WO2025021942A1

WO2025021942A1 - Ultrasound responsive vesicles containing lipid-polyamino acid conjugates

Info

Publication number: WO2025021942A1
Application number: PCT/EP2024/071180
Authority: WO
Inventors: Philippe Bussat; Samir Cherkaoui
Original assignee: Bracco Suisse SA
Current assignee: Bracco Suisse SA
Priority date: 2023-07-27
Filing date: 2024-07-25
Publication date: 2025-01-30
Anticipated expiration: 2026-01-27

Abstract

The present invention generally relates to the field of ultrasound contrast-agents (USCA). In particular, it relates to an ultrasound responsive vesicle comprising an inner core and an outer layer, wherein said inner core comprises a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof, and said outer layer comprises a lipid-polyamino acid conjugate of formula (I). It further relates to a suspension comprising a plurality of said ultrasound responsive vesicles and to said suspension for use in an in vivo imaging method.

Description

ULTRASOUND RESPONSIVE VESICLES CONTAINING LIPID-POLYAMINO

ACID CONJUGATES

Technical field

The invention relates to an ultrasound responsive ("US-responsive") vesicle stabilized by a lipid-polyamino acid conjugate, to an aqueous suspension comprising a plurality of said ultrasound responsive vesicles and to its use as medicament and contrast agent.

Background of the invention

Ultrasound for medical diagnostics is receiving wide acceptance in the clinics thanks to its tolerability, cost effectiveness, portability, safety, absence of ionizing radiation and ability to monitor dynamic processes in real time. In fact, ultrasound contrast agents (UCAs), consisting of a gas core encapsulated by a stabilizing shell (often referred as gas-filled microbubbles (MB)), are nowadays well established as blood pool agents to monitor blood perfusion and velocity in the macro- and microcirculation.

A well-known strategy to improve the blood circulation of these gas-filled microbubbles consists in modifying their outer shell with hydrophilic polymers, such as polyethylene glycol (PEG), able to prevent the adsorption of plasma proteins to the microbubbles outer shell and interfere with recognition and uptake by the reticuloendothelial system (RES).

Despite these benefits, repeated administration of PEGylated gas-filled microbubbles has been reported to lead to immunogenic response known as "accelerated blood clearance (ABC) phenomenon" (Fix, 2018) (Thi, 2020).

The ABC phenomenon describes the observation that PEGylated MBs (PEG-MB) show an extended blood circulation upon initial injection, but they are rapidly cleared from the blood following subsequent injections. It has been reported that an anti-PEG IgM is produced after the first dose of PEG-MB and the IgM antibodies bind to PEG-MB to enhance the uptake in the liver and reduce the blood half-life at the second dose.

Recently, many works have been addressed to develop alternative polymers that mimic the physicochemical properties of PEG without compromising therapeutic pharmacokinetics. Among these alternative polymers, polyaminoacids structures are very promising.

The present invention seeks to provide lipid-based ultrasound responsive vesicles with improved long-circulating properties as compared to conventional vesicles stabilized by PEGylated lipids.

Ultrasound responsive vesicles incorporating lipid-polyamino acid conjugates in accordance with the present invention advantageously showed improved properties such as increased circulation time due to the reduce clearance phenomenon. Summary of the invention

An aspect of the invention relates to an ultrasound responsive vesicle comprising an inner core and an outer layer, wherein said inner core comprises a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof, and said outer layer comprises a lipid-polyamino acid conjugate of formula I:

Formula I wherein:

L is an amphiphilic lipid residue comprising two lipophilic hydrocarbon chains;

Ri is -H or -COCH3;

R₂ is (CH₂)X-COOH or (CH₂)_X-CO-NHR₃;

R3 is -H or (C1-C4) alkyl substituted with one or more hydroxy groups;

X is 0-4 and n is comprised between 2 and 500.

In an embodiment said lipid-polyamino acid conjugate is a lipid-polyglutamic acid conjugate of formula II

Formula II

Wherein

Ri is -H or -COCH3 and n is comprised between 2 and 500.

Preferably said amphiphilic lipid residue comprising two lipophilic hydrocarbon chains is a phospholipid, preferred being l,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), l,2-Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) or 1,2-Distearoyl- sn-glycero-3-phosphorylethanolamine (DSPE).

In a preferred embodiment, said lipid-polyglutamic acid conjugate is selected from a compound of formula III (DPPE -PGA(diol) conjugate)

Formula III

Or a compound of formula IV (DMPE -PGA(diol) conjugate)

Formula IV wherein n is comprised between 2 and 500 monomer subunits, preferably n is lower than 150, still more preferably lower than 130, still more preferably lower than 110, still more preferably lower than 90, still more preferably lower than 70, still more preferably lower than lower than 50, still more preferably lower than 40, up to 2.

In an embodiment said physiologically acceptable compound is a fluorinated compound, preferably is a perfluorinated compound, preferably selected from C4F10, C3F8, SFe or a mixture thereof.

In an embodiment said outer layer further comprises an additional stabilizing material, preferably comprising a phospholipid. In another embodiment said outer layer further comprises a fatty acid.

In another aspect the invention relates to a suspension comprising a plurality of US-responsive vesicles as above defined and a pharmaceutically acceptable liquid carrier.

In a still another aspect the invention relates to a suspension as above defined for use as medicament.

In a further aspect the invention relates to an aqueous suspension as defined above for use in an in vivo imaging method.

Another aspect of the invention relates to a vial comprising: a precursor of the suspension as defined above in the form of a freeze- dried product and a freeze-drying protecting component; and a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof.

Detailed description of the invention

The present invention generally relates to an ultrasound responsive vesicle stabilized by a lipid-polymer conjugate and filled with a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof.

The Applicant observed that incorporating specific lipid-polyamino acid conjugates in the shell of ultrasound responsive vesicles advantageously improved the in vivo circulating properties of said vesicles as compared to conventional gas-filled vesicles coated with PEGylated lipids.

The expression "ultrasound (US) responsive vesicle" indicates a core-shell structure that can change its physicochemical properties in response to an external US stimulus.

According to the present invention, said US-responsive vesicles can be gas-filled vesicles or liquid-filled vesicles (e.g. when the core comprises a gas precursor in liquid form).

Examples of US-responsive gas-filled vesicles are microbubbles and nanobubbles.

Examples of US-responsive liquid-filled vesicles are nanodroplets and nanoemulsions.

Preferably said US-responsive vesicle is selected from the group consisting of gas- filled microbubbles, gas-filled nanobubbles or liquid-filled nanodroplets, more preferably said US-responsive vesicle is a gas-filled microbubble.

Ultrasound responsive gas-filled micro- or nano- bubbles The expression "gas-filled bubbles" generally refers to bubbles of gas bounded, at the gas/liqu id interface, by a very thin envelope (film) involving a stabilizing amphiphilic material, typically a phospholipid, disposed at the gas to liquid interface. Said gas-filled bubbles are suitable as contrast agents in ultrasound imaging techniques, known as Contrast-Enhanced Ultrasound (CEUS) Imaging, or in therapeutic applications, e.g. in combination with ultrasound mediated drug delivery.

