CA2125027C - Stable microbubble suspensions as enhancement agents for ultrasound echography - Google Patents

Abstract

Disclosed are injectable suspensions of gas filled microbubbles in an aqueous carrier liquid usable as contrast agents in ultrasonic echography. The suspensions comprise amphipathic compounds of which at least one may be laminarized phospholipid as a stabilizer of the microbubbles against collapse with time and pressure. The concentration of phospholipids in the carrier liquid is below 0.01 % wt but is at least equal to or above that at which phospholipid molecules are present solely at the gas microbubble-liquid interface. Also disclosed is a method of preparation of the stable suspensions of air or gas filled microbubbles.

Description

wo 94/09829 212 a 0 2 7 pcr/Eps3/o29l5 - SI'ABLE MICROBUBBLE SUSPENSIONS AS ENHANCF~MF~T
AGENTS POR ULTRASOUND ECHOGRAPHY
s Technical Field The invention relates to infectable suspenslons of gas filled 0 microbubbles in an aqueous carrier comprising arnphipathic co~npounds of which at least one is a phospholipid stabilizer of the microbubbles agalnst-~ollapse with time and pressure. The phospholipid st~hili7er may be in a l~m~ r or l:~m1n:~r form. The inv~r~ffon also ~E~.lses a method of m~kln~ stable suspensions of 15 microbubbles usable as contrast agents in ultrasonic echography.

~ack~ound of I~ Uon Use of suspen~on~ of gas microbubbles in a carrier l~quid as eificient ultr~sound reflee~ s is well Icnown in the art. The developm~nt of microbubWe sUspçnc~Qns as echopharm~ce~ltic ls for enh~nctoment of ultrasound im~ followed early obseIv~t1on-s that rapid i~ ,e.lOUS in~ertion~s can cause solnh~li7~-i gases to come out of solution form~ng ~ubbles. Due to their subst:~nt~l difference in acoustic lmpedance relative to blood. these intravascular gas bubbles are found to be excellent reflectors of ultrasound. In~ec~n~ into the blood-stream of liv~ng or~n~sm~;
suspensions of gas microbubbles in a carrier liquid strongly reinforces ultrason~c echography 1m~in~. thus enh:~ncing the vis~ sat~on of internal organs. Since im~n~ of ogans and deep seated tissue can be crucial in est~hli~h~n~ medical Ai~gJlosi.~ a lot of effort is devoted to the development of stable suspensions of highly concentrated gas microbubbles which at the same time would be simple to prepare and ~iminister. would contain a m~nimum of inactive species. would be c~p~l~le of long storage and simple distribution. Many ~ttempt~s towards a solution which will satis~y these criteria have been made. howe~fel. none have provided a completely .s~ff-sf~ctory result.

WO 94/09829 PCI~/EP93J02915 It has been known from EP-A-0 077 752 (Schering) that suspensions of gas microbubbles can be made by mi~ing an aqueous - solution of a surf~c~nt with a sol~lffsrl of a v~scosity enh~ncer as a 5~hili~r. I~e gas b~h~les are introduced into the .~ e by forcing S the mixtllre of reagents and air through a small aperture. A
sll~p~n~iorl of CO2 microbuhhles may be obta~ned by addition of an acid to a ml~re obt~ne~l from a solution collt~ ..~ a surfattant and sodium bicarbonate and a solution of the viscosity enh~ncer.
inE the components ho~ve~,e,. is to be ca~ied out.~ust before use and the solution is to be consumed/in,~ected immerii~tely upon preparation. The disclosed surf~et~n~ (tensides) comprise lecithins;
esters and ethers of fatty acids and fatty alcohols with polyoxyethylerle and polyoxyethylated polyols like sorbitol. glycols and glycerol. cholesterol; and polyoxy-ethylene-polyoxypropylene 15 polymers. Disclosed concentration of tensides in the suspen~iorl is between 0.01% and 10% wt and a preferred range is daimed to be between 0.5% to 5%. The viscosity enh~3ncing and stabili7in~
compounds include for instance mono- and polysaccharides (glucose, lactose, sucrose, dextran, sorbitol); polyols, e.g. glycerol, 20 poly~lycols: and polypeptides ~ke proteins, gel~tin, o~ypolygel~in, pl~ ut~l and the like. Ihe total amount of viscosity enh~ncing agent is limited to 0.5 and 50%. Use of polyo~y~ropylene-polyoxyethylene polymers (eg. Pluronic~' F-68) as viscosity ~nh~ncing agent has also been ~li-c~ oser~l~ In the preferred e,Y~mrle, 25 equivalent volllmefi of tenside. a 0.5% by weight aqueous fiol~ s)r of Pluronlc~Z9F-68 (a polyo.-y~ lene- polyo,~ethylene copolymer3, and the YiScosity enh~ncer (a 10% lactose solution) are vigorously sh~kerl together under sterile cont~ on~ to provide a suspe~on of microbubbles. The suspension obtained lasted over 2 mlnutes and 30 contained close to 50% of bubbles with a size below 50 ~m.
Accoriil~g to the docllment up to 50% of surf~ct~nt~ and/or viscosity ~nh~n~in~ agents may be employed. hcw~:vel, specific ~y~mples use between 1% and 4% of Pluronic~) F~8.

Easy-to-produce aqueous suspensions usable as im~in agents in ultrasonic echography are disclosed in W0-91/15244 (Schneider et. al.). The suspensions contain film forming surfactants in-l~min:~r and/or lamellar form and, optionally, hydrophilic stabilizers. The l~min~rized surf~ct~nts can be in the wo 94/09829 212 5 0 2 7 - pcr/Ep93~o2sl5 form of liposomes i.e. microscopic vesicles, generally spherically shaped. These vesicles are usually formed of one or more con~entrically arranged bi-molecular layers of amph~pathic c~ ds l.e. compounds with a hydroph~lic and a ~y~o~hobic 5 moleffes. The molecllles in the bilayers are or~n~se~l so that the l~.l.o~hobic moIeties are in f~c-lnE rel~t1o~Cl~lr~ the ~y~l~o~hilic moie~es po~nffn~- toward the water phase. The suspe~sior~s are obtained by exposing the l~mln~llzed surf~ct~r~ts to air or a gas prior to or after ~llm~nf~ ~ith an aqueous phase. Co~ lon of 0 fllm forming s~ c~nt~ into l~mell~r form is carried out according to va~ious liposome forming technlques including hlgh pressure homogenisation or sonicatlon under acoustic or ultrasonic frequencies. The concentration of phospholipids claimed is be~w~ e.l 0.01% and 20% and the concentration of microbubbles is between 108 and lO9 bubbles/ml. The mlcrobubble suspensions rem~ine~l st~ble for months. The concentration of phospholipids in ~m~le l is O.S%.

