Freeze-dried phospholipid microbubble contrast agent and preparation method thereof

Abstract

The invention discloses a freeze-dried phospholipid microbubble contrast agent and a preparation method thereof. The freeze-dried phospholipid microbubble contrast agent comprises perfluoropropane, sodium distearoyl phosphatidyl glycerol, dipalmitoyl phosphatidylcholine and an excipient, wherein the molar ratio of the sodium distearoyl phosphatidyl glycerol to the dipalmitoyl phosphatidylcholine is (3.5-4.5): 1. According to the invention, sodium distearoyl phosphatidyl glycerol and dipalmitoyl phosphatidylcholine are used as phospholipid membrane materials to form a stable microbubble structure. The preparation method is investigated and optimized, and the preparation process is optimized and the quality and stability of the product are ensured by screening and researching the variety and dosage of the phospholipid membrane material, the dosage of the tert-butyl alcohol, the dosage of the mannitol, the pH value and the like.

Description

Technical Field

[0001] The invention relates to the technical field of medical ultrasound contrast agents, and in particular to a freeze-dried phospholipid microbubble contrast agent and a preparation method thereof. Background Art

[0002] In the detection and imaging of human microvascular and tissue perfusion, ultrasound detection has the advantages of good imaging effect, real-time, simple operation, no ion radiation, no damage, and wide application. At present, the research and development of ultrasound contrast agents is of great significance to improve the accuracy and reliability of ultrasound detection. Summary of the invention

[0003] The purpose of the present invention is to provide a freeze-dried phospholipid microbubble contrast agent and a preparation method thereof to meet the needs of clinical application.

[0004] To achieve the above purpose, the technical solution provided by the present invention is:

[0005] A freeze-dried phospholipid microbubble contrast agent comprises perfluoropropane, sodium distearoylphosphatidylglycerol (DSPG-Na), dipalmitoylphosphatidylcholine (DPPC) and an excipient; sodium distearoylphosphatidylglycerol and dipalmitoylphosphatidylcholine form a stable microbubble structure; wherein the molar ratio of sodium distearoylphosphatidylglycerol to dipalmitoylphosphatidylcholine is 3.5-4.5:1;

[0006] Preferably, the molar ratio of sodium distearoylphosphatidylglycerol to dipalmitoylphosphatidylcholine is 4:1.

[0007] In order to optimize the above technical solutions, the specific limitations adopted also include:

[0008] Furthermore, the freeze-dried phospholipid microbubble contrast agent contains 28-30 mg of perfluoropropane and 7.5-25.0 mg of excipient per 0.25 g; preferably, it contains 29 mg of perfluoropropane.

[0009] Furthermore, the excipient is mannitol.

[0010] Furthermore, the filling volume of the freeze-dried phospholipid microbubble contrast agent before freeze-drying is 0.25 to 1.0 g.

[0011] The present invention also provides a method for preparing a freeze-dried phospholipid microbubble contrast agent, comprising the following steps:

[0012] Take part of the tert-butyl alcohol aqueous solution in the prescription amount, add the prescription amount of distearoylphosphatidylglycerol sodium (DSPG-Na) and dipalmitoylphosphatidylcholine (DPPC), and stir to dissolve at 56-65°C;

[0013] Add injection water of the same temperature and stir evenly;

[0014] After the drug solution cools down to room temperature, add the prescribed amount of excipients, stir to dissolve, and then add water to the full amount;

[0015] The freeze-dried phospholipid microbubble contrast agent is obtained through filtering, filling, freeze drying and packaging.

[0016] Preferably, the solvent used in the preparation is tert-butanol, which is removed during the freeze-drying process, and the weight proportion of the solvent is 5-15%.

[0017] Specifically, the amount of tert-butanol aqueous solution in the prescription is 45-55% by weight, preferably 50% by weight; the amount of water for injection at the same temperature added is 85-95% by weight, preferably 90% by weight.

[0018] Furthermore, the filtration is to filter the drug solution through a 0.22 μm microporous filter membrane for sterilization.

