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 a lyophilized composition comprising an amphiphilic material and a mixture of lyoprotective components, which can be reconstituted to prepare a suspension of gas-filled microvesicles with a calibrated size useful for diagnostic or therapeutic applications. It also relates to a method for preparing such a lyophilized composition.

Description

Technical Field

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

Background Art

[0002] Calibrated-size microbubbles (CMV) are a new generation of gaseous microbubbles having a narrow, calibrated and controlled size distribution (average size 3 - 8 μm) compared to commercially available polydisperse microbubble ultrasound contrast agents (USCA). 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 at a reasonable production rate (about 60 million bubbles per minute) and at a concentration of suspended microbubbles acceptable for subsequent use (e.g., 3×10 8 individual CMV / mL to 4×10 8 individual 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 CMV such as gas-filled microbubbles, in particular by using microfluidic techniques.

[0004] Despite the fact that the aqueous suspension of the calibrated microvesicles is highly stable at room temperature for several weeks, this stability can pose some constraints for the development of pharmaceutical products where a longer shelf life is generally desired. 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, which is advantageous for storing pharmaceutical products in a dry form for several months. In fact, lyophilized 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 a lyophilized form, which is 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 lyophilized 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 lyophilized 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 now found that such initial properties can be maintained to an acceptable extent after the lyophilization process by using an appropriate mixture of lyoprotectants.

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 provides a suspension of calibrated gas-filled microvesicles when reconstituted using a pharmaceutically acceptable solution in the presence of a biocompatible gas. The lyoprotectant component is a mixture of at least two lyoprotectant components, and the reconstituted suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of less than 1.2.

[0011] In a preferred embodiment of the present invention, the mixture of lyoprotectant components comprises a polymer, preferably a hydrophilic polymer, more preferably a polyglycol, and a polyol or a saccharide.

[0012] In an even more preferred embodiment, the mixture comprises polyethylene glycol (PEG) and sorbitol or PEG and sucrose.

[0013] In a preferred embodiment, the reconstituted suspension of calibrated microvesicles is characterized by a GSD of at least 1.2 or less, preferably at least 1.15, for example up to 1.1.

[0014] In one embodiment of the present invention, the reconstituted suspension of calibrated microvesicles is at least 2.0×10 8 CMV / mL, preferably 2.25×10 8 CMV / mL, more preferably 2.5×108 CMV / mL, and is characterized by a concentration up to 5.5×10 8 CMV / mL.

[0015] According to a further aspect, the present invention relates to a method for preparing a lyophilized composition for the preparation of a reconstituted suspension of calibrated gas-filled microbubbles, the following steps: a. preparing a suspension of calibrated gas-filled microbubbles comprising a mixture of cryoprotective components; b. lyophilizing the calibrated microbubble suspension.

[0016] A further aspect of the present invention relates to a lyophilized composition for preparing a suspension of calibrated gas-filled microbubbles, the lyophilized composition comprising the following steps: a. preparing a first suspension of calibrated gas-filled microbubbles by a flow focusing process (the suspension further comprising a mixture of cryoprotective components); and b. lyophilizing the suspension.

[0017] A further aspect of the present invention relates to a method for preparing an injectable contrast agent comprising a suspension of gas-filled microbubbles, the 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

[0018]

Figure 1

Figure 2

Figure 3

[0019] The expression "gas-filled microbubble" generally refers to a gas bubble whose boundary is created at the gas / liquid interface by a very thin envelope (film) containing a stabilizing amphiphilic material (typically a phospholipid) placed at the interface between the gas and the liquid. The above calibrated gas-filled microbubbles are suitable as contrast agents in ultrasonic imaging techniques (known as contrast-enhanced ultrasound (CEUS) imaging), or in therapeutic applications combined with, for example, drug delivery mediated by ultrasound.

[0020] These stabilized bubbles (dispersed in a suitable physiological solution) are generally referred to in the art by various specialized terms, typically depending on the stabilizing material used in their preparation; these terms include, for example, "microspheres", "microbubbles", "microcapsules" or "microballoons", and are herein collectively referred to as "gas-filled microbubbles" (or, in short, "microbubbles").

[0021] The term "calibrated", when referring to gas-filled microbubbles, specifically refers to a microbubble suspension having highly calibrated microbubbles (CMVs) 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.

[0022] In this specification and the claims, the term "calibrated" is used interchangeably with "size-controlled", "monodisperse" or "single-sized" microbubbles.

[0023] 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 (approximately 60 million bubbles per minute) (Figures 2 and 3).

[0024] 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.

[0025] The size distribution of the above-mentioned calibrated microbubbles is typically characterized by a geometric standard deviation (GSD) value of at least 1.2 or less, preferably at least 1.1, for example up to 1.05.

[0026] 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.

