Perfluoroolefin-containing aerated microbubbles

Abstract

The present invention relates to a gas-filled microbubble suspension comprising an outer layer of a stabilizing material surrounding a gaseous core comprising a perfluoroolefin.

Description

Technical Field

[0001] The present invention relates to a suspension of gas-filled microvesicles whose gaseous core contains a perfluorinated olefin. Background of the Invention

[0003] Suspensions of gas-filled microvesicles (typically dispersed in a suitable physiologically acceptable solution) have been used for decades for ultrasound contrast imaging (CEUS) and more recently for therapy. The gas is typically encapsulated or encapsulated in a layer of a stabilizing material comprising, for example, an emulsifier, an oil, a thickener, or a sugar. Gas-filled microvesicles are commonly referred to in the art using various terms, depending on the stabilizing material used in their preparation; these terms include, for example, "microspheres," "microbubbles," "microcapsules," or "microballoons," collectively referred to herein as "gas-filled microvesicles" (abbreviated as "microbubbles" or "GfM"). The term "GfM" used in the art refers to microvesicles of micron size (e.g., 1 to 10 microns, typically 2-8 microns) or nanometer size (e.g., 50 to 1000 nanometers, typically 100-400 nanometers).

[0004] GfM suspensions can be produced according to various manufacturing methods.

[0005] Some of these methods, see (e.g., WO94 / 09829 or WO2004 / 069284), require first preparing a solution or emulsion containing the desired stabilizing material and mixing it with a freeze-dried protective component. The preparation is then divided into vials and subjected to a freeze-drying process to remove moisture and / or solvent. Then, an appropriate gas (e.g., fluorinated gas) is injected into the headspace of the vial and sealed. Before use, a suitable liquid (e.g., normal saline, glucose solution, etc.) is introduced into the vial, and the vial is gently shaken to dissolve the freeze-dried product and form microbubbles, thereby easily preparing a microbubble aqueous suspension.

[0006] Other preparation methods include preparing an aqueous suspension of the stabilizing material in contact with a suitable gas (in the headspace of the vial containing the suspension); GfM is prepared by vigorously shaking the vial before use.

[0007] Recently, microfluidic technology (see, for example, WO 2020 / 260423) has made it possible to prepare GfM suspensions with calibrated sizes.

[0008] Fluorinated gases, including sulfur hexafluoride, perfluoropropane, and perfluorobutane, have become the gases of choice for preparing gas-filled microbubbles for ultrasound medical applications due to their low solubility and low diffusivity.

[0009] However, these perfluorinated gases, while providing very stable microbubble suspensions, have relatively long atmospheric lifetimes and high global warming potentials (GWPs). GWP is a term used to describe the relative effectiveness of a single greenhouse gas, taking into account how long it remains active in the atmosphere. The global warming potentials (GWPs) currently used are calculated over a 100-year period. Using carbon dioxide as a reference gas, its 100-year GWP is 1. For example, SF6 has a GWP of 23,500, C4F 10 9200 for C3F8 and 8900 for C3F8 (see, for example, Myhre, G., D. Shindell, F.-M. Bréon, W. Collins, J. Fuglestvedt, J. Huang, D. Koch, J.-F. Lamarque, D. Lee, B. Mendoza, T. Nakajima, A. Robock, G. Stephens, T. Takemura and H. Zhang, 2013: Anthropogenic and Natural Radiative Forcing, in Climate Change 2013: The Physical Science Basis, Contribution of Working Group I to the Fifth Assessment Report of the Intergovernmental Panel on Climate Change). In: Change (Stocker, T. F., D. Qin, G.-K. Plattner, M. Tignor, S. K. Allen, J. Boschung, A. Nauels, Y. Xia, V. Bex and P. M. Midgley (eds.), Cambridge University Press, Cambridge, United Kingdom and New York, NY, USA.

[0010] Applicants have now discovered a class of suitable compounds with relatively low GWP values ​​which can advantageously replace current perfluorinated compounds in the preparation of gas-filled microbubbles. SUMMARY OF THE INVENTION

[0012] One aspect of the present invention relates to a suspension of gas-filled microbubbles comprising an outer layer of a stabilizing material surrounding a gaseous core, wherein the gaseous core comprises perfluoropentene. Preferably, the compound is perfluoro-2-pentene.

[0013] The perfluorinated compound is preferably mixed with air or nitrogen, preferably nitrogen. The volume percentage of the perfluorinated compound is 10% or more. In certain embodiments, the amount of the perfluorinated compound can be 20%, 30%, 35%, 40%, 50% or more, up to, for example, 70%, preferably up to 60%.

