Preparation method for sinapultide phospholipid surfactant liposomes

Abstract

The present invention provides a preparation method for sinapultide phospholipid surfactant liposomes and relates to the technical field of pharmaceutical formulations. In the preparation method, nitrogen is introduced during thin film evaporation to improve the efficiency of the thin film evaporation process. In addition, process parameters of the thin film evaporation process are optimized, which significantly improves the stability of the product and enables the product to meet industry-related requirements for quality indicators such as biological activity, content, and related substances.

Description

A method for preparing cinapeptide phospholipid surfactant liposomes Technical Field

[0001] This application belongs to the field of pharmaceutical formulation technology, specifically relating to a method for preparing cinapeptide phospholipid surfactant liposomes. Background Technology

[0002] Liposomes are bilayer lipid vesicles prepared from lipid materials that can be used as drug carriers. When amphiphilic lipid molecules are dispersed in an aqueous phase, their hydrophobic tails tend to aggregate and avoid the aqueous phase, while their hydrophilic heads are exposed to the aqueous phase, spontaneously forming closed vesicles with a lipid bilayer structure. The main methods for preparing liposomes include solvent injection and thin-film dispersion.

[0003] Solvent injection involves rapidly injecting an organic phase (such as ethanol or ether) containing liposome membrane material into a large aqueous phase containing the drug, followed by ultrasonication to prepare drug-loaded liposomes. A small amount of organic phase injected into a large aqueous phase is rapidly diluted, while the liposome membrane material within the organic phase quickly forms drug-loaded SUV liposomes. Film dispersion involves dissolving the liposome membrane material in a suitable solvent, removing the solvent to form a lipid film, and then hydrating it to form liposomes. During drug encapsulation, lipid-soluble drugs can be added along with the film material, while water-soluble drugs are added along with the hydration solution, ultimately preparing drug-loaded liposomes.

[0004] Neonatal respiratory distress syndrome (NRDS) refers to respiratory distress symptoms occurring in newborns shortly after birth, which can lead to respiratory failure and even death. NRDS primarily affects premature infants, but can also occur in full-term infants. Its causes are related not only to abnormal lung development but also to a deficiency of alveolar surfactant. Sinapultide phospholipid surfactant injection (FDA-approved brand name: Surfaxin, the world's first synthetic lung surfactant) is composed of the synthetic peptide Sinapultide (KL4), phospholipid DPPC, sodium POPG, and palmitic acid PA. It is a product designed based on the 21-amino acid peptide KL4 and tailored to the characteristics of natural human lung surfactant, mimicking human lung surfactant protein SP-B, and is used for the prevention of neonatal respiratory distress syndrome (NRDS).

[0005] The cinapeptide phospholipid surfactant injection acts as a pulmonary surfactant, promoting the formation of a lipid monolayer at the air / liquid interface within the alveoli. The main lipid component of the monolayer is DPPC, which is stabilized through electrostatic interactions between positively charged lysine residues of KL4 and negatively charged polar head groups of phospholipids, thus maintaining the phospholipid layer's sequence stability. PA contributes to the stability of the phospholipid sequence and helps reduce surface tension of the monolayer by improving phospholipid fluidity, membrane properties, and phase behavior. POPG and Na enhance fluidity and promote the conversion of liposomes into monolayers.

[0006] The main components of cinaleptide phospholipid surfactant injection include dipalmitoylphosphatidylcholine (DPPC), sodium palmitole glycerophosphate (POPG, Na), palmitic acid (PA), and cinaleptide (KL4), all of which are poorly soluble in water but soluble in ethanol. Cinaleptide phospholipid surfactant injection is a complex liposome injection and has poor stability. Summary of the Invention

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

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

[0009] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0010] This application provides a method for preparing cinapeptide 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, palmitoylglycerol phosphate sodium salt, palmitic acid and cinapeptide;

[0012] S2. The mixed drug solution is transferred to a thin-film evaporator, and thin-film evaporation is performed under nitrogen gas to remove ethanol; wherein,

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

[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 angle of inclination between the thin-film evaporator and the ground is 45° to 60°.

