Freeze-dried formulations for aerated microbubbles

Abstract

A freeze-dried powder composition comprising a phospholipid and polyethylene glycol, the polyethylene glycol having a percentage of folded polymer chains above a predetermined limit. The composition is suitable for preparing aerated microbubbles.

Description

[0001] This application is a divisional application of the Chinese patent application with an application date of July 5, 2019, application number 201980035252.9, and invention name “Lyophilized preparation for inflated microbubbles”. Technical Field

[0002] The present invention relates to the preparation of gas-filled microbubble formulations for use in ultrasound contrast imaging. Background Art

[0003] The rapid development of contrast agents in recent years has resulted in many different compositions and formulations that can be used for contrast-enhanced imaging and therapy of human or animal organs and tissues.

[0004] One class of contrast agents particularly useful for contrast-enhanced ultrasound imaging ("CEUS" imaging) comprises a suspension of nano- and / or micron-sized gas bubbles dispersed in an aqueous medium. The gas is typically entrained or encapsulated in a membrane layer that includes, for example, an emulsifier, oil, thickener, or sugar. These stable gas bubbles (dispersed in a suitable physiological solution) are commonly referred to in the art by various terms, depending on the stabilizing material used to prepare them, including, for example, "microspheres," "microbubbles," "microcapsules," or "microballoons," but are collectively referred to herein as "gas-filled microbubbles" (or "microbubbles").

[0005] Ultrasound contrast agents ("USCAs") are produced using various manufacturing methods. One method, described, for example, in WO 94 / 09829(i), involves dissolving a mixture of membrane-forming components (e.g., phospholipids and / or fatty acids) and a hydrophilic stabilizing compound (e.g., polyethylene glycol) in an organic solvent; the resulting mixture is then filled into vials and freeze-dried (lyophilized). The vials, which contain a solid lyophilized solid residue (dry cake) at the bottom, are then filled with a suitable gas (e.g., a fluorinated gas) and finally sealed for storage. Prior to use, an aqueous suspension of microbubbles can be easily prepared by injecting a suitable liquid (e.g., saline) into the vial and shaking it to dissolve the solid residue.

[0006] Commercially available USCAs that can be prepared as described above are available from Bracco. (or American ).

[0007] The applicant has now observed that variations in the properties of the polyethylene glycol used in the preparation of the lyophilized "dry cake" may negatively affect the number of gas-filled microbubbles obtained upon reconstitution of the lyophilized powder.

[0008] Specifically, the applicants have observed that different batches of commercially available PEG 4000 (even from the same manufacturer) can have different numbers of folded polymer chains in the polymer material. As the applicants have observed, if the percentage of such folded chains in the polymer material is too low, this can result in an excessive number of vials failing acceptability testing in a production batch. Since a production batch on an industrial scale can contain thousands of vials, discarding even a relatively small number of vials is highly undesirable.

[0009] Based on the above observations, the applicants determined that the polyethylene glycol used in the lyophilized powder formulation for preparing gas-filled microvesicles should have a percentage of folded polymer chains above a predetermined value. Summary of the Invention

[0010] One aspect of the present invention relates to a lyophilized powder composition for preparing gas-filled microvesicles, the composition comprising a phospholipid and polyethylene glycol, wherein the polyethylene glycol has a folded polymer chain percentage greater than 34%, preferably greater than 35%.

[0011] The percentage of folded chains is preferably at least 40%, more preferably at least 42%, even more preferably at least 44%. In a particularly preferred embodiment, the percentage of folded chains is at least 48%.

[0012] In a preferred embodiment, the polyethylene glycol has an average molecular weight (or number average molecular weight, Mn) of at least 4000 g / mol (or Dalton, Da), more preferably at least 4025 g / mol and even more preferably at least 4050 g / mol.

[0013] In another preferred embodiment, the phospholipid is DSPC, DPPG-Na or (preferably) a mixture thereof.

[0014] The composition preferably further comprises a fatty acid, preferably palmitic acid.

[0015] In another aspect of the present invention, the present invention relates to a sealed vial comprising the above composition in contact with a physiologically acceptable gas.

