Lyophilized composition for preparing corrected gas-filled microvesicles

Abstract

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.

Description

Technical Field

[0001] The present invention generally relates to the field of ultrasonic contrast agents (USCAs). In particular, it relates to a lyophilized composition comprising an amphiphilic material and a cryoprotective component, which can be reconstituted to prepare a suspension of gas-filled microbubbles having a calibrated size useful for diagnostic or therapeutic applications. Further, it relates to a method for preparing such a lyophilized composition.

Background Art

[0002] Calibrated-size microbubbles (CMVs) are a new generation of gaseous microbubbles having a narrow, calibrated and controlled size distribution (average diameter size 3 - 8 μm) compared to commercially available polydisperse microbubble ultrasonic contrast agents (USCAs). These calibrated-size microbubbles are designed to enhance imaging sensitivity and improve the efficiency of delivering drugs and genes to specific organs. Calibrated microbubbles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting and flow focusing. In particular, the flow focusing technique produces calibrated microbubbles (typically having a geometric standard deviation (GSD) value of 1.05 - 1.08) in a very reproducible manner with a reasonable production rate (about 60 million bubbles per minute) and a suspension of microbubbles at a concentration acceptable for subsequent use (e.g., 3×10 8 cells of CMV / mL - 4×10 8 cells of CMV / mL).

[0003] Reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and Reference 2 [PCT application number PCT / EP2019 / 055325] are both by the applicant of the present application and describe methods for preparing CMVs such as gas-filled microbubbles, in particular by using microfluidic techniques.

[0004] The aqueous suspension of the calibrated microvesicles is very stable at room temperature for several weeks. However, this stability may pose some constraints for the development of pharmaceutical products where 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 forms, which are reconstituted using 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 adjusted sizes and subsequent freeze-drying using its sucrose solution.

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

[0007] So far, to the knowledge of the applicant, such techniques have not yet been applied to prepare freeze-dried compositions from suspensions of calibrated microvesicles. Summary of the Invention Problems to be Solved by the Invention

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

[0009] The applicant has found here that such initial properties can be maintained to an acceptable extent after the lyophilization process by using an appropriate cryoprotective component at an appropriate concentration.

Means for Solving the Problems

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

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

[0012] In a preferred embodiment, the reconstituted suspension of the 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 present invention, the reconstituted suspension of the calibrated microvesicles is at least 2.0×10 8 individuals of CMV / mL, preferably 2.1×10 8 individuals of CMV / mL, more preferably 2.3×10 8 individuals of CMV / mL, and is characterized by a concentration of up to 5.5×10 8 individuals of CMV / mL.

[0014] According to a further aspect, the present invention relates to a method for preparing a lyophilized composition for preparing a reconstituted suspension of calibrated gas-filled microvesicles, the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing polyglycol as a cryoprotective component; b. Freezing and drying the corrected microvesicle suspension.

[0015] A further aspect of the present invention relates to a lyophilized composition for preparing a suspension of corrected gas-filled microvesicles, said lyophilized composition comprising the following steps: a. Preparing a first suspension of corrected gas-filled microvesicles by a flow focusing process (said suspension further comprising a polyglycol as a cryoprotective component); and b. Freezing and drying said 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 reconstituting the lyophilized composition as defined above with a pharmaceutically acceptable solution in the presence of a biocompatible gas.

Brief Description of the Drawings

[0017]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0018] The expression "gas-filled microbubble" generally refers to a gas bubble whose boundaries are created at the gas / liquid interface by a very thin envelope (film) containing a stabilizing amphiphilic material (typically a phospholipid) placed at the interface of the gas and the liquid. The above-calibrated gas-filled microbubbles are suitable as contrast agents in ultrasonic imaging techniques (known as contrast-enhanced ultrasonic (CEUS) imaging), or in therapeutic applications combined with, for example, drug delivery mediated by ultrasound.

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

[0020] The term "calibrated", when referring to gas-filled microbubbles, in particular refers to a microbubble suspension having highly calibrated microbubbles (CMV) characterized by a size distribution having different sizes from 3 to 8 μm and a geometric standard deviation (GSD) of at least 1.2 or less, preferably at least 1.1, for example up to 1.05.

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

[0022] In the present invention, calibrated gas-filled microbubbles are preferably produced using microfluidic flow focusing technology, where a gas thread converges between two liquid flows within a flow focusing device, and calibrated microbubbles stabilized by phospholipids are formed and collected within an outlet channel. Using this approach, calibrated microbubbles are produced in a highly reproducible manner at a reasonable production rate (about 60 million bubbles per minute) (Figures 1 and 2).

[0023] Depending on the manufacturing process and device parameters, calibrated microbubbles can be obtained with a relatively narrow size distribution of approximately any desired average diameter, such as 3 - 8 μm, preferably about 4 μm.