[0014] Another aspect of the present invention relates to a suspension of gas-filled microbubbles in a physiologically acceptable liquid, wherein the microbubbles are as defined above.

[0015] Another aspect of the invention relates to a vial comprising:

[0016] - a gas-filled microvesicle precursor in the form of a freeze-dried product comprising a stabilizing material capable of forming a stabilizing layer of said microvesicles and a freeze-drying protective component; and

[0017] -Perfluoropentene. Detailed Description of the Invention

[0019] GfM components

[0020] Suitable stabilizing materials for forming the outer layer of GfMs include amphiphilic materials such as phospholipids, biodegradable polymers or biodegradable water-insoluble lipids (e.g., those described in US 5,711,933 and US 6,333,021), proteins (e.g., albumin, hemoglobin described in US 4,276,885 or EP 0324938).

[0021] Preferably, the stabilizing material is an amphiphilic material.

[0022] According to a preferred embodiment, the amphiphilic material for forming the stabilization layer comprises.

[0023] Phospholipids. Phospholipids, like other amphiphilic molecules, are generally capable of forming a stable material film (usually in the form of a monolayer) at the air-water boundary of the final gas-filled microbubble suspension. Such materials are also referred to in the art as "film-forming" materials.

[0024] Phospholipids generally contain at least one phosphate group and at least one (preferably two) lipophilic long-chain hydrocarbon groups.

[0025] Examples of suitable phospholipids include esters of glycerol with one or preferably two (same or different) fatty acid residues and phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such as choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), and inositol (phosphatidylinositol). In the art, esters of phospholipids having only one fatty acid residue are generally referred to as "lyso" forms of phospholipids or "lysophospholipids." The fatty acid residues present in phospholipids are typically long-chain fatty acids, typically containing 12 to 24 carbon atoms, preferably 14 to 22 carbon atoms; the aliphatic chain may contain one or more degrees of unsaturation, or preferably is fully saturated. Examples of suitable fatty acids contained in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Saturated fatty acids, such as myristic acid, palmitic acid, stearic acid, and arachidic acid, are preferably used.

[0026] Other examples of phospholipids are phosphatidic acid, i.e., a diester of glycerol-phosphate with fatty acids; sphingolipids, such as sphingomyelin, i.e., those phosphatidylcholine analogs in which the residues of the fatty acid diester of glycerol are replaced by ceramide chains; cardiolipin, i.e., an ester of 1,3-diphosphatidylglycerol with fatty acids; glycolipids, such as ganglioside GM1 (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids.

[0027] As used herein, the term "phospholipid" includes naturally occurring, semisynthetic or synthetically prepared compounds, which may be used alone or as a mixture.

[0028] Examples of naturally occurring phospholipids are natural phosphatidylcholines (phosphatidylcholine (PC) derivatives), such as conventional soya lecithin or egg yolk lecithin.

[0029] Examples of semisynthetic phospholipids are partially or fully hydrogenated derivatives of naturally occurring phosphatidylcholine. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or sphingomyelin.

[0030] Examples of preferred phospholipids are, for example, dilauroyl-phosphatidylcholine (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), dibehenoyl-phosphatidylcholine (DBPC), 1,2-distearoyl-sn-glycero-3-ethylphosphatidylcholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl -phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidylglycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts Metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoylphosphatidic acid (DMPA) and its alkali metal salts, dipalmitoylphosphatidic acid (DPPA) and its alkali metal salts, distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidylethanolamine (DSPE), dioleoylphosphatidylethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleoylphosphatidylethanolamine amine (DLPE), dimyristoylphosphatidylserine (DMPS), diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP) and distearoylsphingomyelin (DSSP), dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI).

[0031] Suitable phospholipids also include phospholipids modified by attachment of a hydrophilic polymer, such as polyethylene glycol (PEG) or polypropylene glycol (PPG). Preferred polymer-modified phospholipids include "PEGylated phospholipids," i.e., phospholipids conjugated to a PEG polymer. Examples of PEGylated phospholipids are PEGylated phosphatidylethanolamines (abbreviated as "PE-PEGs"), i.e., phosphatidylethanolamines with a hydrophilic ethanolamine moiety attached to a PEG molecule of variable molecular weight (e.g., 300 to 20,000 daltons, preferably 500 to 5,000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG, or DOPE-PEG). For example, DPPE-PEG5000 refers to DPPE to which a PEG polymer having an average molecular weight of approximately 5,000 is attached. An example of DPPE-PEG5000 is the methoxy terminated PEG derivative 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000].