[0016] A thin-film evaporator is a high-efficiency evaporation device that uses a high-speed rotating scraper to disperse a liquid feed into a uniform thin film for material concentration. The heating jacket used in the thin-film evaporator has a honeycomb structure, which enhances the stability of the inner cylinder, improves load-bearing capacity, and provides good heat transfer. The inclination angle of the thin-film evaporator to the ground is 45°–60°, which effectively promotes uniform flow of the liquid feed within the evaporation tubes, improving evaporation efficiency and resulting in better uniformity of the evaporated liquid feed.

[0017] In an optional embodiment, in step S2, before the film evaporation, the temperature of the mixed drug solution is controlled at 28–32 °C. Controlling the temperature of the mixed drug solution within the range of 28–32 °C before film evaporation can improve the flowability and uniformity of the liposome mixed drug solution while ensuring that product quality is not affected by temperature.

[0018] In one 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 to the Tris buffer, the step of heating the Tris buffer to 43–53 °C is further included.

[0020] In one optional embodiment, in step S1, the mixing operation is performed in a liposome forming vessel, which comprises at least three layers of perforated impellers from bottom to top. This impeller design facilitates product mixing, promotes the formation of a uniform and stable liposome solution, and avoids the generation of a large number of bubbles that could affect the subsequent film evaporation feed rate.

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

[0022] In one alternative embodiment, the step of filtering the Tris buffer and the API ethanol solution is further included before adding the heated API ethanol solution to the Tris buffer.

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

[0024] In an optional embodiment, the API ethanol solution contains, by weight percentage, 21.8–22.2% of dipalmitoylphosphatidylcholine, 7.14–7.54% of palmitoylglycerol phosphate sodium salt, 3.8–4.2% of palmitic acid, and 0.7–0.9% of cinapeptide.

[0025] In one optional embodiment, after the film evaporation is completed, the preparation method further includes the step of transferring the evaporated drug solution to a receiving vessel and adding water for injection to determine the weight.

[0026] The conditions for determining the weight include: the temperature of the drug solution is 38-42 ℃, and the stirring speed is 45-55 rpm.

[0027] In one optional embodiment, the Tris buffer solution has a pH of 7.55–7.65 and contains tromethamine and sodium chloride, wherein 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 method for preparing cinapeptide phospholipid surfactant liposomes provided in this application introduces nitrogen gas during the thin film evaporation process, which improves 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 bioactivity, content, related substances and other quality indicators all meet the relevant industry regulations.

[0030] Specifically, cinapeptide phospholipid surfactants have poor stability under high temperature conditions, while the film evaporation temperature is relatively high. In order to avoid the film evaporation process from affecting product stability, the film evaporation parameters and evaporation efficiency are controlled. This can ensure that the film evaporation time is shortened under the condition that ethanol is completely evaporated, thus ensuring that the product quality is qualified.

[0031] (2) The method for preparing cinapeptide phospholipid surfactant liposomes provided in this application uses a thin film evaporator with a honeycomb jacket structure and specific thin film evaporation conditions to further improve the stability of the product.

[0032] (3) The key technologies of the preparation method of cinapeptide phospholipid surfactant liposomes provided in this 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 parameter conditions provided in this application, the stability, bioactivity, content, related substances and other quality indicators of the prepared cinapeptide phospholipid surfactant liposomes all meet the requirements. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

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

[0035] Figure 2 is a schematic diagram of the structure of the liposome forming vessel provided in the embodiments of this application. Detailed Implementation

[0036] To further illustrate the technical means and results adopted by this application to achieve the intended inventive purpose, the following preferred embodiments are used to describe in detail the specific implementation methods, technical solutions, and features according to this application. Specific features, structures, or characteristics in the various embodiments described below can be combined in any suitable form.

