WO2025073838A1

WO2025073838A1 - Freeze-drying process

Info

Publication number: WO2025073838A1
Application number: PCT/EP2024/077847
Authority: WO
Inventors: Philippe Bussat; Samir Cherkaoui; David Lazarus
Original assignee: Bracco Suisse SA
Current assignee: Bracco Suisse SA
Priority date: 2023-10-04
Filing date: 2024-10-03
Publication date: 2025-04-10
Anticipated expiration: 2026-04-04

Abstract

The invention relates to the manufacturing of a freeze-dried product for preparing a suspension of gas-filled microvesicles and to a suspension of gas-filled microvesicles prepared by reconstituting said freeze-dried precursor with a physiologically acceptable liquid carrier in the presence of a physiologically acceptable gas. According to the invention, the freeze-dried product is obtained by freeze-drying an emulsion preparation in bulk.

Description

FREEZE-DRYING PROCESS

Technical field

The present invention relates to the manufacturing of a freeze-dried product for preparing a suspension of gas-filled microvesicles and to a suspension of gas-filled microvesicles prepared by reconstituting said freeze-dried product.

Background of the invention

Aqueous suspensions of gas bubbles of nano- and/or micro-metric size dispersed in an aqueous medium are known since a while as contrast agents useful for Contrast Enhanced UltraSound imaging ("CEUS" imaging). The gas is typically entrapped or encapsulated in a film-layer comprising, for instance, emulsifiers, oils, thickeners or sugars. These stabilized gas bubbles (dispersed in a suitable physiological solution) are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, "(micro)spheres", "(micro)bubbles", "(micro)capsules" or "(micro)balloons", globally referred to here as "gas-filled (micro)vesicles" (or "GfV" in short). A class of suitable GfV includes GfV stabilized by amphiphilic materials (typically comprising a phospholipid); where not mentioned differently, in the present description the term GfV will be referred to such class of GfV.

GfV can be produced according to various manufacturing methods. One of these methods, see e.g. WO94/09829, entails the dissolution of an amphiphilic material and of a freeze-drying protecting compound (e.g. polyethyleneglycol) in an organic solvent; the obtained mixture is distributed into vials which are then subjected to a freeze-drying process, to obtain a freeze-dried product. Another method, see e.g. W02004/069284, entails the preparation of a microemulsion of water with a water immiscible organic solvent, said emulsion comprising an amphiphilic material and a freeze-drying protecting compound. As for the previous preparation process, the emulsion is distributed into vials and then subjected to the freeze-drying process, to obtain a freeze-dried product. At the end of the freeze-drying step (typically when the vials are in the freeze-dryer under vacuum), the headspace of the vials (containing the freeze-dried solid product in powder form at the bottom thereof) is then filled with a pharmaceutically acceptable gas (e.g. comprising a fluorinated gas) and finally sealed for storage. Before use, the aqueous suspension of microbubbles is easily prepared by introducing a suitable liquid into the vial (e.g. saline) and gently shaking the vial to dissolve the freeze-dried product.

More recently, suspensions of GfV have also become of interest for therapeutic applications, in combination with ultrasound sonification of a region or organ subjected to the therapeutic treatment.

While therapeutic applications typically require larger amounts of GfV with respect to those needed for the diagnostic applications, the amount of the freeze-dried product contained in the vials is at present calibrated to obtain suspensions with the amounts of GfV normally required for diagnostic applications; it is thus generally necessary to combine the suspensions of GfV from multiple vials in order to achieve the desired amount of GfV which is necessary for such therapeutic applications.

As the amount of freeze-dried product contained into a vial is proportional to the volume of solution/suspension which is introduced in the vial before the freeze-drying process, it would theoretically be sufficient to fill the vials with larger volumes of liquid precursor lipid preparation, in order to obtain a suspension of GfV with the desired total amount of GfV. However, as observed by the Applicant, the filling volume of vials to be subjected to a freeze-drying process is relatively limited. For instance, it has been observed that the maximum filling volume for DIN8R (having an overall capacity of about 11.5 mL) is of about 2 mL, while for DIN20R vials (having an overall capacity of about 26 mL) said maximum filling volume is of about 4 mL. Above such filling volumes, cracks may happen in the vials which are subjected to freeze-drying process. Typically, even 1% of cracked vials is an unacceptable amount for industrial manufacturing batches; accordingly, the maximum filling volumes of the vials is kept below such volumes. These filling volumes correspond typically to a final amount of freeze-dried product in the vial of less than 200 mg for DIN8R vials or of less than 400 mg for DIN20R vials.

Alternatively, it would theoretically be possible to concentrate the liquid precursor lipid preparation before the freeze-drying thereof, or directly preparing more concentrated liquid precursor lipid preparations, in order to have more GfV-forming material in a same volume of liquid precursor lipid preparations. However, as observed by the Applicant, too concentrated liquid precursor lipid preparations may result in reconstituted suspensions of GfV with lower concentrations of GfV and/or uncontrolled size distribution thereof.

The Applicant has now found that it is possible to substantially increase the amount of freeze-dried product contained in a vial by first performing a freeze-drying "in bulk" of the liquid precursor lipid preparation (e.g. by distributing the liquid preparation on a freeze-drying tray and subjecting it to the freeze-drying process) and then filling the vial with the desired amount of freeze-dried product. Such "bulk" freeze-drying of the suspension can be advantageously applied to emulsified liquid precursor lipid preparation.

As observed by the Applicant the freeze-drying in bulk is particularly advantageous when the precursor liquid preparation to be freeze-dried is an emulsion, in particular an emulsion of microdroplets, prepared for instance according to the manufacturing method described in W02004/069284. Summary of the invention

An aspect of the invention relates to a method for manufacturing a freeze-dried product suitable for the preparation of a suspension of gas-filled vesicles, said freeze-dried product comprising (i) an amphiphilic material and (ii) a freeze-drying protecting compound, said method comprising: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a bottom surface of a freeze-drying support having an area of 25 cm² or larger to form an emulsion layer within said freeze-drying support, and c. submitting said emulsion layer to a freeze-drying step to obtain a freeze-dried product.