[0032] In one embodiment, the phospholipid can carry a reactive portion that can then react with a corresponding reactive portion carrying a suitable active ingredient (e.g., a targeting ligand) to bind the active ingredient to the microbubble. Examples of suitable reactive portions include, for example, reactive groups capable of reacting with amino groups bound to the active ingredient, such as isothiocyanate groups (to form a thiourea bond), reactive esters (to form an amide bond), aldehyde groups (to form an imine bond, thereby reducing to an alkylamine bond); reactive groups capable of reacting with thiol groups bound to the active ingredient, such as haloacetyl derivatives or maleimides (to form a thioether bond); reactive groups capable of reacting with carboxyl groups bound to the active ingredient, such as amines or hydrazides (to form an amide bond or an alkylamide bond). Preferably, the amphiphilic compound carrying a reactive moiety is a lipid carrying a hydrophilic polymer, such as those mentioned above, preferably a PEGylated phospholipid, such as DPPE-PEG2000 or DSPE-PEG2000, such as DPPE-PEG2000-maleimide and DSPE-PEG2000-maleimide.

[0033] Particularly preferred phospholipids are DBPC, DAPC, DSPC, DPPC, DMPA, DPPA, DSPA, DMPG, DPPG, DSPG, DMPS, DPPS, DSPS and ethyl-DSPC. The most preferred phospholipids are DPPG, DPPS and DSPC.

[0034] Phospholipid mixtures may also be used, for example mixtures of DPPE and / or DSPE (including PEGylated derivatives), DPPC, DSPC and / or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, ethyl-DSPC and / or ethyl-DPPC.

[0035] The phospholipids can be conveniently mixed with any other compounds, preferably amphiphilic compounds. For example, lipids (such as cholesterol, ergosterol, phytosterols, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate), fatty acids (such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof) or butylated hydroxytoluene and / or other non-phospholipid (amphiphilic) compounds can be optionally added to one or more of the aforementioned phospholipids, for example, preferably in a proportion of less than 50% by weight, more preferably up to 25% or less. As additional compounds to be mixed with the phospholipids, fatty acids are particularly preferred. The fatty acids that can be used in the compositions of the present invention can be saturated or unsaturated, including C esters terminated by carboxylic acid moieties. 10 -C 24 Aliphatic chain, preferably C 14 -C 22 Aliphatic chain, more preferably C 16 -C 20 Aliphatic chain. Examples of suitable saturated fatty acids include capric acid (decanoic acid), lauric acid (dodecanoic acid), myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), stearic acid (octadecanoic acid), arachidic acid (eicosanoic acid), behenic acid (docosanoic acid) and tetracosanoic acid. Preferred saturated fatty acids are myristic acid, palmitic acid, stearic acid and arachidic acid, more preferably palmitic acid. Examples of unsaturated fatty acids include myristoleic acid (cis-9-tetradecenoic acid), palmitoleic acid (cis-9-hexadecenoic acid), sapienic acid (cis-6-hexadecenoic acid), oleic acid (cis-9-octadecenoic acid), linoleic acid (cis-9,12-octadecadienoic acid), linolenic acid (cis-9,12,15-octadecatrienoic acid), gondoic acid (cis-11-eicosenoic acid), cis-11,14-eicosadienoic acid, cis-5,8,11-eicosatrienoic acid, cis-8,11,14-eicosatrienoic acid, cis-11,14,17-eicosatrienoic acid, arachidonic acid (cis-8,11,14,17-eicosatetraenoic acid), and erucic acid (cis-13-docosatrienoic acid).

[0036] According to one embodiment, the amphiphilic material mixture comprises a mixture of DSPC, DPPG and palmitic acid.

[0037] According to an alternative embodiment, the amphiphilic material comprises a mixture of DSPC, DPPE-PEG5000 and palmitic acid, and optionally further comprises a targeting ligand.

[0038] The compositions and GfMs of the invention may optionally comprise a targeting ligand, for example as described in WO2020 / 229642.

[0039] Preparation method

[0040] A suitable method for preparing an injectable gas-filled microbubble suspension comprises reconstituting a freeze-dried product comprising a stabilizing material (capable of forming a stabilizing outer layer) and an aqueous carrier in the presence of a suitable gas or gas mixture. The freeze-dried product is typically obtained by freeze-drying a liquid mixture comprising the amphiphilic material and a freeze-protective component in a suitable solvent. The liquid mixture subjected to the freeze-drying process can be obtained according to methods known in the art, such as those disclosed in WO94 / 09829 or WO2004 / 069284.