[0027] 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 (i.e., relatively smaller size) than one in which the particle sizes are narrowly distributed around the average value.

[0028] 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 measurement device (e.g., Coulter Counter Multisizer 3 equipped with Multisizer 3 software), where each channel corresponds to a predetermined diameter of the microvesicles (e.g., in 0.1 micron units). 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

[0029] Among various commercially available measurement devices, Coulter Counter Multisizer 3 equipped with Multisizer 3 software can calculate and provide GSD values as defined above.

[0030] 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.

[0031] 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.

[0032] 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 (e.g., concentration and size distribution) cannot be maintained and may gradually deteriorate.

[0033] 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.

[0034] 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.

[0035] Surprisingly, it has been found that the initial properties of the CMVs, such as concentration, GSD, and final diameter, can be substantially maintained after a lyophilization process using an appropriate combination of cryoprotective components as compared to a lyophilization process using the same cryoprotective component as a single additive.

[0036] In a first aspect, the present invention provides a lyophilized composition comprising an amphiphilic material and a mixture of cryoprotective components, 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 1.2 or less, preferably at least 1.15, for example up to 1.1.

[0037] As used herein and in the claims, the terms "freeze-drying" and "lyophilization" are used interchangeably, as are the terms "freeze-dried" and "lyophilized".

[0038] Lyophilized composition As used herein, the expression "lyophilized composition" refers to any dried form for the long-term storage of a gas-filled microvesicle preparation obtained by a lyophilization process. The lyophilized composition can comprise one or more active ingredients and a mixture of at least two cryoprotective components.

[0039] As used herein, the expression "active ingredient" refers to a microvesicle stabilizing material, such as an amphiphilic material, which is included in the lyophilized composition together with the cryoprotective components.

[0040] Mixture of lyoprotective components As used herein, the expression "mixture of cryoprotective components" refers to a combination of at least two components suitable for lyophilization, which are included in the microvesicle suspension prior to the lyophilization process.

[0041] The term "cryoprotective component" refers to any compound added to protect the active ingredient during any stage of the lyophilization process. Examples of suitable cryoprotective components are polyethylene glycol (PEG), polyols, saccharides, surfactants, buffers, amino acids, chelate complexes, and inorganic salts.

[0042] According to one embodiment of the present invention, the mixture of the lyoprotectant components is a combination of at least two different compounds suitable for lyophilization, and is selected from the group of polymers, polyols and saccharides. Preferably, the mixture includes a combination of a polyol or saccharide and a polymer.

[0043] In a preferred embodiment of the present invention, one of the components of the mixture of the lyoprotectant components is a polymer, preferably a hydrophilic polymer, more preferably a polyglycol.

[0044] In an even more preferred embodiment, the polyglycol is polyethylene glycol (PEG).

[0045] 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).

[0046] According to the present specification, the expression "molecular weight" indicates the average length of the PEG polymer chain and has a variation of ±10% with respect to the indicated molecular weight.

[0047] According to one embodiment of the present invention, the mixture of the lyoprotectant components preferably includes PEG having a molecular weight included between 2000 - 10000 g / mol, preferably 4000 - 8000 g / mol.

[0048] 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%).

[0049] In one embodiment of the present invention, the mixture of cryoprotective components is added to the suspension of CMV as a solution having a concentration included between 100 mg / mL and 300 mg / mL, preferably between 120 mg / mL and 250 mg / mL, more preferably a concentration close to 200 mg / mL.

[0050] As observed by the applicant, polymer (especially polyglycol, such as PEG) suspensions having a concentration of 150 mg / mL or more (for example 200 mg / mL) can be difficult to handle during industrial processes due to their relatively high viscosity. Therefore, it is preferred to continue using polymer suspensions having a concentration lower than 150 mg / mL.

[0051] To improve the maintenance of the initial properties of the CMV suspension reconstituted after the lyophilization process, the applicant has found that it is preferable to use an appropriate mixture of PEG in combination with a second cryoprotective component at a concentration lower than 150 mg / mL (for example 100 mg / mL).

[0052] In a preferred embodiment of the present invention, the lyophilized composition comprises a mixture of cryoprotective components which is a combination of a polyol and a polymer, preferably a polyglycol.

[0053] In the present 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 is characterized by the general formula HOCH2(CHOH) n CH2OH, where n is an integer from 1 to 6, preferably from 2 to 4. The polyols have different chain lengths, i.e., carbon chains of 4, 5 or 6 carbons. They each have one hydroxyl group attached to each carbon. The polyols can be further distinguished by the relative configuration (stereochemistry) of these hydroxyl groups. Suitable polyols include erythritol, xylitol, sorbitol, lactitol and mannitol.

[0054] 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.

[0055] In an even more preferred embodiment, the polyol is sorbitol or xylitol.