Preferably said freeze-drying support is a freeze-dryer tray.

In an embodiment, said method further comprises the step of: d. portioning said freeze-dried product into each of a plurality of containers while leaving a predetermined headspace volume into each of said containers, thereby obtaining a plurality of said containers comprising said freeze-dried product.

Preferably said method further comprises the steps of: e. saturating the headspace volume of each of said containers with a physiologically acceptable gas; and f. sealing said containers.

The obtained freeze-dried product is suitable for being reconstituted with a physiologically acceptable (aqueous) carrier, to form a suspension of gas-filled vesicles.

Preferably, the layer of emulsion distributed on the freeze-drying support is of at least 2 mm, to allow a homogeneous coverage of the support. More preferably, the thickness is of at least 4 mm. On the other hand, the layer has preferably a thickness of at most 15 mm, more preferably of at most 10 mm in order to allow a rapid (and homogeneous) freezing of the whole volume of the emulsion.

With the above process, the initial emulsion may advantageously undergo the freeze- drying process without the need of portioning it into the single vials. This allows for the subsequent portioning of the desired amounts of freeze-dried product into the vials, which results in the preparation of suspensions of GfV having the desired amounts of GfV suitable for therapeutic applications. Another aspect of the invention thus relates to a sealed container containing a freeze- dried product comprising i) an amphiphilic material and ii) a freeze-drying protecting compound, in contact with a physiologically acceptable gas, wherein said freeze-dried product forms a suspension of gas-filled vesicles upon admixing it with a pharmaceutically acceptable liquid carrier in the presence of the physiologically acceptable gas and wherein said container is obtainable by a method comprising the steps of: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a bottom surface of a freeze-drying support having an area of 25 cm² or larger to form an emulsion layer within said freeze-drying support; c. submitting said emulsion layer to a freeze-drying step to obtain a freeze- dried product; d. portioning said freeze-dried product into each of a plurality of containers thereby leaving a predetermined headspace volume into each of said containers; e. saturating the headspace volume of each of said containers with a physiologically acceptable gas; and f. sealing said containers.

Preferably, the amount of said amphiphilic material of 1.5 mg or higher.

In an embodiment said container is a DIN8R vial containing at least 250 mg of said freeze-dried product suitable for reconstituting a suspension of gas filled microvesicles, preferably at least 300 mg and even more preferably at least 400 mg, up to e.g 800 mg of freeze-dried product.

In an alternative embodiment, said sealed container is a DIN20R vial containing at least 450 mg, preferably 600 mg, more preferably at least 800 mg of said freeze-dried product for reconstituting a suspension of gas filled microvesicles, up to e.g. 2 g.

Figures

Figures 1, 2 and 3 show the comparison between the characteristics of the GfV suspensions obtained freeze-drying the lipid emulsion either in the vials or in bulk.

Figures 4 and 5 show the sampling locations on the trays for the freeze-dried products prepared according to examples 3 and 4, respectively. Detailed description of the invention

The process of the invention has the advantage of manufacturing a freeze-dried product which can be portioned in any desired amount into containers for reconstituting the suspension of GfV, without being limited by a maximum filling volume of vials.

The first step of the process comprises preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, an amphiphilic compound and a freeze-drying protecting compound.

Preferably, as described for instance in W02004/069284, a composition comprising the amphiphilic material may be dispersed in an emulsion of water with a water immiscible organic solvent under agitation, in the presence of the freeze-drying protecting component.

The emulsion may be obtained by submitting the aqueous medium (e.g. water) and the organic solvent, in the presence of the amphiphilic material, to any appropriate emulsion-generating technique known in the art such as, for instance, sonication, shaking, high pressure homogenization, micromixing, membrane emulsification, high-speed stirring or high shear mixing. The freeze-drying protecting component can be added either before or after the formation of the emulsion, e.g. as an aqueous solution comprising such freeze- drying protecting component. Preferably, an organic solution containing one or more amphiphilic materials is prepared and subsequently admixed (emulsified) with an aqueous solution containing the freeze-dried protecting compound. Optionally, other amphiphilic materials may be dispersed in the aqueous solution. Optionally the obtained emulsion can be then subjected to heating (e.g. from 60 to 90°C, preferably 75-85°C) under stirring. Optionally, the obtained emulsion can be diluted with an aqueous solution comprising the freeze-drying protecting component.

The so obtained emulsion typically comprises a plurality of droplets of organic solvent surrounded by a layer of amphiphilic material, dispersed in an aqueous solution comprising the freeze-drying protecting component.

Organic solvents

As used herein, water immiscible organic solvent refers to organic solvents having very poor solubility in water, e.g. of 1 g/L or less, preferably of 0.1 g/L or less, more preferably 0.01 or less, down to e.g. 0.001 g/L.

Suitable water immiscible organic solvents include, for instance, branched or linear alkanes, alkenes, cyclo-alkanes, aromatic hydrocarbons, alkyl ethers, ketones, halogenated hydrocarbons, perfluorinated hydrocarbons or mixtures thereof. Preferred organic solvents are cyclo-alkanes, in particular Ce-Cs cycloalkane, such as cyclohexane, cycloheptane or cyclooctane. Particularly preferred is cyclooctane. The amount of organic solvent in the final emulsion undergoing the freeze-drying step is generally comprised from about 1% to about 50% by volume with respect to the amount of water used for the emulsion. Preferably said amount is from 1% to 25%, more preferably from 2% to 20% and even more preferably from 2% to 15%. In certain embodiments said amount is from 4% to 10%. If desired, a mixture of two or more organic solvents can be used, the overall amount of organic solvent in the emulsifying mixture being within the above ranges.

Amphiphilic materials

Suitable amphiphilic materials comprise a phospholipid. Phospholipids, as other amphiphilic molecules, are generally capable of forming a stabilizing layer of material (typically in the form of a mono-molecular layer) at the organic solvent/water interface, thus forming the desired emulsion containing the droplets of organic solvent dispersed in water. Similarly, amphiphilic materials form a stabilizing film at the gas-water boundary interface in the final suspension of gas-filled vesicles.

