Freeze-dried composition for preparing calibrated gas-filled microvesicles

Abstract

To provide a freeze-dried composition that yields an ultrasound contrast agent upon reconstitution, and a method for preparing the same.SOLUTION: A freeze-dried composition comprising an amphiphilic material and a freeze-drying protecting component, which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein the reconstituted suspension of microvesicles has a geometric standard deviation (GSD) value of at least 1.22 or lower.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates generally to the field of ultrasound contrast agents (USCAs). In particular, it relates to lyophilized compositions comprising an amphiphilic material and a lyoprotectant component, which can be reconstituted to prepare suspensions of gas-filled microvesicles with calibrated sizes useful for diagnostic or therapeutic applications. It also relates to methods for preparing such lyophilized compositions. [Background technology]

[0002] Calibrated-size microvesicles (CMVs) are a new generation of gaseous microbubbles with a narrow, calibrated, and controlled size distribution (mean diameter size 3–8 μm) compared to commercially available polydisperse microbubble ultrasound contrast agents (USCAs). These calibrated-size microbubbles are designed to enhance imaging sensitivity and improve the efficiency of drug and gene delivery to specific organs. Calibrated microvesicles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting, and flow focusing. In particular, flow focusing techniques produce calibrated microvesicles (typically with a geometric standard deviation (GSD) value of 1.05–1.08) in a highly reproducible manner at reasonable production rates (approximately 60 million bubbles per minute) and at concentrations of suspended microvesicles acceptable for subsequent use (e.g., 3 × 10 8 CMV / mL ~4 x 10 8 This allows the production of 1000 CMV / mL.

[0003] Reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and Reference 2 [PCT Application No. PCT / EP2019 / 055325], both by the present applicant, describe methods for preparing CMVs, such as gas-filled microbubbles, in particular by using microfluidic techniques.

[0004] Although aqueous suspensions of calibrated microvesicles are very stable at room temperature for several weeks, this stability may impose some limitations on the development of pharmaceutical products, for which a longer shelf life is generally desirable. Therefore, there is a need to develop long-term storage procedures. Freeze-drying, also known as lyophilization, is a complex and challenging process that is widely used in the pharmaceutical industry, where it is advantageous to store pharmaceutical products in a dry form for several months. In fact, freeze-dried products exhibit better storage stability and can be shipped more easily. Freeze-drying is also used in the field of gas-filled microvesicles to prepare freeze-dried dosage forms, which are reconstituted with an aqueous solvent in the presence of a gas to form a suspension of gas-filled microvesicles.

[0005] Reference 3 [US2017 / 080113 A1-GE Healthcare] describes the preparation of a suspension of microbubbles with controlled size and its subsequent freeze-drying using a sucrose solution.

[0006] Reference 4 [WO97 / 29782 A1 - NICOMED IMAGING A / S] teaches that USCA precursors stable at room temperature (RT) can be prepared by freeze-drying C3F8 microbubbles in the presence of a freeze-drying stabilizer, preferably sucrose.

[0007] To date, to the applicant's knowledge, such a technique has not yet been applied to prepare a freeze-dried composition from a suspension of calibrated microvesicles. Summary of the Invention [Problem to be solved by the invention]

[0008] As observed by the applicant, one of the most challenging issues in the preparation of freeze-dried compositions of calibrated microvesicles relates to the need to avoid substantial alteration of the properties of the initial suspension of calibrated microvesicles, such as concentration, monodispersity or geometric standard deviation (GSD) and / or final mean diameter.

[0009] Applicant has now found that such initial properties can be retained to an acceptable extent after the freeze-drying process by using an appropriate lyoprotectant component at an appropriate concentration. [Means for solving the problem]

[0010] In a first aspect, the present invention relates to a lyophilized composition comprising an amphiphilic material and a lyoprotectant component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein said reconstituted suspension of microvesicles has a geometric standard deviation (GSD) value of at least 1.22 or less.

[0011] In an even more preferred embodiment, the lyoprotectant component is a polymer, preferably a hydrophilic polymer, more preferably a polyglycol, even more preferably polyethylene glycol (PEG).

[0012] In a preferred embodiment, said reconstituted suspension of calibrated microvesicles is characterized by a GSD of at least 1.22 or less, preferably at least 1.21, for example up to 1.10.

[0013] In one embodiment of the invention, the reconstituted suspension of calibrated microvesicles contains at least 2.0 x 10 8 cells / mL, preferably 2.1 x 10 8 CMV / mL, more preferably 2.3 x 10 8 CMV / mL concentration: 5.5 x 10 8 It is characterized by a concentration of up to 100 CMV / mL.

[0014] According to a further aspect, the present invention relates to a method for preparing a freeze-dried composition for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles, comprising the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing polyglycol as a lyoprotectant component; b. freeze-drying the calibrated microvesicle suspension.

[0015] A further aspect of the present invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition comprising the following steps: a. preparing a first suspension of gas-filled calibrated microvesicles by a flow focusing process, said suspension further comprising polyglycol as a lyoprotectant; and b. freeze-drying the suspension.

[0016] A further aspect of the present invention relates to a method for preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, said method comprising the step of reconstituting a lyophilized composition as defined above with a pharmaceutically acceptable solution in the presence of a biocompatible gas. [Brief explanation of the drawings]

[0017] [Figure 1] Typical particle size distribution display. [Figure 2] Schematic representation of the core of the microfluidic flow focusing device. [Figure 3] 1 is an exemplary schematic diagram of a device useful in the process of the present technique. [Figure 4] Effect of PEG4000 and PEG8000 concentrations on CMV yield after lyophilization at two different freezing temperatures, −40°C and −60°C. DETAILED DESCRIPTION OF THE INVENTION

[0018] The expression "gas-filled microvesicles" generally refers to gas bubbles bounded at the gas / liquid interface by a very thin envelope (film) comprising a stabilizing amphiphilic material (typically a phospholipid) placed at the gas-liquid interface. Such calibrated gas-filled microvesicles are suitable as contrast agents in ultrasound imaging techniques (known as contrast-enhanced ultrasound (CEUS) imaging) or in therapeutic applications, for example in combination with ultrasound-mediated drug delivery.

[0019] These stabilized gas bubbles (dispersed in an appropriate physiological solution) are generally referred to in the art by various terms, typically depending on the stabilizing material used in their preparation; these terms include, for example, "microspheres," "microbubbles," "microcapsules," or "microballoons," collectively referred to herein as "gas-filled microvesicles" (or "microvesicles" for short).

[0020] The term "calibrated" (when referring to gas-filled microvesicles) refers in particular to a microvesicle suspension having highly calibrated microvesicles (CMV) having different sizes between 3 and 8 μm and characterized by a size distribution having a geometric standard deviation (GSD) of at least 1.2, preferably at least 1.1, e.g. up to 1.05.

[0021] In this specification and claims, the term "calibrated" is used interchangeably with "size-controlled," "monodisperse," or "single-sized" microvesicles.

