Gas-filled microvesicles for therapeutic use

Abstract

The use of a suspension of gas-filled microbubbles comprising a fatty acid diester of phosphatidylcholine (PC), pegylated phosphatidylethanolamine (PE-PEG) and a fatty acid in a therapeutic method, wherein the suspension is administered as a continuous bolus.

Description

[Technical Field]

[0001] The present invention relates to the field of gas-filled microvesicles, and in particular to the use of such microvesicles for therapeutic treatments. [Background technology]

[0002] The therapeutic use of ultrasound combined with a suspension of gas-filled microvesicles is an emerging modality that is applied in many therapeutic procedures. According to certain procedures, gas-filled microvesicles are systemically injected into the bloodstream. These procedures can be used, for example, in combination with ultrasound to increase vascular permeability, for example, to promote / enhance the delivery of drugs (Reference 1: Wang et al.) or genes, to help break down blood clots (Reference 2: Auboire et al.), to open the blood-brain barrier (Reference 3: Huang et al.), for immunomodulation (Reference 4: Tu et al.), for neuromodulation (Reference 5: Blackmore et al.), for radiosensitization (Reference 6: Deng et al.), or to assist in non-thermal tissue ablation (Reference 7: Arvanitis et al.).

[0003] Because therapeutic treatments can last for many minutes (e.g., from about 10 minutes to over an hour, depending on the location and / or size of the area being treated), it is necessary to administer a continuous suspension of gas-filled microvesicles to the patient throughout the duration of the treatment. Such continuous administration can be achieved by continuous infusion of the suspension or by injecting two or more subsequent boluses of a specific volume of the suspension.

[0004] In general, bolus administration is a relatively simple procedure, involving administering a specific volume of a suspension of gas-filled microvesicles to a subject within seconds, for example, by using a syringe and a catheter placed in a peripheral vein. Infusion is a more complex procedure, requiring the use of dedicated devices, such as an injector or pump, to inject the suspension, longer preparation time before treatment, and the presence of a skilled technician for extended periods (before and after treatment). Furthermore, prolonged infusion must take into account the decantation phenomenon of gas microvesicles in the syringe, requiring dedicated devices (e.g., a rotating syringe) or dedicated procedures (e.g., manual stirring of a saline bag containing the microvesicle suspension). Generally, this represents a substantial additional cost. As observed by the applicant, conventional formulations of gas-filled microvesicles, when administered as a bolus in therapeutic applications, need to be administered at relatively frequent intervals to allow for the presence of a sufficient amount of microvesicles in the anatomical region being treated. For example, Thomas et al. (Reference 8: Thomas et al., 2018) describe the use of SonoVue® to enhance liposome delivery (two boluses administered within 45 seconds). Furthermore, Reference 9 (Dimcevski et al., 2016) discloses the use of SonoVue® in ultrasound-mediated delivery therapeutic treatment with an anticancer drug (gemcitabine); for this purpose, boluses of SonoVue® were injected every 3.5 minutes.

[0005] Reference 10 (Schneider et al.) discloses a suspension of phospholipid-stabilized microvesicles loaded with a mixture of nitrogen and perfluorobutane, obtained by reconstitution of a freeze-dried composition containing PEG4000, DSPC, DPPE-MPEG5000 and palmitic acid, for use as an imaging ultrasound contrast agent. Summary of the Invention [Problem to be solved by the invention]

[0006] Applicant has discovered that by using a suspension of gas-filled microvesicles containing the above ingredients, it is possible to increase the interval between one bolus and subsequent boluses while maintaining substantially the same therapeutic effect as other known formulations administered at shorter intervals. Advantageously, the longer time interval between subsequent bolus administrations allows for a substantial reduction in the total amount of microvesicles that need to be administered for a treatment requiring a particular duration. [Means for solving the problem]

[0007] One aspect of the present invention relates to a suspension of gas-filled microvesicles comprising a fatty acid diester of phosphatidylcholine (PC), PEGylated phosphatidylethanolamine (PE-PEG) and a fatty acid for use in a method for therapeutic treatment of a subject, wherein the suspension is administered as a continuous bolus.

[0008] Preferably, the successive boluses are administered with an interval of at least 4 minutes between the first and second boluses.

[0009] Preferably, the composition comprises DSPC, DSPE-MPEG5000 and palmitic acid. The microbubbles are filled with a substantially water-insoluble, physiologically acceptable gas, preferably a perfluorinated hydrocarbon, more preferably perfluorobutane.

