Freeze-dried composition for preparing calibrated gas-filled microvesicles

Abstract

To provide a freeze-dried composition that yields an ultrasound contrast agent upon reconstitution, and a method for preparing the same.SOLUTION: A freeze-dried composition comprising an amphiphilic material and a freeze-drying protecting component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein the freeze-drying protecting component is a mixture of at least two freeze-drying protecting components and wherein the reconstituted suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) lower than 1.2.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates generally to the field of ultrasound contrast agents (USCAs). In particular, it relates to 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. [Background technology]

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

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

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

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

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

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

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

[0009] The applicant has now found that such initial properties can be maintained to an acceptable extent after the freeze-drying process by using an appropriate mixture of lyoprotectant components. [Means for solving the problem]

[0010] In a first aspect, the present invention relates to a lyophilized composition comprising an amphiphilic material and a lyoprotectant component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein the lyoprotectant component is a mixture of at least two lyoprotectants, and wherein the reconstituted suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of less than 1.2.

[0011] In one 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 sugar.

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

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

[0014] In one embodiment of the invention, the reconstituted suspension of calibrated microvesicles contains at least 2.0 x 10 8 cells / mL, preferably 2.25 x 10 8 CMV / mL, more preferably at a concentration of 2.5 x 10 CMV / mL, 5.5 x 10 8 It is characterized by a concentration of up to 100 CMV / mL.

[0015] According to a further aspect, the present invention relates to a method for preparing a freeze-dried composition for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles, comprising the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing a mixture of lyoprotectant components; b. freeze-drying the calibrated microvesicle suspension.

[0016] A further aspect of the present invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition comprising the following steps: a. Preparing a first suspension of gas-filled calibrated microvesicles by a flow focusing process, said suspension further comprising a mixture of lyoprotectant components: and b. freeze-drying 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 microvesicles, said method comprising the step of reconstituting a lyophilized composition as defined above with a pharmaceutically acceptable solution in the presence of a biocompatible gas. [Brief explanation of the drawings]

[0018] [Figure 1] Typical particle size distribution display. [Figure 2] Schematic representation of the core of the microfluidic flow focusing device. [Figure 3] 1 is an exemplary schematic diagram of a device useful in the process of the present technique. DETAILED DESCRIPTION OF THE INVENTION

[0019] The expression "gas-filled microvesicles" generally refers to gas bubbles bounded at the gas / liquid interface by a very thin envelope (film) comprising a stabilizing amphiphilic material (typically a phospholipid) placed at the gas-liquid interface. Such calibrated gas-filled microvesicles are suitable as contrast agents in ultrasound imaging techniques (known as contrast-enhanced ultrasound (CEUS) imaging) or in therapeutic applications, for example in combination with ultrasound-mediated drug delivery.

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

[0021] The term "calibrated" (when referring to gas-filled microvesicles) refers in particular to a microvesicle suspension having highly calibrated microvesicles (CMV) having different sizes between 3 and 8 μm and characterized by a size distribution having a geometric standard deviation (GSD) of at least 1.2, preferably at least 1.1, e.g. up to 1.05.

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

[0023] In the present invention, calibrated gas-filled microvesicles are preferably produced using a microfluidic flow-focusing technique, in which a gas thread is focused between two liquid streams in a flow-focusing device to form calibrated phospholipid-stabilized microvesicles that are collected in an outlet channel. Using this approach, calibrated microvesicles are produced in a highly reproducible manner at reasonable production rates (approximately 60 million bubbles per minute) (Figures 2 and 3).

[0024] Depending on the manufacturing process and device parameters, calibrated microvesicles can be obtained with a relatively narrow size distribution of about any desired mean diameter, for example between 3 and 8 μm, preferably about 4 μm.

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

[0026] Calibrated microvesicle concentrations (especially when produced using microfluidic flow focusing) are typically 3 × 10 8 ~4×10 8 Contains between 4 x 10 CMV / mL, preferably 4 x 10 8 CMV / mL, close to 3 x 10 8 Not lower than 100 CMV / mL.

[0027] The "geometric standard deviation" (GSD) generally provides an appropriate value for characterizing the breath of size distribution in a population of particles (in certain cases, gas-filled microvesicles). A population of particles with a wide range of sizes will therefore have a larger GSD value (i.e., smaller in size) than one in which particle sizes are narrowly distributed around a mean value.

[0028] Figure 1 shows an example of a size distribution graph (by volume) of a population of gas-filled microvesicles, which can be obtained by determining the volume of gas for each of its channels using a commercially available measurement device (e.g., a Coulter Counter Multisizer 3 with Multisizer 3 software), where each channel corresponds to a given diameter of the microvesicles (e.g., in 0.1 microns). By determining the number of calibrated gas-filled microvesicles in the suspension, their respective diameters and volume distribution in a selected size range (e.g., 3 μm to 6 μm for a 4.5 μm CMV mean diameter), it is possible to calculate the GSD of the CMV distribution by using the following equation 1:

number

number

number

[0029] Among the various commercially available measurement devices, the Coulter Counter Multisizer 3 with Multisizer 3 software is capable of calculating and providing GSD values as defined above.

[0030] For example, a GSD value of 1.2 indicates that approximately 50% of the CMVs fall between 2.5 and 5 μm for a mean diameter of 4 μm; a GSD of 1.05 to 1.08 (<1.1) indicates that approximately 90 to 95% of the CMVs have a size that falls between 2.5 and 5 μm.

[0031] As used herein, the expression "microvesicle concentration" refers to the number of CMVs in a unit of volume, ie, number of CMVs / mL, as determined using a Coulter Counter instrument.

[0032] As observed by the applicant, the calibrated microvesicles obtained by microfluidic flow focusing can be stored at room temperature for several weeks without substantial effect on their main properties, however, after the storage period, the properties of the microvesicles (e.g., concentration and size distribution) may not be maintained and may gradually deteriorate.

[0033] The shelf life of such gas-filled microvesicle suspensions is therefore relatively short for pharmaceutical products, and there is a need to develop long-term storage procedures that are capable of maintaining the initial properties of the CMV, such as concentration, GSD and final diameter, for longer periods of time, e.g., months or years.