[0022] In the present invention, calibrated gas-filled microvesicles are preferably produced using a microfluidic flow-focusing technique, in which a gas thread is focused between two liquid streams in a flow-focusing device to form calibrated phospholipid-stabilized microvesicles that are collected in an outlet channel. Using this approach, calibrated microvesicles are produced in a highly reproducible manner at reasonable production rates (approximately 60 million bubbles per minute) (Figures 1 and 2).

[0023] Depending on the manufacturing process and device parameters, calibrated microvesicles can be obtained with a relatively narrow size distribution of about any desired mean diameter, for example between 3 and 8 μm, preferably about 4 μm.

[0024] The calibrated microvesicle size distribution is typically characterized by a geometric standard deviation (GSD) value of at least 1.20, preferably at least 1.15, such as up to 1.05.

[0025] Calibrated microvesicle concentrations (especially when produced using microfluidic flow focusing) are typically 3 × 10 8 ~4×10 8 Contains between 4 x 10 CMV / mL, preferably 4 x 10 8 CMV / mL, close to 3 x 10 8 Not lower than 100 CMV / mL.

[0026] The "geometric standard deviation" (GSD) generally provides an appropriate value for characterizing the breath of size distribution in a population of particles (in certain cases, gas-filled microvesicles). A population of particles with a wide range of sizes will therefore have a larger GSD value than one in which particle sizes are narrowly distributed around a mean value (i.e., relatively similar in size).

[0027] Figure 1 shows an example of a size distribution graph (by volume) of a population of gas-filled microvesicles, which can be obtained by determining the volume of gas for each of its channels using a commercially available particle analysis instrument (e.g., a Coulter Counter Multisizer 3 with Multisizer 3 software), with each channel corresponding to a given diameter of the microvesicles (e.g., in 0.1 microns). By determining the number of calibrated gas-filled microvesicles in a suspension, their respective diameters and volume distribution in a selected size range (e.g., 3 μm to 6 μm for a 4.5 μm CMV mean diameter), it is possible to calculate the GSD of the CMV distribution by using the following equation 1:

number

number

number

[0028] Among the various commercially available measurement devices, the Coulter Counter Multisizer 3 with Multisizer 3 software is capable of calculating and providing GSD values as defined above.

[0029] For example, a GSD value of 1.2 indicates that approximately 50% of the CMVs fall between 2.5 and 5 μm for a mean diameter of 4 μm; a GSD of 1.05 to 1.08 (<1.1) indicates that approximately 90 to 95% of the CMVs have a size that falls between 2.5 and 5 μm.

[0030] As used herein, the expression "microvesicle concentration" refers to the number of CMVs in a unit of volume, ie, number of CMVs / mL, as determined using a Coulter Counter instrument.

[0031] As observed by the applicant, the calibrated microvesicles obtained by microfluidic flow focusing can be stored at room temperature for several weeks without substantial effect on their main properties, however, after the storage period, the properties of the microvesicles (e.g., concentration and size distribution) may not be maintained and may gradually deteriorate.

[0032] The shelf life of such gas-filled microvesicle suspensions is therefore relatively short for pharmaceutical products, and there is a need to develop long-term storage procedures that are capable of maintaining the initial properties of the CMV, such as concentration, GSD and final diameter, for longer periods of time, e.g., months or years.

[0033] The freeze-drying process is a suitable technique to obtain a dry form of calibrated microvesicles with high stability over time and maintaining their original properties.

[0034] Surprisingly, it has been found that the initial properties of CMV, such as concentration, GSD and final diameter, can be substantially maintained after the freeze-drying process by using an appropriate lyoprotectant component.

[0035] In a first aspect, the present invention provides a lyophilized composition comprising an amphiphilic material and a lyoprotectant component, which, when reconstituted with a suitable aqueous solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, said microvesicles having a GSD value of at least 1.22 or less, preferably at least 1.21, for example up to 1.10.

[0036] In this specification and claims, the terms "freeze-drying" and "lyophilization" are used interchangeably, as are the terms "freeze-dried" and "lyophilized."

[0037] Freeze-dried composition The term "lyophilized composition" as used herein refers to any dry formulation for long-term storage of a gas-filled microvesicle formulation obtained by a freeze-drying process. The freeze-dried composition may comprise one or more active ingredients and a freeze-protecting component.

[0038] As used herein, the expression "active ingredient" refers to the microvesicle stabilizing material, eg, an amphiphilic material, which is included in the lyophilized composition along with the lyoprotectant component.

[0039] Freeze-dried protection ingredient As used herein, the expression "lyoprotectant component" refers to a component suitable for lyophilization and that is included in the microvesicle suspension prior to the lyophilization process.

[0040] The term "lyoprotectant" refers to any compound added to protect an active ingredient during any stage of the lyophilization process. Examples of suitable lyoprotectants are polyethylene glycol (PEG), polyols, sugars, surfactants, buffers, amino acids, chelate complexes, and inorganic salts.

[0041] According to one embodiment of the invention, the lyoprotectant component is selected from the group of polymers, polyols and sugars.

[0042] In one preferred embodiment of the invention, the lyoprotectant component is a polymer, preferably a hydrophilic polymer, more preferably a polyglycol.

[0043] In an even more preferred embodiment, the polymer is polyethylene glycol (PEG).

[0044] Polyethylene glycol (PEG) has its standard chemical meaning. The chemical formula for PEG is HOCH2(CH2OCH2). m CH2OH, where m represents the average number of oxyethylene groups. Typical polyethylene glycols are available in a wide range of average molecular weights, starting from 190-210 g / mol (PEG200; m=4.2) and going up to 7000-9000 g / mol (PEG8000; m=181.4).

[0045] According to this specification, the expression "molecular weight" refers to the average length of the PEG polymer chain, with a variation of ±10% relative to the indicated molecular weight.

[0046] According to one embodiment of the invention, the lyoprotectant is preferably PEG with a molecular weight comprised between 2000 and 10000 g / mol, preferably between 4000 and 8000 g / mol.

[0047] In one embodiment, the lyoprotectant is PEG with a molecular weight of 8000 g / mol (±10%). In another embodiment, the lyoprotectant is PEG with a molecular weight close to 4000 g / mol (±10%).

[0048] In one embodiment of the invention, the lyoprotectant is added to the suspension of CMV as a solution having a concentration comprised between 100 mg / mL and 300 mg / mL, preferably between 120 mg / mL and 250 mg / mL, more preferably close to 200 mg / mL.

[0049] In an even more preferred embodiment, the applicant has found that it is preferable to add PEG to a suspension of CMV as a solution having a concentration of 200 mg / mL in order to improve the retention of the original properties of the CMV suspension reconstituted after the lyophilization process.

[0050] Another parameter that may be considered in the selection of a lyoprotectant component is viscosity.

[0051] The term viscosity as used herein refers to kinematic viscosity (μ, in Pa·s), which is characterized by the resistance to laminar flow of an incompressible fluid (e.g., a CMV suspension before the lyophilization step or reconstituted from the lyophilized product).

