FREEZE-DRIED PRODUCT AND GAS-FILLED MICROVESICLES SUSPENSION

Abstract

A method of manufacturing a suspension of gas-filled microvesicles by reconstituting a freeze-dried product and a suspension obtained according to said method, where the freeze-dried product has been subjected to a thermal treatment

Claims

1.-17. (canceled)

18. A freeze-dried product obtained according to a method comprising: a. preparing a liquid mixture comprising (i) an amphiphilic material comprising a phospholipid and a fatty acid and (ii) a polyethylene glycol as freeze-drying protecting component in a solvent b. freeze-drying the liquid mixture to remove said solvent and obtain a freeze-dried product and c. after completion of the freeze-drying of step b, heating said freeze-dried product at ambient pressure at a temperature higher than 35° C. and lower than the melting point of the polyethylene glycol freeze-drying protecting component, for a period of time of from eight to twenty hours, wherein the freeze-dried product of step b has not been reconstituted prior to step c, and wherein the freeze-dried product is suitable for the preparation of a suspension of stabilized gas-filled microbubbles.

19. (canceled)

20. (canceled)

21. A method of diagnosing which comprises (i) reconstituting the freeze-dried product according to claim 18 with a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas under gentle agitation to obtain a suspension of gas-filled microvesicles; (ii) administering to a patient the suspension of gas-filled microvesicles; and (iii) detecting an ultrasound signal from a region of interest in said patient.

22. The method according to claim 21, wherein the region of interest in said patient is the heart.

23. The method according to claim 21, wherein the region of interest in said patient is the liver.

24. The method according to claim 21, wherein the region of interest in said patient is the urinary tract.

25. A suspension of gas-filled microvesicles comprising a freeze-dried product dissolved in a pharmaceutically acceptable liquid carrier, said freeze-dried product comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), dipalmitoylphosphatidylglycerol (DPPG), palmitic acid and polyethylene glycol 4000 (PEG4000), wherein said suspension has a value of critical pressure (Pc50) of at least 20 kPa, and wherein the microvesicles are filled with a physiologically acceptable gas.

26. The suspension according to claim 25, wherein said suspension has a value of Pc50 of at least 22 kPa.

27. The suspension according to claim 25, wherein said suspension has a value of Pc50 of at least 25 kPa.

28. The suspension according to claim 25, wherein said suspension has a value of Pc50 of from 20 kPa to 32 kPa.

29. The suspension according to claim 25, wherein said physiologically acceptable gas comprises SF.sub.6.

30. The suspension according to claim 25, wherein the weight ratio DSPC/DPPG-Na/palmitic acid is of 4.75/4.75/1.

31. The suspension of claim 30, wherein the weight ratio between DSPC/DPPG-Na/palmitic acid and PEG4000 is of 0.017:1.

32. A suspension of gas-filled microvesicles comprising a freeze-dried product dissolved in a pharmaceutically acceptable liquid carrier, said freeze-dried product comprising 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), palmitic acid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (DPPE-PEG5000) and polyethylene glycol 4000 (PEG4000), wherein said suspension has a value of critical pressure (Pc50) of at least 84 kPa, and wherein the microvesicles are filled with a physiologically acceptable gas.

33. The suspension according to claim 32 having a value of Pc50 of at least 88 kPa.

34. The suspension according to claim 32 having a value of Pc50 of at least 90 kPa.

35. The suspension according to claim 32 having a value of Pc50 of from 84 kPa to 105 kPa.

36. The suspension according to claim 32, wherein said physiologically acceptable gas comprises a mixture of C.sub.4F.sub.10 and nitrogen.

37. The suspension according to claim 32, wherein the weight ratio DSPC/palmitic acid/DPPE-PEG5000 is of 90/7/60.

38. The suspension according to claim 37 wherein the weight ratio between DSPC/palmitic acid/DPPE-PEG5000 and PEG4000 is of 0.0016:1.

39. A method of ultrasound therapeutic treatment which comprises: (i) administering to a patient a suspension of gas-filled microvesicles according to claim 32; (ii) identifying a region of interest in said patient to be submitted to a therapeutic treatment, said region of interest comprising said suspension of gas-filled microvesicles; and (iii) applying an ultrasound beam for therapeutically treating said region of interest; whereby said ultrasound therapeutic treatment is enhanced by the presence of said suspension of gas-filled microvesicles in said region of interest.