[0034] The freeze-drying process is a suitable technique to obtain a dry form of calibrated microvesicles with high stability over time and maintaining their original properties.

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

[0036] In a first aspect, the present invention provides a lyophilized composition comprising an amphiphilic material and a mixture of lyoprotective 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, e.g. up to 1.1.

[0037] In this specification and claims, the terms "freeze-drying" and "lyophilization" are used interchangeably, as are the terms "freeze-dried" and "lyophilized."

[0038] Freeze-dried composition The term "lyophilized composition" as used herein refers to any dry formulation for long-term storage of gas-filled microvesicle formulations obtained by the lyophilization process. The lyophilized composition can comprise one or more active ingredients and a mixture of at least two lyoprotectants.

[0039] As used herein, the expression "active ingredient" refers to the microvesicle stabilizing material, eg, an amphiphilic material, which is included in the lyophilized composition along with the lyoprotectant component.

[0040] Lyoprotectant mixture As used herein, the expression "blend of lyoprotective ingredients" refers to a combination of at least two ingredients suitable for lyophilization and which are contained within the microvesicle suspension prior to the lyophilization process.

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

[0042] According to one embodiment of the present invention, the mixture of lyoprotective components is a combination of at least two different compounds suitable for lyophilization, selected from the group consisting of polymers, polyols and sugars, preferably comprising a combination of a polyol or a sugar with a polymer.

[0043] In one preferred embodiment of the present invention, one of the components of the mixture of lyoprotective 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 for PEG is HOCH2(CH2OCH2). m CH2OH, where m represents the average number of oxyethylene groups. Typical polyethylene glycols are available in a wide range of average molecular weights, starting from 190-210 g / mol (PEG200; m=4.2) and going up to 7000-9000 g / mol (PEG8000; m=181.4).

[0046] According to this specification, the expression "molecular weight" refers to the average length of the PEG polymer chain, with a variation of ±10% relative to the indicated molecular weight.

[0047] According to one embodiment of the invention, the mixture of lyoprotective components preferably comprises PEG with a molecular weight comprised between 2000 and 10000 g / mol, preferably between 4000 and 8000 g / mol.

[0048] In one embodiment, the lyoprotectant is PEG with a molecular weight of 8000 g / mol (±10%). In another embodiment, the lyoprotectant is PEG with a molecular weight close to 4000 g / mol (±10%).

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

[0050] As the applicant has observed, polymer (especially polyglycol, e.g., PEG) suspensions having a concentration of 150 mg / mL or more (e.g., 200 mg / mL) can be difficult to handle during industrial processes due to their relatively high viscosity. Therefore, it is preferable to stick to using polymer suspensions having a concentration lower than 150 mg / mL.

[0051] To improve the retention of the original properties of the reconstituted CMV suspension after the lyophilization process, Applicants have found that it is preferable to use a suitable mixture of PEG in combination with a second lyoprotectant component at a concentration lower than 150 mg / mL (e.g., 100 mg / mL).

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

[0053] In this specification and claims, the term "polyol" has its conventional chemical meaning; it refers to any organic compound having more than two hydroxyl functional groups and has the general formula HOCH2(CHOH) n Polyols are characterized by the formula CH2OH, where n is an integer from 1 to 6, preferably 2 to 4. Polyols vary in chain length, i.e., 4, 5, or 6 carbon chains. They have one hydroxyl group attached to each carbon. Polyols can be further differentiated 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 arts. Sorbitol, or (2R,3R,4R,5S)-hexane-1,2,3,4,5,6-hexol, is a polyhydric alcohol containing a linear carbon chain, with six carbon atoms, each substituted with 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 arts. Xylitol, or (2S,4R)-pentane-1,2,3,4,5-pentol, is a five-carbon sugar alcohol in which all five 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 comprises a mixture of sugars and polymers.

[0059] The term "saccharide" has its standard meaning in chemistry. Sugars, also known as carbohydrates, are molecular compounds made from only three elements: carbon, hydrogen, and oxygen. The simplest sugars are called monosaccharides and are the building units for larger sugars such as disaccharides, trisaccharides, and polysaccharides.

[0060] Preferably, the saccharide is selected from the group of disaccharides, trisaccharides and polysaccharides, more preferably a disaccharide or trisaccharide.

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

[0062] Disaccharides (C 12 H 22 O 11 Disaccharides are sugars consisting of two monosaccharide units joined by a glycosidic bond. This latter is a covalent bond formed by the reaction of the OH group of a second monosaccharide with the anomeric carbon of one cyclic monosaccharide. Disaccharides differ from each other in the monosaccharide components and the specific type of glycosidic bond connecting them. Examples of disaccharides include maltose, lactose, and sucrose. A particularly preferred disaccharide is sucrose.

[0063] Trisaccharides are sugars consisting of three monosaccharides with two glycosidic bonds connecting them. As with disaccharides, each glycosidic bond can be formed between any hydroxyl group on the constituent monosaccharides. Even if all three constituent sugars are identical (e.g., glucose), different bond combinations (regiochemistry) and stereochemistry (α- or β-) result in diastereoisomeric trisaccharides with different chemical and physical properties. Examples of trisaccharides are maltotriose, melezitose, maltotriulose, and raffinose. Raffinose is a particularly preferred trisaccharide.

[0064] Polysaccharides are polymeric sugar 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. A particularly preferred polysaccharide is dextran.

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

[0066] In the present invention, the term "sucrose" has its standard meaning. Sucrose is a disaccharide formed by a glucose unit and a fructose unit 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 has a molecular weight of 342.30 g / mol.

[0067] The initial CMV properties are particularly maintained when a mixture of lyoprotective components is used, characterized in that said mixture of lyoprotective components has a total concentration comprised between 100 mg / ml and 300 mg / ml, preferably between 120 mg / ml and 250 mg / ml, more preferably a total concentration of PEG and polyol or PEG and saccharide of 200 mg / ml.

[0068] In one preferred embodiment, the mixture of lyoprotectant components comprises PEG and polyol or PEG and sugar in a ratio of 2:1 to 2:3, preferably 3:2 to 4:5, more preferably 1:1.

