RU2827409C2 - Lyophilised product and suspension of gas-filled microvesicles - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to production and use of ultrasound imaging techniques with contrast enhancement. Disclosed is a method of producing a lyophilised composition containing (i) an amphiphilic substance comprising a phospholipid and a fatty acid, and (ii) polyethylene glycol as a protective component for lyophilisation, and suitable for preparing a suspension of stabilized gas-filled microvesicles, where the method includes: a) obtaining a liquid mixture comprising said amphiphilic substance and said protective component for lyophilisation in a solvent; b) lyophilizing the liquid mixture to remove said solvent and obtain a lyophilised product; and c) heating said lyophilised product at atmospheric pressure at a temperature higher than 35 °C and up to 50 °C for a period of time from 8 to 20 hours. Following is also proposed: method of making a suspension of gas-filled microvesicles, which involves reconstitution of a lyophilised product obtained according to said method using a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas with gentle stirring, and a diagnostic method, which includes (i) administering to a patient a suspension of gas-filled microvesicles obtained by said method; and (ii) detecting the ultrasound signal from the region of interest in said patient.

EFFECT: use of the group of inventions provides improved characteristics of the suspension of gas-filled microvesicles obtained by reconstitution of the lyophilised product, which is subjected to additional thermal treatment, compared to suspensions obtained from lyophilised products that have not been subjected to such thermal treatment; in particular, the above thermal treatment of the lyophilised product leads to increased pressure resistance of gas-filled microvesicles, which, as a rule, exhibit an increased duration of presence in the bloodstream after injection.

19 cl, 6 tbl, 4 ex

Description

Field of technology to which the invention relates

The present invention relates to a new method for producing a suspension of gas-filled microvesicles by reconstituting a lyophilized product and to a suspension obtained in accordance with said method.

Prerequisites for the creation of an invention

The rapid development of contrast agents in recent years has resulted in a number of different compositions and formulations that are useful for producing contrast-enhanced images of organs and tissues of the human or animal body, as well as for their therapeutic treatment.

A class of contrast agents particularly useful for contrast-enhanced ultrasound imaging (CEUS imaging) includes suspensions of nano- and/or micrometer-sized gas bubbles dispersed in an aqueous medium. The gas is typically entrapped or encapsulated in a film layer comprising, for example, emulsifiers, oils, thickeners or sugars. These stabilized gas bubbles (dispersed in a suitable saline solution) are commonly referred to in the art by various terms, typically depending on the stabilizing agent used to produce them; these terms include, for example, "microspheres", "microvesicles", "microcapsules" or "microbeads", which are collectively referred to in this application as "gas-filled microvesicles" (or "microvesicles").

Ultrasound contrast agents (“USCA”) can be prepared according to various manufacturing processes. One of these processes, see, for example, WO94/09829, involves dissolving an amphiphilic substance (such as a phospholipid and/or a fatty acid) and a lyophilization-protecting compound (such as polyethylene glycol) in an organic solvent; the resulting mixture is then lyophilized, typically after filling into vials, to remove the solvent and produce a lyophilized product. Another process, see, for example, WO2004/069284, involves preparing a microemulsion of water and a water-immiscible organic solvent, said emulsion comprising an amphiphilic substance and a lyophilization-protecting compound. The emulsion is then (after distribution into vials) subjected to a lyophilization step to remove the water and solvent.

The free volume of the vials containing the lyophilized solid product in powder form at the bottom is then filled with a suitable gas (e.g., fluorinated gas) and finally sealed for storage. Before use, an aqueous suspension of the microvesicles is easily prepared by adding a suitable liquid (e.g., saline) to the vial and gently shaking the vial to dissolve the lyophilized product.

A commercially available USCA that can be prepared according to the above method is SonoVue® (or ^Lumason® in the USA) from Bracco.

The Applicant has found that the performance of a suspension of gas-filled microvesicles (especially microvesicles) reconstituted from a lyophilized product can be improved by introducing a final controlled thermal treatment (i.e. heating) at the end of the process of manufacturing the lyophilized solid product.

The essence of the invention

According to one aspect, the present invention relates to a method for the manufacture of a lyophilized composition suitable for the production of a suspension of stabilized gas-filled microvesicles, said composition comprising: (i) an amphiphilic substance capable of stabilizing said gas microvesicles; and (ii) a lyophilization protectant; which comprises:

a. obtaining a liquid mixture comprising said amphiphilic substance and said protective component for lyophilization in a solvent;

b. lyophilizing the liquid mixture to remove said solvent and obtain a lyophilized product comprising said amphiphilic substance and said lyophilization protectant; and

c. heating the said lyophilized product.

Preferably, said heating step comprises heating said product at a temperature above 35°C, more preferably at least 38°C. The heating temperature is preferably below 50°C, more preferably below 48°C.

In some embodiments, the heating step is carried out at atmospheric pressure.

Preferably, the heating step lasts for at least 8 hours, more preferably for at least 12 hours.

According to another aspect, the present invention relates to a lyophilized product obtained according to the manufacturing method described above.

According to another aspect, the present invention relates to a suspension of gas-filled microvesicles obtained by reconstituting a lyophilized product obtained according to the manufacturing method described above, wherein said suspension is obtained by mixing said product with a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas with gentle stirring.

According to a further aspect, the present invention relates to a method for producing a suspension of gas-filled microvesicles stabilized by an amphiphilic substance, which comprises:

a. obtaining a lyophilized product in accordance with the manufacturing method illustrated above; and

b. reconstituting said product by mixing it with a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas with gentle stirring to obtain a suspension of gas-filled microvesicles.

Detailed description of the invention

A suitable method for preparing suspensions of gas-filled microvesicles for injection comprises reconstituting, in the presence of a suitable physiologically acceptable gas, a lyophilized product comprising an amphiphilic substance capable of stabilizing said microvesicles (e.g. by forming a stabilizing layer at the liquid-gas interface), using an aqueous carrier.