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

Examples of suitable phospholipids include esters of glycerol with one or preferably two (equal or different) residues of fatty acids and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such as, for instance, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol). Esters of phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid or "lysophospholipids". Fatty acids residues present in the phospholipids are in general 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 instance, 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 employed.

Further examples of phospholipids are phosphatidic acids, i.e. the diesters of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GM1 (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids. As used herein, the term "phospholipid(s)" includes either naturally occurring, semisynthetic or synthetically prepared compounds that can be employed either singularly or as mixtures.

Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins.

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

Examples of preferred phospholipids are, for instance, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), diarachidoyl-phosphatidylcholine (DAPC), dibeheonyl-phosphatidylcholine (DBPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC), dipentadecanoyl- phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1- palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl- phosphatidylcholine (PSPC), l-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1- palmitoyl-2-oleylphosphatidylcholine (POPC), l-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (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, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroylphosphatidylinositol (DLPI), diarachidoylphosphatidylinositol

(DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI). Suitable phospholipids further include phospholipids modified by linking a hydrophilic polymer, such as polyethyleneglycol (PEG) or polypropyleneglycol (PPG), thereto. Preferred polymer-modified phospholipids include "pegylated phospholipids", i.e. phospholipids bound to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines ("PE-PEGs" in brief) i.e. phosphatidylethanolamines where the hydrophilic ethanolamine moiety is linked to a PEG molecule of variable molecular weight (e.g. from 300 to 20000 daltons, preferably from 500 to 5000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG or DOPE-PEG). For example, DPPE-PEG5000 refers to DPPE having attached thereto a PEG polymer having a mean average molecular weight of about 5000. An example of DPPE-PEG5000 is the methoxy terminated PEG derivative l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 5000].

In an embodiment, the phospholipids may bear a reactive moiety which may then be reacted with a corresponding reactive moiety bearing a suitable active component (e.g. targeting ligand), in order to bind said active component to the microvesicle. Examples of suitable reactive moieties include, for instance, reactive groups capable of reacting with an amino group bound to an active component such as isothiocyanate groups (that will form a thiourea bond), reactive esters (to form an amide bond), aldehyde groups (for the formation of an imine bond to be reduced to an alkylamine bond); reactive groups capable of reacting with a thiol group bound to an active component, such as haloacetyl derivatives or maleimides (to form a thioether bond); reactive groups capable of reacting with a carboxylic group bound to an active component, such as amines or hydrazides (to form amide or alkylamide bonds). Preferably, the amphiphilic material bearing the reactive moiety is a lipid bearing a hydrophilic polymer, such as those previously mentioned, preferably a pegylated phospholipid, e.g. DPPE-PEG2000 or DSPE-PEG2000 such as DPPE- PEG2000-maleimide or DSPE-PEG2000-maleimide.

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

Mixtures of phospholipids can also be used, such as, for instance, mixtures of DPPE and/or DSPE (including pegylated derivates), DPPC, DSPC and/or DAPC with DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, Ethyl-DSPC and/or Ethyl-DPPC.

The phospholipids can conveniently be used in admixture with any other compound, preferably amphiphilic. For instance, 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 derivatives thereof or butylated hydroxytoluene and/or other non-phospholipid (amphiphilic) compounds can optionally be added to one or more of the foregoing phospholipids, e.g. in proportions preferably below 50% by weight, more preferably up to 25% or lower. Particularly preferred as additional compound in admixture with phospholipids are fatty acids. Fatty acids useful in a composition according to the invention, which can be either saturated or unsaturated, comprise a C10-C24, aliphatic chain terminated by a carboxylic acid moiety, preferably a 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 acid, more preferably palmitic acid. Examples of unsaturated fatty acids comprise 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), gondoic (cis-ll-eicosenoic), cis-l l,14-eicosadienoic, cis-5,8,ll-eicosatrienoic, cis-8,ll,14-eicosatrienoic, cisl l, 14, 17-eicosatrienoic, arachidonic (cis-8,l l,14,17-eicosatetraenoic) and erucic (cis-13- docosenoic) acid. Preferred fatty acid is palmitic acid.

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

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

The amount of amphiphilic material, in particular of phospholipids, is generally comprised between about 0.005 and about 1.0% by weight with respect to the total weight of the emulsified mixture. Preferably the amount of phospholipid is comprised between 0.01 and 1.0% by weight with respect to the total weight of the emulsified mixture and more preferably between about 0.05% and 0.5% by weight.

Targeting ligands

Compositions and gas filled vesicles according to the invention may optionally comprise a targeting ligand.

The term "targeting ligand" includes within its meaning any compound, moiety or residue having, or being capable to promote, a targeting activity (e.g. including a selective binding) of the GfV of a composition of the invention towards any biological or pathological site within a living body. Targets with which targeting ligand may be associated include tissues such as, for instance, myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), laminae, connective tissue (including interstitial tissue) or tumors; blood clots; and receptors such as, for instance, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and cytoplasmic receptors for steroid hormones.

The targeting ligand may be synthetic, semi-synthetic, or naturally occurring. Materials or substances which may 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 agents, aptamers, nanofitins and genetic material, including nucleosides, nucleotides and polynucleotides.

The targeting ligand may be a compound per se which is admixed with the other components of the microvesicle or may be a compound which is bound to an amphiphilic molecule (typically a phospholipid) employed for the formation of the microvesicle.

