WO1999058158A1

WO1999058158A1 - Contrast media

Info

Publication number: WO1999058158A1
Application number: PCT/GB1999/001488
Authority: WO
Inventors: Ingrid Henriksen; Liv-Ingrid ØDEGÅRSTUEN; Tove Jacobsen
Original assignee: Nycomed Imaging As; Cockbain, Julian
Priority date: 1998-05-11
Filing date: 1999-05-11
Publication date: 1999-11-18
Also published as: GB9810051D0; AU3839199A; EP1077730A1

Abstract

The invention relates to a process for the preparation of a contrast medium which process comprises: i) obtaining a composition comprising a lipid membrane in a high axial ratio conformation; ii) contacting said composition with an agent serving to release the lipid membrane from the high axial ratio conformation; and iii) agitating said composition in the presence of a biotolerable contrast generator or precursor whereby to form contrast generator or precursor containing lipid-membraned vesicles and to contrast media obtained thereby and to methods of imaging utilising said media.

Description

Contrast media

This invention relates to contrast media, in particular to echogenic contrast media comprising gas containing vesicles for use in ultrasonography.

In diagnostic imaging procedures, contrast media are frequently used to enhance the contrast in the detected images, e.g. to facilitate visualization of particular organs or tissues. In ultrasound imaging, the materials used as contrast agents generally contain or generate gas microbubbles or microballoons .

Such gas microballoons frequently comprise a lipid membrane which encapsulates a biocompatible gas or a gas precursor which generates an echogenic gas before, during or after administration to the patient. In the ultrasound contrast media currently under widespread investigation, the gas or gas precursor is generally a perfluorocarbon such as a perfluoropentane or perfluorobutane.

Such lipid encapsulated microbubbles may be generated by normal emulsification of an aqueous lipid composition and the perfluorocarbon, whereafter the vesicle containing composition may be freeze dried to provide a solid material which can be reconstituted with water immediately prior to use. This procedure is complicated and energy demanding and a noticeable degree of vesicle loss occurs during the freeze drying step. We have now found that lipid-membraned gas- containing vesicles may advantageously be prepared from preformed high axial ratio structures, as a result enabling the preparation of sterilized storage-stable products from which ready-to-use ultrasound contrast media may be prepared in a simple and straightforward manner.

Thus viewed from one aspect the invention provides a process for the preparation of a contrast medium, preferably an ultrasound contrast medium, which process comprises : i) obtaining a composition comprising a lipid membrane in a high axial ratio conformation; ii) contacting said composition with an agent serving to release the lipid membrane from the high axial ratio conformation; and iii) agitating said composition in the presence of a biotolerable contrast generator or precursor therefor (e.g. an iodinated X-ray contrast agent, a paramagnetic chelate, a radioactive material, etc. but preferably a gas or gas precursor) whereby to form contrast agent generator or precursor containing lipid-membraned vesicles . Viewed from a further aspect the invention further provides a kit comprising: a lipid membrane in a high axial ratio conformation; a biotolerable contrast generator or precursor therefor (e.g. an iodinated X-ray contrast agent, a paramagnetic chelate, a radioactive material, etc. but preferably a gas or gas precursor, preferably a fluorinated material) ; and an agent capable of releasing said membrane from the high axial ratio conformation.

In the kit of the invention, the lipid membrane and the agent capable of releasing the membrane from its high axial ratio conformation may be in the same or different compositions; in the former case however the release agent should be in a form which requires chemical or physical activation before it can exert its releasing effect, e.g. it may be encapsulated by a membrane which may be ruptured to release or activate it. However it is more preferred that the release agent is present in a separate composition whereby contacting the lipid membrane containing component and the release agent containing component of the kit serves to release the lipid membrane from its high axial ratio conformation. The gas or gas precursor component may be present in or in the headspace above either or both of the lipid membrane and release agent components. Viewed from a yet further aspect the invention provides the use of a lipid membrane in a high axial ratio confirmation for the preparation of a contrast medium for use in a method of diagnosis which involves administration of said contrast medium and generation of an image, e.g. using a diagnostic imaging modality such as nuclear imaging, light imaging, X-ray imaging, MR imaging or more preferably ultrasound imaging.

Suitable high axial ratio conformations include rods, delices, fibres, ribbons, cylinders, tubules or cochleates, cochleates being preferred. The high axial ratio conformation may be maintained by ionic or other inter/intra molecular bonds, or by molecular spatial conformation (e.g. chirality, rigidity, packaging). In these lipid membrane structures the hydrophilic membrane surfaces are preferably ionically bonded together, for example using polyvalent ions, in particular divalent cations. As an example, a cochleate structure is shown schematically in Figure 1 hereto. The production of these structures, in particular cochleates, is described for example in WO97/30725, WO97/18840, US-A-4871488 , US- A-4078052, W096/25942, Papahadjopoulos et al Biochem Biophys Acta 394 : 483-491 (1975) , Verkleij et al Biochem Biophys Acta 339: 432-437 (1974) , Archibald and Mann Chem. Phys. Lipids 69: 51-54 (1994), Archibald and Mann Biochem Biophys Acta 1166: 154-162, (1993), Tocanne et al Chem. Phys. Lipids 12: 201-219 (1974) and Ververgaert et al Chem Phys Lipids 14: 97-101 (1975) , Nakashima et al. Chem. Letters. 1709-1712 (1984). This last reference relates to chiral ammonium amphiphiles, which are synthetic amphiphiles containing amino acids as the chiral core. These may form helices (with a pure enantiomer) or rods (with a racemic mixture) . Formation of the structures may be reversed by increasing the temperature above Tm (phase transition temperature) or by altering the pH.