[0069] The above mixture of lyoprotective components shows advantageous results when used in the freeze-drying process of calibrated microvesicle suspensions, allowing the preparation of freeze-dried compositions which can then be reconstituted to obtain suspensions of calibrated microvesicles with acceptable properties in terms of concentration and size distribution.

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

[0071] As used herein, the term "amphiphilic material" includes compounds having molecules with a hydrophilic polar head (e.g., a polar or ionic group) that can interact with aqueous media and a hydrophobic organic tail (e.g., a hydrocarbon chain) that can interact with, for example, organic solvents. These compounds therefore generally act as "surfactants," i.e., compounds that can stabilize mixtures of otherwise generally immiscible materials, such as a mixture of two immiscible liquids (e.g., water and oil), a mixture of a gas and a liquid (e.g., gas microbubbles in water), or a mixture of insoluble particles and a liquid (e.g., metal nanoparticles in water).

[0072] Suitable amphiphilic materials include, for example, phospholipids; lysophospholipids; fatty acids, such as palmitic acid, stearic acid, arachidonic acid, or oleic acid; lipids bearing polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone, or polyethylene glycol (PEG), also called "PEGylated lipids"; lipids bearing sulfonated mono-, di-, oligo-, or polysaccharides; cholesterol, cholesterol sulfate, or cholesterol hemisuccinate; tocopherol hemisuccinate; lipids containing ether or ester-linked fatty acids; polymeric lipids; diphosphates. Acetyl; dicetyl phosphate; ceramides; polyoxyethylene fatty acid esters (e.g., polyoxyethylene fatty acid stearates), polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil or ethylene oxide (EO) and propylene oxide (PO) block copolymers; cholesterol glucuronide, lanosterol glucuronide, 7-deoxy-2-methyl-2-propanol Sterol esters of sugar acids, including hydrocholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate, or ergosterol gluconate; esters of alcohols and sugar acids, including lauryl glucuronide, stearoyl glucuronide, myristoyl gluconate, lauryl gluconate, myristoyl gluconate, or stearoyl gluconate; esters of sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, glutamic ... Esters of sugars with fatty acids, including sarcolic acid or polyuronic acid; saponins, including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, or digitoxigenin; glycerol or glycerol monoesters with fatty acids (including glycerol monopalmitate, glycerol monostearate, glycerol monomyristate, or glycerol monolaurate); long-chain alcohols, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, or n-octadecyl alcohol;6-(5-Cholesten-3β-yloxy)-1-thio-β-D-galactopyranoside;Digalactosyl diglyceride;6-(5-Cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-1-thio-β-D-galactopyranoside;6-(5-Cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-1-thio-β-D-mannopyranoside;12-(((7'-Diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid;N-[12-(((7'-Diethylaminocoumarin-3-yl)carbonyl]methylamino]octadecanoic acid

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

[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 whose molecules are capable of forming a stabilizing film of material (typically in the form of a monolayer) at the gas-water interface in the final microbubble suspension. These materials are therefore also referred to in the art as "film-forming phospholipids."

[0075] Examples of suitable phospholipids include esters of one or preferably two (equivalent or different) fatty acid residues and phosphoric acid with glycerol, where the phosphate residue is then linked to a hydrophilic group, such as choline (phosphatidylcholine-PC), serine (phosphatidylserine-PS), glycerol (phosphatidylglycerol-PG), ethanolamine (phosphatidylethanolamine-PE), or inositol (phosphatidylinositol). Esters of phospholipids with only one fatty acid residue are generally referred to in the art as "lyso" forms of phospholipids or "lysophospholipids." The fatty acid residues present in phospholipids are generally long-chain fatty acids, typically containing 12 to 24 carbon atoms, preferably 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably fully saturated. Examples of suitable fatty acids contained in phospholipids include 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 and glycerol-phosphate; sphingolipids such as sphingomyelins, i.e., phosphatidylcholine analogs in which the glycerol diester residue with a fatty acid is replaced by a ceramide chain; cardiolipins, i.e., esters of fatty acids and 1,3-diphosphatidylglycerol; glycolipids, such as ganglioside GM1 (or GM2) or cerebrosides; glycolipids; sulfatides and glycosphingolipids.

[0077] As used herein, the term "phospholipid" includes products of either natural origin, semi-synthetic or synthetic preparation, which may be used either alone or in mixtures.

[0078] Examples of phospholipids of natural origin are natural lecithins (phosphatidylcholine (PC) derivatives), such as typically soybean 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 include, for example, dilauroyl-phosphatidylcholine (DLPC), dimyristoyl-phosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl-phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1,2 distearoyl-sn-glycero-3-ethylphosphocholine (ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC), 1-myristoyl-2-palmito ...-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine (DOPC), 1-myristoyl-2-palmitoyl-phosphatidylcholine (DSPC), dioleoyl- 1-palmitoyl-2-myristoyl-phosphatidylcholine (MPPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PMPC), 1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1-palmitoyl-2-oleylphosphatidylcholine (POPC), 1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl di-glycerol (DAPG) and its alkali metal salts, dimyristoyl phosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoyl phosphatidylglycerol (DPPG) and its alkali metal salts, distearoyl phosphatidylglycerol (DSPG) and its alkali metal salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and alkali metal salts thereof, distearoylphosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and alkali metal salts thereof, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoylphosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidyl-ethanolamine (DAPE), dilinoleylphosphatidylethanolamine (DLPE),Dimyristoylphosphatidylserine (DMPS), diarachidoylphosphatidylserine (DAPS), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoylsphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), and dioleoyl-phosphatidylinositol (DOPI).

[0081] Suitable phospholipids further include phospholipids modified by the attachment of hydrophilic polymers such as polyethylene glycol (PEG) or polypropylene glycol (PPG). Preferred polymer-modified phospholipids include "PEGylated phospholipids," i.e., phospholipids attached to PEG polymers. Examples of PEGylated phospholipids are PEGylated phosphatidylethanolamines (abbreviated "PE-PEG"), i.e., phosphatidylethanolamines in which the hydrophilic ethanolamine moiety is attached to a PEG molecule of variable molecular weight (e.g., 300-20,000 daltons, preferably 500-5,000 daltons), such as DPPE-PEG (or DSPE-PEG, DMPE-PEG, DAPE-PEG, or DOPE-PEG). For example, DPPE-PEG2000 refers to DPPE with a PEG polymer attached thereto having an average molecular weight of approximately 2,000.