[0056] The term "sorbitol" has its conventional meaning in the chemical field. Sorbitol, or (2R,3R,4R,5S)-hexane-1,2,3,4,5,6-hexol, is a polyhydric alcohol containing straight-chain carbons, has 6 carbon atoms, and each is substituted by a hydroxyl functional group. It has a molecular weight of 182.172 g / mol. Sorbitol occurs naturally and is also produced synthetically from glucose. It is an isomer of mannitol.

[0057] The term "xylitol" has its conventional meaning in the chemical field. Xylitol, or (2S,4R)-pentane-1,2,3,4,5-pentol, is a sugar alcohol of 5 carbons, where all 5 carbon atoms of the molecule are bonded to hydroxyl groups. Its molecular weight is 152.146 g / mol.

[0058] In a further embodiment, the lyophilized composition contains a mixture of saccharides and polymers.

[0059] The term "saccharides" 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 units for larger saccharides such as disaccharides, trisaccharides, and polysaccharides.

[0060] Preferably, the saccharides are selected from the group of disaccharides, trisaccharides, and polysaccharides, more preferably disaccharides or trisaccharides.

[0061] Monosaccharides have the general molecular formula (CH2O) nand wherein n can be 3, 5 or 6. Monosaccharides can form cyclic structures by reaction of an OH group and a carbonyl group. These cyclic molecules can subsequently react with another alcohol. Suitable examples of monosaccharides include glucose, fructose and galactose.

[0062] Disaccharides (C 12 H 22 O 11 ) are saccharides consisting of two monosaccharide units linked by a glycosidic bond. The latter is a covalent bond formed by reaction of an OH group of a second monosaccharide and the anomeric carbon of one cyclic monosaccharide. Disaccharides differ from each other in their monosaccharide components and the specific type of glycosidic bond that links them. Examples of disaccharides include maltose, lactose, and sucrose. Particularly preferred among the disaccharides is sucrose.

[0063] Trisaccharides are saccharides consisting of three monosaccharides and having two glycosidic bonds that link them. Similar to disaccharides, each glycosidic bond can be formed between any hydroxyl group on the component monosaccharides. Even if all three component saccharides are identical (e.g., glucose), different combinations of bonds (positional chemistry) and stereochemistry (α- or β-) result in trisaccharides that are diastereoisomers with different chemical and physical properties. Examples of trisaccharides are maltotriose, melezitose, maltotriulose and raffinose. Particularly preferred among the trisaccharides is raffinose.

[0064] Polysaccharides are high molecular weight saccharide molecules consisting of long chains of monosaccharide units linked together by glycosidic bonds. They range in structure from linear to highly branched. Examples of polysaccharides are: starch, cellulose, dextran and chitin. Particularly preferred among the polysaccharides is dextran.

[0065] In a preferred embodiment of the present invention, the saccharide is a disaccharide, more preferably sucrose.

[0066] In the present invention, the term "sucrose" has its standard meaning. Sucrose is a disaccharide formed by glucose and fructose units linked by an acetal oxygen bridge from the hemiacetal of glucose to the hemiketal of fructose. Sucrose has the empirical formula C 12 H 22 O 11 and a molecular weight of 342.30 g / mol.

[0067] The first CMV characteristic is particularly maintained when using a mixture of lyoprotectants, and the mixture of lyoprotectants has a total concentration included between 100 mg / ml and 300 mg / mL, preferably between 120 mg / ml and 250 mg / mL, and more preferably, the total concentration of PEG and polyol or PEG and saccharide is 200 mg / mL.

[0068] In a preferred embodiment, the mixture of lyoprotectants contains PEG and polyol or PEG and saccharide in a ratio of 2:1 to 2:3, preferably 3:2 to 4:5, more preferably 1:1.

[0069] The above mixture of lyoprotectants shows advantageous results when used in the lyophilization process 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 in terms of concentration and size distribution.

[0070] Amphiphilic material Materials suitable for forming a stabilizing layer of gas-filled microvesicles (i.e., microvesicle stabilizing materials) are known in the art. These preferably include amphiphilic materials.

[0071] 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 otherwise, 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).

[0072] 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-bonded 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 palmitoyl homocysteine; 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.

[0073] According to the present invention, the amphiphilic material is preferably a phospholipid.

[0074] 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".

[0075] Examples of suitable phospholipids include esters of glycerol with one or preferably two (equal or different) fatty acid residues and phosphoric acid, where the phosphoric acid residue is then bonded to a hydrophilic group such as, for example, choline (phosphatidylcholine - PC), serine (phosphatidylserine - PS), glycerol (phosphatidylglycerol - PG), ethanolamine (phosphatidylethanolamine - PE), inositol (phosphatidylinositol), etc. Esters of just one fatty acid residue with a phospholipid are generally, in the art, called the "lyso" form or "lysophospholipid" of the phospholipid. The fatty acid residues present within the phospholipid are generally long - chain fatty acids typically containing from 12 to 24 carbon atoms, preferably from 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 exemplified.

