CA2188557A1 - Gas filled microspheres as computed tomography contrast agents - Google Patents

Abstract

Novel gas filled microspheres useful as computed tomography (CT) contrast agents. The microspheres are prepared from a gas and/or a gaseous precursor, and one or more stabilizing compounds.

Description

w0 9~/320~6 218 8 ~ 5 7 r~ . 64,~

GAS FILLED MICROSPHERES
AS COMPUIED TOMOGRAPHY CONTRAST AGENTS
Cross-Reference to Related ~
This is a ,ullLillu.~iu~-in-part of copending U.S. application Serial No.
5 081247,656, filed May 23, 1994, which is related to U.S. application Serial No.
08/116,982, filed Sepember 7, 1993, now allowed, which is a division of U.S.
application Serial No. 07/980,594, filed January 19, 1993, now U.S. Patent 5,281,408, issued January 25, 1994, which is a division of U.S. application Serial No.
07/680,984, filed April 5, 1991, now U.S. Patent 5,205,290, issued April 27, 1993.
The disclosures of each of the above ~ i - ;.".~ are illl,UI~O ' herein by reference in their entirety.
Field of the Invention The preseM invention relates to f~ ..J~;Ii..l.i for computed tulllo~ lly.
More ~al~il ul~uly, the present invention relates to ~:ulll~Oai~iùll~ for computed 15 tomography which comprise gas-filled IlliClUa B~ .v .Id of the In~ention Computed Lulllo~ 4hy (CT) is a diagnostic imaging technique which measures, in its imaging process, the l.~diud~ y of matter. R~iin~Pnriry of matter is typically expressed in Hounsefield Units (HU). iTmlncPfiPI-l Units are a measure of the 20 relative absorption of computed lulllo~ lly X-rays by matter znd is directly wo 95/32006 2 1 8 8 5 5 7 , ~
~IU~JUILiUI~I to electron density. Waoer has arbitrarily been assigned a Yalue of 0 HU, air a value of -1000 HU, and dense cortical bone a value of 1000 HU
Various tissues in the body possess similar densities. Difficulty has been Cll~.UULlt~lC~ in generating by CT visual images of tissues which possess similar S densities and which are proximate each other. For example, it is difficult to generate separate CT images of the ~ Uill;~.~Lil~l (GI) tract and adjacent structures, including, for example, the blood vessels and the Iymph nodes. Accordingly, contrast agentshave been developed in an attempt to change the relative density of different tissues, and thereby improve the diagnostic efficacy of CT.
A commoniy used contrast agent for computed ~OIIIU~ IIY~ ualLi~ulAlly in coMection with scans of the GI tract for increasing the l_Jio ic.~iLy of the bowel lumen, is barium sulfate. Barium sulfate increases electron density in certain regions of the body, and is classifled as a "positive contrast agents."
Currently available CT contrast agents, including barium r....,~
15 such as barium sulfate, suffer from various drawbacks. For example, the viability of CT agents is generally extremely sensitive to ~ , lil r If the ~ ;lAri.~., is too low, little contrast is observed. If the c..,.r. .,:lAli.." is too high, beam hardenmg artifacts result and are observed as streaks in the CT images. In addition, difficuity is generally L~ICUUII;C~C i in visualizing the bowel mucosa with the currently available 20 contrast agents.
Lipid ...".,~ i..,.c, for example, lipid emulsions and/or ~
have been formulated as contrast agents, particularly for the GI tract. Lipids inherently possess an electron density that is lower than water. Accordingly, lipid J~ are capable of decreasing electron density and are generally termed 25 "negative contrast agents".
Lipid r~-...l..~ :~;~-.,~ are capable of providing enhanced vi~ i7~ti-1n in CT scans. However, lipid-based contrast agents also suffer from various drawbacks.
For example, CUIll~ù~i~iulls which comprise iipid alone are generally llnr~ hl~ which limits their use for oral A~ In addition, lipid r. ~ are typically 30 expensive to formulate. Undesirable side effects can also be caused from the high ~....~ .,l~l;..,.~ of lipid which are frequently used in the lipid-based contrast agents to achieve adequate negative contrast in certain regions of the body, for example, the _ _ _ _ , _ , . , . . , ... .. . . .. . . ... ... .... .. ... ... . .. . _ .. _ wo ss/32006 2 1 8 8 5 5 7 , ~
bowel lumen. Patients with ~!allLI~aiiLia, peptic or gastric ulcers, irritable bowel disease, Crohn's disease, or colitis are especially prone to such side effects.
ru~ llulc, lipid-based contrast agents are typically perishable and thus possess a limited shelf-life.
Accordingly, new and/or better contrast agents for CT are needed. The present invention is directed to this, as well as other, important ends.
Brief DescriDtion of the Prior A~rt In U.S. Patent 5,205,290 referred to above, there is disclosed low density I~ lua,ull.,lca serving as contrast agents for computed lulllu~laylly, which are composed of b;r~ synthetic polymers or copolymers prepared from mnnr~mrrc such âS acrylic acid, ~ ylic acid, clllyl~,ll~,il~l;llc, acrylamide, ethylene glycol, N-vinyl-2-~yllulidollc, and the like. In a preferred synthesis protocol, the Illil,lUa,UIl~lC~ are prepared using a heat expansion process in which the Illi~lua~ ,lca, made from an ~.I,A,,.l~hlr polymer or copolymer, contain in their void or cavity, a volatile liquid. The Illiulu~,ull~,lca are then heated, plaa~ illg the Illicluayll~lca and volatilizing the liquid, causing the Illil,lUa~Jll.,lC~ to expand to up to about several times their original size. When the heat is removed, the 11 """~ ;, polymer retains atleast some of its expanded shape. Miclua,ull.,l~a produced by this process tend to be of particularly low density, and are thus said to be preferred.
Volatile liquids useful in the heat expansion process of U.S. Patent 5,205,290 include aliphatic l~yd~ucd~bu~la, such as ethane; clllululluulucall)ulla7 such as CCI~F; tetraalkyl silanes, such as tetramethyl silane; as well as ~ UUlUCdlbUII~, such as those having between 1 and about 9 carbon atoms and between about 4 and about 20 fluorine atoms, especially C~Flo. It is said to be important that the volatile liquid not be a solvent for the Illiclua,ull~lc polymer or copolymer; and that the volatile liquid should have a boiling point that is below the softening point of the -lic~u~ .,lc polymer or copolymer.
The stabilized gaseous precursor filled Illiclu~t ~ used as contrast media in the present invention are ~ i"~ 1r from those of U.S. Patent 5,205,290 in that they are not made from a polymer or copolymer by a heat expansion process, and are not, therefore, subject to the same limitations which require that the volatile 2l 88557 woss/32006 r~l~u~ .Of~

liquid not be a solvent for, and not have a boiling point below the softening point of, the ~ ,;uù~ polymer or copolymer.
D'Arrigo, U.S. Patent Nos. 4,684,479 and 5,215,680 disclose gas-in-liquid emulsions and lipid-coated Illil,lUbU~ , ICù~ .ly~ which are stable and said S to be useful in several fields, including as contrast agents for c~llol,dldio~ld~)lly~ and in the ultrasonic monitoring of local blood flow. However, there is no suggestion that these ~ "~ would be useful as contrast media for computed ~UlUo~;ld~J;Iy.
Quay published application WO 93/û5819 discloses that gases with high Q numbers are ideal for forming stable gases, and that "Illi~,lub~lJl~ ;," of these gases 10 are useful as contrast agents in ultrasound imaging. However, the disclosure is limited to stable gases, rather than their jtAhili77tinn and ~nrs~rS~ innl as in the present invention; although in a preferred r~ ùli;~ described on page 31, sorbitol is used to increase viscosity, which in turn is said to extend the life of a Illi~lu~ubblc in solution.
Also, it is not an essential Ic4uilulll.,~lL of the present invention that the gas involved 15 have a certain Q number or diffusibility factor. Quay contains no suggestion that the gas Illil,lOI)UblJll,~ would be effective as a contrast medium for computed lulllu~la~lly.
Vanderipe published application WO 93/06869 also discloses the use of bubbles of gases and gas mixtures, including L~,.lluu~ucdlbollù, as ultrasound imaging r,.l,~ agents. Again, however, these gas bubbles are not .~ i and 20 there is no suggestion of their use as contrast media for computed lUlllO~ llY
Lanza et al., published application WO 93/20802 discloses acoustically reflective oli~t~lAmPllAr liposomes for ultrasonic image ~ which are mllltilAm-~llAr liposomes with increased aqueous space between bilayers or have liposomes nested within bilayers in a ~ . i. fashion, and thus contain internally 25 separated bilayers. Their use in monitoring a drug delivered in a liposome ad.l.ill;ua,lcd to a patient, is also described. However, there is no suggestion that these liposomes could serve as contrast media for computed ~ulllo~.d~lly.
Widder et a~., published application EP-A-0 324 938 discloses stabilized ububblc-type ultrasonic imaging agents produced from heat-~
30 biocul~ dLiblc proteins, e.g., albumin, hemoglobin, and collagen. Again, hûwever, useof such rnmro~itinnC as contrasl media for computed Lolllo~ld~ully is not described.