[0052] The viscosity of a CMV suspension containing a lyoprotectant component can affect, for example, the process during its manufacture or the administration of a suspension of CMV reconstituted from a lyophilized composition.

[0053] Generally, lower viscosity CMV suspensions are preferred during the manufacturing process to avoid excessive overpressure, for example, within microfluidic flow focusing devices.

[0054] From another perspective, a reconstituted lyophilized composition with a lower viscosity can facilitate its administration, particularly via injection routes (eg, parenteral and intradermal).

[0055] The viscosity of the suspension depends on the concentration and molecular weight of its components; in particular, in this case, on the PEG used as the lyoprotectant component. Specifically, higher concentrations and / or higher molecular weights of the PEG component increase the viscosity of the CMV suspension.

[0056] For example, a solution containing PEG 4000 typically has a relatively lower viscosity value (generally about two to three times lower) than a solution containing an equivalent amount of PEG 8000.

[0057] CMV suspensions containing PEG 4000 at 10% or 20% may therefore offer several advantages in terms of processing or administration of the suspension.

[0058] In this specification and claims, the term "polyol" has its conventional chemical meaning; it refers to any organic compound having more than two hydroxyl functional groups and has the general formula HOCH2(CHOH) n They are characterized by CH2OH, where n is an integer from 1 to 6, preferably from 2 to 4. Suitable polyols include erythritol, xylitol, sorbitol, lactitol and mannitol.

[0059] In the present invention, the polyol is preferably selected from the group of polyols having a carbon chain length of 4 to 6 carbon atoms.

[0060] The term "saccharide" has its standard meaning in chemistry. Sugars, also known as carbohydrates, are molecular compounds made from only three elements: carbon, hydrogen, and oxygen. The simplest sugars are called monosaccharides and are the building units for larger sugars such as disaccharides, trisaccharides, and polysaccharides.

[0061] Amphiphilic Materials Materials suitable for forming the stabilizing layer of gas-filled microvesicles (ie, microvesicle stabilizing materials) are known in the art and preferably include amphiphilic materials.

[0062] As used herein, the term "amphiphilic material" includes compounds having molecules with a hydrophilic polar head (e.g., a polar or ionic group) that can interact with aqueous media and a hydrophobic organic tail (e.g., a hydrocarbon chain) that can interact with, for example, organic solvents. These compounds therefore generally act as "surfactants," i.e., compounds that can stabilize mixtures of otherwise generally immiscible materials, such as a mixture of two immiscible liquids (e.g., water and oil), a mixture of a gas and a liquid (e.g., gas microbubbles in water), or a mixture of insoluble particles and a liquid (e.g., metal nanoparticles in water).

[0063] Suitable amphiphilic materials include, for example, phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid, or oleic acid; lipids bearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone, or polyethylene glycol (PEG), also called "PEGylated lipids"; lipids bearing sulfonated mono-, di-, oligo-, or polysaccharides; cholesterol, cholesterol sulfate, or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids containing ether or ester-linked fatty acids; polymeric lipids; diphosphates. Acetyl; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (e.g., polyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil or ethylene oxide (EO) and propylene oxide (PO) block copolymers; cholesterol glucuronide, lanosterol glucuronide, 7-deoxy-2-methyl-2-propanol Sterol esters of sugar acids, including hydrocholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate, or ergosterol gluconate; esters of alcohols and sugar acids, including lauryl glucuronide, stearoyl glucuronide, myristoyl gluconate, lauryl gluconate, myristoyl gluconate, or stearoyl gluconate; esters of sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, glutamic ... Esters of sugars with fatty acids, including sarcolic acid or polyuronic acid; saponins, including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, or digitoxigenin; glycerol or glycerol monoesters with fatty acids (including glycerol monopalmitate, glycerol monostearate, glycerol monomyristate, or glycerol monolaurate); long-chain alcohols, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecyl alcohol;6-(5-Cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;Digalactosyl diglyceride;6-(5-Cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-Cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7'-Diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid;N-[12-(((7'-Diethylaminocoumarin-3-yl)carbonyl]methylamino]octadecanoic acid

[0023] N-succinyldioleylphosphatidylethanolamine; 1,2-dioleyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol; 1-hexadecyl-2-palmitoylglycerophosphoethanolamine or palmitoylhomocysteine; alkylamines or alkylammonium salts, including at least one of (C; 10 -C 20 ), preferably (C 14 -C 18 ), alkyl chains, such as N-stearylamine, N,N'-distearylamine, N-hexadecylamine, N,N'-dihexadecylamine, N-stearylammonium chloride, N,N'-distearylammonium chloride, N-hexadecylammonium chloride, N,N'-dihexadecylammonium chloride, dimethyldioctadecylammonium bromide (DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary or quaternary ammonium salts, including one or preferably two (C 10 -C 20 ), preferably (C 14 -C 18), acyl chains (linked to the N atom through a (C3-C6) alkylene bridge), such as 1,2-distearoyl-3-trimethylammonium-propane (DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), 1,2-oleoyl-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-3-dimethylammonium-propane (DSDAP); and mixtures or combinations thereof.

[0064] According to the present invention, the amphiphilic material is preferably a phospholipid.

[0065] The term "phospholipid" is intended to encompass any amphiphilic phospholipidic compound whose molecules are capable of forming a stabilizing film of material (typically in the form of a monolayer) at the gas-water interface in the final microbubble suspension. These materials are therefore also referred to in the art as "film-forming phospholipids."

[0066] Examples of suitable phospholipids include esters of one or preferably two (equivalent or different) fatty acid residues and phosphoric acid with glycerol, where the phosphate residue is then linked to a hydrophilic group, such as choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), or inositol (phosphatidylinositol). Esters of phospholipids with only one fatty acid residue are generally referred to in the art as "lyso" forms of phospholipids or "lysophospholipids." The fatty acid residues present in phospholipids are generally long-chain fatty acids, typically containing 12 to 24 carbon atoms, preferably 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably fully saturated. Examples of suitable fatty acids for inclusion in 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.

[0067] Further examples of phospholipids are phosphatidic acids, i.e., diesters of fatty acids and glycerol-phosphate; sphingolipids such as sphingomyelins, i.e., phosphatidylcholine analogs in which the glycerol diester residue with a fatty acid is replaced by a ceramide chain; cardiolipins, i.e., esters of fatty acids and 1,3-diphosphatidylglycerol; glycolipids, such as ganglioside GM1 (or GM2) or cerebrosides; glycolipids; sulfatides and glycosphingolipids.

[0068] As used herein, the term "phospholipid" includes products of either natural origin, semi-synthetic or synthetic preparation, which may be used either alone or in mixtures.

[0069] Examples of phospholipids of natural origin are natural lecithins (phosphatidylcholine (PC) derivatives), such as typically soybean or egg yolk lecithins.

[0070] Examples of semi-synthetic phospholipids are partially or fully hydrogenated derivatives of naturally occurring lecithins. Preferred phospholipids are fatty acid diesters of phosphatidylcholine, ethylphosphatidylcholine, phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol or sphingomyelin.