[0024] The size distribution of the above-mentioned calibrated microbubbles is typically characterized by a geometric standard deviation (GSD) value of at least 1.20 or less, preferably at least 1.15, for example up to 1.05.

[0025] The calibrated microbubble concentration is typically between 3×10 8 ~4×10 8 per mL of CMV, preferably close to 4×10 8 per mL of CMV and not lower than 3×10 8 per mL of CMV.

[0026] The "geometric standard deviation" (GSD) generally provides an appropriate value for characterizing the spread of the size distribution in a population of particles (in a particular case, gas-filled microbubbles). A population of particles with a wide range of sizes thus has a larger GSD value than one in which the particle sizes are narrowly distributed around the average 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 that can be obtained by determining the volume of gas for each of its channels using a commercially available particle analyzer (e.g., Coulter Counter Multisizer 3 equipped with Multisizer 3 software). Each channel corresponds to a predetermined diameter of the microvesicles (e.g., in units of 0.1 micron). By determining the number of calibrated gas-filled microvesicles in the suspension, their respective diameters and volume distributions in a selected size range (e.g., 3 μm to 6 μm for a 4.5 μm CMV mean diameter), the GSD of the CMV distribution can be calculated using the following equation 1:

Number

Number

Number

[0028] Among various commercially available measurement devices, Coulter Counter Multisizer 3 equipped 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 are calibrated to 2.5 - 5 μm for an average diameter of 4 μm; a GSD of 1.05 - 1.08 (<1.1) indicates that approximately 90 - 95% of the CMVs have sizes that are included between 2.5 - 5 μm.

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

[0031] As the applicant has observed, the above - calibrated microvesicles obtained by microfluidic flow focusing can be stored at room temperature for several weeks without substantial impact on their main properties. However, after the above storage period, the properties of the microvesicles (such as concentration and size distribution) cannot be maintained and can gradually deteriorate.

[0032] The storage life of such gas - filled microvesicle suspensions is thus relatively short for pharmaceutical products, and it is necessary to develop long - term storage procedures that can maintain the initial properties of the CMVs, such as concentration, GSD, and final diameter, for longer periods, e.g., several months or several years.

[0033] The lyophilization process is an appropriate method for obtaining the dried form of the calibrated microvesicles while maintaining their initial properties with high stability over the long term.

[0034] Surprisingly, it has been found that the initial properties of the CMVs, such as concentration, GSD, and final diameter, can be substantially maintained after the lyophilization process by using appropriate lyoprotective components.

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

[0036] As used herein and in the claims, the terms "freeze-drying" and "lyophilization" are used interchangeably, as are the terms "freeze-dried" and "lyophilized".

[0037] Lyophilized composition As used herein, the expression "freeze-dried composition" refers to any dried form for the long-term storage of a gas-filled microvesicle preparation obtained by a freeze-drying process. The freeze-dried composition can include one or more active ingredients and a cryoprotectant component.

[0038] As used herein, the expression "active ingredient" refers to a microvesicle stabilizing material, such as an amphiphilic material, which is included in the freeze-dried composition together with the cryoprotectant component.

[0039] Lyoprotectant As used herein, the expression "cryoprotectant component" refers to a component suitable for freeze-drying and is included in the microvesicle suspension prior to the freeze-drying process.

[0040] The term "cryoprotectant component" refers to any compound added to protect the active ingredient during any stage of the freeze-drying process. Examples of suitable cryoprotectant components are polyethylene glycol (PEG), polyols, saccharides, surfactants, buffers, amino acids, chelate complexes, and inorganic salts.

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

[0042] In a preferred embodiment of the present 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 of PEG is HOCH2(CH2OCH2) m CH2OH, where m represents the average number of oxyethylene groups. Typical polyethylene glycols are available with a wide range of average molecular weights starting from 190 - 210 g / mol (PEG200; m = 4.2) to 7000 - 9000 g / mol (PEG8000; m = 181.4).

[0045] As used herein, the expression "molecular weight" refers to the average length of the PEG polymer chain and has a variation of ±10% with respect to the indicated molecular weight.

[0046] According to one embodiment of the present invention, the lyoprotectant component is preferably PEG having a molecular weight included between 2000 - 10000 g / mol, preferably 4000 - 8000 g / mol.

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

[0048] In one embodiment of the present invention, the lyoprotectant component is added to the suspension of CMV as a solution having a concentration included between 100 mg / mL - 300 mg / mL, preferably 120 mg / mL - 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 as a solution having a concentration of 200 mg / mL to the CMV suspension in order to improve the maintenance of the initial properties of the CMV suspension reconstituted after the lyophilization step.

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

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

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

[0053] Generally, during the manufacturing process, a CMV suspension with a lower viscosity is preferred, for example, to avoid excessive overpressure within a microfluidic flow focusing device.

[0054] From another perspective, a reconstituted lyophilized composition having a lower viscosity can facilitate its administration, particularly through injection routes (e.g., parenteral and intradermal).