[0041] For example, according to the method disclosed in WO94 / 09829, the amphiphilic material is dispersed together with a suitable freeze-drying protective component in an organic solvent (e.g., tert-butyl alcohol, dioxane, cyclohexanol, tetrachlorodifluoroethylene or 2-methyl-2-butanol). The dispersion containing the amphiphilic material and the freeze-drying protective component is then freeze-dried to remove the organic solvent, thereby obtaining a freeze-dried product.

[0042] According to an alternative method disclosed in WO2004 / 069284, a composition comprising an amphiphilic material can be dispersed in an emulsion of water and a water-immiscible organic solvent under stirring, preferably in admixture with a freeze-drying protective component. The (micro)emulsion thus obtained, containing the solvent droplets surrounded and stabilized by the amphiphilic material, can then be freeze-dried according to conventional techniques to obtain a freeze-dried material, which can then be used to prepare a suspension of gas-filled microvesicles.

[0043] As defined herein, a freeze-drying protective component is a compound having a cryoprotective and / or freeze-drying protective effect. Suitable freeze-drying protective components include, for example, carbohydrates such as monosaccharides, disaccharides or polysaccharides, for example sucrose, maltose, trehalose, glucose, lactose, galactose, raffinose, cyclodextrin, dextran, chitosan and its derivatives (for example, carboxymethyl chitosan, trimethyl chitosan); polyols such as sugar alcohols, such as sorbitol, mannitol or xylitol; or hydrophilic polymers such as polyoxyalkylene glycols, such as polyethylene glycol (for example PEG2000, PEG4000 or PEG8000) or polypropylene glycol. According to one embodiment, the freeze-drying protective component is polyethylene glycol, preferably PEG4000.

[0044] For the freeze-drying method, a liquid mixture containing the amphiphilic material and the freeze-drying protective component (eg, obtained according to any of the aforementioned preparation methods) is typically sampled into glass vials (eg, DIN4R, DIN8R, or DIN20R), which are then loaded into a freeze dryer.

[0045] The freeze-drying process typically includes an initial step in which the vial is rapidly deep-cooled (e.g., at a temperature of -35°C to -70°C) to freeze the liquid of the mixture and then evacuated (e.g., 0.1-0.8 mbar); in this step (primary drying), substantially all of the frozen liquid (e.g., water and / or solvent) is removed by sublimation, typically up to about 95% of the total liquid volume, preferably up to about 99%. After the primary drying, the residual liquid (including possible interstitial water) can be further removed in a secondary drying process, which is typically carried out at a temperature above room temperature and under vacuum conditions (preferably by maintaining the same vacuum level as during the primary drying). The temperature of the secondary drying is preferably not higher than 35°C. The secondary drying can be stopped when the residual liquid content reaches a desired minimum, for example, a water content of less than 3% (preferably less than 1%) by weight, or a residual solvent content of less than 0.01%, preferably less than 0.08% by weight, relative to the total mass of the remaining freeze-dried product.

[0046] After the freeze drying process is complete, the freeze-dried product can be subjected to an optional additional heat treatment, typically carried out at ambient pressure, such as described in WO2020 / 229642. Preferably, after saturating the vial head space with a suitable gas or gas mixture as defined herein, then plugging the bottle (e.g., with a rubber, such as butyl rubber, stopper) and sealing the bottle (e.g., with a metal, such as aluminum, crimping seal), the sealed bottle is heat treated. In this case, the bottle is preferably taken out of the freeze dryer and placed in a suitable oven for heat treatment. Alternatively, the open bottle (which is preferably placed in a freeze dryer) can also be heat treated, then saturated with a gas or gaseous mixture, and then plugged / sealed.

[0047] Perfluoropentene

[0048] Perfluoropentenes suitable for preparing the GfM according to the present invention include perfluoro-1-pentene, perfluoro-2-pentene or mixtures thereof. Preferably, the perfluoropentene is perfluoro-2-pentene.

[0049] As Applicants have observed, GfMs comprising perfluoropentene in their gaseous core generally exhibit improved pressure resistance relative to GfMs comprising lower molecular weight perfluoroolefins (e.g., C4F8). Even more surprisingly (as shown in the Examples), Applicants have further observed that GfMs comprising perfluoropentene in their gaseous core generally exhibit improved pressure resistance relative to GfMs comprising higher molecular weight perfluoroolefins (e.g., C6F8). 12 ) also exhibit improved pressure resistance. Among the perfluoropentenes, GfM containing perfluoro-2-pentene has been found to have unexpectedly higher pressure resistance, for example relative to GfM containing perfluoro-1-pentene.