w0 95/320~6 5 There is also mentioned a ~ cllLd~iùl~ believed to have been made by Moseley et al. at a 1991 Napa, California meeting of the Society for Magnetic Resonance in Medicine, which is ~IIIIII~AI ;~. 'I in an abstract entitled "~ lubublJlc,. A
Novel MR .S~ y Contrast Agent". The lll;ulubu~bL,~ which are utilized 5 comprise air coated with a shell of human albumin. The stabilized gas-filled iclua~ of the present invention are not suggested, nor is their use as contrast media for computed ~Ulll~Sl4YIlY
Tei et al., ,.,~ .,.,.,i....i patent application disclosure SH0 63-60943 discloses contrast agents for ultrasonic diagnosis comprising a ~ lluuluc ubull 10 emulsion with an emulsion particle size of 1 to 10 ,~m, in which the ~ UUIUC41IJUII is preferably 9 to 11 carbon atoms and the emulsifier may be, for example, a l.I.n~ .;,1 or a nonionic polymeric surfactant such as pOly(u~ ,.llyl. .lc)-pOly(u~y,ulu~yl, ll~) cuuolylll.,l~. The emulsion may be prepared by utilizing a mixer.
There is no suggestion, however, that these p~,lAuulu~4lboll emulsions would be 15 suitable for use as contrast media in computed lulllo~l4ylly.
Knight et al., U.s. Patent 5,049,388 discloses small particle aerosol liposome and liposome-drug culllbill~Liulla for medical use, for example, as systems for delivering drugs to the respiratory tract by inhalation. However, there is no suggestion that these liposomes can be gaseous precursor f~lled or that they might serve as contrast 20 media for computed lull~u~ ully.
Summarv of the Invention The present invention is directed to a contrast medium useful for computed Lu1110014ylly imaging, said contrast medium comprising stabilized gas and gaseous precursor filled llliclua~ll.,lca, wherein the gas may be, for example, air or 25 nitrogen, but may also be derived from a gaseous precursor, for example, ~,lnuOIu~ lL4llc, and the Illil,lUa~ C~ are stabilized by being formed from a stabilizing compound, for example, a l.i~ ;i.lc lipid or polymer. In certain preferred ~lllbOdilll~ a, the b;oc...,.l.~ f lipid comprises a phospholipid which is in the form of a lipid bilayer. A contrast medium in accordance with the present 30 invention comprises a substantially l-.-,-,.-~ as well as aul,uliaill~ly stable suspension of Illil,lUa~ Ca comprising gas and stabili~ing compound. A unique aspect woss/32006 - 6 - P~~
of the preseM invention involYes the use of l,..nuuluL~,~l,u., ~ases which are capable of ,.,,.;,.1~;. ,.~ the integrity, and thus, enhancing the stability, ûf the Illi~lu~,ull.,lc~.
The present invention also concerns a method for preparing stabilized gas-filled ~ lu~Jh.,~c~ for use as computed ~ulllG~ lplly imaging contrast media, 5 comprising the step of agitating an aqueous suspension of a stabilizing cûmpound1 for example, a IJ;~ .lf lipid or polymer, so that stabilized gas-filled Illi~lU~,Ull.lCD
result. Desirably, this step is carried out at a hlll~.,ldiu~c below the gel to liquid crystalline phase transition ~ .ldlUlC of the L.i.~ il.lr lipid so as to achieve a stabilized gas-filled l~lic~u~h~,lc product.
The preseM invention further pertains to a method of providing an enhanced image of an internal region of a patient comprising (i) ~ to the patient one or more of the preseM contrast media, and (ii) scanning the patieM using computed lu.llo~ lly imaging to obtain visible images of the involved regions.
Also ~ by the present inveMion is a method for diagnosing 15 the presence of diseased tissue in a patieM, especially in the ~.~DLIu;l~t~ l regions of the patient, comprising (i) ;~.1ll,;,l;~. . ;.,~ to the patient one or more of the present contrast media, and (ii) scanning the patient using computed tUIllOyla~Jlly imaging to obtain visible images of any diseased tissue in the region.
The present inveMion further relates to a method for preparing in si~l~ in 20 the tissue of a patient a coMrast medium for computed Lul~ ly, the coMrast medium comprising gas-filled Illiclua~ c~. comprising (i) ~ g to the patient gaseous precursor-filled Illi~luaL/II..~, and (ii) allowing the gaseous precursor to undergo a phase transition from a liquid to a gas to provide the gas-filled Ill;~,lU~ll.,lc~.
All of the above aspects of the present invention can be carried out, often with ~ lr attendant advantage, especially with regard to ease of ingestionby a patient, by using gaseous precursors to form the gas of the gas-filled iclu~pll-lca. Once ingested, and upon gas formation in, for example, the LIU;Illt~Lilldl tract, expansion of the gaseous precursor causes an increase in the 30 volume of the contrast medium and impart low density to the ~LIuil~lilldl tract, thereby enhancing computed tomography imaging thereof. These gaseous precursors may be activated by a number of factors, but preferably are Ltlll~ iulc activated, that wo 9s/32006 7 2 l 8 8 5 5 7 . ~ . 4~
is, they are activated by exposure to eleYatéd L~.IIIJ~,..IIUIC Such gaseous precursors are ~ uu~ which, at a selected activation or transition il,llllJ~,ld~UlC, change phases from a liquid to a gas. Activation thus takes place by incFeasing the t.l~ UlC of the compound from a point below, to a point above, the activation or transition 5 i~ UlC Optionally, the contrast medium may further comprise a liquid nuulu~ lbull compound, for example, a ~,llluulu~ bùll, to further stabilize the Illi~lU~ ,lC~ Preferably, the nuulu~lbull liquid is , ' ' by the I~ u~ll"lc~.
The present inveMion also relates to a method for preparing stabilized gas or gaseous precursor filled ll~ u~ lc~ for use as a computed ~UlllUyjld~JIly10 imaging contrast medium. The method comprises agitating an aqueous suspension of a lipid (that is, the lipid stabilizing compound), in the presence of a gas or gaseous precursor, resulting in gas or gaseous precursor filed Illiclu~ ,lca. Desirably,agitation is carried out at a ~tlll~l~ld~U-C below the gel to liquid crystalline phase transition ~ UI~ of the lipid to achieve a preferred product.
Where a gaseous precursor is used, the gaseous precursor filled IlliLloa~Jll.,lc c~ is generally maintained at a ~IU,U~ IIC at which the gaseousprecursor is liquid until ~ n;l~l to the patient. At the time of Alllll;ll;~i~,.l;l~ll the t~ dlUlC may, if desired, be raised to activate the gaseous precursor to form a gas.
The resulting gas filled ll-i~.lu~ ,lc~ are then ad,l.;.l;at.lcd to the patieM.
20 Alternatively, the gaseous precursor filled Illi~,lU~ may, if desired, be ddlllilliaLclcd without raising the ~ UIC, and the gaseous precursor allowed to form a gas as a result of the naturally elevated internal t.lll~,ldlUIc of a patient. The composition may be agitated, if necessary, prior to ~.il.,;,,: l".l;",~
The present invention further pertains to a method of providing an 25 enbanced image of an internal region of a patieM, especially an image of the ~j~LIUiUl~t~ l region of said patient, said method comprising (i) Alllll;l~;~il 1111~ to the patient the foregoing contrast medium, and (ii) scanning the patient using computed tomography imaging to obtain visible images of said region.
The present invention also r ~ , a method for diagnosing the 3û presence of diseased tissue in a patient, especially in the ~ ilUill.~;lldl regions of said patient, said method comprising (i) ddlll;ll;:U~I;llg to the patient the foregoing contrast WO 95B2006 P.

medium, and (ii) scanning the patient using computed ~ulllOgla~lly imaging to obtain visible images of any diseased tissue in the region.
These and other aspects of the invention will become more apparent from the following detailed description when taken in r... j l ~ i.... with the following 5 drawings.
l~rief Description of the Drawir~c It should be noted that, for purposes of making the drawings more readily mrl~ trlorl, only single bilayers are shown. In fact, the ...~ which these drawings illustrate may be either ~llullùla.~.,.a, bilayers"-,lj,,~,l .. 11~., or .. llil--.. l' 10 (~ 1" .IIY, the figures described below should in no way be taken as limiting the present invention to Illi~luaLJll~lca whose envelope or skin is comprised of only a single layer or bilayer of stabilizing compound.
Figure l depicts the ~ of a gas-filled lipid bilayer ~ u~ c with a ~nuu~uua~bu~ that is proximate the hydrophobic tails of the lipids.
Figure 2 depicts the aLabili~aLiull of a gas-filled lipid oli~n~
Illi~lua~ ; with a p~,llluu-u~a~bu~ that is proximate the I-YdIU~JIIUI);~ tails of lipids in a monolayer that is located within a lipid bilayer.
Figure 3 depicts the stabilization of a gas-filled lipid bilayer ~ lua,ull,~c with a ~.,llluo~uca~u~ that is proximate the interior hydrophilic head groups of the 20 lipids.
Figure 4 depicts the aL~b;;i~dLiull of a gas-filled lipid bi~ayer lll;~lvaAull.
with a pc.lluu.u.a.l,ull that is proximate the exterior lIYdIU~JII;I;U head groups of the lipids .
Figure 5 depicts the ~ .;li,,,li"" of a gas-filled lipid monolayer 25 llli~lua,ull~ with a u~,llluu-uca~bul~ that is proximate the interior IIYdIUIJIIUb;~ tails of the lipids.
Figure 6 depicts the stabilization of a gas-filled lipid oli~nl~m~ r U*~ with a p~nuu~u~a~lJu~ that is proximate the l~ydlu,ullobi~ tails of lipids in a monolayer that is located outside of a lipid bilayer.

wo9~2oo6 21 8 8 5 57 r Detailed DescriPtion of the Invention As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to haYe the following meanings.
"Stabilized" refers to Illiclu~yll.lcs which are substantially resistant to that is caused, for example, by the loss of structural or r ~
integrity in the walls of the L~ u~yllclca and/or by the loss of any significant portion of the gas or gaseous precursor which is ~ within the Illiclu~yll~lc.
"Lipid" refers to a syMhetic, ~cl~ yllih~,~iL or naturally-occurring ;. compound which comprises a llydluyllili~ component and a llydluyllulJ;~, 10 c-~mr~ nrnt Lipids include, for example, fatty acids, neutral fats, glycolipids, aliphatic alcohols and waxes, terpenes and steroids.
"Microsphere" refers to a small spherical entity which is ~,LIA~
by the presence of an in~ernal void. Preferred Illi~lU~y l~lcD are formulated from lipids, including the various lipids described herein. In any given Illicluayll~lc. the 15 lipids may be in the form of a monolayer or bilayer, and the mono- or bilayer lipids may be used to form one or more mono- or bilayers. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The lipid Illiclu~yll~
described herein include such entities commonly referred to as liposomes, micelles, bubbles, I~ lub~-lJbl~j, and the like. Thus, the lipids may be used to form a 20 unilamellar Illi~lu~yll~"c (comprised of one monûlayer or bilayer), an oii~o'~ n~l~Ar Illi~lU~yll~lC (comprised of about two or about three Illu~ulAy..~ or bilayers) or a mllltilAmrllAr Illi~lu~yll.lc (comprised of more than about three IllUlloL~y~ orbilayers). The internal void of the Illi~lU~yll~lc~ may be filled with a liquid, including, for example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid or solute material, as desired.
"Liposome" refers to a generally spherical cluster or aggregate of .;l. compounds. including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as lipid Illi~lU~yll~lc~.
"Polymer'' refers to molecules formed from Ihe chemical union of two or more repeating units. Accordingly, included within the term "polymer" are, for woss/32006 2 i 88557 `~

example, dimers, trimers and oligomers. In preferred forrn, the term "polymer" refers to molecules which comprise 10 or more repeating units.
"Semi-synthetic polymer" refers to a naturally-occurring po~ymer that has been chemically modified. Exemplary naturally-occurring polymers include, for 5 example, poly~ - . l,_. ;.l. ~
"Patient" refers to anirnals, includirlg mammals, preferably humans.
The present invention is directed, inter alia, to contrast media comprising stabilized gas filled ~ lua~ ca which are basically bubbles of very small diarneter comprising a "skin" or "envelope" of a stabilizing compound that surrounds 10 or encloses a cavity or void filled with liquid or gas. The stabilizing compound provides integrity to the IlliLlua~ lc such that the I~ ualJll~ a exist for a useful period of time. The stabilized l~ u~ ,lca are pdlLi~uLI~ly suitable for use as contrast agents for computed Lul-lo~ldL-lly (CT). In ~,.,.i.~l;.. ll~ where the stabilizing compound comprises, for example, a lipid, the llliulua~Jh~.~a possess a lower electron 15 density relative to water. This lower electron density imparts highly desirable properties to the contrast agents of the present invention, particularly with respect to CT imaging.
The stabilized l~ lua~ lca of the presem invention comprise a gas and/or a gaseous precursor. Any of the various biocu~ JdLilJlc gas and gaseous 20 precursors may be used in the gas and gaseous precursor filled ~lli..ua~;...ca of the present invention. Preferred gases are gases which are inert and which are 1,;~.~.""1,-~;1,1~, that is, gases which are not injurious to biological function. Preferred gases also have a low solubility and ~irruaibiliLy in aqueous media.
Moreover, it is possible to utilize a gas and a gaseous precursor 25 together. A unique and preferred aspect of the present invention results from the discovery that when a gaseous precursor, for example, a ~ Lluulu~d~ull, is combined with a gas ordinarily used to make the stabilized Illi~lu~Jll.,lca of the present invention.
lua,ull~lca are obtained having an added degree of stability not otherwise obtainable with the gas alone. Thus, it is a preferred aspect of the invention to utilized gaseous 30 precursors which can be activated, for example, upon exposure to elevated L~ dlU~Ca. to form stabilized Illi~lua~ ,lca in the form, for example, of stable ~1 88557 ~ WO 9s/32006 r~l,. . S4~