These stabilized gas-filled bubbles (dispersed in a suitable physiological solution) are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, "spheres", "bubbles", "capsules" or "balloons".

Gas-filled bubbles include microbubbles or nanobubbles. Typically, microbubbles range between 1 and 8 pm while nanobubbles are submicron sized (e.g. lower than 1 pm).

When used as a contrast agent, gas-filled bubbles can be detected by an ultrasound device when they possess acoustical characteristics that differ from the surrounding medium. The microbubbles act as echo-enhancers by increasing ultrasound impedance such that there is a mismatch between the microbubbles and the surrounding tissues. Theragnostic applications are achieved through the mechanisms of cavitation and sonoporation, which are the combined effects of ultrasound and gas-filled bubbles.

It is possible to obtain size-controlled gas-filled bubbles using suitable preparation methods. The expression "size-controlled gas-filled bubbles" refers to an aqueous suspension of US-responsive bubbles characterized by a size distribution having a geometric standard deviation (GSD) of at least 1.2 or lower, preferably of at least 1.1, down to e.g. 1.05.

The term "size-controlled" is used interchangeably with "calibrated", "monodispersed" or "monosize(d)".

Ultrasound-responsive liquid-filled nanodroplets

Liquid-filled nanodroplets are commonly known as PCCAs (Phase-Change Contrast Agents), or acoustically activated nanodroplets and are used in both ultrasound diagnostic and therapeutic delivery.

The term "nanodroplet" indicates an assembly comprising an outer layer and a liquid inner core, said outer layer comprising a stabilizing amphiphilic material (e.g. a phospholipid) and said liquid inner core comprising a gas precursor (in liquid form), such as a liquid fluorinated compound, preferably a perfluorocarbon. In said nanodroplets, the amphiphilic lipid material is oriented in such a way that the hydrophobic portions of the amphiphilic lipid are located at a surface of the fluorinated compound of the inner core. Compositions of said nanodroplets have a z-average diameter comprised between 100 nm and 1000 nm, preferably between 120 and 800 nm, more preferably between 150 and 400 nm.

Owing to Acoustic Droplet Vaporization (ADV) process, the liquid-filled nanodroplets are converted into gas bubbles upon exposure to ultrasound energy beyond a vaporization threshold. In fact, ultrasound act as a remote trigger to promote the vaporization of the droplets in a controllable, non-invasive and localized manner. Thanks to their smaller size compared to conventional microbubbles, nanodroplets display prolonged in vivo circulation, deep penetration into the tissues via the extravascular space. Moreover, below vaporization threshold, they are ultrasonically stable with low acoustic attenuation and can be acoustically vaporized at the location of interest.

Ultrasound-responsive liquid-filled nanodroplets present a real potential as an extravascular ultrasound contrast agent in numerous diagnostic and therapeutic applications including sonopermeabilization and blood brain barrier (BBB) opening.

Said liquid-filled nanodroplets can be "calibrated nanodroplets" indicating a population of nanodroplets having a z-average as above defined and a polydispersity lower than 0.25, preferably lower than 0.20, more preferably lower than 0.15, even more preferably lower than 0.10. Said "calibrated nanodroplets" are preferably obtained through microfluidic technique.

Outer layer

The present invention discloses an ultrasound responsive vesicle stabilized by a lipid-polymer conjugate comprising an amphiphilic lipid residue and a polymer or a monomeric precursor therefor, having an N- and a C-terminal end group, wherein the polymer is selected from the group consisting of a poly-(amino acid), a poly-(amino acid derivative) or a poly-(amino acid analogue) and wherein the amphiphilic lipid residue is covalently attached to the N- or C- terminal end group of the polymer.

The expression lipid-polyamino acid conjugate generally refers to a compound formed by the covalent bonding of at least one lipid moiety with one or more polyamino acid chains. The lipid moieties typically comprise long-chain fatty acids or derivatives thereof, which provide hydrophobic characteristics, while the polyamino acid chains are composed of amino acid monomers linked by peptide bonds, conferring hydrophilic properties and biological functionality.

According to the present invention, the outer layer of said ultrasound responsive vesicle comprises a lipid-polyamino acid conjugate of formula I

Formula I wherein:

L is an amphiphilic lipid residue, comprising two lipophilic hydrocarbon chains;

Ri is -H or -COCH3;

R₂ is (CH₂)X-COOH or (CH₂)_X-CO-NHR₃;

R3 is -H or (C1-C4) alkyl substituted with one or more hydroxy groups;

X is 0-4 and n is comprised between 2 and 500.

Generally, amphiphilic lipid residues suitable for the lipid-polymer conjugates may be selected from a variety of synthetic or naturally occurring lipids, comprising a hydrophobic (e.g. apolar) tail and a hydrophilic polar head group.

Said amphiphilic lipid residue comprises a functional group at its polar head group suitable for covalent attachment to a polymer chain. Examples of suitable polar head groups are primary or secondary amine groups, hydroxyl groups, aldehyde groups, halides or a carboxylic group. The hydrophobic moiety of the amphiphilic lipid residue is typically a hydrocarbon chain, preferably comprising at least 10 carbon atoms, more preferably at least 12 and even more preferably at least 14 carbon atoms, up to e.g. 24 carbon atoms, preferably up to 20. This hydrophobic moiety enables the incorporation of the lipid- polyglutamic conjugates into core-shell structures.

Examples of amphiphilic lipid residues include phospholipids, glycolipids, ceramides, cholesterol and derivatives, saturated or partially unsaturated, branched or straight-chain Cs-Cso mono- or dialkylamines, diacylamines, arylalkylamines, cycloalkylamines, alkanols, aldehydes, carbohalides or fatty acids and the anhydrides thereof.

The amphiphilic lipid residue may be bound to the carboxylic terminal group of the polymer (i.e. repeating unit) through usual ester or amido bonds. According to the disclosed invention, the amphiphilic lipid residue in the lipidpolyamino acid conjugate of formula I is a lipid having two hydrophobic chains, typically alkyl chains, and a polar head group, containing a functional group, as described above.

The Applicant has observed that using lipid-polyamino acid conjugates comprising an amphiphilic lipid residue with two alkyl chains unexpectedly led to US responsive vesicles characterized by substantially better properties than the corresponding vesicles comprising a lipid- polyamino acid conjugate containing an amphiphilic lipid with a single alkyl chain. Suspensions comprising ultrasound responsive vesicles stabilized by an outer shell incorporating lipid-polyglutamic conjugates comprising an amphiphilic lipid with two alkyl chains were characterized by higher values of vesicle concentration (e.g. the number of microbubbles per unit volume of the liquid, typically represented as microbubbles per milliliter (bubbles/mL)) and higher values of Mean Volume Concentration (MVC) (i.e the average volume of gas contained within the microbubbles per unit volume of the liquid (e.g. pL/mL).

An aspect of the invention relates to an aqueous suspension comprising an ultrasound responsive vesicle as above defined. Preferably said ultrasound responsive vesicle comprises a gaseous inner core (e.g. is a gas-filled microbubble).