The lyophilized product is typically obtained by lyophilizing a liquid mixture comprising the said amphiphilic substance and a lyophilization protecting component in a suitable solvent.

The liquid mixture which is subjected to the lyophilization process can be obtained by methods known in the art, for example, those disclosed in WO94/09829 or WO2004/069284.

Obtaining a liquid mixture for lyophilization

For example, according to the method disclosed in WO94/09829, an amphiphilic substance is dispersed in an organic solvent (e.g., tertiary butanol, dioxane, cyclohexanol, tetrachlorodifluoroethylene or 2-methyl-2-butanol) together with a suitable lyophilization protecting agent. The dispersion containing the amphiphilic substance and the lyophilization protecting agent is then lyophilized to remove the organic solvent, thereby obtaining a lyophilized product.

According to an alternative method disclosed in WO2004/069284, a composition comprising an amphiphilic substance may be dispersed in an emulsion of water with a water-immiscible organic solvent under stirring, preferably in admixture with a lyophilisation protectant.

Suitable water-immiscible organic solvents include, for example, branched or linear alkanes, alkenes, cycloalkanes, aromatic hydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons, perfluorinated hydrocarbons, or mixtures thereof.

The emulsion can be prepared by subjecting an aqueous medium and a solvent, in the presence of an amphiphilic substance, to any suitable emulsion preparation method known in the art, such as, for example, sonication, shaking, high-pressure homogenization, micromixing, membrane emulsification, high-speed mixing or high-shear mixing. A lyophilization protectant can be added either before or after the formation of the emulsion, for example in the form of an aqueous solution comprising such a lyophilization protectant. The thus obtained microemulsion, which contains microdroplets of solvent surrounded and stabilized by the amphiphilic substance, is then lyophilized according to conventional methods to obtain a lyophilized substance, which can then be used to prepare a suspension of gas-filled microvesicles.

Amphiphilic substance

According to a preferred embodiment, the amphiphilic substances useful for preparing the above liquid mixtures include a phospholipid. Phospholipids, like other amphiphilic molecules, are typically capable of forming a stabilizing film of material (usually in the form of a monomolecular layer) at the gas-water interface in the final suspension of gas-filled microvesicles, these substances are also referred to in the art as "film-forming" substances.

Phospholipids typically contain at least one phosphate group and at least one, preferably two, long-chain lipophilic hydrocarbon groups.

Examples of suitable phospholipids include esters of glycerol with one or, preferably, two (the same or different) fatty acid residues and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group such as, for example, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol). Esters of phospholipids with only one fatty acid residue are usually referred to in the art as "lyso" forms of phospholipid or "lysophospholipids". The fatty acid residues present in phospholipids are typically long-chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated. Examples of suitable fatty acids included in the phospholipids are, for example, 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 are used.

Other examples of phospholipids are phosphatidic acids, i.e., glycerol-phosphoric acid diesters with fatty acids; sphingolipids such as the sphingomyelins, i.e., those phosphatidylcholine analogues in which the residue of the glycerol-fatty acid diester is replaced by a ceramide chain; cardiolipins, i.e., 1,3-diphosphatidylglycerol fatty acid esters; glycolipids such as the GM1 (or GM2) gangliosides or cerebrosides; glucolipids; sulfatides; and glycosphingolipids.

In the context of the present application, the term “phospholipid(s)” includes naturally occurring, semi-synthetic or synthetically produced compounds which can be used either individually or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives), such as those found in soybeans or egg yolk.

Examples of semi-synthetic phospholipids are partially or fully hydrogenated derivatives of natural lecithins. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or sphingomyelin.

Examples of preferred phospholipids are, for example, 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 (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-oleylphosphatidylcholine (POPC), 1-oleyl-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, Distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, Dioleoyl-phosphatidylglycerol (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, dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidylethanolamine (DSPE), dioleoyl phosphatidyl ethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleoyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoyl sphingomyelin (DSSP), dilauroyl phosphatidylinositol (DLPI), diarachidoyl phosphatidylinositol (DAPI), Dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI).

Suitable phospholipids also include phospholipids modified by the attachment thereto of a hydrophilic polymer such as polyethylene glycol (PEG) or polypropylene glycol (PPG). Preferred polymer-modified phospholipids include “pegylated phospholipids”, i.e. phospholipids linked to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines (in short “PE-PEG”), i.e. phosphatidylethanolamines in which a hydrophilic ethanolamine moiety is linked to a PEG molecule of varying molecular weight (e.g., from 300 to 20,000 daltons, preferably from 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 having a PEG polymer attached thereto having an average molecular weight of about 5000. An example of DPPE-PEG5000 is the methoxy-terminated PEG derivative 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000].

In one embodiment, the phospholipids may comprise a reactive group which can then react with a corresponding reactive group comprising a suitable active component (e.g., a targeting ligand) to bind said active component to the microvesicles. Examples of suitable reactive groups include, for example, reactive groups capable of reacting with an amino group linked to the active component, such as isothiocyanate groups (which form a thiourea bond), reactive esters (to form an amide bond), aldehyde groups (to form an imine bond which must be reduced to an alkylamine bond); reactive groups capable of reacting with a thiol group linked to the active component, such as haloacetyl derivatives or maleimides (to form a thioether bond); reactive groups capable of reacting with the carboxyl group bound to the active component, such as amines or hydrazides (to form amide or alkylamide bonds). Preferably, the amphiphilic compound containing the reactive group is a lipid containing a hydrophilic polymer such as those mentioned above, preferably a PEGylated phospholipid, such as DPPE-PEG2000, such as DPPE-PEG2000-maleimide.

Particularly preferred phospholipids are DAPC, DSPC, DPPC, DMPA, DPPA, DSPA, DMPG, DPPG, DSPG, DMPS, DPPS, DSPS and Ethyl-DSPC. Most preferred are DPPG, DPPS and DSPC.

Mixtures of phospholipids can also be used, such as, 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.