[0037] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0038] The present application will be further described in detail below with reference to specific embodiments. These embodiments should not be construed as limiting the scope of protection claimed in this application. Example 1

[0039] Cinatropin phospholipid surfactant liposomes were prepared according to the following method:

[0040] (1) Preparation of Tris buffer

[0041] Add 80% of the prescribed volume of water for injection at a temperature of 15–30°C to the mixing tank, start stirring, add the prescribed amount of tromethorphan (33.07 g) and sodium chloride (102.6 g), and adjust the pH of the solution to 7.55–7.65 with glacial acetic acid. Then add water for injection to the total volume (13.5 L), continue stirring, and stop stirring after complete dissolution to obtain Tris buffer solution.

[0042] (2) Preparation of API ethanol solution

[0043] 51.6g palmitic acid, 285.4g DPPC, 94.3g POPG, Na and 10.9g KL4 were added to 1.3kg of ethanol in sequence. The temperature was set at 48 ℃±5 ℃ to aid dissolution. Visual inspection was performed until all APIs were completely dissolved to obtain an API ethanol solution.

[0044] (3) Liposome formation

[0045] The Tris buffer prepared in step (1) was passed through two consecutive 0.2 μm filters. The filtrate was placed in the liposome preparation vessel shown in Figure 2 (which includes three perforated stirring paddles from bottom to top). The resulting solution was heated to 48 ± 5 ℃. The API ethanol solution heated to 48 ± 5 ℃ was passed through two consecutive 0.22 μm filters. The filtrate was introduced into the liposome preparation vessel containing the Tris buffer. The stirring speed was set to 70 rpm and stirred for 30 minutes to mix the solution and obtain a mixed drug solution.

[0046] (4) Thin film evaporation

[0047] Before thin-film evaporation, the temperature of the mixed drug solution was controlled at 30±2℃. Then, the mixed drug solution was transferred to the thin-film evaporator (TFE, with a honeycomb heating jacket and an inclination angle of 45° to the ground) shown in Figure 1 by a pump. Thin-film evaporation was carried out under nitrogen gas to remove ethanol, and the drug solution after thin-film evaporation was collected in a receiving tank. The key process parameters of thin-film evaporation are shown in Table 1.

[0048] Table 1 Key process parameters for thin film evaporation in Example 1

[0049]

[0050] (5) Fixed weight

[0051] The drug solution in the receiving vessel 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 well to obtain the cinapeptide phospholipid surfactant liposome of this embodiment. Example 2

[0052] Cinnapteptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (3) of this application, the stirring speed in the liposome preparation tank is 65 rpm and the stirring time is 40 minutes. Example 3

[0053] Cinnapteptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (3) of this application, the stirring speed in the liposome preparation tank is 75 rpm and the stirring time is 20 minutes. Example 4

[0054] Cinnaptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (4) of this application, the key process parameters for thin film evaporation are shown in Table 2:

[0055] Table 2 Key process parameters for thin film evaporation in Example 4

[0056]

[0057] Example 5

[0058] Cinnaptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (4) of this application, the key process parameters for thin film evaporation are shown in Table 3:

[0059] Table 3 Key process parameters for thin film evaporation in Example 5

[0060]

[0061] Comparative Example 1

[0062] The cinapeptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (3) of this application, the liposome preparation vessel shown in Figure 2 is replaced with a commonly used liposome preparation vessel in the field. The manufacturer is Jiangsu Centu Engineering Technology Co., Ltd., and the model is CDYD-100.

[0063] Comparative Example 2

[0064] The cinapeptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (4) of this application, the thin film evaporator shown in Figure 1 is replaced with a thin film evaporator commonly used in the field. The manufacturer is Jiangsu Centu Engineering Technology Co., Ltd., and the model is TCT-0.3.

[0065] Comparative Example 3

[0066] Cinnaptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (4) of this application, the key process parameters for thin film evaporation are shown in Table 4:

[0067] Table 4 Key process parameters for thin film evaporation in Comparative Example 3

[0068]

[0069] Comparative Example 4

[0070] Cinnaptide phospholipid surfactant liposomes were prepared according to the method of Example 1. The difference is that in step (4) of this application, the key process parameters for thin film evaporation are shown in Table 5:

[0071] Table 5 Key process parameters for thin film evaporation in Comparative Example 4

[0072]

[0073] Experimental Example 1

[0074] Key indicators 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.