[0071] Examples of preferred phospholipids include, 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-palmito ...-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl- 1-palmitoyl-2-myristoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-stearoyl-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, diarachidoylphosphatidyl di-glycerol (DAPG) and its alkali metal salts, dimyristoyl phosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoyl phosphatidylglycerol (DPPG) and its alkali metal salts, distearoyl phosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and alkali metal salts thereof, distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and alkali metal salts thereof, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidyl-ethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE),Dimyristoylphosphatidylserine (DMPS), diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), and dioleoyl-phosphatidylinositol (DOPI).

[0072] Suitable phospholipids further include phospholipids modified by the attachment of hydrophilic polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG). Preferred polymer-modified phospholipids include "PEGylated phospholipids," i.e., phospholipids attached to PEG polymers. Examples of PEGylated phospholipids are PEGylated phosphatidylethanolamines (abbreviated "PE-PEG"), i.e., phosphatidylethanolamines in which the hydrophilic ethanolamine moiety is attached to a PEG molecule of variable molecular weight (e.g., 300-20,000 daltons, preferably 500-5,000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG, or DOPE-PEG). For example, DPPE-PEG2000 refers to DPPE with a PEG polymer attached thereto having an average molecular weight of approximately 2,000.

[0073] 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.

[0074] Mixtures of phospholipids may also be used, for example mixtures of DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, ethyl-DSPC and / or ethyl-DPPC with DPPE and / or DSPE (including pegylated derivatives), DPPC, DSPC and / or DAPC.

[0075] For example, the mixture of phospholipids may include phosphatidylcholine derivatives, phosphatidic acid derivatives and pegylated phosphatidylethanolamines, such as DSPC / DPPA / DPPE-PEG, DPPC / DPPA / DPPE-PEG, DSPC / DPPA / DSPE-PEG, DPPC / DPPA / DSPE-PEG, DAPC / DPPA / DPPE-PEG, DAPC / DPPA / DSPE-PEG, DSPC / DSPA / DPPE-PEG, DPPC / DSPA / DSPE-PEG, DSPC / DSPG / DPPE-PEG, DPPC / DSPG / DSPE-PEG.

[0076] According to the present invention, phospholipids can be suitably used in a mixture with any of the above-mentioned amphiphilic compounds. Thus, for example, lipids such as cholesterol, ergosterol, plant sterols, 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 compounds can be optionally added to one or more of the above-mentioned phospholipids, for example, preferably in a proportion ranging from 0 to 50% by weight, more preferably up to 25%. For example, mixtures of amphiphilic materials comprising phospholipids and fatty acids may be advantageously used, including DSPC / DPPG / palmitic acid, DSPC / DPPE-PEG / palmitic acid, DPPC / DPPE-PEG / palmitic acid, DSPC / DSPE-PEG / palmitic acid, DPPC / DSPE-PEG / palmitic acid, DSPC / DPPE-PEG / stearic acid, DPPC / DPPE-PEG / stearic acid, DSPC / DSPE-PEG / stearic acid or DPPC / DSPE-PEG / stearic acid.

[0077] Microvesicles prepared according to the present invention may optionally include a targeting ligand.

[0078] The term "targeting ligand" includes within its meaning any compound, moiety, or residue that has or is capable of promoting targeting activity (including, for example, selective binding) of the microvesicles of the compositions of the invention toward any biological or pathological site in the body. Targets to which a targeting ligand may be associated include tissues, such as myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), thin films, connective tissues (including interstitial tissue), or tumors; blood clots; and receptors, such as cell surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins, and cytoplasmic receptors for steroid hormones.

[0079] The targeting ligand may be synthetic, semi-synthetic, or of natural origin. Materials or substances that can act as targeting ligands include, but are not limited to, proteins, including antibodies, antibody fragments, receptor molecules, receptor-binding molecules, glycoproteins, and lectins; peptides, including oligopeptides and polypeptides; peptidomimetics; sugars, including monosaccharides and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic material, including nucleosides, nucleotides, and polynucleotides.

[0080] The targeting ligand may itself be an amphiphilic compound (mixed with other components of the microvesicle) or may be a compound attached to the amphiphilic molecule (e.g., a phospholipid) used to form the microvesicle.

[0081] gas Suitable gases include biocompatible fluorinated gases, preferably perfluorinated gases. Fluorinated gases include materials containing at least one fluorine atom, such as fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated ketones, such as perfluoroacetone; and fluorinated, preferably perfluorinated ethers, such as perfluorodiethyl ether. Preferred compounds are perfluorinated gases, such as SF6 or perfluorocarbons (perfluorinated hydrocarbons), i.e., hydrocarbons in which all hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable gas-filled microvesicle suspensions.

[0082] The term "perfluorocarbon" includes saturated, unsaturated, and cyclic perfluorocarbons. Examples of biocompatible, physiologically acceptable perfluorocarbons are: perfluoroalkanes, such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (which may be mixed with other isomers, such as perfluoro-n-butane and perfluoro-isobutane), perfluoropentanes, perfluorohexanes, or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g., perfluorobut-2-ene), or perfluorobutadiene; perfluoroalkynes (e.g., perfluorobut-2-yne); and perfluorocycloalkanes (e.g., perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane, and perfluorocycloheptane). Preferred saturated perfluorocarbons are, for example, CF4, C2F6, C3F8, C4F8, C4F 10 , C5F 12 and C6F 14Particularly preferred gases are those that are in gaseous form at room temperature, including SF, C3F8 and C4F 10 Includes:

[0083] One aspect of the present invention therefore relates to a method for preparing a freeze-dried composition for long-term storage of calibrated gas-filled microvesicles, comprising the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing polyglycol as a lyoprotectant component; b. freeze-drying the calibrated microvesicle suspension.

[0084] Preferably, the preparation method of step a) is the microfluidic flow focusing technique described in reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and reference 2 [PCT application number PCT / EP2019 / 055325].

[0085] 2 shows a schematic diagram of the core part 200 of a flow focusing device ("microfluidic chip") useful in the process of the invention. The chip comprises a first supply channel 201 for supplying a gaseous flow 201' and two further orthogonal supply channels 202a and 202b for supplying a liquid flow containing an amphiphilic material.

[0086] The gas flow and the two liquid streams are directed toward the contact zone 203 and then pass through a calibrated orifice 204, shown as a dotted line in FIG. 2 . The calibrated orifice is connected to a calibrated channel 204′, preferably having the same cross-section as the orifice, which is then connected to the initial portion 205 of the outlet channel 206. In an alternative embodiment (not shown), the calibrated orifice 204 may be a nozzle connected directly to the initial portion 205 of the outlet channel 206, i.e., without a calibrated channel in between. Microvesicles 203′ are formed in the calibrated orifice and directed through the calibrated channel 204′ to the initial portion 205 of the outlet channel 206. The hydraulic diameter of the outlet channel is generally larger than that of the calibrated orifice, typically increasing from the initial diameter of the calibrated orifice to the final diameter of the outlet channel 206 and substantially corresponding to the hydraulic diameter of a collection tube (not shown) connecting the flow focusing device to a container for collecting the microvesicle suspension, such as a sealed vial.