Phospholipids are conveniently used in a mixture with any other compound, preferably amphiphilic. For example, lipids such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and their derivatives, or butylated hydroxytoluene and/or other non-phospholipid (amphiphilic) compounds can be optionally added to one or more of the above phospholipids, for example in proportions of preferably less than 50% by weight, more preferably up to 25% by weight or less. Particularly preferred additional compounds in a mixture with phospholipids are fatty acids. Fatty acids useful in the composition of the present invention, which may be either saturated or unsaturated, comprise a _C10 - _C24 aliphatic chain with a terminal carboxylic acid group, preferably _C14 - _C22 and more preferably a _C16 - _C20 aliphatic chain. Examples of suitable saturated fatty acids include capric (n-decanoic), lauric (n-dodecanoic), myristic (n-tetradecanoic), palmitic (n-hexadecanoic), stearic (n-octadecanoic), arachidic (n-eicosanoic), behenic (n-docosanoic) and n-tetracosanoic acid. Preferred saturated fatty acids are myristic, palmitic, stearic and arachidic acids, more preferably palmitic acid. Examples of unsaturated fatty acids include myristoleic (cis-9-tetradecenoic), palmitoleic (cis-9-hexadecenoic), sapienic (cis-6-hexadecenoic), oleic (cis-9-octadecenoic), linoleic (cis-9,12-octadecadienoic), linolenic (cis-9,12,15-octadecatrienoic), gondoaic (cis-11-eicosenoic), cis-11,14-eicosadienoic, cis-5,8,11-eicosatrienoic, cis-8,11,14-eicosatrienoic, cis-11,14,17-eicosatrienoic, arachidonic (cis-8,11,14,17-eicosatetraenoic) and erucic (cis-13-docosenoic) acid.

According to an embodiment, the mixture of amphiphilic substances comprises a mixture of DSPC, DPPG and palmitic acid.

According to an alternative embodiment, said amphiphilic substance comprises a mixture of DSPC, DPPE-PEG5000 and palmitic acid, optionally further comprising a targeting ligand.

Targeting ligands

The compositions and microvesicles of the present invention may optionally include a targeting ligand.

The term "targeting ligand" encompasses within its meaning any compound, fragment or residue that has or is capable of stimulating the target activity (e.g., including selective binding) of the microvesicles of the composition of the present invention with respect to any biological or pathological site in a living organism. Targets to which the targeting ligand can bind include tissues such as, for example, myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), platelets, connective tissue (including interstitial tissue) or tumors; blood clots; and receptors such as, for example, cell surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments and immunoglobulins, as well as cytoplasmic receptors for steroid hormones.

The targeting ligand may be synthetic, semi-synthetic, or of natural origin. Materials or substances that can serve as targeting ligands include, for example, but are not limited to, proteins, including antibodies, antibody fragments, receptor molecules, receptor-binding molecules, glycoproteins, and lectins; peptides, including oligopeptides and polypeptides; peptidomimetics; saccharides, including mono- and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive substances, and genetic material, including nucleosides, nucleotides, and polynucleotides.

The targeting ligand may be a compound per se that is mixed with other components of the microvesicle, or may be a compound that is linked to an amphiphilic molecule (usually a phospholipid) used to form the microvesicle.

In one preferred embodiment, the targeting ligand may be linked to an amphiphilic molecule (e.g., a phospholipid) to form a stabilizing shell of the microvesicle by means of a covalent bond. In such a case, the specific reactive group that should be present in the amphiphilic molecule will depend on the specific targeting ligand that is to be linked thereto, as illustrated in detail above. For covalent linkage to the desired targeting ligand, at least a portion of the amphiphilic compound forming the shell of the microvesicle must therefore contain a suitable reactive group, and linkage of the targeting ligand containing a complementary functional group thereto will be carried out according to known methods, for example, by adding it to a dispersion containing the amphiphilic components of the microvesicle. Preferably, the amphiphilic compound is a lipid comprising a hydrophilic polymer such as those mentioned above, preferably a pegylated phospholipid (e.g. DSPE-PEG2000). In this case, the targeting ligand is coupled to a suitable reactive group on the hydrophilic polymer (e.g. DSPE-PEG2000- _NH2 ), optionally via a linker. The amphiphilic compound can be combined with the desired targeting ligand before the preparation of the microvesicle, and the combination thus obtained can be used to prepare the microvesicle. Alternatively, the coupling of the targeting ligand to the corresponding amphiphilic compound can be carried out during the preparation of the microvesicle (e.g. during the preparation of the intermediate microemulsion by the method described in WO2004/069284). As a further alternative, binding can occur on a formed microvesicle comprising an amphiphilic substance containing a reactive group.

According to an alternative embodiment, the targeting ligand can also be suitably linked to the microvesicle via physical and/or electrostatic interaction. As an example, a functional moiety having high affinity and selectivity for a complementary moiety can be introduced into the amphiphilic molecule, while the complementary moiety is linked to the targeting ligand. For example, an avidin (or streptavidin) moiety (having high affinity for biotin) can be covalently linked to a phospholipid (or a pegylated phospholipid), while the complementary biotin moiety can be included in a suitable targeting ligand, such as a peptide or an antibody. Thus, the biotin-labeled targeting ligand will be linked to the avidin-labeled phospholipid of the microvesicle via the avidin-biotin binding system. Alternatively, both the phospholipid and the targeting ligand may both include a biotin moiety and may subsequently be linked to each other via avidin (which is a bifunctional component capable of linking two biotin moieties). Examples of biotin/avidin linkage of phospholipids and peptides are also disclosed in the above-cited U.S. Patent 6,139,819. Alternatively, van der Waals interactions, electrostatic interactions, and other association processes may be used to associate or link the targeting ligand to the amphiphilic molecules.