[0075] Table 6. Test results of key indicators of the products prepared in Examples 1-5 and Comparative Examples 1-4

[0076]

[0077] Table 6 (continued) Test results of key indicators of products prepared in Examples 1-5 and Comparative Examples 1-4

[0078]

[0079] Table 6 (continued) Test results of key indicators of products prepared in Examples 1-5 and Comparative Examples 1-4

[0080]

[0081] Experiment Example 2

[0082] The stability of the products prepared in Example 1 and Comparative Examples 1-4 was investigated (temperature 5±3℃, humidity 25%), and key indicators were tested. The test results are shown in Table 7.

[0083] Table 7. Stability test results of the products prepared in Example 1 and Comparative Examples 1-4

[0084]

[0085] Table 7 (continued) Stability test results of the products prepared in Example 1 and Comparative Examples 1-4

[0086]

[0087] Table 7 (continued) Stability test results of the products prepared in Example 1 and Comparative Examples 1-4

[0088]

[0089] Table 7 (continued) Stability test results of the products prepared in Example 1 and Comparative Examples 1-4

[0090]

[0091] Table 7 (continued) Stability test results of the products prepared in Example 1 and Comparative Examples 1-4

[0092]

[0093] The above description is merely a preferred embodiment of this application; however, the scope of protection of this application is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this application, based on the technical solution and its improved concept, should be included within the scope of protection of this application.

Claims

1. A method for preparing cinapeptide phospholipid surfactant liposomes, characterized in that, Includes the following steps: 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, palmitoylglycerol phosphate sodium salt, palmitic acid and cinapeptide; S2. The mixed drug solution is transferred to a thin-film evaporator, and thin-film evaporation is performed under nitrogen gas to remove ethanol; wherein, The conditions for the thin-film evaporation include: a feeding rate of 80–150 mL / min for the mixed drug solution, a jacket temperature of 60–80 °C for the thin-film evaporator, a vacuum degree of -0.85 to -1.00 bar, a rotation speed of 500–2000 rpm for the thin-film evaporator, a nitrogen flow rate of 5–15 L / min, and a stirring speed of 65–75 rpm for the receiving tank.

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 angle of inclination between the thin-film evaporator and the ground is 45° to 60°.

3. The preparation method according to claim 1, characterized in that, In step S2, before the film evaporation is carried out, the temperature of the mixed drug solution 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 to the Tris buffer, the step of heating the Tris buffer to 43–53 °C is further included.

5. The preparation method according to claim 1, characterized in that, In step S1, the mixing operation is performed in a liposome forming vessel, which includes at least three perforated stirring paddles from bottom to top. And / or, the mixing conditions include: a stirring speed of 65-75 rpm and a stirring time of 20-40 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 includes filtering the Tris buffer and the API ethanol solution. Optionally, the Tris buffer solution is filtered at least twice, and the filter has a pore size of 0.2 μm. The API ethanol solution is filtered at least twice, and the filter has a pore size of 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 palmitoylglycerol phosphate sodium salt is 7.14-7.54%, the concentration of palmitic acid is 3.8-4.2%, and the concentration of cinapeptide is 0.7-0.9%.

8. The preparation method according to claim 1, characterized in that, After the film evaporation is completed, the preparation method further includes the step of transferring the evaporated drug solution to a receiving vessel and adding water for injection to determine the weight; The conditions for determining the weight include: the temperature of the drug solution is 38-42 ℃, and the stirring speed is 45-55 rpm.

9. The preparation method according to claim 1, characterized in that, The Tris buffer solution has a pH of 7.55–7.65 and contains tromethamine and sodium chloride, with the tromethamine concentration being 2.4–2.6 mg / mL and the sodium chloride concentration being 7.5–7.8 mg / mL.

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