[0076] Further examples of phospholipids are phosphatidic acids, i.e., diesters of fatty acids with 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 with 1,3 - diphosphatidylglycerol; glycolipids such as ganglioside GM1 (or GM2) or cerebrosides; glycosphingolipids; sulfatides and glycosphingolipids.

[0077] 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.

[0078] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives), for example, typically, soy or egg yolk lecithins.

[0079] 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.

[0080] 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 (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), dipalmitoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoyl-phosphatidylinositol (DOPI).

[0081] Suitable phospholipids further include phospholipids modified by the binding of a hydrophilic polymer such as polyethylene glycol (PEG) or polypropylene glycol (PPG) thereto. Preferred polymer-modified phospholipids include "pegylated phospholipids", i.e., phospholipids bound to a PEG polymer. Examples of pegylated phospholipids are pegylated phosphatidylethanolamines (abbreviated as "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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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 can be optionally 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.

[0086] The microvesicles prepared according to the present invention may optionally contain a targeting ligand.

[0087] The term "targeting ligand" includes within its meaning any compound, moiety or residue having or capable of promoting 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 associated 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.

[0088] 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.

[0089] The targeting ligand may itself be an amphiphilic compound (to be mixed with other components of the microvesicle), or a compound bound to an amphiphilic molecule (such as a phospholipid) used in the formation of the microvesicle.

[0090] 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.

[0091] 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 14is included. Particularly preferred gases are those which are in gaseous form at room temperature, such as SF6, C3F8, and C4F 10 is included.

[0092] One aspect of the present invention thus relates to a method for preparing a lyophilized composition for the long-term storage of calibrated gas-filled microvesicles, comprising the following steps: a. preparing a suspension of calibrated gas-filled microvesicles comprising a mixture of lyoprotective components; b. lyophilizing the calibrated microvesicle suspension.

[0093] 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].

[0094] 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 comprising an amphiphilic material.

[0095] The gas flow and two liquid flows are directed towards the contact zone 203 and then pass through a calibrated orifice 204 shown as a dotted line in FIG. 1. 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. The microbubbles 203' are formed within the calibrated orifice and are 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 the hydraulic diameter 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 suspension of microbubbles.

[0096] 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].

[0097] 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.

[0098] Preferably, a suitable aqueous carrier that is physiologically acceptable includes water (preferably sterile water), an aqueous solution such as saline (which may be advantageously buffered so that the final product for injection is not hypotonic), or a solution 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.

[0099] 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 No. PCT / EP2019 / 055325]. The formed gas-filled microvesicles 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 monobutyrin, 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.

[0100] 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].

[0101] During the stabilization stage, 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.

[0102] Examples of HS gases include nitrogen, air, and carbon dioxide, with the latter being particularly preferred due to its higher solubility in water.

[0103] Suitable LS gases are fluorinated gases, preferably perfluorinated gases. The fluorinated gases described previously herein.

[0104] 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 for the production of calibrated microvesicles. A gas stream 302 (e.g., containing a mixture of C4F

[0105] 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.

[0106] 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 materials and any possible residual compounds.

[0107] 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) not-assembled amphiphilic materials and residual compounds.

[0108] According to the present specification, suitable washing techniques include centrifugation, filtration, bubble sorting, and decantation.

[0109] As used herein, the expression "not-assembled amphiphilic materials" 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.

[0110] 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 osmotic pressure regulator described above.

[0111] In a preferred embodiment of the present invention, after the washing operation, a mixture of cryoprotective components is added to the calibrated microvesicle suspension.

[0112] Alternatively, a mixture of cryoprotective components can be added to the liquid stream containing the amphiphilic compound described above during the preparation of microvesicles by microfluidic technology.

[0113] The initial CMV characteristics are particularly maintained when using a mixture of cryoprotective components, and the mixture of cryoprotective components has a total concentration included between 100 mg / ml and 300 mg / mL, preferably between 120 mg / ml and 250 mg / mL, and more preferably the total concentration of PEG and polyol or PEG and saccharides is 200 mg / mL.

[0114] In a preferred embodiment of the present invention, prior to the lyophilization step, the CMV suspension contains a mixture of cryoprotective components at a concentration of 10 - 25%, preferably 14 - 24%, even more preferably 18 - 22% (w / v%).

[0115] The mixture of cryoprotective components represents the greater 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).

[0116] The mixture of cryoprotective components is as described above herein. The mixture shows advantageous results when used in the lyophilization process 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 in terms of concentration and size distribution compared to the initial suspension (before lyophilization).

[0117] For example, using a mixture of cryoprotective components provides the best strategy for substantially maintaining the GSD value after the lyophilization process. According to one embodiment, a GSD value included between 1.16 and 1.18 can be obtained when using a mixture of cryoprotective components compared to the use of a single additive that gives calibrated microvesicles characterized by a higher GSD value.