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

[0084] For example, the mixture of phospholipids may include phosphatidylcholine derivatives, phosphatidic acid derivatives and pegylated phosphatidylethanolamines, such as DSPC / DPPA / DPPE-PEG, DPPC / DPPA / DPPE-PEG, DSPC / DPPA / DSPE-PEG, DPPC / DPPA / DSPE-PEG, DAPC / DPPA / DPPE-PEG, DAPC / DPPA / DSPE-PEG, DSPC / DSPA / DPPE-PEG, DPPC / DSPA / DSPE-PEG, DSPC / DSPG / DPPE-PEG, DPPC / DSPG / DSPE-PEG.

[0085] According to the present invention, phospholipids can be suitably used in a mixture with any of the above-mentioned amphiphilic compounds. Thus, for example, lipids such as cholesterol, ergosterol, plant sterols, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and their derivatives, or butylated hydroxytoluene and / or other non-phospholipid compounds can be optionally added to one or more of the above-mentioned phospholipids, for example, preferably in a proportion ranging from 0 to 50% by weight, more preferably up to 25%. For example, mixtures of amphiphilic materials comprising phospholipids and fatty acids may be advantageously used, including DSPC / DPPG / palmitic acid, DSPC / DPPE-PEG / palmitic acid, DPPC / DPPE-PEG / palmitic acid, DSPC / DSPE-PEG / palmitic acid, DPPC / DSPE-PEG / palmitic acid, DSPC / DPPE-PEG / stearic acid, DPPC / DPPE-PEG / stearic acid, DSPC / DSPE-PEG / stearic acid or DPPC / DSPE-PEG / stearic acid.

[0086] Microvesicles prepared according to the present invention may optionally include a targeting ligand.

[0087] The term "targeting ligand" includes within its meaning any compound, moiety, or residue that has or is capable of promoting targeting activity (including, for example, selective binding) of the microvesicles of the compositions of the invention toward any biological or pathological site in the body. Targets to which a targeting ligand may be associated include tissues, such as myocardial tissue (including myocardial cells and cardiomyocytes), membranous tissues (including endothelium and epithelium), thin films, connective tissues (including interstitial tissue), or tumors; blood clots; and receptors, such as cell surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins, and cytoplasmic receptors for steroid hormones.

[0088] The targeting ligand may be synthetic, semi-synthetic, or of natural origin. Materials or substances that can act as targeting ligands include, but are not limited to, proteins, including antibodies, antibody fragments, receptor molecules, receptor-binding molecules, glycoproteins, and lectins; peptides, including oligopeptides and polypeptides; peptidomimetics; sugars, including monosaccharides and polysaccharides; vitamins; steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic material, including nucleosides, nucleotides, and polynucleotides.

[0089] The targeting ligand may itself be an amphiphilic compound (mixed with other components of the microvesicle) or may be a compound attached to the amphiphilic molecule (e.g., a phospholipid) used to form the microvesicle.

[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, which are known to form particularly stable gas-filled microvesicle 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 mixed with other isomers, such as perfluoro-n-butane and perfluoro-isobutane), perfluoropentanes, perfluorohexanes, or perfluoroheptanes; perfluoroalkenes, such as perfluoropropene, perfluorobutenes (e.g., perfluorobut-2-ene), or perfluorobutadiene; perfluoroalkynes (e.g., perfluorobut-2-yne); and perfluorocycloalkanes (e.g., perfluorocyclobutane, perfluoromethylcyclobutane, perfluorodimethylcyclobutanes, perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane, perfluorodimethylcyclopentanes, perfluorocyclohexane, perfluoromethylcyclohexane, and perfluorocycloheptane). Preferred saturated perfluorocarbons are, for example, CF4, C2F6, C3F8, C4F8, C4F 10 , C5F 12 and C6F 14Particularly preferred gases are those that are in gaseous form at room temperature, including SF, C3F8, and C4F 10 Includes:

[0092] One aspect of the present invention therefore relates to a method for preparing a freeze-dried composition for long-term storage of calibrated gas-filled microvesicles, comprising the following steps: a. Preparing a suspension of calibrated gas-filled microvesicles containing a mixture of lyoprotectant components; b. freeze-drying 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] 2 shows a schematic diagram of the core part 200 of a flow focusing device ("microfluidic chip") useful in the process of the invention. The chip comprises a first supply channel 201 for supplying a gaseous flow 201' and two further orthogonal supply channels 202a and 202b for supplying a liquid flow containing an amphiphilic material.

[0095] The gas flow and the two liquid streams are directed toward the contact zone 203 and then pass through a calibrated orifice 204, shown as a dotted line in FIG. 1 . The calibrated orifice is connected to a calibrated channel 204′, preferably having the same cross-section as the orifice, which is then connected to the initial portion 205 of the outlet channel 206. In an alternative embodiment (not shown), the calibrated orifice 204 may be a nozzle connected directly to the initial portion 205 of the outlet channel 206, i.e., without a calibrated channel in between. Microvesicles 203′ are formed in the calibrated orifice and directed through the calibrated channel 204′ to the initial portion 205 of the outlet channel 206. The hydraulic diameter of the outlet channel is generally larger than that of the calibrated orifice, typically increasing from the initial diameter of the calibrated orifice to the final diameter of the outlet channel 206 and substantially corresponding to the hydraulic diameter of a collection tube (not shown) connecting the flow focusing device to a container for collecting the microvesicle suspension, such as a sealed vial.

[0096] In the initial part 205 of the outlet channel of the device, and preferably also in the contact zone 203 and at the calibrated orifice 204, the temperature of the microvesicles is controlled as described in reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and reference 2 [PCT application number PCT / EP2019 / 055325].

[0097] liquid flow The aqueous liquid stream for preparing calibrated gas-filled microvesicles according to the method of the present invention comprises an amphiphilic material (as defined above) dispersed in an aqueous carrier at a concentration of, for example, 5.0 to 20 mg / mL, preferably 7.5 to 15 mg / mL.