Description

EXAMPLES

Materials

[0101] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0102] DPPG-Na: 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)

[0103] DPPE-PEG5000: 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt)

[0104] PEG4000=Polyethylenglycol (MW=4000 g/mol)

Measurement of Pressure Resistance (Pc50)

[0105] The resistance to pressure of gas-filled microvesicles was evaluated using an in-house developed pressure nephelometer. Briefly, the microvesicles suspension was introduced into a spectrophotometer sample cell (airtight and connected to a pressurization system). The optical density (absorbance at 700 nm) of the suspension is continuously recorded while linearly increasing the pressure applied to the sample in the cell from atmospheric pressure (760 mmHg, 101.3 kPa,) to an over pressure of two bars (2280 mmHg, 303.9 kPa), at a rate of about 4 mmHg/s (533 Pa/s).

[0106] The Pc50 parameter (“critical pressure”) characterizing each suspension identifies the overpressure (with respect to atmospheric pressure) at which the absorbance of the microvesicles suspension drops to half of its initial value.

Example 1

Preparation of Freeze-Dried Product (Batches 1a-1b)

[0107] The procedure illustrated in the working examples of WO 94/09829 was used for preparing two different batches (1a-1b) each consisting of several vials containing the freeze-dried product.

[0108] Briefly, DSPC, DPPG-Na and palmitic acid in a weight ratio of 4.75/4.75/1 were first dissolved in hexane/ethanol (8/2, v/v) at a concentration of about 5 g/L and the solvents were evaporated under vacuum. The solid residue was admixed with PEG4000 in a weight ratio of about 0.017:1, the mixture was dissolved in tert-butanol at around 60° C. and the clear solution was used to fill respective DIN8R vials (with a corresponding volume containing about 25 mg of the mixture). The vials were then rapidly cooled at −45° C. and then subjected the vacuum for removing the frozen solvent by sublimation. The temperature was then raised (above room temperature, not higher than 35° C.) and the remaining solvent was evaporated, down to a final amount of less than 0.5% by weight. At the end of the freeze-drying process, the ambient of the freeze-dryer was saturated with SF.sub.6 at atmospheric pressure and the vials (containing the solid freeze-dried product in contact with SF.sub.6) were stoppered and sealed.

[0109] The two batches were used for the subsequent heat treatment experiments.

Example 2

Preparation of Freeze-Dried Product (2a-2h)

[0110] The procedure illustrated in the working examples of WO2004/069284 was used for preparing eight different batches (2a-2h) each consisting of several vials containing the freeze-dried product.

[0111] Briefly, an emulsion of cyclooctane and water (about 1.5/100 v/v) containing about 90 mg/l of DSPC, 7 mg/l of palmitic acid, 60 mg/l of DPPE-PEG5000 and 100 g/l of PEG4000 is prepared (Megatron MT3000, Kinematica; 10′000 rpm) and sampled into DIN8R vials (about 1 ml/vial).

[0112] The vials were cooled at -50° C. under vacuum and then subjected to lyophilization, followed by secondary drying above room temperature until complete removal of water and solvent (less than 0.5% by weight), as described in example 1. At the end of the freeze-drying process, the headspace of the vials is saturated with a 35/65 mixture of C.sub.4F.sub.10/N.sub.2 and the vials are stoppered and sealed.

[0113] The different batches (1a to 1h) were used for the subsequent heat treatment experiments.

Example 3

Effect of the Heat Treatment on Batches Manufactured According to Example 1

[0114] The vials of the various batches prepared according to examples 1 were submitted to different heat treatments and the effect on the characteristics of the reconstituted suspensions of gas-filled microvesicles were observed.

Experiment 3.1

[0115] The vials of batch 1a were submitted to a heating temperature of 48° C. for a time ranging from 8 hours to one week (five vials for each group). The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured. Results are reported in the following table 1.

TABLE-US-00001 TABLE 1 Batch 1a Heating time Pc50 (kPa) 48° C. mean value No heating 14.4  8 hours 24.8 16 hours 25.5 24 hours 28.7 48 hours 28.8 One week 32.0

[0116] As inferable from the above results, a substantial increase in the pressure resistance can be observed after 8 hours of heat treatment, with respect to the untreated freeze-dried samples. Such pressure resistance slightly increases in time, up to a maximum after one week of treatment. However, as observed by the Applicant, a too long heating time (e.g. after 24 hours and particularly above 48 hours) may negatively impact on other characteristics of the gas-filled microvesicles, such as their total number, the total volume of gas and/or their mean size.