[0010] Another aspect of the present invention relates to gas-filled microvesicles containing the above components in a relative molar ratio (PC / PE-PEG / palmitic acid) of 60-90 / 2-10 / 5-30, preferably 70-80 / 3-9 / 10-25, more preferably about 72-78 / 5-8 / 15-20. [Brief explanation of the drawings]

[0011] [Figure 1]Figure 2 shows the results of the extravasation experiment in Example 2a (high frequency, high sound pressure), where column a shows the effectiveness of bolus treatment with Sonovue®, column b shows the effectiveness of bolus treatment with Definity®, and column c shows the effectiveness of bolus treatment with the formulation of the present invention. [Figure 2] 1 shows the results of the extravasation experiment in Example 2b (low frequency, low sound pressure), where column a shows the effectiveness of bolus treatment with Definity® and column b shows the effectiveness of bolus treatment with a formulation according to the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0012] As mentioned above, the present invention is based on the discovery that certain formulations of gas-filled microvesicles can be administered as successive boluses at advantageously longer time intervals relative to known formulations.

[0013] As applicants have observed, prior art formulations may work well when injected as a bolus at relatively short intervals, but as such intervals increase (e.g., 4 minutes or more), the effectiveness of the treatment may be compromised.

[0014] Applicant has found that microvesicle formulations comprising fatty acid diesters of phosphatidylcholine, PEGylated phosphatidylethanolamine and fatty acids (preferably DSPC, DPPE-MPEG5000 and palmitic acid) and filled with a gas comprising a fluorinated gas (preferably a mixture of nitrogen and perfluorobutane) can be administered as successive boluses at intervals of 4 minutes or more between each other without substantially compromising the effectiveness of the therapeutic treatment, such intervals preferably being at least 5 minutes, e.g., up to 15 minutes, preferably up to 10 minutes.

[0015] Advantageously, the use of longer administration bolus intervals can substantially reduce the total amount of injected material as well as the number of administrations required while maintaining the effectiveness of the therapeutic treatment.

[0016] formulation Examples of fatty acid diesters of phosphatidylcholine include dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2 distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine ( ... DPPC, DAPC, DSPC, and DOPC are preferred, with DSPC being particularly preferred.

[0017] Examples of PEGylated phosphatidylethanolamines include phosphatidylethanolamines in which the hydrophilic ethanolamine moiety is linked to a polyethylene glycol (PEG) molecule of variable molecular weight (e.g., 1000-5000 g / mol), such as PEGylated dipalmitoylphosphatidyletheramine (DPPE-PEG) or distearoylphosphatidylethanolamine (DSPE-PEG). Preferably, the PEG is methoxy-terminated PEG (also referred to as MPEG). Unless otherwise indicated, the term "PE-PEG" as used herein also includes PE-MPEG compounds. PE-PEGs (or PE-MPEGs) with a molecular weight of approximately 5000 g / mol, such as DPPE-MPEG5000 or DSPE-MPEG5000, are preferred.

[0018] Examples of fatty acids are (C 10 -C 22)-Alkyl carboxylic acids, such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid; preferably saturated fatty acids, such as myristic acid, palmitic acid, stearic acid, and arachidic acid; more preferably palmitic acid.

[0019] In an optimized formulation, the above components are preferably in a relative molar ratio (PC / PE-PEG / palmitic acid) of 60-90 / 2-10 / 5-30 (i.e., 60-90% molar amount of PC, e.g., DSPC; 2-10% molar amount of PE-PEG, e.g., DPPE-PEG5000; and 5-30% molar amount of fatty acid, e.g., palmitic acid; the total amount of components is 100%), more preferably 70-80 / 3-9 / 10-25, and even more preferably about 72-78 / 5-8 / 15-20.

[0020] Particularly preferred are formulations containing DSPC, DPPE-PEG5000 and palmitic acid in the above relative molar ratios.

[0021] Preparation of microvesicles The microvesicles of the present invention can be prepared according to any method known in the art.Typically, the preparation method involves preparing a dry powdered material containing the composition of the present invention, preferably by lyophilization (freeze-drying) of an aqueous or organic suspension containing the composition.Then, the microvesicles can be obtained by reconstituting the lyophilized preparation in an aqueous carrier under gentle stirring in the presence of gas.