[0019] The freeze drying process is as follows: the product is put into a box; it is frozen to below -40±2°C and kept warm for 3 hours, a vacuum pump is turned on to evacuate the front box to 20±5Pa, and the temperature is raised and sublimated according to a set curve; perfluoropropane gas is refilled into the freeze drying front box, and the plug is pressed after reaching 1 atmosphere.

[0020] Furthermore, the temperature rise sublimation curve is: the heat transfer oil is set to heat up to -15±2°C in 4h and maintained for no less than 8 hours; the heat transfer oil is set to heat up to 40±2°C in 2h and maintained for 5 to 8 hours.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The freeze-dried phospholipid microbubble contrast agent of the present invention has good stability, adopts distearoylphosphatidylglycerol sodium (DSPG-Na) and dipalmitoylphosphatidylcholine (DPPC) as phospholipid membrane materials to form a stable microbubble structure, and mannitol is used as an excipient to maintain the stability of the preparation.

[0023] The present invention investigates and optimizes the preparation method, optimizes the preparation process by screening and studying the type and amount of phospholipid membrane materials, the amount of tert-butyl alcohol, the amount of mannitol, pH, etc., and ensures the quality and stability of the product.

[0024] The freeze-dried phospholipid microbubble contrast agent of the invention has high safety, and perfluoropropane is used as a common gas source for ultrasound contrast agents, which has high safety.

[0025] The freeze-dried phospholipid microbubble contrast agent of the present invention has a wide range of applications, has important applications in the detection and imaging of human microvessels and tissue perfusion, and helps to improve the accuracy and reliability of ultrasonic detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1: Graph showing the change in resistance of the eutectic point of the freeze-dried phospholipid microbubble contrast agent of Example 7 of the present invention during the cooling process. DETAILED DESCRIPTION

[0027] The above contents of the present invention are further described in detail below in the form of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments, and all technologies realized based on the above contents of the present invention belong to the scope of the present invention.

[0028] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0029] For the sake of simplicity, this document only specifically discloses some numerical values ​​and optional ranges. However, any lower limit can be combined with any upper limit to form an unambiguous range; and any lower limit can be combined with other lower limits to form an unambiguous range. Similarly, any upper limit can be combined with any other upper limit to form an unambiguous range; the optional items in the optional range can also be combined arbitrarily.

[0030] Unless otherwise specified, the terms used in this application have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the parameters mentioned in this application can be measured using various measurement methods commonly used in the art.

[0031] Example 1 Selection of phospholipid membrane material type and dosage

[0032] One of the main purposes of this prescription screening is to obtain a phospholipid microbubble ultrasound contrast agent with a longer liver parenchyma imaging time. Previous studies have shown that the retention of microbubbles in the liver parenchyma is mainly related to the macrophages present in the liver, and positively charged materials are more easily engulfed by macrophages because the cell membrane of macrophages is usually negatively charged. There is an electrostatic attraction between positively charged materials and macrophage membranes, which facilitates the interaction and proximity between the material and the cell membrane, thereby increasing the possibility of engulfment.

[0033] Common negatively charged phospholipids include phosphatidylserine (PS), phosphatidylglycerol (PG), etc.

[0034] Since the solubility of phosphatidylserine (PS) in water is very low, it has a certain solubility in ethanol and methanol. However, during the freeze-drying process, methanol or ethanol does not exist in the freeze-dried layer in a frozen state, but exists in the frozen skeleton in the form of residual liquid. When the ethanol in the prescription is increased to 20%, the cake will collapse and it is almost impossible to dry. Therefore, PG phospholipids were selected.

[0035] Phospholipids with saturated hydrophobic chains are used in the preparation of microbubbles for the following reasons:

[0036] First, saturated hydrophobic chains can provide better stability; the structure of saturated chains is more regular and the interaction between molecules is stronger, making the phospholipid membrane stronger and more stable, which helps to maintain the structural integrity of the microbubbles.

[0037] For example, in the in vivo environment, unsaturated chains are prone to oxidation reactions, resulting in changes in the properties of the phospholipid membrane, while saturated chains can avoid this and allow microbubbles to exist more stably.