[0087] In the initial part 205 of the outlet channel of the device, and preferably also in the contact zone 203 and at the calibrated orifice 204, the temperature of the microvesicles is controlled as described in reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and reference 2 [PCT application number PCT / EP2019 / 055325].

[0088] liquid flow The aqueous liquid stream for preparing calibrated gas-filled microvesicles according to the method of the present invention comprises an amphiphilic material (as defined above) dispersed in an aqueous carrier at a concentration of, for example, 5.0 to 20 mg / mL, preferably 7.5 to 15 mg / mL.

[0089] Suitable aqueous carriers, preferably physiologically acceptable, include water (preferably sterile water), aqueous solutions such as saline (which may be advantageously buffered so that the final product for injection is not hypotonic), or solutions of one or more osmolytes, including salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g., glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols, etc.), chitosan derivatives such as carboxymethylchitosan, trimethylchitosan, or gelling compounds such as carboxymethylcellulose, hydroxyethyl starch, or dextran.

[0090] In an alternative embodiment, an additional oil phase may be added to incorporate therapeutic hydrophobic substances into the microvesicles. To this end, two additional conduits may be provided within the device to supply the desired oil phase, as described, for example, in Reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and Reference 2 [PCT Application No. PCT / EP2019 / 055325]. The formed gas-filled microvesicles thus have an oil film disposed at the interface between the gas and the stabilizing layer of amphiphilic material, and can be loaded with the desired therapeutic agent. Suitable oils may include any biocompatible oil that is liquid at room temperature, for example, saturated or unsaturated (C2-C3) 18 ) Mono-, di-, or tri-esters of glycerol with alkyl chains (including homo- or hetero-alkyl esters (allkylesters)), such as glycerol monobutyrin, glycerol monolinoleate, 1,2-dihexanoylglycerol, 1,2 dioctanoylglycerol, 1,2-dioleyl-sn-glycerol, triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, and mixtures thereof; or natural oils, such as soybean oil, olive oil, safflower seed oil, sunflower seed oil, peanut oil, and mixtures thereof.

[0091] mixed gas flow The newly formed microvesicles contain a gas selected from those previously set forth, preferably a mixture of a gas that is highly soluble in water ("HS gas") and a gas that is sparingly soluble in water ("LS gas"), as contemplated in Reference 2 [PCT Application No. PCT / EP2019 / 055325].

[0092] During the stabilization step, most of the highly soluble gases dissolve rapidly in water, while the less soluble ones remain trapped in the dense layer of amphiphile, typically with some residual amount of HS-soluble gases dispersed therein.

[0093] Examples of HS gases include nitrogen, air, and carbon dioxide, the latter being particularly preferred due to its higher solubility in water.

[0094] Suitable LS gases are fluorinated gases, preferably perfluorinated gases, as previously described herein.

[0095] In one preferred embodiment of the present invention, CO2 / C4F is mixed in a volume ratio of 80 / 20 to 90 / 10, for example 85 / 15. 10 Gas-filled microvesicles containing can be prepared by a gas mixing device similar to that shown schematically in FIG.

[0096] Figure 3 shows an example of a microfluidic flow focusing device used to produce calibrated microvesicles. A gas stream 302 (e.g., CF 10 and CO2) and liquid stream 301 (containing an amphiphilic material, e.g., phospholipids, fatty acids, or mixtures thereof) are fed into microfluidic chip 303 and passed through orifice 304 to produce microvesicles. The microvesicle suspension is preferably heated at atmospheric pressure with a gas (e.g., CF4). 10) is collected in a pre-filled vial 305. A venting device (e.g., needle 306) is preferably used to equalize excess pressure created by the liquid filling of the vial. At the end of collection of the microvesicle suspension, the venting device is preferably removed and the container is preferably sealed to avoid further gas exchange with the external atmosphere.

[0097] According to a preferred embodiment of the present invention, after the recovery step, the calibrated microvesicles obtained by the microfluidic flow focusing method are treated using appropriate washing techniques to remove non-assembled amphiphilic material and possible residual compounds.

[0098] As used herein, the term "washing" refers to any operation performed on a freshly prepared microvesicle suspension and completed to remove (or substantially reduce the amount of) unassembled amphiphilic material and residual compounds.

[0099] According to this specification, suitable washing techniques include centrifugation, filtration, bubble sorting and decantation.

[0100] As used herein, the expression "not-assembled amphiphilic material" refers to amphiphilic molecules that are present in the calibrated microvesicle suspension at the end of the preparation process but that do not form the stabilizing layer of the gas-filled microvesicles.

[0101] As used herein, "residual compounds" refers to any possible additive substances added to the amphiphilic material solution during the preparation of the microvesicles, such as the osmolality adjusting agents previously described.

[0102] In one preferred embodiment of the present invention, a lyoprotectant component is added to the calibrated microvesicle suspension after the washing operation.

[0103] Alternatively, the lyoprotectant component can be added to the liquid stream containing the amphiphilic compound described above during the preparation of the microvesicles by microfluidic techniques.

[0104] The original CMV properties are particularly maintained when polyethylene glycol is used as the lyoprotectant component, characterized in that said polyethylene glycol is added to the CMV suspension as a solution having a total concentration comprised between 100 mg / ml and 300 mg / mL, preferably between 120 mg / mL and 250 mg / mL (more preferably the total PEG concentration is 200 mg / mL).

[0105] In one preferred embodiment of the present invention, prior to the lyophilization step, the CMV suspension contains polyethylene glycol as a lyoprotectant component at a concentration between 12 and 25%, preferably between 14 and 24%, and even more preferably between 18 and 22% (w / v%).

[0106] The lyoprotectant component represents the majority of the final lyophilized formulation, which typically represents at least 90%, preferably 94% to 99.7%, more preferably 99.5%, and up to 99.9% (w / w).

[0107] The lyoprotectant component is as described herein above. The use of polyethylene glycol as a lyoprotectant component has shown advantageous results when used in the lyophilization process of calibrated microvesicle suspensions, allowing the preparation of lyophilized compositions which can then be reconstituted to obtain suspensions of calibrated microvesicles with acceptable properties in terms of concentration and size distribution compared to the initial suspension (before lyophilization).

[0108] For example, the addition of polyethylene glycol as a protective component provides the best strategy for substantially maintaining the GSD value after the freeze-drying process, compared to the addition of polyols or sugars. According to one embodiment, GSD values comprised between 1.20 and 1.22 can be obtained with polyethylene glycol, compared to the use of polyols or sugars, which provide calibrated microvesicles characterized by higher GSD values (up to 1.34).

[0109] In one preferred embodiment, the use of polyethylene glycol as a lyoprotectant component makes it possible to significantly improve the yield of calibrated microvesicles after reconstitution of the lyophilized product, by more than 60%, e.g., 68%, higher than that obtained with the use of polyols or sugars.