[0022] Preferably, as disclosed in International Patent Application WO2004 / 069284, the composition comprising a mixture of phospholipids and fatty acids can be dispersed in an emulsion of water-immiscible organic solvents (such as branched or straight-chain alkanes, alkenes, cycloalkanes, aromatic hydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons, perfluorinated hydrocarbons or mixtures thereof) and water, under stirring, preferably mixed with lyophilization additives (such as carbohydrates, sugar alcohols, polyglycols, polyoxyalkylene glycols and mixtures thereof; preferably, polyethylene glycol is used as a lyophilization agent, particularly PEG4000).The emulsion can be obtained by subjecting the aqueous medium and solvent to any suitable emulsion-forming technique known in the art in the presence of phospholipids and fatty acids, such as ultrasonication, shaking, high-pressure homogenization, fine mixing, membrane emulsification, flow-focus emulsification, high-speed stirring or high-shear mixing. Preferably, an organic solution containing phospholipids and fatty acids is first prepared; separately, PEGylated phospholipids are dissolved in an aqueous solution containing a lyophilizing agent; then, the organic phase and the aqueous phase are mixed and emulsified as described above. The microemulsion thus obtained may be diluted with a solution containing a lyophilizing agent. The microemulsion, which contains microdroplets of solvent surrounded by a stabilizing layer containing phospholipids and fatty acids, is then lyophilized according to conventional techniques to obtain a lyophilized material.

[0023] The lyophilized product is generally in the form of a powder or cake and can be stored (typically in a sealed vial) in contact with the desired gas. The lyophilized additive (e.g., PEG 4000) typically represents the largest amount of the composition (e.g., about 500 to about 1000 times the amount of the microvesicle components (i.e., PC / PE-PEG / palmitic acid) by weight). The product is easily reconstituted in a suitable physiologically acceptable aqueous liquid carrier, such as saline, by gentle shaking.

[0024] gas Any biocompatible gas, gas precursor, or mixture thereof may be used to form the microvesicles of the present invention (also identified herein as a "microvesicle-forming gas").

[0025] Gas can include, for example, air; nitrogen; oxygen; carbon dioxide; hydrogen; nitrous oxide; rare gas or inert gas, such as helium, argon, xenon or krypton; low molecular weight hydrocarbon (for example, containing up to 7 carbon atoms), for example, alkane such as methane, ethane, propane, butane, isobutane, pentane or isopentane, cycloalkane such as cyclobutane or cyclopentane, alkene such as propene, butene or isobutene, or alkyne such as acetylene; ether; ketone; ester; halogenated gas, preferably fluorinated gas, for example, or halogenated, fluorinated or perfluorinated low molecular weight hydrocarbon (for example, containing up to 7 carbon atoms); or any mixture of the above.When halogenated hydrocarbon is used, preferably at least a portion, more preferably all, of the halogen atoms in the compound are fluorine atoms.

[0026] Fluorinated gas is preferred, and perfluorinated gas is particularly preferred.Fluorinated gas includes material that contains at least one fluorine atom, such as fluorinated hydrocarbon (organic compound that contains one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated ketone, such as perfluoroacetone; and fluorinated, preferably perfluorinated ether, such as perfluorodiethyl ether.Preferred compound is perfluorinated gas, such as SF6 or perfluorocarbon (perfluorinated hydrocarbon), that is, hydrocarbon that all hydrogen atom is replaced by fluorine atom, which is known to form particularly stable microbubble suspension, for example, as disclosed in EP0554213, which is incorporated herein by reference.

[0027] The term "perfluorocarbon" includes saturated, unsaturated, and cyclic perfluorocarbons. Examples of biocompatible, physiologically acceptable perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane (e.g., perfluoro-n-butane, optionally mixed with other isomers, such as perfluoroisobutane), perfluoropentane, perfluorohexane, or perfluoroheptane; perfluoroalkenes, such as perfluoropropene, perfluorobutene (e.g., perfluorobut-2-ene), or perfluorobutadiene; perfluoroalkynes (e.g., perfluorobut-2-yne); and perfluorocycloalkanes (e.g., perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutane, perfluorotrimethylbutane, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentane, perfluorocyclohexane, perfluoromethylcyclohexane, and perfluorocycloheptane). Preferred saturated perfluorocarbons are, for example, CF4, C2F6, C3F8, C4F8, C4F 10 and C5F 12 and preferably C4F 10 is.