[0098] Suitable aqueous carriers, preferably physiologically acceptable, include water (preferably sterile water), aqueous solutions such as saline (which may be advantageously buffered so that the final product for injection is not hypotonic), or solutions of one or more osmolytes, including salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials (e.g., glucose, sucrose, sorbitol, mannitol, glycerol, polyethylene glycols, propylene glycols, etc.), chitosan derivatives such as carboxymethylchitosan, trimethylchitosan, or gelling compounds such as carboxymethylcellulose, hydroxyethyl starch, or dextran.

[0099] In an alternative embodiment, an additional oil phase may be added to incorporate therapeutic hydrophobic substances into the microvesicles. To this end, two additional conduits may be provided within the device to supply the desired oil phase, as described, for example, in Reference 1 [WO2018 / 041906 A1 - BRACCO SUISSE SA] and Reference 2 [PCT Application No. PCT / EP2019 / 055325]. The formed gas-filled microvesicles thus have an oil film disposed at the interface between the gas and the stabilizing layer of amphiphilic material, and can be loaded with the desired therapeutic agent. Suitable oils may include any biocompatible oil that is liquid at room temperature, for example, saturated or unsaturated (C2-C3) 18 ) Mono-, di-, or tri-esters of glycerol with alkyl chains (including homo- or hetero-alkyl esters (allkylesters)), such as glycerol monobutyrin, glycerol monolinoleate, 1,2-dihexanoylglycerol, 1,2 dioctanoylglycerol, 1,2-dioleyl-sn-glycerol, triacetin, tributyrin, tricaproin, tricaprylin, tricaprin, and mixtures thereof; or natural oils, such as soybean oil, olive oil, safflower seed oil, sunflower seed oil, peanut oil, and mixtures thereof.

[0100] mixed gas flow The newly formed microvesicles contain a gas selected from those previously set forth, preferably a mixture of a gas that is highly soluble in water ("HS gas") and a gas that is sparingly soluble in water ("LS gas"), as contemplated in Reference 2 [PCT Application No. PCT / EP2019 / 055325].

[0101] During the stabilization step, most of the highly soluble gases dissolve rapidly in water, while the less soluble ones remain trapped in the dense layer of amphiphile, typically with some residual amount of HS-soluble gases dispersed therein.

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

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

[0104] In one preferred embodiment of the present invention, CO2 / C4F is mixed in a volume ratio of 80 / 20 to 90 / 10, for example 85 / 15. 10 Gas-filled microvesicles containing can be prepared by a gas mixing device similar to that shown schematically in FIG.

[0105] Figure 3 shows an example of a microfluidic flow focusing device used for the production of calibrated microvesicles. Gas flow 302 (e.g., CF 10 and CO2) and liquid stream 301 (containing an amphiphilic material, e.g., phospholipids, fatty acids, or mixtures thereof) are fed into microfluidic chip 303 and passed through orifice 304 to produce microvesicles. The microvesicle suspension is preferably heated at atmospheric pressure with a gas (e.g., CF4). 10) is collected in a pre-filled vial 305. A venting device (e.g., needle 306) is preferably used to equalize excess pressure created by the liquid filling of the vial. At the end of collection of the microvesicle suspension, the venting device is preferably removed and the container is preferably sealed to avoid further gas exchange with the external atmosphere.

[0106] According to one preferred embodiment of the present invention, after the recovery step, the calibrated microvesicles obtained by the microfluidic flow focusing method are treated using appropriate washing techniques to remove non-assembled amphiphilic material and possible residual compounds.

[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) unassembled amphiphilic material and residual compounds.

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

[0109] As used herein, the expression "not-assembled amphiphilic material" refers to amphiphilic molecules that are present in the calibrated microvesicle suspension at the end of the preparation process but that do not form the stabilizing layer of the gas-filled microvesicles.

[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 osmolality adjusting agents previously described.

[0111] In one preferred embodiment of the present invention, a mixture of lyoprotective ingredients is added to the calibrated microvesicle suspension after the washing procedure.

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

[0113] The initial properties of CMV are particularly maintained when a mixture of lyoprotective components is used, characterized in that said mixture of lyoprotective components has a total concentration comprised between 100 mg / ml and 300 mg / ml, preferably between 120 mg / ml and 250 mg / ml, more preferably a total concentration of PEG and polyol or PEG and saccharide of 200 mg / ml.

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

[0115] The mixture of lyoprotective components represents the majority of the final lyophilized formulation, which typically represents at least 90%, preferably 94% to 99.7%, more preferably 99.5%, and up to 99.9% (w / w).

[0116] The mixture of lyoprotective components is as described herein above and shows advantageous results when used in the freeze-drying process of a calibrated microvesicle suspension, allowing the preparation of a freeze-dried composition which can then be reconstituted to obtain a suspension of calibrated microvesicles with acceptable properties in terms of concentration and size distribution compared to the initial suspension (before freeze-drying).

[0117] For example, the use of a mixture of lyoprotective ingredients provides the best strategy for substantially maintaining the GSD value after the lyophilization process. According to one embodiment, a GSD value comprised between 1.16 and 1.18 can be obtained using a mixture of lyoprotective ingredients compared to the use of a single additive, which results in calibrated microvesicles characterized by a higher GSD value.

[0118] In one preferred embodiment, the use of a mixture of lyoprotective components can significantly improve the yield of calibrated microvesicles after lyophilization by an increase of 38% when compared to the use of a single lyoprotective component.

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

[0120] As applicants have observed, freeze-drying parameters may be selected to further optimize the properties (eg, yield, size, GSD) of the reconstituted suspension of microvesicles.

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

[0122] In a further preferred embodiment the freeze-drying pressure is preferably below 0.5 mbar, preferably below 0.2, for example around 0.1 mbar.

[0123] A further aspect of the present invention relates to a freeze-dried composition for preparing a suspension of calibrated gas-filled microvesicles, said freeze-dried composition comprising the following steps: a. Preparing a first suspension of gas-filled calibrated microvesicles by a flow focusing process comprising the steps of: wherein the suspension further comprises a mixture of lyoprotective ingredients; and b. freeze-drying the suspension.

