the mixing operation is performed in a liposome forming tank, and the liposome forming tank includes at least 3 layers of stirring paddles with holes from bottom to top; And / or, the mixing conditions include: a stirring speed of 65 to 75 rpm and a stirring time of 20 to 40 min.

6. The preparation method according to claim 1, characterized in that: Before adding the heated API ethanol solution to the Tris buffer, the method further comprises filtering the Tris buffer and the API ethanol solution; Optionally, the Tris buffer is filtered at least twice, and the pore size of the filter is 0.2 μm; The API ethanol solution is filtered at least twice, and the pore size of the filter is 0.22 μm.

7. The preparation method according to claim 1, characterized in that: In the API ethanol solution, the concentration of dipalmitoylphosphatidylcholine is 21.8-22.2% by weight, the concentration of palmitoleoylglycerophosphoglycerol sodium salt is 7.14-7.54% by weight, the concentration of palmitic acid is 3.8-4.2% by weight, and the concentration of sinapeptide is 0.7-0.9% by weight.

8. The preparation method according to claim 1, characterized in that: After the film evaporation is completed, the preparation method further comprises the steps of transferring the drug solution after the film evaporation to a receiving tank and adding water for injection to determine the weight; The weight determination conditions include: the temperature of the liquid medicine is 38-42° C., and the stirring speed is 45-55 rpm.

9. The preparation method according to claim 1, characterized in that: The pH value of the Tris buffer is 7.55-7.65, and it contains tromethamine and sodium chloride, the concentration of tromethamine is 2.4-2.6 mg / mL, and the concentration of sodium chloride is 7.5-7.8 mg / mL.

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