According to an embodiment, the vesicles concentration of said aqueous suspension is comprised between lxlO⁹ vesicles/mL and 3xl0⁹ vesicles/mL.

According to another embodiment, the MVC of said suspension is comprised between 4 and 10 pL/mL.

For instance, in ultrasound-mediated diagnostic applications suitable values of vesicle concentration and MVC are those necessary to obtain an improved imaging quality.

In a preferred embodiment, said amphiphilic lipid residue comprises two hydrophobic chains (i.e. lipophilic hydrocarbon chains), preferably having a length of at least 14 carbons atoms.

In a still preferred embodiment, said amphiphilic lipid residue is a phospholipid, preferred being a phosphatidyl ethanolamine derivative containing a reactive amino group.

Still more preferably said phospholipid is selected from 1,2-Dimyristoyl-sn-glycero- 3-phosphorylethanolamine (DMPE), l,2-Dipalmitoyl-sn-glycero-3- phosphorylethanolamine (DPPE) or l,2-Distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE).

Typically, the polymer part of the lipid-polyamino acid conjugates is formed by a polyamino acid, preferably a polyamino acid derivative or a polyamino acid analogue. A polyamino acid derivative is a polymer, comprising respective amino acid repeating units, to which one or more substituents are attached. A polyamino acid analogue as herein disclosed is a polymer, wherein the carbon atom chain length of the amino acid repeating units is reduced or prolonged.

According to formula I of the present invention, n indicates the number of monomer subunits of the polyamino acid part, which is comprised between 2 and 500 monomer subunits, preferably between 20 and 150.

Preferably n is lower than 150, still more preferably lower than 130, still more preferably lower than 110, still more preferably lower than 90, still more preferably lower than 70, still more preferably lower than lower than 50, still more preferably lower than 40, up to 2.

Preferably, the polymer part of the lipid-polyamino acid conjugates is formed by a poly(glutamic acid) derivative consisting of glutamic acid repeating units, more preferably said glutamic acid repeating units are functionalized with a dihydrossipropilamide substituent ("PGA(diol) derivative").

Preferably, in the lipid-polyamino acid conjugate of formula I R.2 is (CH2)x-CO-NHR.3 wherein

R.3 is a C3 alkyl chain substituted with two hydroxy groups;

X is 2 and n is comprised between 2 and 500.

In a preferred embodiment said lipid-polyamino acid conjugate is a lipidpolyglutamic acid conjugate of formula II

Formula II

Wherein

L is an amphiphilic lipid residue comprising two lipophilic hydrocarbon chains;

Ri is -H or -COCH3; n is comprised between 2 and 500. Preferably said amphiphilic lipid residue comprises two lipophilic hydrocarbon chains having a length of at least 14 carbons atoms. More preferably said amphiphilic lipid is a phospholipid, preferred being a phosphatidyl ethanolamine derivatives containing a reactive amino group.

Preferably, said lipid-polyglutamic acid conjugate is selected from a compound of formula III (DPPE -PGA(diol) conjugate)

Formula III or a compound of formula IV (DMPE -PGA(diol) conjugate)

Said lipid-polyglutamic acid ("lipid-PGA(diol)") conjugates can be prepared according to methods known in the art.

For instance, a suitable method for the preparation of lipid-polyglutamic conjugates according to this invention can be found in WO2002098951.

Lipid-polyamino conjugates of Formula I can also be prepared as described in WO2023156629A1, which is hereby incorporated by reference.

According to an embodiment, the outer layer of said ultrasound-responsive vesicles further comprises an additional stabilizing material.

Suitable examples of additional stabilizing materials for forming the outer layer of the ultrasound-responsive vesicles include amphiphilic materials, such as phospholipids, biodegradable polymers or biodegradable water-insoluble lipids (such as those described, for instance, in US5,711,933 and US 6,333,021), proteins (e.g. albumin, haemoglobin, as described for instance US4,276,885 or EP 0324938).

Preferably the stabilizing material is an amphiphilic material.

According to a preferred embodiment, amphiphilic materials useful for forming the stabilizing layer comprise a phospholipid. Phospholipids, as other amphiphilic molecules, are generally capable of forming a stabilizing film of material (typically in the form of a mono-molecular layer) at the core-water boundary interface in the final vesicles suspension, these materials are also referred to in the art as "film-forming" materials.

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

Examples of suitable phospholipids include esters of glycerol with one or preferably two (equal or different) residues of fatty acids and with phosphoric acid, wherein the phosphoric acid residue is in turn bound to a hydrophilic group, such a, for instance, choline (phosphatidylcholines - PC), serine (phosphatidylserines - PS), glycerol (phosphatidylglycerols - PG), ethanolamine (phosphatidylethanolamines - PE), inositol (phosphatidylinositol). Esters of phospholipids with only one residue of fatty acid are generally referred to in the art as the "lyso" forms of the phospholipid or "lysophospholipids". Fatty acids residues present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22; the aliphatic chain may contain one or more unsaturations or is preferably completely saturated. Examples of suitable fatty acids included in the phospholipids are, for instance, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Preferably, saturated fatty acids such as myristic acid, palmitic acid, stearic acid and arachidic acid are employed.

Further examples of phospholipid are phosphatidic acids, i.e. the diesters of glycerol-phosphoric acid with fatty acids; sphingolipids such as sphingomyelins, i.e. those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain; cardiolipins, i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid; glycolipids such as gangliosides GM1 (or GM2) or cerebrosides; glucolipids; sulfatides and glycosphingolipids.

As used herein, the term "phospholipid(s)" includes either naturally occurring, semisynthetic or synthetically prepared compounds that can be employed either alone or as mixtures.

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

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

Examples of preferred phospholipids are, for instance, dilauroylphosphatidylcholine (l,2-Dilauroyl-sn-glycero-3-phosphocholine, DLPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoyl-phosphatidylcholine (DPPC), diarachidoyl- phosphatidylcholine (DAPC), distearoyl-phosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dibehenoyl-phosphatidylcholine (DBPC), 1,2 Distearoyl-sn- glycero-3-Ethylphosphocholine (Ethyl-DSPC), di pentadeca noyl-phosphatidylcholine (DPDPC), l-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC), l-palmitoyl-2-myristoyl- phosphatidylcholine (PMPC), l-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC), 1- stearoyl-2-palmitoyl-phosphatidylcholine (SPPC), 1 -pa I mitoyl-2-oley I phosphatidylcholine (POPC), l-oleyl-2-palmitoyl-phosphatidylcholine (OPPC), dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts, diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal salts, dioleoylphosphatidylglycerol (DOPG) and its alkali metal salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal salts, distearoyl phosphatidic acid (DSPA), diarachidoylphosphatidic acid (DAPA) and its alkali metal salts, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), distearoyl phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine (DOPE), diarachidoylphosphatidylethanolamine (DAPE), dil inoleyl phosphatidylethanolamine (DLPE), dimyristoyl phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP), dilauroyl-phosphatidylinositol (DLPI), diarachidoylphosphatidylinositol (DAPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI), distearoylphosphatidylinositol (DSPI), dioleoylphosphatidylinositol (DOPI).

