CA2222186A1 - Gas emulsions stabilized with fluorinated ethers having low ostwald coefficients - Google Patents

Abstract

Long lasting gas emulsions for ultrasound and magnetic resonance imaging contrast enhancement utilize low Ostwald coefficient fluoromono- and fluoropolyether compounds. Gas emulsions comprising microbubble preparations are disclosed wherein the microbubbles comprise fluoroethers such as perfluorodiglyme (CF3(OCF2CF2)2OCF3), perfluoromonoglyme (CF3OCF2CF2OCF3), perfluorodiethylether C2F5OC2F5, perfluoroethylmethylether CF3OC2F5, perfluorodimethylether CF3OCF3, as well as CF3OCF2OCF3 and fluoropolyethers CF3(OCF2)2OCF3, CF3(OCF2)3OCF3, and CF3(OCF2)4OCF3.

Description

W O 96140281 PCTnUS~G/O~C~8 GAS EMULSIONS STABTT ~7~n WITH FLUORINATED
~;l~KS HAVING LOW OSTWALD COEFFICIENTS
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention includes a method for prcL)a~ g stable, long-lived gas em~ ions for ulllasu~ld contrast çnh~n~ment and other uses, and to colll~o~iLions of the gas ~mlll~ione so ~r~d. Additionally, the present invention includes ~JlCl;Ul;~iUl~ for ~iC~ hlg such emulsions.

2. Back~round of the Art UlLIasoulld teçhn- logy provides an hll~o~ and more economical :~ltern~tive to im~in~ techniques which use ionizing radiation. While numerous collvc-,lio~l im~ging technologies are available, e.g., m~gnlotic ~c30ll~ulce im~ging (~aRI), co~ P.;~cl tomography (CT), and positron emission tomography (PET), each of these techniques use extremely w~ ive eqnirment Moreover, CT and PET utilize inni7in~ radiation. Unlike these techniques, ultrasound im~ging e4~ l is relatively inc~cnsivc. Moreover, ultrasound im~ginf~ does not use ionizing nq~ ticn UlLIaSOulld im~in~ makes use of ~lirr~ .~.lces in tissue density and composition that affect the reflection of sound waves by those tissues. Images are es~eçi~lly sharp where there are distinct v~ri~tion~ in tissue density or collll~lci,~ibility, such as at tissue int~rf~es Tlllr. riiçes bcLwccll solid tissues, the ~l~elet~l system, and various organs and/or tumors are readily imaged with ultrasound.
Accoldi~ly, in many im~ging applications ull~asound ~ rc,.llls suitably without use of colllla~l erlh~n~onn~nt agents; however, for other applications, such as vicn~li7~tion of flowing blood, there have been ongoing efforts to develop such agents toprovide contrast enhancement Oneparticularly ci~nific~nt application for such collLla~l agents is in the area of perfusion im~ging Such ultrasound contrast agents could improve im~ging of flowing blood in the heart mnc-,le, kidneys, liver, and other tissues. This, in turn, would f~ te lcscalcl~ gn~-si~, surgery, and therapy related to the imaged tissues. A blood pool contr~et agent would also allow im~ging on the basis of blood content (e.g., tumors and infl~mecl tissues) and would CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 aid in the vi~ li7~tion of the placenta and fetus by enhancing only the m~tPrnzll circulation.
A variety of ultrasound contrast enhancement agents have been proposed.
The most sllcc~s~ful have generally consisted of dispersions of small bubbles of gas that can be injected h~Lldvenously. The bubbles are injected into the bloodstream of a living body to be imaged thereby providing an emulsion in the flowing bloodthat is of a dirr~,re~lL density and a much higher cOlllp.~ ibility than the surrounding fluid tissue and blood. As a result, these bubbles can easily be imaged with ultrasound.
UllrolLulldLely, the creation of bubbles that are effective ultrasound sc~LL~,.e.~
in vivo has been difficult. Several ç~rl~n~tions are a~nL. First, such bubbles tend to shrink rapidly due to the diffusion of the trapped gas into the surrounding liquid. This is especially true of bubbles cu..l;~ g air or its component gases (such as nitrogen) which are highly soluble in water. It might be expected that bubblelifetime could be i",~,ov~;d by simply hl~le~illg the size of the bubbles so more gas needs to escape before the bubbles di~ e~. This approach has proven m~:~ticf~rtory, however, becau~e bubbles larger than about 10 ~Lm in ~ mPtPr arecleared from the blood~L,~" by the lungs, preventing their further circllls~tion-Additionally, larger bubbles are not capable of circlll~tin~ through smaller blood vessels and capillaries.
Microbubbles with s~ti~f~ctory in vivo ~clrollll~lce should also posses advantageous biological ch~-t~ tics. First, the compounds m~king up the gas inside the microbubbles should be bioco",~dlible. Ultimately, the microbubbles cu,.l~ g the gas phase will decay and the gas phase will be released into the blood either as a dissolved gas or as submicron droplets of the con~iPn~ed liquid.
Therero~e, the gases will primarily be removed from the body through lung le~i,dlion or through a combination of l~,~hdlion and other metabolic pillhwdys in the reticuloendothelial system. Even when bubble per~i~tPnre is sufficient to allow for several passes through the circulatory system of an animal or human, microbubble uptake by the reticuloen~lothPli~l phagocytic cells of the liver can limit the ~rre-;liv~"ess of the co"l,~L agent. Adverse immlmP system reactions can also reduce the in vivo lifetimes of the bubble, and should be avoided. For ~mple, "naked" microbubbles have been shown to produce adverse responses such as the activation of complement (See, for example, K.A. Shastri et al. (1991) Undersea CA 02222l86 l997-ll-25 W O 96/407.~1 PCT~US96/09068 Biomed. Res., 18, 157). However, as known in the art, these undesired le;,~o,lses may be reduced through the use of a~ ol)l;ate ~n~ps~ ting agents.
Accordingly, efforts to improve the in vivo lifetime, of microbubbles have included the use of stability, and hence the various en~ pslll~ting m~teri~l~ For S ;n.~t~nre, gelatins or alburnin lmicrospheres that are initially formed in liquid ~ ,u~ l ,ion, and which entrap gas during soli~ific?~tiQn, have been used. The use of surf~rt~nt~ as stabilizing agents for gas bubble dispersions has also been explored, as in U.S. Patent Nos. 4,466,442 to E~ilm~nn et al., and 5,352,436 to Wheatley et al.
Some sllrf~t~nt-cc~ l;llg COll~ 7l ~nh~n~m~nt agents entrap gas bubbles in the aqueoùs core of liposomes as in U.S. Patent No. 5,334,381 to Unger and U.S. Patent No. 4,900,540 to Ryan et al.
Recently, the affects of the c;lllldpped gas on bubble lifetime has received considerable ~ ntion Aside from air and its co".~v,lents, various noble gases such as k~y~loll and argon have been used. ~tt-ontion has now focused on bioco,..~ ible gases which have low water solubilities. Low solubility has been shown theoretically to be an hll~ ll factor in gas bubble stability. In Epstein and Plesset, On the Stability of Gas Bubbles in Liquid-Gas Solutions, (1950) J. Chem.
Phys. 18(11), 1505-1509, the rate of gas bubble ~hnnk~ge was derived as a function of gas density, solubility, and dirru~ivily in the surrounding medium. The stability of liquid-liquid emnleion~ has also been shown to increase with the de.,lea~illgsolubility of the dispersed phase (K~b~lnrv and Sh~ k;", Ostwald Ripening Theory: Applic~tion~ to Fluorocarbon Emulsion Stability, A~vances in Colloid andInterface Science, 38:69-97, 1992).
With certain simplifying ~u~ lions, the Epstein and Plesset formula leads to the formula for bubble lircli"lc (~) given by Quay in U.S. Patent 5,393,524:

T CY p/DC (1) where p is the density of the e.lllal,~cd gas, D is the dirru~iviLy of the gas in the ~ul,vullding ~e~ ll, and C is the solubility of the gas in the surrounding m~ m Based on this f~rm~ Quay forms bubbles using gases scle~l~;d on the basis of being a gas at atmospheric pl~cs~ule and body telll~ lulc (37~C) and having reduced water solubility, higher density, and ~,duced gas dirru~ivily in solution in co,l,~u;son to air. In the same vein, Schn~ r et al. in EP0554213Al disclose gases chosen on the basis of low water solubility and high molecular weight.

WO 96/40281 PCTrUS96/09068 Specifically disclosed gases include SF6, and SeF6, as well as various perfluorinated hydrocarbons.
Although reduced water solubility and diffusivity can affect the rate at which the gas leaves the bubble (as originally predicted by Epstein and Plesset), the Quay S and Srhnpi~ler gas selection criteria are inaccurate in that they result in the incl~ n of certain lm~llit~hle gases and the exclusion of certain optimally suitable gases. For example, in U.S. Patent No. 5,393,524, Quay suggests choosing microbubble gases based on a calculation of the Q value for the proposed gas, v~ nl:

Q = 4 x 1o-7 X p/DC, (2) p is the gas density (kg/m3), C is the water solubility of the gas (M), and D is the dirrll~ivily of the gas in solution (cm2/s). Quay teaches that the Q value should be at least 30 to be a useful gas for ulLlasOu-ld contrast ~nh~nr~mPnt A simple estim~tç using lil.. <~l.. ~ water solubility data (E. Wilhelm, R Battino, and R.J.
Wilcock, Chemical Reviews, 1977, v. 77, p. 219) shows that the Q values of virtually all known gases (with the exception of hydrogen and helium) approach or exceed this value. At 25 degrees C, oxygen, for example, has a Q of 20, and nitrogen has a Q of 35. The Quay ~ rlosllre, therefore, provides little gni~l~nre for the sel~ctiol~ of effective microbubble gases.
MG.CjO~e1, the Quay Q coefficient criterion as well as S~hnçi-l.or's disclosure in EP0554213Al fail to cQn~ r certain major causes of bubble shrinkage, namely, the effects of bubble surface t~n~ion, sllrf~rt~nt~ and gas osrnotic effects, and the potential for filling g~ contlt~n~ti~n into a liquid. Namely, the partial ~ies~ule of the filling gas must be high enough to oppose the excess Laplace OV~ ;S~ inside the bubbles. If the saLulal~d vapor plC~ iiS low the filling gas may condense into liquid and ccllLI~l ability will be lost. Accordingly, a need exists in the art for stabili~d contr~t Pnl~ r~.. ~1 agents that are bioco.. p~;hle, easily piepaled, and provide superior in vivo contr~t ~nh~nr~m~nt in ultrasound im~gin~ A need also exists for microbubble pl~ OlS and mtothntl~ to prepare and use such cOllLIasl ~nh~nrlom~nt agents.

CA 02222l86 l997-ll-25 SummarY of the Invention The present invention utilizes low Ostwald coefficient fluoroether compounds to provide long lasting gas çnmll~ir,n~ comprising microbubble ~ .~d~ions for ulll~soulld and m~gnPtic lesonS~i.ce im~ging contrast Pnh~nc~ement. When microbubble pL~ald~ions are ple~ d using the compounds of the present invention,~ longer lasting images of the heart and other intPrn~l organs may be obtained than has been before possible. In this invention, gas emulsions comprising a previously unron~ ered class of compounds which combine a reduced water solubility without a significantly reduced ~ d~ed vapor ~l~,S:jul~ (and thus surprisingly low Ostwald coefficients) are ~ rlosP~1 The high vapor ~les~ additionally helps to reduce the loss of con~ il due to the filling gas co~len~tion into liquid. These compounds are the fluorinated mono- and polyethers. When perfluoropolyethers are co",~ ,d withtheir perfluorocarbon analogues with the same number of carbon atoms, adding ether oxygen does not affect the vapor ~re~ G eignifir~ntly, whilst the water solubility decreases by a factor of a~ruAi",ately 2-3. This is uneApe-;LGd and surprising in that the co~ ion of hy~oc~lJoll to ethers results in .cignific~nt increases in water solubility.
Thus, a gas emulsiom for ulll~soulld contrast enhancçnnent cnmrri~in~ a plurality of gas bubbles in a liquid medium, with the gas colll~lisil1g a fluoromono-or fluoropolyether, or a llliAIUlG thereof is disclosed. In some embo-lim~nt~, the gas cornrri~çe a compound having an Ostwald coefficient of less than about 100 x 10-6 at 37 degrees C, leading to espeçi~lly long in vivo contrast rnh~nr~ml?nt Vapor of perfluorodiethylether, perfluorodimethylether, perfluoromethylethylether,perfluorornonoglyme, perfluorodiglyme,C4Fl003,C5FI204,C6F~4O5 have been found to be especially advantageous.
The gas bubbles of the present invention may be surrounded by a ~ulra.;~-layer which preferably comprises a first and a second surfactant, the first ~llrf~rt~nt con~ tin~ es~nti~lly of a phospholipid or ",ixlu,~ of phospholipids having at least one acyl chain which comprises at lea~st 10 carbon atoms, and comprising at least about 5% w/w of total sllrf~r,t~nt, with the second sl~rf~rt~nt being more watersoluble than the first surfactant. Most preferably, the first sl~ rt~nt compri~çs a phnsph~ti~lylcholin~ ~,vith one or more acyl chains, at least one chain comprising 12 l:o 18 carbon atoms, and said second sllrf~r,t~nt comrri~çc a phosphatidylcholine with one or more acyl chains, at least one chain compri~ing 6 to 12 carbon atoms.