Alternatively, the phospholipid may be modified with a protein suitable for specific binding to the Fc domain of immunoglobulin (Ig), such as protein A, protein G, protein A/G or protein L. According to an alternative embodiment, the targeting ligand may be a compound that is mixed with the components forming the microvesicle for eventual incorporation into the microvesicle structure, such as, for example, a lipopeptide, as described, for example, in International Patent Applications WO 98/18501 or 99/55383.

Alternatively, a microvesicle may first be prepared that includes a compound (lipid or polymer-modified lipid) having a suitable group capable of interacting with the corresponding complementary moiety of the targeting ligand; the desired targeting ligand is then added to the microvesicle suspension to bind to the corresponding complementary moiety on the microvesicle.

Examples of suitable specific targets that microvesicles can be directed to are, for example, fibrin and the GPIIbIIIa binding receptor on activated platelets. In fact, fibrin and platelets are commonly present in "thrombi", i.e. clots that can form in the bloodstream and cause vascular obstruction. Suitable binding peptides are disclosed, for example, in the above-cited US Patent 6,139,819. Other binding peptides specific for targeting fibrin are disclosed, for example, in International Patent Application WO 02/055544.

Other examples of important targets include receptors in unstable plaques and tumor-specific receptors such as the kinase domain region (KDR) and the VEGF (vascular endothelial growth factor)/KDR complex. Binding peptides suitable for KDR or the VEGF/KDR complex are disclosed, for example, in International Patent Applications WO 03/74005, WO 03/084574 and WO2007/067979. In one embodiment, the target peptide is a dimeric peptide-phospholipid conjugate (lipopeptide) as described in WO2007/067979.

Protective component for lyophilization

As defined in the present application, a lyophilization protecting agent is a compound with a cryoprotective and/or lyoprotective effect. Suitable lyophilization protecting agents include, for example, carbohydrates, for example a mono-, di- or polysaccharide, such as sucrose, maltose, trehalose, glucose, lactose, galactose, raffinose, cyclodextrin, dextran, chitosan and its derivatives (e.g. carboxymethyl chitosan, trimethyl chitosan); polyols, for example sugar alcohols such as sorbitol, mannitol or xylitol; or hydrophilic polymers, for example a polyoxyalkylene glycol, such as polyethylene glycol (e.g. PEG2000, PEG4000 or PEG8000) or polypropylene glycol. According to an embodiment, said lyophilization protecting agent is polyethylene glycol, preferably PEG4000. PEG4000 in the context of the present application has its usual meaning known in the art and means polyethylene glycol having a molecular weight of about 4000 g/mol, typically with a deviation of +/- 10% from the stated value.

Lyophilization process

For the lyophilization process, a liquid mixture containing an amphiphilic substance and a lyophilization protective component (obtained, for example, according to any of the previously illustrated manufacturing methods) is typically dispensed into glass vials (e.g. DIN8R or DIN20R), which are loaded into a lyophilizer.

The lyophilization process typically includes an initial step (primary drying) in which the vials are subjected to rapid deep cooling (e.g. at temperatures between -35°C and -70°C) to freeze the liquid(s) of the mixture and then subjected to vacuum conditions (e.g. 0.1-0.8 mbar); during the primary drying, a significant portion of the frozen liquid(s) (e.g. water and/or solvents) is removed by sublimation, typically up to about 95% of the total liquid, preferably up to about 99%. After the primary drying, the residual liquid (including any intervening water) may then be removed in a secondary drying process, which is typically carried out at a temperature above room temperature, under vacuum (preferably by maintaining the same vacuum as during the primary drying). The temperature during the secondary drying preferably does not exceed 35°C. The secondary drying may be stopped when the residual liquid(s) content reaches a desired minimum value, for example less than 3% (preferably less than 1%) by weight of water based on the total weight of the residual lyophilized product, or, for example, less than 0.01% by weight, preferably less than 0.08% for the residual solvent(s).

After completion of the lyophilization process (i.e. cessation of heating and removal of the vacuum), the lyophilized product may be subjected to an additional step of heat treatment according to the present invention, at atmospheric pressure. In the context of the present application, the term "atmospheric pressure" refers to the normal value of atmospheric pressure (i.e. about 103.12 kPa at sea level, typically between 95 and 104 kPa). Preferably, the heat treatment is carried out on a tightly closed vial, after saturating the free volume of the vials containing the lyophilized product with a suitable physiologically acceptable gas, and then sealing (e.g. with a rubber stopper, e.g. butyl rubber) and hermetically sealing (e.g. with a metal, e.g. aluminum, crimp seal) the vials. In this case, the vials are preferably removed from the lyophilizer and placed in a suitable oven for heat treatment. Alternatively, such heat treatment can be carried out on an open vial (preferably stored in a lyophilizer), which is then gassed and then stoppered/sealed.

_{Examples of} suitable physiologically acceptable gases include, for example, fluorinated gases such as _SF6 , _C3F8 , _C4F10 , optionally mixed with air or nitrogen.

In one embodiment, SF ₆ gas is used in combination with a mixture of amphiphiles comprising DSPC, DPPG and palmitic acid as defined above.

In another embodiment _{, C4F10} gas, or a mixture of _C4F10 with _nitrogen , is used in combination with a mixture of amphiphiles comprising DSPC, DPPE-PEG5000 and palmitic acid, optionally further comprising a targeting ligand as defined above.

Other components

Other components, such as excipients or additives, may either be present in the dry composition for obtaining microvesicles or may be added together with the aqueous vehicle used for its reconstitution, optionally participating (or only partially participating) in the formation of the stabilizing shell of the microvesicle. These include, for example, pH regulators, osmolality regulators, viscosity enhancers, emulsifiers, fillers, etc., and they can be used in conventional amounts.

Suspensions of gas-filled microvesicles

Suspensions of gas-filled microvesicles can then be prepared by reconstituting the lyophilized product using a physiologically acceptable (aqueous) carrier, with gentle stirring. Suitable physiologically acceptable (aqueous) carriers include, for example, water for injection, physiological saline or glucose solution, optionally containing excipients or additives as illustrated above.