[0028] It may also be advantageous to use a mixture of any of the above gases in any ratio. For example, the mixture may contain a conventional gas, such as nitrogen, air, or carbon dioxide, and a gas that forms a stable microbubble suspension, such as sulfur hexafluoride or the perfluorocarbons listed above. Examples of suitable gas mixtures can be found, for example, in WO 94 / 09829, which is incorporated herein by reference. The following combinations are particularly preferred: a mixture of gases (A) and (B), where gas (B) is a fluorinated gas selected from those exemplified above, including mixtures thereof, and (A) is selected from air, oxygen, nitrogen, carbon dioxide, or mixtures thereof. The amount of gas (B) can represent about 0.5% to about 95% v / v of the total mixture, preferably about 5% to 80%.

[0029] Particularly preferred gases are SF6, C3F8, and C4F 10 or mixtures thereof, which may be mixed with air, oxygen, nitrogen, carbon dioxide or mixtures thereof. 10 is particularly preferred, and C4F 10 Even more preferred is a mixture of nitrogen and HCl, preferably in a 35 / 65 (v / v) ratio.

[0030] Preparation kits and administration The microvesicle suspension of the present invention can be stored as such or, preferably, in the form of a lyophilized precursor that can be reconstituted with an aqueous carrier. According to the present invention, the precursor of the microvesicle suspension is therefore preferably stored in a dried, powdered form and can therefore be advantageously packaged in a two-component diagnostic and / or therapeutic kit, preferably for administration by injection. The kit preferably comprises a first container containing the lyophilized precursor composition in contact with a selected microvesicle-forming gas, and a second container containing a physiologically acceptable aqueous carrier, particularly saline, for reconstituting the microvesicle suspension. The two-component kit can comprise two separate containers or a dual-chamber container. In the former case, the containers are preferably vials sealed with conventional septa, through which the carrier liquid can be injected, optionally using a prefilled syringe, while the vial containing the lyophilized residue is sealed by a septum. In such cases, the syringe used as the container for the second component is also used to inject the microvesicle suspension. In the latter case, the dual-chamber container is preferably a dual-chamber syringe, and once the lyophilized product has been reconstituted, it can be mixed appropriately or gently shaken and the container can be used directly to inject the suspension of microvesicles.

[0031] The reconstituted suspension thus contains gas-filled microvesicles with a stabilizing layer containing the aforementioned components (PC, PE-PEG, and fatty acid), preferably in the relative molar ratios indicated above, preferably containing a mixture of nitrogen and perfluorobutane (65 / 35 v / v). Typically, the reconstituted gas-filled microvesicles have a median volume concentration (Dv50) of about 2.5-4 microns, preferably 3.0-3.5, and a number-average diameter (Dn) of about 1-2 microns, preferably 1.2-1.8. Typically, substantially all microvesicles (e.g., at least 98%, preferably 99%, more preferably 100%) have a diameter of less than 8 microns. Generally, the amount of 2-8 micron gas-filled microvesicles can be about 5% to about 40% (preferably 10-30%) of the total number of microvesicles in the suspension. Depending on the volume of reconstitution, the concentration of gas-filled microvesicles in the suspension can be about 1 x 10 8 ~Approx. 2×10 9 Microvesicles / mL.

[0032] The microvesicle suspensions of the present invention can be used in combination with ultrasound in a variety of therapeutic techniques to increase vascular permeability, including, for example, enhanced / enhanced pharmaceutical delivery (e.g., to enhance the therapeutic efficacy of drugs or genes in the treatment of tumors or neurological disorders), thrombus disruption, immunomodulation, neuromodulation, radiosensitization, or non-thermal tissue ablation.

[0033] According to a preferred embodiment, an effective amount of microvesicles is administered to a patient, typically by injection of a suspension thereof, preferably as a continuous bolus, preferably in combination with a pharmaceutical agent (e.g., an anti-tumor drug). The administration of the suspension of microvesicles may be either concurrent or subsequent to the administration of the pharmaceutical agent.

[0034] As used herein, the phrase "bolus administration" refers to a single intravascular injection of a specific volume of a gas-filled microvesicle suspension, for example, using a syringe and a catheter placed in a peripheral vein. A bolus administration typically occurs within a few seconds (e.g., 5 to 120 seconds, preferably within 60 seconds), while a "subsequent" or "repeated" bolus refers to a repeated bolus injection after a specific period of time, for example, according to the present invention, after at least 4 minutes.