foams, which can be utilized as effective ïOw density contrast agents for compuoed ~u---o~ y.
Stabilized Illi,lUDlJIll,lCD made with gaseous precursors have several advantages. First, as the gases generated from gaseous precursors tend to be insoluble S and relatively non-diffusible, these gases can be stabilized for use as contrast media for computed ~u---o~ y. Because the gases are relatively stable, less stabilizing compound is necessary than would be required for more soluble and diffusible gases, such as nitrogen or air. In general, a thicker walled skin or envelope of stabilrzing compound, for example, a thick walled ~ u~ c, is necessary to stabilize gases 10 such as air or nitrogen. While thick walled Illi~lUDIJlll,lC~ filled with air, nitrogen or other gases can be used as CT contrast agents, the thick walls of such llliclua~raise the effective density of the contrast medium, which may in turn limit the e~cLi~ D of the contrast medium. ru~Ll~ lul~, thick walled llliClUDIJ;l.,lCD may be relatively lI~ for oral ingestion, or may be difficult to metabolize following 15 illild~.,llUUD injection. With the gaseous precursors used in the present invention, for example, a ~ UUlU~UIJUll~ the stabilizing cnmrmln~l~ can be less rigid and the resulting Illil,lUD~UII.I~,;. can be thinner walled and easier to m~t~l~nli7~, yet still possess sufficient stabilizing compound to stabilize the III;~,IUDIJlI.,IC.
As is described in more detail further below, the stabilized III;CIUD~ D
20 used in the present inYention may be formed simply by agitation of the stabilizing compound in an aqueous Cll~ilUlUl~.,llL and in the presence of a gas andlor gaseous precursor. Where a gaseous precursor is used, the gaseous precursor filled Illi~lUa~ ,lC contrast medium which has been prepared, before A~llll;ll ~ll~<l;lll- to a patient, is desirably maintained at a L~ LUIC at which the gaseous precursor is 25 liquid. At the time of A~l~.,;":.l,dl;.. it can be pre-shaken and then ingested as a pre-formed foam. AlternatiYely, the contrast medium can be ingested as a suspension to form a foam in siru within, for exarnple, the stomach and ~ Uill;~.Lilldl tract of a - patient. The bowel motility serYes to mix the gaseous precursor within the stabilizing compound and the increase in i~ ,ldLUlC serYes to form the gas filled IlliClUD~
30 based foam in siru within the bowel. A preferred ~Ludi~ L described in detailfurther below involves ill,nl~)UldLill~ a suitable viscosity modifying agent, for example, a natural and semi-natural gum, cellulose or synthetic polymer, for example, wos~/32006 2 1 88557 ~.,. 16l,, pol,~ ul. In the presence of such a viscosity modifying agent and the stabilizing compound, the gas bubbles as they are generated are coated with these rnmro~ln~c and become stabilized through this coating process, whereby the contrast medium of the present invention is formed.
Thus, the l.. i.lu~l-.lc, are formed from, or created out of, a matrix of stabilizing r.~ ,u~ which permit the gas filled Illi lU:l,UIl-lC:~ to be established and thereafter retain their size and shape for the period of time required to be useful in computed Lulllo~ imaging. These stabilizing rr/mrollnrl~ include those which have a l~ u~llub;~/llydlu~Jllili~ character which allows them to form bilayers, and thus 10 ~ u~ ," in the presence of water. Thus, water, saline or some other water-based medium, often referred to hereafter as a diluent, is an important aspect of the stabilized gas and gaseous precursor filled Illi~lUaull--c contrast agents of the present invention, p~lLi~ul~lly in ~" I,o-l;,-- .,l~ involving Illil.lU~ which comprise bilayers.
The stabilizing compound may be a mixture of compounds which 15 contribute various desirable attributes to the stabilized Illi~lu*~ ca. For example, compounds which assist in the dissolution or dispersion of the r",..l- ,....,~l stabilizing compound have been found ii~ Lr,..v~.~. The gas, which can be a gas at the time the Illi-lUa~ C::~ are made, or can be a gaseous precursor which, in response to an activator, such as elevated L.,.l~.ldlulc, is ~l~u~rullll~ from the liquid phase to the gas 20 phase. The various aspects of the stabilized gas and gaseous precursor filled contrast agents of the preseM invention will now be described, starting with the gases and gaseous precursors.
Gases and Gaseous Precursors The Illi~lu~ ca of the present invention are essentially stabilized 25 bubbles which r~ r a gas and/or a gaseous precursor. The gases and/or precursors thereto provide the ~UIIIIJU~;liUll~ with increased negative density. This increases their errc~Li~ as contrast agents for CT.
Preferred gases are gases which are extremely stable. The term stable gas, as used herein, refers to gases which are substantially inert and which are30 I,jo~""~ , that is, gases which are not injurious to biological functions and which will not result in any degree of u.,- r ~ lr toxicity, including allergenic responses W095/32006 21 88557 r~

and disease states. Preferred also are gases which have low solubility and/or dirruaii~ :;Ly in aqueous media. Gases, such as ~llluùlu~ bu~la. are less diffusible and are relatively insoluble in aqueous media. Accordingly, they are easier to stabilize into the form of bubbles in aqueous media.
Preferable gases include those selected from the group consisting of air, noble gases, such as helium, neon, argon and xenon, carbon dioxide, nitrogen, fluorine, oxygen, sulfur-based gases, such as sulfur l,. .An,.l..;,lf and sulfur', lluuluu_~bulla, p~nuulu~,~lbull gases, and mixtures thereof. Preferred gases are ~ UUIUU~IIJUI~ gases. Exemplary ~ L~UIUU~IIIJUII gases include, for 10 example, ~..lluu.u~ .llluulu.ll~.-." u~llluu~u~u~ , u~llluu~ubuLul~, p .llluulu.,yl,lubu~ll~ and mixrures thereof. Also preferred are mixrures of different types of gases, such as a ~..lluoluu~llbull gas and another type of gas, such as oxygen.
The gases discussed in Quay, published application WO 93/05819, including the high "Q" factor gases described therein, may be used also. The disclosures of Quay, published application WO 93/05819 are illUUl,UUI~ herein by reference in their entirety. In addition, p ~-IIIAr,ll ;;l gases and gases of isotopes, such as "O, may be used. It is Cu~ that contrast media which comprise these latter gases may also be used in commection with other diagnostic techniques, such as Magnetic Resonance Imaging (MRI).
Other gases, including the gases PYPmrlifiPd above, would be readily apparent to one skilled in the art based on the present disclosure.
In certain ,u_-~iuul~-ly preferred r~ u~ . a precursor to a gaseous substance is ;... ~ rl in the Illi~luD,ull..ca. Such precursors include materials which are capable of being converted to a gas in vivo. Exemplary precursors arc 25 materials which are liquids at room ~tlll~J~ldlUI~: and which, after being a.l...i. ia;..~d to a patient, undergo a phase transition to a gas in vivo. Preferably, the gaseouS
precursor is b;ocu...~._~il,l~, and the gas produced in vivo is ll;-~r~ IC also.Exemplary of suitable gaseous precursors are of the ~..lluulu~,~.l,u. a. As the artisan will appreciate, a particular u~lluu-u~_lbù-~ may exist in the liquid state when the 30 llli~luaull~l~a are first made, and are thus used as a gaseous precursor, or the p.llluoluu~ ull may be used directly as a gas. Whether the perfluorocarbon is used as a liquid or a gas generally depends on its liquid/gas phase transition ~tlllU~ UI~, or .. .. , _ .. 2 1 ~8557 wo 95/3~006 boiling point. For example, a preferred p.ln~ulu.~l.l,ull, ~ lluulu~llL~u~c, has a liquid/gas phase transition ~C~ .ldLu.c or boiling point of 29.5C. This means that uulu~llL~lc will be a liquid at room ~ ,U.IdLUlC (about 25C), but will become agas within the human body, the normal t lll~,ld~Ulc of which (37C) is above theS transition t~lll}~.ld~UlC or boiling point of ~lnUulUIJ~lltdllC. Thus, under normal p~nuulu~ Ldllc is a gaseous precursor. As a further example, there are the homologs of ~ uulu~ llL~ulc~ namely ~ UUIUbUL~U~ amd ~lnuulullcA~u~c.
The liquid/gas transitiûn of u.,llluulubuLd-lc is 4C and that of l).lnuulullcAhll.~ is 57C. Thus, p~nuululJuLhllc is potentially useful as a gaseous precursor, althûugh 10 more likely as a gas, whereas ~.lnUUlUIll,Adlle would likely be useful as a gaseûus precursor only because of its relatively high bûiling point.
A wide variety of materials can be used as gaseous precursors in the present ~ ,ù~ . It is only required that the maoerial be capable of UII;I~I~;U AI~; a phase transition to the gas phase upon passing through the appropriate ~tlll~J.ld~UlC.
15 Suitable gaseous precursors include, for example, IlcAdlluulu~ ,.u~lc, isopropyl acetylene, allene, ~t~ldnuuludllene, boron trifluoride, 1,2^butadiene, 2,3-butadiene, 1,3-butadiene, 1 ,2,3-trichloro-2-fluoro-1 ,3-butadiene, 2-methyl-1 ,3-butadiene, hexafluoro-1,3-butadiene, butadiyne, 1-11uuo-ub.lLdllc, 2-ll..l~ylbui~ulc, dc.dlluulubuLdllc, 1-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1-butene, perfluoro-20 1-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne, butyl nitrate, I-butyne, 2-butyne, 2-Chloro-1,1,1,4,4,4-l-cAdlluulul,uiy.lc, 3-methyl-1-butyne, perfluoro-2-butyne, 2-bromo-butyraldehyde, carbonyi sulfide, ".. ~.. ~.. ;1.;1, .
~Y~IOIJ~ dllc, methyl-iyl,lul,uid.-c, u. ldlluulu~y~lulJuidllc, I,..nuolu~y~lùllu.~l¢, 3-~I-IU-U~Y~IULJ~ IC~ P~ UU~U~Y~ , U~LdnUUIU~YI-IUY~ C~ ~Y~IUI)IU~ IC~
25 ~..lluo~u~y~lu~ulJdllc, 1,2-dimethyl-cy.,lu~,.u~ ,,1,1-du~ yl~,yulu~lu~....~, 1,2-d;lll~illyl~y~lu~lu~d~c, cLl~yl~y~lu~Jluy~ e~ lll.,illyl-y.luplu,u llc, diacetylene, 3-ethyl-3-methyl diaziridine, l~l~l-trifluorol~ , dimeLhyl amine, lCAdnUUlUdilll.illyldlllillC, di~ yl~.llyl~ullillc~ bis-(du~l.il,yl~l.u,~l.i..c)amine, ,u..lluu~ul~cAd~lc, ~.lnuu~ul~.~Lhllc, 2,3-dimethyl-2-norbornane, 30 ~cllluulu~:lilll.illyldlllillc, dimethyloxonium chloride, 1,3-dioxolane-2-one, 4-methyl-1,1,1,2-LtLldnuUlU..IldllC, 1,1,1-~inuolu--illdllC, 1,1,2,2 ..ildlluolu~,illdl~e, 1,1,2-trichloro-1.2,2-trifluoroethane, 1,1-.liulllolu.il....lc, 1,1-dichloro-1,2,2,2-wo 9~/32006 2 1 8 8 ~ 57 r ~ U~.~r.'~4 L~i~aLluu~u~l d..e, 1 ,2-dilluulu~l~a~ -ChlOrO- 1 ~ 1 ,2,2,2-~lLdrluulu~llGllc. 2-chloro-l, l-dinuulu~llallc~ l, l -dichloro-2-nuulu.l-au-~, l -chloro- 1,1 ,2,2-LcLldlluulu~l a.le, 2-chloro-l,l-dinuu~u~lla~ I-Iu-u.l-.~, Chlulu~ Ldnuulu~llallc, LluluLIinuulu~ a~ nuOIu.Llldllt~ AdnUUlU~llGllC~ u~,..-Lanuulu~ ulc~
S ll;Ll.~J,u .l,An,..,.u~llà~ rluu~u~dlyldlll;ll~ ethyl vinyl ether, I,l-dichloroethane, 1,1-dichloro-1,2-din~u.u.;l al.c, 1,2-dilluolu.Llldll~ methane, ilinU~-"~ Ir~ YI~IIIUIi~IC~l~iIIUUI~ YIIIUUIi~Ie~
b~ul~lùdinuu~u~ iLI~ , blulllunuululll~llall~ bromochlu-unuulu~ al-c, lUlll~llaUlC, ~ lU~U-li[lUul- ' Ulll~;llallC, ClllUI- ' Ull~ ilalle, lû ~lllulunuu~u~ lalle~ ~I-luluLlinucllulll~llallc~ IulU~linUUlUUl.lldllc, ~lilJ~u~-ù~linuu~u~l,,l-a~-e, ~li-,lllulu~linu~lulll~lldlle~ l-lulunuuluul~lla ~linuulu~ aul~ .linuU.-~ r, nuululll~lllallc, jori~lll. llAIlr, i~ri~,i,in,~ ulll~dlallc~ ULlinUUlU111~11511C, Il;~luauLlinuululll~llaul~
LcLlalluulu~ ilallc~ L-i~,l-lulunùululll.ll~ulc, Llilluu~u~ a.l., 2-~ Ll~ylbuLdll~ methyl 15 ether, methyl isopropyl ether, methyllactate, ~.l.llyllliLI;~ yl~ulrl~, methyl vinyl ether"lr..l.. .llA~., nitrous oxide, 1,2,3-ll..ll- l Al~r-Lliualbu~yliu acid-2-llydlu~yLlilll~ 1-nonene-3-yne, 1,4-pPnt~iiPnP. n-pentane, p.llluu~u~ Ldllc, 4-amino-4-l....l.yl~.l.La..-2-one, 1-pentene, 2-pentene (cis), 2-pentene (trans), 3-bromopent-l-ene, ~.llluu~u,u..~L-l-ene, tetrachlu-u,ul-Llldli- acid, 2,3,6-20 ~liul~llyl~;u~lidil~c, propane, 1,1,1,2,2,3-llcAdnuulu~ulu,uallc, 1,2 el,u.,y~.vuallc~ 2,2-dinuuluylul~allc~ 2-alllillU,ulU~all~. 2-~hlOlul~lu~uallc~ heptafluoro-l-l-;,lul~-uudl~c, LalluOIu-l-lliLIuDu~lu~allC, ~.llluu-u,ulu,ua--c, propene, Il~.~dnuulu~Jlu~allc,lrl,L2.3,3-hexafluoro-2,3-di,,l.lu.u,ulu~al.c, I-ulllulu~ulu,uall~, chlulu~lul,allc-(trans), 2-clllolulJlu~Jallc~ 3-lluulu,ululJall~;~ propyne, 3,3,3-~linUUlUylu,uyl~, 3-fluulu~lylcllc, 25 sulfur (di)-~if ~n...,.;.i. (SIFlo)~ 2,4-~ nin~t~ nP~ Llinuul~ ", L-inuu.ul.. llyl peroxide, L~inuu.u.. l~yl sulfide, tungsten llr-An~ , vinyl acetylene and vinyl ether.
In certain preferred r~ , a gas, for example, air or a p~.nuulu~all,oll gas, is combined vvith a liquid ,u.llluu-uca~l/u--. such as 30 IJ~lnuuluoLiylluulllide (PFOB), ~lnuulud~alin, perfluoroir~iPf~lin ,u~,.nuoluu~Lyliodidc, ~ uu~uLliulu~ylalllillc, and l~llluu~uLlil~uLyldlllill~.