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

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

In an embodiment the additional stabilizing material, e.g. phospholipids, or the lipid-PGA(diol) conjugate may bear a reactive moiety which may then be reacted with a corresponding reactive moiety bearing a suitable active component (e.g. targeting ligand), in order to bind said active component to the US-responsive vesicles. Examples of suitable reactive moieties include, for instance, reactive groups capable of reacting with an amino group bound to an active component such as isothiocyanate groups (that will form a thiourea bond), reactive esters (to form an amide bond), aldehyde groups (for the formation of an imine bond to be reduced to an alkylamine bond); reactive groups capable of reacting with a thiol group bound to an active component, such as haloacetyl derivatives or maleimides (to form a thioether bond); reactive groups capable of reacting with a carboxylic group bound to an active component, such as amines or hydrazides (to form amide or alkylamide bonds).

The phospholipids can conveniently be used in admixture with any other compound, preferably amphiphilic. For instance, lipids such as cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate or ascorbyl palmitate, fatty acids such as myristic acid, palmitic acid, stearic acid, arachidic acid and derivatives thereof or butylated hydroxytoluene and/or other non-phospholipid (amphiphilic) compounds can optionally be added to one or more of the foregoing phospholipids, e.g. in proportions preferably below 50% by weight, more preferably up to 25% or lower. Particularly preferred as amphiphilic additional compound in admixture with phospholipids are fatty acids. Fatty acids useful in a composition according to the invention, which can be either saturated or unsaturated, comprise a C10-C24, aliphatic chain terminated by a carboxylic acid moiety, preferably a C14-C22 and more preferably a C16-C20 aliphatic chain. Examples of suitable saturated fatty acids include capric (n- decanoic), lauric (n-dodecanoic), myristic (n-tetradecanoic), palmitic (n-hexadecanoic), stearic (n-octadecanoic), arachidic (n-eicosanoic), behenic (n-docosanoic) and n- tetracosanoic acid. Preferred saturated fatty acids are myristic, palmitic, stearic and arachidic acid, more preferably palmitic acid. Examples of unsaturated fatty acids comprise myristoleic (cis-9-tetradecenoic), palmitoleic (cis-9-hexadecenoic), sapienic (cis-6-hexadecenoic), oleic (cis-9-octadecenoic), linoleic (cis-9,12-octadecadienoic), linolenic (cis-9,12,15-octadecatrienoic), gondoic (cis-ll-eicosenoic), cis-11,14- eicosadienoic, cis-5,8,ll-eicosatrienoic, cis-8,ll,14-eicosatrienoic, cis-11, 14,17- eicosatrienoic, arachidonic (cis-8,ll,14,17-eicosatetraenoic) and erucic (cis-13- docosenoic) acid.

According to an embodiment, said outer layer further comprises a phospholipid.

According to another embodiment, said outer layer comprises a mixture of amphiphilic materials comprising a phospholipid and a fatty acid. Preferably said fatty acid is palmitic acid.

According to an alternative embodiment, said outer layer further comprises a targeting ligand.

Inner core

The disclosed ultrasound-responsive vesicle comprises an inner core comprising a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof that allows for activation of the vesicle upon application of ultrasound (US), said inner core being in gaseous or liquid form at room temperature (i.e. 20°C to 25°C)

Any physiologically acceptable (biocompatible) gas, gas precursor or mixture thereof may be employed to fill the above ultrasound responsive vesicles.

In an embodiment said gaseous inner core comprises a physiologically acceptable gas, being preferably a fluorinated compound.

Physiologically acceptable gases refer to compounds having a boiling point below room temperature (RT; 25 °C), that are in a gaseous form at Standard Ambient Temperature and Pressure (SATP), namely at 25 °C and 1 atm (101.325 kPa) (i.e. highly volatile compounds).

In the present description and claims, the term "fluorinated compounds" refers to a group of fluorine-containing compounds derived from hydrocarbons by partial or complete substitution of hydrogen atoms with fluorine atoms, which can be gas or liquid at room temperature.

Gaseous fluorinated compounds include materials which contain at least one fluorine atom such as, for instance, fluorinated hydrocarbons (organic compounds containing one or more carbon atoms and fluorine); sulfur hexafluoride; fluorinated, preferably perfluorinated, ketones; and fluorinated, preferably perfluorinated, ethers. Preferred compounds are perfluorinated gases, such as SF₆ or perfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons where all the hydrogen atoms are replaced by fluorine atoms, which are known to form particularly stable gas-filled vesicles suspensions.

The term "perfluorocarbon" includes saturated, unsaturated, and cyclic perfluorocarbons. Suitable examples of biocompatible, physiologically acceptable gaseous perfluorocarbons are 1,1, 1,2, 3, 3, 3 heptafluoropropane, 1,1, 1,2, 2, 3- Hexafluoropropane, 1,1,1,2,3,3-Hexafluoropropane, 1,1, 1,3, 3, 3- Hexafluoropropane,

1.1.1.2.2.3.3.4.4 Nonafluorobutane, l,l,l,3,3,3-Hexafluoro-2-(trifluoromethyl)propane,

1.1.1.2.2.3.3.4 Octafluorobutane or a mixture thereof.

Suitable examples of perfluorocarbons (in a gaseous form at SATP), are perfluorocyclopropane, perfluoropropane, perfluorocyclobutane, perfluorobutane, perfluoroisobutane or a mixture thereof.

In an embodiment said gaseous perfluorocarbon is preferably perfluorobutane (boiling point -2°C) or perfluoropropane (boiling point -37°C).

In a further embodiment said gaseous fluorinated compound is a perfluoro olefin, selected from C4-C6 perfluoro olefins, preferably C4-C5, more preferably C5 perfluoro olefins. Specific examples include perfluoro-2-butene, perfluoro-l-pentene, perfluoro-2- pentene, or mixtures thereof. More preferably the perfluoro olefin is perfluoro-2- pentene.

Particularly preferred gases are SFe, C3F8 and C4F10.

In another embodiment, said liquid inner core comprises a gas precursor in liquid form, said gas precursor being selected from liquid fluorinated compounds having a boiling point above room temperature (RT; 25 °C) that are in a liquid form at Standard Ambient Temperature and Pressure (SATP), namely at 25 °C and 1 atm (101.325 kPa), or gaseous fluorinated compounds having a boiling point below room temperature (RT; 25 °C), that are in a gaseous form at Standard Ambient Temperature and Pressure (SATP), namely at 25 °C and 1 atm (101.325 kPa) (i.e. highly volatile compounds).

The expression "gas precursor in liquid form" refers to a compound, typically a fluorinated compound, which is comprised in the inner core of an ultrasound responsive vesicle in a liquid state and which, upon exposure to an external stimulus, such as ultrasound, can be activated to transition to a gaseous state, resulting in a phase change that can be utilized for diagnostic or therapeutic applications.

Despite the boiling point of said gas precursor (i.e. above or below room temperature at Standard Ambient Temperature and Pressure), the core of said vesicles remains liquid due to several factors, like physicochemical properties associated with nanoscale confinement and the stability provided by the surfactant shell.