[0016] The gas is preferably fluorinated gas, more preferably sulfur hexafluoride.

[0017] According to another aspect, the present invention relates to a suspension of gas-filled microbubbles obtained by dispersing said lyophilized powder composition in a physiologically acceptable liquid, preferably a 0.9% w / v NaCl solution, in the presence of a gas.

[0018] According to another aspect, the present invention relates to a method for preparing a lyophilized composition comprising a phospholipid, polyethylene glycol and optionally a fatty acid, said method comprising:

[0019] a. dissolving the phospholipid, optionally a fatty acid and polyethylene glycol in a solvent;

[0020] b. freezing the solution, and

[0021] c. removing the solvent by lyophilization;

[0022] The percentage of folded polymer chains of the polyethylene glycol is 40% or higher.

[0023] According to another aspect, the present invention relates to a method of ultrasound imaging, comprising:

[0024] d. administering to the patient an effective amount of the suspension of gas-filled microbubbles;

[0025] e. sending an ultrasound signal to the patient's body part;

[0026] f. Collecting echo signals from the body part. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Examples of a "smooth" lyophilized cake (1a) and a "rough" lyophilized cake (1b) are given;

[0028] Figure 2 The graph depicts the relationship between the percentage of folded polymer chains and the number of rejected vials per production batch;

[0029] Figure 3 A descriptive MDSC thermogram of the second heating cycle of the polyethylene glycol sample is shown. DETAILED DESCRIPTION

[0030] (or ) is a sterile, pyrogen-free, lyophilized powder formulation stored in septum vials. The lyophilized powder contains polyethylene glycol 4000 (PEG4000, 24.56 mg), distearoylphosphatidylcholine (DSPC, 0.19 mg), sodium dipalmitoylphosphatidylglycerol (DPPG-Na, 0.19 mg), and palmitic acid (0.04 mg). The headspace of each vial is filled with sulfur hexafluoride (SF6). After reconstitution with 5 mL of sterile saline, SonoVue / Lumason becomes a milky white, homogeneous suspension containing microbubbles (also called "microspheres" or "microbubbles") filled with sulfur hexafluoride.

[0031] Lyophilized powders can be prepared using the process described in the aforementioned patent application WO 94 / 09829. Generally, this method requires dissolving PEG 4000, phospholipids, and palmitic acid in a suitable solvent (such as dioxane, cyclohexanol, 2-methyl-2-butanol, tetrachlorodifluoroethane, or tert-butanol) in weight ratios substantially corresponding to their intended use in the final lyophilized product. For example, the solution may contain 22-28 parts by weight of polyethylene glycol, 0.15-0.25 parts by weight of DSPC, 0.15-0.25 parts by weight of DPPG-Na, and 0.02-0.06 parts by weight of palmitic acid. The resulting solution is then filled into glass vials, which are rapidly frozen (e.g., at -45°C) and then subjected to the lyophilization process. On an industrial scale, each production batch typically includes several thousand vials. At the conclusion of the freeze-drying step, the headspace of the vials containing the lyophilized residue (in the form of a solid cake) is saturated with SF6 gas, and the vials are sealed with rubber stoppers. The vials thus obtained can be stored for at least two years.

[0032] However, as observed by the applicant, the use of PEG with a folded chain content below a predetermined limit may negatively affect the quality of the lyophilized cake in the vials of a production batch.

[0033] For example, as Ginés et al. 2 As mentioned above, polyethylene glycol (PEG) is a semicrystalline hydrophilic polymer that contains varying proportions of amorphous and ordered crystalline phases in the solid state, depending on its synthesis and thermal history. In the crystalline region, the polymer chains exist as both extended and folded chains, particularly in PEG with a molecular weight of approximately 4000-6000 g / mol. As observed by Ginés et al., the folded chain content in PEG 4000 is generally higher when the molten sample is cooled at room temperature compared to when the same sample is quenched in an ice bath or immersed in liquid nitrogen.