CA 02222186 1997-ll-2~
W O 96/40281 PCT~US96/09068 Moreover, in a broad aspect the present invention provides microbubble precursors and methods of forming gas emulsions. Those skilled in the art will appreciate that the microbubble ~lcp~lions of the present invention may be ed using a number of dirLlelll techniques. For example, microbubbles may be formed using the disclosed fluoroether compounds in conjunction with powders,protein microspheres, spray dried microspheres, void co~ particles, particulates, liposomes, saturated sugar solutions, etc. Each of these structural materials may further be used to provide dried microbubble precursors when a fluoroether is dispersed therein. Upon addition of a liquid lllediulll, preferably water, gas emulsions may be formed.
In a preferred embo~im~-nt the microbubbles are produced by spray drying a liquid fc-rm~ tion coll~ g a biocompatible membrane-forming material to form a microsphere powder ther~flulll, combining the microspheres with the low Ostwald coefficient fluoroether compounds as disclosed herein, and mixing an aqueous phase with the powder. The microsphere powder subst~nti~lly dissolves in the aqueous phase to form microbuWles. Preferably, the microbubbles are coated with a monolayer of surfactant.
Further, the present invention provides for methods of im~gin~, including harmonic llltr~cnic im~ing, using the disclosed gas emulsions.
Other objects, r~Lu,es and advantages of the present invention will be ay~ lll to those skilled in the art from a con~ r~tion of the following detaileddescription of plcrcll~,d e~mrl~ry embo~ thereof taken in conjunction with the Figures which first will be described briefly.

Brief Des~fiyLion of the Figures Figure 1 is a graph of the in vivo pulsed Doppler signal intensity as a function of time from two fluoroether gas emulsions acGu~dhlg to the present invention versus air.
S Figures 2a, 2b and 2c are graphical ~ res~ lions of the decay of ultrasound signals over time following injection of gas emulsion contrast media into a rabbit.
Each individual graphical le~le3~,~lL~Lion is arranged in such a way that microbubble epaldlions comprising fluoroethers are colllyal~d to prior art microbubble e~a-aLions comprising fluorocarbon analogues.

CA 02222l86 l997-ll-2S
W O 961402~1 PCT~US96/09068 Figures 3a, 3b, and 3c each show two ul~l~soulld images of a pig heart before the injection of the bubble contrast media (Fig. 3a), l min (Fig. 3b) and 6 min (Fig.
3c) after injection. In the Figures, the top image (other than the control images) is g~ led using a microbubble prc~dLion comprising a perfluoropolyether, C5F,204, ~vhile the bottom image was gcllcl~led using a microbubble ~rc~Lion compri~in~
perfluorohçY~n~, C6F,4.

Detailed Desc~ ion of the Invention I. General As used herein, microbubbles are considered to be bubbles of gas in an aqueous me~ m having a ~ mPt~r b~lwccll about 0.5 and 300 ,um, preferably having a ~ meter no more than about 200, lO0, or 50 ~m. Microbubbles may or may not have a layer or coating at the gas/liquid intl-rf~ce. If present, the coating rnay be one or more molecules thick. Additionally, m-icrobubbles may be trapped by a bimolecular layer (as in the case of 1mi1~m~ r liposomes), or may be trapped by several layers of bilayers (m111ti1~m~ r vesicles). The microbubbles of the present invention may also be ~ ded by more p~ shell-like structures such as denaLuled proteins.
As ~mll1~io~ are generally char~rteri7~(l as a dispersion of two or more immi~cihle fluids stabilized by a surfactant int~rf.qre, the surfactant co~ g embo-1im~nt~ of the present invention are in essence gas emulsions, with the discol.lilluous phase of the emulsion being a g~, rather than a liquid. Consequently, 1he term "gas em111~io~", as used herein, comprises a dispersion of a plurality of microbubbles of gas in an aqueous ."~-1;..." with or without a surfactant interface.
That is, the gas emulsions of the present invention are simply microbubble pl~cpaudLions comprising a fluoroether.
For intravascular use, o~ lu ll bubble size is ~lett--rmin~(l by two col~ ,Lmg concerns. ~m~11er bubbles are effective in circ1~1~ting through small blood vessels and capillaries, but ullldsoulld echogenicity is strongly dependent upon bubble size.
Suitable microbubbles for vascular ull,as(Julld co~ enh~nrem~nt are therefore preferably about 1 - lO ~m in ~i~met~or~ with 3-5 ~m especially preferred.

W O 96/40281 PCT/US96/0~0~8 II. Selecting microbubble ~ases and ~as combinations The short lifetime of most microbubble pl~dlions is caused in part by the increased gas ~les~ inside the bubble, which results from the surface tension forces acting on the bubble. This elevated intPrn~l plei~UlC increases as the diarneter of the bubble is reduced. The increased intPrn~l gas pressure forces the gas inside the bubble to dissolve, resl~lting in bubble collapse as the gas is forced into solution. The Laplace equation, ~P=2c~/r, (where ~P is the increased gas ~le,~u-c inside the bubble, c~ is the surface tension of the bubble film, and r is the radius of the bubble) describes the ple~uc exerted on a gas bubble by the surrounding bubble surface or film. The Laplace 1J1e~U1C iS inversely ~l~ullional to the bubble radius, thus, as the bubble shrinks, the Laplace plc~ e increases, h~clea~ing the rate of diffusion of gas out of the bubble and the rate of bubble shrinkage.
Quay's formula for bubble lifetime (Equation 1) ignores this factor.
Difr~ .l conclusions regarding gas suitability result when one considers the effect of the bubble T.~rl~-~e ~le~ ; in conj~l ;lion with the fact that the blood naturally contains certain gases, such as nitrogen, at near ~tmosphPric prc~ule. More specifically, it leads to the conclusion that a gas ~- ixlu~c of a "~lilll~U ~ modifier gas"
such as nitrogen, or air, or another gas n~t lr~lly abundant in the blood, in combination with a "gas osmotic agent" of low water solubility and high vapor plc:i~ule results in o~lilllulll bubble lifetime. Some embo~limPnt~ of such gas ...ixlu.~s are described in co-pending U.S. Patent Application Serial Nos.
08/099,951; 08/284,083; and 08/395,680 herein incol~,oldled by reference.
The stabilizing influence of proper gas combin~ti- n~ can be understood more readily through a discussion of certain hypothetical bubbles in aqueous solution.
The bubbles ~ c~ e(1 may all be considered to be surrounded by a layer of surface tension re~ ring s~rf~c-t~nt However, the effects of gas or gas combinations with differing solubilities, s~rf~t~nt membrane layer permeabilities, and e~ctPrn~l concentrations will be considered.
The physical interactions of the pll-ll~ modifier gas, secondary osmotic agent, and medium can be incul~o.dlcd into a general theory of bubble behavior.
In a solution co..l;.;..i..g a relatively high collc~,.-L,dlion of the ~filll~ modifier gas (as co...~ed to the concel-lldlion in solution of the gas osmotic agent), bubble lives can be ~lPtPrminP~l theoretically as a function of certain physical characteri~ti~s of the secondary gas osmotic agent.

W O 96/40281 PCT~US~6/0~068 _9_ Consider a microbubble of radius r, co, .~ g two ideal gases: air (nitrogen) (na moles) and osmotic agent (nF lmoles). The microbubble is in an infinite water met1illm, which contains no osmotic agent and is saturated with an infinite supply c,f air. Air is much more soluble in water and dirruses quickly out of the S microbubble. Treating the microbubble in a manner analogous to a se.l.i~cl.. eable membrane, we may consider that the çh--mir~l potential of air in the microbubbleis the sarne as in the infinity, whereas the chemical potential of the fluorocarbon in ~dhe microbubble is higher than that in the infinity. Mechanical equilibration to the plei,~ul~, gradient across the interface is ~llme-l to be fast. Thus, it is the diffusion of osmotic agent out of the microbubble that clet~?~mines the microbubble lifetime.
The ple;,~ul~ inside the microbubble is the sum of the partial ples~ules of the air and the fluorocarbon:

pb=pb+pb Because air is very soluble in the water .. e~ .. , and ~lirr~es into and out a,f the bubble quickly, net mass flow of air is small, and the partial ~re~ of the air inside the rnicrobubble is approxim~tely equal to the atmospheric air l~ie,,~
applied to the water .,.~1;..", This means that the excess Laplace plc;~:~w~ is due to osmotic agent only:

pb=20 = F RT
r 4~

(4) Furth~ ....u.e, the steady-state diffusional mass flow J (mol/s) of the osmotic agent from a spherical particle into the medium with zero concentration in the metlillm is equal to:

J=4~cr2D CF,subs r (S) Here D is the osmotic agent-in-water diffusion coefficient, and cFSL,bS"~is the equilibrium ~l s~ r~e osmotic agent-in-water concel-LIdlion. We assume the L~r;~ce osmotic agent concentration in water to be in equilibrium with the W O 96/40281 PCT~US96/09068 fluorocarbon in the microbubble. Because the vapor is unde.sdLuldl~d, the ~ub~ulrdce concentration of the microbubble osmotic agent is lower than its ~dLuldled concentration, and is related to the internal osmotic agent vapor ~ u-e as follows:

..
C --C (T) PF C (T) 2a (6) From Equations 4, S, and 6, it follows that:

d ~=3DRT--dt PF,~

RT--10Note that the combination PF~ is ~lim~n~ nlecs and has within it the ratio of the ~nll--,-~ecl osmotic agent vapor p~ UI~ to the corresponding equilibrium osmotic agent water solubility. This ratio is known as the Ostwald coefficient (often denoted "L"). The square of the microbubble radius decreases with time at a rate~u~u~lional to the Ostwald coefflci~nt of the gas osmotic agent. Accordingly, gas 15osmotic agents with low Ostwald coefflci~nt~ provide superior bubble longevity.
The Ostwald coçffiçient of the gas osmotic agent is preferably less than about 500 x 10-6, 100 x 10-6, or 50 x 10-6, most preferably less than about 40 x 10-6, 30 x 10-6, 20 x 10-6, 10 x 10-6, 5 x 10~, or 1 x 10-6.

CA 02222l86 l997-ll-25 W O 96J40281 PCT~US96/09068 fjlling gas b.p., ~C c PF.~ at~n t~ o6 ~2 -183 31110 :~i SF6 b8 23.5 sgso CF~ -128 159 5172 C2F6 -78 26.2 1273 CF30CF, -59 10.9 932 n-C3F, -37 6.8 583 CF3OC2F5 -21.5 3.9 316 n-C<F~o -2 2.2 212 C2FsOC2Fs 1 1.9 73 CF,OC2F~OCF3 17 1.16 36 n-C5Fl2 29 0.84 66 CF30C2F~OCIFs 38.5 0.55 9.0 n-C6F,4 57 0.27 24 C3F,OC3F, 56 0.30 6.7 CF3O(CF2CF2O)2CF3 64 0.20 0.9 (, ~
T. M. Reod, 111, in: Fluorine Chemistry, J. H. Simons, Ed., V. 5, Acsdemic Press, New York and London, 1964, p. 133; A. A. Woolf, J. Fluorine Chem., 63 (1993) 19; V. V. Berenblit, Yu. P. Dolnakov, V. P. Sass, L.
N. Scnyushov, and S. V . Sokolov, Zh. Org. TChim., 10 (1974) 2031, and ~
If not present in Refs. 1, cslculated with the model of D. D. Lawson, J. Moscanin, K. V. Scherer, Ir, T. F.
Terrarlova, and J. D. Ingham, J. Fluorine Chem., 12 (1978) 221.
t~'~ The first four values as reporbed by E. Wilhelm, R Bsttino, and R ~. Wilcock, Chem. Rev., 77(1977) 219.
The others estimsted as described in: A. S. Kabalnov, K. N. Maksrov and E. V. S' ' ' ' ~.. , J. Fluorine Chem., 50 (1990) 271.