An ;~lr...~ a stable echogenic suspens'on is ~1~seloseA
20 in WO-92/11873 (Beller et. ai.). Af,ueous l,r~ t1sn~ de-~e~etl to absorb and ~~b~ e microbubbles for use as an ech~raphic co~ astlng agent are made with pol~A~ lene/polyo~y~ Jylene polymers and negatively charged phospholipids such as phosphatidylgl~c~ol. phosphatidyl~nositol, phosphatidylçth~nol-2s amine. phosphatidylserine as well as their lysoforms. Theconcentra~on range of phosrho!~ e in the preparz~tior~s may be between 0.01% and 5% by volume or weight. howc;ver.
prepar~tion~s w,ith 1% of ~lir~lmitoylphosrh~titlyl glycerol (DPPG) are spe~fio~lly disclosed and claimed. Isl addition to the ne~tively 30 charged phospholipids the compos~tions must cont~n between 0.1% and 10% of polymeric material (Pluronlc~ ~-68). The total amount of solutes in the ~ ations is between 5.1% and 10.4%.
The conc.ontration of the microbubbles is no~ reported. hc,~e~, according to the results given it may be es~im~te~l to be about 107 35 bubbles/ml. The ~:t~b11ity of the suspe~s~on~ is reported to be better than that of EP-A-O 077 752.

Although the prior art compositions have merit, they still suffer several drawbacks which hamper their practical use. Firstly.

some prior art compositions have relatively short life spans and secondly, they have a relatively low initial bubble count e.g. between and 105 bubbles/ml. lhis m~kes reproducibility and analysis of echographic tests made with such compos~tinns fairly ~l~mclllt. In 5 addition, some te-~hn~ques produce bubbles in a ~ide range of diameters (up to 50 ~lm) which lJlG~e~l~; their use as echographic agents in ce~ ~1 aprli~t~onc (e.g. echo~raphy of the left heart).

The need for stable formulations of microbubbles which will o resist pressure variat~ons in the blood streams and have a good sheIf lIfe is further amplified by poor stab~lity of some of the state-of-the-art compositions. Microbubble formulations whose distribution and storage would not present problems are particularly important.
Another drawback is that many of the heretofore known compositions contain a high amount of different solutes such as poly~ners, phospholipids, electrolytes, and other which render their practical use more and more ~llmc~ t For example, lt is 20 known ~hat use of polyo~ylene/ polyo,~yyropylene polymers (Pluronic~Z9) with par~icular pa~ents may cause unple~nt side effects (see for instance G. M. Vercellotti et. al. Blood (19821 59.
1299). Preparations with a high phosrhol~ri~l content in certain cases may also be undesirable. In any event, compositions with a 25 high degree of various solutes are ~tlmin~ctered reluctantly and their wide spread use is beoo-m~n~ corl-si~lered to be undesirable. In fact, the trend in the pharmaceutical industry is to reduce concentrations of active and inactive lngre li-ont~ in various me~
or pharmaceutical formulations to their lowest possible levels and 30 elim~n~te from the preparations ~ .yl~ g that is not necess~
Finding alternative methods and form~ t~n~ more effective compositions co~ ues to be i~lLant. This is part1clll~rly so with microbubble suspensions used in echography since here the ingredients have no curative effect and should lead to the least 35 possible after consequences. Hc,we~ , as stated above. the state of the ar~ preparations with typical concentrations in the range of 1~~
and 4% by weight and the te~hings of prior art discourage use of reduced amounts of phospholipids and other non-phospholipid additives. The reason for the discouragement is most probably WO 94/09829 212 ~ 0 2 7 PCr/EP93/02915 hidden in the fact that in the course of the routine experimentation further reduction in concentration of the - ingredients never produced suspen~Qnc which were stable enough to have any pr~ c~l use or encourage iurther tinkering in the lower end of the known range.

Sl~mm~ry of the i~ Uon The present invention is based on the unexpected fin~l~n~
lO that very stable suspen~on-c of a gas filled microbubbles comprising at least 107 microbubbles per mlllilitre may be obtained using phospholipids as s~h~li7~rs even if very low conc~~ ffons thereof are employed. The suspenslons usable as contrasting agents in ultrasonic echography are obtained by suspen~lin~ in an aqueous 5 carrier at least one phospholipid as a s~hil~ser of the microbubbles ~nst collapse with time and pressure, the concentration of the phospholipids being below 0.01% wt. but equal to or higher than that at which the phospholipid molecllles are present solely at the gas microbubble-liquid interface.
It was quite ~~n~spected to dlscover that as ne~ hle amounts of the phosrho~ri~l surf ~ smt~s involved here (used alone or wlth a relaU~el~ small proportions of other ~mrhirh1les) can so effectively st~h~l~7e mic,ol,~bles. It is post~ e~l that. ln the 25 ~rc~e~ce of other amph~r~th~c co~ ds (suc-h- as Pluronic~) the mutual cohesion be~,.ee.l fit~h~l~7~r molec~les is decreased and formation of monomolec~ r phospholipid hlms is inhibited.
Hu~vel. in the absence of large amounts of other amph~rh~l~c agents, the llnhin~lered intermolecular binding forces (electrostatic 30 interaction or hydrogen bonAin~ be-~eell phospholipid molecules are sufficient to ensure forrn~tion of stable fllm-like structures st~h~ in~ the bubbles against c~ rse or coalescence.

According to the i~ on, suspen~iQn~ of high microbubble 35 concentration, high stability, long storage capaci~r and ease of preparation may be obtained even if the concentrations of surf~c~nts and other additives in the suspens~ons are kept well below the levels used in the state-of-the-art formulations. The amount of phospholipids used in the compositions of the invention WO 94/09829 ~ / ~ 5 PCI/EP93/02915 may be as low as about that only neeessz~y for forn~ orl of a single monol~yer of the surf~ct~nt around the gas microbubbles while the - concentration of the bubbles in the suspen~on is n~ t~ined above .. 107 microbubbles per m~lltl~re. In the present invention, s mlcrobubbles w~th a liposome-lllce double layer of surf~ct~nt (~as i~lled liposomes) are not likely to exist and have not been observed.