[0034] As observed by the applicants, the folded chain content of PEG materials (typically PEG 4000) used as stabilizing compounds in formulations for preparing gas-filled microbubbles can vary significantly from one industrial batch to another, even for materials with the same nominal molecular weight (i.e., 4000 g / mol), even if they come from the same manufacturer.

[0035] The applicants have now surprisingly discovered that the use of PEG having too low a percentage of folded polymer chains can negatively impact the quality of the lyophilized cake contained in a batch of vials manufactured using such PEG. Specifically, if the percentage of folded polymer chains in the PEG falls below a predetermined limit, the number of reject vials in the batch (i.e., vials whose lyophilized cake does not meet predetermined specifications) becomes excessively high.

[0036] Among the various criteria for determining the quality of a lyophilized cake, an important one is the physical properties of the cake. In particular, the applicant has determined that there is a relatively good correlation between the physical properties of the cake and the quality of the gas-filled microbubble suspension produced by reconstitution of the cake with a physiologically acceptable liquid. As the applicant has observed, a cake having a relatively smooth surface appearance (e.g., see Figure 1 a) generally provide more microbubbles upon reconstitution, whereas those with a relatively rough surface appearance (e.g. see Figure 1 b) significantly fewer microbubbles are formed upon reconstitution. Furthermore, as observed by the present applicants, microbubble suspensions formed from dry cakes with relatively smooth surfaces have higher MVCs (microbubble volume concentration, i.e., the total amount of gas contained in the reconstituted microbubbles per mL of suspension). Typically, due to the aforementioned lower mass, gas-filled microbubble suspensions obtained from reconstitution of rough dry cakes are less effective for CEUS imaging.

[0037] In particular, as observed by the applicant, when the percentage of folded chains is 34% or less of the total polymer chains, e.g. Figure 2 As shown, the number of rejected vials (containing "coarse" dry cake) is typically greater than 16%. Applicants have therefore determined that to reduce the number of rejected vials, PEG with a folded chain percentage greater than 34%, preferably greater than 35%, should be used. To further reduce the rejection rate of vials, the folded chain percentage in the PEG is preferably at least 40% of the total polymer chain. More preferably, the folded polymer chain percentage should be greater than 42%, and even more preferably greater than 44%, with 48% being particularly preferred. While there is generally no upper limit to the folded chain percentage (typically, PEG with a nominal Mn of about 6000 g / mol can have a folded chain percentage of up to about 100%), PEG with a nominal Mn of about 4000 (e.g., up to 4600 g / mol) typically has a folded chain percentage of less than 80%, and typically 75% or less. The folded chain percentage is preferably about 70% or less, more preferably about 65% or less, and even more preferably 60% or less.

[0038] Determination of the percentage of folded chains

[0039] The percentage of folded chains in a polymeric material can be determined by methods known in the art, preferably by differential scanning calorimetry (DSC). A preferred method (also used in the following examples) is modulated differential scanning calorimetry (MDSC) performed, for example, using a DSC-Q2000 system (TA instruments, New Castle, DE USA).

[0040] Details of the MDSC method are described in the working examples.

[0041] Briefly, the sample was put into a heating / cooling / heating cycle by applying a temperature modulation amplitude (eg, 0.16°C per 30 seconds) within a predetermined temperature range (eg, 20-70°C) at a constant temperature rate (eg, 2°C / min).

[0042] During the second heating cycle, the fraction of polymer material present in the folded form can be characterized. In fact, polyethylene glycol has the inherent characteristic of splitting into two peaks during the second heating cycle, the peak with the lower melting temperature corresponding to the melting of PEG with folded chains, and the peak with the higher melting temperature corresponding to the melting of PEG with unfolded chains. Figure 3 A typical MDSC thermogram of PEG is shown, wherein the first and second melting peaks obtained during the second heating cycle are identified. Specifically, Figure 3 Peak A (at about 57° C.) corresponds to the melting of PEG with a folded chain, while peak B (at about 61° C.) corresponds to the melting of PEG with an unfolded chain. The area of each peak corresponds to the fraction of each PEG with a folded or unfolded chain.