[0038] Secondly, saturated hydrophobic chains help control the size and uniformity of microbubbles; their regular structure is conducive to the formation of a uniform phospholipid membrane, thereby obtaining microbubbles of relatively uniform size.

[0039] Furthermore, saturated chain phospholipids generally have more defined and predictable physicochemical properties; this allows for more precise control of process parameters during the preparation of microbubbles, improving the consistency of microbubble quality and performance.

[0040] In summary, the main PG phospholipids that can be used are DPPG and DSPG. Considering that the structure of microbubbles is a bubble structure formed by a phospholipid monolayer membrane wrapping the gas, the length of the phospholipid hydrophobic chain directly affects the difficulty of the internal gas to overflow from the phospholipid membrane, thereby affecting the stability of the microbubbles. In theory, microbubbles composed of the long-chain phospholipid DSPG will have better stability than those composed of the short-chain phospholipid DPPG.

[0041] (1) DSPG-Na dosage screening

[0042] Table 1: DSPG-Na dosage screening

[0043]

[0044]

[0045] Note: Perfluoropropane is charged into the freeze dryer after freeze drying, and is pushed out of the box after reaching 1 atmosphere of pressure.

[0046] In the process of DSPG-Na dosage research, it was found that as the DSPG-Na dosage increased from 40mg to 80mg, its number concentration was positively correlated with the DSPG-Na dosage, and as the DSPG-Na dosage increased from 40mg to 100mg, its volume concentration was positively correlated with the DSPG-Na dosage. As the DSPG-Na dosage increased, its 2-4μm volume percentage increased, and when the DSPG-Na dosage increased to 80mg, it no longer increased. The DSPG-Na dosage is tentatively set at 80mg DSPG-Na / 100 sticks;

[0047] (2) Introducing other types of phospholipids

[0048] When only DSPG-Na is present, both the number concentration and volume concentration of the foaming rate are too low, and it is proposed to introduce other types of phospholipids to improve the foaming rate.

[0049] Table 2: Introduction of other types of prescription compositions

[0050]

[0051]

[0052] Note: Perfluoropropane is charged into the freeze dryer after freeze drying, and is pushed out of the box after reaching 1 atmosphere of pressure.

[0053] Table 3: Foaming rate results of introducing other phospholipids

[0054]

[0055]

[0056] Experimental conclusion:

[0057] Judging from the state of the samples obtained after re-dissolving and shaking, among the 11 samples of Prescription 1 to Prescription 11, except for Prescription 3 and Prescription 9, the other samples had many super-large bubbles visible to the naked eye; the presence of such super-large bubbles will make the quantitative aspiration of the drug solution inaccurate, and may also be dangerous after injection into the human body.

[0058] Among the nine samples with many large bubbles, eight samples except for prescription 6 were samples doped with DPPE-PEG5000, indicating that although the doping of DPPE-PEG5000 can increase the production of microbubbles, it will produce many super-large bubbles. In addition, among the eight samples doped with DPPE-PEG5000, the volume distribution of seven samples (prescription 1, prescription 2, prescription 4, prescription 7, prescription 8, prescription 10, and prescription 11) showed double peaks. The possible reason for this is that the phospholipids did not form a uniform phospholipid membrane when forming the membrane, and some phospholipids formed the membrane alone, resulting in two phospholipid membranes in the solution to form bubbles separately.

[0059] Although DPPE-PEG5000 is used as an excipient in Definity, some problems were found in the experiment (large bubbles are easily generated and the volume distribution is prone to double peaks). In addition, although the PEG polymer contained in DPPE-PEG5000 phospholipids is biocompatible, it is not degraded in the human body. Therefore, considering these aspects, DPPE-PEG5000 is no longer considered as an essential excipient in subsequent prescription screening.

[0060] In the experiment, the two samples of formula 3 and formula 9 that produced less large bubbles were found. The amount of bubbles produced by formula 3 was much greater than that of formula 9. The components of the two were DSPG and DPPC, and DSPG and DSPC, respectively. The main reason for this is that the phase transition temperature of DPPC is lower than that of DSPC (41°C and 55°C, respectively), and the lower phase transition temperature is more conducive to the formation of microbubbles.