[0035] The total amount of microvesicles for each injection volume of suspension (bolus) was 6 x 10 5 ~2×10 9 Microvesicles / kg of patient, preferably 1 x 10 7 ~1×10 9 microvesicles / kg, even more preferably 2 x 10 7 ~4×10 8 Microvesicles / kg.

[0036] The volume of each infusion bolus is adapted to the required total amount of microvesicles to be infused (usually dependent on the particular treatment being performed), taking into account the concentration of microvesicles in the infusion suspension and the patient's weight. As a general rule, approximately 1 x 10 8 ~Approx. 2×10 9 For a concentration of microvesicles in suspension of microvesicles / mL, the bolus volume may vary from 0.01 to 120 mL, preferably from 0.2 to 60 mL, and even more preferably from 0.4 to 20 mL.

[0037] Although the present composition can be administered as a continuous bolus at any time interval, the applicant has observed that the preparation can be used in therapeutic treatment in combination with ultrasound by administering it at a longer interval than known microvesicle preparations. In particular, the above-mentioned time interval between two continuous boluses can be at least 4 minutes, preferably 5 minutes, for example, up to 15 minutes, preferably up to 10 minutes. On the other hand, those skilled in the art will recognize that by using the preparation of the present invention with a shorter interval between boluses and continuous boluses, the effectiveness of treatment will nevertheless be improved compared to the effectiveness of other known preparations of gas-filled microvesicles injected under the same conditions (i.e., microvesicle concentration and time interval).

[0038] Suitable ultrasonic irradiation of the body part to be treated in the presence of microvesicles provides the desired enhanced therapeutic effect, for example, by increasing the therapeutic efficacy of the drug administered in combination with a suspension of gas-filled microvesicles. Ultrasound irradiation can be carried out under conventional conditions. For example, ultrasonic irradiation at frequencies of 0.1 MHz to 15 MHz, preferably 0.15 to 5 MHz, and more preferably 0.2 to 2.5 MHz can be used. The acoustic pressure can be 50 kPa to 10 MPa, preferably 100 kPa to 5 MPa, and more preferably 100 kPa to 3.5 MPa. The pulse length is adapted to the specific treatment and the organ being treated and can vary, for example, from 1 μs to 120 seconds, preferably up to 60 seconds, and more preferably up to 1 second.

[0039] The following examples help to further illustrate the invention. [Example]

[0040] JPEG0007743415000001.jpg88166

[0041] Example 1 Preparation of experimental formulation (F01) The procedure described in the Examples of WO2004 / 069284 was used to prepare an experimental formulation (F01) for use in subsequent experiments.

[0042] Briefly, emulsions of cyclooctane and water (approximately 1.5 / 100 v / v) containing approximately 90 mg / l DSPC, 7 mg / l palmitic acid, 60 mg / l DPPE-PEG5000, and 100 g / l PEG4000 were prepared (Megatron MT3000, Kinematica; 10 000 rpm) and sampled into DIN8R vials (approximately 1 ml / vial).

[0043] The vials were cooled under vacuum at -50°C and then subjected to lyophilization, followed by secondary drying above room temperature until water and solvent were completely removed (less than 0.5% by weight). At the end of the lyophilization process, the headspace of the vials was filled with CF 10 The vial was saturated with a 35 / 65 mixture of HCl / N2 and the vial was stoppered and sealed.

[0044] Before the experiment, the vial was reconstituted with 1 ml of saline.

[0045] Example 2 Therapeutic efficacy of experimental formulation F01 General Setup CY(c)5.5 Mono NHS ester was prehydrolyzed overnight at room temperature using a basic buffer (100 mM phosphate, 150 mM saline, pH 8). Anesthetized male Sprague-Dawley rats were administered an intravenous infusion of hydrolyzed Cy(c)5.5 (Cy5.5h) to achieve an initial plasma concentration of 3.3 μM. Subsequently, continuous bolus injections of gas-filled microvesicles (as specified in the Examples below) were administered, and the left hindlimb muscle was subjected to therapeutic ultrasound irradiation (frequency, acoustic pressure, and pulse characteristics as specified in the Examples below).

[0046] Boluses were spaced 5 min apart, and the total duration of the procedure ranged from 10 to 20 min.