Generally these high axial ratio structures may be produced by the action of polyvalent counterions, e.g. trivalent or divalent cations such as Mg²⁺ or more preferably Ca²⁺, on small lipid vesicles. Negatively charged polyvalent counterions, e.g. carbonates, phosphates, oxalates, citrates or sulphates may be used with lipids which are positively charged. The high axial ratio structures generally appear as water- insoluble particulate structures.

The release agents which may be used to release the lipid membrane from the high axial ratio conformation may be any material which competes with the divalent cations for binding to the membrane surface or, more preferably, any material which competes with the membrane surface for binding to the divalent cations. Typically, the release agent will be a physiologically tolerable chelating agent, in particular a calcium or magnesium chelating agent, e.g. a aminopolycarboxylic acid such as EDTA, DTPA, DTPA-BMA, DOTA, D03A, TMT, PLED, DPDP or EGTA, most preferably EDTA. Examples of suitable chelating agents may be found in the literature, especially the published patent applications of Nycomed, Salutar, Sterling Winthrop, Schering, Bracco, Squibb, Guerbet and Mallinckrodt , relating to diagnostic contrast agents, in particular for MR and nuclear imaging. Alternatively, the release agent for use with negatively charged lipids may be a precipitating agent capable of precipitating ions (e.g. Mg²⁺ or Ca²⁺) used to form the high axial ratio structure. Suitable precipitating agents include phosphate, citrate, oxalate, sulphate or carbonate ions. The release agent for use with positively charged lipids may be a precipitating agent capable of precipitating ions (e.g. phosphate, citrate, oxalate, sulphate or carbonate) used to form the high axial ratio structure. Suitable precipitating agents include Mg²⁺ or Ca²⁺ .

Also included within the term "release agents" are factors which cause a change in an environmental parameter, e.g. pH, temperature or ionic strength, which serves to alter the high axial ratio conformation and thereby release the lipid membrane for vesicle formation.

The lipid from which the lipid membrane is formed may comprise a single lipid or a mixture of lipids. Preferably at least one negatively charged lipid is used. Examples of suitable lipids include phospholipids such as lecithins (i.e. phosphatidylcholines) or derivatives thereof e.g. PEG derivatives, for example natural lecithins such as egg yolk lecithin or soya bean lecithin and synthetic or semisynthetic lecithins such as dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine , distearoylphosphatidylcholine or diacetylenic phosphatidylcholines; phosphatidic acids; phosphatidylethanolamines ; phosphatidylserines ; phosphatidylglycerols ; phosphatidylinositols ; cardiolipins; sphingolipids such as sphingomyelin; mixtures of any of the foregoing and mixtures with other lipids such as cholesterol. Further suitable lipids include glycolipids such as ganglioside GM1 and GM2 ; glucolipids; sulfatides; glycosphingolipids; galactosphingolipids; lipids bearing polymers such as polyethyleneglycol , chitin, hyaluronic acid or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate and cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether and ester-linked fatty acids; polymerized lipids; diacetyl phosphate; cardiolipin; phospholipids with short chain fatty acids of 6-8 carbons in length; synthetic phospholipids with asymmetric acyl chains (e.g. with one acyl chain of 6 carbons and another acyl chain of 12 carbons) ; lipids with an oligopeptide or polypeptide as a hydrophilic headgroup; 6- (5-cholesten-3.beta. -yloxy) -1-thio- .beta. - D-galactopyranoside, digalactosyldi-glyceride, 6- (5- cholesten-3.beta . -yloxy) hexyl-6-amino-6-deoxy-l-thio- .beta. -D-galactopyranoside. 6- (5-cholesten-3.beta. - yloxy) hexyl-6-amino-6-deoxyl-l-thio- .beta. -D- mannopyranoside; 12- ( ( (7 ' -diethylaminocoumarin-3- yl) carbony)methylamino) -octadecanoic acid; N-[12-(((7'- diethylaminocoumarin-3-yl) carbonyl) methyl - amino) octadecanoyl] -2-aminopalmitic acid; cholesteryl (4 ' -trimethylammonio) butanoate; N-succinyl- dioleoylphosphatidylethanolamine; mono-, di- and triglycerides such as 1 , 2-dioleoyl-sn-glycerol ; 1,2- dipalmitoyl -sn-3-succinylglycerol ; 1 , 3-dipalmitoyl-2- succinylglycerol ; l-hexadecyl-2-palmitoylglycerophospho- ethanolamine; and palmitoylhomocysteine; glutamate or polyglutamate-based amphiphiles and/or combinations thereof. Mention may also be made of cationic lipids such as DOTMA, N- [1- (2 , 3-dioleoyloxy) propyl] -N,N,N- trimethylammonium chloride: DOTAP, 1 , 2-dioleoyloxy-3- (trimethylammonio) propane; and DOTB, 1 , 2-dioleoyl-3- (4 " - trimethyl-ammonio) butanoyl-sn-glycerol ; DSTAP, 1,2- distearoyl-3-trimethylammonium-propane and of carbohydrate-bearing lipids.