Table 1. Ostwald coefficients and vapor pl~ ul'eS at 25 degrees C

Table 1 shows the solnhilities, vapor ~lei,~ules, and Ostwald coefficients of several co~ oullds, in~ tling certain bioco~ ble fluorocarbons. Table 1 illu~lldl~s that perfluorobutane and pel~luor~ ~le~ which are gases at body telll~c~dlulG and atmospheric ~/le;~:iUl'G, and which are co~ "~ tecl as bubble gases by Quay and SchnP;(ler, have low Ostwald coefficients, and therefore also p~,.rOllll suitably as gas osmotic agents in conjullclion with a primary modifier gas. However, the abilit,v to consider c~n~ te compounds which are liquids at body tenl~Gldl lre and ~ttnosrheric plGs~ule allows the selection of certain optimal low Ostwald coefficient compounds that have not previously been considered in any way suitable for microbubble pl~dldLions.
It should be remembered that Equation 7 is valid for bubbles col~ g gas combin~tion~ where one of the gases is already present in the blood~lle~ll, and where that gas (the ~Iplilll~ modifier gas") can diffuse across the gas/liquid CA 02222l86 l997-ll-25 W O 96/40281 PCT~US~6/090C8 interface much faster than the other gas (the "gas osmotic agent") in the combination. Only then is the partial pressure of the gas osmotic agent in the bubble equal to only the Laplace ple~ulG rather than the total ~lGi7iiUI~ inside the bubble. Because the T ~pl~r,e plcs~ulc may be less than 1 ~tmosph~re (at least for a large yG~cGlllage of a bubble's lifetime) it is possible to use gas osmotic agents that are liquids at body ~e~ clalule and atmospheric pLei,.,ule. Such compounds would not form bubbles at all without the additional ple3Gllce of the primary modifier gas.
On the other hand, although the gas osmotic agent can be a liquid at body lcllll)clalule~ its saluld~ed vapor ~ i'iUlc must be large enough so that the Laplace ples~,ule does not imm~ tely force the gas osmotic agent in the bubble to colldensG- into a liquid. The salulaled vapor ~ Ulc of the gas osmotic agent is preferably larger than appl~x;~ t~ly 100 torr. Perfluorinated hydrocarbons, previously co..~k~ ted as microbubble filling gases have generally correlated water solubilities and salulaled vapor plci7~ S. That is, choosing a fluorocarbon with reduced water solubility also meant choosing a fluorocarbon with leluced ~alulalcd vapor ~l~i7-jUlc.
In this invention, we disclose a previously unconsidered class of compounds which combine a reduced water solubility without a significantly reduced salulaled vapor ple~ ulc, and thus these compounds have surprisingly low Ostwald coefficients. These compounds are the flllorin~t~cl mono- and polyethers. Fhl~-rin~t~(l mono- and polyethers are known to be safe and non-toxic. It is also known in the art (D.D. Lawson et al., ~ Fluorine Chem. 12, p 221 (1978)) that these compounds have a very high vapor pleij~ulc and low boiling point at a given number of carbon atoms. Thus, the boiling point and salu,aled vapor ~rc~ c of a fluorinated polyether are almost the same as those of its fluorocarbon analogue with the same carbon number.
However, the water solubility, and thus the Ostwald coefficient, of the fluoroethers is lower than that of the fluorocarbon analogues - the value de.;,~ases by a factor of 2 - 3 with each oxygen atom added. Normally, it would be c~e-iled that the ~ lition of an oxygen atom capable of hydrogen bonding to water would lead to an increase in solubility. It has been found cx~cl;...rnt~lly that especially long lived co"l,a~l ~nh~nr.t~m~-.nt gas ennlll~ic)n~ may be ~,cp~cd when the gas bubbles contain air or nitrogen mixed with a fluoromono- or W O 96J4~281 PCT~US96/09068 polyether. Accordingly, perfluorodiglyme, CF3(0CF2CF2)20CF3, perfluoromonoglyme, CF30CF2CF2CF3, perfluorodiethylether, C2F50C2F5, perfluoroethylmethylether, CF30C2F5, perfluorodimethylether, CF30CF3, and perfluoropolyethers such as CF30CF20CF3, CF3(0CF2)20CF3, CF3(0CF2)30CF3, and CF3(0CF2)40CF3 have been found to be especially suitable gas osmotic ~ agents.
A wide variety of flllorin~te~1 ethers have the above described ~rop~lies which make them especially suitable as gas osmotic agents for stabilizing gas ~m~ ions. Dep~?n-ling on the number of carbon atoms, the fl~lorin~ter1 ethers nnay be either gases or liquids at body le~ dLule and atmospheric plc;,~ul~.
Those flllo~ ecl ethers which are gases at body L~ dLul~; and ~tTno~pheric iUl~ are also useful as the sole ~5dseuus co,l,~ol~e"l of a gas etnlll~ion udLion. A ~ mn-lifier gas, though i"ll"ovulg the efficacy of gas emlll~inn~ made with all gas osmotic agents, is not required if the fluorinated ether used is a gas at body t~ ; n~u-~ and atmospheric ple~u,e. Fu~ ore, usefill fluorinated ether osmotic agents may be either completely or only partially fluorin~te~l Some of the partially hydrogenated fluorinated ethers which are useful as gas osmotic agents accoldi,lg to the present invention are:
CH3CH20CF2CHF2, CH3CH20CF2CF3, CHF2CH20CF2CHF2, CF3CH20CF2CH2F, CF3CH20CH2CF3, CF3CH20CF2CHF2, CHF2CH20CF2CF3, CF3CH20CF2CF3, CH30CH2CF2CHF2, CH30CH2CF2CF3, CH30CF2CF2CHF2, CH30CF2CHFCF3, CH30CF2CF2CF3, CHF2OCH2CF2CHF2, CHF2OCH2CF2CF3, CF30CH2CF2CHF2, CF30CH2CF2CF3, CH30CH(CF3)2, CH30CF(CF3)2, CHF20CH(CF3)2, CH30CH2CHF2, CH30CF2CH2F, CH30CH2CF3, CH30CF2CHF2, CHF20CH2CHF2, CHF20CF2CH2F, CHF20CH2CF3, CHF20CHFCF3, CF30CH2CHF2, CH30CF2CF3, CF30CH2CF3, alld CF30CHFCF3.
Once a suitable low Ostwald coefficient gas is chosen, preferably a flu-)rin~ted ether, microbubbles incol~ldli"g the gas may be formed in a varietyof ways, both with and without a shell or surfactant int~ l layer, as is described in detail below.

III. Microbubble form~tic n and ~llcay~ulation Microbubble ~l~d~ion methods include the form~tion of particulate microspheres through the ultrasonication of albumin or other protein as described CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 in E~u~e~l Patent Applications 0,359,246 and 0,633,030 by Molecular Bio~y~L~ ls, Inc.; the use of ten~i-les and viscosity increasing agents as described in U.S. Patent No. 4,446,442; lipid coated, non-liposomal, microbubbles as is described in U.S. Patent No. 4,684,479; liposomes having c..Lld~ped gases as is S described in U.S. Patent Nos. 5,088,499 and 5,123,414; the use of ~ l,iccompounds as is described in U.S. Patent No. 5,445,813; the use of lipid ~.l~ell~ions as is described in PCT published application WO 96/08234; the use of l~llhl~;;~cd surf~ct~nt~ as described in U.S. Patent Nos. 5,271,928 and 5,380,519; the use of microparticulates as described in U.S. Patent Nos.
0 4,442,843, 5,141,738 and 4,657,756; and the use of albumin particulate microspheres as is described in U.S. Patent No. 4,718,433. The disclosure of each of the foregoing patents and applications is hereby incorporated by reference.
It will further be appreciated by those skilled in the art that the gas em~ ion~ of the present invention include pl~udlions of free gas microbubbles comprising fluoroethers. That is, in selected embo~lim~nt~ the gas emulsions of the present invention may be formed without the use of a surfactant as describedin U.S. Patent Nos. 5,393,524 and 5,049,688 which are incul~uldled herein by 1cr~.~ce.
In plcfc.led embo~lim~ntQ the microbubble ~ ~alions may be p.~ d using sonication. Sonic~tiorl can be accompli~hP~l in a number of ways. For Px~mple, a vial colll;~ g a sllrf~ct~nt solution and gas in the h~ p~ce of the vial can be sonicated through a thin membrane. Preferably, the membrane is less than about 0.5 or 0.4 mm thick, and more preferably less than about 0.3 or even 0.2 mm thick, i.e., thinner than the wavelength of ultrasound in the material, in order to provide acceptable tr~n~mi~ion and ...i..;..~ membrane hP~tin~ The mPnnhr~n~ can be made of m~t~ri~l~ such as rubber, Teflon, mylar, urethane, s~h~ d film, or any other sQnic~lly Ll~lu~e~lL synthetic or natural polymer film or film forming m~tPri~l The soni~tion can be done by contacting or even dc~ ing the membrane with an ultrasonic probe or with a focused ultrasound "beam." The ultrasonic probe can be disposable. In either event, the probe can be placed against or ~.~s~.led through the membrane and into the liquid. Once the soniC~tion is accomrli~hPd the microbubble solution can be withdrawn from and vial and delivered to the patient.

W O 96140~81 PCTnUS96/0~068 Sonication can also be done within a syringe with a low powe}
ultrasonically vibrated a~l,i,dling assembly on the syringe, similar to an inkjet printer. Also, a syringe or vial may be placed in and s()n~ te~l within a low power ultrasonic bath that focuses its energy at a point within the container.
Mechanical form~tion of microbubbles is also cont~lrnrlated. For ~ example, bubbles can be forrned with a mechanical high shear valve (or double syringe needle) and two syringes, or an a~halor assembly on a syringe. Even simple ch~king may be used. The ehrinkin~ bubble techniques described below a~re particularly suitable for meeh~nic~lly formed bubbles, having lower energy input than sonicated bubbles. Such bubbles will typically have a diameter much larger than the llltim~t~ly desired biOc~-...pn~ihle im~ping agent, but can be made to shrink to an a~-~-iate size in accol-l~,ce with the present invention.
In another method, microbubbles can be formed through the use of a liquid osmotic agent emlll~ion ~u~ dlulal~d with a modifier gas at elevated ples~ule introduced into in a ~--.r~ ." solution. This pro~l~ctiQn method works similarly to the opening of soda pop, where the gas foams upon release of l~,S:iUl~ forming the bubbles.
In another method, bubbles can be formed similar to the foaming of shaving cream, with perfluorobutane, freon, or another like material that boils when ple~:iUl~. iS released. However, in this methl-cl it is desirable that the ~nlll~ifif-d liquid boils sufficiently low or that it contain numerous bubble nl-clt~tion sites so as to prevent ~u~ g and su~e,~lu,dlion of the aqueous phase. This ~u~ l;Qn will lead to the g~.,eldLion of a small number of large bubbles on a limited number of nllele~tion sites rather than the desired large n~lmber of small bubbles (one for each droplet).
In the alternative, a lyophili7~d cake of sllrf~t~nt and bulking reagents produced with a fine pore or void-co--l;1;--;--g SI~UC;~Ul~i can be placed in a vial with a sterile solution and a head spaced with an osmotic gas mixture. The solution can be frozen rapidly to produce a fine ice crystal structure and, therefore, upon lyophili7~tion produces fine pores (voids where the ice crystalswere removed).
~lt~rn~tively, any dissolvable or soluble void-forming structures or materials, such as powdered and gr~n~ te~l sugars, may be used. It is not n~C~ that such structural m~teri~l~ define a plurality of voids prior to the CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 addition of a liquid mediurn. Further, while it is preferable that the void-forming structures comprise a surfactant, this is not required for practicing the present invention. In this emborlimPnt where the void-forming m~t~?ri~l is not made from or does not contain surfactant, both s~rf~ct~nt and liquid are supplied into the container with the structures and the desired gas or gases. Upon .ec~ l;on these voids trap the osmotic gas and, with the dissolution of the solid cake or powder, form microbubbles with the gas or gases in them.
In still another method, dry void-co.,~ .;.,g particles or other structures (such as hollow spheres or ho.~Gyco-..bs) that rapidly dissolve or hydrate, preferably in an aqueous solution, e.g., albumin, microfine sugar crystals, hollow spray dried sugar, salts, hollow ~u.r~ l spheres, dried porous polymer spheres, dried porous hyaluronic acid, or ~ul~LilulGd hyaluronic acid spheres, or even commercially available dried lactose microspheres can be stabilized with a gas osmotic agent. Moreover, while dGn~lul~d protein microspheres are not particularly soluble, they are co--ly~Lible with the present invention an may beused as void-co..~ g ~l u-;lu es in accor~-ce with the te~rhing.c herein.
Accordingly, in a broad aspect the present invention provides microbubble precursor compositions comprising:
a structural m~feri~l defining a plurality of voids;
a gas or gas ~~ixLu e comprising a fluoroether dispersed in said voids, and a sl~rf~ct~nt wh~,.cin said structural m~tçri~l, said gas or gas llliXLulci and said sllrf~t~nt are together adapted to form microbubbles upon addition of a liquid to said co..l;~
It will be appreciated that, as used herein, the term "structural material"
shall be held to mean any material rl~fining a plurality of voids that promotes the formation of bubbles upon combination with a liquid medium. Such structural m~tt?ri~l~, which include both void-co..l~il.i..g and void forming structures may be soluble or insoluble in an aqueous environment. Exemplary structural materials that are co...~ ihle with the present invention include, but are not limited to, spray dried powders, powdered or gr~n--l~te~l sugars, protein microspheres including denatured proteins microspheres, lyophili7~-1 cakes, lyophylized powders, salts, hollow ~--- ri1- L;l~L spheres, dried porous polymerspheres and dried porous hyaluronic acid. In particularly ~..crc~l~,d emborli...e..
the ~L U;LUldl m~t~ri~l compri~es a surfactant.