In one preferred embodiment, the targeting ligand may be bound to an amphiphilic molecule (e.g. a phospholipid) forming the stabilizing envelope of the microvesicle through a covalent bond. In such a case, the specific reactive moiety that needs to be present on the amphiphilic molecule will depend on the particular targeting ligand to be coupled thereto, as illustrated in detail above. In order to covalently bind a desired targeting ligand, at least part of the amphiphilic material forming the microvesicle's envelope shall thus contain a suitable reactive moiety and the targeting ligand containing the complementary functionality will be linked thereto according to known techniques, e.g. by adding it to a dispersion comprising the amphiphilic components of the microvesicle. Preferably, the amphiphilic material is a lipid bearing a hydrophilic polymer, such as those previously mentioned, preferably a pegylated phospholipid (e.g. DSPE-PEG2000). In this case, the targeting ligand is linked to a suitable reactive moiety on the hydrophilic polymer (e.g. DSPE-PEG2OOO-NH2), optionally through a linker. The amphiphilic material may be combined with the desired targeting ligand before preparing the microvesicle, and the so obtained combination may be used for the preparation of the microvesicle. Alternatively, the targeting ligand may be linked to the respective amphiphilic material during the preparation of the microvesicle (e.g. in the intermediate microemulsion preparation of the process described in W02004/069284). As a further alternative, the binding may take place on the formed microvesicle comprising an amphiphilic material bearing a reactive moiety.

In an embodiment, an avidin (or streptavidin) moiety (having high affinity for biotin) may be covalently linked to a phospholipid (or to a pegylated phospholipid). Streptavidin- labelled microvesicles may be used for subsequently binding to biotin-labelled counterparts, e.g. a biotin-labelled antibody, for instance in buoyancy assisted cell separation (BACS) methods.

According to an embodiment, the targeting ligand may be associated with the microvesicle via physical and/or electrostatic interactions. As an example, a functional moiety having a high affinity and selectivity for a complementary moiety may be introduced into the amphiphilic molecule, while the complementary moiety will be linked to the targeting ligand. For instance, an avidin (or streptavidin) moiety may be covalently linked to a phospholipid (or to a pegylated phospholipid) while the complementary biotin moiety may be incorporated into a suitable targeting ligand, e.g. a peptide or an antibody. The biotin-labelled targeting ligand will thus be associated with the avidin-labelled phospholipid of the microvesicle by means of the avidin-biotin coupling system. Alternatively, both the phospholipid and the targeting ligand may be provided with a biotin moiety and subsequently coupled to each other by means of avidin (which is a bifunctional component capable of bridging the two biotin moieties). Examples of biotin/avidin coupling of phospholipids and peptides are also disclosed in the above cited US 6,139,819. Alternatively, van der Waal's interactions, electrostatic interactions and other association processes may associate with or bind to the targeting ligand to the amphiphilic molecules.

Alternatively, the phospholipid may be modified with a protein suitable for specific coupling to Fc domain of Immunoglubulin (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 which is admixed with the components forming the microvesicle, to be eventually incorporated into the microvesicle structure, such as, for instance, a lipopeptide as disclosed e.g. in International patent Applications WO 98/18501 or 99/55383.

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

Examples of suitable specific targets to which the microvesicles may be directed are, for instance, fibrin and the GPIIbllla binding receptor on activated platelets. Fibrin and platelets are in fact generally present in "thrombi", i.e. coagula which may form in the blood stream and cause a vascular obstruction. Suitable binding peptides are disclosed, for instance, in the above cited US 6,139,819. Further binding peptides specific for fibrin- targeting are disclosed, for instance, in International patent application WO 02/055544.

Other examples of targets include receptors in vulnerable plaques and tumor specific receptors, such as kinase domain region (KDR) and VEGF (vascular endothelial growth factor)/KDR complex. Binding peptides suitable for KDR or VEGF/KDR complex are disclosed, for instance, in International Patent application WO 03/74005, WO 03/084574 and W02007/067979. In an embodiment, the targeting peptide is a dimeric peptidephospholipid conjugate (lipopeptide) as described in W02007/067979.

Freez-drying protecting component

Suitable freeze-drying protecting components include, for instance, carbohydrates, e.g. a mono- di- or poly-saccharide, such as sucrose, maltose, trehalose, glucose, lactose, galactose, raffinose, cyclodextrin, dextran, chitosan and its derivatives (e.g. carboxymethyl chitosan, trimethyl chitosan); polyols, e.g. sugar alcohols such as sorbitol, mannitol or xylitol; or hydrophilic polymers, e.g. polyoxyalkyleneglycol such as polyethylene glycol (e.g. PEG2000, PEG4000 or PEG8000) or polypropylenglycol. According to an embodiment said freeze-drying protecting component is polyethylene glycol, preferably PEG4000. PEG4000 as used herein has its normal meaning in the field, indicating a polyethyleneglycol having a molecular weight of about 4000 g/mole, in general with a variation of +/- 10% around said value.

Freeze-Drying process

According to the invention, an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, an amphiphilic material and a freeze-drying protecting component (prepared for instance as described above), is poured onto a freeze-drying support for the subsequent freeze-drying step.

According to the present invention, a freeze-drying support can be any vessel or container suitable for carrying the emulsion to be freeze-dried. For instance, a stainless- steel freeze-drying tray, typically used as support in small (e.g. laboratory scale) or larger (e.g. pilot or industrial scale) freeze-dryers, can be used. Alternatively, disposable trays could be used such as Lyoguard® freeze-drying trays (Gore®).

Typically, said freeze-drying support is in the form of a continuous single surface. This means it constitutes a single piece of equipment, and the term "area" refers to the bottom surface area of this individual support.

Optionally, the freeze-drying support can be divided into a plurality of sub-units preferably interconnected by each other (e.g. segmented into smaller compartments by a mesh-like or grid structure).

The size of the freeze-drying support (typically a tray) will depend on the size of the freeze-dryer; the size can thus typically be 25 cm² or larger, 50 cm² or larger, 75 cm² or larger, 100 cm² or larger, 200 cm² or larger, 300 cm² or larger, 400 cm² or larger, 500 cm² or larger, 600 cm² or larger, 700 cm² or larger, 800 cm² or larger, 900 cm² or larger, 1000 cm² or larger, up to about e.g. 2 m² or 3 m², and in some cases up to about 4 m² or 5 m². Said emulsion is thus arranged on said bottom surface of said freeze-drying support, thereby forming an even emulsion layer thereupon.