[0043] Therefore, the fraction of polymer chains that are folded, f(folded), can be determined as follows:

[0044] Formula 1

[0045]

[0046] Among them A 折叠 and A 未折叠 The normalized heat flow integrals (melting enthalpy, joules per gram of sample) representing the folded and unfolded PEG forms are calculated as the area under each peak of the MDSC thermogram of the PEG sample during the second heating cycle. Therefore, the percentage of folded polymer chains in the PEG sample is: f(folded) x 100.

[0047] As shown in the experimental section, although there is a certain correlation between the percentage of folded chains and the average molecular weight of polyethylene glycol (generally, the larger the molecular weight of the polymer, the greater the percentage of folded chains), this correlation is not necessarily linear. This finding suggests that although molecular weight plays a role in the number of folded chains, it is not the only determining factor, as the number of folded chains can also be affected by the thermal history of the polymer sample (for example, including parameters of the sample production / cooling process, storage conditions or aging). However, the applicant has observed that the average molecular weight (or number average molecular weight, Mn) of the polyethylene glycol is preferably at least 4000 g / mol, more preferably at least 4025 g / mol and even more preferably at least 4050 g / mol. Particularly preferred are polyethylene glycols with an Mn of 4075 or higher (e.g., 4100 or higher). Although in principle there are no higher Mn values that affect the number of folded chains, the molecular weight of the polymer should be compatible with the viscosity of the final suspension of gas-filled microbubbles. Therefore, it is preferred that the Mn is 6200 g / mol or lower, more preferably 5000 g / mol or lower, and even more preferably 4400 g / mol or lower.

[0048] The molecular weight of polyethylene glycol can be determined conventionally, preferably by hydroxyl value titration (OHV), for example according to DIN 53240. The average molecular weight (or number average molecular weight, Mn) of PEG can then be easily calculated from the OHV, for example: Mn=112'220 / OHV.

[0049] Determination of dry cake appearance

[0050] The physical appearance of the dry cake can be checked visually. Visual inspection is preferably performed by using a semi-automatic inspection machine with a suitable lighting and mirror system (e.g., Seidenader M10063 semi-automatic machine). In practice, the dry cake is illuminated from the top with a suitable lighting system, while the bottom of the dry cake is observed for its transparency through a mirror below. The operator thus checks the appearance of the dry cake as shown on the mirror and determines whether the vial is acceptable or unacceptable based on the following criteria. A freeze-dried cake that passes the acceptance test has a substantially smooth appearance ("smooth dry cake"), a uniform surface, and a plurality of crystalline structures throughout the dry cake (see Figure 1 a). On the other hand, the dry cakes that failed the acceptance test had a noticeably rough appearance ("rough dry cake") with cracks and / or large spots appearing in the dry cake (see Figure 1 b).

[0051] Using this acceptance test, the applicants have determined, as described in detail in the Examples below, that the number of rejected vials in a production batch is inversely proportional to the percentage of folded chains in the polyethylene glycol used to prepare each batch.

[0052] When tested by reconstitution with saline, the smooth dry cake generally formed more microbubbles in suspension and the volume of gas contained in the microbubbles was higher.

[0053] The reconstituted suspension may be administered to a patient during a routine CEUS procedure, in which the patient or a body part is submitted for ultrasound examination and echo signals are collected and analyzed.

[0054] The following examples help to further describe the present invention.

[0055] Example

[0056] Example 1

[0057] Determination of the percentage of folded chains by MDSC

[0058] a. Calibration of equipment and systems

[0059] All MDSC experiments were performed in a Tzero TM technology (which can directly measure heat capacity) and The temperature was measured using a DSC-Q2000 system (TA Instruments, New Castle, DE USA) with the TA2000 option (which allows for the superposition of sinusoidal temperature fluctuations on a conventional linear temperature ramp).

[0060] Conveniently operate at temperatures from -90°C to 550°C using the refrigerated cooling attachment (RCS90) with a two-stage refrigeration system.

[0061] Data acquisition and processing were performed with the help of the Universal Analysis Software package.