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

[0056] For example, a solution containing PEG4000 typically has a relatively lower viscosity value (generally, about one-half to one-third) than an equal amount of a solution containing PEG8000.

[0057] CMV suspensions containing PEG4000 at 10% or 20% can thus provide several advantages from the perspective of suspension processing or administration.

[0058] In this specification and the claims, the term "polyol" has its conventional chemical meaning; it refers to any organic compound having more than two hydroxyl functional groups, and is represented by the general formula HOCH2(CHOH) n 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 above 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 the chemical field. Saccharides, also called carbohydrates, are molecular compounds made up of only three elements: carbon, hydrogen and oxygen. The simplest saccharides are called monosaccharides and are the building blocks for larger saccharides such as disaccharides, trisaccharides and polysaccharides.

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

[0062] As used herein, the term "amphiphilic material" includes compounds having a molecule with a hydrophilic polar head (e.g., a polar or ionic group) capable of interacting with an aqueous medium and a hydrophobic organic tail (e.g., a hydrocarbon chain) capable of interacting with, for example, an organic solvent. These compounds thus generally act as "surfactants", i.e., compounds that can stabilize mixtures that are generally immiscible materials in other ways, such as mixtures of two immiscible liquids (e.g., water and oil), mixtures of a gas and a liquid (e.g., gas microbubbles in water), or mixtures 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 carrying polymers such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), also called "pegylated lipids"; lipids carrying sulfonated mono-, di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate or cholesteryl hemisuccinate; tocopheryl hemisuccinate; lipids containing ethers or ester-linked fatty acids; polymeric lipids; diacetyl phosphate; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (e.g., polyoxyethylene stearate), polyoxyethylene aliphatic alcohols, polyoxyethylene aliphatic alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol ricinoleate, ethoxylated soy sterols, ethoxylated castor oil or ethylene oxide (EO) and propylene oxide (PO) block copolymers; sterol esters of saccharic acids including cholesteryl glucuronide, lanosterol glucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide, cholesteryl gluconate, lanosterol gluconate, or ergosterol gluconate; esters of alcohols and saccharic acids including lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoyl gluconate, or stearoyl gluconate; esters of fatty acids and saccharides including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid or polyuronic acid; saponins including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, or digitoxigenin; glycerol monoesters with glycerol or 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)-octadecanoyl]-2-aminopalmitic acid; N-Succinyldioleylphosphatidylethanolamine; 1,2-Dioleoyl-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 (C; 10 -C 20 ), preferably (C 14 -C 18 ), alkyl chains, for example, 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), for example, 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, the molecules of which are capable of forming a stabilizing film of the material (typically in the form of a monolayer) at the gas-water interface in the final microbubble suspension. Thus, these materials are also referred to in the art as "film-forming phospholipids".

[0066] Examples of suitable phospholipids include esters of one or preferably two (equal or different) fatty acid residues and phosphoric acid with glycerol, where the phosphoric acid residue is then bound to a hydrophilic group such as, for example, choline (phosphatidylcholine - PC), serine (phosphatidylserine - PS), glycerol (phosphatidylglycerol - PG), ethanolamine (phosphatidylethanolamine - PE), inositol (phosphatidylinositol), etc. An ester of just one fatty acid residue with a phospholipid is generally, in the art, called the "lyso" form or "lysophospholipid" of the phospholipid. The fatty acid residues present within the phospholipids are generally long - chain fatty acids typically containing 12 to 24 carbon atoms, preferably 14 to 22; the aliphatic chains may contain one or more unsaturations or, preferably, are completely saturated. Examples of suitable fatty acids contained in phospholipids are, for example, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. 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 - phosphoric acid; sphingolipids such as sphingomyelins, i.e., phosphatidylcholine analogs in which the residue of the diester of glycerol with a fatty acid is replaced by a ceramide chain; cardiolipins, i.e., esters of fatty acids and 1,3 - diphosphatidylglycerol; glycolipids, for example, ganglioside GM1 (or GM2) or cerebrosides; glycosphingolipids; sulfatides and glycosphingolipids.

[0068] As used herein, the term "phospholipid" includes products prepared either naturally - derived, semi - synthetic or synthetic, which can be used either alone or as a mixture.

[0069] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives), for example, typically, soy 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 are, for example, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), dilaidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipalmitadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-oleoyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, dilaidoyl phosphatidyl-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 its alkali metal salts, distearoyl phosphatidic acid (DSPA), dilaidoyl phosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), distearoyl-phosphatidylethanolamine (DSPE), dioleoyl phosphatidylethanolamine (DOPE), dilaidoyl phosphatidylethanolamine (DAPE), dilinoleoyl phosphatidylethanolamine (DLPE),Dimyristoylphosphatidylserine (DMPS), Dipalmitoylphosphatidylserine (DPPS), Distearoylphosphatidylserine (DSPS), Dioleoylphosphatidylserine (DOPS), Dipalmitoylsphingomyelin (DPSP), and Distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI).