[0110] In this specification and claims, the term lyophilization has its standard meaning in the pharmaceutical arts. The lyophilization process consists of drying a pre-frozen liquid product at low pressure or vacuum and low temperature. The primary purpose is to remove liquid from the product to provide a lyophilized product suitable for long-term storage.

[0111] As applicants have observed, freeze-drying parameters may be selected to further optimize the properties (eg, yield, size, GSD) of the reconstituted suspension of microvesicles.

[0112] In one preferred embodiment of the present invention, the freezing temperature of the above method for long-term storage of calibrated gas-filled microvesicles is in the range of -30°C to -70°C, preferably -30°C to -60°C.

[0113] In a further preferred embodiment, the negative pressure applied during the freeze-drying process is preferably below 0.5 mbar, preferably below 0.2, for example around 0.1 mbar.

[0114] A further aspect of the present invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition comprising the following steps: a. preparing a first suspension of gas-filled calibrated microvesicles by a flow focusing process; and b. freeze-drying the suspension.

[0115] A further aspect of the present invention relates to a method for preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, said method comprising the step of reconstituting a lyophilized composition obtained as described above, comprising an amphiphilic material and a lyoprotectant component, with a pharmaceutically acceptable solution in the presence of a biocompatible gas.

[0116] The freeze-dried composition can then be reconstituted with a suitable pharmaceutically acceptable (aqueous) solution in the presence of a biocompatible gas, thus providing a suspension of calibrated gas-filled microvesicles, wherein the microvesicles have a GSD of at least 1.22 or less, preferably at least 1.21, for example up to 1.10.

[0117] In the present invention, a pharmaceutically acceptable (aqueous) solution is water, typically sterile, pyrogen-free water (to minimize the possibility of contamination in the final reconstituted product), an aqueous solution such as saline (which may be advantageously buffered so that the final product for injection is not hypotonic), or an aqueous solution of one or more osmolytes such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.

[0118] Lyophilized compositions are typically reconstituted with a volume of aqueous solution similar to the volume of the suspension that is subjected to the lyophilization process, and therefore the concentration of the lyoprotectant component in the reconstituted suspension is substantially the same as that in the initial suspension.

[0119] Another embodiment of the present invention therefore relates to a reconstituted CMV suspension having a GSD of at least 1.22 and a polyethylene glycol concentration of 12-25%, preferably 14-24%, even more preferably 18-22%.

[0120] These amounts of lyoprotectant component are typically higher than the amounts in prior art gas-filled microvesicle preparations, which are generally less than 10% (w / w).

[0121] Surprisingly, it has been found that the reconstituted suspension of calibrated microvesicles substantially maintains the original properties of the calibrated microvesicles as characterized before the freeze-drying process and is therefore suitable for subsequent pharmaceutical applications.

[0122] In one embodiment of the invention, the reconstituted suspension of calibrated microvesicles contains at least 2.0 x 10 8 cells / mL, preferably 2.1 x 10 8 CMV / mL, more preferably 2.3 x 10 8 CMV / mL concentration: 5.5 x 10 8 It is characterized by a concentration of up to 100 CMV / mL.

[0123] As stated above, the expression "microvesicle concentration" refers to the number of microvesicles in a unit of volume, ie, number of CMVs / mL, as determined using a Coulter Counter instrument.

[0124] Typically, the calibrated microvesicle concentration (%) measured after reconstitution of the freeze-dried composition of the invention with an appropriate aqueous solution allows the determination of the microbubble yield after said reconstitution compared to the microvesicle concentration measured before the freeze-drying process.

[0125] In the present invention, the yield of calibrated microvesicles after reconstitution of the freeze-dried composition of the present invention is at least 50%, preferably at least 55%, more preferably at least 60%, even more preferably at least 65%, for example up to 85%, preferably 90%, more preferably 95%, even more preferably 100%.

[0126] The expression "calibrated microvesicle yield" refers to the ratio between the microvesicle concentration measured before lyophilization and the microvesicle concentration measured after reconstitution of the lyophilized product with a physiologically acceptable solution (in short, "post-lyophilization") (see Equation 2): Equation 2: CMV yield after lyophilization (%) = (CMV concentration after lyophilization / mL) / (CMV concentration before lyophilization / mL)

[0127] Purpose Microvesicles prepared by the methods of the present invention may be used in a variety of diagnostic and / or therapeutic techniques, including ultrasound and magnetic resonance in particular.

[0128] Diagnostic methods include any method that allows for enhanced visualization of a portion or part of an animal's (including human) body through the use of gas-filled microvesicles, including imaging for preclinical and clinical research purposes. Various imaging techniques can be used in ultrasound applications, including, for example, fundamental and harmonic B-mode imaging, pulsed or phase-inversion imaging, and fundamental and harmonic Doppler imaging; three-dimensional imaging techniques can be used if desired.

[0129] Microvesicles of the present invention may 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 composition, the tissue or organ being imaged, and / or the imaging technique selected. This general concentration range may, of course, vary depending on the specific imaging application, e.g., in cases where signals can be observed at very low doses, such as color Doppler or power pulse inversion.

[0130] In one embodiment, the diagnostic method comprises: (i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of the lyophilized product obtained by the process of the invention; and (ii) detecting an ultrasound signal from a region of interest in the patient.

[0131] According to one embodiment, the suspension of gas-filled microvesicles comprises an amphiphilic material and a lyoprotectant component.

[0132] Reconstitution of the lyophilized product is preferably performed with a physiologically acceptable gas (e.g., CF 10 This is done by dispersing it in a physiologically acceptable aqueous carrier (e.g., saline) in the presence of HCl, with gentle stirring.

[0133] Other potential diagnostic imaging applications include scintigraphy, optical imaging, and X-ray imaging (including X-ray phase contrast imaging).

[0134] Another aspect of the invention relates to the use of a suspension of microvesicles reconstituted from a lyophilized product according to the invention in a method of therapeutic treatment.

[0135] Therapeutic techniques include any method of treating a patient (as defined above), including the use of ultrasound and gas-filled microvesicles in combination, either by themselves (e.g., in ultrasound-mediated thrombolysis, high intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization), or in combination with a therapeutic agent (i.e., for ultrasound-mediated delivery, e.g., delivery of a drug or bioactive 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, immune or autoimmune disease therapy, or for use as a vaccine); thus, the presence of gas-filled microvesicles may provide a therapeutic effect by itself or upon specific activation by various physical methods (e.g., including ultrasound-mediated delivery), or may enhance the therapeutic effect of the applied ultrasound, e.g., by imparting or contributing to impart a biological effect in vitro and / or in vivo.

[0136] Microvesicles of the present invention can typically be administered for therapeutic purposes at a concentration of about 0.01 to about 5.0 μL of gas per kg of patient, depending, for example, on the respective composition, the type of subject being treated, the tissue or organ being treated, and / or the therapeutic method being used.