Suspensions with high microbubble concentrations e.g.
between 109 and 1010 bubbles/ml of relatively high stability and lO long storage capacity may be ~ d even ii- the concentration of the phospholipid surfactants are kept well below the levels known in the art. Suspensions with as little as 1 llg of phospholipids per ml may be prepared as long as the amount of the surfactants used is not below that which is necess~y for formation of a single monolayer of the lipids around the gas microbubbles and as long as they are produced according to one of the methods herein disdosed.

Calculations have shown that for bubble conc~llu~ttons of 20 10~ bubbles/ml depending on the size distribution of the microbubbles this conce -t.ation may be as low as 1 llg/ml or 0.0001%, ho~NC~ . the phospholipid concentrations between 0.0002% and up to 0.01% are preferred. More preferably the conc~ dtlon of the phosphollrs(ls in the stable suspen~is)ns of 2s microbubbles of the ~lv~ on is be~,ell 0.001% and 0.009%.
Although further reduction of the amount of phosrhol1rlds in the suspension is possible. suspensions prepared with less than 0.0001% wt. are unstable, their total bubble count is low and their echographic response upon in~ection is not s~t~.sf~ctory. On the 30 other hand, suspensions prepared with more than 0.01% of phospholipids upon in.~ection do not perform better i.e. their stability and echographic response do not further improve with the concentration. Thus. the h~her concentrations may only increase the probability of undesirable side effects as set out in the 35 discussion of the prior art. It is t~ ly postulated that only the segments of the surfactants which are in the lamellar or laminar form c~n effectively release molecules organized properly to stabilize the bllbbles. This may exrl~in why the concentration of the wo 94~09829 212 5 0 2 7 Pcr/EPs3/o29l~

surf~c~nt may be so low without ~mr~ring the s~h~lity of the gas bl l~h~

The suspen~ons of the in.~ in-1 offer important advantages s over the co.~ osit~Qn~ of the prior art not only bec~uce of the low phospholipld C~ t~.lt but also bec~t~e the total amount of in~ected ~soll1te-~ l.e.- lipids and/or synthetic polymers and other additives is bc~- ~ I.OOO and 50.000 t~mes lower than heretofore. This is achieved without any loss of microbubble concentration i.e.
0 echogenicity or stability of the product. In addition to ~he very low concentration of sblutes~ the invention provides suspensions which may contain only the microbubbles whose contribution to the echographic signal is relat~vely ~ c~nt i.e. susp~nsion~ which are free of any microbubbles which do not actively participate in 5 the 1m~jng process.

Nee-lless to say that wlth such low concentrat~ons of solutes in the in~ectable composition of the invention probability of undesirable side effects is greatly reduced and ~lim~n~tt~on of the 20 injected agent is s~fic~ntly i~lu~ed.

Ihe microbubble suspensions with low phospholipid eontet~t of the invent~on may be prepared from the fllm forming phocrhol~r~-ls whose structure has been mo~l~flefl in a conv~lient 2S m~nn~r e.g. by freeze-d~ying or spray~ying soltlffons of the crude phospholipids in a suitable solvent. Prior to formation of the suspen~ion by dispersion in an aqueous carrier the freeze dried or spray dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier the 30 powdered phospholiplds whose structure has been disrupted will form lamellarized or l~min~rized se~nen~ which wlll st~hfli~e the microbubbles of the gas dispersed therein. Con~eniently. the suspensions with low phospholipid co~tent of the inventlon may also be prepared with phospholipids which were lamellarized or 3s l~min~rized prior to their cont~ctin~ with air or another gas.
Hence, contacting the phospholipi-l-s with air or another gas may be carried out when the phospholipids are in a dry powder form or in the form of a dispersion of l~min~rized phospholiplds in the aqueous carrier.

The term lamellar or lz~min~r form indicates that the surfac~nts are in the form of thin fllms or sheets involving one or - more molecular layers. In this form. the surfactant molecules organize in structures ~im~l~r to that e~tj~l..g in liposome vesic~es.
s As described in WO-A-9 1/15244 COI1~Ve1 slon of film forming surfac~nts into lamellar form can easily be done by any liposome fo~ lg method for in~nce by high pressure homogerl~-s~tion or by sonication under acoustical or ultrasonic frequencies. The collv~lslon into lamellar form may also be performed by co~ting 0 microparticles ~10 ~Lm or less) of a hydrosoluble carrier solld (NaCl. sucrose. lactose or other carbohydrates~ w~th a phospholipid with subsequent dlssolution of the coated carrier in an aqueous phase. Simllarly, insoluble partides. e.g. glass or resin mlcrobeads may be co~te-l by moistening in a solution of a phospholipid in an 15 organic solvent following by evaporation of the solvent. The lipid coated microbeads are thereafter contacted with an aqueous carrier phase, whereby liposomic vesicles wlll form in the carrier phase. Also, phospholirirl~ can be l~mell~rized by he~tin~ ~li~tly above critical temperature ~c~ and gentle stirring. The critical 20 temperature is the temperature of gel-to-liquid transition of the phospholipids.

Practically. to produce the low phospholipid content suspensions of microbubbles accor~ g to ~he invention, one may 2s start with liposome suspensions or solutions prepared by any known technique as long as the liposomic vesicles are "unloaded", i.e. they do not have encapsulated therein any foreign materiaI but the aqueous phase of the solution itself.

~0 The introduction of air or gas into a liposome solution can be effected by usual ~ne~n~. injection i.e. forcing air or gas through tiny orifices into the liposome solution, or simply dissolving the gas in the solution by applying pressure and then suddenly rele~ing the pressure. Another way is to agitate or sonicate the liposome 3s solution in the presence of air or another physiologically acceptable gas. Also one can generate the formation of a gas within the solution of liposomes ltself, for in~t~nce by a gas rele~n~ chem1c~1 reacffon, e.g. decomposing a dissolved carbonate or bicarbonate by acid.

wo 94/09829 2 I 2 5 ~ 2 7 PCI/EP93/02915 When 1~mtn~r~zed surf~ct~nts are suspended in an aqueous liquid carrier and air or another gas Is lntroduced to provide - microbubbles, it is thought that the microbubbles become pro~ ly su~ ded and st~h~llce~l by a monomo1ecular layer s of surfact~nt molecules and not a bilayer as in the case of liposome vesicles. This structural ,e~,al~gement of the surf~ct~nt molecules can be activated meçh~n~cally (agitation) or thermally. The requtred ener~r is lower in the presence of coheston rele~ n~
agents, such as Pluronic~!9. On the other hand, presence of the l0 cohesion rele~s~nE agents in the microbubble formulations reduces the natural af~inity be~e,l phospholipid molecules having as a direct consequence a reduced stabllity of the microbubbles to external pressures (e.g. above 20-30 Torr).