For said reasons, said ultrasound responsive vesicles having a liquid core filled with a highly volatile fluorinated compound are referred to as "metastable", because they are stable as droplets at room conditions (i.e. SATP) and physiological conditions (typically a temperature from 36.5 to 37.5 °C and a pressure of 120/80 mm/Hg), meaning that they do not spontaneously expand into gas bubbles without being submitted to an external acoustic energy. For instance, after their administration, the sole exposition of said vesicles filled with a highly volatile fluorinated compound to the body temperature and physiological pressure do not cause their activation and conversion in microbubbles. Additional energy is thus required to trigger this phenomenon after administration, such as ultrasound stimulus provided by a medical device.

In other words, the liquid core of the vesicles of this invention is characterized by the ability to remain in a liquid state despite the low boiling point of the core substance. Upon exposure to an external stimulus, such as ultrasound, said vesicles can be activated to transition to a gaseous state, resulting in a phase change that can be utilized for diagnostic or therapeutic applications

Suitable examples of liquid fluorinated compounds are 1-Fluorobutane, 2- Fluorobutane, 2,2-Difluorobutane, 2,2,3,3-Tetrafluorobutane, 1,1, 1,3,3- Pentafluorobutane, 1,1, 1,4, 4, 4- Hexafluorobutane, 1,1, 1,2, 4, 4, 4- Heptafluorobutane, 1,1,2,2,3,3,4,4-Octafluorobutane, 1,1,1,2,2-Pentafluoropentane, 1, 1,1, 2, 2, 3,3,4-

Octafluoropentane, 1,1,1,2,2,3,4,5,5,5-Decafluoropentane, 1, 1,2, 2, 3, 3, 4, 4, 5, 5,6, 6- Dodecafluorohexane, or a mixture thereof.

Preferably said gaseous fluorinated compound are perfluorinated compounds,

Suitable examples of liquid perfluorocarbons are perfluoropentane, perfluorohexane, perifluoroheptane, perfluorooctane, perfluorononane, perfluorodecalin, perfluorooctylbromide (PFOB), perfluoro-15-crown-5-ether (PFCE), perfluorodichlorooctane (PFDCO), perfluorononane (PFN), and l,l,l-tris(perfluorotert- butoxymethyl)ethane (TPFBME), or a mixture thereof.

In an embodiment said perfluorocarbon is preferably perfluoropentane (PFP) (boiling point 29°C) or perfluorohexane (PFH) (boiling point 57°C).

It may also be advantageous to use a mixture comprising any of the above gases or gas precursors in any ratio. For instance, the mixture may comprise a conventional gas, typically a non-fluorinated gas, such as nitrogen, air or carbon dioxide and a gas forming a stable microvesicle suspension, such as sulfur hexafluoride or a perfluorocarbon as indicated above. Examples of suitable gas mixtures can be found, for instance, in WO 94/09829, which is herein incorporated by reference.

Particularly preferred gases are SFe, CsFs, C4F10 or mixtures thereof, optionally in admixture with air, oxygen, nitrogen, carbon dioxide or mixtures thereof, e.g. a 35/65 (v/v) mixture of perfluorobutane or perfluoropropane and nitrogen.

As used herein, the "pressure resistance" of US-responsive gas-filled vesicles is defined by the Pc50 parameter measured on a suspension of US-responsive gas-filled vesicles; as known, an increasing overpressure applied on a suspension of US-responsive gas-filled vesicles results in the progressive reduction of the population of vesicles with respect to the initial one (measured at atmospheric pressure), due to the collapse of the vesicles. The Pc50 of a suspension of gas-filled vesicles identifies the value of applied overpressure (with respect to atmospheric pressure) at which the absorbance of the suspension drops to half of the absorbance of the suspension measured at atmospheric pressure. Reduction of the absorbance of a suspension of US-responsive gas-filled vesicles is related to the reduction of the initial population of gas-filled US-responsive gas-filled vesicles, whereby the initially milky suspension (typical of high concentration of microvesicles) becomes more and more transparent under increasing pressure (reduced concentration of US-responsive gas-filled vesicles). The higher the Pc50 values, the higher the resistance to pressure (e.g. blood pressure or ultrasound pressure) of vesicles, and typically the longer the circulation time of the US-responsive gas-filled vesicles once administered.

Method of Manufacturing

Gas-filled vesicles

A suitable method for preparing aqueous suspensions of gas-filled vesicles comprises the reconstitution, in the presence of a suitable gas or gaseous mixture, of a freeze-dried product comprising a stabilizing material (capable of forming a stabilizing outer layer) with an aqueous carrier. The freeze-dried product is typically obtained by freeze-drying a liquid mixture comprising said amphiphilic material and a freeze-drying protecting component in a suitable solvent. The liquid mixture which undergoes the freeze-drying process can be obtained according to methods known in the art, as disclosed e g. in WO94/09829 or WO2004/069284.

For instance, according to the process disclosed by WO94/09829, the amphiphilic material is dispersed into an organic solvent (e.g. tertiary butanol, dioxane, cyclohexanol, tetrachlorodifluoro ethylene or 2-methyl-2-butanol) together with a suitable freeze-drying protecting component. The dispersion containing the amphiphilic material and the freeze-drying protecting component is then subjected to freeze-drying to remove the organic solvent thus obtaining a freeze-dried product.

According to the process disclosed in W02004/069284, a composition comprising an amphiphilic material may be dispersed in an emulsion of water with a water immiscible organic solvent under agitation, preferably in admixture with a freeze-drying protecting component. The so obtained emulsion, which contains of solvent surrounded and stabilized by the amphiphilic material, is then freeze-dried according to conventional techniques to obtain a freeze-dried material, which can then be used for preparing a suspension of gas-filled vesicles.

As defined herein, a freeze-drying protecting component is a compound with cryoprotective and/or lyoprotective effect. Suitable freeze-drying protecting components include, for instance, carbohydrates, e.g. a mono- di- or poly-saccharide, such as sucrose, maltose, trehalose, glucose, lactose, galactose, raffinose, cyclodextrin, dextran, chitosan and its derivatives (e.g. carboxymethyl chitosan, trimethyl chitosan); polyols, e.g. sugar alcohols such as sorbitol, mannitol or xylitol; or hydrophilic polymers, e.g. polyoxyalkyleneglycol such as polyethylene glycol (e.g. PEG2000, PEG4000 or PEG8000) . According to an embodiment said freeze-drying protecting component is polyethylen glycol, preferably PEG4000.

For the freeze-drying process, the liquid mixture containing the amphiphilic material and the freeze-drying protecting component (obtained e.g. according to either of the previously illustrated manufacturing processes), is typically sampled into glass vials (e.g. DIN4R, DIN8R or DIN20R) which are loaded into a freeze-dryer.