[0072] Suitable phospholipids further include phospholipids modified by the binding thereto of a hydrophilic polymer such as polyethylene glycol (PEG) or polypropylene glycol (PPG). Preferred polymer-modified phospholipids include "pegylated phospholipids", i.e., phospholipids bound to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines (abbreviated "PE-PEG"), i.e., phosphatidylethanolamines in which the hydrophilic ethanolamine moiety is bound to a PEG molecule of variable molecular weight (e.g., 300 to 20,000 daltons, preferably 500 to 5,000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG or DOPE-PEG). For example, DPPE-PEG2000 refers to DPPE to which a PEG polymer having an average molecular weight of about 2000 is attached.

[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, such as DSPS, DPPS, DSPA, DPPA, DSPG, DPPG, ethyl-DSPC and / or ethyl-DPPC, and mixtures of DPPE and / or DSPE (including pegylated derivatives), DPPC, DSPC and / or DAPC can also be used.

[0075] For example, the mixture of phospholipids may include phosphatidylcholine derivatives, phosphatidic acid derivatives and pegylated phosphatidylethanolamine, 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, the phospholipid can be suitably used in admixture with any of the amphiphilic compounds mentioned above. Thus, for example, lipids such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and their derivatives, or butylated hydroxytoluene and / or other non-phospholipid compounds may optionally be added to one or more of the aforementioned phospholipids, for example, preferably in the range of 0 to 50% by weight, more preferably up to 25%. For example, mixtures of amphiphilic materials containing phospholipids and fatty acids, 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, can be advantageously used.

[0077] The microvesicles prepared by the present invention may optionally contain a targeting ligand.

[0078] The term "targeting ligand" includes within its meaning any compound, moiety or residue that has or can promote the targeting activity (including, for example, selective binding) of the microvesicles of the compositions of the present invention towards any biological or pathological site in vivo. Targets to which a targeting ligand may be relevant include tissues such as myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissue (including endothelium and epithelium), thin membranes, connective tissue (including stromal 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 of synthetic, semi-synthetic or natural origin. Materials or substances that can act 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 monosaccharides and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic materials including nucleosides, nucleotides, and polynucleotides.

[0080] The targeting ligand may itself be an amphiphilic compound (to be mixed with other components of the microvesicle), or it may be a compound bound to an amphiphilic molecule (such as a phospholipid) used in the formation of 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, and are known to form particularly stable gas-filled microbubble 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 a mixture with other isomers such as perfluoro-n-butane, perfluoro-isobutane), perfluoropentanes, perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (such as perfluorobut-2-ene) or perfluorobutadiene; perfluoroalkynes (such as perfluorobut-2-yne); and perfluorocycloalkanes (such as 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 およびC6F 14It includes. Particularly preferred gases are those which are in gaseous form at room temperature, such as SF6, C3F8 and C4F 10 It includes.

[0083] One aspect of the present invention thus relates to a method for preparing a lyophilized composition for long-term storage of calibrated gas-filled microvesicles, comprising the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing a polyglycol as a lyoprotectant component; b. Lyophilizing 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] Figure 2 shows a schematic view of the core part 200 of a flow focusing device ("microfluidic chip") useful in the process of the present 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 two liquid flows travel towards the contact zone 203 and then pass through a calibrated orifice 204 shown as a dotted line in FIG. 2. The calibrated orifice is preferably connected to a calibrated channel 204' having the same cross-section as the orifice and then to the first portion 205 of the outlet channel 206. In an alternative embodiment (not shown), the calibrated orifice 204 may be a nozzle directly connected to the first portion 205 of the outlet channel 206, i.e., without an intervening calibrated channel. Microbubbles 203' are formed within the calibrated orifice and directed through the calibrated channel 204' towards the first portion 205 of the outlet channel 206. The hydraulic diameter of the outlet channel is generally larger than that of the calibrated orifice and typically increases from the initial diameter of the calibrated orifice to the final diameter of the outlet channel 206, substantially corresponding to the hydraulic diameter of a collection tube (not shown) that connects the flow focusing device to a container, such as a sealed vial, for collecting the microbubble suspension.

[0087] In the first portion 205 of the outlet channel of the device, and preferably also in the contact zone 203 and in the calibrated orifice 204, the temperature of the microbubbles 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 flow for preparing calibrated gas-filled microbubbles by the method of the present invention contains 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] Preferably, suitable aqueous carriers that are 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 containing one or more osmotic regulators. Osmotic regulators include salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (such as glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols, etc.), chitosan derivatives such as carboxymethyl chitosan, trimethyl chitosan or gelling compounds such as carboxymethyl cellulose, hydroxyethyl starch or dextran.