Heat treatment

According to the present invention, the lyophilized product (contained in the respective vials at the end of the lyophilization process) is advantageously subjected to an additional final thermal treatment (or heat treatment) step.

As stated above, the heat treatment is preferably carried out on the lyophilized product in sealed vials already containing a physiologically acceptable gas; alternatively, it can be carried out on the lyophilized product in vials before filling them with gas and sealing them. In the first case, the heat treatment can be carried out either in a lyophilizer or, preferably, in a separate heating device (e.g., an oven). In the second case, the heating step is preferably carried out in a lyophilization apparatus; then the atmosphere is saturated with the desired gas and the vials are hermetically sealed.

As noted by the applicant, the said heat treatment of the lyophilized product unexpectedly leads to improved characteristics of the suspension of gas-filled microvesicles obtained upon reconstitution of the lyophilized product, compared to suspensions obtained from lyophilized products that have not been subjected to such heat treatment.

The applicant, in particular, noted that such treatment leads to increased resistance of the obtained microvesicles to pressure.

The lyophilized product is preferably heated at a temperature above 35°C (for example, 36°C), more preferably at a temperature of 38°C or higher. The maximum heat treatment temperature generally depends on the substances contained in the lyophilized product. For example, such a temperature should be lower than the melting point of the substance used as a lyophilization additive, which is the component that forms the majority of the mass of the lyophilized product (usually has a mass of 50 to more than 600 times the mass of the active components that form the stabilizing layer of the microvesicles). For example, PEG4000 has a melting point of 53-58°C. According to an embodiment, the heating temperature is preferably 50°C or lower. Preferred temperatures for heat treatment are from 38°C to 45°C.

The duration of the heat treatment generally depends on the temperature of the treatment; generally, the higher the temperature, the shorter the heating duration. Since the substances forming the shell of the gas-filled microvesicles (in particular phospholipids) may undergo a decomposition reaction when exposed to excessive temperatures for too long a period of time (with possible negative consequences for the characteristics of the recovered microvesicles), the duration of the heat treatment should not be increased unnecessarily. Although a treatment duration of about 8 hours may be sufficient (especially in combination with temperatures above 45°C, for example 48°C), the duration of the heat treatment is preferably 12 hours, for example up to 20 hours, more preferably 14-18 hours. Although longer heat treatments may well be used in specific cases (especially in combination with temperatures below 45°C, preferably below 42°C), the Applicant has found that this results in only minor, if any, further improvement in the characteristics of the final gas-filled microvesicles; such an extended duration is therefore in most cases unnecessary and is usually inconvenient from the point of view of economy in production on an industrial scale.

In some embodiments, the lyophilized product comprises a mixture of a phospholipid and a fatty acid as defined above, in a mixture with a protective component for lyophilization. It has been proven that the thermal treatment of the invention is particularly effective for improving the characteristics of gas-filled microvesicles comprising such a mixture of components.

According to one embodiment, the lyophilized product comprises DSPC, DPPG and palmitic acid in combination with a lyophilization protective component (e.g. polyethylene glycol, such as PEG4000). Said lyophilized product is preferably heated at a temperature of about 40°C to 48°C, in particular about 45°C (+/- 3°C), for at least eight hours, preferably for about 18 hours (+/- 4 hours).

According to another embodiment of the invention, the lyophilized product comprises DSPC, DPPE-PEG5000 and palmitic acid in combination with a lyophilization protective component (e.g. polyethylene glycol, such as PEG4000). Said lyophilized product is heated at a temperature of about 36°C to 45°C, in particular about 39°C (+/- 3°C), for at least eight hours, preferably for about 15 hours (+/- 5 hours).

According to another embodiment, said mixture of DSPC, DPPE-PEG5000 and palmitic acid further comprises a targeting lipopeptide, for example as described in WO2007/067979.

As indicated above, the heat treatment of the lyophilized product of the present invention results in increased resistance of the gas-filled microvesicles to pressure. Preferably, the microvesicles with increased resistance to pressure generally exhibit an increased duration of presence in the bloodstream after injection.

The pressure resistance of gas-filled microvesicles can be assessed by determining the empirical parameter “Pc50” or “critical pressure”.

As explained in detail in the experimental section, the Pc50 value of a gas-filled microvesicle suspension defines the value of the applied excess pressure (relative to the atmospheric pressure) at which the absorptivity of the microvesicle suspension drops to half the absorptivity of the suspension measured at atmospheric pressure, wherein said applied excess pressure results in a significant decrease in the microvesicle population compared to the initial one (at atmospheric pressure). In fact, the decrease in the absorptivity of the microvesicle suspension is associated with a decrease in the initial population of gas-filled microvesicles, resulting in an initially milky suspension (high concentration of microvesicles) becoming increasingly transparent with increasing pressure (decreasing concentration due to the destruction of microvesicles). The higher the Pc50 values, the higher the resistance of the microvesicles to pressure. For ultrasound diagnostic applications, a minimum Pc50 value of at least 12 kPa is desirable for gas-filled microvesicles, preferably at least 13 kPa (about 100 mmHg), more preferably at least 14 kPa (105 mmHg). For ultrasound therapeutic applications, which typically require a longer residence time in the bloodstream, a minimum Pc50 value of at least 55 kPa (about 412 mmHg), preferably at least 70 kPa (about 525 mmHg), more preferably at least 80 kPa (about 600 mmHg) is desirable, while higher Pc50 values are even more preferred.

Typically, the heat treatment of the lyophilized product of the present invention allows to increase the Pc50 of the reconstituted suspension of microvesicles by at least 5 kPa, preferably at least 8 kPa and more preferably at least 10 kPa compared to the Pc50 of the reconstituted suspension obtained from the lyophilized product that has not been subjected to such heat treatment. Such an increase in Pc50 can be up to 15 kPa, and in some embodiments up to 25 kPa.