Preferably the lipid comprises an amphiphilic phospholipid material in particular a material which consists essentially of phospholipid predominantly comprising molecules with net charges. Desirably at least 75%, preferably substantially all of the phospholipid material consists of molecules bearing a net overall charge under conditions of preparation and/or use, which charge may be positive or, more preferably, negative. Representative positively charged phospholipids include esters of phosphatidic acids such as dipalmitoylphosphatidic acid or distearoyl-phosphatidic acid with aminoalcohols such as hydroxyethylenediamine. Examples of negatively charged phospholipids include naturally occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and cardiolipins . The fatty acyl groups of such phospholipids will typically each contain about 14-22 carbon atoms, for example as in palmitoyl and stearoyl groups. Lyso forms of such charged phospholipids are also useful in accordance with the invention, the term "lyso" denoting phospholipids containing only one fatty acyl group, this preferably being ester-linked to the 1- position carbon atom of the glyceryl moiety. Such lyso forms of charged phospholipids may advantageously be used in admixture with charged phospholipids containing two fatty acyl groups .

Thus, preferably the lipid will comprise phospholipids, sphingolipids or derivatives thereof, particularly phosphatidylcholines and/or phosphatidyl serines; DOTMA or DSTAP.

Phosphatidylserines represent particularly preferred phospholipids for use in contrast agents according to the invention and preferably constitute a substantial part, e.g. at least 80% of the initial phospholipid content thereof, for example 85-92%. Preferred phosphatidylserines include saturated (e.g. hydrogenated or synthetic) natural phosphatidylserine and synthetic or semi-synthetic dialkanoylphosphatidylserines such as distearoylphosphatidylserine , dipalmitoylphosphatidyl - serine and diarachidoylphosphatidylserine .

Especially preferred for use in contrast agents according to the invention are negatively charged lipids alone or in combination with phosphatidylcholines, e.g. phosphatidyl serines or phosphatidic acid or phosphatidyl glycerol in ad mixture with phosphatidylcholine; if phosphatidyl serine is present in the mixture, it preferably comprises at least 75% of the total lipid content.

Any biocompatible gas may be employed in the contrast agents of the invention, it being appreciated that the term "gas" as used herein includes any substances (including mixtures) substantially or completely in gaseous (including vapour) form at the normal human body temperature of 37°C. The gas may thus, for example, comprise air; nitrogen,- oxygen; carbon dioxide; hydrogen; nitrous oxide; an inert gas such as helium, argon, xenon or krypton; a sulphur fluoride such as sulphur hexafluoride, disulphur decafluoride or trifluoromethylsulphur pentafluoride; selenium hexafluoride; an optionally halogenated silane such as methylsilane, dimethylsilane or tetramethylsilane; a low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms), for example an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentane, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne; an ether such as dimethyl ether; a ketone; an ester; a halogenated low molecular weight hydrocarbon (e.g. containing up to 7 carbon atoms) ; or a mixture of any of the foregoing. At least some of the halogen atoms in halogenated gases advantageously are fluorine atoms. Thus biocompatible halogenated hydrocarbon gases may, for example, be selected from bromochlorodifluoromethane, chlorodifluoromethane , dichlorodifluoromethane , bromotrifluoromethane , chlorotrifluoromethane , chloropentafluoroethane , dichlorotetrafluoroethane , chlorotrifluoroethylene , fluoroethylene, ethylfluoride, 1 , 1-difluoroethane and perfluorocarbons . Representative perfluorocarbons include perfluoroalkanes such as perfluoromethane, perfluoroethane, perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture with other isomers such as perfluoroisobutane) , perfluoropentanes, perfluorohexanes and perfluoroheptanes; perfluoroalkenes such as perfluoropropene, perfluorobutenes (e.g. perfluorobut-2- ene) and perfluorobutadiene; perfluoroalkynes such as perfluorobut-2-yne; and perfluorocycloalkanes such as perfluorocyclobutane , perfluoromethylcyclobutane , perfluorodimethylcyclobutanes , perfluorotrimethylcyclobutanes, perfluorocyclopentane, perfluoromethylcyclopentane , perfluorodimethylcyclopentanes , perfluorocyclohexane , perfluoromethylcyclohexane and perfluorocycloheptane . Other halogenated gases include methyl chloride, fluorinated, e.g. perfluorinated, ketones such as perfluoroacetone and fluorinated, e.g. perfluorinated, ethers such as perfluorodiethyl ether.

It may be advantageous in contrast agents of the invention to employ fluorinated gases such as sulphur fluorides or fluorocarbons (e.g. perfluorocarbons) which are known to form particularly stable microbubble suspensions. Gas mixtures based on considerations of partial pressures both inside and outside the microbubbles and consequent osmotic effects on microbubble size, e.g. as described in WO-A-95/03835, may if desired be employed, for example a mixture of a relatively blood-soluble gas such as nitrogen or air and a relatively blood-insoluble gas such as a perfluorocarbon. The processes of the present invention may be used in the formation of preparations as described in WO-A- 98/17324, the contents of which are incorporated herein by reference, which comprise a diffusable component capable of inward diffusion into the dispersed gas phase to promote temporary growth thereof, said preparations being especially suitable for cardiac imaging. Suitable contrast e.g. gas precursors and diffusable components include gases/vapours, volatile liquids and solids and any biocompatible component capable of gas generation in vivo, i.e. at body temperature and physiological pH. Suitable diffusable components include all the "gases" previously listed herein as suitable for employment in the contrast agents of the invention. Representative examples of gas precursors include inorganic and organic carbonates and bicarbonates, and nitrogen-generating substances such as pyrazolines, pyrazoles, triazolines, diazoketones, diazonium salts, tetrazoles and azides.