[0118] In a preferred embodiment, the use of a mixture of cryoprotective components can significantly improve the yield of vesicles corrected after lyophilization by 38% increase compared to the use of a single cryoprotective component.

[0119] In this specification and the claims, the term lyophilization has its standard meaning in the field of pharmaceutical technology. The lyophilization process consists of drying a pre-frozen liquid product at low pressure or in a vacuum and at low temperature. The main purpose is to remove the liquid from the product to provide a lyophilized product suitable for long-term storage.

[0120] 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 vesicles.

[0121] In a preferred embodiment of the present invention, the freezing temperature of the above method for long-term storage of calibrated gas-filled vesicles is in the range of -30°C to -70°C, preferably -30°C to -60°C, and even more preferably -40°C.

[0122] In an even more preferred embodiment, the pressure of lyophilization is preferably 0.5 mbar or less, preferably 0.2 or less, for example around 0.1 mbar.

[0123] A further aspect of the present invention relates to a lyophilization composition for preparing a suspension of calibrated gas-filled vesicles, said lyophilization composition comprising the following steps: a. Preparing a first suspension of calibrated gas-filled vesicles by a flow focusing process comprising the following steps, wherein said suspension further comprises a mixture of cryoprotective components; and b. Lyophilizing said suspension, and can be obtained by a process comprising the steps.

[0124] A further aspect of the present invention relates to a method of preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, wherein the method comprises reconstituting the lyophilized composition obtained as described above, comprising an amphiphilic material and a mixture of cryoprotective components, with a pharmaceutically acceptable solution in the presence of a biocompatible gas.

[0125] The lyophilized 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 1.2 or less, preferably at least 1.15, for example up to 1.1.

[0126] 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.

[0127] The lyophilized composition is typically reconstituted with an aqueous solution of the same volume as the volume of the suspension undergoing the lyophilization process. Thus, the concentration of the cryoprotective component in the reconstituted suspension is substantially the same as that in the original suspension. For this reason, an excessive amount of polymer (typically a polyglycol, such as PEG), for example an amount greater than 150 mg / mL (e.g., 200 mg / mL), should preferably be avoided to avoid an excessive viscosity of the suspension to be administered.

[0128] Surprisingly, the reconstituted suspension of calibrated microvesicles has been found to substantially maintain the initial properties of the calibrated microvesicles characterized prior to the lyophilization process and is suitable for subsequent pharmaceutical use.

[0129] The reconstituted suspension of the calibrated microvesicles is characterized by calibrated microvesicles having a GSD of at least 1.2 or less, preferably at least 1.15, for example up to 1.1.

[0130] In one embodiment of the invention, the reconstituted suspension of the calibrated microvesicles is at least 2.0×10 8 particles of CMV / mL, preferably 2.25×10 8 particles of CMV / mL, more preferably 2.5×10 8 particles of CMV / mL, and is characterized by a concentration of up to 5.50×10 8 particles of CMV / mL.

[0131] As described above, the expression "microvesicle concentration" refers to the number of microvesicles, i.e., the number of MB / mL, within a volume unit determined using a Coulter Counter instrument.

[0132] Typically, the concentration (%) of the calibrated microvesicles measured after reconstituting the lyophilized composition of the invention with a suitable aqueous solution enables determination of the yield of the reconstituted microbubbles compared to the microvesicle concentration measured before the lyophilization step.

[0133] In the present invention, the yield of the calibrated microvesicles after reconstitution of the lyophilized composition of the 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%.

[0134] The expression "yield of calibrated microvesicles" refers to the ratio between the microvesicle concentration measured before lyophilization and the microvesicle concentration measured after lyophilization and redispersion (see Equation 2): Equation 2: CMV yield after lyophilization (%) = (CMV concentration / mL after lyophilization) / (CMV concentration / mL before lyophilization)

[0135] The expression "GSD ratio" refers to the ratio of the GSD value measured for CMV before lyophilization to the GSD value after lyophilization (see Equation 3): Equation 3: GSD ratio = (GSD before lyophilization) / (GSD after lyophilization)

[0136] The monodispersity of the CMV system after the lyophilization process can be monitored by the value of the GSD ratio. For example, good monodispersity is evaluated when the GSD value after lyophilization is similar to that before lyophilization, resulting in a GSD ratio close to 1. Generally, the higher the GSD ratio (i.e., the closer it is to 1), the more it indicates the maintenance of the initial monodispersity in the CMV distribution after lyophilization.

[0137] 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.

[0138] 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.

[0139] The microvesicles according to the present invention are typically administered at a gas concentration of about 0.01 to about 1.0 μL per kg of the patient, depending, for example, on their respective composition, the tissue or organ to be imaged, and / or the selected imaging technique. 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.