The size of the Illi~lUa~ .l-a can be adjusted, if desired, by a variey ûf procedures including, for example, l~ ;r.. ~ vortexing, extrusion, filtration, sonication, l.. ,.~.. ;,,.l;.~,. repeated cycles of freezing and thawing cycles, extlusion under pressure through pores of defined size, and similar methods.
For illUdV~Uldl use, the Illi.lUa,Jll.,lCa preferably have diameters of less than about 30 ~Lm, and more preferably, less than about 12 ~m. For targeted illLIdvda~ul~u use including, for example, binding to certain tissue, such as cancerous tissue, the Illi.lU~ C, can be a;~ irl~ ly smaller, for example, less than 100 mm in diameter. For enteric or ~;daLlUillt.~lilldl use, the Illi.lUD,Ull~,lC, can be S;~ll.rl~ ly larger, for example, up to a millimeter in size. Preferably, the llli~lUa~ c~ are sized to have diameters between about 20 f~m and 100 ~Lm.
Tabulated below is a iisting of a series of gaseous precursors which undergo phase transitions from liquid to gas at relatively close to normal human body t~ ld~UlC (37C) or below. Also listed in the table are the srzes, in diameter, of emulsified droplets that would be required to form a Ill;~lua,ull.,lc of a maximum size of about 10 ~m.

Physical C~ t..iali.a of Gaseous Precursors and Diameter of r ~ ~ ~ Droplet to Forrn a 10 /Im ~Ii~. ~ . ' I
Diameter (llm) of emulsified droplet Molecular Boiling Point to make 10 micron 20 Compound Weight (' C) Density Ill;~.lual~ll, lc perfluoro- 288.04 29.5 1.7326 2.9 pentane 1- 76.11 32.5 6.7789 1.2 fl UUl Ulhl dllC
25 2-methyl- 72.15 27.8 0.6201 2.6 butane (isopentane)

2-methyl-1- 70.13 31.2 0.6504 2.5 butene 30 2-methyl-2- 70.13 38.6 0.6623 2.5 butene .. ...... . .

wo 95/32006 r ~ ~ F 1 Physical Cl~ a of Gaseous Precursors and Diameter of r - Droplet to Form a 10 llm ~L~, c Diameter (~m) of emulsified droplet Molecular Boiling Point to make 10 micron Compound Weight ( C) Density ~ ,lua~ c l-butene-3- 66.10 34.0 0.6801 2.4 yne-2-methyl

3-methyl-1- 68.12 29.5 0.6660 2.5 butyne 5octafluoro- 200.04 -5.8 1.48 2.8 .lulJu~llc decafluoro- 238.04 -2 1.517 3.0 butane hexafluoro- 138.01 -78.1 1.607 2.7 10etbane ~Source: Chemical Rubber Company Handbook of Chemistry and Physics Robert C.
Weast and David R. Lide. eds. CRC Press. Inc. Boca Raton, Florida. (1989 - 1990).
It is part of the present invention to optimize the utility of the ua~ .c~ by using gases of limited solubility. Limited solubility, as used herein, 15 refers to the ability of the gas to diffuse out of the I~ lva~ ca by virtue of its solubility in the aulluulldill~ aqueous medium. A greater solubility in the aqueous medium imposes a gradient with the gas in the llliclualJll.,lc such that the gas will have a tendency to diffuse out of the llli~lua~ lc. A lesser solubility in the aqueous medium will decrease the gradient between the llli~lua~ and the interface such that 20 the diffusion of the gas out of the Illi~lua,ull.,lc will be i~npeded. Preferably, the gas entrapped in the l"i.,ua~ lc has a solubility less than that of oxygen, namely, 1 part - gas in 32 parts water. See Mûtl?eson Gas Da~a Book, Matheson Company, Inc.(1966). More preferably, the gas entrapped in the llliLlua~ .c possesses a solubility in water less than that of air; and even more preferably, the gas entrapped in the 25 Illi~lua,ull~.lc possesses a solubility in water less than that of nitrogen.

~, 88557 9~2006 nt~ C~ . ' One or more stabilizing c~ are employed to form tile IlliClU~ C~, and to assure continued ~ of the gases or gaseous precursors. EYen for relatively insoluble, non-diffusible gases, such as S u.,.nuulu~lu,u~lc or sulfur ll~.Adlluu~iLit, improved lu;~lu~ ,lc ,UIC,U~IIdliOI~ are obtained when one or more stabilizing ~,, ,,I,,J....ri~ are utilized in the formation of the gas 2nd gaseous precursor filled Illi-,lU~UIl..lC~. These compounds maintain the stability and the integrity of the Illi.lu~Lll"lL~ with regûrd to their size, shape and/or other attributes.
A wide variety of stabilizing compounds can be employed in the contrast media of the present invention. When combined with a gas and/or a gaseous precursor, the stabilizing CU~ )UUIId~ are capable of promoting the formation, and improving the stability, of the Illil,lU~,UIl.lU~. The stabilized ll~ic~u~,ull~lc~ of the present invention are substantially resistant to dF,,l~li-l;FII~ as measured by the loss of 15 Illiclu~ll.,lc structure or I ~ Al~l gas or gaseous precursor for a useful period of time. Typically, the l~iclu~,ull~lc~ are capable of retaining at ieast about 90 percent by volume of its original structure for a period of at least about two or three weeks under normal ambient conditions, although it is preferred that this period be at least about a month, more preferably, at least about two months, even more preferably, at least 20 about six months, and more preferably, about a year, and still more preferably about three years. Thus, the III;LIU~,UII~,IC~ of the present invention possess long shelf-lives, even under adverse conditions, including elevated t~llllJ~ldlUlC~ and pressures.The stability of the Illi.lu,~ of the present invention is A~ r at least in part, to the materials from which the ~l~ic~u~,ull.,lc~ are made, and it is often 25 not necessary to employ additional stabilizing additives, although it is optional, and sometimes preferred, to do so. Such additional stabilizing agents and their ld' Ltli~LiC~ are explained in more detail below.
In preferred '~ , the stabilizing compounds comprise l-ir~.,.,.l.rl;l,l~ lipid compounds and/or polymeric compounds, with lipids being 30 preferred. Preferably, the lipids or polymers are inert. Because of the ease of f~ including the ability of producing the ~liclu~ .c~ just prior to a.~ ", the IlliClU~)Il.lC~ can be made ~ull~ ily on site.

WO 95132006 2 1 8 8 5 5 7 r~ r s ~
lg -r . . - LiPids A wide variety of ~ c...,.l -~;l.le lipids can be used as the stabilizing compound. Suitable lipids include, for example, Iysolipids, I.~ f~ , such as yllcl~yl~ yl~llùlill~a with both saturated and I ~ lipids including S ~lic~ yll-l.. -~l-l-- ;-lyl.ll li-.~ yli~uyl~ ;-lyl~,llolillc;, y ll ll ~ Iy l--l--oli---- ~ lul Uy ~ y l~l--o~
dipah..;~uyll.l...~l.l.-~;.lyl.,llolil.~ (DPPC) and d;~ uyll.l...~ ;.lyl~lloliul~ (DSPC);
yllU Iyll~lliLly1~ . .- `, such as ~liùli~--y 1¦-~ lyl~ A I ,r~l . . "; "r uyll~l~fl~lll ~ - ;.lyl. ~ and ~liy_lllliluyllll ~f,~l.~ ;,lyll . I ~
10 yllu~yllf~Lhlyl~ yl~u~yl"Lidylglycerols; pllOayll_tidy' ~,; crhin~ftliriric, such as ~yll..l~ullly.,lill, glycolipids, such as ganglioside GMl and GM2; ~1.,. ~I;~.;.i~
sulfatides; ~Iy~ll~l.l.;..~,l~lirific, If~ i l; acid; palmitic acid; stearic acid;
AIA~ -; acid; oleic acid; lipids bearmg polymers, including such polymers as pol~.l.yl~ 51y-ul, chitin, hyaluronic acid or pOIyVillylyylluli~ull~;~ lipids bearing 15 sulfonated mono-, di-, oligo- or poly~,..l"..id~, .IIùI.,i.luls and cholesterol Il. .11;~11~ . '.IA~., Lu~uyll~luls and tocopherol I , lipids with ether and ester-linlced fatty acids; pOlylll.liL.~ lipids; diacetyl phosphate; dicetyl phosphate;
:,L alylA-.Il;.... cardiolipin; ~.1,..~,1,.~1;~.;,1~ with short chain fatty acids (C6 to C8);
synthetic ~ ,f~l;~,;.i~ with a~yl-u-~ i- acyl chains, for example, a first acyl chain of 20 C6 and a second acyl chain of C12; ceramides; yulyu~y~,l-yl.l-e fatty acid esters, pUlyU~y.Lllyl~ fatty alcohols, pc,lyu,.~.llyl.l.~ fatty alcohol ethers, yolyu~.~.lly' sorbitan fatty acid esters, glycerol yoly.Ll.yl...c glycol oxystearate, glycerolpoly..l.yl~ glycol ricinoleate, sterols, ~ w-y' ~ soybean sterols, ~LIIu~y' ' castor oil, polyu~y.Llly~ -yolyu~yyluyyl~llc; polymers, and polyui~y~llyl~ ; fatty acid2~ stearates; sterol aliphatic acid esters including cholesoerol sulfaoe, cholesoerol butyraoe, cholesoerol isobutyrate, cholesterol palmitate. cholesterol stearaoe, lanosterol acetate, ergosoerol palmitaoe, and phytosoerol n-butyraoe; sterol esoers of sugar acids including - cholesterol ~lul,u~u~ide, lanosterol ~lul,ulullid~, 7-d~hYLIIV~IIUIiS~t~IUI ~IU~UIUII;d~ ergosterol ~lu~ulullid~, cholesterol gluconate, 30 lanosterol gluconate, and ergosoerol gluconate; esters of sugar acids and alcohols including lauryl y,lUuulull;dcl SoearOYI ~IU~UIUII;~, myristoyl ~lu-,u-u"i.i~. Iauryl gluconate. myristoyl gluconate, and soearoyl gluconate; esoers of sugars and aliphatic w0 95/32006 ` r~