The diffusable component or gas precursor/generator may be present in the form of an emulsion and appropriate emulsifiable compounds may, for example, be selected from the various lists of emulsifiable low boiling liquids given in WO-A-94/16379 , the contents of which are incorporated herein by reference.

Specific examples of emulsifiable components include aliphatic ethers such as diethyl ether; polycyclic oils or alcohols such as menthol, camphor or eucalyptol ; heterocyclic compounds such as furan or dioxane; aliphatic hydrocarbons, which may be saturated or unsaturated and straight chained or branched, e.g. as in n-butane, n-pentane, 2-methylpropane, 2 -methylbutane, 2 , 2-dimethylpropane, 2 , 2-dimethylbutane, 2,3- dimethylbutane, 1-butene, 2-butene, 2-methylpropene, 1, 2 -butadiene, 1, 3 -butadiene, 2 -methyl-1-butene, 2- methyl -2-butene, isoprene, 1-pentene, 1 , 3-pentadiene, 1, 4-pentadiene, butenyne, 1-butyne, 2-butyne or 1,3- butadiyne; cycloaliphatic hydrocarbons such as cyclobutane, cyclobutene, methylcyclopropane or cyclopentane; and halogenated low molecular weight hydrocarbons (e.g. containing up to 7 carbon atoms) . Representative halogenated hydrocarbons include dichloromethane, methyl bromide, 1 , 2-dichloroethylene, 1, 1-dichloroethane, 1-bromoethylene, 1-ch oroethylene, ethyl bromide, ethyl chloride, 1-chloropropene, 3- chloropropene, 1-chloropropane, 2-chloropropane and t- butyl chloride. Advantageously at least some of the halogen atoms are fluorine atoms, for example as in dichlorofluoromethane, trichlorofluoromethane, 1,2- dichloro-1, 2 -difluoroethane, 1 , 2-dichloro-l , 1, 2 , 2- tetrafluoroethane, 1,1, 2-trichloro-l, 2,2- trifluoroethane, 2-bromo-2-chloro-l, 1, 1-trifluoroethane, 2-chloro-l, 1, 2-trifluoroethyl difluoromethyl ether, 1- chloro-2,2 ,2-trifluoroethyl difluoromethyl ether, partially fluorinated alkanes (e.g. pentafluoropropanes such as 1H, 1H, 3H-pentafluoropropane, hexafluorobutanes, nonafluorobutanes such as 2H-nonafluoro- t-butane, and decafluoropentanes such as 2H, 3H-decafluoropentane) , partially fluorinated alkenes (e;g. heptafluoropentenes such as 1H, 1H, 2H-heptafluoropent-1-ene, and nonafluorohexenes such as 1H, 1H, 2H-nonafluorohex-1-ene) , fluorinated ethers (e.g. 2 , 2 , 3 , 3 , 3 -pentafluoropropyl methyl ether or 2,2,3,3,3 -pentafluoropropyl difluoromethyl ether) and, more preferably, perfluorocarbons . Examples of perfluorocarbons include perfluoroalkanes such as perfluorobutanes, perfluoropentanes, perfluorohexanes (e.g. perfluoro-2- methylpentane) , perfluoroheptanes, perfluorooctanes, perfluorononanes and perfluorodecanes; perfluorocycloalkanes such as perfluorocyclobutane, perfluorodimethyl-cyclobutanes, perfluorocyclopentane and perfluoromethylcyclopentane; perfluoroalkenes such as perfluorobutenes (e.g. perfluorobut-2-ene or perfluorobuta-1, 3-diene) , perfluoropentenes (e.g. perfluoropent-1-ene) and perfluorohexenes (e.g. perfluoro-2-methylpent-2-ene or perfluoro-4-methylpent- 2-ene) ; perfluorocycloalkenes such as perfluorocyclopentene or perfluorocyclopentadiene; and perfluorinated alcohols such as perfluoro- -butanol . Such emulsions may also contain at least one surfactant in order to stabilise the dispersion. In general a wide range of surfactants may be useful, for example selected from the extensive lists given in EP-A- 0727225, the contents of which are incorporated herein by reference. Representative examples of useful surfactants include fatty acids (e.g. straight chain saturated or unsaturated fatty acids, for example containing 10-20 carbon atoms) and carbohydrate and triglyceride esters thereof, phospholipids (e.g. lecithin) , fluorine-containing phospholipids, proteins (e.g. albumins such as human serum albumin), polyethylene glycols, and block copolymer surfactants

(e.g. polyoxyethylene-polyoxypropylene block copolymers such as Pluronics, extended polymers such as acyloxyacyl polyethylene glycols, for example polyethyleneglycol methyl ether 16-hexadecanoyloxy-hexadecanoate, e.g. wherein the polyethylene glycol moiety has a molecular weight of 2300, 5000 or 10000) , and fluorine-containing surfactants (e.g. as marketed under the trade names Zonyl and Fluorad, or as described in WO-A- 9639197 , the contents of which are incorporated herein by reference) . Particularly useful surfactants include phospholipids comprising molecules with net overall negative charge, such as naturally occurring (e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially or fully hydrogenated) and synthetic phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, phosphatidic acids and/or cardiolipins .