[0062] A Tzero aluminum crucible (ref 901683.901) and a Tzero aluminum lid (ref 901671.901) (both obtained from TA instruments) were used to hold the sample to be tested, and the crucible was sealed by a Tzero press (ref 901600.901).

[0063] The DSC system was calibrated using metallic indium, including temperature and heat flow. In practice, approximately 5 mg of indium was weighed, flattened, and transferred to a Tzero crucible, which was then sealed with a Tzero lid using a Tzero press. The calibration scan was performed between 100°C and 180°C at a constant temperature rate of 10°C / min. The specifications for indium were as follows: melting enthalpy of 28.71 J / g ± 0.5 J / g and melting onset temperature of 156.6 ± 0.25°C, respectively.

[0064] b. Sample Preparation and MDSC Measurement

[0065] Four different batches of PEG4000 were characterized as follows.

[0066] The PEG4000 flakes of each sample were crushed with a pestle, and 5 mg ± 0.1 mg of the sample was weighed in a Tzero crucible using a microbalance XP26 (Mettler Toledo), and the crucible was then sealed with a Tzero lid by a Tzero press. An empty Tzero crucible of the same weight was similarly prepared and used as a reference compared to the empty sample crucible.

[0067] As summarized in Table 1 below, MDSC measurements were performed on each crucible containing a PEG4000 sample by performing a heating / cooling / heating cycle at a constant temperature rate of 2°C / min within the temperature range of 20-70°C. Only a heating modulation signal was applied (temperature modulation amplitude of 0.16°C per 30-second temperature modulation cycle). Nitrogen was used as a purge gas at a flow rate of 50 ml / min.

[0068] Table 1: MDSC heating / cooling / heating cycle

[0069]

[0070]

[0071] The following parameters were determined on the MDSC thermogram using Universal Analysis software:

[0072] - Peak temperature and melting enthalpy of the first heating cycle;

[0073] - the peak temperature and crystallization enthalpy of the first cooling cycle; and

[0074] - Peak temperature of the second heating cycle and melting enthalpy of folded and extended chains.

[0075] During the second heating cycle, the percentage of polymer material present in the folded form was characterized and calculated according to Equation 1. This process was repeated for three different samples for each batch of PEG 4000. Details of the measurements and results are given in Tables 2-5 below.

[0076] Table 2: Main characteristics of MDSCs of PEG4000 batch 1 (comparative example)

[0077]

[0078] Table 3: Main characteristics of MDSCs from the second batch of PEG4000

[0079]

[0080] Table 4: Main characteristics of MDSCs from batch 3 of PEG4000

[0081]

[0082] Table 5: Main characteristics of MDSCs from batch 4 of PEG4000

[0083]

[0084] Table 6 below summarizes the MDSC measurement results for various PEG batches and their respective number average molecular weights (Mn) as provided by the supplier.

[0085] Table 6: Folded chain % and average Mn of PEG4000

[0086] PEG4000 batch number Folded chain% Mn (g / mol) Batch 1 (comparative example) 34 3997 Batch 2 43 4107 Batch 3 49 4159 Batch 4 54 4154

[0087] The results reported in Tables 2-6 show that batch 1 (only 34% of folded polymer chains) does not meet the requirements of the present invention, while the other three batches of PEG 4000 meet these requirements. In addition, it can be observed that the Mn of batch 1 is less than 4000, while the Mn of all other batches is greater than 4000 g / mol, specifically at least 4050 g / mol.

[0088] Example 2

[0089] Preparation of freeze-dried cake

[0090] The process for preparing the lyophilized cake essentially follows the description in the examples of WO 94 / 09829. Briefly, DSPC, DPPG-Na, and PA were first dissolved in hexane / ethanol (8 / 2, v / v) at a concentration of approximately 5 g / L in a weight ratio of 4.75 / 4.75 / 1, and the solvent was evaporated under vacuum. The residue was mixed with PEG4000 at a weight ratio of approximately 0.017:1, the mixture was dissolved in tert-butyl alcohol at approximately 60°C, and the transparent solution was filled into individual DIN8R vials (each volume containing approximately 25 mg of the mixture). The vials were then rapidly cooled at -45°C before the final freeze-drying step. At the end of the freeze-drying process, the ambient conditions of the lyophilized product were saturated with SF6 at normal pressure, and the vials (containing the solid lyophilized cake in contact with SF6) were sealed with rubber stoppers.