According to an embodiment (for example, when the lyophilized product comprises DSPC, DPPG, palmitic acid and PEG4000), the microvesicle suspension reconstituted from the lyophilized product subjected to heat treatment according to the present invention has a Pc50 value of at least 20 kPa, preferably at least 22 kPa and more preferably at least 25 kPa.

According to another embodiment (for example, when the lyophilized product comprises DSPC, DPPE-PEG5000 and palmitic acid in combination with a lyophilization protective component, for example a polyethylene glycol such as PEG4000), the microvesicle suspension reconstituted from the lyophilized product subjected to heat treatment according to the present invention has a Pc50 value of at least 75 kPa, preferably at least 80 kPa and more preferably at least 90 kPa.

Pharmaceutical kit, administration and methods of use

Vials containing the lyophilized product may preferably be packaged as a two-component diagnostic and/or therapeutic kit, preferably for administration by injection. The kit preferably comprises a vial containing the lyophilized product and a second container (e.g., a syringe barrel) containing a physiologically acceptable aqueous carrier for reconstitution.

The microvesicles of the present invention can be used in various diagnostic and/or therapeutic methods, including, in particular, ultrasound.

Thus, an aspect of the invention relates to the use in a diagnostic method of a suspension of microvesicles reconstituted from a lyophilized product subjected to heat treatment according to the present invention.

Diagnostic methods include any method in which the use of gas-filled microvesicles improves visualization of an area or part of the body of an animal (including humans), including imaging for preclinical and clinical research purposes. For ultrasound applications, various imaging techniques may be used, including, for example, fundamental and harmonic B-mode imaging, pulsed or phase inversion imaging, and fundamental and harmonic Doppler imaging; three-dimensional imaging techniques may be used if desired.

The microvesicles of the present invention can typically 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. This general concentration range can, of course, vary depending on specific imaging applications, for example, where signals can be observed at very low doses, such as in color Doppler or pulse power inversion.

In one embodiment, said diagnostic method comprises

(i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstituting a lyophilized product obtained in accordance with the method of the present invention; and

(ii) detection of an ultrasound signal from a region of interest in a specified patient.

According to an embodiment, said suspension of gas-filled microvesicles comprises DSPC, DPPG, palmitic acid and PEG4000,

The reconstitution of the lyophilized product is preferably carried out by dispersing it in a physiologically acceptable aqueous carrier, such as a physiological solution, in the presence of a physiologically acceptable gas, such as SF ₆ , with gentle stirring.

Said microvesicle suspension preferably has a Pc50 value of at least 20 kPa, more preferably at least 22 kPa and even more preferably at least 25 kPa.

In one embodiment, the diagnostic method includes cardiac ultrasound imaging, in particular to opacify the left ventricular chamber and improve the delineation of the left ventricular endocardial border in adult patients with suboptimal echocardiograms.

In another embodiment, the said diagnostic method includes ultrasound imaging of the liver, in particular for characterizing focal liver lesions in adults and children.

In another embodiment, said diagnostic method includes ultrasound imaging of the urinary tract, in particular for evaluating suspected or known vesicoureteral reflux in pediatric patients.

In another embodiment of said diagnostic method, said suspension of gas-filled microvesicles comprises DSPC, DPPE-PEG5000, palmitic acid, optionally a targeting lipopeptide and PEG4000. Preferably, the targeting lipopeptide is a VEGF/KDR targeting lipopeptide, for example as described in WO2007/067979.

Other possible diagnostic imaging applications include scintigraphy, light imaging, and X-ray imaging, including X-ray phase contrast imaging.

Another aspect of the invention relates to the use in a method of therapeutic treatment of a suspension of microvesicles reconstituted from a lyophilized product subjected to heat treatment according to the present invention.

Therapeutic methods include any method of treating (as defined above) a patient that involves the combined use of ultrasound techniques and gas-filled microvesicles per se (e.g. in ultrasound thrombolysis, high-intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization) in combination with a therapeutic agent (i.e. ultrasound-mediated delivery, e.g. for delivering a drug or biologically active compound to a selected site or tissue, e.g. in tumor therapy, gene therapy, infectious disease therapy, metabolic disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immunological or autoimmune disease therapy, or for use as a vaccine), wherein the presence of the gas-filled microvesicles may itself provide a therapeutic effect or be capable of enhancing the therapeutic effects of the ultrasound techniques used, e.g. by causing them, or when they are responsible for causing them, biological effect in vitro and/or in vivo, either independently or upon specific activation by various physical methods (including, for example, ultrasound-mediated delivery).

The microvesicles of the present invention can typically be administered for therapeutic purposes at a concentration of from about 0.01 to about 5.0 μl of gas per kg of patient, depending, for example, on their respective composition, the type of subject being treated, the tissue or organ being treated, and/or the therapeutic method being employed.

In one embodiment, said method of therapeutic treatment using ultrasound comprises:

(i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstituting a lyophilized product obtained in accordance with the method of the present invention;

(ii) identifying a region of interest in said patient to be therapeutically treated, said region of interest comprising said suspension of gas-filled microvesicles; and

(iii) the use of an ultrasound beam for therapeutic treatment of the said area of interest;

whereby said therapeutic ultrasound treatment is enhanced by the presence of said suspension of gas-filled microvesicles in said region of interest.

In one embodiment, said gas-filled microvesicle suspension comprises DSPC, DPPE-PEG5000, palmitic acid, optionally a targeting lipopeptide and PEG4000.

The reconstitution of the lyophilized product is preferably carried out by dispersing it in a physiologically acceptable aqueous carrier, for example, a physiological solution, in the presence of a physiologically acceptable gas, for example, a mixture of C ₄ F ₁₀ and nitrogen, with gentle stirring.

Said microvesicle suspension preferably has a Pc50 value of at least 84 kPa, more preferably at least 88 kPa and even more preferably at least 90 kPa, such as up to about 105 kPa.