[0061] Therefore, after comprehensive consideration, DSPG-Na and DPPC were selected as the further optimized phospholipid combination among these phospholipid combinations. (3) Screening of DSPG-Na and DPPC dosage

[0062] According to the introduction of other types of phospholipids, DSPG-Na and DPPC were selected as the further optimized phospholipid combination, and the effects of DSPG-Na and DPPC in different ratios on the microbubble characteristics were investigated.

[0063] Table 4: DSPG-Na and DPPC dosage screening formula composition

[0064]

[0065]

[0066] Note: Perfluoropropane is charged into the freeze dryer after freeze drying, and is pushed out of the box after reaching 1 atmosphere of pressure.

[0067] Table 5: Results of DSPG-Na and DPPC dosage screening experiment

[0068]

[0069] Experimental conclusion:

[0070] As the amount of DSPG-Na increased and the amount of DPPC decreased, the sample number concentration, volume concentration and volume percentage of 2-4 μm all showed a downward trend.

[0071] As the dosage of DSPG-Na increased and the dosage of DPPC decreased, the microbubbles were more stable in the animals (inspected by longer liver angiography time) and the activated preparations were not easily stratified.

[0072] Considering the foaming rate, the stability of microbubbles in animals and the stratification of samples after activation, the molar ratio of DSPG-Na to DPPC of 4:1 was selected as the optimal ratio.

[0073] Example 2 Screening of tert-butyl alcohol dosage

[0074] Compared with some organic solvents such as methanol, ethanol, isopropanol, acetone, n-butanol, or dioxane (these solvents are not easy to freeze, and unqualified products are obtained due to solvent boiling and agglomeration collapse during sublimation drying), tert-butanol is easy to freeze and can remain frozen in the first stage of drying, and form a loose and porous structure, which reduces the flow resistance of water vapor, which is conducive to the evaporation of water.

[0075] This time, a tert-butanol / water mixed solution was selected as the solvent. This solvent has a relatively low melting point, and the volatility of tert-butanol is not very strong. In addition, it has good solubility for most phospholipids.

[0076] Table 6: Screening of tert-butyl alcohol dosage

[0077]

[0078] Experimental results:

[0079] Phospholipids will not precipitate when the drug solution contains only 5-15% tert-butyl alcohol, but phospholipids with a higher proportion of tert-butyl alcohol will easily precipitate. Note: tert-butyl alcohol is removed during the freeze-drying process.

[0080] Embodiment 3 the dosage selection of mannitol

[0081] Selection of mannitol dosage: The effect of different dosages of mannitol on the microbubble characteristics of the product was investigated, and it was found that the dosage of mannitol had a certain effect on the foaming rate of the product, and when the dosage of mannitol was large, the foaming rate was low. Taking into account the product appearance and foaming rate, the mannitol of Qingdao Mingyue Seaweed Group was selected, and its mannitol concentration was set at 3% to 8%, that is, 7.5 to 25 mg / tube.

[0082] Table 7: Mannitol dosage screening prescription composition

[0083]

[0084]

[0085] Table 8: Mannitol dosage screening test results

[0086]

[0087] From the results of the mannitol dosage screening experiment, it can be seen that when the mannitol dosage is below 2%, the product does not take shape. When the mannitol dosage is 3% to 8%, the product appearance is better, and the foaming rate is higher than 10% to 20%. The mannitol concentration can be set at 3% to 8%, that is, 7.5 to 25 mg / tube.

[0088] Example 4 pH screening

[0089] Considering that the human body's tolerance range for pH is 4 to 9, and phospholipids are easily hydrolyzed under alkaline conditions, the pH range of this product's solution is investigated to be 4.5 to 8.5.

[0090] Table 9: pH screening formulation composition

[0091]

[0092]

[0093] Table 10: pH screening experiment results

[0094]

[0095] The experimental results show that when the pH value of this product is between 4.5 and 8.5, the foaming rate is relatively high at pH 5.5 to 8.5. In order to ensure the quality of the sample, the pH value of this product should be controlled between 5.5 and 8.5.