W O 96)40281 PCT~US~6/0~0~8 -17-Preferably, gas emulsion compositions incorporating low Ostwald coefficient gases are ~,~cd by spray drying an aqueous dispersion which contains a hydrophilic monomer or polymer or combination thereof. This plrocedure is also described in detail in co-pending U.S. Patent Application Serial No. 08/405,477. In this case, a bubble forming composition is formed by spray drying an aqueous dispersion of a hydrophilic moiety such as starch, preferably also including a surfactant, to form a structual material. More particlularly, form a powder of dry, hollow, al.~rox,l"ately microspherical porous shells of a~lo~ t~ly 1 to 10 ~m in diameter, with shell thill~n~ ;e~ of a~rox,,,lalely 0.2 ,um. Commercially available spray dryers are well known to those in the art,and suitable settings for any particular starch/snrf~t~nt dispersions can be readily ~l~ternnin~d through standard empirical testing, with due reference to the examples that follow. After formation, the desired gas is made to p~rmP~te the structural m~teri~l or dry microspheres by placing the microspheres into a vial,ev~ tin~ the air, and replacing it with the desired gas or gas lllixlul~,.
The hy~Lv~hilic moiety in the solution to be spray dried can, for e~mrle, be a carbohydrate, such as glucose, lactose, or starch. Polymers such as PVA or PVP are also cc"lt;",~lated. Various starches and derivatized ~ hes have been found to be especi~lly suitable. Particularly p,~r~.,ed ~l~cl1es for use in formation of microbubbles include those with a molecular weight of greater than about 500,000 daltons or a d~ se equivalency (DE) value of less than about 12. The DE value is a ~u~u,lil~liv~ measurement of the degree of starch polymer hydrolysis. It is a measure of re~ cing power col~ a~ed to a dextrose standard of 100. The higher the DE value, the greater the extent of starch hydrolysis. Such ~lcfe,l~,d starches include food grade vegetable starches of the type commercially a~allable in the food industry, including those sold under the tr~1em~rk~ N-LOK
and CAPSULE by National Starch and Chemical Co., (Bridgc;w~ler, NJ);
d~.iv~li~;d ~l~ches, such as hy~ ,xy~lhyl starch (available under the tr~fiem~rk~
H:ETASTARCH and HESPAN from du Pont ph~rm~euticals~ M-Hydlc~xy~lhylstarch from Ajinimoto, Tokyo, Japan). However, due to particularly advantageous stabilization char~ct~-ri.ctics, starches with a molecular weight of 500,000 or above are ~c;fel~ed (Note that short chain starches spray dry well and may be used to produce microbubbles in acco,dance with the present invention.) The hydrophilic monnmer or polymer is present in this CA 02222l86 l997-ll-2~

embodiment of the precursor solution at a range of about 0.1% to 10% w/v of solution, with about 1% to 5% w/v having been found to be especially suitable.
Preferably, the aqueous dispersion also includes an optional surfactant or mixture of surf~rt~nt~, provided at about 0.01% to 20% w/v of solution. Many S surf~ct~nt~ and s lrf~ct~nt lllixlu~es are known and may be used. SllrfRrt~nt~ may be selected from the group con~i~ting of phospholipids, phosphocholines, lysophospholipids, nonionic s~rf~rt~nt~, neutral or anionic s~rf~ct~nt~, fluorinated surf~rt~nt~, which can be neutral or anionic, and combinations of such emulsifying or foaming agents. Other specific examples of sllrf~ct~nt~ include block copolymers of polyoxy~ ylene and polyoxyethylene (an example of such class of compounds is Pluronic, such as Pluronic F-68), sugar esters, fatty alcohols, aliphatic amine oxides, hyaluronic acid aliphatic esters, hyaluronic acid aliphatic ester salts, dodecyl poly(ethyleneoxy)ethanol, nollyl~hc;lloxy poly(ethyleneoxy)ethanol, d~livdli;~d starches, hydroxy ethyl starch fatty acid esters, salts of fatty acids, commercial food vegetable starches, dextran fatty acid esters, sorbitol fatty acid esters, gelatin, serum albumins, and combin~ticn~
thereof. Also colllc;ll~lated are polyl~xyt;lllylene fatty acids esters, such aspolyoxy~(llylene ~le~dl~s, polyoxy~lhylene fatty alcohol ethers, polyoxyethylated solbi~ fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated ~oyl,eall sterols, ethoxylated castor oils, and the hydrogenated d~ivdlives thereof. In addition, nonionic alkylglucosides such as Tweens'l9, Spans~ and Brijs'lD are also within the scope of the present invention. The Spans include so~ tetraoleate, sorbitan tetrastearate, sorbitan tristearate, sorbitan trip~lmit~t~o, soll,ilall trioleate, and soll~ilal~ distearate. Tweens include polyoxyc;lhylene sorbitan tristearate, pOlyOxy~lllyleIle solbil~n trip~lmit~te, polyoxyethylene sorbitan trioleate. TheBrij family is another useful category of m~trri~l~, which includes polyuxyt;lhylene 10 stearyl ether. Anionic surfactants, particularly fatty acids (or their salts) having 6 to 24 carbon atoms, may also be used. One example of a suitable anionic surfactant is oleic acid, or its salt, sodium oleate. Also suitable are cationic surfactants and their salts, such as dodecyltrimethylarnmonium chloride.
It will be appreciated from the foregoing that a wide range of ~--, can be used. Indeed, virtually any s -rf~ct~nt (including those still to be W O 96J40281 PCT~US~G/'030~8 _19_ developed) or surfactant combination can be used in the present invention. The ~ Ihllwll slrf~rt~nt for a given application can be d~r..lli..ed through ~nnrirics~1 studies that do not require undue c~ nt~tion. Consequently, one practicing 1he art of the present invention could select a surfactant primarily based S ~,lo~ lies such as bioco.. -~ bility.
It has been found especially suitable for the solution to contain a llli~lW~, of surfiqct~nt~ inr111-1ing a hydlopllobic rhnsrho1ipid as a first surfactant and at least one ~ ition~l more hyd~ l~ilic second surfactant. Preferably, the hy~o~hobic phospholipid has at least one acyl chain with a total of at least about 10 carbon atoms (e.g. a ~ ec~noyl phospholipid). In some embo(1im~nt~, the phospholipid first s11rf~rt~nt will have acyl chains from about l0 or 14 to about 20 or 24 carbon atoms. For t;~ JlC, fiir~1mitoylphosphatidylcholine (Comrri~ing two acyl chains, each comrri~in~ l6 carbon atoms) may be used.
The acyl chain may be hydrogen~ted or fl11orin~te~1 Other rhc slr~holipid head lS groups are also con~ lated. For example, the phosphatidy1~erine~, phosph~ti~1ylglycerols, or phosphatidylethanol~nnines will have l,l~c.lies suited to the present invention. Combinations of such phosphnlipids can also comprise the "first s11rf~r,t~qnt " as can n~t~lr~lly derived phospholipid products such as egg or soy lecithin, or lung s11rf~rt~nt~ In ~ 1itinll, the phospholipid first s11rf~rtzlnt may be suppl~ P~l with other highly water insoluble sl1rf~rt~nt~ such as sucrose di-, tri-, and tetra-esters. Cholesterol may also supplennt-nt the firstsurfactant, and has been found useful in promoting stability when provided in a range from about 0.0l to 0.5 w/w cholesterol to phospholipid. Preferably, the acyl chains of the phospholipid are saturated, although uns~lu,dLed acyl groups are also within the scope of the present invention. The first surfactant is preferably provided in a range from about 0.005% to 20% w/v of the solution, rnost preferably in the range of 0.02% to 10% w/v.
It has been found to be advantageous to use a phospholipid mixture compri~inp~ a rcl~Liv~ly hydrophobic long acyl chain phospholipid in combinationwith a shorter chain rhn~rh-lipid which is more hydrophilic than the first phospholipid. As a specific example, a first phosrholipid having acyl chains with 12 or 14 carbon atoms may be provided with a second phospholipid as a co-ct~nt having acyl chains with eight or ten carbon atoms.

CA 02222186 1997-11-2~
WO 96/40281 PCT~US96/09068 -20-It has been found particularly advantageous to provide phospholipid comprising 12 carbon atom acyl chains as either the first or second surfactants.For example, a phospholipid with 12 carbon atom acyl chains may comprise the first surfactant, and a sugar ester or Pluronic compound can comprise the secondsurfactant. As another option, a phospholipid with 16 carbon atom acyl chains may comprise the first surfactant, and a phospholipid with 12 carbon atom acyl chains may comprise the second snrf~rt~nt The spray dried product llltim~t~ly produced is a more effective bubble producer if an infl~tinp agent, preferably a fluorocarbon such as Freon 113, is dispersed in the starch/surfactant solution described above. The infl~ting agentcan be any material that will turn to a gas during the spray drying process. Theinfl~ting agent is dispersed throughout the surfactant solution, using, for in~t~nr,e7 a commercially available microfluidizer at a ple~ule of about 5000 to 15,000 psi. This process forms a conventional emulsion comprised of submicron droplets of water immiscible Freon (or other infl~ting agent) coated with a monomolecular layer of snrf~rt~nt Dispersion with this and other techniques are common and well known to those in the art.
The inclusion of an infl~ting agent in the solution to be spray-dried results in a greater ultrasound signal per gram of spray-dried powder by forming a greater number of hollow microspheres. The infl~ting agent nucleates steam bubble formulation within the ~lo...i~l droplets of the solution entering the spray dryer as these droplets mix with the hot air stream within the dryer. Suitable infl~ting agents are those that :iu~ dLulale the solution within the atomized droplets with gas or vapor, at the elevated l~ lalul~ of the drying droplets (approximately 100~C). Suitable agents include:
1. Dissolved low-boiling (below 100~C) solvents with limited miscibility with aqueous solutions, such as methylene chloride, acetone and carbon lllficle used to saturate the solution at room telllpt;ldlul~,.
2. A gas, e.g. CO2 or N2, used to saturate the solution at room tc;lll~cld~u~e and elevated pl~:s~u~e (e.g. 3 bar). The droplets are then su~cl~dluldled with the gas at 1 atmosphere and 100 ~C.
3. Emulsions of immi~cihle low-boiling (below 100 ~C) liquids such as Freon 113, perfluorop~.,l~le, perfluoroh~ n~, perfluorobutane, pentane, butane, FC-ll, FC-llBl, FC-llB2, FC-12B2, FC-21, FC-21Bl, FC-21B2, CA 02222186 1997-11-2~
W o 96J'~028~ PCT~US96/09068 FC-31Bl,FC-113A,FC-122,FC-123,FC-132,FC-133,FC-141,FC-141B,FC-142,FC-151,FC-152,FC-1112,FC-1121 and FC-1131.
Infl~ting agents are added to the starch/surfactant solution in quantities of about 0.5% to 10% v/v of the sllrf~ct~nt solution. Approximately 3% v/v infl~ting agent has been found to produce a spray dried powder which forms suitable microbubbles. The infl~ting agent is subst~nti~lly ev~l,ol~Led during the spray drying process and thus is not present in the final spray-dried powder in more than trace qll~ntitiee Other optional components of this solution are various salts or other agents within the aqueous phase. Such agents may advantageously include conventional viscosity modifiers, buffers such as phosphate buffers or other convention~l biocomp~tible buffers or pH adjusting agents such as acids or bases, osmotic agents (to provide isotonicity, hyperosmolarity, or hyposmolarity).
Preferred solutions have a pH of about 7 and are isotonic. These additional ingredients each typically comprise less than 5% w/v of solution. Examples of suitable salts include sodium phosphate (both monobasic and dibasic), sodium chloride, calcium phosphate, and other physiologically-acceptable salts.
After spray drying, the various individual components of the microspheres preferably comprise the following ~.opo-Lions of the final spray dried product in % by weight:

Hydrophilic ~LI-l~;Luldl m~t~riz~l 1% to 100%
Surfactant 0% to 90%
Salts, buffer, etc. 0% to 90%
In particularly preferred embodiments, the composition has the following ~ropo"ions in % by weight:

Hydropl~ilic structural m~teri~l 10% to 60%
Surfactant 0.1% to 10%
Salts, buffer, etc. 10% to 60%

As mentioned above, the desired gas is made to perme~te the dry microspheres by placing the microspheres into a vial, which is placed in a CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 vacuum chamber to evacuate the air. The air is then replaced with the desired gas or gas mixture. The gas will then diffuse into the voids of the spheres.
Diffusion can be aided by pl~s~ulc or vacuum cycling. The vial is then crimp sealed and preferably sterilized with gamma radiation or heat.
Preferably, the first primary modifier gas (which may be air or any of its component gases such as nitrogen) and the second osmotic stabilizer gas (preferably having low Ostwald coefficient) are respectively present in a molar ratio of about 1:100, 1:75, 1:50, 1:30, 1:20, or 1:10 to about 1000:1, 500:1, 250:1, 100:1, 75:1 or 50:1. In a particularly ~lcr.~led embodiment, the gas is nikogen that has been saturated with perfluorodiglyme at 20 degrees C.

IV. p~ in~ and use It will be appreciated that kits can be plep~d for use in m~king the microbubble ~ cudLions of the present invention. These kits can include a container enclosing the gas or gases described above for forming the microbubbles, the liquid, and the s lrf~,f~nt The coll~aillcl can contain all of the sterile dry col,lpo~ , and the gas, in one chamber, with the sterile aqueous liquid in a second chamber of the same container. Alternatively, the surfactant may be solubilized in the liquid prior to adding.
Accordingly, in a broad aspect the present invention provides a method for plC~dl;llg a gas emulsion c~ g:
providing a col~ . having therein a structural material defining a plurality of voids, a ~"~ r~ and a gas or gas llliXLulc comprising a fluoroetherdispersed in said voids;
adding an aqueous liquid to said container; and, ~tlmi~ing said structural material, said surfactant and said aqueous liquid, thereby forming a gas emulsion in said collldillcl, said gas t?mlll.cion comprising bubbles of said gas or gas llli~s~ule surrounded by a layer of the surfactant.
"
Suitable two-chamber vial containers are available, for example, under the tr~-lPtn~rk~ WHEATON RS177FLW or S-1702FL from Wheaton Glass Co., (Millville, NJ). Another example is provided by the B-D HYPAK Liquid/Dry 5+5 ml Dual Chamber prefilled syringe system (Becton Dickinson, Franlclin WO 96~'~0281 PCT/US96/09068 Lakes, NJ; described in U. S. Patent 4,613,326). The advantages of this system include:
1. Convenience of use;
2. The aqueous-insoluble gas osmotic agent is sealed in by a chamber of aqueous solution on one side and an extremely small area of elastomer sealing the needle on the other side; and 3. a filtration needle such as Monoject #305 (Sherwood Medical, St. Louis, MO) can be fitted onto the syringe at the time of m~nnf~ctllre to ensure that no undissolved solids are injected.
The use of the two chamber syringe to form microbubbles is described in Example VIII.
It may be appreciated by one of ordin~ skill in the art that other t~,vo-chamber reco~ n systems capable of combining the spray dried powder with the aqueous solution in a sterile manner are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed bet~veen the water-insoluble osmotic gas and the envilo,ll"G"l, to increase shelf life of the product. VVhere a material nf cçs~s.. y for forming the microbubbles is not already present in the co~ ;-,e~, it can be packaged with the other components of the kit, preferably in a form or c~l";~ fradapted to f~cilit~te ready combLnation with the other co~ ollents of the kit.
Examples of particular uses of the microbubbles of the present invention include perfusion im~ging of the heart, the myocardial tissue, and dete. ",i~ ;on of perfusion char~cte~ ri~tics of the heart and its tissues during stress or exercise tests, or perfusion defects or changes due to myocardial infarction. Similarly, myocardial tissue can be viewed after oral or venous ~-lmini~tration of drugs designfd to h~,~ase the blood flow to a tissue. Also, vi~ li7~tion of changes inmyocardial tissue due to or during various inl~,v~lllions, such as COlOll~h,y tissue vein grafting, coronary angioplasty, or use of thrombolytic agents (TPA or skeptokinase) can also be rnh~nrerl As these col~ l agents can be ~mini~trred cc llvc;lliently via a p~ ;ldl vein to enh~n~e the vi.~ i7~tion of the entire circulatory system, they will also aid in the diagnosis of general vascular pathologies and in the ability to monitor the viability of pl~nt~l tissue ultrasonically .

CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 In a particularly l,rere~ d embodiment, the present invention provides for a method for harmonic ultrasound im~ginp using the disclosed gas emulsions as contrast agents. The bubbles of the present invention are especially useful in harmonic im~ging methods such as those described in co-pending United States S patent application 08/314,074. By optimi7ing the ability of the disclosed microbubbles to transform the frequency of the ultrasonic radiation to which they are subjected (the filn~l~ment~l), im~gin~ is enh~n~etl Thus, the present invention advantageously provides for the use of microbubbles capable of generating h~rmcrlics at medically useful ultrasound exciting amplitudes.
It should also be emphasized that the present invention have applications beyond ultrasound im~ging Indeed, the invention is sufficiently broad to encompass the use of phospholipid-cc,..~ g gas emulsions in any system, including nonbiological applications.
It will further be understood that other components can be included in the microbubble form~ tinn~ of the present invention. For example, osmotic agents, stabilizers, chelators, buffers, viscosity modulators, air solubility modifiers, salts, and sugars can be added to modify the microbubble suspensions for m~xi",u", life and contrast enhancement ~;rr~;livelless. Such considerations as sterility,isotonicity, and biocompatibility may govern the use of such conventional additives to injectable compositions. The use of such agents will be understood to those of ordh.~ y skill in the art and the specific quantities, ratios, and types of agents can be determined empirically without undue ~ l;lllent~tion.
Any of the microbubble plepdldLions of the present invention may be a~1mini~t~red to a ~,~,lL~bld~e, such as a bird or a m~mm~l, as a contrast agent for ultrasonically im~ging portions of the ~v~lLebldLe. Preferably, the vertebrate is a human, and the portion that is imaged is the v~eclll~tllre of the vertebrate. In this embor1imPnt, a small quantity of microbubbles (e.g., 0.1 ml/Kg [2 mg/Kg spray-dried powder] based on the body weight of the vertebrate) is introduced intravascularly into the animal. Other quantities of microbubbles, such as from about 0.005 ml/Kg to about 1.0 ml/Kg, can also be used. ~mzlging of the heart, arteries, veins, and organs rich in blood, such as liver and kidneys can be ultrasonically imaged with this technique.

W O 96~40281 PCT~US96/09068 V. Examples The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of plGr~led methods of practicing the present invention and are not limitin~ of the scope ofthe invention or the claims appended hereto.

Example I
Pl. ~dldlion of microbubbles throu~h sonication Microbubbles with an average number weighted size of S microns were ple~dled by sonication of an isotonic aqueous phase CO~ i"g 2% Pluronic F-68 and 1% sucrose stearate as surf~ct~nt~, air as a modifier gas and perfluorohex~ne as the gas osmotic agent.
In this e~.;,..ent 1 3 ml of a sterile water solution co..~ .P 0 9% NaCl, :2% Pluronic F-68 and 1% sucrose stearate was added to a 2.0 ml vial. The vial had a rPm~ining head space of 0.7 ml initially co.~ g air. Air sdluldl~d with perfluorohexane vapor (220 torr of perfluorohexane with 540 torr of air) at 25 degrees C was used to flush the h~ p~re of the vial. The vial was sealed with a thin 0.22 rnm polytetrafluoroethylene (PTFE) septum. The vial was turned holiGolll~lly, and a 1/8" (3 mm) sonication probe ~ rhed to a 50 watt sonicator rnodel VC50, available from Sonics & Materials was pressed gently against the septum. In this position, the septum s~dles the probe from the solution. Power was then applied to the probe and the solution was sonicated for 15 seconds, ~orming a white solution of finely divided microbubbles, having an average number weighted size of 5 microns as measured by Horiba LA-700 laser light scdlL~;lillgparticle analyzer.

Example II
Spray dryin~ of phospholipid-co..~ solution One liter of the following solution was ~repdled in water for injection: 2.0%
w/v Maltrin M-100 maltodextrin (Grain Procee~inE Corp. Mll~c~tine7 IA), 0.95% w/v sodium chloride (~llinrl~rodt, St. Louis, MO), 1.0% Superonic F-68 (Serva, ~Teiclrlherg, Germany), 1.0% w/v Ryoto Sucrose Stearate S-1670 (Mitsubishi-KaseiFood Corp., Tokyo, Japan), and 0.5% Lipoid E-100-3 hydrog~on~ted phospholipid (Ludwigshafen, Germany).

This solution was then spray dried in a Niro Atomizer Portable Spray Dryerequipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark) employing the following settinge hot air flow rate 39.5 CFM
inlet air temp. 245~C
outlet air temp. 100~C
atomizer air flow 350 liters/min liquid feed rate 1 liter/hr The dry, hollow spherical product had a diameter between about 1 ~M and about 15 ~4M and was collected at the cyclone separator as is standard for this dryer.
Aliquots of powder (250 mg) were weighed into 10 ml tubing vials, evacuated and sparged with perfluorohexane-saturated nitrogen at 13~C and sealed. The nitrogenwas s~luldl~d with perfluorohexane by passing it through three perfluorohexane filled gas washing bottles immersed in a 13~C water bath.
Upon lecon~liLulion with 5 ml of water for injection, numerous bubbles were observed by light microscopy, ranging in size from 1 to 20 microns. The fact that many approximately 1 micron bubbles could be observed for an appreciable time demon~L,dles the added stability gained by including a phospholipid in the formula as an additional non-Newtonian viscoelastic surfactant.

Example III
Perfluorodi~lyme ~as emulsion with sucrose ester/Poloxamer surfactant One liter of each of the following two solutions was ple~.d with the following ingredients for injection:
Solution 1:

3.9% w/v m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan) 3.25% W/V Sodium chloride (M~llinr~rodt, St. Louis, MO) 2.83% W/V Sodium phosphate, dibasic (M~llinckrodt, St. Louis, MO) 0.42% w/v Sodium phosphate, monobasic (~llinr~rodt, St. Louis, MO) Solution 2:
2.11% W/V Poloxamer 188 (BASF, P~i~ly, N~

W O 96/~028~ PCT/US96/09068 0.32% w/v Ryoto Sucrose Stearate S-1670 (Mitsubishi-Kasei Food Corp., Tokyo, Japan) 0.16% w/v Ryoto Sucrose Stearate S-570 (Mitsubishi-Kasei Food Corp., Tokyo, Japan) Solution 2 was added to high shear mixer and cooled in an ice bath. A coarse suspension of 30 ml of 1,1,2-trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, NJ) was made in the 1 liter of solution 2. This suspension was f!m~ if ietl using a Microfluidizer (Microfluidics Corporation, Newton, MA; model h~-~llOF) at 10,000 psi, 5~C for S passes. The resulting emulsion was added to solution 1. This mixture was then spray dried in a Niro Atomizer Portable Spray Dryer equipped with a two fluid ~tomi7Pr (Niro Atomizer, Copenhagen, Denmark) employing the following settings:
hot air flow rate 31 CFM
inlet air temp. 370 ~C
outlet air temp. 120 ~C
mi7~r air flow 290 liters/min emulsion feed rate 1.5 liter/hr The dry, hollow spherical product had a ~ mçter between about 1 ~M and albout 15 ~M and was collected at the cyclone separator as is standard for this dryer.
Aliquots of powder (200 mg) were weighed into 10 ml tubing vials, sparged with perfluorodiglyme-~luldled nitrogen at 20~C and sealed. The nitrogen was s~LulaLed with perfluorodiglyme by passing it through three perfluorodiglyme filled gas washing bottles hlllncl~ed in a 20~C water bath. The amount of perfluorodiglyme vapor per vial was 12-14 mg.
The vials were l~con~ uled with 5 ml water for injection after inserting an 18-gauge needle as a vent to relieve ~.es~ as the water was injected, forming approxirnately 6 x 108 bubbles per ml which were stable in vitro for several days.