The freeze-drying process generally includes an initial step where the vials are rapidly deep-cooled (e.g. at temperatures of from -35°C to -70°C) to freeze the liquid(s) of the mixture and then subjected to vacuum (e.g. 0.1-0.8 mbar); during this step (primary drying), the substantial totality of the frozen liquid(s) (e.g. water and/or solvents) is removed by sublimation, typically up to about 95% of the total amount of liquid, preferably up to about 99%. After the primary drying, residual liquid (including possible interstitial water) can be further removed during the secondary drying, which is typically conducted at a temperature higher than room temperature, under vacuum (preferably by maintaining the same vacuum applied during the primary drying). The temperature during the secondary drying is preferably not higher than 35°C. The secondary drying can be stopped when the residual content of the liquid(s) reaches a desired minimum value, e.g. less than 3% (preferably less than 1%) by weight of water with respect to the total mass of residual freeze-dried product, or e.g. less than 0.01% by weight, preferably less than 0.08%, for residual solvent(s).

After completion of the freeze-drying process, the freeze-dried product may undergo an optional additional thermal treatment, typically under ambient pressure, as described for instance in WO2020/229642. Preferably the thermal treatment is performed on the sealed vial, after saturating the headspace of the vials with a suitable gas or gaseous mixture as defined herein and then stoppering (e.g. with a rubber, such as butyl rubber, stopper) and sealing (e.g. with a metal, such as aluminium, crimp seal) the vials. In this case, the vials are preferably removed from the freeze-drier and introduced in a suitable oven for the thermal treatment. Alternatively, such thermal treatment can be performed on the open vial (which are preferably kept into the freeze- dryer), which are then saturated with the gas or gaseous mixture and then stoppered/sealed.

Liquid-filled vesicles

Ultrasound responsive vesicles having an inner core in liquid form (at SATP) according to this invention can be prepared according to methods known in the art.

For instance, a suitable method for the preparation of liquid-filled nanodroplets can be found in Melich, 2020.

According to a process disclosed in WO2022101365, liquid-filled nanodroplets can also be prepared by microfluidic technique by using a method comprising the following steps: a) Preparing an aqueous phase; b) Preparing an organic phase, wherein i) said aqueous phase comprises a lipid-polyamino acid conjugate of Formula I and the organic phase comprises a gas precursor or ii) said organic phase comprises a lipid-polyamino acid conjugate of Formula I and a gas precursor. c) Injecting said aqueous phase into a first inlet and said organic phase into a second inlet of a microfluidic cartridge, thereby mixing said aqueous phase and said organic phase in a mixing device of the microfluidic cartridge, wherein the operating pressure into said microfluidic cartridge is lower than 7000 kPa, to obtain an aqueous suspension of calibrated US-responsive nanodroplets having a core in liquid form, and d) Collecting the aqueous suspension of calibrated US-responsive nanodroplets having a core in liquid form from the exit channel of the microfluidic cartridge. Preferably, said gas precursor is a fluorinated compound, more preferably is a perfluorinated compound, preferred being perfluorohexane and perfluoropentane.

Preferably wherein said calibrated US-responsive nanodroplets having a core in liquid form (i.e. liquid-filled) have a z-average diameter comprised between 100 nm and 1000 nm and a polydispersity lower than 0.20.

According to an alternative method disclosed in WO2023118387A1, which is here incorporated by reference, the above mentioned microfluidic method can also be used to prepare liquid-filled nanodroplets starting from a gaseous fluorinated compound.

Preferably, said gaseous fluorinated compound is a perfluorocarbon, preferred being perfluorobutane and perfluropropane.

Advantageously, said calibrated US-responsive liquid-filled nanodroplets can be freeze-dried obtaining a freeze-dried composition comprising i) a lipid-polyamino acid conjugate of Formula I, ii) a gas precursor in liquid form and iii) a freeze-drying protecting component which, upon reconstitution with a pharmaceutically acceptable liquid carrier, provides a suspension of US-responsive liquid-filled nanodroplets.

In another words, said freeze-dried composition acts as freeze-dried precursor of said suspension of US-responsive liquid-filled nanodroplets, which may be advantageously comprised in a vial, further comprising a freeze-drying protecting component.

Freeze-dried compositions of calibrated US-responsive liquid-filled nanodroplets can be prepared as described in WO2024/033540.

For instance, a method for preparing said freeze-dried composition comprises the steps of: a) preparing an initial suspension comprising i) a plurality of US-responsive liquid-filled nanodroplets comprising lipid- polyamino acid conjugate of Formula I and a a gas precursor in liquid form and ii) a freeze-drying protecting component; and b) freeze-drying said initial suspension wherein said freeze-dried composition upon reconstitution with a pharmaceutically acceptable liquid carrier, provides a suspension of US-responsive liquid-filled nanodroplets, wherein the amount of said fluorinated compound is from 50% to 100% of the amount of fluorinated compound comprised in the initial suspension of step a).

Suitable pharmaceutically acceptable (aqueous) liquid carrier may be water, typically sterile, pyrogen free water (to prevent as much as possible contamination in the final reconstituted product), aqueous solutions such as saline (which may advantageously be balanced so that the final product for injection is not hypotonic), or aqueous solutions of one or more tonicity adjusting substances such as salts or sugars, sugar alcohols, glycols or other non-ionic polyol materials.

Use of ultrasound responsive vesicles

An aspect of the present invention relates to a suspension comprising an ultrasound responsive vesicle as defined above for use as medicament.

An embodiment relates to said aqueous suspension for use in an ultrasound- mediated treatment.

Another aspect relates to a suspension comprising an ultrasound responsive vesicle as defined above for use in a diagnostic and/or therapeutic treatment.

An aspect relates to a suspension comprising an ultrasound responsive vesicle as defined above for use in an ultrasound-mediated method of diagnosis in vivo.

A further aspect relates to a suspension comprising an ultrasound responsive vesicle as above defined for use as an ultrasound contrast agent.

A still further aspect of the invention relates to the use of a suspension comprising an ultrasound responsive vesicle as above defined as contrast agent for ultrasound imaging.

A further aspect relates to a suspension comprising an ultrasound responsive vesicle as defined above for use in an in vivo imaging method.

Another aspect relates to a diagnostic imaging composition comprising as imaging agent a suspension comprising a US-responsive vesicle as defined above.

The suspension of US-responsive vesicle according to the invention may be used in a variety of diagnostic and/or therapeutic techniques.

Diagnostic methods, and in particular imaging methods, include any method where the use of vesicles allows enhancing the visualisation of a portion or of a part of an animal (including humans) body, including imaging for preclinical and clinical research purposes. A variety of imaging techniques may be employed in ultrasound applications, for example including fundamental and harmonic B-mode imaging, pulse or phase inversion imaging and fundamental and harmonic Doppler imaging; if desired three- dimensional imaging techniques may be used.

For instance, US-responsive vesicles according to the invention may typically be administered in a diagnostically effective amount, depending e.g. on their respective composition, the tissue or organ to be imaged and/or the chosen imaging technique. Thel concentration may of course vary depending on specific imaging applications, e.g. when signals can be observed at very low doses such as in colour Doppler or power pulse inversion. In an embodiment said method of diagnosing comprises administering to a patient a suspension of US-responsive vesicles according to the invention; and detecting an ultrasound signal from a region of interest in said patient.