[0137] In one embodiment, the above method of ultrasound therapeutic treatment comprises: (i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of the lyophilized product obtained by the process of the invention; (ii) identifying a region of interest in the patient to be exposed to a therapeutic treatment, the region of interest containing the suspension of gas-filled microvesicles; and (iii) applying an ultrasound beam to therapeutically treat the region of interest; The therapeutic treatment of the ultrasound is thereby enhanced by the presence of the suspension of gas-filled microvesicles in the region of interest.

[0138] The following examples will help to further illustrate the invention. [Example]

[0139] [Example 1] Preparation of gas-microvesicles Gas-filled microvesicles were synthesized using a commercially available microfluidic flow focusing device (CU4553.007 N30 design, Micronit Microfluidics, NL) mounted in a commercially available chip holder (Micronit microfluidics, Fluidic Connect PRO Chip Holder with 4515 Inserts). The microvesicle-forming channel had a width of 19 μm. The chip and its holder were placed in an optically transparent, temperature-controlled water bath mounted on an inverted microscope equipped with a 20x magnification objective (Olympus, LMPLAN 20x) and a CCD camera (Lumenera, LM156M). The temperature of the constant temperature bath was set to 50°C.

[0140] The amphiphilic material in the liquid stream was DSPC:DPPE-PEG5000, with a respective molar ratio of 9:1.

[0141] The material was added to a 20 mg / mL mixture of chloroform and methanol (volume ratio) at 60 °C with stirring until the amphiphilic material was completely dissolved. The solvent was then evaporated under reduced pressure, and the resulting film was dried overnight under reduced pressure. The dried material was then redispersed in saline (0.9% NaCl) at a concentration of 15 mg / mL for 30 minutes at 60 °C with stirring. The dispersion was then sonicated using a tip sonicator (Branson Sonifier 250) to homogenize the material. The preparation was then filtered using a polycarbonate filter (0.45 μm pore size), cooled to room temperature, and degassed.

[0142] CO2 / C4F in a volume ratio of 85 / 15 10Gas-filled microvesicles containing CO2 and CF4 were prepared using a gas mixing device similar to the one shown schematically in Figure 3. Briefly, two gas containers were filled with CO2 and CF4, respectively. 10 The gas flow of each gas was controlled by the respective mass flow controllers: (i) EL-Flow: F200CV-002-RAD-11-K (for CO2) and (ii) Low-ΔP-Flow: F-200DV-RAD-11-Z (for C4F 10 The gas flow rate was adjusted by a mass flow controller (both gas controllers were from Bronkhorst, Ruurlo, The Netherlands). The mass flow controller was controlled by a customized software program implemented in Matlab (Mathworks) installed on a personal computer to set and maintain the desired mixture ratio. A pressure sensor (PSE530-M5-L; SMC Corp., Tokyo, Japan) measured the actual pressure of the gas mixture in the outlet channel leading to the microfluidic chip; a gas pressure of 2 bar was used for microvesicle formation. The liquid co-flow rate was controlled by a separate mass flow controller (Mini Cori Flow: M13V14I-MAD-11-KS; Bronkhorst, Ruurlo, The Netherlands). A liquid co-flow rate of approximately 150 μL / min was used to operate the flow-focusing device in the jet regime to produce microvesicles with a diameter (mode) of approximately 4 μm.

[0143] [Example 2] Preparation of the Lyophilized Composition First, 3 mL of the calibrated microvesicle suspension obtained by the microfluidic flow focusing method described in Example 1 was placed in a Pyrex tube (Pyrex disposable tube 12 × 75 mm) and CF4 10The calibrated microvesicle suspension was then centrifuged (Sigma 3-16 centrifuge) at 64 g for 6 minutes, and the infranatant was aspirated using a syringe equipped with a needle. The remaining 200 μL of washed microvesicles were redispersed with 3 mL of a solution containing a lyoprotectant component, as detailed in the Examples below.

[0144] The calibrated microvesicles, suspended in a solution of lyoprotectant components, were then aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a freeze-dryer.

[0145] The vials were cooled to temperatures between -30°C and -60°C (as detailed in the Examples below) and lyophilized under vacuum for approximately 1 hour. At the end of the procedure, the lyophilized composition was obtained as a white, homogeneous, dry solid. Then, pure CF4 was added to the headspace. 10 Filled with.

[0146] [Example 3] Effect of lyoprotectant components on the properties of calibrated microvesicles Table 2 lists the lyoprotectant ingredients tested.

[0147] JPEG2025118721000005.jpg78166

[0148] The above materials were used at various concentrations at a freezing temperature of -60°C in the formulation of the lyophilized compositions exemplified in Example 2. Table 3 illustrates the compositions and concentrations of the lyoprotectant components tested (1A, 1B, 2A, 2B, S3-S13).

[0149] JPEG2025118721000006.jpg109166

[0150] Characterization After the lyophilization process, each lyophilized composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas to obtain a calibrated, stable microvesicle suspension. Thus, the lyophilized compositions were reconstituted with a volume of aqueous solution similar to the volume of the suspension undergoing the lyophilization process. Thus, the resulting concentrations of the lyoprotectant components in the reconstituted suspensions were 93, 75, and 188 mg / mL, respectively, starting from solutions with concentrations of 100 and 200 mg / mL.

[0151] After reconstitution, the reconstituted calibrated microvesicle suspension was allowed to sit on the bench for 5 minutes before being characterized using a Coulter Counter Multisizer 3 equipped with a 30 μm aperture tube to measure microvesicle size, geometric standard deviation (GSD), concentration and yield after the freeze-drying process.

[0152] result The main results of the characterization of the reconstituted calibrated microvesicle suspensions are reported in Table 4.

[0153] The GSD values and concentrations of the microvesicles were measured before and after the freeze-drying process to assess the effectiveness of the freeze-protectant components in maintaining the original properties of the calibrated microvesicles.

[0154] JPEG2025118721000007.jpg160166

[0155] Considering the GSD values and microvesicle yields after freeze-drying, the results clearly showed that adding polyethylene glycol to the calibrated microvesicle suspension before the freeze-drying process is much more efficient than adding polyols or sugars in terms of maintaining the microvesicle properties.

[0156] In particular, freeze-drying of the calibrated microvesicles in polyethylene glycol (PEG4000 and PEG8000) solutions was able to improve the GSD values and / or microvesicle yields.

[0157] Furthermore, adding polyethylene glycol solution at a concentration of 200 mg / mL to a CMV suspension produced better results than adding polyethylene glycol at a concentration of 100 mg / mL. In particular, a CMV suspension containing PEG 4000 at a concentration of approximately 200 mg / mL (1B) was characterized by a GSD of 1.22 and a remarkable CMV yield of 63%. Even more advantageously, a solution of 200 mg / mL PEG 8000 (2B) allowed a GSD of 1.20 and a CMV yield of 64% to be achieved.