acids including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid, and polyuronic acid; saponins, including Gl,. ~ mil:l~enin 1-- i ~ , oleanolic acid, and ~ .,; glycerol dilaurate, glycerol trilaurate, glycerol ~ir~ t~t~. glycerol and glycerol esters, 5 including glycerol ~ glycerol distearate, glycerol tristearate, glycerol dilllyl , glycerol Llilllyl , long chain alcohols of, for example, about 10 to about 30 carbon atoms, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and n-octadecyl alcohol; alkyl ~ , alkyl l:' ,' and alkyl phosphites; 6-(5-cholesten-3,B-yloxy)-l-thio-,B-D-~ .L~y-~-lu~;d~,. di~ uDyl-10 di~ idL, 6-(5-cholesten-3,~-yloxy)hexyl-6-amino-6-deoxy-l-thio-~-D-gaL.~,~u~y-~u-o-side; 6-(5-cholesten-3~-yloxy)hexyl-6-amino-6-deoxy-1-thio-cY-D-..l~llllu~yl~llo~i.h"
12-(((7'-di,,.llyl~.l.;.ln~u.llll- ;l,-3-yl)carbonyl)l~ .l.yl~....i.lo)o~ ,;. acid;
N-[12-(((7'-di~ ylAIll;ll~u~ i.,-3-yl)carbonyl).l.~l.yl~llllillù)u~ dc~lluyl]-2-amin palmitic acid; ~lw~ lyl(4~ Llly;~ ùlli~)butanoate; N-~u~,il-yldiul~,vyl-15 pllu~ idyl~ l;l", 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succrnyl-glycerol; 1,3-dilJ~lll-iluyl-2-~uu~illyl~ly~ ul; 1-hexadecyl-2-pal.--iluyl~ly~cl~r' l~ -r~ , and pal,uiLuyll-u---u"y~
Suitable lipid compounds include also lipids typically used to make mixed micelle systems, such as l~.ylllill~.,llyl~ bromide;
20 C~.iyl~lilll~..llylA~ bromide; --yli~lylllilll~Lllyl~ -l~lll~ll;ll~ll bromide;
alkyldimethylb~,~yl~ chloride (where alkyl is, for example, Cl2~ C~ or C16);
b~ yl~ llyldodc~yl- l"l"l";'l'll blUlllid~/~.lllUlid~:, b.,.~yldull.,.llyll~ u~.,yl - l .. l .. .l l;l ', . ' bromide/chloride;
yldill.~.llyl~~ ,yl~ l; - l- bromide/chloride; c.,Lyldi..-~,llyl.,Lllyl~ """..,;~
25 bromide/chloride; and "~;iyl~Jylidil,iulll bromide/chloride.
Suitable lipids for use in the present ru~ include also lipids carrying a net charge, for example, anionic and/or cationic lipids. Exemplary cationic lipids include, for example, N-[1-(2,3-di~l~uylu~y)propyl]-N,N,N-iliull~lly' chloride (DOTMA); 1 ,2-dioleoyloxy-3-(l~ ill..,il.y lcu.u.lulno)propane (DOTAP);30 1,2-dioleoyl-e-(4'-~ l....llyL..I-lllul.io)butanoyl-sn-glycerol(DOTB); and lipids bearing cationic polymers, such as polylysine and polyarginine. In general the molar ratio of cationic lipid to non-cationic lipid in the Illi~.lU~ may be, for example, 1:1000, wo 95~ 20 0 6 ^ 2 1 - . ~ l / L ~
1:100, preferably, between 2:1 to l:10, more preferably in the range between l:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationic lipid to mole amount non-cationic lipid, e.g., DPPC). A wide variety of lipids may comprise the non-cationic lipid when cationic lipid is used to construct the III;~IV~ C. Preferably, this non-S cationic lipid is dipal..-i~uyl~ ;,lyll,l-ol;.lr" ~i~).dl~liluylllllr.~ lyl~
or diulcuy~ lyl~ ;l.--,nl-..,;", In lieu of the cationic lipids as described above, lipids bearing cationic polymers, such as polylysine or pûly~.~, . as well as alkyl ~' l' , alkyl r~ ~ ' , and alkyl phosphites, may also be used to construct the Illi~,lUo~U~
It has been ~ul,uli~ ;ly and lmPYr~rt~tily found tbat the stability of the ~icluolulll,lc~ can be substantially improved by ill~ulr ~ a small amount, for example, about l to about 10 mole percent of the total lipid, of a negatively charged lipid. It is believed that the negatively charged lipids enhance stability by reducing the tendency ûf the Illil_lU~ lCD to rupture by fusing together. It is believed that this is 15 achieved, at least in part, by the formation of a negatively charged layer from the negatively charged lipid on the outer surface of the --iulu~h.,~c. The negatively charged Illi-,lua~ll.,lc is then repulsed by other, similarly negatively chargedIlli~,lUo~ C~. This repulsion prevents contact between l--i~-" ' CO which typically leads to a rupture of the walls of the Illi-,luo~ ca and ~ of the contacting 20 Illi~,lUD,UIl~lCo into larger Illi lU~ ,lC:>.
Suitable negatively charged lipids include, for example, lipids containing free carboxy (CO,~) groups, such as pllo~!)ll.,lidylO~ -li l;. acid, such as di~ uyll.l...~l.l.-~;.li~ acid, and fatty acids. In certain preferred r~ the lipid comprises di~ l---iluyll~ ly'~ and l.l...~ acid in a total 25 amount of from about 0.5 to about 30 mole percent. In certain other preferred~lllI,odilll~,llLO, the lipid comprises dipalllliLuyll.l...~ yl~llùlillc and di~ uyll.l...~ ;.lyl~l~oline, in an amount of from about 70 to about 100 mole percent.
As noted above, it is desirable, in certain rll.l.o.i;~ . to include as 30 stabilizing cnmrollnri~ lipids bearing polymers Preferably, the polymer is covalently bound to the lipid and has a molecular weight of from about 400 to about 100,000.
Exemplary polymers include llydlu~ ilic polymers, such as poly(eLllyl.,.lc~ly,,ol) 2 1 8~557 W0 95/32006 p~

(PEG), poly(~ ylluli ii.._), pulyu~u~ and poiysorbate and poly(~i-lyl.~l.,ullvl).
Preferred among the PEG polymers are PEG 2000, PEG 5000 and PEG 8000, which have molecular weights of 2000, 5000 and 8,000 l~,a~..Li~.ly. Other suitable polymers, II~ i~ul~llilic and otherwise, will be readily apparent to those skilled in the art 5 based on the present disclosure. Polymers which may be illcvl~vldlcd via alkylation or acylation reactions with a lipid are particularly useful for improving the stability of the lipid ~ Exemplary lipids which bear hydrophilic polymers include, for example, vii~,dlllliLvyll,l.n~ i.lyl, ~ ,..;... PEG, diole~,yl~ ;.ly~ ,..,nl-.";PEG and Liiat~ yll~ lyl~ -PEG
In addition to, or instead of, the lipid compounds discussed above, the present lipid c.",.l...~;l;....~ may comprise an aliphatic carboxylic acid, for example, a fatty acid Preferred fatty acids include those which conLain about S to about 22carbon atoms in the aliphatic group. The aliphatic group can be either linear orbranched. Exemplary saturated fatty acids include, for example, (iso)lauric, 15 (iso)myristic, (iso)palmitic and (iso)stearic acids. Exemplary l ' fatty acids include, for example, lauroleic, physeteric, myristoleic, r~lmirnl~oir, ~..lur.~,lilli~" and oleic acid. Suitable fatty acids include also, for example, fatty acids in which the aliphatic group is an isoprenoid or prenyl group. In addition, c~llboi~ ' bearing polymers may be used in the present lipid c~ Cd-bul~y~' bearing lipids 20 are described, for example, in U.S. Patent No. 4,310,505, the disclosures of which are hereby illl.Ul~J~ ' ' by reference herein, in their entirety.
Preferred lipids are ~ l.l.nl;l,;~c including DPPC, DPPE, DPPA and DSPC, with DPPC being preferred.
Other lipid LUIIIIJVUllVis for use in the present f-..,.~ ;n..~. in addition to 25 those ~ .liri. (l above, would be apparent in view of the present disclosure.Preferably, lipids are selected to optimize certain desirable properties of the ~,J,"l,n~;li.",~. including stability and half-life. The selection of suitable lipids in the preparation of the present ~ in addition to the lipids r~ llir~ above.
would be apparent to one skilled in the art and can be achieved without undue 30 ~All~ .;"....l~lin..~ based on the present disclosure.
As discussed in detail below, a wide variety of methods are available for the ~IC~ dLiUII of I~ luaiJll..ca including, for example, shaking, drying, gas-woss~2006 2 1 8 8 5 57 r ~ 4~
inct~ ri~m spray drying, and the like. Preferably, the Illiclua~ are preparedfrom lipids which remain in the gel state, this being the t~ UIc at which a lipid bilayer converts from the gel state to the liquid crystalline state. See, for example, Chapman et al., J. Biol. Chem. 1974 249, 2512-25~1, the disclosures of which areS hereby il-cu~uvl~-Lcd by reference herein, in their entirety. The following table lists L. i..,llL~ , lipids and their phase transition t~ tu Saturated Diacyl-sn-Glycero-3-Ph~. . ' - ' ' Main Chain Phase Transition T; , .llL~
Carbonâ in Acyl Main Phase Transition Chains Temperature C
1 ,2-(12:0) -1 .0 1,2-(13:0) 13.7 1,2-(14:0) 23.5 1,2-(15:0) 34.5 1,2-(16:0) 41.4 1,2-(17:0) 48.2 1,2-(18:0) 55.1 1,2-(19:0) 61.8 1,2-(20:0) 64.5 1,2-(21:0) 71.1 1,2-(22:0) 74.0 1,2-(23:0) 79.5 1,2-(24:0) 80.1 25 See, e.g., Derek Marsh, CRC Handbook of Lipid Bilayers, p. 139 (CRC Press, Boca Raton, FL 1990).
The lipid material or other stabilizing compound used to form the iulua~ .ca is also preferably flexible, by which is meaM, in the context of gas and gaseous precursor filled Illi~lua~ lca, the ability of a structure to alter its shape, for ~1 88557 WO 95/3tO06 r~ . r F IJ, example, in order to pass through an opening having a size smaller than the Illi-,l u~ c .
R r . . . , ' ' ' Polvmers As noted above, the stabilizing compûund can also comprise a 5 I~ ",~ ;I.If polymeric compound. The polymers can be naturally-occurring, semi-synthetic or synthetic. Exemplary natural polymers include, for example, uvly ~ C, such as arabinans, fructans, fucans, galactans, ~ .LUlull~ glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, . Al l.. L" .. 1 gald.LUL~lUIu~c, pectic acid, amylose, pullulan, glycogen, alllylulJ.,..Lill, cellulose, dextran, pustulan, chitin, agarose, keratin, I,l~u~dlu;L~ul, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch and various other natural hulllv~olyll.~,l or Il.,~lu~uly~ll.,l~ such as those containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, Iyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, cl~ihllllva~, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, yrosine, asparagine, glutamine, aspartic acid, glutamic acid, Iysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, III~IIUIUIVII;~, acid, ~1". V~
lr, and neuraminic acid, and naturally occurring derivatives thereof.
Exemplary semi-synthetic polymers include ~IbuAylll~,,llyl~cllulOse~
llydlu~yl~ llyl~llulose, ll~d-u~yl~luL~ylllu,iilyll,~llulose, methylcellulose, and u~y~ ulose.
Exemplary synthetic polymers include ~oly~Ll,y.~.l.,~, such as, for example, polyethylene glycol, polyu,~,.llylull~" and pol~il.y~llc t~,~c~ L~L~
25 polyl,.u~yl~ , such as, for example, POIYIJIUL/YII~IIC glycol, polyu-cLl.~ , such as, for example, polyvinyl alcohol (PVA), polyvillyl~llloride and polyvillylyyllvlidullc~
polyamides, such as, for example, nylon, polystyrene, polylactic acids, fluorinated llydlu~llJull~, such as, for example, polyLcLl~lnuulu~.llylull~, and ~oly.,l~,.llyllll~Lll,l~,ly' , and derivatives thereof. Methods for the preparation of polymer-based IlliclualJllclc~ will be readily apparent to those skilled in the art, once armed with the present disclosure, and when coupled with illrullll~Livll known in the wo 9sl32006 2 1 8 8 5 5 7 ~ 4~

art, such as the ;..r,...,.-~i...l set forth in Unger, U.S. Patent No. 5,Z05,290, the disclosures of which are hereby ill~UllJ~ ' ' by reference herein in their entirety.
Preferably, the polymer possesses a relatively high water binding capacity. When used, for example, in the GI region, a polymer having a high water 5 binding capacity can bind large amounts of free water. This enables the polymer to carry a large volume of liquid through the GI tract, thereby filling and distending the tract. The filled and distended Gl tract permits enhanced CT imaging of the region.
In addition, where imaging of the GI region is desired, the polymer is preferably not substantially degraded in, and absorbed from, the Gl region. Thus, 10 ", ~ ,., and absorption within the Gl tract is preferably minimized to avoid removal of the contrast agent. This also avoids the possible formation of gas within the Gl tract from such f~ .., For imaging the GI region, preferred polymers are capable of displacing air and ~ g the formation of large air bubbles within the contrast medium.
Particularly preferred ~ .o~ of the present invention include i-lua~llc~c~ wherein the stabilizing compound from which the stabilized gas and gaseous precursor filled Ill;~lU~ C:~ are formed comprises three ~ (1) a neutral lipid, for example, a nonionic or ~wiLt..iv..;. Iipid, (2) a negatively charged lipid, and (3) a lipid bearing a hydrophilic polymer. Preferably, the amouM of the 20 negatively charged lipid will be greater than I mole percent of total lipid present, and the amount of lipid bearing a hydrophilic polymer will be greater than 1 mole percent of total lipid present. It is also preferred that the negatively charged lipid be a rl~ u~ 1;. acid. The lipid bearing a llydlu~llilil polymer will desirably be a lipid covalently bound to the polymer and the polymer will preferably have a weight average 25 molecular weight of from about 400 to about 100,000. The hydrophilic polymer is preferably selected from the group consisting of pOiy-illyl-ll ~;ly~ol (PEG), poly~.u~yl.l..~;ly.ùl, polyvinylalcohol, and polyvi--yl~yl-ulidone and cu~Jolylll~l~
- thereof. The PEG or other polymer may be bound to a lipid, for example, DPPE, through a covalent linkage, such as through an amide, carbamate or amine lirlkage.
30 Alternatively, ester, ether, thioester, thioamide or disulfide (thioester) lirlkages may be used with the PEG or other polymer to bind the polymer to, for example, cholesterol or other ~I~u~ul~uli~ ls. Where the hydrophilic polymer is PEG, a lipid bearing such a ... . . _ . _ . _ _ . . .... ..