Contrast agents comprising microbubbles of a perfluoroalkane such as perfluorobutane stabilised by phosphatidylserine, are surprisingly stable in size following intravenous administration to a subject, and do not exhibit the previously noticed tendency of microbubbles of such gases to grow uncontrollably as a result of inward diffusion of blood gases such as oxygen, nitrogen and carbon dioxide, instead rapidly reaching a maximum size beyond which further growth is not observed.

The high axial ratio structures may thus for example be formed by adding a physiologically tolerable divalent cation, e.g. using a magnesium or more preferably a calcium salt such as a halide, carbonate, nitrate, or phosphate to an aqueous vesicular composition comprising the lipid or lipid mixture. Such a vesicular composition may readily be prepared by passing an aqueous lipid mixture through small diameter pores, e.g. in a filter membrane or a series of membranes, for example with pores having a diameter in the range 0.02 to 5μm, especially 0.05 to 2μm, e.g. about 0.1 μm. Vesicles may also be produced by the thin film vaporization technique, sonication, homogenization, microfluidation, French press, the transient pH jump technique, cosolubilizer removal technique, detergent depletion technique, reversed phase vaporization, different injection methods such as ethanol injection or ether injection or other methods available for those skilled in the art. Prior to formation of the high axial ratio structures, the lipid or lipid mixture is preferably sterilised e.g. by autoclaving (e.g. at 121°C for 15 minutes) .

The divalent cation is preferably added at a molar (cation to lipid) ratio of 1:0.01 to 1:20, more preferably 1:0.1 to 1:10, especially 1:0.25 to 1:5, relative to the lipid (or more preferably relative to the anionic lipid) . Addition is preferably above the lipid phase transition temperature, e.g. 30 to 95°C, especially 50 to 80°C. The precipitate may be recovered and dried if desired; alternatively it may be used as formed or simply concentrated by decanting some of the supernatant fluid.

Release of the lipid from the high axial ratio conformation may be performed before or simultaneously with agitation with the gas or gas precursor to form the gas or gas-precursor containing vesicles. Release from the high axial ratio conformation may be achieved for example by adding an agent such as EDTA which competes to bind the divalent cations, preferably an excess of such an agent, e.g. a mole ratio of 1.5 to 15, preferably 2 to 10, more preferably 3 to 8 of the release agent relative to the divalent cations. The release agent is preferably allowed to react with the high axial ratio structure for a period of 15 minutes to 4 hours, more preferably 30 minutes to 2 hours before the released membrane is agitated to form the contrast generator or precursor containing vesicles. Addition of release agent may conveniently be at ambient or elevated temperatures, e.g. temperatures in the range 10 to 80°C, preferably 15 to 30°C.

The divalent cations and the release agent are preferably added to the lipid in aqueous solution,- however if desired they may be added in dry form.

Where the release agent is an environmental factor, such as pH, temeprature or ionic strength, means (such as acid, alkali, heat etc.) to alter one or more of these parameters may be added or applied to the lipid in high axial ratio conformation. Preferably the local environment of the high axial ratio structure is altered for a period of 15 minutes to 4 hours, more preferably 30 minutes to 2 hours before the released membrane is agitated to form the contrast generator or precursor containing vesicles. A change in local temperature may serve to release lipid as the ambient temperature is adjusted to above or below, usually above Tm (the phase transition temperature of the lipid membrane) . Changes in pH and ionic strength can release lipid as a result of a change in the charge of the lipids in the high axial ratio structure. Thus, addition of an acid to the lipid may alter its structure causing lipid release as may addition of a salt.

The contrast generator or precursor containing vesicles may be prepared by any of the methods known in the art for making vesicles; suitable methods include sonication, shaking, agitation, mixing e.g. by rotor stator etc. Typically, the released lipid is agitated in the presence of the gas or gas precursor, e.g. using a sonicator or cap-mixer or by simple manual shaking. Vesicle sizes will generally be in the range 1 to 50μm, more preferably 1 to lOμm, and if desired a size separation may be effected before administration of the vesicular composition, or the composition may be extruded through small pores, e.g. in a filter membrane, e.g. with pore sizes of 0.5 to lOμm, especially 0.5 to 5 μm. The contrast generator or precursor containing vesicles may be in the form of liposomes, microspheres, emulsion droplets etc.

The composition is preferably sterilized, e.g. by autoclaving (for example at 121°C for 15 minutes) , before gas filled vesicle formation. This may for example involve heat sterilization of the separate lipid membrane and release agent components and of the contrast generator or precursor component if this is present in a separate container. Particularly preferably, the kit of the invention comprises a multicompartment , e.g. two compartment, container, containing lipid membrane in a high axial ratio confirmation (e.g. in an aqueous medium) in one compartment and release agent (e.g. in aqueous solution) in a separate compartment and with the gas or gas precursor in one or both of these compartments or in a separate compartment . Such a container may be heat sterilized after filling and sealing. Such filled multicompartment containers form a further aspect of the present invention. Alternatively the lipid membrane and gas or gas precursor may be in a sealed container provided with a septum through which the release agent may be introduced.