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

[0125] The freeze-dried composition can then be reconstituted with a suitable pharmaceutically acceptable (aqueous) solution in the presence of a biocompatible gas, thus providing a suspension of calibrated gas-filled microvesicles, wherein the microvesicles have a GSD of at least 1.2, preferably at least 1.15, e.g., up to 1.1.

[0126] In the present invention, a pharmaceutically acceptable (aqueous) solution is water, typically sterile, pyrogen-free water (to minimize the possibility of contamination in the final reconstituted product), an aqueous solution such as saline (which may be advantageously buffered so that the final product for injection is not hypotonic), or an aqueous solution of one or more osmolytes such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.

[0127] Lyophilized composition is typically reconstituted with aqueous solution of the same volume as the suspension that undergoes lyophilization process.Therefore, the concentration of lyoprotective component in the reconstituted suspension is substantially the same as that in the initial suspension.For this reason, excessive amount of polymer (typically polyglycol, for example PEG), for example, more than 150mg / mL (for example, 200mg / mL), should preferably be avoided to avoid excessive viscosity of the suspension that is administered.

[0128] Surprisingly, the reconstituted suspension of calibrated microvesicles was found to substantially maintain the original properties of the calibrated microvesicles as characterized before the freeze-drying process, making them suitable for subsequent pharmaceutical applications.

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

[0130] In one embodiment of the invention, the reconstituted suspension of calibrated microvesicles contains at least 2.0 x 10 8 cells / mL, preferably 2.25 x 10 8 CMV / mL, more preferably 2.5 x 10 8 CMV / mL concentration: 5.50 x 10 8 It is characterized by a concentration of up to 100 CMV / mL.

[0131] As stated above, the expression "microvesicle concentration" refers to the number of microvesicles in a unit of volume, ie, number of MB / mL, as determined using a Coulter Counter instrument.

[0132] Typically, the calibrated microvesicle concentration (%) measured after reconstitution of the freeze-dried composition of the invention with an appropriate aqueous solution allows the determination of the microbubble yield after said reconstitution compared to the microvesicle concentration measured before the freeze-drying process.

[0133] In the present invention, the yield of calibrated microvesicles after reconstitution of the freeze-dried composition of the present invention is at least 50%, preferably at least 55%, more preferably at least 60%, even more preferably at least 65%, for example up to 85%, preferably 90%, more preferably 95%, even more preferably 100%.

[0134] The expression "calibrated microvesicle yield" 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 after lyophilization / mL) / (CMV concentration before lyophilization / mL)

[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 freeze-drying) / (GSD after freeze-drying)

[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 assessed when the GSD value after lyophilization is similar to that before lyophilization, resulting in a GSD ratio close to 1. Generally, a higher GSD ratio (i.e., closer to 1) indicates the maintenance of the initial monodispersity in the CMV distribution after lyophilization.

[0137] Purpose Microvesicles prepared by the methods of the present invention may be used in a variety of diagnostic and / or therapeutic techniques, including ultrasound and magnetic resonance in particular.

[0138] Diagnostic methods include any method that allows for enhanced visualization of a portion or part of an animal's (including human) body through the use of gas-filled microvesicles, including imaging for preclinical and clinical research purposes. Various imaging techniques can be used in ultrasound applications, including, for example, fundamental and harmonic B-mode imaging, pulsed or phase-inversion imaging, and fundamental and harmonic Doppler imaging; three-dimensional imaging techniques can be used if desired.

[0139] Microvesicles of the present invention may typically be administered at a concentration of about 0.01 to about 1.0 μL of gas per kg of patient, depending, for example, on their composition, the tissue or organ being imaged, and / or the imaging technique selected. This general concentration range may, of course, vary depending on the specific imaging application, e.g., in cases where signals can be observed at very low doses, such as color Doppler or power pulse inversion.

[0140] In one embodiment, the diagnostic method comprises: (i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of the lyophilized product obtained by the process of the invention; and (ii) detecting an ultrasound signal from a region of interest in the patient.

[0141] According to one embodiment, the suspension of gas-filled microvesicles comprises an amphiphilic material and a mixture of lyoprotectant 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 agitation in the presence of a physiologically acceptable gas (e.g., SF6).

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

[0144] Another aspect of the invention relates to the use of a suspension of microvesicles reconstituted from a lyophilized product according to the invention in a method of therapeutic treatment.

[0145] Therapeutic techniques include any method of treating a patient (as defined above), including the use of ultrasound and gas-filled microvesicles in combination, either by themselves (e.g., in ultrasound-mediated thrombolysis, high intensity focused ultrasound ablation, blood-brain barrier permeabilization, immunomodulation, neuromodulation, radiosensitization), or in combination with a therapeutic agent (i.e., for ultrasound-mediated delivery, e.g., delivery of a drug or bioactive compound to a selected site or tissue, e.g., in tumor therapy, gene therapy, infectious disease therapy, metabolic disease therapy, chronic disease therapy, degenerative disease therapy, inflammatory disease therapy, immune or autoimmune disease therapy, or for use as a vaccine); thus, the presence of gas-filled microvesicles may provide a therapeutic effect by itself or upon specific activation by various physical methods (e.g., including ultrasound-mediated delivery), or may enhance the therapeutic effect of the applied ultrasound, e.g., by imparting or contributing to impart a biological effect in vitro and / or in vivo.

[0146] Microvesicles of the present invention can typically be administered for therapeutic purposes at a concentration of about 0.01 to about 5.0 μL of gas per kg of patient, depending, for example, on the respective composition, the type of subject being treated, the tissue or organ being treated, and / or the therapeutic method being used.

[0147] In one embodiment, the above method of ultrasound therapeutic treatment comprises: (i) administering to a patient a suspension of gas-filled microvesicles obtained by reconstitution of the lyophilized product obtained by the process of the invention; (ii) identifying a region of interest in the patient to be exposed to a therapeutic treatment, the region of interest containing the suspension of gas-filled microvesicles; and (iii) applying an ultrasound beam to therapeutically treat the region of interest; The therapeutic treatment of the ultrasound is thereby enhanced by the presence of the suspension of gas-filled microvesicles in the region of interest.