~VO 95/32006 1 ~I/U~

polymer can be referred to as being "PEGylated". The lipid bearing a hydrophilicpolymer is preferably ~lir~l Yl~ YI II~ PEG 5000 (DPPE-PEG
5000), which means a dipal-.~;iuyll l~n~ yl~ l; r lipid having a PEG
polymer of a mean average molecular weight of about 5000 attached thereto.
Preferred r ltr,lll;,~ of the prese~t invention include ~ic~u*~ c~
which comprise, for example, about 77.5 mole percent di~ iLvyll.lln~ ylcl,oli,,c(DPPC), about 12.5 mole perceM of d;,u~ uyll~ acid (DPPA), and about 10 mole percent of di~ uyl~ ly~ ";l ~-PEG 5000. Such in a ratio of mole ~ c~ t~ of 82:10:8 are preferred also. The DPPC
10 component is ~,wi~lcliul~ic and therefore, effectively neutral, since the ,UllUD,UI~ii-lyl portion is negatively charged and the choline portion is positively charged. The DPPA
component, which is negatively charged, is added to enhance ct~hili7~rinn in accordance with the mechanism described above regarding negatively charged lipids.
The third cnmrnn~nl DPPE-PEG 5000, provides a PEGylated material bound to the 15 lipid membrane or skin of the ,,.i,,.u~l,l..,~c by the DPPE moiety, with the PEG moiety free to surround the ll~il lu~ c membrane or skin, and thereby form a physical barrier to various enzymatic and other . l~,. .."~ agents in the body whose function is to degrade such foreign materials. It is also theorized that the PEGylated material is able to defeat the action of the IllllLlu~ull.,r~ of the human immune system, which 20 would otherwise tend to surround and remove the foreign object. The result is an increase in the time during which the stabilized ~ u~lul~,lcs can exist, in vivo, and therefore function as CT contrast agents.
~xiliar~ Stabilizin~.C~ ~ ' It is also i, l.l.l-lrf~ to be a part of the present invention to prepare ~5 stabilized gas and gaseous precursor filled Ill;clu~ull~lc~ using materials in addition to the bin~u,~ il,lr lipids and polymers described abûve, provided that the ll~iclu~,ull~lc~
so prepared meet stability and other criteria set forth herein. These materials may be basic and r",..l- ,....~1 and thus, can form the primary basis for creating or establishing the stabilized gas and gaseous precursor filled ~liu~u~ On the other hand, they 30 may be auxiliary, and therefore act as subsidiary or ~ ".y agents which either enhance the functioning of the basic stabilizing compound or rnmrrJ lnr~ or else w095/32006 2 188557 r~l" ~133 contribute some desired property in addition to that afforded by the basic stabilizing compound.
However, it is : ,' ' that difficulty may be ~ u~ l in ~1. ~. .11.;.1,..~ whether a particular compound is a basic or an auxiliary ageM, since the 5 r".. Il... .~ of the compound in question is generally determined empirically, or by the results produced with respect to producing stabilized ~ ,' ca. For example, the simple ~.. l,;.. -~i.. of a 1,;.~ .. I.-;.l.lc lipid and waoer or saline, when shaken, will often give a cloudy solution subsequent to ' Vill~ for ~ 11 Such a cloudy solution may function as a contrast agent, but is aesthetically ub;~Liul~lc and may 10 imply instability in the form of ulldiaaOl~d or ~ d lipid particles. Thus, propylene glycol may be added to remove this cloudiness by facilitating dispersion or dissolution of the lipid particles. The propylene glycol may also function as a thickening agent which improves Illiclua~ c formation and ~ I..,. by increasing the surface tension on the Ill;~lua~ ,lc membrane or skin. It is possible that the 15 propylene glycol further functions as an additional layer that coats the membrane or skin of the Ill;~,lua,ull~,lc, thus providing additional ~I_l,;lj,_l;,", Basic and auxiliary materials for use in the ,UlC~ iUII of stabilized Ill;~,lua~ . I would be apparent to one skilled in the art based on the present disclosure. Such materials include ~o~ lLiul.~l surfactants which are disclosed, for 20 example, in D'Arrigo, U.S. Patents Nos. 4,684,479 and 5,215,680, the disclosures of which are illCUI,UI ' 1 herein by reference, in their entirety.
Additional auxiliary and basic stabilizing compounds include such agents as oils, for example, peanut oil, canola oil, olive oil, safflower oil, corn oil, or any other oil which is commonly known to be ingestible. Another auxiliary and basic 25 stabilizing compound is trehalose.
It has been found that the gas and gaseous precursor filled Illi~lua~ ,L~:a used in the present invention may be controlled according to size, solubility and heat stability by choosing from among the various additional or auxiliary stabilizing agents described herein. These ageMs can affect these parameters of the I~ uaL~ ca not 30 only by their physical interaction with the lipid coatings, but also by their ability to modify the viscosity and surface tension of the surface of the gas and gaseous precursor filled Ill;~lua~ll.,lca. Accordingly, the gas and gaseous precursor filled wo 95/32006 1 - 2~ -u~ ca may be favorably modified and further stabilized, for example, by the addition of a viseosity modifier, ineluding, for example, udllJoll~l' and the ~llu~ b~yldLcd and sulfonated derivatives thereof, polyethers, ineludimg polyethers having a moleeular weight of, for example, from about 400 to about 100,000 and di-5 and trihydroxy alkanes and their polymers having a moleeular weight of, for example,about 200 to about 50,000; c~lula;ryillg and/or solubilizmg agents, ineluding, for example, aeaeia, eholesterol, .1; ~ , glyeerol ~ t- - `, lanolin aleohols, leeithin, mono- and di~,ly.,~,lid~ oleie aeid, oleyl aleohol, poloxamer, ~olyu~y.,l-yl.,ll., 50 stearate, polyoxyl 35 eastor oil, polyoxyl 10 oleyl 10 ether, polyoxyl 20 eetostcaryl ether, polyoxyl 40 stearate, 3Joly ' 20, ~u;,ri~ul' ' 40, polysorbate 60, polysorbate 80, propylene glyeol diae~tate, propylene glyeolt~ , sodium lauryl sulfate, sodium stearate, sorbitan I , sorbitan IllUI10-l , sorbitan mnnor~l , sorbitan , stearie aeid, trolamine, and ~Il.U ~i~yill~ wax; suspending and/or viseosity-inereasing agents, ineluding, for 15 example, agar, alginie aeid, aluminum ...u bentonite, magma, earbomer 934P, cd~buAy..l~,l.ylccllulose~ ealeium and sodium and sodium 12, ~ .. ~,. -,.
eellulose, dextran, gelatin, guar gum, loeust bean gum, veegum, ll~l~u~y~d~yl eellulose, ll~d~u~y~u~Jyllll.llyll,cllulose, m~P~ ~-aluminum-silieate, methyleellulose, peetin, polyethylene oxide, povidone, propylene glyeol alginate, 20 silieon dioxide, sodium alginate, tragaeanth, xanthan gum, ~-d-~glyeerol and mannitol; synthetic suspending agents, including, for example, pul~llyl~ ;y~,ul (PEG), polyvi..yl~,y..ulidu.l~ (PVP), polyvinylalcohol (PVA), ~lyl~lu,uylcllc glycol and pOlyaul' ' and materials which raise the tonicity of the uulll,uOai~iul1S, including, for example, sorbitol, ~u~yl~ 51y~ùl and glycerol.
25 AqueDPs Diluents As mentioned earlier, where the Illiulua~ are lipid in nanure, a particularly desired component of the stabilized Illiulua~)ll.lc~ is an aqueous ~llVilUIllll~llL of some kind, which induces the lipid, because of its l~y-llu~llubic/llydlu~ nature, to form Illiulua~ lca, which is a highly stable 3û Cul-rl~uldLiull in such an .llVilUlllll.,lli. The diluents which can be employed to create such an aqueous Cll~ilUlllll~llL include, but are not limited to, water, either deionized or w095132006 21885~7 r~"~ 3 containing any number of dissolved salts which will not interfere with the creation and of the stabilized ~ LU~,U~ a or their use as CT agents, and normal saline and physiological saline.
~hods o~
The stabilized gas and gaseous precursor filled ~ lu~,uh~.ca used in the present inveMion may be prepared by a number of suitable methods. These are described below separately for gas filled Illic~u*)ll~,lc~, gaseous precursor filled .;.lU*JII.,I.,i" and both gas and gaseous precursor filled Illi~u~
Methods of r.~"~. Usin~ a Gas A preferred ~.. ,1.. ,ll;,.,. .,i comprises the steps of agitating an aqueous solution containing a stabilizing compound, preferably a lipid, in the presence of a gas at a t.~ LUlC below the gel to liquid crystalline phase transition t lll~ UlC of the lipid to form gas filled Ill;.lU~ll.,IC,. The term agitating, and variations thereof, as used herein, means any motion that shakes an aqueous solution such that gas is 15 introduced from the local ambient ~llvilulull.,llL into the aqueous solution. The shaking must be of sufficient force to result in the formation of Illi~lU~,UII-I-~, ~ull;~ulolly stabilized Illi~lua~ ,lc~. The shaking may be by swirling, such as by vortexing, side-to-side, or up and down motion. Different types of motion may be combined. Also,the shaking may occur by shaking the container holding the aqueous lipid solution, or 20 by shaking the aqueous solution within the container without shaking the container itself.
Further, the shaking may occur manually or by machine. MPrh~nir:
shakers that may be used include, for example, a shaker table such as a VWR
Scientific (Cerritos, CA) shaker table, or a Wig-L-Bug- Shaker from Dental Mfg. Ltd., 25 Lyons, Ill., which has been found to give excellent results. It is a preferred - e,l.l,o.lu.-.,l.~ of the present invention that certain modes of shaking or vortexing be used to make stable III;~IU~ lCS within a preferred size range. Shaking is preferred, and it is preferred that this shaking be carried out using the Wig-L-Bug- mPrh~nir~l shaker. In accordance with this preferred method, it is preferred that a IC~;,UlUUli;ll~
30 motion be utilized to generate the gas and gaseous precursor filled Ill;.lU~.ll.,IC,. It is 2~ 88557 wo 95/32006 1~ t eYen more preferred that the motion be ~ lu~.~Lil~t, in the form of an arc. It is still mûre preferred that the mûtiûn be lc~ luu.lLi-~ in the form of an arc between about 2 and about 2û, and yet further preferred that the arc be between about 5 and about 8. It is most preferred that the motion is IC~ilJlUCr~iill~ between about 6 and about 5 7, most ,u~lLiculdlly about 6.5. It is c" "~ l that the rate of lcu;~lu~lliùll, as well as tne arc thereof, is ~ Licul~lly important in A~ the amount and sr~e of the gas filled Illiulu~ ca formed. Preferably, the number of lc~i,ulu~:~Livlra or full cycle r~5rill~ir~n~, is from about lO00 to about 20,000 per minute. More preferably, the number of lc~i~JlU~,~Liul~ or oscillations is from about 5000 to about 8000. The Wig-~Bug', referred to above, is a m~rh:~nirA~I shaker which provides 2000 pestle strikes every lO seconds, i.e., 6000 r~5f`ill~rir~nc every minute. Of course, the number of oscillations is dependent upon the mass of the contents being agitated, with the Iarger the mass, the fewer the number of r,crili~tir,nc Another means for producing shaking includes the action of gas emitted under high velocity or pressure.
lS It will also be understood that preferably, with a larger volume of aqueous solution, the total amount of force will be cullca~ull-iill~;ly increased.
Vigorous shaking is defined as at least about 60 shaking motions per minute, and is preferred. Vortexing at about 60 to 300 revolutions per minute is more preferred.
Vortexing at about 300 to 1800 revolutions per minute is even more preferred.
The formalion of gas Fllled Illi~lua~ll.lca upon shaking can be detected visually. The . ..". . .,ll,.l;(~l~ of lipid required to form a desired stabilized Illi~lua~ull~lc level will vary depending upon the type of lipid used, and may be readily determined by routine ~ ;IIII For example, in preferred rllll~o~ , the c....~ ;IIAI
of l,2-dipal...iL~Jyll-l,.-~l-l.-~;-lyl.l.oline (DPPC) used to form stabilized Illiclu,~ll..c~
25 according to the methods of the present invention is about O.l mg/ml to about 30 mg/ml of saline solution, more preferably from about 0.5 mg/ml to about 20 mg/ml of saline solutiûn~ and even more preferably from about l mg/ml to about lO mg/ml of saline solution. The . u,. .1..ll;..l. of dia;..l-uyl~l-ua~l.,lidylcholine (DSPC) used in preferred embodiments is about 0. l mg/ml to about 30 mg/ml of saline solution, more 30 preferably from about û.5 mg/ml to about 20 mg/ml of saline solution, and even more preferably from about 1 mg/ml to about lO mg/ml of saline solutiorl.