Experiment 3.2

[0117] In a second experiment, the vials of batch 1b were heated at temperatures of 40° C., 45° C. and 49° C. for time periods of 12, 16 or 20 hours (three vials for each group, for a total of 27 vials). The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured. Results are reported in the following table 2.

TABLE-US-00002 TABLE 2 Batch 1b Pc50 (kPa) Heating time T (° C.) mean value 12 hours 40 22.8 16 hours 40 23.6 20 hours 40 23.7 12 hours 45 25.5 16 hours 45 22.5 20 hours 45 22.4 12 hours 49 24.1 16 hours 49 24.4 20 hours 49 24.9

[0118] As inferable from the above data, a substantial increase in the pressure resistance is obtained with the heat treatment of the freeze-dried products (with respect to the initial value of about 14 kPa). While a higher increase of Pc50 may generally be observed for treatments at 49° C., treating at this temperature may however negatively impact on other characteristics of the reconstituted gas-filled microvesicles, particularly on the mean size values.

Example 4

Effect of the Heat Treatment on Batches Manufactured According to Example 2

[0119] The vials of the various batches (2a-2h) prepared according to example 2 were submitted to different heat treatments and the effect on the characteristics of the reconstituted suspensions of gas-filled microvesicles were observed.

[0120] Experiment 4.1

[0121] The vials of batch 2a were submitted to a heating temperature of 40° C. or 45° C. for 16 hours or not heated. The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured. Results are reported in the following table 3.

TABLE-US-00003 TABLE 3 Batch 1a Heating T Pc50 (kPa) for 16 h mean value No heating 66.1 40° C. 84.8 45° C. 78.8

[0122] As inferable from the above data, a substantial increase in the pressure resistance is obtained upon heat treatment also for batches manufactured according to the procedure of example 2.

Experiment 4.2

[0123] The vials of batch 2b were submitted to a heating temperature of 40° C. for a time ranging from 16 to 88 hours, or not heated. The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured. Results are reported in the following table 4.

TABLE-US-00004 TABLE 4 Batch 2b Heating time Pc50 (kPa) T = 40° C. mean value No heating 82.1 16 hours 99.0 40 hours 103.6 64 hours 98.6 88 hours 102.5

[0124] As inferable from the above data, a substantial increase in the pressure resistance is obtained upon heat treatment at 40° C. A duration of the treatment of 16 h is generally considered sufficient, also for avoiding possible negative effects caused by longer thermal treatments on other characteristics of the microvesicles (e.g. increase of large size microvesicles in the reconstituted suspension).

Experiment 4.3

[0125] The vials of batches 2c-2g were submitted to a heating temperature of 40° C. for a period of 16 hours, or not heated. The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured. Results are reported in the following table 5.

TABLE-US-00005 TABLE 5 Batches 2c-2g (40° C., 16 h) Thermal Pc50 (kPa) Batch No. Treatment mean value 2c No 70.6 2c Yes 93.7 2d No 74.1 2d Yes 94.3 2e No 69.6 2e Yes 94.2 2f No 62.8 2f Yes 79.8 2g No 55.4 2g Yes 81.3

[0126] As inferable from the above table, for the suspensions of microvesicles reconstituted from the various batches an increase in pressure resistance of more than 15 kPa or more and up to about 25 kPa is obtained after heat treatment of the freeze-dried products.

Experiment 4.4

[0127] The vials of batch 2h were submitted to a heat treatment at 38° C. for a time ranging from two to 24 hours. The product in the vial was then reconstituted with 5 ml of saline and the characteristics of the microvesicles in the suspension were measured.

[0128] Results are reported in the following table 6.

TABLE-US-00006 TABLE 6 Batch 2h Heating time Pc50 (kPa) (h) at 38° C. mean value 0 63.19 2 73.33 4 74.66 6 79.33 8 80.26 12 82.66 16 79.73 24 83.06

[0129] As inferable from the above table, an increasing pressure resistance of the microvesicles in the reconstituted suspension is obtained upon heating the freeze-dried material for an increasing time, up to 8-12 hours at 38° C. Further heating of the material (16 or 24 hours) does not substantially further increase the pressure resistance.