The term "even emulsion" refers to an emulsion that is uniformly mixed and distributed, with consistent composition and texture throughout the entire surface of the freeze-dying support. Said emulsion layer is continuous and homogenous, i.e. covering the entire surface of the freeze-drying support without variations in its distribution.

Typically, the emulsion is distributed on the freeze-drying support as a relatively thin layer, preferably with a thickness of from about 2 mm to about 10 mm, more preferably from about 3 mm and 8 mm and even more preferably from about 4 mm and 6 mm.

The volume of emulsion distributed on the freeze-drying support is dependent on the bottom surface area of said support considering the preferred maximum thickness of the emulsion layer. Preferably the emulsion volume is at least 25 mL.

In an alternative embodiment, step b) of the disclosed method comprises distributing a volume of at least 25 mL or higher of said emulsion on a bottom surface of a freeze- drying support to form an emulsion layer within said freeze-drying support.

Furthermore, said emulsion is characterized by consistent thickness, ensuring uniform distribution over the tray's surface.

The process of the invention allows to distribute much larger volumes of emulsion on a single freeze-drying support, as compared to the limited volumes of emulsion allowed in single vials, e.g. as mentioned before the 4 mL maximum volume for DIN20R vials. In particular, the present process allows to distribute at least 25 mL of emulsion on said freeze-drying support, more preferably at least 50 mL and even more preferably at least 100 mL of emulsion on a single freeze-drying support. The possibility of distributing larger volumes of emulsion on the freeze-drying support will depend on the size of the support; for instance, for larger supports such volume can be up to 300mL , 500 mL, 1000 mL, 2000 mL or up to e.g. 5000 mL for larger supports.

The freeze-drying support containing the emulsion is then inserted into the freezedrier and subjected to a conventional freeze-drying procedure. The freeze-drying process generally includes an initial step (freezing step) where the sample is rapidly deep-cooled (e.g. at temperatures of from -35°C to -70°C) to freeze the liquid(s) of the sample. Then the frozen sample is subjected to vacuum (e.g. 0.1-0.8 mbar) at a fixed temperature (e.g. -30°C to -5°C) during the primary drying step, the substantial totality of the frozen liquid(s) is removed by sublimation, typically up to about 95% of the total amount of liquid, preferably up to about 99%. After the primary drying, residual liquid (including possible interstitial water) can be further removed during the secondary drying, which is typically conducted at a temperature higher than room temperature, under vacuum (preferably by maintaining the same vacuum applied during the primary drying). The temperature during the secondary drying is preferably not higher than 35°C. The secondary drying can be stopped when the residual content of the liquid(s) in the sample reaches a desired minimum value, e.g. less 3% (preferably less than 1%) by weight of water with respect to the total mass of residual freeze-dried product, or e.g. less than 0.01% by weight, preferably less than 0.008%, for residual solvent(s).

After completion of the freeze-drying process the freeze-dried product may undergo an optional additional thermal treatment step, as described for instance in WO 2020/229642.

The freeze-dried product can then be portioned in any desired amount in containers such as vials of different volumes, e.g. DIN8R, DIN20R of DIN50R.

According to the invention, at the end of the preparation method the total amount of the freeze-dried product contained in the containers is composed by a part comprising an amphiphilic material, and a part comprising a freeze-drying protecting compound. The freeze-drying protecting component usually represents the larger amount of the final freeze-dried preparation, wherein it is typically at least 90%, preferably between 94% and 99.7%, more preferably 99,5%, up to 99.9% (w/w).

Preferably the amount of amphiphilic material (e.g. lipid) per container (e.g. vial) is 1.50 mg or higher, more preferably 2 mg or higher, more preferably 4 mg or higher, still more preferably 5 mg or higher, up to e.g. 20 mg.

The headspace of said containers (e.g. vials) may be filled with a suitable physiologically acceptable gas having low solubility in water, e.g. a perfluorinated gas, preferably a perfluorinated hydrocarbon such as CsFs or C4F10, preferably in admixture with air or nitrogen, and the vial finally stoppered. Alternatively, the vials may be stoppered without filling with the gas (i.e. leaving an air atmosphere in the vial), which may then be inserted subsequently by replacing the air of the headspace (e.g. by means of a gas-filled syringe and a needle).

An alternative aspect of the invention relates to a method for manufacturing a freeze- dried product, suitable for the preparation of a suspension of gas-filled vesicles, said freeze-dried product comprising (i) an amphiphilic material and (ii) a freeze-drying protecting compound, said method comprising: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a freeze-drying support to form an emulsion layer within said freeze-drying support; c. submitting said emulsion layer to a freeze-drying step to obtain a freeze- dried product; d. portioning said freeze-dried product into each of a plurality of containers while leaving a predetermined headspace volume into each of said containers, thereby obtaining a plurality of said containers comprising said freeze-dried product; e. saturating the headspace volume of each of said containers with a physiologically acceptable gas; and f. sealing said containers.

Suspension of aas-filled microvesicles

The suspension of gas-filled microvesicles can then be prepared by reconstituting the freeze-dried product with a physiologically acceptable (aqueous) carrier, under gentle agitation. Suitable physiologically acceptable (aqueous) carriers include, for instance, water for injection, saline or glucose solutions, optionally containing excipients or additives including for instance pH regulators, buffers, osmolality adjusters, viscosity enhancers, emulsifiers and bulking agents.

As observed by the Applicant, the number of gas-filled vesicles per vial obtained after reconstitution increases linearly as a function of the amphiphilic material amount per container (e.g. vial).

In particular, suspensions of gas-filled microvesicles characterized by suitable properties for diagnostic and therapeutic applications, including high GfV concentration (particles/mL), Dv (volume distribution of the GfV), and MVC (Microvesicles Volume Concentration, which is the total gas volume per mL of suspension), can be achieved by reconstituting the disclosed freeze-dried product in the containers for pharmaceutical use.