w09s/32006 2 1 88557 ~ '"~

In addition to the simple shaking methods described above, more elaborate methods can also be employed. Such elaborate methods include, for example, liquid crystallme shaking gas instillation processes and vacuum drying gas instillation processes, such as those described in copending U.S. application Serial No.
5 08/076,250, filed June 11, 1993, which is ill~Ul~J~ ' ' herein by reference, in its entirey. When such processes are used, the stabilized llliclua,ull~ which are to be gas filled, may be prepared prior to gas installation using any one of a variety of .,ol~ lLiulldl liposome ,UII. ' ,~/ techniques which will be appareM to those skilled in the art. These techrliques include freeze-thaw, as well as techrliques such as0 sonication, chelate dialysis, Il.,.,..,~,...;,-li.),. solvent infusion, lllicl.~ ..".l~;ri, ~ ."
u~ forrnation, solvent \~ French pressure cell technique, controlled detergent dialysis, and others, each involving preparing the ll~iclu~LJll~lc~ in various fashions. See, e.g., Madden et al., Chemistry and Physics of Lipids. 1990 53, 37 'L6, the disclosures of which are hereby illCUI~/I ' ' herein by reference in their entirey.
The gas filled ~ u~yll~lc~ prepared in accordance with the methods described above range in size from below a micron to over 100 ~m in size. In addition, it will be noted that after the extrusion and . ~ .;l;,-li..., procedures, the agitation or shaking step yields gas and gaseous precursor filled Illi-,lU~lll~lC~ with cllh~t:~n~ lly no or minimal residual anhydrous lipid phase in the remainder of the 20 solution. (Bangham, A.D., Standish, M.M, & Watkins, J.C. (1965) J. Mol. Biol. Vol.
13, pp. 238-252 (1965). The resulting gas filled ~;clu~ c~ remain stable on storage at room ~IlllJ,,.dLulc for a year or even longer.
The size of gas filled ll~iClU~LJll~lC.~ can be adjusted, if desired, by a variety of procedures, including IlliClU ~ .., vortexing, extrusion, filtration,25 sonication, 1~ 6. ..:,~l;.. repeated freezing and thawing cycles, extrusion under pressure through pores of defined size, and similar methods. It may also be desirable to use the Illil,lU:l,UIl~lC:l of the present invention as they are formed, without any attempt at further ,.,.~ l;..., of the size thereof.
The gas filled Illi~ lU~L~ lC~ may be sized by a simple process of 30 extrusion through fllters; the filter pore sizes control the size ,l;~l, ;l ,,.li, ,ll of the resulting gas filled Illi,lU*/Il.,lC~. By using two or more cascaded or stacked set of filters, for example, a 10 ~m filter followed by an 8 ~m filter, the gas filled ~1 88557 WO 95132006 I ~_ / .a.. '~ ~ ~3 III;~IU~ I.IC~ can be selected to have a very narrow size .~ i.". around 7 to 9 ~m.
After filtration, these stabilized gas filled III;LIU~ IC~ remain stable for over 24 hours.
The sizing or filtration step may be a~ ., .".1,1;~1.. I by the use of a filter 5 assembly when the suspension is remoYed from a sterile vial prior to use, or more preferably, the filter assembly may be ;II~UI~ ' ' into the syringe itself during use.
The method of srzing the Illi~lU~,UIl~lC~ Will then comprise using a syringe comprising a barrel, at least one filter, and a needle; and will be carried out by a step of extracting which comprises extruding said Illi~lU~ lC:~ from said barrel through said filter fitted 10 to said syringe between said barrel and said needle, thereby sizing said Ill;.lU~ .IC~
before they are adlll;l.,~-.lcd to a patient in the course of using the Ill;~luaull.lc~ as CT
contrast agents in accordance with the present invention. The step of extracting may also comprise drawing said ll.;.lu~ll..c~ into said syringe, where the filter will function in the same way to size the Ill;.lU~Il.IC~ upon entrance into the syringe.
15 Another alternative is to fill such a syringe with l,lic.u~ll.,c~ which have already been sized by some other means, in which case the filter now functions to ensure that only i.,lu~ within the desired size range, or of the desired maximum size, are ly adlll;ll;~..lcd by extrusion from the syringe.
In preferred i.."l.o.l;~ , the solution or suspension of l.l;.lu~ is 20 extruded through a filter and is heat sterilized prior to shaking. Once gas filled Ill;.lU~ .,IC~ are formed, they may be filtered for sizing as described above. These steps prior to the formation of gas and gaseous precursor filled l~lic~u~ .lcs provide the advantages, for example, of reducing the amount of unhydrated stabilizing compound, and thus providing a ~;tll;r..a..~ly higher yield of gas filled III;~IUD~JIII.I.....
25 as well as and providing sterile gas filled III;~IU~ IC~ ready for A~llll;ll;~IAI;llll to a patient. For example, a mixing vessel such as a vial or syringe may be filled with a filtered stabilizing compound, especially lipid ~ r~ n and the suspension may then be sterilized within the mixing vessel, for example, by au uclavillg. Gas may beinstilled into the lipid suspension to form gas filled Ill;-lu~ ,lc~ by shaking the sterile 30 vessel. Preferably, the sterile vessel is equipped with a filter positioned such that the gas filled III;~IU~UII~lC~ pass through the filter before contacting a patient.

W0 95/32006 2 1 8 8 5 5 7 I~

The first step of this preferred method, extruding the solution of stabilizing compound through a filter, decreases -the amount of unnydrated compound by brcalcing up the dried compound and exposing a greater surface area for hydration.
Preferably, the filter has a pore size of about 0.1 to about 5 ~m, more preferably, 5 about 0.1 to about 4 ~m, eYen more preferably. about 0.1 to about 2 ~m, and still more preferably, about 1 ~m. Unhydrated compound, especially lipid, appears as amorphous clumps of non-unifortn size and is ~
The second step, ~ ;.". provides a c~ that may be readily ad.. i. ia~ cd to a patient for CT imaging. Preferably, ~ ;l;,~l;.". is 10 ~ by heat at~,liliL~iùll, preferably, by ~uLucl~vill~ the solution at a Lc~ c of at least about 100C, and more preferably, by dU~UCl~Vil~ at about 100C to about 130C, even more preferably, about 110C to about 130C, still more preferably, about 120C to about 130C, and even more preferably, about 130C.
Preferably, heating occurs for at least about 1 minute, more preferably, about l to 15 about 30 minutes, even more preferably, about 10 to about 20 minutes, and still more preferably, about 15 minutes.
If desired, alternatively, the first and second steps, as outlined above, may be reversed, or only one of the two steps can be used.
Where aLcl iliL LLiull occurs by a process other than heat ~t~ at a 20 ~ llllr which would cause rupture of the gas filled l~liclua~ Ir~ ll may occur subsequent to the formation of the gas filled ~ lua~ll.,~ca, and is preferred. For example, gamma radiation may be used before andlor after gas filled llliclua,ull~,lca are formed.
~Iethods of P~"al~iu.. Usin~ â Gaseous Precursor 25 In addition to the aru~ ;.. ". d ~ ,l.~.l;.. ,. l~. one can also use gaseous precursors contained in the llliclua,ull.,lc~ which, upon activation, for example, by t.,~ Lulc, light, or pH, or other properties of the tissues of a host to which it is ~dlll;ll;aL~.cd, undergo a phase transition from a liquid entrapped in the Illi~.lU~
to a gaseous state, expanding to create the stabilized, gas-filled llliclua~ ca of the 30 present invention. This technique is described in detail in copending patent applications Serial Nos. 08/160.232. filed November 30, 1993 and 081159,687, filed 21 88557 ~=
WO 95/32006 1 ~"~