[0140] 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.

[0141] According to one embodiment, the suspension of gas-filled microvesicles comprises an amphiphilic material and a mixture of cryoprotective components.

[0142] Reconstitution of the lyophilized product is preferably carried out by dispersing it in a physiologically acceptable aqueous carrier (e.g., saline) with gentle stirring in the presence of a physiologically acceptable gas (e.g., SF6).

[0143] Possible other diagnostic imaging applications include scintigraphy, optical imaging, and X-ray imaging (including X-ray phase contrast imaging).

[0144] 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.

[0145] The therapeutic technique includes any treatment method for 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 (i.e., delivery mediated by ultrasound, for example, for the delivery of a drug or 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 for use as a vaccine). Thus, the presence of gas-filled microbubbles can, for example, provide the therapeutic effect itself by giving or being a factor in giving 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), or can enhance the therapeutic effect of the applied ultrasound.

[0146] 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 patient, depending, for example, on each composition, the type of subject being treated, the tissue or organ being treated, and / or the therapeutic method used.

[0147] In one embodiment, the above method of therapeutic treatment with ultrasound is (i) administering to the patient a suspension of gas-filled microbubbles obtained by reconstituting a 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.

[0148] The following examples will help to further illustrate the present invention.

Example

[0149] [Example 1] Preparation of gas-filled 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.

[0150] The amphiphilic material in the liquid flow was DSPC:DPPE-PEG5000, and the respective molar ratios were 9:1.

[0151] 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 overnight under reduced pressure. 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.

[0152] 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 10 respectively. The gas flows of each gas were adjusted 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 are 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, a flow focusing device in the jet regime was operated to produce microbubbles having a diameter (mode) of about 4 μm.

[0153] [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 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. The effective concentration of the lyoprotectant was 188 mg / mL after redispersion of the remaining washed microvesicles.

[0154] Then, the calibrated microvesicles suspended in the lyoprotectant solution were aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a lyophilizer.

[0155] 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, pure C4F was filled into the headspace. 10 was filled.

[0156] [Example 3] Effect of lyoprotective components on the properties of calibrated microvesicles Table 1 lists the lyoprotectants tested.

[0157] JPEG0007712877000004.jpg78166

[0158] The above materials were used at various concentrations as single components or their mixtures at a temperature of -60 °C in the preparation of the lyophilized composition shown in Example 2. Table 2 lists various examples of single components (S1 - S13) and their mixtures (M1 - M9).

[0159] JPEG0007712877000005.jpg153166

[0160] 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 and stable microvesicle suspension. The concentration of the mixture of lyoprotective components was 188 mg / mL before and after lyophilization.

[0161] 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.

[0162] Results The main results of the characterization of the reconstituted and calibrated microvesicle suspension are reported in Tables 3 and 4.

[0163] The GSD values and concentrations of the microvesicles were measured before and after the lyophilization process to evaluate the effectiveness of the lyoprotective components in maintaining the initial characteristics of the calibrated microvesicles.

[0164] Considering the GSD values and the yield of microvesicles after lyophilization, the results clearly show that adding a mixture of lyoprotective components to the calibrated microvesicle suspension is more effective in maintaining the characteristics of the microvesicles than adding a single component.

[0165] JPEG0007712877000006.jpg126166

[0166] From the results shown in Table 4, it is clear that the lyophilization efficiency was improved using a mixture of lyoprotective components. In particular, the lyophilization of calibrated microvesicles in a mixture of polyethylene glycols (PEG4000 and PEG8000) and polyols (i.e., xylitol, sorbitol, and mannitol) enabled an improvement in the yield of microvesicles after lyophilization. For example, the use of mixture M1 enabled a 17% increase in the yield of microvesicles after lyophilization compared to the use of S1A. Even more advantageously, the use of M2 enabled a higher increase (38%) in the CMV yield compared to formulation S2A. Furthermore, the lyophilization of CMV in mixture M2 enabled the obtaining of a better GSD value (1.17), which was lower than those obtained using the single lyoprotective components of formulations S2A and S3A (1.21 and 1.33, respectively).

[0167] A similar trend was observed after lyophilizing calibrated microvesicles in a mixture of polyethylene glycols (PEG4000 and PEG8000) and saccharides (i.e., maltose, sucrose, raffinose, dextran 6000). For example, it was found that the CMV yield after lyophilization in mixture M5 was 51%, which was a 25% increase compared to formulation S2A. Furthermore, it was found that the GSD value using mixture M5 was 1.17, which was significantly lower than those of the single lyoprotective components S2A and S6 (1.21 and 1.28, respectively).

[0168] JPEG0007712877000007.jpg181166

[0169] [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.

[0170] 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.