[0096] Example 5 Preparation Study

[0097] (1) Order of adding raw materials and auxiliary materials

[0098] Experimental description: The raw material of this product is perfluoropropane gas, which is inflated into the box after the liquid medicine is freeze-dried. The materials used in the preparation of the liquid medicine are all auxiliary materials.

[0099] The final preparation sequence is as follows: take the prescription amount of 50% tert-butyl alcohol aqueous solution, add the prescription amount of DSPG-Na and DPPC, stir and dissolve at 56℃~65℃, add water for injection at the same temperature to 90% of the total amount, stir evenly, wait for the liquid to cool to room temperature, add the prescription amount of mannitol, stir and dissolve, and add water to the full amount. The liquid is filled at 0.25g / tube, placed in a freeze dryer for freeze drying, and after freeze drying, filled with perfluoropropane gas, and the rubber plug is pressed out of the box.

[0100] (2) Phospholipid dissolution method

[0101] Take 50% of the prescribed amount of tert-butyl alcohol, add the prescribed amount of phospholipids (DSPG-Na, DPPC), and dissolve at 56°C.

[0102] Reason: tert-Butanol is volatile and has a high freezing point (25.7°C; it will solidify at a slightly lower room temperature). Mix tert-Butanol with an equal amount of water to prevent it from solidifying at room temperature.

[0103] The phase transition temperature of DSPG-Na is usually in the higher temperature region, about 55℃-60℃.

[0104] The phase transition temperature of DPPC is relatively clear, at around 41°C.

[0105] In the preparation process of new drug preparations containing phospholipids, such as liposomes, nanolipid carriers, and nanoemulsions, the phase transition temperature of phospholipids is a very important parameter. Film formation is the most important factor in the formation of preparations, and the film formation temperature must be higher than the phase transition temperature of phospholipids. Therefore, this study investigated the effect of phospholipid dissolution temperature (35℃, 45℃, 56℃) on the product.

[0106] Table 11: Phospholipid dissolution temperature study

[0107]

[0108] The sample quantity concentration and volume concentration are both high at 56°C, and this product is intended to dissolve phospholipids at 56°C.

[0109] (3) Product nitrogen filling research

[0110] Experimental description: Investigate the effect of nitrogen filling and non-nitrogen filling on the liquid medicine of this product, to confirm whether the liquid medicine needs nitrogen filling protection during the filling process. In addition, place the sample for 8 hours to investigate the stability of the liquid medicine.

[0111] Experimental method: Prepare the liquid medicine according to the prescription process, filter it through a 0.22μm microporous membrane, and divide it into two parts after preparation, one part is filled with nitrogen for protection, and the other part is not filled with nitrogen for protection. Place it at a constant temperature of 25℃, and take samples for determination at 0h, 2h, 4h, 6h, and 8h. The experimental results are shown in Table 12.

[0112] Table 12: Solution stability study and the effect of nitrogen filling on the solution

[0113]

[0114] Experimental conclusion:

[0115] There is no obvious difference between the liquid medicine filled with nitrogen and not filled with nitrogen. During the filling process, the liquid medicine does not need nitrogen protection.

[0116] There is no obvious change in the color, clarity, pH and related substances of the solution within 8 hours. The solution of this product is stable within 8 hours.

[0117] Example 6 Filtration Study

[0118] Experimental overview: The filter membrane materials are generally: PTFE (polytetrafluoroethylene), water-based PES (polyethersulfone), water-based MCE (mixed cellulose ester), organic nylon 6, organic nylon 66, PVDF (polyvinylidene fluoride). The commonly used filter membrane materials in the filter element on the market are: PTFE, PVDF, PES. Now we will investigate these three filter membranes to provide a basis for the selection of filter membranes.

[0119] Experimental method: The liquid medicine was prepared according to the prescription process, divided into three parts, and filtered through PTFE, PVDF, and PES filter membranes (all 0.22μm) respectively to examine the changes in product properties, pH, and phospholipid content before and after filtration.