[0090] In an alternative embodiment, an additional oil phase may be added to incorporate the therapeutic hydrophobic substance into the microvesicles. To achieve this, 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 number PCT / EP2019 / 055325]. The gas-filled microvesicles formed thus have a film of oil disposed at the interface between the gas and the stabilizing layer of the amphiphilic material and can carry the desired therapeutic agent. Suitable oils may include any biocompatible oil that is liquid at room temperature, for example, mono-, di- or tri-esters of saturated or unsaturated (C2 - C 18 ) alkyl chains with glycerol (including homo- or hetero-alkyl esters), such as glycerol monobutyrate, glycerol monolaurate, 1,2 - dihexanoyl glycerol, 1,2 - dioctanoyl glycerol, 1,2 - dioleoyl - 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 shown previously. Preferably, the gas is a mixture of a gas that is very soluble in water ("HS gas") and a gas that is poorly soluble in water ("LS gas"), as expected in reference 2 [PCT application number PCT / EP2019 / 055325].

[0092] During the stabilization phase, most of the very soluble gas rapidly dissolves in water, while the less soluble ones remain trapped in the dense layer of the amphiphilic compound, and typically, a residual amount of a portion of the HS solubility gas is dispersed therein.

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

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

[0095] In a preferred embodiment of the present invention, gas-filled microvesicles containing CO2 / C4F in a volume ratio of 80 / 20 to 90 / 10, for example 85 / 15, can be prepared by a gas mixing device similar to that schematically shown in FIG. 3. 10 FIG. 3 shows an example of a microfluidic flow focusing device used to produce calibrated microvesicles. A gas stream 302 (e.g., containing a mixture of C4F

[0096] and CO2) and a liquid stream 301 (containing an amphiphilic material, e.g., a phospholipid, a fatty acid, or a mixture thereof) are supplied to a microfluidic chip 303, and microvesicles are produced through an orifice 304. The microvesicle suspension is preferably at atmospheric pressure with a gas (e.g., C4F 10 and CO2 mixture). 10) is collected into the vial 305 which is pre-filled. A venting device (e.g., needle 306) is preferably used to equalize the overpressure generated by the liquid filling of the vial. At the end of the 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 suitable washing techniques to remove the not-assembled amphiphilic material and any 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) the not-assembled amphiphilic material and residual compounds.

[0099] According to the present 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 do not form the stabilizing layer of the gas-filled microvesicles.

[0101] As used herein, "residual compound" refers to any possible additive substance added to the amphiphilic material solution during the preparation of the microvesicles, such as the osmotic pressure regulator described above.

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

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

[0104] The initial CMV characteristics are particularly maintained when polyethylene glycol is used as the lyoprotectant, and the polyethylene glycol is added to the CMV suspension as a solution having a total concentration included between 100 mg / ml and 300 mg / mL, preferably between 120 mg / ml and 250 mg / mL (more preferably the total concentration of PEG is 200 mg / mL).

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

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

[0107] The above lyoprotectant is as described above herein. The use of polyethylene glycol as a lyoprotectant shows advantageous results when used in the lyophilization step of the calibrated microvesicle suspension, and a lyophilized composition can be prepared and then reconstituted to obtain a suspension of calibrated microvesicles having acceptable characteristics from the viewpoints of concentration and size distribution compared to the initial suspension (before lyophilization).

[0108] For example, the addition of polyethylene glycol as a cryoprotectant provides the best strategy for substantially maintaining the GSD value after the lyophilization process compared to the addition of polyols or saccharides. According to one embodiment, a GSD value included between 1.20 and 1.22 can be obtained when using polyethylene glycol, as compared to the use of polyols or saccharides that give calibrated microvesicles characterized by higher GSD values (up to 1.34).

[0109] In a preferred embodiment, by using polyethylene glycol as a cryoprotectant for lyophilization, it is possible to significantly improve the yield of calibrated microvesicles after reconstitution of the lyophilized product to a value exceeding 60%, for example a value of 68%, which is higher than that obtained with the use of polyols or saccharides.

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

[0111] As observed by the applicant, the lyophilization parameters may be selected to further optimize the properties (such as yield, size, GSD) of the reconstituted suspension of microvesicles.

[0112] In a 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 an even more preferred embodiment, the negative pressure applied during the lyophilization process is preferably 0.5 mbar or less, preferably 0.2 or less, for example around 0.1 mbar.

[0114] A further aspect of the present invention relates to a lyophilized composition for preparing a suspension of calibrated gas-filled microbubbles, said lyophilized composition comprising the following steps: a. preparing a first suspension of calibrated microbubbles filled with gas by a flow focusing process; and b. lyophilizing said suspension, and can be obtained by a process comprising these steps.