[0050] Preferably, the perfluoropentene is mixed with another gas, typically a non-fluorinated gas such as air, oxygen, carbon dioxide or nitrogen, preferably nitrogen.

[0051] Although mixing a perfluorinated gas with increasing amounts of a non-fluorinated gas (e.g., nitrogen) may gradually reduce the pressure resistance of the GfM, it still has the advantage of reducing the total amount of fluorinated gas used to prepare the GfM. In addition, the advantage of mixing with a second gas (usually nitrogen, which has a relatively low boiling point) is that perfluorinated alkenes (e.g., perfluoropentene) with boiling points close to or slightly above room temperature can be used to prepare the GfM. For example, a gas mixture containing 50% (v / v) perfluoropentene (boiling point 26°C-29°C) and nitrogen can be prepared. It is worth noting that GfM containing a relatively low percentage of perfluorinated alkenes (e.g., 20% by volume perfluoropentene and 80% nitrogen) maintains relatively high pressure resistance values.

[0052] As used herein, the "pressure resistance" of GfMs is defined by the Pc50 parameter measured on a GfMs suspension; as is known, an increased overpressure applied to a GfMs suspension causes the number of microbubbles to gradually decrease relative to the initial number (measured at atmospheric pressure) due to the collapse of the microbubbles. As explained in detail in the experimental section, the Pc50 of a gas-filled microbubble suspension determines the value of the applied overpressure (relative to atmospheric pressure) at which the absorbance of the suspension drops to half the absorbance of the suspension measured at atmospheric pressure. The decrease in the absorbance of the GfMs suspension is associated with a decrease in the initial number of gas-filled microbubbles, whereby an initially emulsified suspension (typically a high concentration of microbubbles) becomes increasingly transparent under increasing pressure (the GfMs concentration decreases). The higher the Pc50 value, the higher the pressure resistance of the microbubbles (e.g., blood pressure or ultrasound pressure), and generally, the longer the circulation time of the GfMs once administered.

[0053] The Uses of GfM

[0054] The GfM suspension according to the invention may be used in various diagnostic and / or therapeutic techniques, including in particular ultrasound.

[0055] Therefore, one aspect of the present invention relates to the use of a microvesicle suspension according to the present invention in a diagnostic method.

[0056] Diagnostic methods include any method that uses gas-filled microbubbles to enhance visualization of parts or regions of the body of animals (including humans), including imaging for preclinical and clinical research purposes. Various imaging techniques can be used in ultrasound applications, such as fundamental and harmonic B-mode imaging, pulsed or phase-inversion imaging, and fundamental and harmonic Doppler imaging; if desired, three-dimensional imaging techniques can be used.

[0057] Microbubbles according to the present invention can generally be administered at a concentration of about 0.01 to about 1.0 μL of gas per kg of patient, depending, for example, on their respective composition, the tissue or organ to be imaged, and / or the imaging technique selected. Of course, this general concentration range may vary depending on the specific imaging application; for example, when used in color Doppler or power pulse inversion imaging, signals may be observed at very low doses.

[0058] In one embodiment, the diagnostic method comprises:

[0059] (i) administering to a patient a suspension of gas-filled microbubbles according to the present invention; and

[0060] (ii) detecting an ultrasound signal from a region of interest of the patient.

[0061] Another aspect of the present invention relates to the use of the microbubble suspension according to the present invention in a method of treatment.

[0062] Therapeutic techniques include any method of treating a patient (as defined above) that includes the use of ultrasound in combination with gas-filled microbubbles themselves (e.g., in ultrasound-mediated thrombolysis, high-intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization) or in combination with a therapeutic agent (i.e., ultrasound-mediated delivery, e.g., for the delivery of drugs or biologically active compounds to selected sites or tissues, such as in tumor therapy, gene therapy, infectious disease therapy, metabolic disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immune or autoimmune disease therapy, or as a vaccine), whereby the presence of the gas-filled microbubbles can itself provide a therapeutic effect or can enhance the therapeutic effect of the applied ultrasound, for example, by exerting or being responsible for exerting a biological effect in vitro and / or in vivo, either by themselves or by specific activation by various physical methods (including, for example, ultrasound-mediated delivery).

[0063] Microvesicles according to the present invention can typically be administered for therapeutic purposes at a concentration of about 0.01 to about 5.0 μL of gas per kg of patient, the specific concentration depending on, for example, their respective composition, the type of subject being treated, the tissue or organ being treated, and / or the treatment method being employed.