As already mentioned, to prepare the low phospholipid content suspensions of the inventlon, in place of phospholipid solutions. one may start with dry phosrholirids which may or may not be lamellarized. When l~me11~tized, such phospholipids can be obtained for instance by dellydlaU~g l~posomes. i.e. liposomes which have been prepared normally by means of conventional techn~ques in the form of aqueous solu~ions and thereafter dehydrated by usual means. One of the methods for dehydrat~ng - liposomes is freeze-drying (lyoph~1~7~t~0n), i.e. the llposome soIution. preferably con~n~nE ll~d~ lic compounds. is f~ozen and dried by evaporation (s~ m~ffon) under reduced ~-ess~lre.

In another approach, non-lamellarized or non-1~m~n~rized ~hosrho1ipids may be o~ ed by dissolving the phospholipid in an organic solvent and drying the solution without going through liposome form~tior1. In other words. this can be done by dissolving the phospholipids in a suitable organic solvent together with a hy(ilO~hilic stabiliser subs~ance e.g. a polymer l~ke PVP. PVA, PEG, etc. or a compound soluble both in the organic solvent and water and freeze-drying or spray-drying the solution. Further ex~mrles of 3s the hydrophilic stabiliser compounds soluble in water and the organic solvent are malic acld. glycolic acid, maltol and the like.
Any suitable organic solvent may be used as long as its boiling point is sufficiéntly low and its melting point is sufflciently high to facilitate subsequent drying. Typical organic solvents would be for WO 94/09829 212 5 0 2 7 PCI'/EP93/02915 ~nst:~nce ~lioY:~ne, cyr,lo~ ol, ter~ary bllt:~nol~ tetrachlorodifluoro ethylene (C2C14F2) or 2-methyl-2-butanol how~.,~. tertiaTy butanol, 2-methyl-2-butanol and C2Cl4F2 are preferred. In this variant the criteria used for selection of the hydrophilic stabiliser is lts 5 solubility in the organic solvent of choice. The suspensions of microbubbles are produced from such powders using the same steps as with powders of the l~min~rized phosp~Qlir~ic.

S1mll~rly, prior to effecting the freeze-drying of pre-o lamellarized or pre-l~m~n~rlzed phospholipid solutions, a hydrophilic stabiliser compound is dissolved in the solution.
el. here the choice of the l~dr~l~hilic s~h~l~sers is much greater since a carbohydrate like lactose or sucrose as well as a hydloL)hilic polymer like dextran, starch, PVP, PVA. PEG and the 15 l~ke may be used. This is useful in the present invention since such hydrophllic compounds also atd ln homogenl-cin~ the mtcrobubbles size distribution and enh~nce stabil~ty under storage. Act~lly m ~k~ng very dilute aqueous soluttons (0.0001 - 0.01% by welght) of freeze-dried phosphol~ri~l~s st~h~ e~1 with, for instance. a 10:1 to 20 1000:1 weight ratio of poIyethyleneglycol to lipid erl~hles to produce aqueous mtcrobubbles suspçnslons counting 109-101~
bubbles/ml (size distribution mainly 0.5 - 10 ,uml which are st~hle, wtthout signlficant observable ch~nge. even when stored for prolonged periods. Ihis is obtatned by s~mrle dtssolutton of the air-25 stored dried l~min~r~zed phosrholiri~ls without ~h~kin~ or anyviolent agitat~on. The freeze-dry~ng technlque under reduced pressure is ve~y useful because it pe,lL~lts, restoration of the pressure above the dried powders with any physlologically acceptable gas, i.e. nitrogen, CO2, argon~ meth~ne, freons, SF6, CF4, 30 etc., whereby after redispersion of the phospholipids processed under such conditions susp~ns~o~s of microbubbles cont~1n~ng the above gases are obt~ine~l It has been found that the surf~ct~nt.s which are convenient 35 in this invention can be selected from ~ phipathlc compounds capable of fonning stable films in the presence of water and gases.
The preferred surfactants include the lecithins (phosphatidylcholine) and other phospholipids, inter alia phosphatidic acld (PA), phosphatidylinositol phosphatidyl-'~ 2 1 ~
wo 94/09829 ~ 2J ~J ~ 5 Pcr/EPs3Jo29l5 ethanol~m1ne (PE), phosrh~tidylserlne (PS). phosphatidylglyce,ol(PG), cardiolipin (CL), sphingomyelins. F,~mrles of suitable phosph-~1iFi~-s are natural or synthetic lecltl~ s, such as egg or soya bean lecithln, or saturated synthetic lecithins, such as, s dimyristoylphosphatidylcholine. dipalmitoylphosphatidylcholine.
~liste~royiphosphatidylcholine or diarachidoylphosphatidyl~holine or unsaturated synthetic lecithins, such as dioleylphosphatidyl choline or dilinoleylphosphP~tlylcholine, with saturated lec1thlns being preferred.

Additives like cholesterol and other substances can be added to one or more of the foregoing lipids in proportions r~n~in~ from zero to 50% by weight. Such addiffves may include other non-phospholipid surfac~nt~ that can be used in ~lm~ re with the 5 fllm forming surfactants and most of which are known. For instance. compounds like polyo~y~ropylene glycol and polyo~yethylene glycol as well as various copolymers thereof, phosphatidylglycel ol, phosrhP~ c acid, dicetylphosphate. fatty acids. ergosterol. phytosterol, sitosteroI. lanosterol. tocopherol.
o~yl E~ te~ ascolb.~i r~lm~t~te and butyiated hydroxy-toluene.
The amount of these non-fllm fo,~ g surf~çt~nt~ are usually up to 50% by ~veight of the total amount of sur-f~ç~nts but ~.~felably betw~e" 0 and 30%. Again this mepn~ that the concf, .f . ~tion of the various additives in the low phospholipid content susp~ns~oI-c Of 25 the invention are in the range of 0-0.05% which is more than one hundred times less than in the co~osiffons hl.~n so ifar.