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

[0116] Then, the lyophilized composition can be reconstituted with a suitable pharmaceutically acceptable (aqueous) solution in the presence of a biocompatible gas, thus providing a suspension of calibrated gas-filled microbubbles, wherein said microbubbles 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, the pharmaceutically acceptable (aqueous) solution is water, typically sterilized, pyrogen-free water (to prevent 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 osmotic regulators such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.

[0118] The lyophilized composition is typically reconstituted with an aqueous solution having a volume similar to the volume of the suspension undergoing the lyophilization process. Thus, the concentration of the lyoprotectant component in the reconstituted suspension is substantially the same as that in the original suspension.

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

[0120] These amounts of cryoprotective components are typically higher than those in the preparation of prior art gas-filled microvesicles which are generally less than 10% (w / w).

[0121] Surprisingly, the reconstituted suspension of calibrated microvesicles was found to substantially maintain the initial properties of the calibrated microvesicles characterized prior to the lyophilization process and is thus suitable for subsequent pharmaceutical use.

[0122] In one embodiment of the present invention, the above reconstituted suspension of calibrated microvesicles is at least 2.0×10 8 CMV / mL, preferably 2.1×10 8 CMV / mL, more preferably 2.3×10 8 CMV / mL, characterized by a concentration up to 5.5×10 8 CMV / mL.

[0123] As described above, the expression "microvesicle concentration" refers to the number of microvesicles, i.e., the number of CMV / mL, within a volume unit determined using a Coulter Counter instrument.

[0124] Typically, the concentration (%) of calibrated microvesicles measured after reconstituting the lyophilized composition of the present invention with a suitable aqueous solution enables determination of the yield of microbubbles after reconstitution compared to the microvesicle concentration measured prior to the lyophilization process.

[0125] In the present invention, the yield of calibrated microvesicles after reconstitution of the lyophilized 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 "yield of calibrated microvesicles" 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 (for short, "after lyophilization") (see Equation 2): Equation 2: CMV yield after lyophilization (%) = (CMV concentration / mL after lyophilization) / (CMV concentration / mL before lyophilization)

[0127] Use The microvesicles prepared by the method of the present invention may be used in various diagnostic and / or therapeutic techniques, particularly including ultrasound and magnetic resonance.

[0128] Diagnostic methods include any method that can enhance the visualization of a portion or part of the body of an animal (including humans) by the use of gas-filled microvesicles, including imaging for preclinical and clinical research purposes. Various imaging techniques can be used in ultrasound applications, for example, including basic and harmonic B-mode imaging, pulse or phase inversion imaging, and basic and harmonic Doppler imaging; three-dimensional imaging techniques can be used as needed.

[0129] The microvesicles according to 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 respective composition, the tissue or organ being imaged and / or the imaging technique selected. This general concentration range can of course vary depending on the specific imaging application, for example, when 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 reconstituting a lyophilized product obtained by the process of the present invention; and (ii) detecting an ultrasonic 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] The reconstitution of the lyophilized product is preferably carried out by dispersing it in a physiologically acceptable aqueous carrier (such as saline) with gentle stirring in the presence of a physiologically acceptable gas (such as C4F 10 ).

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

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

[0135] The therapeutic technique includes any treatment method of a patient (as defined above), which includes, as such, the combined use of ultrasound and gas-filled microbubbles (for example, in thrombolysis mediated by ultrasound, high-intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization), or in combination with a therapeutic agent (that is, delivery mediated by ultrasound, for example, for the delivery of a drug or a bioactive compound to a selected site or tissue, for example, in tumor treatment, gene therapy, treatment of infectious diseases, treatment of metabolic diseases, treatment of chronic diseases, treatment of degenerative diseases, treatment of inflammatory diseases, treatment of immune or autoimmune diseases, or in use as a vaccine). Thus, the presence of gas-filled microbubbles can provide the therapeutic effect itself, or enhance the therapeutic effect of the applied ultrasound, for example, by providing or contributing to a biological effect in vitro and / or in vivo, by itself, or upon specific activation by various physical methods (including, for example, delivery mediated by ultrasound).

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

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

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

Examples

[0139] [Example 1] Preparation of gas-microvesicles Gas-filled microbubbles 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 microbubble formation 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 thermostatic bath was set at 50 °C.

[0140] The amphiphilic material in the liquid stream was DSPC:DPPE-PEG5000, and the respective molar ratios were 9:1.

[0141] The materials were added to a 2:1 (volume ratio) mixture of chloroform / methanol at a concentration of 20 mg / mL at the above molar ratio and stirred at 60 °C until the amphiphilic materials were completely dissolved. Then, the solvent was evaporated under reduced pressure, and the resulting film was dried under reduced pressure overnight. Then, the dried material was redispersed in saline (0.9% NaCl) (at a concentration of 15 mg / mL) with stirring at 60 °C for 30 minutes. Then, the dispersion was sonicated using a tip sonicator (Branson Sonifier 250) to uniformly disperse the materials. Then, the preparation was filtered using a polycarbonate filter (0.45 μm pore size), cooled to room temperature, and degassed.