[0047] In Examples 2a and 2b below, the ability of gas-filled microvesicles of the present invention (F01, containing a mixture of DSPC, DPPE-MPEG5000, and palmitic acid) to induce Cy5.5h extravasation was compared under identical conditions with Sonovue® (a mixture of DSPC, DPPG, and palmitic acid) and Definity® (a mixture of DPPC, DPPA, and DPPE-MPEG5000) under different ultrasound conditions (frequency 0.5 MHz to 1.6 MHz; acoustic pressure 170 kPa to 800 kPa).

[0048] The therapeutic effect of the combination of gas-filled microvesicles and ultrasound was evaluated by measuring the extravasation level of Cy5.5h in the hindlimb muscles 120 minutes after injection. This was achieved by quantitative measurement of Cy5.5h fluorescent signal ex vivo using near-infrared fluorescence imaging (Fluobeam® 700 Fluoptics Grenoble) on fresh tissue. Treatment efficacy was considered positive if the mean fluorescent signal ratio between the treated muscle and the control muscle (right hindlimb, not exposed to ultrasound irradiation) was higher than 1.5.

[0049] Example 2a For each animal (see "General Setup" section above), four successive bolus infusions of F01, Sonovue, or Definity were administered, with approximately 6 × 10 7 The microvesicles were administered at a concentration of 100 microvesicles / kg of animal, with an interval of 5 minutes between each other.

[0050] Five animals were tested for each formulation.

[0051] The left hindlimb muscles were subjected to ultrasonic treatment at 800 kPa, 1.6 MHz, 1 ms ON-4 sec OFF for 20 minutes.

[0052] As can be seen from the results in Figure 1 (column c), the fluorescence signal in muscles treated with ultrasound in combination with F01 was at least twice that measured in control muscles. In contrast, SonoVue or Definity gas-filled microvesicles failed to induce Cy5.5h extravasation when each was injected for 5 min under identical conditions (columns a and b, respectively).

[0053] Example 2b Two successive boluses (5 minutes apart) were administered using F01 and Definity, each approximately 1 x 10 9 The same experiment as in Example 2a was repeated by administering microvesicles at a concentration of 100 mg / kg of animal.

[0054] Three and four animals were tested on F01 and Definity, respectively.

[0055] The left hindlimb muscles were subjected to ultrasonic treatment at 170 KPa and 0.5 MHz (1 ms ON-4 sec OFF) for 10 minutes.

[0056] The results in Figure 2 show that the fluorescence signal in muscles treated with F01 and ultrasound (column b) was more than double that measured in control muscles. In contrast, Definity gas-filled microvesicles failed to induce Cy5.5h extravasation when injected for 5 min each under identical conditions (column a).

[0057] The above experimental results demonstrate the greater efficacy of the composition of the present invention (relative to commercially available microvesicle compositions) in increasing vascular permeability when administered as a continuous bolus in combination with ultrasound treatment at different conditions of ultrasound irradiation.

[0058] JPEG0007743415000002.jpg164166

Claims

1. A suspension of gas-filled microvesicles comprising a fatty acid diester of phosphatidylcholine (PC), PEGylated phosphatidylethanolamine (PE-PEG) and palmitic acid for use in a therapeutic method, the suspension is administered as at least two subsequent boluses spaced at least 4 to 10 minutes apart; the relative molar ratio of PC / PE-PEG / palmitic acid is 60-90 / 2-10 / 5-30; Suspension.

2. 2. The suspension of claim 1, The at least two successive boluses are administered with an interval of at least 5 minutes between each other. Suspension.

3. 2. The suspension of claim 1, The ratio is 70 to 80 / 3 to 9 / 10 to 25; Suspension.

4. A suspension according to any one of claims 1 to 3, the gas-filled microvesicles comprise DSPC, DPPE-MPEG-5000 and palmitic acid; Suspension.

5. A suspension according to any one of claims 1 to 4, the gas-filled microvesicles comprise a fluorinated gas; Suspension.

6. A suspension according to any one of claims 1 to 5, The method comprises applying ultrasound. Suspension.

7. 7. The suspension of claim 6, the method comprising increasing vascular permeability in a subject receiving the therapeutic method; Suspension.

8. A suspension according to any one of claims 1 to 7, The total amount of microvesicles for each injection volume of suspension is 6 x 10 per kg of subject receiving said therapeutic method. ５ ~2 x 10 ９ microvesicles, Suspension.

9. A suspension according to any one of claims 1 to 8, The concentration of microvesicles in the injection suspension was 1 × 10 ８ ~2 x 10 ９ microvesicles / mL, Suspension.

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