[0091] The above preparation process was carried out using each of the four batches of PEG4000 (Batches 1-4) characterized in Example 1, thereby obtaining four batches (Batches 1-4, respectively) of several thousand vials each, each containing 25 mg of freeze-dried solid material in the form of a dry cake at its bottom.

[0092] Example 3

[0093] Vial / dry cake inspection

[0094] Each batch obtained by the preparation method described in Example 2 was checked as follows to check whether there was any unqualified lyophilized cake.

[0095] According to the process described above, each batch of vials was inspected using a Seidenader M10063 semi-automatic machine and classified as "acceptable vials" containing a smooth dry cake (SCV, e.g. Figure 1 a) or "rejected vials" containing rough dry cake (RCV, such as Figure 1 b).

[0096] The inspection results implemented according to the above process are shown in Table 7 below and recorded in Figure 2 middle.

[0097] Table 7: Relationship between the number of unqualified vials and the % folded chains in PEG4000

[0098] Production batch % of unqualified vials Folded chain % in PEG4000 Batch 1 (comparative example) 16 34 Batch 2 8.9 43 Batch 3 5.6 49 Batch 4 3.7 54

[0099] From the results shown in Table 7, it can be inferred that the percentage of defective vials in production batch 1 (in which the composition had only 34% folded chains in the PEG4000) was higher than the defective amounts in other batches of the present invention made using PEG4000 with a higher folded chain percentage.

[0100] Example 4

[0101] Reconstituted vial of gas-filled microbubble suspension

[0102] Six vials were sampled from each set of "acceptable vials" and "rejected vials" (as defined above in Example 3) to characterize the microvesicles obtained by reconstitution of the dry cake contained in said vials.

[0103] Various parameters of the gas-filled microbubble suspension, such as MVC (microbubble volume concentration) and the total number of microbubbles in the suspension, were measured using a Coulter Counter Multisizer 3 with a 30 μm pore tube. Briefly, 50 μL of the microbubble suspension was diluted in 100 mL of 0.9% NaCl solution using an analysis volume of 100 μL.

[0104] The measurement results (average values for each group of vials) are given in Table 8 below.

[0105] Table 8: Characteristics of gas-filled microbubbles prepared from smooth or rough dry cakes

[0106] Vial Type MVCμL / mL Microbubble concentration (ml / mL) Acceptable (smooth dry cake) 6.4 <![CDATA[4.38×10 8 ]]> Unqualified (rough dry cake) 3.2 <![CDATA[3.87×10 8 ]]>

[0107] From the results shown in Table 8, it can be inferred that the microbubbles obtained by reconstitution of the smooth dry cake showed a larger total volume of gas entrained in the microbubbles (specifically, twice as much), and there were also more microbubbles in the suspension (specifically, 0.5×10 8 ).

[0108] References

[0109] 1 International Patent Application WO 94 / 09829(Bracco International)

[0110] 2 Gines et al., "ThermalCharacterization Of Polyethylene GlycolsApplied in the Pharmaceutical Technology Using Different Scanning Calorimetryand Hot Stage Microscopy", Journal of Thermal Analysis, Vol. 46 (1996) 291-304.

[0111] In particular, the present invention also relates to the following items:

[0112] Item 1. A freeze-dried powder composition for preparing gas-filled microbubbles, the composition comprising phospholipids and polyethylene glycol, wherein the percentage of folded polymer chains of the polyethylene glycol is greater than 34%.

[0113] Item 2. The composition of Item 1, wherein the percentage of the folded chains is at least 40%.

[0114] Item 3. The composition of Item 1, wherein the percentage of the folded chains is at least 42%.

[0115] Item 4. The composition of Item 1, 2 or 3, wherein the polyethylene glycol is PEG4000 having a number average molecular weight (Mn) of at least 4000 g / mol.