One ml of the resl-ltin~ microbubble suspension was injected intravenously into an a~ploxil-lately 3 kg rabbit instrllmP-nte~l to monitor the Doppler ultrasound signal of its carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA, model ES-10-20) connçcte~l to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF Doppler signal to a LeCroy 9410 oscilloscope (LeCroy, (~h~stnllt Ridge, NY). The root mean square (RMS) voltage of ~e signal con~ Led CA 02222l86 l997-ll-2~
W O 96/40281 PCT~USg6/09068 by the oscilloscope was transferred to a computer and the resultant curve fitted to obtain peak echogenic signal intensity and half-life of the microbubbles in blood.
Signals before contrast were less than 0.1 volts RMS.
60 seconds post injection, signal intensity was 1.1 V rms, with a decay constant of appro~im~tely .00859 s~' F~mple IV
Perfluorodiglvme ~as emulsion with phospholipid/Poloxamer surfactant One liter of each of the following two solutions was ~l~.d with the following ingredients for injection:
Solution 1:

36 g m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan) 30 g Sodium chloride (M~llin~ rodt, St. Louis, MO) 26 g Sodium phosphate, dibasic (~llinckrodt, St. Louis, MO) 3.9 g Sodium phosphate, monobasic (M~llin~ rodt, St. Louis, MO) Solution 2:
4.5 g Poloxamer 188 (BASF, P~ y, NJ) 4.5 g Dip~lmitnyl phosph~ti(lylcholine (Avanti Polar Lipids, ~l~b~eter, AL) Solution 2 was added to high shear mixer and cooled in an ice bath. A coarse ~u~ sion of 30 ml of 1,1,2-trichlcl~ ll;nuoroethane (Freon 113; EM Science, Gibbstown, NJ) was made in the 1 liter of solution 2. This ~u~ ion was emlll.cified using a Microfluidizer (Microfluidics Corporation, Newton, MA; model M-llOF) at 10,000 psi, 5~C for 5 passes. The res-lltinp emulsion was added to solution 1. This lllixlule was then spray dried in a Niro Atomizer Portable Spray Dryer equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark) employing the following settin~c hot air flow rate 31 CFM
ir~et air temp. 325 ~C
outlet air temp. 120 ~C
~lomi~;l air flow 290 liters/min emulsion feed rate 1.5 liter/hr W O 96140281 PCT~US96~9~68 The dry, hollow spherical product had a tli~mrtrr between about 1 ~4M and about 15 ,uM and was collected at the cyclone separator as is standard for this dryer.
Aliquots of powder (200 mg) were weighed into 10 ml tubing vials, sparged with perfluorodiglyme-s~luldled nitrogen at 20~C and sealed. The nitrogen was salu,dLed with perfluorodiglyme by passing it through three perfluorodiglyme filled gas washing bottles immersed in a 20~C water bath. The amount of perfluorodiglyme vapor per vial was 12-14 mg.
The vials were reco~ with S ml water for injection after inserting an 18-gauge needle as a vent to relieve plc~ul~ as the water was injected, forming a~,ploxi~ tely 3 x 108 bubbles per ml which were stable in vitro for several days.

One ml of the resulting microbubble suspension was injected intravenously into an a~ro~ Lely 3 kg rabbit instr lm~nted to monitor the Doppler ultrasound signal of its carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA; model ES-10-20) connected to a System 6 Doppler flow module (Triton Technology Inc.) fed the RE~ Doppler signal to a LeCroy 9410 oscilloscope (LeCroy, Ch~strlllt Ridge, NY). The root mean square (RMS) voltage of the signal co~ uledby the oscilloscope was Lld~lsr~ ,d to a coll~ and the reslllt~nt curve fitted to obtain peak echogenic signal h~ ily and half-life of the microbubbles in blood.
Signals before colllldsl were less than 0.1 volts RMS.
60 seconds post injection, signal hll~ ily was 0.4 V rms, with a decay colls~ll of appru~ tely .01835 s-l Example V
Perfluorodi~l,vme gas emulsion with phospholipid llli~lulci surfactant One liter of each of the following two solutions was ~re~d with the following ingredients for injection:
- 30 Solution 1:

36 g m-HES hy~ y~lhylstarch (Ajinimoto, Tokyo, Japan) 30 g Sodium chloride (M~llinckrodt, St. Louis, MO) 26 g Sodium phosphate, dibasic (M~llinckrodt, St. Louis, MO) 3.9 g Sodium phosph~t~7 monobasic (M~llinrLrodt, St. Louis, MO) Solution 2:

CA 02222186 1997-11-2~

4.8 g Dipalmitoyl phosphatidylcholine (Avanti Polar Lipids, ~l~h~et~r, AL) 3.4 g Dioctanoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) 5Solution 2 was added to high shear mixer and cooled in an ice bath. A
coarse suspension of 30 ml of 1,1,2-trichlo~ ;nuoroethane (Freon 113; EM Science, Gibbstown, NJ) was made in the 1 liter of solution 2. This suspension was emllleified using a Microfluidizer (Microfluidics Colyo.dlion, Newton, MA; modelM-llOF) at 10,000 psi, 5~C for 5 passes. The resulting emulsion was added to solution 1. This llli~We was then spray dried in a Niro Atomizer Portable Spray Dryer equipped with a two fluid atomizer (Niro Atomizer, Copenhagen, Denmark) employing the following setting~e hot air flow rate 31 CFM
inlet air temp. 325 ~C
outlet air temp. 120 ~C
~tomi7~r air flow 290 liters/min emulsion feed rate 1.5 liter/hr The dry, hollow spherical product had a rli~m~ter between about 1 ~4M and about 15 ~M and was collected at the cyclone sey~u~lol as is standard for this dryer.
Aliquots of powder (200 mg) were weighed into 10 ml tubing vials, sparged with perfluorodiglyme-sdLwaL~d nitrogen at 13~C and sealed. The nitrogen was salwdt~dwith perfluorodiglyme by passing it through three perfluorodiglyme filled gas washing bottles immersed in a 13~C water bath. The amount of perfluorodiglyme vapor per vial was 12-14 mg.
The vials were lcico~ le~l with S ml water for injection after inserting an 18-gauge needle as a vent to relieve yl~ ule as the water was injected, forming approximately 2 x 108 bubbles per ml which were stable in vitro for several days.
One ml of the resl-lting microbubble ~u~yellsion was injected intravenously into an approximately 3 kg rabbit instrl-mentçcl to monitor the Doppler ultraeound signal of its carotid artery. A 10 MHz flow cuff (Triton Technology Inc., San Diego, CA, model ES-10-20) conn~cte~l to a System 6 Doppler flow module (Triton Technology Inc.) fed the RF doppler signal to aLeCroy 9410 oscilloscope (LeCroy,Ch~stm-t Ridge, NY). The root mean square (RMS) voltage of the signal computed by the oscilloscope was transferred to a co.nyu~er and the reeult~nt curve fitted to CA 02222l86 l997-ll-25 W O 96/40281 PCTrUS96/09068 obtain peak echogenic signal intensity and half-life of the microbubbles in blood.
Signals before contrast were less than 0.1 volts RMS.
60 seconds post injection, signal intensity was 0.2 V rms, with a decay constant of a~ uxilllately .00387 s-l.
s ~ Example VI
BiocomPatibility of Gas Emulsions Prepared from Mixed Lon~-Chain/Short-Chain Phospholipids One liter of the following emulsion was pl'~,d for spray-drying as described in Example II:

3.6% w/v m-HES hydLoxy~ ylstarch (Ajinimoto, Tokyo, Japan) 3.0% w/v Sodium chloride (~llinr~rodt, St. Louis, MO) 2.6% w/v Sodium phosphate dibasic (~llin~rodt, St. Louis, MO) 0.39% w/v Sodium phosphate monobasic (~l~llinr~rodt, St. Louis, MO) 0.22% w/v Dip~lmitoylphosphatidylcholine (Syngena Ltd., Cambridge, MA) 0.31% w/v Dioctanoylphosphatidylcholine (Avanti Polar Lipids Inc., ~l~h~t.or, AL) 3.0% v/v 1,1,2-Trichlorotrifluoroethane (Freon 113, EM Science, Gibbstown, NJ) At these ratios of ~lip~lmitoylphosphatidylcholine to dioctanoylphosphatidylcholine the surf~t~ntc form mixed micelles only. Upon leco~ n with 5 ml water, approximately 51 million gas emulsion droplets per ml were observed, ranging in size from 1 to 20 microns. The first order decay constant of the echogenic signal of the gas emulsion in rabbits at a dose of 5 mg/kg was det~rminecl to be .0029 gl. This cc.l.e~ullds to an intravascular half-life of 4 ...i...~l~s The gas emulsion was assayed for complement activation using an in-vitro C3a diagnostic kit supplied by Quidel Corp. (San Diego, CA). No dirrelence bc;lw~en the gas emulsion and ~e ne~s~live control (saline) were observed, indicating that the gas emulsion does not activate complement. It is well known that naked microbubbles activate complement.

Sample Tested ~C3a] (n~/ml) Zymosan (positive control) 43403 Saline (negative control) 604 gas emulsion 412 CA 02222186 1997-11-2~

The gas emulsion was also assayed for changes in hemodynamics in anesthetized dogs at a dose of 20 mg/kg. No changes in mean arterial ~rcs~u,e orpulmonary artery ~ UIC were observed. These results indicate that no hemodynamic effects are observed with the gas emulsion at 10-100 times the clinically relevant dose.

Time (,.li~ s) Mean Arterial Pulmonary Artery Plcs~ulc (mmHg) Pressure (mmHg) 0 109.4 13.3 1 109.2 14.2 2 1 10.4 14.1 1 15.0 14.3 117.9 15.7 111.0 13.2 120.9 13.6 Thus7 excellent efficacy and bioco,~hlibility are provided in the same gas emulsion formnl~tion.

Example VII
Microbubble formation using two chamber vial 800 mg of spray dried powder was weighed into the lower chamber of a 20 ml Wheaton RS-l 77FLW two çll~mbt?r vial. The vial was flushed with perfluorohexane-~ lcd nitrogen at 13~C before hlse-lillg the interchamber seal.
The upper chamber was filled with 10 ml sterile water for injection. The upper chamber stopper was inserted so as to elimin~te all air bubbles in the upper chamber. Upon depression of the upper stopper, the hl~.cl1all,ber seal was forced into the lower chamber, allowing the water to flow into the lower chamber and reco"sliluLc the powder. Numerous stable microbubbles were formed as demonstrated by light microscopy. This procedure demon~l.ale~ the convenience of this form of p~-k~gin~ and the elimin~tion of the need to provide a vent to elimin~te ples~ule buildup when the aqueous phase is added to the powder.