In another embodiment, a suspension of gas-filled microvesicles according to the invention can be advantageously used in a method for separating cells, typically by buoyancy (also known as buoyancy-activated cell sorting "BACS"). The method can be useful for separating a desired cell type from other cells in a physiological fluid (e.g., blood or plasma). In one embodiment, the separation method comprises labeling a desired cell to be separated with a suitable labeled antibody capable of binding to a specific (and selective) receptor on said cell. The microvesicles according to the invention are then added to a suspension of cells to be separated (including those bearing the labeled antibody); after mixing with the cell suspension, the microvesicles bind via a ligand to a labeling moiety linked to the antibody/cell construct, allowing the cells to be separated by buoyancy (see, e.g., WO 2017/117349). For example, the labeled antibody is a biotinylated antibody, wherein the biotin residue is capable of binding to a corresponding moiety, such as, for example, an avidin, neutravidin or streptavidin residue, on gas-filled microvesicles. The increased pressure resistance allows the microvesicles of the invention to be used in a wide variety of cell separation methods.

The following examples will help to further illustrate the invention.

EXAMPLES

Materials

DSPC: 1,2-distearoyl-sn-glycero-3-phosphocholine

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

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

PEG4000=polyethylene glycol (MW=4000 g/mol)

Pressure resistance measurement (Pc50)

The pressure stability of gas-filled microvesicles was assessed using a proprietary pressure nephelometer. Briefly, a suspension of microvesicles was introduced into the sample cell of the spectrophotometer (air-tight and connected to a pressurization system). The optical density (absorption at 700 nm) of the suspension was continuously recorded while linearly increasing the pressure applied to the sample in the cell from atmospheric pressure (760 mmHg, 101.3 kPa) to an overpressure of two bars (2280 mmHg, 303.9 kPa), at a rate of about 4 mmHg/sec (533 Pa/sec).

The parameter Pc50 (“critical pressure”), which characterizes each suspension, defines the excess pressure (relative to atmospheric pressure) at which the absorption capacity of the microvesicle suspension drops to half of its initial value.

Example 1

Obtaining lyophilized product (Batches 1a-1b)

The procedure illustrated in the working examples of WO 94/09829 was used to prepare two different batches (1a-1b), each consisting of several vials containing the lyophilised product.

Briefly, DSPC, DPPG-Na and palmitic acid in a weight ratio of 4.75/4.75/1 were first dissolved in a hexane/ethanol mixture (8/2, v/v) at a concentration of about 5 g/L and the solvents were evaporated under vacuum. PEG4000 was added to the solid residue in a weight ratio of about 0.017:1, the mixture was dissolved in tert-butanol at a temperature of about 60 °C and the clear solution was used to fill corresponding DIN8R vials (with an appropriate volume containing about 25 mg of the mixture). The vials were then rapidly cooled to -45 °C and then subjected to vacuum conditions to remove the frozen solvent by sublimation. The temperature was then raised (above room temperature but not higher than 35 °C) and the remaining solvent was evaporated to a final amount of less than 0.5 wt%. At the end of the lyophilization process, the environment of the lyophilizer was saturated with SF ₆ at atmospheric pressure, and the vials (containing the solid lyophilized product in contact with SF ₆ ) were stoppered and hermetically sealed.

These two batches were used for subsequent heat treatment experiments.

Example 2

Obtaining lyophilized product (2a-2h)

The procedure illustrated in the working examples of WO2004/069284 was used to prepare eight different batches (2a-2h), each consisting of several vials containing the lyophilised product.

Briefly, an emulsion of cyclooctane and water (about 1.5/100 v/v) containing about 90 mg/L DSPC, 7 mg/L palmitic acid, 60 mg/L DPPE-PEG5000 and 100 g/L PEG4000 was prepared (Megatron MT3000, Kinematica; 10,000 rpm) and samples were collected in DIN8R vials (about 1 ml/vial).

The vials were cooled at -50°C under vacuum and then lyophilized, followed by secondary drying at above room temperature until water and solvent were completely removed (less than 0.5% by weight), as described in Example 1. At the end of the lyophilization process, the free volume of the vials was saturated with a mixture _{of C4F10 / N2} in a ratio of 35/65 and the vials were stoppered and hermetically sealed.

Different batches (1a - 1h) were used for subsequent heat treatment experiments.

Example 3

Effect of heat treatment on batches obtained according to Example 1

Vials containing different batches obtained according to Example 1 were subjected to different heat treatments and the effect on the characteristics of the reconstituted suspensions of gas-filled microvesicles was observed.

Experiment 3.1

Vials of batch 1a were heated at 48°C for periods 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 microvesicle characteristics in suspension were measured. The results are presented in the following Table 1.

Table 1
Party 1a Heating time
48°C Pc50 (kPa)
average value Without heating 14.4 8 hours 24.8 16 hours 25.5 24 hours 28.7 48 hours 28.8 One week 32.0

As follows from the above results, a significant increase in pressure resistance can be observed after 8 hours of heat treatment, compared to untreated lyophilized samples. This pressure resistance increases slightly with time, up to a maximum after a week of treatment. However, as noted by the applicant, too long a heating time (e.g. after 24 hours and especially after 48 hours) can negatively affect other characteristics of the gas-filled microvesicles, such as their total number, total gas volume and/or their average size.

Experiment 3.2

In the second experiment, vials of lot 1b were heated at 40°C, 45°C, and 49°C for 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 microvesicle characteristics in suspension were measured. The results are presented in the following Table 2.

Table 2
Party 1b Heating time T (°C) Pc50 (kPa)
average 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

As can be seen from the data above, a significant increase in pressure resistance is obtained by heat treatment of the lyophilized products (compared to the initial value of about 14 kPa). While a higher increase in Pc50 is typically observed at a treatment temperature of 49°C, treatment at this temperature may, however, negatively affect other characteristics of the reconstituted gas-filled microvesicles, especially the mean size values.

Example 4

Effect of heat treatment on batches produced according to Example 2

Vials containing different lots (2a-2h) obtained according to Example 2 were subjected to different heat treatments and the effect on the characteristics of the reconstituted suspensions of gas-filled microvesicles was observed.