[0142] CO2 / C4F at a volume ratio of 85 / 15 10Gas-filled microbubbles containing [were prepared using a gas mixing device similar to that schematically shown in Figure 3. Briefly, two gas containers were filled with CO2 and C4F respectively. 10 The gas flow of each gas was regulated by 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 ). Both gas controllers were from Bronkhorst, Ruurlo, The Netherlands. The mass flow controllers were controlled by a customized software program implemented in Matlab (Mathworks) installed on a personal computer to set and maintain the desired mixing ratio. A pressure sensor (PSE530-M5-L; SMC Corp., Tokyo, Japan) measured the actual pressure in the gas mixture in the outlet channel leading to the microfluidic chip; a gas pressure of 2 bar was used for microbubble formation. The liquid co-flow rate was controlled by using a separate mass flow controller (Mini Cori Flow: M13V14I-MAD-11-K-S; Bronkhorst, Ruurlo, The Netherlands). Using a liquid co-flow rate of about 150 μL / min, the flow focusing device in the jet regime was operated to produce microbubbles with a diameter (mode) of about 4 μm.

[0143] [Example 2] Preparation of lyophilized composition First, 3 mL of the calibrated microbubble suspension obtained by the microfluidic flow focusing method described in Example 1 was placed into a Pyrex tube (Pyrex disposable tube 12 × 75 mm) with C4F in the tube 10It was transferred without further addition. Then, the calibrated microvesicle suspension was centrifuged at 64 g for 6 minutes (Sigma 3-16 centrifuge), and the infranatant was aspirated using a syringe equipped with a needle. The remaining 200 μL of washed microvesicles were redispersed using 3 mL of a solution containing a lyoprotectant as detailed in the following examples.

[0144] Then, the calibrated microvesicles suspended in the solution of the lyoprotectant were aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a lyophilizer.

[0145] The vials were cooled at a temperature of -30 °C to -60 °C (detailed in the following examples) 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, the headspace was filled with pure C4F 10 was filled.

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

[0147] JPEG0007712878000004.jpg78166

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

[0149] JPEG0007712878000005.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 reconstituted and stable microvesicle suspension. Thus, the lyophilized composition was reconstituted with an aqueous solution of the same volume as the volume of the suspension undergoing the lyophilization process. Thus, the resulting concentration of the lyoprotectant in the reconstituted suspension started from solutions with concentrations of 100 mg / mL and 200 mg / mL and was 93.75 mg / mL and 188 mg / mL, respectively.

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

[0152] Results The main results of the characterization of the reconstituted and calibrated microvesicle suspension are reported in Table 4.

[0153] The GSD values and concentrations of the microvesicles were measured before and after the lyophilization process to evaluate the effectiveness of the lyoprotectant in maintaining the initial characteristics of the calibrated microvesicles.

[0154] JPEG0007712878000006.jpg160166

[0155] Considering the GSD values and the yield of microvesicles after lyophilization, the results clearly showed that adding polyethylene glycol to the calibrated microvesicle suspension before the lyophilization process was much more efficient than adding polyols or saccharides in terms of maintaining microvesicle characteristics.

[0156] In particular, lyophilization of the calibrated microvesicles in solutions of polyethylene glycol (PEG4000 and PEG8000) was able to improve the GSD value and / or the microvesicle yield.

[0157] Furthermore, when a polyethylene glycol solution was added to the CMV suspension at a concentration of 200 mg / mL, better results were obtained than when polyethylene glycol was added at a concentration of 100 mg / mL. In particular, the CMV suspension (1B) containing PEG4000 at a concentration of about 200 mg / mL was characterized by a GSD of 1.22 and a remarkable CMV yield of 63%. Even more advantageously, when a solution of PEG8000 at 200 mg / mL (2B) was used, it was possible to reach a GSD of 1.20 and a CMV yield of 64%.

[0158] [Example 4] Characterization of the properties of calibrated microvesicles after lyophilization at different freezing temperatures The preparation of the lyophilized composition was further investigated by evaluating different freezing temperatures at which the freshly prepared calibrated microvesicles were cooled at the beginning of the lyophilization procedure.

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

[0160] At the end of the lyophilization process, each lyophilized composition was reconstituted with 1.5 mL of an aqueous solution in the presence of a biocompatible gas in order to obtain a calibrated and stable microvesicle suspension. The lyophilized composition was thus reconstituted with an aqueous solution of the same volume as the volume of the suspension that had undergone the lyophilization process. Thus, the resulting concentrations of the lyoprotectant in the reconstituted suspension were 93, 75 mg / mL and 188 mg / mL, starting from solutions of the lyoprotectant at concentrations of 100 mg / mL and 200 mg / mL, respectively.