[0120] Table 13: Filter membrane selection study

[0121]

[0122] Experimental results: Among the three filter membranes, PES and PTFE have no obvious adsorption, PVDF may have a small amount of adsorption, and the pH of the drug solution has no obvious change before and after filtration. Since PTFE takes a long time to filter, PES filters faster. PES can be selected as the filter material for this batch.

[0123] Example 7 Freeze-drying process study

[0124] According to the measurement of the eutectic point of the liquid medicine, the eutectic point of this product is about -11°C. During the experiment, the temperature of the product is lowered, and the eutectic point of the product is determined by the change in the resistance of the liquid medicine during the cooling process. Figure 1 shown.

[0125] Freeze drying is generally divided into the following steps

[0126] (1) Pre-freezing

[0127] In general, the product is pre-frozen at 20°C below the eutectic point, usually at -40°C.

[0128] (2) Primary drying

[0129] The primary drying temperature should be lower than the eutectic point of the product. The primary drying temperature of this product can be set to -15°C. The filling volume of this product is only 0.25g / tube, which is packaged in a 2ml vial. The height of the drug solution is relatively low. According to the general lyophilization time of 1mm for 1h, the primary drying time of this product can be set to 5-8h.

[0130] (3) Secondary drying

[0131] This product is intended to be stored at room temperature. The secondary drying can be set at an accelerated temperature under room temperature, i.e. 40°C. The secondary drying time is mainly determined by the residual moisture of the product. Generally, the residual moisture of freeze-dried preparations is ≤5%.

[0132] (4) Press the plug out of the box

[0133] According to the above theoretical research, the initial freeze-drying parameters are set:

[0134] Table 14: Freeze-drying curve study

[0135]

[0136]

[0137] Freeze-drying curve 1: The non-uniformity of the freeze-drying plate layer. Some samples were not completely dried at one time, resulting in the collapse of some samples.

[0138] Freeze-drying curves 2 and 3: All samples were white loose lumps. The primary drying of the products was completed, and the secondary drying was 5 to 8 hours. The moisture content of the products was <5%, and the residual tert-butyl alcohol was <0.05%.

[0139] The freeze-drying curve of this product can be set as follows:

[0140] The product is put into the box, frozen to below -40±2℃ and kept warm for 3 hours, the vacuum pump is turned on to evacuate the front box to 20±5Pa, and the temperature is raised and sublimated according to the following curve:

[0141] Set the temperature of the heat transfer oil to -15±2℃ in 4h and maintain it for no less than 8 hours;

[0142] Set the temperature of the heat transfer oil to 40±2℃ within 2h and maintain it for 5 to 8 hours;

[0143] Fill the freeze-drying box with perfluoropropane gas, and press the plug after reaching 1 atmosphere. Tighten the lid after taking out of the box. Pack.

[0144] Example 8

[0145] A freeze-dried phospholipid microbubble contrast agent, during the preparation process, strictly controls the operating conditions and parameters of each step to ensure the quality and stability of the product.

[0146] Table 15: Example 8 prescription composition table

[0147]

[0148]

[0149] A freeze-dried phospholipid microbubble contrast agent, the preparation process is as follows:

[0150] (1) Preparation before preparation: Preparation of 50% tert-butyl alcohol aqueous solution (g / g): Take a certain amount of tert-butyl alcohol and an equal amount of water and mix them evenly for use.

[0151] (2) Preparation of drug solution: Take 50% of the prescribed amount of tert-butyl alcohol, add the prescribed amount of DSPG-Na and DPPC, and stir to dissolve at 56-65°C. After the phospholipid is dissolved, add water of the same temperature to 90% of the total amount. After the drug solution is cooled, add the prescribed amount of mannitol, stir to dissolve, and measure the pH.

[0152] (3) Sterile filtration: After the drug solution passes through two 0.2 μm microporous filter membranes, the intermediate is taken for inspection.

[0153] (4) Filling: Determine the filling volume based on the intermediate phospholipid content, fill into 2 mL borosilicate controlled injection bottles, half-fill with rubber stoppers, place on trays, and send to the freeze dryer for freeze drying according to the freeze drying procedure.