W O 96)40281 PCT~US96/09068 Example VIII
Microbubble fornnation usin~ two chamber syrin~e One hundred mg of spray dried powder was weighed into a S ml+ S ml S HYPAK Liquid/Dry dual channber syringe (Becton Dickinson, Franklin Lakes, NJ) and shaken into the powder (needle end) channber. The hlh,.;l~alllber seal was then positioned just above the bypass çh~nn~l A 5 ~4M filter-co~ -g needle was then fitted on the syringe. The powder-co..l~ g chamber was then filled with the gas osmotic agent by placing the assembly in a vacuum chamber, ev~c~l~ting and refilling the charnber with the gas osmotic agent, perfluorohexane-saturated nitrogen at 13~C. The filter needle allows the evacuation and refilling of the atmosphere in the powder-co..l;l;..;-.~ chamber. A sealing needle cover was then placed on theneedle. The liquid chamber was then filled with 4 ml water for injection and theplunger was seated using a temporarv vent (wire inserted between the glass syringe barrel and the plunger so as to eli.. i.~ all air bubbles.
To recon~tihlt~, the needle sealing cover was removed to elimin~te ~res~ule buildup in the powder chamber. The plunger was then depl~s~ed, forcing the interchamber seal to the bypass position which allowed the water to flow around the h~ cl~ lber seal into the powder-co~ chamber. The plunger motion was stopped when all the water was in the powder chamber. The syringe was ~git~ted to dissolve the powder. Excess gas and any large bubbles were expelled by holding ~e syringe, needle end up, and further dt;~.e~ g the plunger. The solution c~ ;ll;llg numerous stabilized microbubbles (as observed by light microscopy) was then expelled from the syringe by deple.,~illg the plunger to its limit.
Example IX
In vivo efficacv of fluoroether co..l~;";"~ ~as emulsions versus air and fluoroalkane co..~ as emulsions One liter of the dispersion A was prepared and spray dried as described in Example III, and one liter of dispersions B and C were pr~a,~d and spray dried as described in Example V.

A. Sucrose Ester Microbubble Form~ tion ("AF0145" in Table) CA 02222186 1997-11-2~
W O 96/40281 PCT~US96/09068 36 g of m-HES hy-lloxy~ ylstarch (Ajinimoto, Tokyo, Japan) 30 g of Sodium chloride (Mallincrodt, St. Luis, MO) 26 g Sodium phosphate dibasic (Mallincrodt, St. Luis, MO) 3.9 g of Sodium phosphate, monobasic (Mallincrodt, St. Luis, MO) 4. 5 g of Sucrose ester 11025003 (Alliance Ph~rm~(~e~ltical Corp., San Diego, CA) 19.5 g of Poloxamer 188 (BASF, P~il,~ly, NJ) 30 ml of 1, 2, 2 - Trichlolollifluoroethane (Freon 113, EM Science, Gibbstown, NJ) Water: for injection: 490 ml B. Phospholipid Mixture Microbubble Formulation ("24b" in Table) 36 g of m-HES hydloxy~ ylstarch (Ajinimoto, Tokyo, Japan) 30 g of Sodium chloride (Mallincrodt, St. Luis, MO) 26 g Sodium phosphate dibasic (Mallincrodt, St. Luis, MO) 3.9 g of Sodium phosphate, monobasic (Mallincrodt, St. Luis, MO) 4.5 g of Dimiristoyl phosphatidylcholine (Avanti Polar Lipids, ~l~h~et.or, .~l~b~m~) 4.5 g of Dioctanoyl phosph~tidylcholine (Avanti Polar Lipids, Al~h~ter, bslm~) 5.8% v/v perfluorl h~n~ (3M) Water: for injection: 490 ml C. Phospholipid Mixture Microbubble Formulation ("24f" in Table) 36 g of m-HES hydroxyethylstarch (Ajinimoto, Tokyo, Japan) 30 g of SodiD chloride (Mallincrodt, St. Luis, MO) 26 g SodiD phosphate dibasic (Mallincrodt, St. Luis, MO) 3.9 g of SodiD phosph~tç, monobasic (Mallincrodt, St. Luis, MO) 3.4 g of Dimiri~toyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, ~l~h~m~) 4.8 g of Dioctanoyl phosphatidylcholine (Avanti Polar Lipids, ~ h~et~r, ~l~h~m~) 5.8% v/v perfluorohexane (3M) Water: for injection: 490 ml 100 mg samples of the spray-dried powder were placed in 10-ml vials and gassed by perfluoroether-air mixture repeated evacuation-gassing cycles with thehelp of a syringe needle equipped with a three-way valve. As the filling gases, perfluorodimethyl ether (85%, Exfluor Research, Texas, Austin), perfluoro(methylethyl ether) (80%, Exfluor Research, Texas, Austin), perfluoro(diethyl ether) ( 90%, Strem Chemicals, Nev~/l,ul y~ol L, MA), n-perfluoro~lopalle and n-perfluorobutane (97%, PCR Incol~o,dled) were used. The amount of perfluoroether and fluorocarbon vapors per vial is shown in the Table.

CA 02222l86 l997-ll-25 W O 96~40281 PCT/US96/09068 After recon~titlltin~ with 5 ml of water, the bubbles were formed, which were stable in vitro for several days. Their echogenic propelties in vivo were evaluated using a Pulsed Doppler Signal Enhancement Rabbit Model as described in Example III.
The properties of the bubble dispersions sllmm~rized in the table below.

CA 02222186 1997-ll-2~

Sample Powder Filling Amount of Doppler Doppler No. ga~ osmotic ~ignal, signal, filling gas V, 100 s 300 8 per vial, after after mg/atm injection injection 1 24~ CF30CF3 16.5mg/0.26 0.3 0.1 atm 2 24~ CF30C2Fs 38 mg/ 0.46 0.8 0.2 atm 3 24f n-ClF8 49.7mg/0.65 0.6 0 atm 4 24~ air - o o 24b C2FsOC2Fs 48.2mg/0.46 1.25 0.6 atm 6 24b n-C~Flo 50 mg/0.51 1.0 0.5 atm 0 7 AF0145 CF30C2Fs 41.7 mg/0.50 0.75 0.1 atm 8 AF0145 air - o o All the perfluoroether samples gave a significant ultrasound signal up to 300 s after injection into the bloodstream. The same preparations filled with air did not show any echogenicity 5 s after injection. Furthermore, perfluoroether-filled samples had a 20-30~ better efficacy than their fluorocarbon analogues with the same number of carbon atoms, even when applied in smaller quantities. The figure illustrates the pulsed Doppler signal in volts as a function of time for experiments 1 and 2 shown in the above table.
Example X
In vivo echoqenicity of heart and liver after administration of fluoroether qas emulsion versus fluoroalkane containinq qas emulsion Samples 2 and 3 as shown in the Table of Example IX
were injected into an ear vein of a rabbit, after which the ultrasound scattered signal was measured by an ACUSON 128XP
instrument with a 7 MHz transducer. Just after injection, both compositions led to a substantial contrasting-out of the blood vessels and the heart. This contrast gradually (at the timescale of several minutes) vanished and was replaced by contrasting out of liver, which lasted for 10 min with perfluorobutane (sample 3) and ~15 min with perfluoro(methyl ethyl ether) (sample 2).

CA 02222186 1997-11-2~

The present invention provides a stable gas dispersion or emulsion that is suitable for use as ultrasound and magnetic resonance imaging (MRI) contrast enhancement agents wherein the bubbles have a prolonged longevity in 5 vivo. Typical ultrasound contrast enhancement agents only exhibit contrast enhancement potential for approximately one pass through the arterial system, or a few seconds to about a minute. Accordingly, such agents are not generally circulated past the aorta in a patient following intravenous injection. By comparison, stable contrast agents prepared in accordance with the present invention continue to demonstrate contrast enhancement duration sufficient for multiple passes through the entire circulatory system o~ a patient following intravenous injection. In vivo bubble lives of several minutes are easily demonstrated. Such lengthening of contrast enhancement potential during ultrasound is highly advantageous. In addition, the contrast enhancement agents of the invention provide superior imaging. For example, clear, vivid, and distinct images of blood flowing through the heart, liver, and kidneys are achieved. Thus small, nontoxic doses of the compositions of the present invention can be administered in a peripheral vein and used to enhance images of the entire body.
EXAMPLE XI
In vivo efficac~ of perfluoroether-containinq qas emulsions versus Perfluoroalkane-containina qas emulsions:
~rabbit model One liter of the dispersion D was prepared and spray dried as described in Example V:
Composition of Dispersion D:
43. 2 g of m-HES Hydroxyethylstarch (Ajinimoto, Tokyo, Japan) 31.32 g of sodium phosphate dibasic (Mallincrodt, St.
Louis, MO) 4.68 g of sodium phosphate monobasic (Mallincrodt, St.
Louis, MO) -W O 96/40281 PCTrUS96/09068 1.2 g of Poloxamer 188 (BASF, Parsipany, NJ) 6 g of dimyristoyl phosphatidylcholine (Avanti Polar Lipids, Alabaster, Alabama) 61.2 g of per~luorohexane (3M) 44.4 g of sodium chloride (Mallincrodt, St. Louis, MO) Water for injection: 945 g 200 mg samples of the spray dried powder were placed in 20-ml vials and gassed by an osmotic agent- nitrogen mixture, preliminary prepared in an 1 L air bag. The vials with powder were repeatedly evacuated and ~illed with the mixture of an osmotic agent and nitrogen under the total pressure of 1 atm; the partial pressure of the osmotic agent amounted to 0.13~0.03 atm. The osmotic agents studied are listed in Table III below.
Table III Osmotic Agents Used in mixtures with nitrogen with the Powder D

Formula (name) Source boiling point, ~C Time of decay of the Doppler signal to baseline, s, at PF=
~- 13~0.03 atm n-C4Flo 97%, PCR iul~ul~Juldled -2 300 (p~.nuùi~l~uLh.lC) CF3-O-CF2CF2-O-CF3 99 %, Exfluor 17 400 (p.,.nuvlu.l.onoglyme) Research, Austin, lX
n-C5FI2 97%, PCR in~ u-_~,1 29 400 (Fi~,.nuu.~.". ~) CF3-(OCF2)3OCF3 95%, custom synthesis 59 1200 (C5F~204) n-C6FI4 98%, 3M 57 600 (p~,-nuU-uh~ IC) CF3-(OCF2CF2)2OCF3 99%, Exfluor 64 >1800 (p~,,nuulu.liglyme) Research, Austin, TX

After reco.,~ .g the powder with 10 ml of water, the bubbles were formed. Their echogenic pLo~llies in vivo were evaluated using a Pulsed Doppler Signal F.nh~n~.ement Rabbit Model, as described in Example III, with the dirreL~;-lce W O 96/40281 PCT~US96/O~OG8 lhat the injected dose was reduced to 0.2 ml ( ca. L mg of dry powder per kg of rabbit). Figures 2a, 2b, and 2c colllpdle the decay of ulkasound signal over time for dirr~lent filling gases at close partial ~les~u-~s. The data are arranged in pairs so that microbubble ~lc~ dlions comprising perfluoroethers (thick lines) are colll~ d directly with their perfluorocarbon analogues (thin lines). From the graphs it is evident that perfluoroether-filled bubbles have a longer persistenee in the blood~l,.,dlll than their fluorocarbon analogues.

Example XII
In vivo efficacy of perfluoroether-co"l~;,.;.. ~ gas emulsions versus perfluoroalkane-co.~ ir-g ~as emulsions: pi~ model Powder D was prepared as described in Lxample XI and filled with perfluornh~n~-N2 mixture (28mg of osmotic agent per vial, partial ~ Ure 0.16 atm) and C5Fl2O4 -N2 llli~ , (22 mg of osmotic agent per vial, partial ~ U~ 0.12atm). After reconstituting the powder with 10 ml of water, the bubbles were formed.
~nestheti7ed pigs (14-16 kg) were fitted with indwelling ç~thloter~s in the femoral artery and femoral and jugular veins for hemodynamic mn..;lr..;i~ and contrast agent ~ ministration. p~.x~.."s3l short-axis cardiac images at the level of the papillary mllscles were obtained using an HP Sonos 2500 Ultrasound m~ehine Ilmages were acquired in the Second ~rmonic mode with a wide bandwidth linear phased array probe emitting at 2 MHz and receiving at 4 MHz. ~m~ging was intermittçnt (gated), triggered at end-diastole of every cardiac cycle. 0.5 mL of recon~LiluL~d collLI~xl agent was diluted with 0.5mL sterile saline and infused over 1 min via the jugular vein.
Figures 3a, 3b and 3c r~leselll an image of the heart before infusing the contrast agent (3a), one minute (3b) and six min~tes (3c) after injection.
Sul~llial contrast of the heart is evident (Fig. 3b) for both filling gases one minute after injection. However, while there is still a great deal of tissue contrast in the image obtained using the microbubble p.~ udlion comprising a perfluoroether at six ~ es (Fig. 3c), the contrast in the image obtained using a microbubble e~.,.l;on comprising perfluorohexane has declined m~rke-lly. This demon~lldlt;s that perfluoropolyether-filled microbubbles clearly provide clinically useful contrast irnages for an exten~led period.