[0171] At the end of the lyophilization process, each lyophilized composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas in order to obtain a calibrated and stable microvesicle suspension. The lyophilized composition is thus reconstituted with an aqueous solution of the same volume as the volume of the suspension that has undergone the lyophilization process. Thus, the concentration of the lyoprotectant component in the reconstituted suspension is substantially the same as the concentration in the original suspension.

[0172] 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.

[0173] Table 5 reports the GSD values and microvesicle yields measured after lyophilization for each microvesicle suspension obtained after reconstitution of the lyophilized compositions containing different lyoprotectant components frozen at different temperatures.

[0174] Results The results confirmed that the use of a mixture of lyoprotectant components improves the lyophilization performance at any of the tested freezing temperatures compared to the use of a single component. For example, as shown in Table 5, CMV lyophilized in mixture M2 was characterized by the lowest GSD value at any of the tested freezing temperatures, and the results previously obtained in Example 3 were confirmed.

[0175] JPEG0007712877000008.jpg155166

[0176] [Example 5] Effect of the concentration of lyoprotective components on the properties of calibrated microvesicles To evaluate the lyophilization efficiency in terms of maintaining the GSD value and microvesicle yield after lyophilization, the concentration of the mixture of lyoprotectant components was also tested.

[0177] For this test, a mixture of cryoprotective components was compared to the corresponding cryoprotective components alone. In particular, the following cryoprotective components were evaluated: i) PEG4000 and sorbitol (in equal amounts), compared to PEG4000 alone. ii) PEG4000 and xylitol (in equal amounts), compared to PEG4000 alone. iii) PEG8000 and sorbitol (in equal amounts), compared to PEG8000 alone. iv) PEG8000 and xylitol (in equal amounts), compared to PEG8000 alone.

[0178] Different solutions in the concentration range of 50 mg / ml to 250 mg / ml were prepared for each of the tested formulations.

[0179] Then, calibrated microvesicles suspended in different solutions of cryoprotective components were aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a freeze dryer.

[0180] The vials were cooled at -60 °C or -40 °C (PEG8000 mixture) and -60 °C (PEG4000 mixture) for 1 hour at atmospheric pressure, and then primary dried at -20 °C and 0.1 mbar. 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.

[0181] Results Considering the CMV yield (Equation 2) and GSD ratio (Equation 3) after lyophilization, the effect of the concentration of cryoprotective components on the characteristics of calibrated microvesicles was tested.

[0182] i) PEG4000 and sorbitol (in equal amounts), compared to PEG4000 alone. Table 6 reports a comparison of the CMV yield and GSD ratio after lyophilization for the FD protection mixture (PEG4000 and sorbitol) and PEG4000 alone at increasing concentrations.

[0183] Considering the CMV yield, the results showed that the increase in the improvement of lyophilization efficiency was obtained for both the mixture and the single component using a lyoprotectant concentration of 50 mg / mL to 200 mg / mL. Furthermore, the results showed that at the tested freezing temperatures, substantially similar CMV yields were obtained for the mixture (PEG4000 and sorbitol) and PEG4000 alone (≤200 mg / ml).

[0184] In contrast, regarding the GSD ratio, a difference between the single component and the mixture was observed. In particular, when frozen at -60 °C, the GSD ratio yielded systematically increased values for the mixture of PEG4000 and sorbitol compared to PEG4000 alone.

[0185] JPEG0007712877000009.jpg87166

[0186] ii) PEG4000 and xylitol (in equal amounts), compared to PEG4000 alone. Table 7 reports a comparison of the CMV yield and GSD ratio after lyophilization for the FD-protecting PEG4000 and xylitol mixture and PEG4000 alone at increasing concentrations.

[0187] JPEG0007712877000010.jpg97166

[0188] Similar to the previous (case i), the results regarding the GSD ratio showed an improved lyophilization efficiency for the mixture of PEG4000 and xylitol when frozen at -60 °C, resulting in better GSD ratio values compared to PEG4000 alone. In contrast, the CMV yield was found to be higher for the mixture only at low concentrations (50 - 100 mg / mL).

[0189] iii) PEG8000 and sorbitol (in equal amounts), compared to PEG8000 alone Table 8 reports a comparison of the CMV yield and GSD ratio after lyophilization for each tested concentration by comparing formulations containing a mixture of PEG8000 and sorbitol with formulations containing PEG8000 alone.

[0190] JPEG0007712877000011.jpg98166

[0191] Considering the CMV yield, the results showed that an increase in the improvement of lyophilization efficiency was obtained for both the mixture and the single component at both freezing temperatures using concentrations of the lyoprotectant component from 50 mg / mL to 250 mg / mL.

[0192] However, at a freezing temperature of -60 °C, the CMV yield increased only for the mixture at low concentrations (50 - 100 mg / mL), but the values were found to be substantially similar for both the mixture and the single component at higher concentrations (especially ≥ 200 mg / mL).