In a preferred embodiment, said suspension of gas-filled vesicles obtained by reconstitution a freeze-dried product comprising an amount of amphiphilic material (e.g. lipid) of 1.50 mg or higher, is characterized by a concentration (particle/mL) higher than 1.5xl0¹⁰ GfV/vial. Higher amounts of amphiphilic materials in the container will typically result in a higher number of gas-filled microvesicles in the reconstituted suspension.

Pharmaceutical kit, administration and methods of use

The container containing the freeze-dried product can be packaged in a two- components diagnostic and/or therapeutic kit, preferably for administration by injection. The kit preferably comprises the container (e.g. a vial) containing the freeze-dried product and a second container (e.g. a syringe barrel) containing the physiologically acceptable aqueous carrier for reconstitution.

The GfV obtained by reconstituting the freeze-dried product may be used in a variety of diagnostic and/or therapeutic techniques, including in particular ultrasound. Diagnostic methods include any method where the use of the gas-filled microvesicles allows enhancing the visualisation of a portion or of a part of an animal (including humans) body, including imaging for preclinical and clinical research purposes. A variety of imaging techniques may be employed in ultrasound applications, for example including fundamental and harmonic B-mode imaging, pulse or phase inversion imaging and fundamental and harmonic Doppler imaging; if desired three-dimensional imaging techniques may be used.

GfV may typically be administered in a concentration of from about 0.01 to about 1.0 pL of gas per kg of patient, depending e.g. on their respective composition, the tissue or organ to be imaged and/or the chosen imaging technique. This general concentration range may of course vary depending on specific imaging applications, e.g. when signals can be observed at very low doses such as in colour Doppler or power pulse inversion.

As an example, a method of diagnosing comprises

(i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of a freeze-dried product obtained according to the process of the invention; and

(ii) detecting an ultrasound signal from a region of interest in said patient.

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

Reconstitution of the freeze-dried product is preferably made by dispersing it into a physiologically acceptable aqueous carrier, e.g. saline, in the presence of a physiologically acceptable gas, under gentle agitation.

Alternatively, a GfV suspension prepared by reconstituting the above freeze-dried product can be used in a method of therapeutic treatment of a suspension of microvesicles.

Therapeutic techniques include any method of treatment (as above defined) of a patient which comprises the combined use of ultrasounds and gas-filled microvesicles either as such (e.g. in ultrasound mediated thrombolysis, high intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromudulation, radiosensitization) or in combination with a therapeutic agent (i.e. ultrasound mediated delivery, e.g. for the delivery of a drug or bioactive compound to a selected site or tissue, such as in tumor treatment, gene therapy, infectious diseases therapy, metabolic diseases therapy, chronic diseases therapy, degenerative diseases therapy, inflammatory diseases therapy, immunologic or autoimmune diseases therapy or in the use as vaccine), whereby the presence of the gas-filled microvesicles may provide a therapeutic effect itself or is capable of enhancing the therapeutic effects of the applied ultrasounds, e.g. by exerting or being responsible to exert a biological effect in vitro and/or in vivo, either by itself or upon specific activation by various physical methods (including e.g. ultrasound mediated delivery).

GfV can typically be administered for therapeutic purposes in a concentration of from about 0.01 to about 5.0 pL of gas per kg of patient, depending e.g. from their respective composition, the type of subject under treatment, the tissue or organ to be treated and/or the therapeutic method applied.

For instance, said method of ultrasound therapeutic treatment comprises:

(i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of a freeze-dried product obtained according to the process of the invention;

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

(iii) applying an ultrasound beam for therapeutically treating said region of interest; whereby said ultrasound therapeutic treatment is enhanced by the presence of said suspension of gas-filled microvesicles in said region of interest.

Furthermore, a GfV suspension prepared by reconstituting the above freeze-dried product can be 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 type of cells from other cells in a physiological liquid (e.g. blood or plasma). In an embodiment, the separation method comprises labelling a desired cell to be separated with a suitable labelled antibody capable of binding to a specific (and selective) receptor on said cell. The microvesicles of the invention are then added to the suspension of cells to be separated (including those bearing the labelled antibody); once admixed to the suspension of cells, the microvesicles associate through the ligand with the labelling residue bound to antibody/cell construct thus allowing separation of the cells by buoyancy (see e.g. WO 2017/117349). For instance, the labelled antibody is a biotinylated antibody, where the biotin residue is capable of associating with a respective moiety, such as for instance an avidin, neutravidin or streptavidin residue on a gas-filled microvesicles.

The following examples will help to further illustrate the invention.

EXAMPLES

Materials:

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

DPPG: l,2-dipalmitoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt) DPPE-PEG5000: l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy(polyethylene glycol)-5000], ammonium salt PEG4OOO: polyethyleneglycol 4000

Example 1

Freeze-drying in vials - Comparative

An organic phase was prepared by dissolving 50 mg of DSPC/palmitic acid blend (8/2 molar ratio) in 8 mL of cyclooctane at 75 °C.

An aqueous phase was prepared by dissolving 5 g of PEG4000 and 33.4 mg of DPPE- PEG5000 in 45 mL of milliQ water.

The organic phase was emulsified in the aqueous phase using the Megatron MT3000 at 9500 rpm during 5 min. The emulsion was then heated to 80 °C for 1 hour under stirring. After cooling to room temperature, the emulsion was divided in four parts.

Emulsion A : no dilution - 1.44 mg/mL lipids (DSPC/Palmitic acid/DPPE-PEG5000) Emulsion B : two-fold dilution with PEG4000 10% solution - 0.72 mg/mL lipids Emulsion C : three-fold dilution with PEG4000 10% solution - 0.48 mg/mL lipids Emulsion D : four-fold dilution with PEG4000 10% solution - 0.36 mg/mL lipids Each emulsion was then sampled in vials (0.75 mL or 1.5 mL in DIN8R vials and 3 mL in DIN20R vials).