W O 96/40281 PCT~US96/09068 The foregoing description details certain preferred embo~1iment~ of the present invention and describes the best mode contemplated. It will be appreciated, however, that no matter how detailed the foregoing appears in text, the invention can be practiced in many ways and the invention should be construed in accordance with the appended Claims and any equivalents thereof.

Claims

WHAT IS CLAIMED IS:
1. A gas emulsion for ultrasound contrast enhancement comprising a plurality of gas bubbles in a liquid medium, said gas bubbles comprising a fluoroether.
2. The gas emulsion of Claim 1 wherein said gas bubbles comprise a perfluoroether.
3. The gas emulsion of Claim 1 wherein said fluoroether is selected from fhe group consisting of CH3CH2OCF2CHF2, CH3CH2OCF2CF3, CHF2CH2OCF2CHF2, CF3CH2OCF2CH2F, CF3CH2OCH2CF3, CF3CH2OCF2CHF2, CHF2CH2OCF2CF3, CF3CH2OCF2CF3, CH3OCH2CF2CHF2, CH3OCH2CF2CF3, CH3OCF2CF2CHF2, CH3OCF2CHFCF3, CH3OCF2CF2CF3, CHF2OCH2CF2CHF2, CHF2OCH2CF2CF3, CF3OCH2CF2CHF2, CF3OCH2CF2CF3, CH3OCH(CF3)2, CH3OCF(CF3)2, CHF2OCH(CF3)2, CH3OCH2CHF2, CH3OCF2CH2F, CH3OCH2CF3, CH3OCF2CHF2, CHF2OCH2CHF2, CHF2OCF2CH2F, CHF2OCH2CF3, CHF2OCHFCF3, CF3OCH2CHF2, CH3OCF2CF3, CF3OCH2CF3, CF3OCHFCF3, CF3OCF2OCF3, CF3(OCF2)2OCF3, CF3(OCF2)3OCF3, CF3(OCF2)4OCF3 and mixtures thereof.
4. The gas emulsion of Claim 1 wherein the fluoroether is selected from the group consisting of perfluorodiethylethers, perfluorodimethylethers, perfluorodiglymes, perfluoromethylethylethers, and perfluoromonoglymes.
5. The gas emulsion of Claim 1, wherein the gas bubbles additionally comprise air or nitrogen.
6. The gas emulsion of Claim 1 wherein the gas bubbles are surrounded by a surfactant layer.
7. The gas emulsion of Claim 6 wherein the surfactant layer comprises a surfactant selected from the group consisting of nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof.
8. The gas emulsion of Claim 6, wherein said surfactant layer comprises a non-Newtonian surfactant.
9. The gas emulsion of Claim 6 wherein said surfactant layer comprises a compound selected from the group consisting of phospholipids, fatty acids, blockcopolymers and sugar esters.

10. The gas emulsion of Claim 6, wherein the surfactant layer comprises at least a first and a second surfactant, the first surfactant consisting essentially of a phospholipid or mixture of phospholipids having at least one acyl chain which comprises at least 10 carbon atoms, and comprising at least about 5% w/w of total surfactant, and wherein the second surfactant is more water soluble than the first surfactant.
11. The gas emulsion of Claim 10, wherein said second surfactant is selected from the group consisting of fatty acids, salts of a fatty acids, a sugar esters of fatty acids, polyoxypropylene-polyoxyethylene copolymers, nonionic alkylglucosides, polysorbate, and combinations thereof.
12. The gas emulsion of claim 1 wherein said gas bubbles are free gas bubbles.
13. The gas emulsion of Claim 1 wherein said gas bubbles further comprise microspheres.
15. The gas emulsion of Claim 13 wherein said microspheres comprise a protein.
16. The gas emulsion of Claim 1 wherein said gas bubbles further comprise a liposome.
17. The gas emulsion of Claim 1 wherein said gas bubbles are formed through the solubilization of void containing structures.
18. The gas emulsion of Claim 1 wherein said gas bubbles are formed by altering the pressure on an emulsified liquid comprising an aqueous phase and said fluoroether.
19. The gas emulsion of Claim 1 wherein said microbubbles are formed through sonication.
20. A method for forming a gas emulsion comprising the steps of:
providing a container having therein a structural material defining a plurality of voids, a surfactant and a gas or gas mixture comprising a fluoroether dispersed in said voids;
adding an aqueous liquid to said container; and, admixing said structural material, said surfactant and said aqueous liquid, thereby forming a gas emulsion in said container, said gas emulsion comprising bubbles of said gas or gas mixture surrounded by a layer of the surfactant.
21. The method of Claim 20 wherein said structural material is substantially water soluble.
22. The method of Claim 20 wherein said fluoroether is selected from the group consisting of CH3CH2OCF2CHF2, CH3CH2OCF2CF3, CHF2CH2OCF2CHF2, CF3CH2OCF2CH2F, CF3CH2OCH2CF3, CF3CH2OCF2CHF2, CHF2CH2OCF2CF3, CF3CH2OCF2CF3, CH3OCH2CF2CHF2, CH3OCH2CF2CF3, CH3OCF2CF2CHF2, CH3OCF2CHFCF3, CH3OCF2CF2CF3, CHF2OCH2CF2CHF2, CHF2OCH2CF2CF3, CF3OCH2CF2CHF2, CF3OCH2CF2CF3, CH3OCH(CF3)2, CH3OCF(CF3)2, CHF2OCH(CF3)2, CH3OCH2CHF2, CH3OCF2CH2F, CH3OCH2CF3, CH3OCF2CHF2, CHF2OCH2CHF2, CHF2OCF2CH2F, CHF2OCH2CF3, CHF2OCHFCF3, CF3OCH2CHF2, CH3OCF2CF3, CF3OCH2CF3, CF3OCHFCF3, CF3OCF2OCF3, CF3(OCF2)2OCF3, CF3(OCF2)3OCF3, CF3(OCF2)40CF3 and mixtures thereof 23. The method of Claim 20 wherein the fluoroether is selected from the group consisting of perfluorodiethylethers, perfluorodimethylethers, perfluoromethylethylethers, perfluoromonoglymes and perfluorodiglymes.
24. The method of Claim 20 wherein said structural material comprises said surfactant.
25. The method of Claim 20 wherein the surfactant is selected from the group consisting of phospholipids, phosphocholines, lysophospholipids, nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof.
26. The method of Claim 20 wherein said material is selected from the group comprising void-containing structures and soluble void-forming structures.
27. The method of Claim 20 wherein said structural material is selected from the group consisting of sugars, spray dried microspheres, lyophilized powders, lyophilized cake, powdered and granulated sugars, protein microspheres, and dried porous hyaluronic acid.
28. A microbubble precursor composition comprising:
a structural material defining a plurality of voids;

a gas or gas mixture comprising a fluoroether dispersed in said voids; and a surfactant, wherein said material, said gas or gas mixture and said surfactantare together adapted to form microbubbles upon addition to said container of a liquid.

29. The composition of Claim 28 wherein said structural material is substantially water soluble.
30. The composition of Claim 28 wherein said fluoroether is selected from the group consisting of CH3CH2OCF2CHF2, CH3CH2OCF2CF3, CHF2CH2OCF2CHF2, CF3CH2OCF2CH2F, CF3CH2OCH2CF3, CF3CH20CO2CHF2, CHF2CH2OCF2CF3, CF3CH2OCF2CF3, CH3OCH2CF2CHF2, CH3OCH2CF2CF3, CH3OCF2CF2CHF2, CH3OCF2CHFCF3, CH3OCF2CF2CF3, CHF2OCH2CF2CHF2, CHF2OCH2CF2CF3, CF3OCH2CF2CHF2, CF3OCH2CF2CF3, CH3OCH(CF3)2, CH3OCF(CF3)2, CHF2OCH(CF3)2, CH3OCH2CF2, CH3OCF2CH2F; CH3OCH2CF3, CH3OCF2CHF2, CHF2OCH2CHF2, CHF2OCF2CH2F, CHF2OCH2CF3, CHF2OCHFCF3, CF3OCH2CHF2, CH3OCF2CF3, CF3OCH2CF3, CF3OCHFCF3, CF3OCF2OCF3, CF3(OCF2)2OCF3, CF3(OCF2)3OCF3, CF3(OCF2)4OCF3 and mixtures thereof.
31. The composition of Claim 28 wherein the fluoroether is selected from the group consisting of perfluorodiethylethers, perfluorodimethylethers, perfluoromethylethylethers, perfluoromonoglymes and perfluorodiglymes.
32. The composition of Claim 28 wherein said structural material comprises said surfactant.
33. The composition of Claim 28 wherein the surfactant is selected from the group consisting of phospholipids, phosphocholines, lysophospholipids, nonionic surfactants, neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof.
34. The composition of Claim 28 wherein said structural material is selected from the group of void-containing structures and water soluble void-forming structures.

35. The composition of Claim 28 wherein said structural material is selected from the group consisting of spray dried microspheres, granulated and powdered sugars, lyophilized powders, lyophilized cakes, protein microspheres and dried porous hyaluronic acid.
36. The composition of Claim 35 wherein said structural material comprises spray dried microspheres and said spray dried microspheres comprise a compound selected from the group consisting of starches, derivatized starches and sugar esters.
37. A method for ultrasonically imaging an object or body comprising the steps of:
introducing the contrast medium of Claim 1 into said object or body; and, imaging at least a portion of said object or body.
38. The method of Claim 37 wherein said imaging step comprises ultrasonic harmonic imaing.
39. A method for magnetic resonance imaging an object or body comprising the steps of:
introducing the contrast medium of Claim 1 into said object or body; and, imaging at least a portion of said object or body.
40. A microbubble composition for use in imaging comprising a plurality of microbubbles in a biocompatible liquid medium wherein said microbubbles compriseat least one fluoroether gas osmotic agent and at least one modifier gas.
41. The microbubble composition of Claim 40 wherein said fluoroether gas osmotic agent comprises a perfluoroether.
42. The microbubble composition of Claim 40 wherein said fluoroether gas osmotic agent is selected from the group consisting of CH3CH2OCF2CHF2, CH3CH2OCF2CF3, CHF2CH2OCF2CHF2, CF3CH2OCF2CH2F, CF3CH2OCH2CF3, CF3CH2OCF2CHF2, CHF2CH2OCF2CF3, CF3CH2OCF2CF3, CH3OCH2CF2CHF2, CH3OCH2CF2CF3, CH3OCF2CF2CHF2, CH3OCF2CHFCF3, CH3OCF2CF2CF3, CHF2OCH2CF2CHF2, CHF2OCH2CF2CF3, CF3OCH2CF2CHF2, CF3OCH2CF2CF3, CH3OCH(CF3)2, CH3OCF(CF3)2, CHF2OCH(CF3)2, CH3OCH2CHF2, CH3OCF2CH2F, CH3OCH2CF3, CH3OCF2CHF2, CHF2OCH2CHF2, CHF2OCF2CH2F, CHF2OCH2CF3, CHF2OCHFCF3, CF3OCH2CHF2, CH3OCF2CF3, CF3OCH2CF3, CF3OCHFCF3, CF3OCF2OCF3, CF3(OCF2)2OCF3, CF3(OCF2)3OCF3, CF3(OCF2)4OCF3 and mixtures thereof.
43. The microbubble composition of Claim 40 wherein said microbubbles further comprise microspheres.
44. The microbubble composition of Claim 43 wherein said microspheres comprise a protein.
45. The microbubble composition of Claim 40 wherein said microbubbles further comprise a liposome.
46. The microbubble composition of Claim 40 wherein said microbubbles are formed through the solubilization of void containing structures.
47. The microbubble composition of Claim 40 wherein said microbubbles are formed by altering the pressure on an emulsified liquid comprising an aqueous phase and said fluoroether gas osmotic agent.
48. The microbubble composition of Claim 40 wherein said microbubbles are formed through sonication.
49. The microbubble composition of Claim 40 wherein said microbubbles further comprise a surfactant layer.
50. The microbubble composition of Claim 49 wherein said surfactant layer comprises a compound selected from the group consisting of nonionic surfactants,neutral surfactants, anionic surfactants, neutral fluorinated surfactants, anionic fluorinated surfactants and combinations thereof.
51. The microbubble composition of Claim 49, wherein said surfactant layer comprises a non-Newtonian surfactant.
52. The microbubble composition of Claim 49 wherein said surfactant layer comprises a compound selected from the group consisting of phospholipids, fatty acids, block copolymers and sugar esters.
53. The microbubble composition of Claim 40 wherein said modifier gas comprises oxygen.
54. The microbubble composition of Claim 40 wherein said modifier gas comprises nitrogen.