[0064] In one embodiment, the ultrasound treatment method comprises:

[0065] (i) administering a suspension of gas-filled microbubbles according to the present invention to a patient;

[0066] (ii) identifying a region of interest in the patient to be treated, the region of interest comprising the gas-filled microbubble suspension; and

[0067] (iii) applying an ultrasound beam to treat the region of interest;

[0068] The ultrasound treatment is thereby enhanced by the presence of the gas-filled microbubble suspension at the area of ​​interest.

[0069] In another embodiment, the gas-filled microbubble suspension of the present invention can be advantageously used in a method for separating cells, which is typically achieved by buoyancy (also known as buoyancy-activated cell sorting, "BACS"). This method can be used to separate cells of the desired type from other cells in a physiological fluid (such as blood or plasma). In one embodiment, the separation method comprises labeling the desired cells to be separated with a suitable labeled antibody that is capable of binding to specific (and selective) receptors on the cells. The microbubbles of the present invention are then added to a suspension of cells to be separated (including cells with labeled antibodies); once mixed with the cell suspension, the microbubbles bind to the labeled residues bound to the antibody / cell construct via a ligand, thereby allowing the cells to be separated by buoyancy (see, for example, WO 2017 / 117349). For example, the labeled antibody is a biotinylated antibody, wherein the biotin residue is capable of binding to a corresponding portion (such as an avidin, neutravidin or streptavidin residue on the gas-filled microbubble). The improved pressure resistance allows the microbubbles of the present invention to be used in a variety of cell separation methods.

[0070] The following embodiments will help to further illustrate the present invention. Example

[0071] Material

[0072] DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

[0073] DPPG-Na: 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)

[0074] DPPE-PEG5000: 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt)

[0075] PEG4000 = polyethylene glycol (MW = 4000 g / mol)

[0076] Palmitic acid

[0077] SF6: sulfur hexafluoride

[0078] C4F 10 : Perfluorobutane

[0079] C3F6: Perfluoropropylene

[0080] 1-C4F8: Perfluoro-1-butene

[0081] 2-C4F8: Perfluoro-2-butene

[0082] 1-C5F 10 :Perfluoro-1-pentene

[0083] 2-C5F 10 :Perfluoro-2-pentene

[0084] 1-C6F 12 :Perfluoro-1-hexene

[0085] m-2-C6F 12 :Perfluoro(4-methyl-2-pentene)

[0086] Example 1

[0087] Preparation of vials containing freeze-dried product in contact with various gases (Formulations C1 and 1a-1o)

[0088] The procedure described in the working examples of WO 94 / 09829 was used to prepare the first batch of vials containing the freeze-dried product in contact with the gaseous headspace. Briefly, DSPC, DPPG-Na, and palmitic acid were first dissolved in hexane / ethanol (8 / 2, v / v) at a weight ratio of 4.75 / 4.75 / 1 to a concentration of approximately 5 g / L, and the solvent was then evaporated under vacuum. The solid 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 clear solution was used to fill the corresponding DIN8R vials (each volume containing approximately 25 mg of the mixture). The vials were then rapidly cooled at -45°C and then vacuum-treated to remove the frozen solvent by sublimation. The temperature was then raised to above room temperature, and the remaining solvent was evaporated to a final content of less than 0.5% (weight percentage). At the end of the freeze-drying process, the freeze-drying machine environment was saturated with SF6 at atmospheric pressure, and the vials were then stoppered and sealed. Thermal post-treatment was performed by heating the vials at about 45°C for about 16 hours as described in WO2020 / 229642.

[0089] The vials were then divided into different groups of approximately 3 vials each.

[0090] The first group (C1, comparative group) remained as is, while in the other groups, the headspace of the vials was replaced with a perfluoroolefin as defined in Table 1 or a mixture of a perfluoroolefin and nitrogen.

[0091] Table 1: Composition of the vial headspace

[0092] preparation Composition of the headspace of the vial (volume) C1 (control group) <![CDATA[100%SF6]]> 1a <![CDATA[100%C3F6]]> 1b <![CDATA[100%2-C4F8]]> 1c <![CDATA[100%1-C4F8]]> 1d <![CDATA[50%C3F6+50%N2]]> 1e <![CDATA[50%2-C4F8+50%N2]]> 1f <![CDATA[50%2-C5F 10 +50%N2]]> 1g <![CDATA[20%C3F6+80%N2]]> 1h <![CDATA[20%2-C4F8+80%N2]]> 1i <![CDATA[20%1-C4F8+80%N2]]> 1j <![CDATA[20%2-C5F 10 +80%N2]]> 1k <![CDATA[20%1-C5F 10 +80%N2]]> 1l <![CDATA[10%1-C6F 12 +90%N2]]> 1m <![CDATA[20%1-C6F 12 +80%N2]]> 1n <![CDATA[20%m-2-C6F 12 +80%N2]]> 1o <![CDATA[35%m-2-C6F 12 +65%N2]]>