Besides the lipid, release agent and gas or gas precursor, the compositions may contain other physiologically tolerable components, carriers or excipients, e.g. buffers, surfactants, osmolality and/or viscosity modifiers, and pH adjusting agents.

The contast medium, following vesicle generation, may be administered by conventional means, e.g. by infusion or injection into the vasculature, and at conventional dosages, e.g. containing 0.001 - 2% w/w, preferably 0.005 to 0.8 % w/w, most preferably about 0.01 to 0.1% w/w lipid.

Imaging may then be performed using conventional ultrasound techniques, although second harmonic imaging procedures may generally be preferred.

Viewed from a further aspect the present invention thus also provides a method of contrast enhanced imaging, preferably ultrasound imaging, of a human or non human animal (e.g. mammalian) subject, said method comprising:

(i) generating contrast generator or presursor (preferably gas or gas precursor) containing vesicles using a lipid membrane in a high axial ratio confirmation; (ii) administering said vesicles into said subject, preferably into the vasculature thereof; and

(iii) generating an image (preferably an ultrasound image) of at least a part of said subject containing said vesicles. The contrast media of the invention may be used in other diagnostic imaging modalities besides ultrasonography, e.g. mri, X-ray imaging, nuclear imaging, and light imaging, and such uses form further aspects of the invention. The contrast media of the invention may be used in physiological mapping, where the emphasis is on obtaining information relating to a selected physiological parameter within the animal body, e.g. temperature or pH, rather than a structural visualisation of part of the body. Such methods are described in PCT/GB99/01100 , the contents of which are incorporated herein by reference .

The invention is also applicable to the generation of ultrasound or other gas or gas-precursor containing vesicular contrast agents from other pre-formed lipid membrane conformations than high axial ratio conformations. Thus viewed from a further aspect the invention provides a process for the preparation of a contrast medium which process comprises obtaining an aqueous composition comprising an organised lipid membrane structure, optionally contacting said composition with an agent serving to destabilise said structure, and agitating said composition in the presence of a biotolerable contrast generator or precursor (preferably a gas or gas-precursor) whereby to form contrast generator or precursor containing membraned vesic1es . In this aspect of the invention, the pre-formed organised lipid membrane structure may comprise lipid monolayers or multilayers which may or may not provide a membrane envelope, ie. a continuous external surface for a vesicle. Thus the membrane structures may for example be sheets, folded or coiled sheets (e.g. cochleates), rods, helices, fibres, tubules, ribbons, cylinders or vesicles. The benefit of the process is that the contrast generator or precursor containing vesicles may be more readily formed from such pre-formed structures than from simple lipid, water and gas or gas-precursor mixtures .

In this aspect of the invention, the agitation of the composition serves not only to introduce the gas or gas-precursor into the interior of the vesicular product but also to rearrange the organised lipid membrane structures so as to produce vesicles of appropriate dimensions. In general this will involve a reduction in maximum dimension from the organised structure to the vesicular product. In this way the energy requirement for vesicle formation and the lipid wastage may be reduced. The agitation to induce vesicle formation is particularly preferably effected at a temperature below the lipid phase transition temperature, e.g. up to 20C° or more preferably up to 10C° below the lipid transition temperature .

Thus viewed from a further aspect the invention provides a process for the preparation of a contrast medium which comprises obtaining (optionally with heating) a dispersion of a lipid membrane forming material and water at a temperature below the phase transition temperature of the lipid whereby to form ordered lipid membrane structures, e.g. vesicles, and, optionally after sterilization (e.g. by autoclaving) and filtration, homogenizing the resultant composition (e.g. by sonication) whereby to yield contrast generator or precursor (e.g. gas or gas-precursor) containing vesicles.

Using this process the number of microbubbles of contrast enhancing size (e.g. 3-5 μm) is increased significantly above that achieved where agitation to form vesicles is effected above the lipid phase transition temperature.

Viewed from a still further aspect the invention provides a process for the preparation of microbubbles which process comprises: i) obtaining a composition comprising a lipid membrane in a high axial ratio conformation; ii) contacting said composition with an agent serving to release the lipid membrane from the high axial ratio conformation; and iii) agitating said composition whereby to form said microbubbles.

The present invention will now be described further with reference to the following non-limiting Examples and the attached drawings, in which:

Figure 1 is a schematic diagram of a cochleate; Figure 2 is a photomicrograph of gas filled vesicles produced according to the invention;

Figure 3 is a schematic representation of the procedure used to prepare the vesicles of Figure 2; and Figure 4 is a schematic representation of the procedure used to produce a container containing a ready-to-use ultrasound contrast medium.

EXAMPLE 1

Materials

Propylenglycol (1 , 2-Propanediol extra pure), Merck

Calcium chloride-Dihydrate p. a., Merck TES (N-tris Hydroxymethyl -Methyl -2 -aminoethane-sulfonic acid) , Sigma Chemical Co

Titriplex^® III (EDTA disodium 99%) p. a., Merck

H-EPS Na (Hydrogenated Egg Phosphatidylserine Sodium) ,

NOF Corporation Perfluorobutane (PFB) , West Florida Ordnance, USA

Stock solutions:

EDTA diNa 0.18M / TES lOmM, pH = 7.4 (Procedures 1-4)

EDTA diNa 0.9M / TES 5mM, pH = 8.2 (Procedures 5-6)