It should also be mentioned that another feature of the suspensions of the invention is a relatively "high" gas entrapping 30 c~p~e~t~ of the microbubbles i.e. high ratio between the amount of the surfactant and the total amount of the entrapped gas. Hence, with suspens~ons in which the microbubbles have sizes in the l to 5 ~lm range. it is tentaffvely estim~te~l that the weight ratio of phospholipids present at the gas bubble-liquid interface to the 35 volume of entrapped gas under s~3ntl~rd conditions is be~eell 0. l mg/ml and 100 mg/ml.

In pracffce all in~ectable compositions should also be as far as possible isotonlc wlth blood. Hence, before in~ection, small wo 94/09829 21 2 ~'j 0 2 7 pcr/Eps3/o2sl5 amounts of isotonic agents may also be added to the suspensions of the invention. The isotonlc agents are physiological solutions commonly used ~n medicine and they comprise aqueous saline sol~tion (0.9% NaCll. 2.6% glyc~ol solllffon 5% dextrose solution.
5 etc.

The l~ Uon further concerns a method of m~k~n~ stable suspensions of microbubbles according to claim 1 usable as contrast agents in ultrasonlc echography. R~ic~lly. the method o comprises adapting the concentration of the phospholipids in the suspension of microbubbles st~h~l~7ed by said phospholipids to a selected value within the limits set forth in the ~'~i~s. Usually. one will start with a microbubble suspenslon con~ining more phospholiplds than the value desired and one will reduce the S amount of said phospholipids relatively to the volume of gas or air entrapped in the microbubble. vnthout subs~nti~lly reducing the count of echogenerating bubbles. This can be done. for instance. by removing portions of the carrier liquid con~ain~n~ phosrholiri~l~
not directly involved at the air/liquid interface and diluting the 20 suspension with more fresh carrier liquid. For doing this. one may create wlt~i~ ~he suspensicr region (a) where the echogenerating bl~h~le~s will ~ther and region (b) where said bubbles are strongly diluted. Then the liquid in region (b) can be withdrawn by separation by usual ~ne~qn~ (de~nt~tion. ~ir~oninf~. etc.) and a 2s comparable volume of fresh carrier liquid is supplied for replenls~ment to the suspension. This operation can be repeated one or more times. whereby the content in phospholipids not directly involved in st~hi'i7ing the bubbles will be progressively reduced.
It is generally not desirable to achieve complete removal of the phospholipid molecules not present at the bubble gas/liquid interface as some lmh~l~nce from equilibrium may result i.e. if the depletion is advanced too far. some surfactant molecules at the 35 gas/liquid interface may be set free with consequent bubble destab~l'7~t~on. Experiments have shown that the concentration of phospholipids in the carrier liquid may be decreased down to within the neighborhood of the lower limit set forth in the ~ im.s without s~gnihc~t changes in properties and adverse effects. This WO 94/09829 212 5 0 2 7 PCI/EP93/0291~i means that, ~ctl1~11y, the optimal phospholipid concentration (within the given limits) will be rather dictated by the type of - app110~tion i.e. if relatively high phospholipid concentrations are ~-lmiss1hle, the ldeal conc~tration value w~ll be near the upper 5 limit of the range. On the other hand, if depending on the condition of the r~t~ent to be ~ osed, the absolute value of phospho1ir~-1s must be further reduced, this can be done without adverse effects regarding microbubble count and echogenic eificiency.

An embodiment of the method comprises select1ng a film forming surf~ct~nt and optior~11y aJ,~ Ung it into 1~mell~r form using one of the methods known in the art or disclosed hereinbefore. The surf~ nt is then contacted wlth air or another gas and a~im1Yed with an aqueous liquld carrier in a closed container wh~eL.y a suspension of microbubbles will form. The suspension is allowed to stand for a while and a layer of gas filled microbubbles formed is left to rise to the top of the corlt~iner. The lower part of the mother llquor is then removed and the 20 s11~t n~nt layer of mic~ ,hle~ washed with an aqueous solution sa~u~ted with ~he gas used in preparation of the microbubbles.
This w~sh1nE can be repeated several times until s11hst~nt~ y all unused or free surf~nt molect11~s are removed. Unused or free molectlles m~nC all ~'t~cts~nt molec~lles that do not par~ p~te in 25 format~on of the st~hllicln~ morlomolecular layer around the gas microbubbles.

In addition to provlding the low phosphol~pid content suspensions, the w~hln~ te~-hn~que oi~ers an ad~ c)n~1 advantage 30 in that it allows further purlfication of the suspensior~ of the invention, i.e. by removal of all or ~1most all microbubbles whose contribution. to the echographic response of the in~ected suspen~ion is relatively inci~~ n~. The purifi~ffon thus provides suspensions co~ lsing only pGslUvely selected microbubbles, i.e.
3s the microbubbles which upon injection will participate equally in the reflection of echographic sign~1-s. This leads to susp.onsio~
cont~ining not only a very low concentration of phospholipids and other additives, but free from any microbubbles which do not actively particlpate in the ~m~n~ process.

' ~ 212~027-wo 94/09829 pcr/Eps3/o2s In a v~ul~.t of the method, the surfq~ q-nt which optionally may be in lqmell~r form, is q~lm1~efl with the aqueous liquid carrier - prior to cont~q~ng with air or another gas.
..
s Brief descripffon of dl~w~ s Figure 1 is grqrh~c~ql presentq-tioll of echoeraphic responses as a function of the microbubble concentration for a freshly prepared suspension accoldlllg to the lnvention.