[0158] [Example 4] Characterization of the properties of calibrated microvesicles after freeze-drying at different freezing temperatures The preparation of the freeze-dried compositions was further investigated by evaluating different freezing temperatures to which freshly prepared calibrated microvesicles were cooled at the beginning of the freeze-drying procedure.

[0159] The formulation process was carried out as described above in Example 2, except that the vials were cooled at different freezing temperatures as reported in Table 5.

[0160] At the end of the freeze-drying process, each freeze-dried composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas to obtain a calibrated stable microvesicle suspension. The freeze-dried compositions were therefore reconstituted with a volume of aqueous solution similar to the volume of the suspension that underwent the freeze-drying process. Thus, the resulting concentrations of the freeze-protecting component in the reconstituted suspension were 93, 75, and 188 mg / mL, respectively, starting from solutions of the freeze-protecting component at concentrations of 100 mg / mL and 200 mg / mL.

[0161] After reconstitution, the reconstituted calibrated microvesicle suspension was allowed to sit on the bench for 5 minutes before being characterized using a Coulter Counter Multisizer 3 equipped with a 30 μm aperture tube to measure microvesicle size, geometric standard deviation (GSD), concentration and yield after the freeze-drying process.

[0162] Table 5 reports the GSD values and CMV yields of microvesicle suspensions obtained after reconstitution of lyophilized compositions containing different lyoprotectant components. Formulations 2A, 2B, and S3A were selected to test different freezing temperatures.

[0163] result The results confirmed that adding PEG 8000 to the calibrated microvesicle suspensions improved freeze-drying performance at any tested freezing temperature compared with the use of sorbitol. For example, the use of 100 mg / mL PEG 8000 was far more advantageous in terms of GSD and CMV yield than the use of 100 mg / mL sorbitol, which was unable to efficiently maintain the initial CMV state. Indeed, CMV suspensions containing approximately 100 mg / mL sorbitol (S3A) were characterized by low CMV yields, up to 6%, and high GSD values ranging from 1.27 to 1.38.

[0164] Furthermore, these results confirmed that the CMV suspension containing PEG8000 at a concentration of approximately 200 mg / mL (2B) was characterized by a lower GSD value and significantly higher CMV yield when compared to the CMV suspension containing PEG8000 at approximately 100 mg / mL (2A). For example, in the former case, the CMV yield was found to be between 56 and 68%, while in the latter case, a higher CMV yield of 45% was found at -40 °C. Furthermore, the GSD value was also lower for formulation 2B, with a value below 1.20, confirming the superior performance of the 200 mg / mL PEG8000 solution in maintaining the initial properties of CMV.

[0165] JPEG2025118721000008.jpg161166

[0166] [Example 5] Effect of the concentration of cryoprotectants on the properties of calibrated microvesicles The concentration of the lyoprotectant component was also tested to evaluate the lyophilization efficiency in terms of maintaining the GSD value and microvesicle yield after lyophilization.

[0167] For this study, PEG 4000 and PEG 8000 were selected as the lyoprotectants because they showed the most promise among those tested in this study. Different solutions ranging in concentration from 50 mg / mL to 250 mg / mL were prepared for each tested formulation.

[0168] The calibrated microvesicles suspended in different solutions of lyoprotectant components were then aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a lyophilizer.

[0169] Two different freezing temperatures were tested. The vials were cooled to -60°C and -40°C and freeze-dried under vacuum for approximately 1 hour. At the end of the procedure, the freeze-dried composition was obtained as a white, homogeneous, dry solid. Then, pure CF4 was added to the headspace. 10 Filled with.

[0170] result JPEG2025118721000009.jpg76166

[0171] JPEG2025118721000010.jpg74166

[0172] The results showed a linear relationship between the final microvesicle yield after lyophilization and the total concentration of the lyoprotectants. As shown in Figure 4, increasing improvements in lyophilization efficiency were obtained using lyoprotectant concentrations between 50 mg / mL and 200 mg / mL for both PEG4000 and PEG8000.

[0173] The above results indicate that increasing the concentration of the PEG solution has a generally positive effect on the GSD value and / or CMV yield.

[0174] References 1. WO2018 / 041906 A1 - BRACCO SUISSE SA 2. PCT application number PCT / EP2019 / 055325 3. US2017 / 080113 A1 - GE Healthcare 4. WO97 / 29782 A1 - NICOMED IMAGING A / S

Claims

1. 1. A lyophilized composition comprising an amphiphilic material and a lyoprotectant component, which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, The reconstituted suspension of microvesicles has a geometric standard deviation (GSD) value of at least 1.22 or less. Lyophilized composition.

2. 2. The freeze-dried composition of claim 1, the lyoprotectant component is a polymer; Lyophilized composition.

3. 3. The freeze-dried composition of claim 2, the polymer is a polyglycol;

4. 4. The freeze-dried composition of claim 3, The polyglycol is more preferably polyethylene glycol (PEG). Lyophilized composition.

5. 5. The freeze-dried composition according to claim 1, The reconstituted suspension of calibrated microvesicles contains at least 2.0 x 10 ８ characterized by a concentration of microvesicles / mL; Lyophilized composition.

6. 6. The freeze-dried composition according to any one of claims 1 to 5, The reconstituted suspension of calibrated microvesicles contains a lyoprotectant component at a concentration of 12-25% (w / v%). Lyophilized composition.

7. 7. A freeze-dried composition according to claim 1, wherein the freeze-dried composition is obtained by reconstituting the freeze-dried composition with a pharmaceutically acceptable solution in the presence of a biocompatible gas. A suspension of gas-filled microvesicles.

8. 8. A suspension of gas-filled microvesicles according to claim 7, the lyoprotectant component is present in a concentration of 14% to 22%; A suspension of gas-filled microvesicles.

9. 1. A method for preparing a freeze-dried composition for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles, comprising: a. Preparing a suspension of calibrated gas-filled microvesicles containing polyglycol as a lyoprotectant; and b. Lyophilizing the calibrated microvesicle suspension; Including, method.

10. 10. The method of claim 9, The preparation method of step a. is a microfluidic flow focusing technique; method.

11. 10. The method of claim 9, The calibrated microvesicles obtained according to step a. have a size distribution characterized by a geometric standard deviation (GSD) of 1.22 or less; method.

12. 10. The method of claim 9, the lyoprotectant component has a total concentration comprised between 100 mg / ml and 300 mg / mL, preferably between 120 mg / ml and 250 mg / mL, more preferably a total concentration of 200 mg / mL; method.

13. A freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, comprising: The freeze-dried composition is prepared by the following steps: a. preparing a first suspension of gas-filled calibrated microvesicles by a flow focusing process, said suspension further comprising a lyoprotectant component; and b. freeze-drying the suspension; can be obtained by a process comprising Lyophilized composition.

14. 1. A method for preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, the method comprising: The method comprises the step of reconstituting a freeze-dried composition as defined in any one of claims 1 to 6 with a pharmaceutically acceptable solution in the presence of a biocompatible gas. method.

15. 15. The method of claim 14, The suspension of calibrated microvesicles is characterized by a GSD of 1.22 or less. method.

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