[0093] Example 2

[0094] Preparation of vials containing freeze-dried product in contact with various gases (Formulations C2 and 2a-2f)

[0095] The procedure shown in the working example of WO2004 / 069284 was used to prepare a second batch of vials containing the freeze-dried product in contact with the gaseous headspace. Briefly, an emulsion (Megatron MT3000, Kinematica; 10,000 rpm) 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 was prepared and sampled into a DIN8R vial (approximately 0.75 mL / vial).

[0096] The vials were cooled at -50°C under vacuum and then dried once, followed by a second drying at a temperature above room temperature until the water and solvent were completely removed (less than 0.5% by weight), as described in Example 1. At the end of the freeze drying process, the vials were dried with a 35 / 65 mixture of C4F 10 The headspace of the vial was saturated with a mixture of N2 and the vial was then stoppered and sealed. Thermal post-treatment was performed by heating the vial at about 38°C for about 16 hours as described in WO2020 / 229642.

[0097] The vials were then divided into different groups of approximately 3 vials each.

[0098] The first group (C2, comparative group) remained as is, while in the other groups, the headspace of the vials was replaced with a perfluoroolefin as defined in Table 2 or a mixture of a perfluoroolefin and nitrogen.

[0099] Table 2: Composition of the vial headspace

[0100]

[0101]

[0102] Example 3

[0103] Preparation and Characterization of Gas-Filled Microbubble Suspensions (Suspensions C1 and 1a-1o)

[0104] A suspension of gas-filled microbubbles was obtained from the preparation shown in Example 1 by redispersing the corresponding freeze-dried product in 5 ml of physiological saline.

[0105] Using a Coulter counter Multisizer TM 3 (pore size 30 μm, size range 0.7 to 18 μm) to characterize the particle size distribution, total number of microbubbles, and the total amount of gas encapsulated within the microbubbles in the suspension.

[0106] The pressure resistance of gas-filled microbubbles is measured using a pressure turbidimeter (pressure nephelometer) developed internally. In short, the microbubble suspension of dilution is introduced into a spectrophotometer sample cell (airtight and connected to a pressurized system). The optical density (absorbance at 700nm) of the suspension is continuously recorded, while at a rate of about 4mmHg / s (533Pa / s), the pressure applied to the sample in the sample cell is linearly increased to the overpressure of two bars (2280mmHg, 303.9kPa) from atmospheric pressure (760mmHg, 101.3kPa).

[0107] The Pc50 parameter ("critical pressure") characterizing each suspension determines the overpressure (relative to atmospheric pressure) at which the absorbance of the microbubble suspension drops to half its initial value.

[0108] The suspensions prepared from the different formulations of Example 1 exhibited substantially similar concentrations, particle size distributions, and gas contents. However, the pressure resistance of the microbubbles containing selected perfluoroolefins (particularly perfluoropentene) was significantly improved compared to Comparative Formulation 1. On the other hand, as shown in Table 3, the formulations containing perfluoropropylene as the gas exhibited relatively low pressure resistance.

[0109] Table 3: Characteristics of gas-filled microbubbles

[0110] preparation Headspace composition Pressure resistance (Pc50, mmHg) C1 (comparison) <![CDATA[100%SF6]]> 308 1a <![CDATA[100%C3F6]]> 122 1b <![CDATA[100%2-C4F8]]> 322 1c <![CDATA[100%1-C4F8]]> 287 1d <![CDATA[50%C3F6+50%N2]]> 125 1e <![CDATA[50%2-C4F8+50%N2]]> 341 1f <![CDATA[50%2-C5F 10 +50%N2]]> 511 1g <![CDATA[20%C3F6+80%N2]]> 121 1h <![CDATA[20%2-C4F8+80%N2]]> 306 1i <![CDATA[20%1-C4F8+80%N2]]> 272 1j <![CDATA[20%2-C5F 10 +80%N2]]> 458 1k <![CDATA[20%1-C5F 10 +80%N2]]> 376 1l <![CDATA[10%1-C6F 12 +90%N2]]> 239 1m <![CDATA[20%1-C6F 12 +80%N2]]> * 1n <![CDATA[20%m-2-C6F 12 +80%N2]]> 304 1o <![CDATA[35%m-2-C6F 12 +65%N2]]> *

[0111] *The suspension is not stable enough to measure its pressure resistance

[0112] As can be inferred from the above table, formulations containing perfluoropentenes, particularly perfluoro-2-pentene, exhibit particularly improved pressure resistance compared to similar formulations containing SF6. Furthermore, formulations containing perfluoropentenes exhibit higher pressure resistance than formulations containing similar compositions of other perfluoroolefin gases having lower or higher molecular weights. Furthermore, it can be observed that formulations containing perfluoro-2-pentene unexpectedly exhibit higher pressure resistance than similar formulations containing perfluoro-1-pentene.