Suspens~ons and the method of m~k~n~ low phospholipid ~ont.ont suspensions of the invention will be further illustrated by the following ~rqmples:
Example 1 M-11tilqmellar vesicles (MLVs~ were prepared by dissolving 240 mg of diarachidoylphosphatidylcholine (DAPC. from Avanti Polar Lipids) and 10 mg of flirqlm~toyl-phosphqt~lic acid (DPPA
20 acid form. from Avanti Polar Lipids) in 50 ml of hexane/ethq-nol (8/2. v/v) then evaporating the solvents to dryness in a round-bottomed flask using a lo~y eva~ at~l. The residual lipid film was dried in a vacuum dess~c-q-tor. After addition of water (5 ml), the suspension was ~ncl~hqte~l at 90~C for 30 minutes under 25 q~itqt~on. nle result~ng MLVs were extruded at 85~C through a 0.8 llm polycarbonate fllter (Nuclepore~'3. 2.6 ml of the resulting MLV
preparation were added to 47.4 ml of a 167 mg/ml solution of dextran 10'000 MW (Fluka) in water. The resulting solution was thoroughly m~xe-l, transferred in a 500 ml round-bottom flask, 30 frozen at -45~C and lyophilised under 0. l Torr. Complete sublimation of the ice was obtained overnight. Thereafter. air pressure was restored in ~he ev~c~l~te~l con~iner. Various amounts of the resulting powder were introduced in glass vials (see table) and the vials were closed with rubber stoppers. Vacuum was 35 applied via a needle through the stopper and the air removed from vials. Upon evacuation of air the powder was e~posed to sulfur hexafluoride gas SF'6.

--W0 94/09829 212 5 0 ~ 7 - PCr/EP93/02915 Bubble suspçn~ons were obtained by in.~ecting in each vial 10 ml of a 3% glycerol solution in water (through the stopper) - followed by gentle m~x~ng. The res~ n~ microbubble suspensions were counted using a hem~ me~er. The mean bubble size (in 5 volume~ was 2.2 llm.

I~IY weight Phosrholiri~l conc.Concentration (mg/mlJ ~g per ml)(bubbles/ml) O.S 8 9.0x 106 16 1.3 x 107 81 7.0x 107 161 1.4 x 108 Preparations were inlected to rabbits (via the ~ugular vein) as well as min~pigs (via the ear vein) at a dose of 1 ml/Skg. In vivo lO echographic measurements were performed using an Acuson XP128 ultrasound system (Acuson CoIp. USA~ and a 7 MHz sector transducer. The ~nim~lfi were :ln~esthe~sed and the transducer was positioned and then fi~ced in place on t_e left side of the chest providing a view of the right and left ventricles of the heart in the case of rabbit and a lon~ lin~l four~h~mher view in the case of the miniI~g. The preparat~on co~lt~ 0.5 mgtml dry weight gave slight opacification of the right as well as the left ventricle in rabbits and in min1r~. Ihe opaci~tion~ hc,~ . ~as super~or w~h the 1. 5 and 10 mg/ml prepar~t~o~

Example 2 Lyophilis~tes were prepared as described in F~mple 1 with 25 air (instead of SF6) in the gas phase. The lyorh~ ~es were then suspended ~n 0.9% saline (instead of a 3% glycerol solution).
Similar bubble concentrations were obtained, Ho~ever. after injection in the rabblt or the m~nlpig the persistence of the effect was shorter e.g. 10-20 s instead of 120 s. Moreover. in the minipig 30 the opacification of the left ventricle was poor even with the 10 mg/ml preparation.

WO 94/09829 - 2 12 5 0 2 7 PCI'/EP93/0291~

F~mrle 3 - MLV liposomes were prepared as described in F~y~mple 1 using 240 mg of DAPC and 10 mg of DPPA (molar ratio 95: 5). I~ro s m~ litres of this preparation were added to 20 ml of a polyetllyleneglycol (PEG 2~000) solution (82.5 mg/ml). After mix~n for 10 min at room tempe.~Lule. the resulting soluffon was frozen during 5 min at -45~C and lyophilised durlng 5 hours at 0.2 mbar.
The powder obtained (1.6 g~ was transferred into a glass vial o equlpped with a rubber stopper. ~e powder was exposed to SF6 (as described in Example 1) and then dissolved in 20 ml of distilled water. The suspension obtained showed a bubble concentraffon of 5 x 109 bubbles per ml with a median diameter in volume of 5.5 ~Lm. This suspension was introduced into a 20 ml syringe. the syringe was dosed and left in the horizontal position for 24 hours. A white layer of bubbles could be seen on the top of solution in the syringe. Most of the liquid phase (-16-18 ml~ was e~acuated while the syringe was maintained in the horizontal pos~t~o~ and an equivalent volume of fresh. SF6-saturated. water 20 was introduced. The syringe was then sh~k~n for a wh~le in order to homogenise the bubbles in the aqueous phase. A second decantation was performed under t-h-e same conditions after 8 hours followed by three further dec~n~tions performed in four hour l-lt~,vals. The final bubble phase (batch P145) was suspended 2s in 3 ml of distilled water. It co~lta~ned 1.8 x 109 bubbles per ml with a median diameter in volume of 6.2 ~m. An aliquot of this suspension (2 ml) was lyophilised during 6 hours at 0.2 mbar. The resulting powder was dissolved in 0.2 ml of tetrahydrofuran/water (9/1 v/v) and the phospholipids present in this solution were 30 analysed by HPLC using a light scattering detector. This solution contained 0.7 mg DAPC per ml thus corresponding to 3.9 ~g of phospholipids per 108 bubbles. A Coulter counter analysis of the ~ctt~l bubble size distribution in batch P145 gave a total surface of 4.6 x 107 1lm2 per 108 bubbles. Asst7m1n~ that one molecule of 35 DAPC will occupy a surface of 50 A2, one can calculate that 1,3 ~g of DAPC per 108 bubbles would be necessaly to form a monolayer of phospholipids around each bubble. The suspension P145 was than left at 4~C and the concentration of gas bubbles measured on a regular basis. After 10 days, the product looked as good as after its WO 94/09829 21 2 .5 0 2 7 PCI/EP93/02915 preparation and still contalned 1-1.2 x 109 bubbles per ml. The e~c~ional stabillty was found very surprlsing considering the ~l~e~ely low amount of phospholiritls in the suspen~ion s The experlment described above was repeated on a second batch of m~crob~ bles using a shorter dec~nt~t~on time in order to collect preferably larger bubbles (batch P132). The median meter in volume obp~ne~l was 8.8 tlm and the total surface determined with the Coulter counter ~;vas 22 x 108 ~1m2 per 108 0 bubbles. The calc~ t~or showed that 6 ~lg DAPC for 108 bubbles would be necess~ry to cover this bubble population with a monolayer of DAPC. The ~ctll~l amount of DAPC determined by HPLC was 20 ~lg per 108 bubbles. Taking into account the difflculty of obt~n~n~ precise estim~tes of the total surface of the bubble population, it appears that within the experimental error. the results obtz~ne~l are c~nc~ctent with cov~ age of the microbubbles with one phospholipid layer.