The vials were placed on the freeze-drier (Lyobeta 35 - Telstar) shelves cooled to - 50°C and then freeze-dried (primary drying -20°C - 0.2 mBar for 16 hours). At the end of the freeze-drying process, the vials were stoppered and sealed and the headspace was replaced by a C4F10/N2 mixture (35/65 v/v). The vials were finally heated at 38 °C for 16 hours.

Vials were redispersed in saline (two-fold the initial volume of emulsion) and gas- filled vesicles were characterized in size and concentration (Coulter counter Multisizer 3).

Table 1 below lists the various comparative preparations, identifying in particular the total amount of lipids for each vial and the redispersion volume.

Table 1 - Sampling and redispersion of the vials - comparative

Example 2

Freeze-drying in bulk

An emulsion was prepared according the procedure of Example 1.

After cooling at room temperature, the emulsion was diluted four times with PEG4000 10% solution (0.36 mg/mL lipid, same as emulsion D of example 1). The diluted emulsion (212 mL) was then poured into a stainless-steel tray (35 x 28 cm), with a layer thickness of about 2 mm. The tray was then placed in freeze-dryer shelf (-50°C) and freeze dried as in example 1. At the end of the freeze-drying process, the freeze-dried product was sampled in different amounts in various vials (see table 2). The vials were stoppered and sealed, and the headspace was replaced by a C4F10/N2 mixture (35/65 v/v). The vials were finally heated at 38 °C for 16 hours.

The content of the vials was redispersed in saline and the gas-filled vesicles were characterized in size and concentration (Coulter counter Multisizer 3). Table 2 below lists the various preparations, identifying in particular the total amount of lipids for each vial and the redispersion volume. Table 2 - Sampling and redispersion in the vials

Figures 1, 2 and 3 show the comparison between the results of Examples 1 and 2. As inferable from figure 1, for the preparations of Example 2 (dots) the number of GfV per vial increases linearly as a function of lipid amount per vial. To the contrary, for the comparative preparations of example 1 (squares), the GfV concentration reaches a plateau around 1.5xl0¹⁰ GfV/vial. Thus, according to the invention it is possible to increase the amount of GfV contained in a vial, with the only limitation of the redispersion volume in the vial. To the contrary, according to previous method, even by increasing the amount of lipids in the vial (i.e. increasing the volume of emulsion subjected to the freeze-drying process), the total amount of GfV in the vial is substantially limited.

Similarly, figures 2 and 3 show that according to the present method, the size (Dv) of GfV and the % of GfV larger than 8 microns, remain constant in the various preparations, while both values increase with the increase of lipid amounts for the comparative preparation method.

Similar results are obtained with other phospholipids containing formulations prepared according to the above method, e.g. as described in WO2020/27816. In particular, Example 3 of WO2020/27816 was repeated (formulation with 1 % molar ratio of DPPE-PEG5000 - preparation 3d - table 3) with the difference that after STV coupling and dilution of the emulsion (final volume 240 mL), the emulsion was poured onto a stainless-steel tray (35 x 28 cm). The tray was then placed in freeze-drier shelves (-50°C). After 1 hour, the emulsion was freeze-dried as in example 1. At the end of freeze-drying, the powder was recovered and sampled in various vials. The vials were stoppered and sealed, and the headspace was replaced by a C4F10/N2 mixture (35/65 v/v). The vials were finally heated at 38 °C for 16 hours. Vials were redispersed in saline and microbubbles were characterized in size and concentration (Coulter counter Multisizer 3). Results are summarized in table 3.

Table 3 - Sampling, redispersion of the vials and microbubble concentration

*maximum amount for freeze-drying in vial

Example 3

Homogeneity of the in bulk freeze-dried cake

An organic phase was prepared by dissolving 25 mg of DSPC/palmitic acid blend (8/2 molar ratio) in 4 mL of cyclooctane at 75 °C.

An aqueous phase was prepared by dissolving 5 g of PEG4000 and 16.6 mg of DPPE- PEG5000 in 45 mL of milliQ water.

The organic phase was emulsified in the aqueous phase using the Megatron MT3000 at 8500 rpm during 4 min. The emulsion was then heated to 80 °C for 1 hour under stirring.

Two emulsions were prepared according to the previous protocol and mixed together. The obtained emulsion was diluted 5-fold with PEG4000 10% solution (final volume ~500 mL). The emulsion was poured into stainless-steel tray (35 x 28 cm), with a layer's thickness of about 5 mm. The tray was then placed in the freeze-drier shelves (-50°C). After 1 hour, the emulsion was freeze dried as in example 1.

At the end of the freeze-drying, three samples of 75 mg of powder were collected in the center of the tray (positions 1, 2, 3 in fig. 4), three samples in the front corner of the tray (4, 5 , 6 in fig. 4) and 3 samples in the rear corner of the tray (7, 8 and 9 in fig. 4). Each of the 75 mg sample was introduced into a respective DIN8 vial.

The vials were stoppered and sealed, and the headspace was replaced by a C4F10/N2 mixture (35/65 v/v). The vials were heated at 38 °C for 16 hours. Vials were redispersed in saline and microbubbles were characterized in size and concentration (Coulter counter Multisizer 3). Table 4 shows the Dv (distribution in volume of the GfV), the GfV concentration (particles/mL) and the MVC (Microvesicles Volume Concentration, i.e. the total amount of gas entrapped in the GfV per mL of suspension). Table 4 - Microbubble characteristics as a function of sampling location

The above results show the substantial homogeneity of the reconstituted GfV suspensions, independently from the location of the freeze-dried product on the tray.

Example 4

Alternative preparation process - inhomogeneity of the in bulk freeze-dried cake

189.1 mg of DSPC, 189.1 mg of DPPG and 39.7 mg of palmitic acid were dissolved in a mixture of Hexane/isopropanol/water (85/15/1 in volume) at a concentration of about 5 g/L. After solvent evaporation, the residue is admixed with PEG4000 (25 g). The mixture was dissolved in tert-butanol (175 g) at 60 °C to obtain a clear solution.