NaOH and HCl for pH adjustments

Methods

1% Propyleneglycol was heated to 70°C and 0.1% H-

EPS Na was added during stirring. The lipid dispersion was further stirred for 45 minutes at 70 °C. Maintaining the temperature at 70°C, the dispersion was extruded 3 times through filter membranes having successive pore diameters of lμm, 0.8μm and 0.6μm. Thereafter, the dispersion was extruded 3 times through filter membranes having successive pore diameters of 0.4μm, 0.2μm and O.lμm. The resulting dispersion of mainly unilamellar vesicles was stirred at 70°C, and CaCl₂ was added in a mole ratio of approx. 1:0.25 (Ca : HEPS Na) . The white calcium-phospholipid precipitate was further stirred for 1 hour at 70°, and then cooled to room temperature. An excess of EDTA/TES was added in different ratios (according to the below procedures) , and the composition was homogenized on a cap-mixer for 45 seconds with PFB in the headspace .

Procedure 1 :

A 3x molar excess (EDTA : Ca mole ratio) of EDTA was added to the warm cochleate dispersion, and maintained at 70°C for 30 min. PFB was added and the dispersion was homogenized on a cap-mixer while warm.

Procedure 2 : As procedure 1, but homogenized after cooling to room temperature .

Procedure 3 :

A 3x molar excess (EDTA : Ca mole ratio) of EDTA was added to the warm cochleate dispersion, PFB added and homogenized immediately.

Procedure 4 :

A 3x molar excess (EDTA : Ca mole ratio) of EDTA was added to the cooled cochleate dispersion, PFB added and homogenized immediately.

Procedure 5 :

A 7.5x molar excess (EDTA : Ca mole ratio) of EDTA was added to the cooled cochleate dispersion, PFB added and homogenized immediately.

Procedure 6 :

A 7.5x molar excess (EDTA : Ca mole ratio) of EDTA was added to the cooled cochleate dispersion, this was left on the bench for 2 hours, PFB added and homogenized on a cap-mixer.

Results All of the above procedures resulted in formation of stabilized PFB microbubbles, as shown by visual and microscopic examination of the product. The best yield was seen with the largest excess of EDTA combined with swelling for some time after addition (procedure 6) . Microscopy showed that the latter procedure gave a population of microbubbles of mainly l-10μm, which was confirmed by Coulter counter measurement. In comparison, procedure 1 resulted in a broader population of microbubbles (Mainly l-50μm by microscopy - see Figure 2) .

EXAMPLE 2

Two samples of a dispersion of phospholipid (hydrogenated egg-phosphatidyl serine sodium, 4g) , water (800 mL) , propylene glycol (12g) and glycerol (35g) were heated, one to 60°C and one to 80°C (ie. below and above the lipid phase transition temperature) . The mixtures was then autoclaved (121°C for 15 minutes) , filtered through a 20 μm filter and homogenized by sonication. The results of particle size analysis (by Coulter counter) showed a two-fold increase in microbubble content in the size range 3-5 μm for dispersions prepared at 60 °C compared with dispersions prepared at 80°C.

EXAMPLE 3

Materials

D-Sphingosine from bovine brain Cerebrosides, approximately 99% (TLC) , SIGMA Methanol p. a. > 99.8%, Merck

Perfluorobutane (PFB) , West Florida Ordnance, USA

Distilled water

10 μm polycarbonate membrane filter, Poretics

Stock solutions : HCL, 1 mM

Stock 1: Sphingosine 1.6 mg/mL in methanol

Stock 2: Sphingosine 6.3 mg/mL in methanol Methods

Fibres in 12.5% methanol were prepared using two concentrations of Sphingosine:

A. 0.2 mg/mL Sphingosine: 250 μL stock 1 was added to a test tube and 1.75 mL water was added to it. The tube was inverted once to mix, and left overnight at room temperature .

B. 0.8 mg/mL Sphingosine: 125 μL stock 2 was added to a test tube and 0.875 mL water was added to it. The tube was inverted once to mix, and left overnight at room temperature .

After recrystallisation, the fibers were isolated by filtration through a 10 μm membrane filter, and washed with 2 x 2 mL water. The precipitate was resuspended by shaking the filter in a test tube with 1 mL water.

Production of microbubbles before and after acidification was attempted by homogenisation in a cap- mixer for 45 seconds with PFB in headspace of the vial.

Results

Visually, the recrystallised fibers appeared as white clouds in the test tube. Examination by optical microscopy showed the presence of a network of thread- like structures, both in solution and on the membrane filter after washing with water.

Cap-mixing of the fibres in the presence of PFB produced only a few microbubbles, which disappeared within a few minutes. Acidification of the fiber suspension was performed either by adding 0.2 mL 1 mM HC1 to the cap-mixed suspension, or by adding 1 mL 1 mM HCL directly to the filter with the fibers and shaking. The precipitate disappeared and a clear solution was formed. Cap-mixing of the first solution in presence of PFB resulted in a grey/white dispersion, and microbubbles in the size range of 5-50 μm could be seen in a microscope. Cap- mixing of the second solution resulted in a milky white suspension and the formation of foam. Microbubbles mainly between 5-50 μm could be seen in the microscope. The presence of microbubbles were further shown by filling the dispersion in a 2 mL syringe and increasing the pressure until the dispersion appeared clear. Only a few microbubbles around 10 μm could now be seen in the microscope, in addition to non-gas filled particles below 5 μm. Fibres produced from procedures A and B gave similar results, but B appeared to give a higher yield of microbubbles after acidification and cap-mixing.