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

[0149] [Example 1] Preparation of gas-filled microvesicles Gas-filled microvesicles were synthesized using a commercially available microfluidic flow focusing device (CU4553.007 N30 design, Micronit Microfluidics, NL) mounted in a commercially available chip holder (Micronit microfluidics, Fluidic Connect PRO Chip Holder with 4515 Inserts). The microvesicle-forming channel had a width of 19 μm. The chip and its holder were placed in an optically transparent, temperature-controlled water bath mounted on an inverted microscope equipped with a 20x magnification objective (Olympus, LMPLAN 20x) and a CCD camera (Lumenera, LM156M). The temperature of the constant temperature bath was set to 50°C.

[0150] The amphiphilic material in the liquid stream was DSPC:DPPE-PEG5000, with a respective molar ratio of 9:1.

[0151] The material was added to a 20 mg / mL mixture of chloroform and methanol (volume ratio) at 60 °C with stirring until the amphiphilic material was completely dissolved. The solvent was then evaporated under reduced pressure, and the resulting film was dried overnight under reduced pressure. The dried material was then redispersed in saline (0.9% NaCl) at a concentration of 15 mg / mL for 30 minutes at 60 °C with stirring. The dispersion was then sonicated using a tip sonicator (Branson Sonifier 250) to homogenize the material. The preparation was then filtered using a polycarbonate filter (0.45 μm pore size), cooled to room temperature, and degassed.

[0152] CO2 / C4F in a volume ratio of 85 / 15 10Gas-filled microvesicles containing CO2 and CF4 were prepared using a gas mixing device similar to the one shown schematically in Figure 3. Briefly, two gas containers were filled with CO2 and CF4, respectively. 10 The gas flow of each gas was controlled by the respective mass flow controllers: (i) EL-Flow: F200CV-002-RAD-11-K (for CO2) and (ii) Low-ΔP-Flow: F-200DV-RAD-11-Z (for C4F 10 The gas flow rate was adjusted by a mass flow controller (both gas controllers were from Bronkhorst, Ruurlo, The Netherlands). The mass flow controller was controlled by a customized software program implemented in Matlab (Mathworks) installed on a personal computer to set and maintain the desired mixture ratio. A pressure sensor (PSE530-M5-L; SMC Corp., Tokyo, Japan) measured the actual pressure of the gas mixture in the outlet channel leading to the microfluidic chip; a gas pressure of 2 bar was used for microvesicle formation. The liquid co-flow rate was controlled by a separate mass flow controller (Mini Cori Flow: M13V14I-MAD-11-KS; Bronkhorst, Ruurlo, The Netherlands). A liquid co-flow rate of approximately 150 μL / min was used to operate the flow-focusing device in the jet regime to produce microvesicles with a diameter (mode) of approximately 4 μm.

[0153] [Example 2] Preparation of the Lyophilized Composition First, 3 mL of the calibrated microvesicle suspension obtained by the microfluidic flow focusing method described in Example 1 was placed in a Pyrex tube (Pyrex disposable tube 12 × 75 mm) and CF4 10The microvesicle suspension was then centrifuged at 64 g for 6 minutes (Sigma 3-16 centrifuge), and the supernatant was aspirated using a syringe equipped with a needle. The remaining 200 μL of washed microvesicles was redispersed with 3 mL of a solution containing a lyoprotectant, as detailed in the Examples below. The effective concentration of the lyoprotectant was 188 mg / mL after redispersion of the remaining washed microvesicles.

[0154] The calibrated microvesicles, suspended in a solution of lyoprotectant components, were then aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a freeze-dryer.

[0155] The vials were cooled to temperatures between -30°C and -60°C (as detailed in the Examples below) and lyophilized under vacuum for approximately 1 hour. At the end of the procedure, the lyophilized composition was obtained as a white, homogeneous, dry solid. Then, pure CF4 was added to the headspace. 10 Filled with.

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

[0157] JPEG2025118720000005.jpg78166

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

[0159] JPEG2025118720000006.jpg153166

[0160] Characterization After the freeze-drying process, each freeze-dried composition was reconstituted with 1.5 mL of aqueous solution in the presence of a biocompatible gas to obtain a calibrated stable microvesicle suspension. The concentration of the mixture of cryoprotective components was 188 mg / mL before and after freeze-drying.

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

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

[0163] The GSD values and concentrations of the microvesicles were measured before and after the freeze-drying process to assess the effectiveness of the freeze-protectant components in maintaining the original properties of the calibrated microvesicles.

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

[0165] JPEG2025118720000007.jpg126166

[0166] The results shown in Table 4 clearly demonstrate that freeze-drying efficiency was improved using a mixture of lyoprotectants. In particular, freeze-drying calibrated microvesicles in a mixture of polyethylene glycol (PEG 4000 and PEG 8000) and polyols (i.e., xylitol, sorbitol, and mannitol) allowed for improved microvesicle yield after freeze-drying. For example, the use of mixture M1 allowed for a 17% increase in microvesicle yield after freeze-drying compared to the use of S1A. Even more advantageously, the use of M2 allowed for a higher increase in CMV yield (38%) compared to formulation S2A. Furthermore, freeze-drying CMV in mixture M2 allowed for a better GSD value (1.17), lower than that obtained using the single lyoprotectants of formulations S2A and S3A (1.21 and 1.33, respectively).

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

[0168] JPEG2025118720000008.jpg181166

[0169] [Example 4] Characterization of the properties of calibrated microvesicles after freeze-drying at different freezing temperatures The preparation of the freeze-dried compositions was further investigated by evaluating different freezing temperatures to which freshly prepared calibrated microvesicles were cooled at the beginning of the freeze-drying procedure.

[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 to obtain a calibrated, stable microvesicle suspension. The lyophilized composition was then reconstituted with a volume of aqueous solution similar to the volume of the suspension that underwent the lyophilization process. Thus, the concentration of the lyoprotectant in the reconstituted suspension was substantially identical to that in the initial suspension.

[0172] After reconstitution, the reconstituted calibrated microvesicle suspension was allowed to sit on the bench for 5 minutes before being characterized using a Coulter Counter Multisizer 3 equipped with a 30 μm aperture tube to measure microvesicle size, geometric standard deviation (GSD), concentration and yield after the freeze-drying process.