Another aspect of the invention relates to the use in a method of therapeutic treatment of a suspension of US-responsive vesicles according to the invention.

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

For instance, US-responsive vesicles according to the invention can typically be administered a therapeutically effective amount, depending e.g. from their respective composition, the type of subject under treatment, the tissue or organ to be treated and/or the therapeutic method applied.

In an embodiment said method of ultrasound therapeutic treatment comprises:

• administering to a patient a suspension of US-responsive vesicles according to the invention;

• identifying a region of interest in said patient to be submitted to a therapeutic treatment, said region of interest comprising said suspension of US-responsive vesicles; and

• applying an ultrasound beam for therapeutically treating said region of interest;

• whereby said ultrasound therapeutic treatment is enhanced by the presence of said suspension of US-responsive vesicles in said region of interest.

The following examples will help to further illustrate the invention.

EXAMPLES

The following materials are employed in the subsequent examples: Table 1 Materials

Example 1

Preparation of gas-filled microbubbles stabilized by lipid-polyglutamic acid conjugates

The procedure illustrated in the working examples of WO2004/069284 was used for preparing an experimental formulation for use in subsequent experiments.

An organic phase was prepared by dissolving 5 mg of DSPC/palmitic acid lipid blend in 0.8 mL cyclooctane at 75 °C. PEG4000 (1 g) was dissolved in 8 g of milliQ water. PGA(diol) derivatives (e.g.

DMPE-PGA(diol) and DPPE-PGA(diol)) were solubilized in 1 mL of milliQ water. After complete solubilization, the solution of derivative was added to the aqueous PEG solution. Due to the low solubility of DPPE-PGA(diol) derivatives, the aqueous phase was heated to 80°C for 15 min to allow complete solubilization and the emulsification was carried out with the hot aqueous phase. The organic phase was then emulsified in the aqueous phase at 8'000 rpm for 1'30" using Polytron PT3100.

The obtained emulsion was heated for 1 hour at 80 °C with mixing. After cooling to room temperature, the emulsion was diluted two-fold with a PEG4000 10% solution.

The diluted emulsion was sampled in DIN8R vials (1 mL emulsion/vial). The stoppers were positioned.

The vials were placed in the freeze dryer on the shelves precooled to -50°C and subjected to lyophilization, followed by a secondary drying above room temperature until complete removal of water and solvent.

At the end of the freeze-drying process, the vials were stoppered under vacuum, then crimped and gassed with C4F10/N2 (35/65 v/v) mixture. Finally, gassed vials are placed in an oven set at 38 °C for 16 hours.

The specific type and amounts of materials used in said suspensions are summarized in Table 2.

Table 2 Compositions used in the following examples

Example 2

Effect of the nature of PGA-diol derivatives on the oas-filled microbubble features Three vials of each freeze-dried batch were redispersed with 2 mL saline/vial to measure microbubbles size and concentration (Coulter counter), pressure resistance of microbubbles and Zeta potential of microbubbles (Nanosizer ZSP).

The size distribution and concentration of microbubbles were measured using a Coulter counter Multisizer3 fitted with a 30 pm aperture (dilution 50 pL of MB suspension in 100 mL of NaCI 0.9 % solution - analytical volume = 100 pL). Parameters such as the mean diameter in number and in volume (DN and DV, respectively in pm), the total concentration of microbubbles (Conc.T. in MB/mL), the concentration of MB>2pm (in MB/mL), the total microbubble surface (Surf.T. in pm²/mL) and the total microbubbles volume (MVC in pL/mL) were obtained.

Microbubble zeta potential was measured using the Nanosizer ZSP (Malvern instruments). The sample (20 or 50 pL of sample) was diluted in 1 mL saline solution at ImM. The result was the mean of three measurements. Results

Table 3 Characteristics of US responsive vesicle after reconstitution of the freeze-dried product

As inferable from Table 3, the use of a lipid-PGA(diol) conjugate comprising an amphiphilic lipid residue with a single alkyl chain (e.g. C14-PGA(diol)24) led to less stable US-responsive microbubbles compositions.

During the preparation of the US-responsive vesicles, the emulsification step was significantly affected by using a lipid-PGA(diol) conjugate comprising an amphiphilic lipid with a single alkyl chain, resulting in less stable emulsions. In addition to this, after freeze-drying and redispersion, the concentration of gas-filled microvesicles comprising a lipid-PGA(diol) conjugate comprising an amphiphilic lipid with a single alkyl chain, was substantially low (Table 3).

On the other hand, emulsions obtained by using a lipid-PGA(diol) conjugate comprising an amphiphilic lipid residue with two alkyl chains were rather stable independently on the molar ratio used and on the amphiphilic lipid conjugated to the PGA(diol), e.g. DPPE or DMPE.

Microbubbles characteristics (i.e. size distribution and concentration) were found to be in optimal ranges. Almost no large microbubbles were observed in these batches. Finally, the MB pressure resistance was found to be around 600 mmHg.

Furthermore, the influence of the molar ratio was studied using DPPE-PGA(diol)48 derivative. The microbubble size was similar whatever the molar ratio and a slight increase in MB concentration was observed with the increase of molar ratio.

Example 3

Quantitative determination of lipid-PGA(diol) conjugates and additional stabilizing materials after preparation of US-responsive vesicles.

In order to evaluate the incorporation of the lipid -PGA(diol) conjugates in the outer shell of the US-responsive vesicles, their contents were determined in both native and washed vials (n = 2 per batch). For native MBs, quantification of the different ingredients was carried out using HPLC-ELSD (High Performance Liquid Chromatography coupled with Evaporative Light Scattering Detection) analytical procedure following solubilization of the freeze-dried powder in THF/water 1/1 (2 mL) mixture.

For washed MBs, the vial was firstly redispersed in 2 ml saline. The microbubble suspension was transferred in 5mL-glass tube and the headspace was purged by C4F10/N2 (35/65 v/v) mixture. The microbubble suspension was then centrifuged (180g I 10 min). The infranatant was discarded and the microbubbles (supernatant) was redispersed in 1.5 mL of fresh milliQ water. The procedure was repeated except the volume for final redispersion was 0.5 mL (final volume 0.55 mL). For HPLC-ELSD assay, 500 pL of washed microbubbles suspension was diluted with 500 pL of THF. Results

Table 4 Amounts of of lipid-PGA(diol) conjugates and additional stabilizing materials after preparation

All results are summarized in Table 4. Initial contents were determined directly in vials (2 vials per batch) and compared to the theoretical amounts. As inferable from Table 4, the amounts of lipid-PGA(diol) conjugates and additional stabilizing materials present in the vials after preparation of US-responsive vesicles were substantially similar to the theoretical one for all batches.

A substantially better incorporation of the lipid-PGA(diol) conjugates in the outer shell of a US-responsive vesicle is achieved by using lipid-PGA(diol) derivatives wherein the lipid portion comprises C-chains longer than 14 carbon atoms.