Experiment 4.1

Batch 2a vials were heated at 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 microvesicle characteristics in suspension were measured. The results are presented in the following Table 3.

Table 3
Party 1a T heating
within 16 hours Pc50 (kPa)
average value Without heating 66.1 40°C 84.8 45°C 78.8

As follows from the above data, a significant increase in pressure resistance is obtained by heat treatment also for the batches obtained in accordance with the procedure of Example 2.

Experiment 4.2

Batch 2b vials were heated at 40°C for periods ranging from 16 to 88 hours, or were not heated. The product in the vial was then reconstituted with 5 ml of saline and the microvesicle characteristics in suspension were measured. The results are presented in the following Table 4.

Table 4
Party 2b Heating time
T=40°C Pc50 (kPa)
average value Without heating 82.1 16 hours 99.0 40 hours 103.6 64 hours 98.6 88 hours 102.5

As follows from the above data, a significant increase in pressure resistance is obtained by heat treatment at 40°C. A treatment duration of 16 hours is generally considered sufficient, also to avoid possible negative effects caused by longer heat treatment on other characteristics of the microvesicles (e.g., an increase in the number of large-sized microvesicles in the reconstituted suspension).

Experiment 4.3

Vials of lots 2c-2g were heated at 40°C for 16 hours or not heated. The product in the vial was then reconstituted with 5 ml of saline and the microvesicle characteristics in suspension were measured. The results are presented in the following Table 5.

Table 5
Batches 2c-2g (40°C, 16 hrs.) Batch No. Heat treatment Pc50 (kPa)
average 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

As can be seen from the above table, for the microvesicle suspensions reconstituted from different batches, an increase in pressure resistance of more than 15 kPa or more, up to about 25 kPa, was obtained after heat treatment of the lyophilized products.

Experiment 4.4

Vials of lot 2h were heat treated at 38°C for two to 24 hours. The product in the vial was then reconstituted with 5 ml of saline and the microvesicle characteristics in suspension were measured. The results are presented in the following Table 6.

Table 6
Party 2h Heating time
(hour) at 38°C Pc50 (kPa)
average 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

As can be seen from the table above, an increase in the pressure resistance of the microvesicles in the reconstituted suspension was achieved by heating the lyophilized material for an increasing period of time, up to 8-12 hours, at 38°C. Further heating of the material (16 or 24 hours) did not substantially increase the pressure resistance.

Claims (24)

1. A method for producing a lyophilized composition suitable for producing a suspension of stabilized gas-filled microvesicles, said composition comprising: (i) an amphiphilic substance comprising a phospholipid and a fatty acid; and (ii) polyethylene glycol as a protective component for lyophilization; which comprises: a) obtaining a liquid mixture comprising said amphiphilic substance and said protective component for lyophilization in a solvent; b) lyophilizing the liquid mixture to remove said solvent and obtain a lyophilized product; and c) after completion of the lyophilization of step b) heating said lyophilized product at atmospheric pressure at a temperature above 35°C and up to 50°C for a period of time from eight to twenty hours. 2. The method according to claim 1, wherein said liquid mixture comprises said amphiphilic substance and said protective component for lyophilization, dispersed in an organic solvent. 3. The method according to claim 1, wherein said liquid mixture comprises said amphiphilic substance and said protective component for lyophilization, dispersed in an aqueous emulsion of a water-immiscible solvent and water. 4. The method according to claim 1, wherein said heating step is carried out at a temperature of 38°C or higher. 5. The method according to claim 1, wherein said heating step is carried out at a temperature of 40°C or higher. 6. The method according to claim 1, wherein said phospholipid comprises 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 (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-oleyl-phosphatidylcholine (POPC), 1-oleyl-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, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoylphosphatidic acid (DMPA) and its alkali metal salts, dipalmitoylphosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoyl phosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl phosphatidyl ethanolamine (DSPE), dioleoyl phosphatidyl ethanolamine (DOPE), diarachidoyl phosphatidylethanolamine (DAPE), dilinoleoyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylserine (DSPS), dioleoyl phosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP) and distearoyl sphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI) or dioleoyl-phosphatidylinositol (DOPI). 7. The method according to claim 1, wherein said fatty acid comprises capric (n-decanoic), lauric (n-dodecanoic), myristic (n-tetradecanoic), palmitic (n-hexadecanoic), stearic (n-octadecanoic), arachidic (n-eicosanoic), behenic (n-docosanoic) or n-tetracosanoic acid. 8. The method according to claim 1, wherein said amphiphilic substance comprises DSPC, DPPG and palmitic acid. 9. The method according to claim 1, wherein said amphiphilic substance comprises DSPC, DPPE-PEG5000 and palmitic acid. 10. The method according to claim 8, wherein said heating step is carried out at a temperature of from 40°C to 48°C for at least 8 hours. 11. The method according to claim 9, wherein said heating stage is carried out at a temperature from 36°C to 45°C. 12. The method according to claim 1, wherein said heating in step c) is carried out for at least twelve hours. 13. The method according to any of the preceding claims, wherein said liquid mixture is collected in glass vials which are loaded into a lyophilizer. 14. The method according to claim 13, which comprises, after completion of step b), saturating the free volume of the vials containing the lyophilized product with a physiologically acceptable gas and then sealing and sealing the vials. 15. A method for producing a suspension of gas-filled microvesicles, which comprises reconstituting a lyophilized product obtained in accordance with the method of any one of claims 1-14, using a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas with gentle stirring. 16. A diagnostic method that includes (i) administering to the patient a suspension of gas-filled microvesicles obtained by the method of claim 15; and (ii) detection of an ultrasound signal from a region of interest in a specified patient. 17. The method of claim 16, which includes ultrasound imaging of the heart. 18. The method of claim 16, which includes ultrasound imaging of the liver. 19. The method of claim 16, which includes ultrasound imaging of the urinary tract.