[0154] (5) Freeze drying:

[0155] The product is put into the box, frozen to below -40±2℃ and kept warm for 3 hours, the vacuum pump is turned on to evacuate the front box to 20±5Pa, and the temperature is raised and sublimated according to the following curve:

[0156] Set the temperature of the heat transfer oil to -15±2℃ in 4h and maintain it for no less than 8 hours;

[0157] Set the temperature of the heat transfer oil to 40±2℃ in 2h and maintain it for 6 hours;

[0158] Fill the freeze-drying box with perfluoropropane gas, and press the plug after reaching 1 atmosphere. Tighten the lid after taking out of the box. Pack.

[0159] Prepare three batches of samples to study product quality

[0160] Table 16: Batch Analysis Summary

[0161]

[0162]

[0163] In summary, the freeze-dried phospholipid microbubble contrast agent of the present invention has good stability, wide application, optimized preparation method and high safety.

[0164] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession, without departing from the scope of the technical solution of the present invention, according to the technical essence of the present invention, any simple modification, equivalent replacement and improvement made to the above embodiment still falls within the protection scope of the technical solution of the present invention.

Claims

1. A freeze-dried phospholipid microbubble contrast agent, characterized in that: The invention comprises perfluoropropane, sodium distearoylphosphatidylglycerol, dipalmitoylphosphatidylcholine and excipients, wherein the sodium distearoylphosphatidylglycerol and dipalmitoylphosphatidylcholine form a stable microbubble structure; wherein the molar ratio of sodium distearoylphosphatidylglycerol to dipalmitoylphosphatidylcholine is 3.5-4.5:

1.

2. The freeze-dried phospholipid microbubble contrast agent according to claim 1, characterized in that: The freeze-dried phospholipid microbubble contrast agent contains 28-30 mg of perfluoropropane and 7.5-25.0 mg of excipients per 0.25 g.

3. The freeze-dried phospholipid microbubble contrast agent according to claim 1, characterized in that: The excipient is mannitol.

4. The freeze-dried phospholipid microbubble contrast agent according to claim 1, characterized in that: The filling volume of the freeze-dried phospholipid microbubble contrast agent before freeze-drying is 0.25 to 1.0 g.

5. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: Take part of the tert-butyl alcohol aqueous solution in the prescription amount, add the prescription amount of distearoylphosphatidylglycerol sodium (DSPG-Na) and dipalmitoylphosphatidylcholine (DPPC), and stir to dissolve at 56-65°C; Add injection water of the same temperature and stir evenly; After the drug solution cools down to room temperature, add the prescribed amount of excipients, stir to dissolve, and then add water to the full amount; The freeze-dried phospholipid microbubble contrast agent is obtained through filtering, filling, freeze drying and packaging.

6. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to claim 5, characterized in that: The solvent used in the preparation is tert-butyl alcohol, which is removed during the freeze-drying process, and the weight proportion of the solvent is 5-15%.

7. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to claim 5, characterized in that: The part of the tert-butanol aqueous solution in the prescription is 45-55% of the weight of the tert-butanol aqueous solution in the prescription; and the injection water at the same temperature is added to 85-95% of the total weight.

8. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to claim 5, characterized in that: The filtration is to filter the drug solution through a 0.22 μm microporous filter membrane for sterilization.

9. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to claim 5, characterized in that: The freeze drying process is as follows: the product is put into a box; it is frozen to below -40±2°C and kept warm for 3 hours, a vacuum pump is turned on to evacuate the front box to 20±5Pa, and the temperature is raised and sublimated according to a set curve; perfluoropropane gas is refilled into the freeze drying front box, and the plug is pressed after reaching 1 atmosphere.

10. The method for preparing the freeze-dried phospholipid microbubble contrast agent according to claim 9, characterized in that: The temperature rise sublimation curve is: set the heat transfer oil to heat up to -15±2℃ in 4h and keep it for no less than 8 hours; set the heat transfer oil to heat up to 40±2℃ in 2h and keep it for 5 to 8 hours.

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