[0193] In contrast, at a freezing temperature of -40 °C, the mixture of PEG8000 and sorbitol showed improved values for both the CMV yield and the GSD ratio compared to PEG8000 alone.

[0194] As shown in Table 8, the advantage of using a mixture of PEG8000 and sorbitol instead of PEG8000 alone was also confirmed by the value of the GSD ratio. In particular, higher GSD values were evaluated when CMV was lyophilized in solutions of PEG8000 and sorbitol at all tested concentrations and both freezing temperatures, showing improved monodispersity compared to CMV suspensions containing an equal amount of PEG8000.

[0195] v) PEG8000 and xylitol (in equal amounts), compared to PEG8000 alone. Table 9 reports a comparison of the CMV yield and GSD ratio after lyophilization for each tested concentration by comparing formulations containing a mixture of PEG8000 and xylitol with formulations containing PEG8000 alone.

[0196] JPEG0007712877000012.jpg95166

[0197] As indicated by the increasing values of the CMV yield, an improvement in the lyophilization efficiency was obtained for both the mixture and the single component using a concentration of the lyoprotectant component of 50 mg / mL to 250 mg / mL. The results showed that at a freezing temperature of -60 °C, the values of the CMV yield were particularly improved for the mixture at low concentrations (<100 mg / mL).

[0198] The GSD ratio was found to be improved in the mixture at all concentrations and both freezing temperatures.

[0199] Similar to case iii), at a freezing temperature of -40 °C, the mixture of PEG8000 and xylitol had improved values of both the CMV yield and the GSD ratio compared to PEG8000 alone, and its improved lyophilization efficiency was confirmed.

[0200] Analysis of all the results revealed that the use of a mixture of FD protectant components gave an increased improvement in the lyophilization efficiency when compared to a single FD protectant component.

[0201] In particular, when comparing S1A with M1 (PEG4000 + sorbitol) and M9 (PEG4000 + xylitol), it was observed that the improvement in the value of the GDS ratio was evaluated for the mixture when frozen at -60 °C.

[0202] From the comparison between S2A (PEG8000) and the mixtures M2 (PEG8000 + sorbitol) and M3 (PEG8000 + xylitol (Xyilitol)), an increase in the improvement of the freeze-drying efficiency of the mixtures was evaluated when frozen at -40 °C, and an increase in all the tested parameters (CMV yield, GSD, GSD ratio) was found to be observed. However, at a freezing temperature of -60 °C, the CMV yield was found to be the same or slightly higher for the mixtures compared to the single FD protective component, but the GSD ratio and GSD value showed improvement only for mixtures M2 and M3, especially at high concentrations of cryoprotectant (≧200 mg / mL).

[0203] Cited 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. A lyophilized composition comprising (i) an amphiphilic material containing a phospholipid and (ii) a lyoprotectant component, which provides a suspension of calibrated gas-filled microvesicles when reconstituted with a pharmaceutically acceptable solution in the presence of a biocompatible gas, wherein the lyoprotectant component is a mixture of at least two lyoprotectant components having a concentration of 10 to 25% (w / v%) and comprising polyethylene glycol having a molecular weight included between 4000 and 8000 g / mol, and a polyol selected from sorbitol or xylitol, wherein the reconstituted suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of less than 1.2, lyophilized composition.

2. The lyophilized composition according to claim 1, The reconstituted suspension of the corrected microvesicles is characterized by a concentration of 2.5×10 ８ microvesicles / mL, lyophilized composition.

3. Obtained by reconstituting the lyophilized composition according to claim 1 or 2 with a pharmaceutically acceptable solution in the presence of a biocompatible gas, suspension of gas-filled microvesicles.

4. A method for preparing the lyophilized composition according to claim 1 or 2 for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles having a geometric standard deviation (GSD) of less than 1.2, comprising: a. preparing a suspension of calibrated gas-filled microvesicles comprising a mixture of lyoprotectant components having a geometric standard deviation (GSD) of less than 1.2 and comprising polyethylene glycol having a molecular weight included between 4000 and 8000 g / mol, and a polyol selected from sorbitol or xylitol; and b. lyophilizing the calibrated microvesicle suspension, comprising method.

5. The method according to claim 4, wherein the preparation method of step a. comprises a microfluidic flow focusing technique, method.

6. The method according to claim 4, wherein the mixture of lyoprotectant components has a total concentration included between 120 mg / ml and 250 mg / mL, method.

7. The method according to claim 6, wherein the concentration is 200 mg / mL, method.

8. A method for preparing an injectable contrast agent comprising a suspension of gas-filled microvesicles, wherein the method comprises the step of reconstituting the lyophilized composition defined in claim 1 or 2 with a pharmaceutically acceptable solution in the presence of a biocompatible gas, method.

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