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

[0174] result The results confirmed that the use of a mixture of lyophilization components improved lyophilization performance at any tested freezing temperature when 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 tested freezing temperature, confirming the results previously obtained in Example 3.

[0175] JPEG2025118720000009.jpg155166

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

[0177] For this study, mixtures of lyoprotectants were compared to the corresponding lyoprotectants alone. In particular, the following lyoprotectants were evaluated: i) PEG4000 and sorbitol (included 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 ranging in concentration from 50 mg / ml to 250 mg / ml were prepared for each tested formulation.

[0179] The calibrated microvesicles suspended in different solutions of the lyoprotectant component were then aliquoted into DIN8R glass vials (1.5 mL suspension / vial) and transferred into a freeze-dryer.

[0180] The vials were cooled at ambient pressure for 1 hour at -60°C or -40°C (PEG 8000 mixture) and -60°C (PEG 4000 mixture), followed by primary drying at -20°C and 0.1 mbar. At the end of the procedure, the lyophilized compositions were obtained as white, homogeneous, dry solids. Then, pure CF4 was added to the headspace. 10 Filled with.

[0181] result The effect of the concentration of the lyoprotectant component on the properties of the calibrated microvesicles was tested, taking into account the CMV yield (Equation 2) and GSD ratio (Equation 3) after lyophilization.

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

[0183] Considering CMV yield, the results showed that increasing improvements in lyophilization efficiency were obtained using lyoprotectant concentrations between 50 mg / mL and 200 mg / mL for both the mixture and the single components. Furthermore, the results showed that substantially similar CMV yields were obtained for the mixture (PEG4000 and sorbitol) and PEG4000 alone (≤200 mg / mL) at the freezing temperatures tested.

[0184] In contrast, differences between the single components and the mixtures were observed as far as the GSD ratios were concerned: especially when frozen at −60° C., the GSD ratios yielded systematically increased values for the PEG4000 and sorbitol mixtures compared to PEG4000 alone.

[0185] JPEG2025118720000010.jpg87166

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

[0187] JPEG2025118720000011.jpg97166

[0188] As before (Case i), the results regarding the GSD ratio showed improved lyophilization efficiency of the PEG 4000 and xylitol mixture when frozen at -60 °C, resulting in better GSD ratio values than PEG 4000 alone. Unlike that, 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 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] JPEG2025118720000012.jpg98166

[0191] Considering CMV yield, the results showed that increasing improvements in lyophilization efficiency were obtained using concentrations of lyoprotectant components between 50 mg / mL and 250 mg / mL at both freezing temperatures for both mixtures and single components.

[0192] However, at a freezing temperature of −60°C, CMV yields were found to increase only for mixtures at low concentrations (50–100 mg / mL), while values were substantially similar at higher concentrations (especially ≥200 mg / mL) for both mixtures and single components.

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

[0194] The advantage of using a mixture of PEG8000 and sorbitol instead of PEG8000 alone was also confirmed by the GSD ratio values, as shown in Table 8. Notably, higher GSD values were observed when CMV was lyophilized in a solution of PEG8000 and sorbitol at all tested concentrations and both freezing temperatures, indicating improved monodispersity compared to CMV suspensions containing an equivalent amount of PEG8000.

[0195] v) PEG8000 and xylitol (included in equal amounts) compared to PEG8000 alone. Table 9 reports a comparison of 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] JPEG2025118720000013.jpg95166

[0197] Improved lyophilization efficiency, as indicated by increasing CMV yield values, was obtained using lyoprotectant concentrations between 50 mg / mL and 250 mg / mL for both the mixture and the single components. Results showed that at a freezing temperature of -60°C, CMV yield values were particularly improved for the mixture at low concentrations (<100 mg / mL).

[0198] The GSD ratio was found to be improved in the mixtures 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 showed improved values for both CMV yield and GSD ratio compared to PEG8000 alone, confirming its improved freeze-drying efficiency.

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

[0201] In particular, when comparing S1A with M1 (PEG4000 + sorbitol) and M9 (PEG4000 + xylitol), an improvement in the GDS ratio values was observed when frozen at -60°C for the mixtures.

[0202] Comparison between S2A (PEG 8000) and mixtures M2 (PEG 8000 + sorbitol) and M3 (PEG 8000 + xylitol) revealed that an increased improvement in the lyophilization efficiency of the mixtures was assessed when frozen at -40°C, with an increase in all tested parameters (CMV yield, GSD, GSD ratio) being observed. However, at a freezing temperature of -60°C, CMV yield was found to be similar or slightly higher for the mixtures compared to the single cryoprotectant components, while GSD ratio and GSD values only resulted in an improvement for mixtures M2 and M3, especially at higher concentrations of cryoprotectant (≥200 mg / mL).

[0203] 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 comprising a phospholipid and (ii) a lyoprotectant component, which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, the lyoprotective component has a concentration of 10-25% (w / v%) and is a mixture of at least two lyoprotective components including polyethylene glycol having a molecular weight comprised between 4000 and 8000 g / mol, and a sugar, the sugar being sucrose or maltose; The reconstituted suspension of calibrated gas-filled microvesicles has a geometric standard deviation (GSD) of less than 1.

2. Lyophilized composition.

2. 2. The freeze-dried composition of claim 1, The reconstituted suspension of calibrated microvesicles was 2.5 x 10 ８ characterized by a concentration of microvesicles / mL; Lyophilized composition.

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

4. A method for preparing a freeze-dried composition according to any one of claims 1 to 3 for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles, comprising: a. preparing a suspension of calibrated gas-filled microvesicles comprising a mixture of lyoprotective components including polyethylene glycol having a molecular weight comprised between 4000 and 8000 g / mol and a sugar, wherein the sugar is sucrose or maltose; and b. Lyophilizing the calibrated microvesicle suspension; Including, method.

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

6. 6. The method of claim 4 or 5, the mixture of lyoprotective components has a total concentration comprised between 120 mg / ml and 250 mg / mL; method.

7. The method of claim 6, The total concentration is 200 mg / mL; method.

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

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