[0116] Item 5. The composition of Item 4, wherein the Mn is at least 4025 g / mol.

[0117] Item 6. The composition of any one of Items 1-5, wherein the phospholipid is DSPC, DPPG-Na or a mixture thereof.

[0118] Item 7. The composition of any one of Items 1-6, wherein the composition further comprises a fatty acid.

[0119] Item 8. The composition of Item 7, wherein the fatty acid is palmitic acid.

[0120] Item 9. The composition of Item 8, comprising 22-28 parts by weight of PEG4000, 0.15-0.25 parts by weight of DSPC, 0.15-0.25 parts by weight of DPPG-Na and 0.02-0.06 parts by weight of palmitic acid.

[0121] Item 10. The composition of Item 9, comprising 24.56 mg of PEG4000, 0.19 mg of DSPC, 0.19 mg of DPPG-Na and 0.04 mg of palmitic acid.

[0122] Item 11. A sealed vial comprising the lyophilized powder composition of any one of items 1-10 in contact with a physiologically acceptable gas.

[0123] Item 12. The sealed vial of Item 11, wherein the gas is fluorinated gas.

[0124] Item 13. A method for producing a freeze-dried composition comprising a phospholipid and polyethylene glycol, comprising:

[0125] a. dissolving the phospholipid and the polyethylene glycol in a solvent to obtain a solution;

[0126] b. freezing the solution; and

[0127] c. removing the solvent by lyophilization;

[0128] The percentage of folded polymer chains of the polyethylene glycol is greater than 34%.

[0129] Item 14. The method of Item 13, wherein the composition further comprises a fatty acid, and step a further comprises dissolving the fatty acid in the solvent.

[0130] Item 15. A gas-filled microbubble suspension obtained by dispersing the lyophilized powder composition of any one of Items 1 to 10 in a physiologically acceptable liquid in the presence of a gas.

Claims

1. A lyophilized powder composition in the form of a lyophilized cake contained in a vial for preparing gas-filled microvesicles, the composition comprising 22-28 parts by weight of polyethylene glycol, 0.15-0.25 parts by weight of DSPC, 0.15-0.25 parts by weight of DPPG-Na, and 0.02-0.06 parts by weight of palmitic acid, wherein the polyethylene glycol has a number average molecular weight Mn of at least 4050 g / mol.

2. The composition of claim 1, wherein the polyethylene glycol has a number average molecular weight Mn of at least 4075 g / mol.

3. The composition of claim 1, wherein the polyethylene glycol has a number average molecular weight Mn of at least 4100 g / mol.

4. The composition of claim 1, wherein the polyethylene glycol has a number average molecular weight Mn of at most 4400 g / mol.

5. The composition according to claim 1, comprising 24.56 mg of PEG4000, 0.19 mg of DSPC, 0.19 mg of DPPG-Na and 0.04 mg of palmitic acid.

6. A sealed vial comprising the lyophilized powder composition according to any one of claims 1 to 5 in contact with a physiologically acceptable gas. The sealed vial according to claim 6 , wherein the gas is fluorinated gas.

8. A method for producing a lyophilized powder composition in the form of a lyophilized cake contained in a vial, the lyophilized powder composition comprising 22-28 parts by weight of polyethylene glycol, 0.15-0.25 parts by weight of DSPC, 0.15-0.25 parts by weight of DPPG-Na, and 0.02-0.06 parts by weight of palmitic acid, the method comprising: a. dissolving the DSPC, DPPG-Na, palmitic acid and the polyethylene glycol in a solvent to obtain a solution; b. freezing the solution; and c. removing the solvent by lyophilization; The number average molecular weight Mn of the polyethylene glycol is at least 4050 g / mol.

9. The method of claim 8, wherein the composition further comprises a fatty acid, and the step a. further comprises dissolving the fatty acid in the solvent.

10. A suspension of gas-filled microbubbles obtained by dispersing the lyophilized powder composition according to any one of claims 1 to 5 in a physiologically acceptable liquid in the presence of a gas.

Images