[0113] Example 4

[0114] Preparation and Characterization of Gas-Filled Microbubble Suspensions (Suspensions C2 and 2a-2j)

[0115] A suspension of gas-filled microbubbles was obtained from the preparation shown in Example 2 by redispersing the corresponding freeze-dried product in 5 ml of physiological saline.

[0116] The various gas-filled microbubble suspensions were characterized according to the procedure set forth in Example 3. The suspensions exhibited substantially similar concentrations, size distributions, and gas contents. Table 4 shows the pressure resistance measurements for each formulation.

[0117] Table 4: Characteristics of gas-filled microbubbles

[0118] preparation Headspace composition (vol) Pressure resistance (Pc50, mmHg) C2 <![CDATA[35%C4F 10 +65%N2]]> 623 2a <![CDATA[100%C3F6]]> 151 2b <![CDATA[100%2-C4F8]]> 295 2c <![CDATA[50%2-C5F 10 +50%N2]]> 464 2d <![CDATA[35%2-C5F 10 +65%N2]]> 418 2e <![CDATA[20%2-C5F 10 +80%N2]]> 369 2f <![CDATA[35%1-C5F 10 +65%N2]]> 319 2g <![CDATA[10%1-C6F 12 +90%N2]]> 230 2h <![CDATA[20%1-C6F 12 +80%N2]]> * 2i <![CDATA[20%m-2-C6F 12 +80%N2]]> 304 2j <![CDATA[35%m-2-C6F 12 +65%N2]]> *

[0119] *The suspension is not stable enough to measure its pressure resistance

[0120] From the table above, it can be inferred that although the pressure resistance of microbubbles containing perfluoroolefins is generally lower than that containing C4F 10 The pressure resistance of the microbubbles was not significantly different from that of the microbubbles, but the values ​​were still within the acceptable range for diagnostic / therapeutic uses of such microbubbles. Similar to the results of the previous examples, formulations containing perfluoropentenes exhibited higher pressure resistance than formulations with similar compositions containing other perfluoroolefin gases of lower or higher molecular weight. Furthermore, formulations containing perfluoro-2-pentene unexpectedly exhibited higher pressure resistance than similar formulations containing perfluoro-1-pentene.

Claims

1. A suspension of gas-filled microbubbles comprising an outer layer of stabilizing material surrounding a gaseous core, wherein the gaseous core comprises perfluoropentene.

2. The suspension according to claim 1, wherein the perfluoropentene is selected from perfluoro-1-pentene, perfluoro-2-pentene, or a mixture thereof.

3. The suspension according to claim 2, wherein the perfluoroolefin is perfluoro-2-pentene.

4. The suspension according to any one of the preceding claims, wherein the perfluoropentene is mixed with nitrogen. The suspension according to claim 4 , wherein the volume percentage of the perfluorocarbon is 10% to 70%. The suspension according to claim 4 , wherein the volume percentage of the perfluorocarbon is 10% to 60%.

7. The suspension according to any one of the preceding claims, wherein the stabilizing material comprises a phospholipid.

8. The suspension of claim 7, wherein the stabilizing material further comprises a fatty acid.

9. A vial comprising: - a gas-filled microvesicle precursor in the form of a freeze-dried product comprising a stabilizing material capable of forming said microvesicle stabilizing layer and a freeze-drying protective component; and - Gaseous components comprising perfluoropentene.

10. The vial of claim 9, wherein the perfluoroolefin is selected from perfluoro-1-pentene, perfluoro-2-pentene, or a mixture thereof.

11. The vial of claim 10, wherein the perfluoroolefin is perfluoro-2-pentene.

12. The vial according to any one of the preceding claims 8 to 11, wherein the perfluoroolefin is mixed with nitrogen.

13. The vial according to claim 12, wherein the volume percentage of the perfluoroolefin is 10% to 70%.

14. The vial according to claim 12, wherein the volume percentage of the perfluoroolefin is 10% to 60%.

15. The vial according to any one of claims 9 to 14, wherein the stabilizing material comprises a phospholipid.

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