EXAMPLE 4

Materials :

1, 2-distearoyl-3-trimethylammonium-propane (DSTAP) ;

Avanti Polar Lipids, Inc., USA Na₃(C₆0₇H₈), Sodium Citrate CaCl₂, Calcium chloride

Water for injection (WFI)

Methods

10 mg DSTAP (1 , 2-distearoyl-3-trimethylammonium- propane) dispersed in 2 mL WFI was precipitated by addition of 500 μL sodium citrate 100 mg/mL to form cochleate structures . The precipitation was reversed by addition of 1 mL calcium chloride, 100 mg/mL. Microbubbles were formed by homogenisation in a cap- mixer for 45 seconds with PFB in the headspace of the vial .

Results

The above procedure resulted in formation of stabilised PFB microbubbles of 1-30 μm, confirmed by Coulter Counter measurement .

Claims

Claims :

1. A process for the preparation of a contrast medium which process comprises: i) obtaining a composition comprising a lipid membrane in a high axial ratio conformation; ii) contacting said composition with an agent serving to release the lipid membrane from the high axial ratio conformation; and iii) agitating said composition in the presence of a biotolerable contrast generator or precursor whereby to form contrast generator or precursor containing lipid-membraned vesicles.

2. A process as claimed in claim 1 wherein the high axial ratio conformation is a rod, helix, fibre, ribbon, tubule, cylinder or cochleate.

3. A process as claimed in claim 2 wherein the high axial ratio conformation is a cochleate.

4. A process as claimed in any of the preceding claims wherein the release agent is a chelating agent .

5. A process as claimed in claim 4 wherein the chelating agent is a calcium or magnesium chelating agent selected from the group consisting of EDTA, DTPA, DTPA-BMA, DOTA, D03A, TMT, PLED, DPDP and EGTA.

6. A process as claimed in any one of claims 1 to 3 wherein the release agent is a factor which causes a change in an environmental parameter.

7. A process as claimed in claim 6 wherein the environmental parameter is pH, temperature or ionic strength.

8. A process as claimed in any one of the preceding claims wherein the contrast generator or precursor is a gas or gas precursor.

9. A process as claimed in any one of the preceding claims wherein the lipid membrane comprises phosphatidylcholine and/or one or more negatively charged lipids selected from phosphatidyl serines, phosphatidic acid and phosphatidyl glycerol .

10. A process as claimed in any one of claims 1 to 8 wherein the lipid membrane comprises one or more positively charged lipids selected from DOTMA, DOTAP, DOTB and DSTAP.

11. A process as claimed in any one of the preceding claims wherein the lipid membrane is held in a high axial ratio conformation by polyvalent ions.

12. A process as claimed in any one of the preceding claims wherein the lipid membrane in a high axial ratio conformation is obtained by adding polyvalent ions to an aqueous vesicular composition comprising a lipid or lipid mixture.

13. A process as claimed in claim 12 wherein the polyvalent ions are added at molar, ion to lipid, ratios of 1:0.1 to 1:10, preferably 1:0.25 to 1:5.

14. A process as claimed in any of the preceding claims wherein the released lipid is agitated with a gas or gas precursor to form gas or gas precursor containing lipid- membraned vesicles.

15. A process as claimed in any one of the preceding claims wherein the lipid-membrane vesicles are: i) optionally subjected to size separation or extruded through pores; ii) sterilised; and iii) combined with other physiologically tolerable carriers, excipients or components.

16. A process as claimed in claim 15 wherein in step iii) the vesicles are combined with additional carriers, excipients or components to a lipid content of 0.001-2% w/w.

17. A kit comprising: a lipid membrane in a high axial ratio conformation; a biotolerable contrast generator or precursor; and an agent capable of releasing said membrane from the cochleate conformation.

18. The use of a lipid membrane in a high axial ratio conformation for the preparation of a contrast medium for use in a method of diagnosis which involves administration of said contrast medium and generation of an image .

19. A method of contrast enhanced imaging of a human or non human animal subject, said method comprising:

(i) generating contrast generator or precursor containing vesicles using a lipid membrane in a high axial ratio conformation;

(ii) administering said vesicles into said subject, preferably into the vasculature thereof; and

(iii) generating an image of at least a part of said subject containing said vesicles.

20. A method as claimed in claim 19 being a method of ultrasound imaging.

21. A method as claimed in claim 20 wherein the vesicles contain gas or a gas precursor.

22. A process for the preparation of a contrast medium which process comprises obtaining an aqueous composition comprising an organised lipid membrane structure, optionally contacting said composition with an agent serving to destabilise said structure, and agitating said composition in the presence of a biotolerable contrast generator or precursor whereby to form contrast generator or precursor containing membraned vesicles.

23. A process as claimed in claim 22 wherein agitation of the composition serves to introduce the contrast generator or precursor into the vesicles and to rearrange the organised lipid membrane structures so as to produce the vesicles.

24. A process for the preparation of microbubbles which process comprises: i) obtaining a composition comprising a lipid membrane in a high axial ratio conformation; ii) contacting said composition with an agent serving to release the lipid membrane from the high axial ratio conformation; and iii) agitating said composition whereby to form said microbubbles.

25. A Contrast medium prepared by a process according to any one of claims 1 to 16 and 22 to 23.