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

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

[0163] Results From the results, it was confirmed that adding PEG8000 to the calibrated microvesicle suspension can improve the lyophilization performance at any of the tested freezing temperatures compared to using sorbitol. For example, the use of 100 mg / mL of PEG8000 was much more advantageous in terms of GSD and CMV yield than the use of 100 mg / mL of sorbitol (which could not efficiently maintain the initial CMV state). Indeed, the CMV suspension (S3A) containing sorbitol at about 100 mg / mL was characterized by a low CMV yield of up to 6% and a high GSD value included between 1.27 and 1.38.

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

[0165] JPEG0007712878000007.jpg161166

[0166] [Example 5] Effect of lyoprotectant concentration on the properties of calibrated microvesicles To evaluate the lyophilization efficiency from the perspective of maintaining the GSD value and microvesicle yield after lyophilization, the concentration of the lyoprotectant was also tested.

[0167] For this test, since PEG4000 and PEG8000 emerged as the most promising among those tested in this study, they were selected as cryoprotective components. Different solutions in the concentration range of 50 mg / mL to 250 mg / mL were prepared for each of the tested formulations.

[0168] The calibrated microvesicles suspended in different solutions of cryoprotective 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 at -60 °C and -40 °C 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, the headspace was filled with pure C4F 10 was filled.

[0170] Results JPEG0007712878000008.jpg76166

[0171] JPEG0007712878000009.jpg74166

[0172] The results showed a linear relationship between the final microvesicle yield after lyophilization and the total concentration of the cryoprotective components. As shown in Figure 4, an increase in the improvement of lyophilization efficiency was obtained for both PEG4000 and PEG8000 using cryoprotective component concentrations of 50 mg / mL to 200 mg / mL.

[0173] The above results indicate that an increase in the concentration of the PEG solution generally has a 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

**Claim 1** A lyophilized composition comprising (i) an amphiphilic material containing a phospholipid and (ii) polyethylene glycol (PEG) having a molecular weight between 2000 and 10000 g / mol, which provides a suspension of calibrated gas-filled microvesicles when reconstituted using a pharmaceutically acceptable solution in the presence of a biocompatible gas, wherein the reconstituted suspension of microvesicles has a geometric standard deviation (GSD) value of 1.22 or less, and the polyethylene glycol (PEG) has a concentration of 12 to 25% (w / v%); lyophilized composition. **Claim 2** The lyophilized composition according to claim 1, wherein the polyethylene glycol (PEG) has a molecular weight between 4000 and 8000 g / mol; lyophilized composition. **Claim 3** The lyophilized composition according to claim 2, wherein the polyethylene glycol (PEG) is selected from PEG having a molecular weight of 4000 g / mol (±10%) or PEG having a molecular weight of 8000 g / mol (±10%); lyophilized composition. **Claim 4** The lyophilized composition according to any one of claims 1 to 3; The reconstituted suspension of the corrected microvesicles is characterized by a concentration of at least 2.0×10 ８ microvesicles / mL, lyophilized composition. **Claim 5** The lyophilized composition according to any one of claims 1 to 4, wherein the polyethylene glycol (PEG) has a concentration of 14 to 24% (w / v%); lyophilized composition. **Claim 6** A suspension of gas-filled microvesicles obtained by reconstituting the lyophilized composition according to any one of claims 1 to 5 using a pharmaceutically acceptable solution in the presence of a biocompatible gas; suspension of gas-filled microvesicles. **Claim 7** The suspension of gas-filled microvesicles according to claim 6, wherein the polyethylene glycol (PEG) is present at a concentration of 12% to 25%; suspension of gas-filled microvesicles. **Claim 8** A method for preparing the lyophilized composition of claim 1 for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles having a geometric standard deviation (GSD) of 1.22 or less, comprising a. preparing a suspension of calibrated gas-filled microvesicles containing polyethylene glycol having a molecular weight between 2000 and 10000 g / mol as a cryoprotective component, wherein the calibrated microvesicles have a size distribution characterized by a geometric standard deviation (GSD) of 1.22 or less; and b. lyophilizing the calibrated microvesicle suspension; comprising method. **Claim 9** The method according to claim 8, wherein The preparation method of step a is a microfluidic flow focusing technique, Method.

10. The method according to claim 8 or 9, wherein the lyoprotectant component has a total concentration included between 100 mg / ml and 300 mg / mL, Method.

11. The method according to claim 10, wherein the lyoprotectant component has a total concentration included between 120 mg / ml and 250 mg / mL, Method.

12. The method according to claim 11, wherein the lyoprotectant component has a total concentration of 200 mg / mL, Method.

13. A method for preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, The method includes the step of reconstituting the lyophilized composition defined in any one of claims 1 to 5 using a pharmaceutically acceptable solution in the presence of a biocompatible gas, Method.

14. The method according to claim 13, wherein the suspension of calibrated microvesicles is characterized by a GSD of 1.22 or less, Method.

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