Echographic m ~u~ nt.s perfo~ned with different w~s~etl bubble pIepa~ffq'l~i showed that upon ~ aUon the lower phase gives a much weaker e-~ho~ d~l~C signal than the upper phase or a freshly ~ ar~d sample. On a first sight this seemed normal as the white layer on the top of the syringe collt~ined the mayority of the - gas microbubbles al,yway~ How~-e~ as shown in Fig. 1 the bubble 2s count showed a surpr~singly high microbubble pop~ ffon in the lower layer too. Only upon Coulter measurement it became apparent that the microbubbles had a size below 0.5 ~lm, which ~n~lic~te~s that with small bubbles even when in high concentration.
there ls no adequate reflection of the ultrasound A four fold dilution of the preparation P132 in a 3% glycerol solution was in~ected in the minipi~ (0.2 ml/kg). The preparation of washed bubbles con~in~ng 2.5 x 107 bubbles per ml and 5 llg of phospholipids per ml provided excellent opacification in the left and right ventricle with outstanding endocardial border deline~tion Good opaciflcation was also obtained by in~ecting to a minipig an aliquot of preparation P145 (diluted in 3~/0 glycerol) colles~onding to 0.2 ~lg of phospholipids per kg. Contrast was even detectable in the left ventricle after injection of 0.02 llg/kg.

~ wo 94/09829 212 5 ~ 2 7 PCI'/EP93J0291~

Furthermore, in the renal artery the existence of a contrast effect could be detected by pulsed Doppler at phosphQlir)id doses as low - a_ 0.005 11g/kg.
.
It follows that as long as the ~ .~ed phospholipids are arranged in a single mono1~yer around the ga~ microbubbles the sUspenC~o~lc produced ~ill have adequate stability. Thus providing an explanation for the present unexpected finding and demonstrating that the amount of phosrholip~ds does not have to o be greater than that required for formation of a monolayer around the microbubbles present in the susp~nc~orl.

Example 4 A solution cor~ ning 48 mg of DAPC and 2 mg of DPPA in hexane/ethanol 8/2 (v/v) was prepared and the solvent evaporated to dryness (as described ln F~m~le 1). 5 mg of the resulting powder and 375 mg of polyethyleneglycol were dissolved in 5 g of tert-butanol at 60~C. The clear ~oll-ffo~ was then rapidly cooled to ~5~C and lyorh~ l. 80 mg of the lyorh~ te was introduced in a glass v~al and the powder exposed to SF6 (see F'Y~mrle 1). A 3%
glyce.ol solution (10 ml) was then introduced in the vial and the lyoph~lis~e dissolved by gentle swl~ g. The resulttng suspension had 1.5 x 108 bubbles per ml with a me~ n diameter (in volume) of 9.5 llm. This solution was ir~ected to a rabbit providing outstanding views of the right and left ventricle. Even a ten fold dilution of this s~ sion showed strong contrast enh~ncemen~

Ex~nple 5 The procedure of ~y~mrle 4 ~ras repeated except that the initial dissolution of the phosphol~p~ in hexane/ethanol solution was omitted. In other words, crude phosphollrids were dissolved.
3s together with polyethylene glycol in tertiary butanol and the solution was freeze-dried thereafter. the residue was suspended in water. Several phospholipids and combin~tions of phospholipids with other lipids were investigated in these experiments. In the results shown in the next table the phospholipids were dissolved in 40 a tertia~ butanol solution co.-t~ ng 100 mg/ml of PEG 2'000. The ~ wo 94/09829 2 1 2 S O .? ~ pcr~Eps3/o29ls residues ob~ ne~ ter freeze d~ying were saturated ~lth SF6 (see ~mrle 1). then dissolved in distilled water at a conce..~.~tion of - lOO mg d~y weight per ml.
,.
I~pid m~ re Conc. in tert- Bubbleconc. Metli~n diam (welght ratio) bt-t~nol(m~/ml) (x 109/ml) (llm) DSPC 2 1.3 1 0 DAPC/DPPG (100/4) 2 3.8 7 DSPC/Chol (2/l) 6 0.1 40 DAPC/Plur F68 (2/1) 6 0.9 15 DAPC/Palm. ac. (60/1) . 2 0.6 11 DAPC/DPPA (lOO/4) - 1 2.6 8 DAPC/Chol/DPPA (8/1/l) 8 1.2 19 DAPC/DPPA (100/4)~ 5 2.4 18 L~end DAPC = d~ doylrhos~h~dyl choline DSPC = dlstc~uyl ~!h~ yl chollne 10 DPPG = ~l~r~ toylrhQsph~t~dyl glycerol (acld form) DPPA ~ ~ J)~ h~s~k4~ acld Chol = rh~
Palm. ac. = ~lm~ ac~d Plur F68 ~ Pl~".lc ~9F-68 15 In ~s ~ , CF4 was usod as gas 1nstead of SF6 In all cases the Susp~nc~cn~s obt~1ne-1 showed high microbubble concentrations indic~t1n~ that the ~nitial conversion of phosphollpids lnto liposomes was not necessary. These 20 suspen~ions were diluted in 0.15 M NaCl and ln~ected to m~nir~
as descr~bed in F~mrle 3. In all cases outst~n~lin~ oracific~tlon of the right and left ventricles as well as good delineation of the endocardial border were obtained at doses of 10-50 llg of lipids per kg body weight or less.
Example 6 PEG-2000 (2 g). DAPC (9.6 mg) and DPPA (0.4 mg) were dissolved in 20 ml of tertiary butanol and the solution was freeze dried 30 overnight at 0.2 mbar. Ihe powder obtained was exposed to SF6 and then dissolved in ~0 ml of distilled water. The suspension cont~1n1ng 1.4 x lO9- bubbles per ml (as determined by --wo 94/09~29 2 1 2 ~ 0 2 7 PCr/EP93/02915 hemacytometry) was introduced into a 20 ml syringe..which was closed and left in ho~ ~l position for 16 hours. A white layer of bubbles could be seen on top of the solution. ~he lower phase (16-18 ml) was discarded while n :~int~inin~ the sy~inge horizontally.
s An equivalent volume of fresh SF6-saturated distilled water was asplrated in the syr~nge and the bubbles were homogenised in the aqueous phase by agitation. Two different populations of microbllhbles i.e. large-sized and medium-sized were obtained by repeated decA~ t~on~ over short periods of time. the large bubbles o being collected after only 10-15 min of decantation and the medlum sized bubbles being collected after 30-45 min. These decantations were repeated 10 times in order to obtain narrow bubble size distributions for the two types of populations and to elimin~te all phospholip~tls which were not associated with the 5 microbubbles. All phases cont~inlng large bubbles were pooled ("large-sized bubbles"J. ~simil~rly the fractions co~t~lning medium sized bubbles were combined ("medium-sized bubbles"). Aliquots of the two bubble populations were lyorhllise-l and then analysed by HPLC in order to determine the amount of phosrhol~r~ present 20 in each fr~tion. The large-sized bubble fraction contained 2.5 x 107 bubbles per ml with a median diameter in number of 11.3 llm and 13.7 ~lg phospholipids per 107 bubbles. This result is in excellent agreement with the theoretical amount. 11.5 llg per 107 bubbles. c~c~ te~l ass~ g a mo~ol~yer of phosphol~rl~l.s around 25 each bubble and a surface of 50 A per phosrholiri~ molecule. The me~ m-sized bubble fr~f t~on cont~ne-l 8.8 x 108 bubbles per ml with a median diameter in number of 3.1 llm and 1.6 ~lg phosphollpids per 107 bubbles. The latter value is again in e~cellent agreement with the theoret~cal amount. 1.35 llg per 107 30 bubbles. These results further indicate that the stability of the microbubble suspensions herein disclosed is most probably due to formation of phospholipid monolayers around the microbubbles.