15 DIN8R vials were filled with 0.25 mL/vial of the clear solution (control vials). The remaining of the solution was poured onto stainless-steel tray (35 x 28 cm).

The vials and the tray were placed in the Freeze-Dryer (Lyobeta 35 Telstar) shelves at -50 °C and then freeze-dried according to the previously described procedure.

After freeze-drying, the vials are closed and sealed, and the headspace was replaced with SF6. Five vials were redispersed with 5 mL saline per vial and microbubbles characteristics were determined.

Several samples of 25 mg of powder were collected in various points of the tray and placed in DIN8R vials as indicated in figure 4. The vials were closed and sealed, and the headspace was replaced with SF6. Vials were redispersed with 5 mL saline per vial and microbubbles characteristics were determined.

Results are summarized in table 5, showing the GfV concentration of particles with a diameter larger than 2 microns (particles>2 pm /mL) and the MVC (Microvesicles Volume Concentration, i.e. the total amount of gas entrapped in the GfV per mL of suspension). Table 5 - Microbubbles characteristics - comparison freeze-drying in vials vs. different locations on tray

While the characteristics of the GfV in the 15 vials filled with the solution and then subjected to freeze-drying were substantially homogeneous, a high heterogeneity of the

GfV reconstituted from solution subjected to freeze-drying on the tray was observed. In particular, the suspensions obtained by reconstituting the freeze-dried product from the tray show a great variability in the number of bubbles as well as in the total volume of gas entrapped therein, depending on the location of freeze-dried product on the tray. Solutions containing amphiphilic materials seem thus less suitable for undergoing a freeze- drying process in bulk with respect to emulsions (as those of examples 2 or 3, for instance).

References

WO1994/09829

WO2004/069284

US 6,139,819

WO98/18501

WO99/55383

WO 02/055544

W02003/74005 W02020/229642W02017/1 17349

WO2020/ 127816

Claims

1. A method for manufacturing a freeze-dried product suitable for the preparation of a suspension of gas-filled vesicles, said freeze-dried product comprising (i) an amphiphilic material and (ii) a freeze-drying protecting compound, said method comprising: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a bottom surface of a freeze-drying support having an area of 25 cm² or larger to form an emulsion layer within said freeze-drying support, and c. submitting said emulsion layer to a freeze-drying step to obtain a freeze- dried product.

2. The method according to claim 1, wherein the freeze-drying support is a freeze-dryer tray.

3. The method according to any of the preceding claims, wherein the emulsion layer distributed on the freeze-drying support has a thickness of at least 2 mm.

4. The method according to any of the preceding claims wherein the aqueous- organic emulsion undergoing the freeze-drying step comprises an amount of an organic solvent of from 1% to 25% by volume with respect to water.

5. The method according to any of the preceding claims, wherein said amount of organic solvent is of from 2% to 15%.

6. The method according to claim 5, wherein said amount of organic solvent is of from 4% to 10%.

7. The method according to any of the preceding claims, wherein said amphiphilic material comprises a phospholipid.

8. The method according to any of the preceding claims, wherein said organic solvent has a solubility in water of 1 g/L or less.

9. The method according to claim 8, wherein said solubility is of 0.1 g/L or less.

10. The method according to any of the preceding claims further comprising the step of: d. portioning said freeze-dried product into each of a plurality of containers while leaving a predetermined headspace volume into each of said containers, thereby obtaining a plurality of said containers comprising said freeze-dried product.

11. The method according to claim 10 further comprising the steps of: e. saturating the headspace volume of each of said containers with a physiologically acceptable gas; and f. sealing said containers.

12. A method for manufacturing a freeze-dried product suitable for the preparation of a suspension of gas-filled vesicles, said freeze-dried product comprising (i) an amphiphilic material and (ii) a freeze-drying protecting compound, said method comprising: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a freeze-drying support to form an emulsion layer within said freeze-drying support; c. submitting said emulsion layer to a freeze-drying step to obtain a freeze- dried product; d. portioning said freeze-dried product into each of a plurality of containers while leaving a predetermined headspace volume into each of said containers, thereby obtaining a plurality of said containers comprising said freeze-dried product; e. saturating the headspace volume of each of said containers with a physiologically acceptable gas; and f. sealing said containers.

13. The method according to claims 10 - 12, wherein said container is a vial.

14. A method for preparing a suspension of gas-filled vesicles which comprises: i . preparing a freeze-dried product according to claims 11 or 12, and ii. reconstituting said freeze-dried product by admixing it in the container with a pharmaceutically acceptable liquid carrier in the presence of the physiologically acceptable gas to form said suspension of gas-filled vesicles.

15. A sealed container containing a freeze-dried product comprising i) an amphiphilic material and ii) a freeze-drying protecting compound, in contact with a physiologically acceptable gas, wherein said freeze-dried product forms a suspension of gas-filled vesicles upon admixing it with a pharmaceutically acceptable liquid carrier in the presence of the physiologically acceptable gas and wherein said container is obtainable by a method comprising: a. preparing an aqueous-organic emulsion comprising an aqueous medium, an organic solvent, said amphiphilic material and said freeze-drying protecting compound; b. distributing said emulsion on a bottom surface of a freeze-drying support having an area of 25 cm² or larger to form an emulsion layer within said freeze-drying support; c. submitting said emulsion layer to a freeze-drying step to obtain a freeze- dried product; and d. portioning said freeze-dried product into each of a plurality of containers thereby leaving a predetermined headspace volume into each of said containers.

16. The sealed container according to claim 15 containing an amount of amphiphilic material of 1.5 mg or higher.

17. The sealed container according to claim 15 or 16, wherein said container is a DIN8R vial containing at least 250 mg of said freeze-dried product.

18. The sealed container according to claim 17, wherein said DIN8R vial contains at least 300 mg of said freeze-dried product

19. The sealed container according to claim 15 or 16, wherein said container is a DIN20R vial containing at least 450 mg of a freeze-dried product.

20. The sealed container according to claim 19, wherein said DIN20R vial comprises at least 500 mg of said freeze-dried product.