Example 4

Effect of multi-iniections on microbubbles circulation time

The effect of multi-injections on microbubbles circulation time was studied by echography in rat left ventricle opacification model as described in Fix, 2018.

For this study, a microbubbles composition stabilized by DSPC/palmitic acid/DPPE- PGA(Diol)129 (74.1/18.5/7.4) was administered to Sprague-Dawley rats (female, Janvier Labs) and an Acuson Sequoia 512 clinical imaging system equipped with a 7 MHz linear array transducer was used (15L8, Siemens, Mountain View, CA, USA).

The microbubbles compositions were administered through multiple injections over time using a semi-automatic injection system (catheter in rats left caudal vein).

A trans-thoracic short-axis view of the heart was obtained and a qualitative evaluation was performed on diastole images taken at 50", 1'30", 2'30", 3'30", 5', 7'30", 10' and 15' after each injection (first one at day 1, second at day 8, third at day 15 and fourth at day 22).

Results

After the first injection microbubbles stabilized by a lipid-polyglutamic derivative showed an extended blood circulation. Furthermore, while a significant acceleration of blood clearance was observed after the second administration, almost all blood circulation time was nevertheless recovered after the 4th injection (e.g. ultrasound responsive vesicles visible (after 10-15 min)

As used herein the expression circulation time refers to the duration for which ultrasound responsive vesicles remain within the bloodstream after being injected into the body. Example 5

Comparative example

The ultrasound responsive vesicles of the present invention were compared with microbubbles stabilized by a PEGylated lipid shell (composition similar to the marketed Definity).

For this purpose, a control batch (pegylated microbubbles) and a composition comprising the polyamino acid derivatives of the present invention were prepared according to the procedure disclosed in Example 4 of the patent US5585112A, replacing the gas (i.e. C4F10) with C3F8.

To prepare the control batch, 1.6 mg of DPPC (2.18 pmoles), 0.18 mg of DPPA (0.27 pmoles) and 1.2 mg of DPPE-PEG5000 (0.21 pmoles) were placed in a 25mL- balloon and dissolved in Chloroform/methanol (2/1 v/v) mixture. The solvents were then evaporated to obtain a lipid blend. This blend was dried under vacuum (0.2 mBar) at 25°C overnight.

The lipid blend was redispersed in 4 mL of aqueous phase (saline 0.9 % with 63 mg/mL of glycerol and 50 mg/mL of propylene glycol) at 65°C with mixing for 30 min. After cooling to room temperature, the solution was sampled in vials DIN2R (1.5 mL solution I vial - 2 vials). After sealing, the headspace of the vials was replaced by C3F8.

The microbubbles suspension was obtained by mixing using Vialmix™ for 45 seconds.

A similar procedure was performed to obtain a suspension of microbubbles stabilized by lipid-polyamino acid derivatives according to this invention. For these purposes, the above-described method was followed except that 2.24 mg (0.21 pmoles) of DPPE-PGA(diol)48 were used instead of DPPE-PEG5000.

After cooling to room temperature, both formulations were characterized using Coulter counter Multisizer3 (MB size and concentration).

Results

Table 5 Characteristics of microbubbles comprising lipid-polyamino acid conjugates compared with microbubbles comprising a PEGylated lipid shell

As inferable from the results, both the control batch comprising microbubbles stabilized by PEGylated-lipid and the disclosed composition comprising microbubbles stabilized by lipid-polyamino acid derivatives were characterized by similar sizes after the preparation using a known preparation method.

References

1. Fix et al, Ultrasound in Medicine & Biology, 44, 2018, 1266-1280

2. Thi et al, Polymers, 2020, 12, 298

3. EP22382135.6

4. W02002098951

5. US5,711,933

6. US 6,333,021

7. US 4,276,885

8. EP 0324938

9. WO9409829

10. W02004069284

11. WO2020229642

12. Melich et al, International Journal of Pharmaceutics, 587, 2020, 119651

13. WO2022101365

14. WO2023118387A1

15. W02024033540

16. WO2020229642

17. US5585112A

Claims

1) An ultrasound responsive vesicle comprising an inner core and an outer layer, wherein said inner core comprises a physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof, and said outer layer comprises a lipid-polyamino acid conjugate of formula I:

Formula I wherein :

L is an amphiphilic lipid residue comprising two lipophilic hydrocarbon chains;

Ri is -H or -COCH3;

R₂ is (CH₂)X-COOH or (CH₂)_X-CO-NHR₃

R3 is -H or (Ci-C4)alkyl substituted with one or more hydroxy groups;

X is 0-4 and n is comprised between 2 and 500.

2) The ultrasound responsive vesicle according to claim 1, wherein said lipid-polyamino acid conjugate is a lipid-polyglutamic acid conjugate of formula II

Formula II

Wherein

Ri is -H or -COCH3 and n is comprised between 2 and 500.

3) The ultrasound responsive vesicle according to claim 2, wherein n is lower than 150.

4) The ultrasound responsive vesicle according to any of the preceding claims, wherein said amphiphilic lipid residue is a phospholipid.

5) The ultrasound responsive vesicle according to claim 4, wherein said phospholipid is selected from l,2-Dimyristoyl-sn-glycero-3-phosphorylethanolamine (DMPE), 1,2- Dipalmitoyl-sn-glycero-3-phosphorylethanolamine (DPPE) or 1,2-Distearoyl-sn- glycero-3-phosphorylethanolamine (DSPE).

6) The ultrasound responsive vesicle according to any of the preceding claims, wherein said lipid-polyglutamic acid conjugate is selected from a compound of formula III (DPPE -PGA(diol) conjugate)

or a compound of formula IV (DMPE -PGA(diol) conjugate)

Formula IV wherein n is comprised between 2 and 500 monomer subunits. 7) The ultrasound responsive vesicle according to claim 6, wherein n is lower than 150.

8) The ultrasound responsive vesicle according to any of the preceding claims, wherein said physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof is a fluorinated compound.

9) The ultrasound responsive vesicle according to claim 8, wherein said fluorinated compound is a perfluorinated compound.

10) The ultrasound responsive vesicle according to claim 9, wherein said perfluorinated compound is selected from C4F10, C3F8, SFe or a mixture thereof.

11) The ultrasound responsive vesicle according to any of the preceding claims, wherein said outer layer further comprises an additional stabilizing material.

12) The ultrasound responsive vesicle according to claim 11, wherein said additional stabilizing material comprises a phospholipid.

13) The ultrasound responsive vesicle according to claim 11 and 12, wherein said additional stabilizing material comprises a fatty acid.

14) An aqueous suspension comprising a plurality of ultrasound responsive vesicles as defined in claims 1-13 and a pharmaceutically acceptable liquid carrier.

15) An aqueous suspension as defined in claim 14 for use as medicament.

16) An aqueous suspension as defined in claim 14 for use in an ultrasound mediated treatment.

17) A vial comprising:

- a precursor of the aqueous suspension as defined in claim 14 in the form of a freeze-dried product and a freeze-drying protecting component; and

- physiologically acceptable compound selected from the group consisting of a gas, a gas precursor in liquid form or a mixture thereof.