Claims (22)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. An injectable suspension of gas filled microbubbles in an aqueous carrier liquid, usable as contrast agent in ultrasonic echography, comprising at least 10 7 microbubbles per millilitre and amphipathic compounds at least one of which is a phospholipid stabilizer of the microbubbles against collapse, characterized in that the concentration of the phospholipids in the carrier liquid is below 0.01% by weight while being equal to or above that at which the phospholipid molecules are present solely at the gas microbubble-liquid interface.

2. The injectable suspension of claim 1, in which the concentration of microbubbles per millilitre is between 10 8 and 10 10.

3. The injectable suspension of claim 1, in which the concentration of phospholipids is above 0. 00013% wt.

4 . The injectable suspension of any preceding claim, in which the liquid carrier further comprises water soluble poly- and oligo-saccharides, sugars and hydrophilic polymers as stabilizers, said stabilizers selected from polyethylene glycols.

5 . The injectable suspension of any preceding claim, in which the phospholipids are at least partially in lamellar or laminar form and are lecithins selected from phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol phosphatidylinositol, cardiolipin and sphingomyelin.

6. The injectable suspension of claims 4 or 5, further containing substances affecting the properties of phospholipids selected from phosphatidylglycerol, phosphatidic acid, dicetylphosphate, cholesterol, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene.

7. The injectable suspension of claims 1, 2 or 3, in which the phospholipids are in the form of powders obtained by freeze-drying or spray-drying.

8. The injectable suspension of claim 1, containing about 10 8 - 10 9 microbubbles per millilitre with the microbubble size between 0.5 -10 µm showing little or no variation under storage.

9. The injectable suspension of claim 1, in which the liquid carrier further comprises up to 50% by weight non-laminar surfactants selected from fatty acids, esters and ethers of fatty acids and alcohols with polyols selected from polyalkylene glycols, polyalkylenated sugars and other carbohydrates, and polyalkylenated glycerol.

10. The injectable suspension of any preceding claim, in which the microbubbles are filled with SF6, CF4, freons or air.

11. A method of making suspensions of air or gas filled microbubbles comprising selecting at least one film forming surfactant, converting the surfactant into a powder, contacting the powder with air or another gas and admixing the powder surfactant with an aqueous liquid carrier to form said suspension, characterised by introducing the suspension into a container, forming a layer of the gas filled microbubbles in the upper part of the container, separating the layer of the microbubbles formed, and washing the microbubbles with an aqueous solution saturated with the microbubble gas.

12. The method of claim 11, in which prior to converting into the powder, the film forming surfactant is at least partially lamellarized.

13. The method of claim 12, in which prior to contacting with air or another gas the partially lamellarized surfactant is admixed with the aqueous liquid carrier.

14. The method of claims 12 or 13, in which the liquid carrier further contains stabiliser compounds selected from hydrosoluble proteins, polypeptides, sugars, poly- and oligo-saccharides and hydrophilic polymers.

15. The method of claim 12, in which the conversion is effected by coating the surfactant onto particles of soluble or insoluble materials leaving the coated particles for a while under air or a gas, and admixing the coated particles with an aqueous liquid carrier.

16. The method of claim 12, in which the conversion is effected by sonicating or homogenising under high pressure an aqueous solution of film forming lipids, this operation leading, at least partly, to the formation of liposomes.

17. The method of claim 16, in which prior to contacting of at least partially lamellarized surfactant with air or another gas the liposome containing solution is freeze-dried.

18. The method of claims 16 and 17, in which the aqueous solution of film forming lipids also contains viscosity enhancers or stabilisers selected from hydrophilic polymers and carbohydrates in weight ratio relative to the lipids comprised between 10:1 and 1000:1.

19. A method of preparation of a suspension of air or gas filled microbubbles comprising a film forming surfactant, a hydrophilic stabiliser and an aqueous liquid carrier, characterised by dissolving the film forming surfactant and the hydrophilic stabiliser in an organic solvent, freeze drying the solution to form a dry powder, contacting the powder with air or another gas and admixing said powder with the aqueous carrier.

20. The method of claim 19, in which the hydrophilic stabiliser is polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, glycolic acid, malic acid or maltol.

21. The method of claims 19 or 20, in which the organic solvent is tertiary butanol, 2-methyl-2-butanol or C2Cl4F2.

22. A method of making an injectable suspension of gas-filled microbubbles according to claim 1, which comprises suspending laminarized phospholipids, and optionally other additives, in an aqueous carrier liquid, said phospholipids having been in contact with said gas prior or after being suspended, under conditions such that a concentration of said microbubbles sufficient to provide an echographic response is formed in the suspension, allowing a portion of said phospholipids to form a stabilization layer around said bubbles and thereafter depleting the carrier liquid of the excess of phospholipids not involved in microbubble stabilization.