November 30, 1993 both of which are ill~Ul,U~ ' herein by reference in their eMirety.
The preferred method of actiYating the gaseous precursor is by exposure to elevated L~ . Activation or transition t.ll~ , and like terms, refer to 5 the boiling point of the gaseous precursor which is the r , ~ l r at which the liquid to gaseous phdse transition of the gaseous precursor takes place. Useful gaseousprecursors are those materials which have boiling points in the range of about -100C
to 70C. The activation Lu~ dLul~ is particular to each gaseous precursor. An activation ~ r of about 37C, or about human body ~ r, is preferred lO for gaseous precursors of the present invention. Thus, in preferred form, a liquid gaseous precursor is activated to become a gas at 37'C. Elowever, the gaseous precursor may be in liquid or gaseous phase for use in the methods of the present invention.
The methods of preparing the CT imaging contrast agents of the present 15 invention may be carried out below the boiling point of the gaseous precursor such that a liquid is illUVI~l ' into a Illi~lu~l~h.,~. In addition, the methods may be performed at the boiling point of the gaseous precursor such that a gas is ill~ul~oldLr;d into a Illil,lU~ C. For gaseous precursors having low ~ J.ldiUlc boiling points,liquid precursors may be emulsified using a llli~lunuidi ~l device chilled to a low 20 t~ ldLul~. The boiling points may also be depressed using solvents in liquid media to utilize a precursor in liquid form. Further, the methods may be performed where the ~r~ ,ldLul~ is increased throughout the process, whereby the process starts with a gaseous precursor as a liquid and ends with a gas.
The gaseous precursor may be selected so as to form the gas in situ in 25 the targeted tissue or fluid, in vivo upon entering the patient or animal, prior to use, during storage, or during IlldllUrdLLUlC. The methods of producing the L~ .,ldLul~:-activated gaseous precursor-filled l.li.lu~ lc~ may be carried out at a L~ ldLulC
below the boiling point of the gaseous precursor. In this ,.,.1..~.1;,.,. .1l the gaseous precursor is entrapped within a llliLlUa~ll..C such that the phase transition does not 30 occur during Illdllurit~Lul~. Instead, the gaseous precursor-filled Illi~lU:~lll.,lC:~ are l-dl~urd~Lul~d in the liquid phase of the gaseous precursor. Activation of the phase transition may take place at any time as the Lr;lll~,ldLul~ is allowed to exceed the WO 95/32006 ~ 6~33 ~ 35 ~
boiling point of the precursor. Also, knowing the amount of liquid in a droplet of liquid gaseous precursor, the size of the ~ u"ul-.lc, upon attaining the gaseous state may be ~iPtPrminp~i Altematively, the gaseous precursors may be utilized to create stable 5 gas-filled IlI;L~U~ lC~ which are pre-formed prior to use. In this ~ o~ ..; the gaseous precursor is added to a container housing a suspending and/or stabilizing medium at a tLlllU. l~llUlC below the liquid-gaseous phase transition ~ lllG of the respective gaseous precursor. As the ~I III~JI IGiUUC is then exceeded, and an emulsion is fomned between the gaseous precursor and liquid solution, the gaseous precursor 10 undergoes transition from the liquid to the gaseous state. As a result of ihis heating and gas fommation, the gas displaces the air in the head space above the liquid suspension so as to fomm gas-filled spheres which entrap the gas of the gaseous precursor, ambient gas (e.g. air), or coentrap gas state gaseous precursor and ambient air. This phase transition can be used for optimal mixing and ~ II of the CT
15 imaging contrast medium. For example, the gaseous precursor, p~nuulubuL~llc, can be entrapped in the l,;. c~ . stabilizing compound, and as the hl..,u~ Lu.G is raised, beyond 4C, which is the boiling point of ~,lnuulubuL~Ilt, p~,lriuulul~uL~Ile gas is entrapped in ~..i..u,lul~lc,. As an additional example, the gaseous precursornuu-ubuLd-.c can be suspended in an aqueous suspension containing cl~ulairyillg and 20 stabilizing agents, such as glycerol or propylene glycol, and vortexed on a c.~". . . ;-i vortexer. Vortexing is ~ at a ~L~ iUlC low enough that the gaseous precursor is liquid and is continued as the ~LIIIU~I~IiUlC of the sample is raised past the phase transition ~LIII!)~IdLUlG from the liquid to gaseous state. In so doing, the precursor converts to the gaseous state during the IlliLlU ... -I`iri. ~lil... process. In the 25 presence of the .:IU~)IU~JI stabilizing agents, stable gas-filled Ill;LIual)ll.,lca result.
Accordingly, the gaseous precursors may be selected to form a gas-filled lui.lu"u~.lc in vivo or may be designed to produce the gas-filled ~ u~L~ll, lc in situ, during the ,.. ~.. ,.r,., 1.. , ;,.g process, on storage, or at some time prior to use.
As a further I ,.,I,oili,.,. .ll of this invention, by pre-fomming the gaseous 30 precursor in the liquid state into an aqueous emulsion, the maximum size of the lububble may be estimated by using the ideal gas law, once the transition to thegaseous state is Pffech~P~ For the purpose of making gas-filled IlliLlua~ lca from wossl32oo6 2 1 88557 , ", . 1-~ --gaseous precursors, the gas phase is assumed to form ;~ A, v~ y and substantially no gas in the newly formed l..iulu~lul,~,lc has been depleted due to diffusion jMO the liquid, which is generally aqueous in nature. Hence, from a known liquid volume in the emulsion, one would be able to predict an upper limit to the size of the gas-filled 5 Illi.ll . ' c.
Pursuant to the present invention, an emulsion of a stabilizing compound such as a lipid, and a gaseous precursor, containing liquid droplets of defined size may be r, i, such that upon reaching a specific It,..l.. .AIlllC, the boiling point of the gaseous precursor, the droplets will expand into gas-filled ~ lu~L.c~ of defuled10 size. The defined size represents an upper limit to the actual size because factors such as gas diffusion into solution, loss of gas to the dLlllU:l~JIl.lC, and the effects of increased pressure are factors for which the ideal gas law cannot account.
The ideal gas law and the equation for calculating the increase in volume of the gas bubbles on transition from the liquid to gaseous states is as follows:
PV = nRT
where P = pressure in aLIllu~
V = volume in liters n = moles of gas 20 T = ~ IdLUlC in K
R = ideal gas constant = 22.4 L dLIllo~,ll..c, deg~l mole-l With knowledge of volume, density, and ~ lc of the liquid in the emulsion of liquids, the amount (e.g. number of moles) of liquid precursor as well as the volume of liquid precursor, a pnon, may be calculated, which when converted to a 25 gas, will expand into a ..li.lu~ll.lc of known volume. The calculated volume will reflect an upper limit to the size of the gas-filled ~ v~ , assuming ;Il~ .. u"~expansion into a gas-filled Illi~lU~ .lC and negligible diffusion of the gzs over the time of the expansion.
Thus, for ~L~;I;~diiu.. of the precursor in the liquid state in an emulsion 3û wherein the precursor dropleL is spherical, the volume of the precursor droplet may be determined by the equation:
Volume (sphere) = 4/3 1rr3 21 88~57 wo ss/3200 where r = ra'dius of the sphere Thus, once the volume is predicted. and knowing the density of the liquid at the desired ~~ Lul~, the amount of liquid (gaseous precursor) in the - 5 droplet may be ~ r~rmin~-~i In more descriptive terms, the following can be applied:
V,3 = 4/3 7T(r,3) by the ideal gas law, PV=nRT
reveals, vx,5 = nRT/Pe3 or, (A) n = 4/3 [7rr333] P/RT
amount n = 4/3 [7rr~33 P/RT]-MWn Converting back to a liquid volume 15 (B) Vljq = [4/3 [7Tr~33] P/RT]-MWn/D]
where D = the density of the precursor Solving for the diameter of the liquid droplet, (C) diameer/2 = [3147r [4/3-[~r~33] P/RT] MWn/D]' 3 which reduces to 20 Diameter = 2[[re33] P/RT [MWn/D]]"3 As a further means of preparing ~ ,lua~ll.lc~ of the desired size for use as CT imaging contrast agents in the present invention, and with a knowledge of the volume and especially the radius of the stabilizing compound/precursor liquid droplets, one can use ~u~ ly sized filters in order to size the gaseous precursor droplets to 25 the ~ylU~ tL diameter sphere.
A Ic~lca~ ive gaseous precursor may be used to form a ~ ,lUayll-lC
of defined size, for example, 10 ~m diameter. In this example, the lllic~u~ lc is formed in the blOOda~ l of a human being, thus the typical t.,lll~ UlC would be 370C or 310 K. At a pressure of I dll.loa~ll.lc and using the equation in (A), 7.54 x 30 10-'7 moles of gaseous precursor would be required to fill the volume of a 10 ~m diameter llli~lua~ll.lc.

21 8855~ -wo 95/32006 ~ 33 Using the above calculated amount of gaseous precursor, and 1-nuuluiJuLdllc, which possesses a molecular weight of 76.11, a boiling point of 32.5C
and a density ûf 0.7789 grams/mL I at 20C, further r~lr~ inn~ predict that 5.74 x 10-'5 grams ûf this precursor would be required for a 10 ~m Illk,lU~
5 F51".~ funher, and armed with the knowledge ûf the density, equation (B) further predicts that 8.47 x 10 16 mL of liquid precursor are necessary to form a i lu~ c with an upper limit of 10 ~Lm.
Finally, using equation (C), an emulsion of lipid droplets with a radius of 0.0272 ~Lm or a ,UllC;~JUlldillg diameter of 0.0544 ~m need be formed to make a 10 gaseous precursor filled Illil,lU~ with an upper limit ûf a 10 ~m llliulua~ lc.
An emulsion of this panicular size could be easily achieved by the use of an d~lU~l 'y sized filter. In addition, as seen by the size of the filter necessary to form gaseous precursor droplets of defined size, the size of the filter would also suffice to remove any possible bacterial ~r.,,lA,.,;,.,,,,I~ and, hence, can be used as a sterile 15 filtration as well.
This ~,.,.I,ùll;,., 1l for preparing gas-filled llliclu~ lca used as CT
imaging contrast agents in the methods of the present invention may be applied to all gaseous precursors activated by ~ ldlUl~. In fact, depression of the freezing point of the solvent system allows the use gaseous precursors which would undergo liquid-to-20 gas phase transitions at t~,llll..ldLul~ below 0C. The solvent system can be selectedto provide a medium for suspension of the gaseous precursor. For example, 20%
propylene glycol miscible in buffered saline exhibits a freezing point depression well below the freezing point of water alone. By increasing the amount of propylene glycol or adding materials such as sodium chloride, the freezing point can be depressed even 25 further.
The selection of d~lU~ solvent systems may be determined by physical methods as well. When substances, solid or liquid, herein referred to as solutes, are dissolved in a solvent, such as water based buffers for example, the freezing point is lowered by an amount that is dependent upon the Culll~O~i~iull of the 30 solution. Thus, as defined by Wall, one can express the freezing point depression of the solvent by the following equation:
InxD = In (I - xù) = ~H~U5/R(1/T~- I/T) wo ss/32006 r~

where:
x, = rnole fraction of the solvent xb = mole fraction of the solute ~HiUs = heat of fusion of the solvent S To = Normal freezing point of the solvent The normal freezing point of the solvent results from solving the equation. If xb is small relative to x" then the above equation may be rewritten:
xb = AH,U,/R[T- To/T~T] z ~Hfus~TlRTD2 The above equation assumes the change in t~ lGlUlC ~T is small compared to T2.
10 The above equation can be simplified further assuming the c~ ;r". of the solute (in moles per thousand grams of solvent) can be expressed in terms of the molality, m.
Thus, Xb =m/[m + 1000/m,l = mMa/1000 where:
15 Ma = Molecular weight of the solvent, and m = molality of the solute in moles per 1000 grams.
Thus, ~ for the fraction Xb:
~T = [M,RTO2/lO00~H",s]m or AT = K,m, where K,=M,RTJ2/lOOOAH,Us K, is referred to as the molal freezing point and is equal to 1.86 degrees per unit of molal ,~ .,I,,.I;r~l~ for water at one d~lllU~ lC pressure. The above equation may be used to accurately determine the molal freezing point of gaseous-precursor filled IlliClU~ lC solutions used in the present invention.
Hence, the above equation can be applied to estimzte freezing point d~ ca~iull~ and to determine the a~JIJlu~ l;..ll5 of liquid or solid solute necessary to depress the solvent freezing LclllLJ.laLulc to an d,U~lU~lidt~ value.
Methods of preparing the Lclll,u.,ldlulc activated gaseous precursor-filled l~liclu~ c~ include:
(a) vortexing an aqueous suspension of gaseous precursor-filled ll~iclu~ cs used in the present invention; variations on this method include optionall~ duLuclGvillg before shaking, optionally heating an aqueous suspension of wo95~2006 2 ~ 88557 ~ r~

gaseous precursor and lipid, optionally Yenting the vessel containing the optionaily shaking or permitting the gaseous precursor ~ ua~ ca to form v ly and cooling down the gaseous precursor filled ..;.-u~lu~ Sll~rl ncirm, and optionaliy extruding an aqueous suspension of gaseous precursor and lipid through 5 a filter of about 0.22 ~m, al.."~ ly, filtering may be performed during in vivo 1;..,. of the resulting ~ lua~ll.,.ca such that a filter of about 0.22 ~Lm is employed;
(b) a Illi~lU' I` ri -li ll method whereby an aqueous suspension of gaseous precursor-filled Illi~lua~ ,lca of the present invention is emulsified by agitation 10 and heated to form Illi~lualJll.lc~ prior to ~ ;. " to a patient; and (c) forming a gaseous precursor in lipid suspension by heating, and/or agitation, whereby the less dense gaseous precursor-filled Illi~lua~ ,lca float to the top of the solution by expanding and displacing other Illi.lua~ .a in the vessel andventing the vessel to release air; and (d) in any of the above methods, utilizing a sealed vessel to hold the aqueous suspension of gaseous precursor and stabilizimg compound such as lipid, said suspension being maintained at a tclll~ Lul~ below the phase transition ~ UIC of the gaseous precursor, followed by dULU~IdVill~ to move the J,ld~Ul~ above the phase transition t.~ ul~, optionally with shaking, or 20 permitting the gaseous precursor Illi~luaL~ll.,lca to forln alJullldl~cvualy, whereby the expanded gaseous precursor in the sealed vesâel increases the pressure in said vessel, and cooling down the gas-filled Illi~lua~ lc s~l~r~ncir~n, after which shaking may also take place.
Freeze drying is useful to remove water and organic materials from the 25 stabilizing ~-, - I u~ prior to the shaking gas instillation method. Drying-gas instillation methods may be used to remove water from Illi~lua~ ,. By pre-entrapping the gaseous precursor in the dried Illi~lua~ll. l~a (i.e. prior to drying) after warming, the gaseous precursor may expand to fill the Illi~lua~)ll.,l~. Gaseous precursors can also be used to fill dried Illi~lua~ a after they have been subjected to 30 vacuum. As the dried Illi~lua~ll.lca are kept at a ~ J,ld~UI~ below their gel state to liquid crystalline ICIII~Id~UI~, the drying chamber can be slowly filled with the gaseous precursor in its gaseous state, e.g. ~llluulubuidll~ can be used to fill dried woss/32006 21 88557 r~ 4,j lU~ C~ composed of di!)alllli~uy~ n~ lylcholine (DPPC) at ~cll~ .lc~
between 4C (the boiling point of ~llluu~ubuLallc) and below 40C, the phase transition t~ .la~ulc of the bi~c~ lipid. In this case, it would be most preferred to fill the ~ u~l~l-.,lc~ at a t~ iUlC about 4C to about 5C.
Preferred methods for preparing the ~IIl~).laLulc activated gaseous precursor-filled Illi~lv~ll.,lc~ comprise shaking an aqueous solution having a stabilizmg compound such as a ~ ' lipid in the presence of a gaseous precursor at a 1" r~ below the gel state to liquid crystalline state phase trarlsition ~Clll~ UIC
of the lipid, and below the liquid state to gas state phase transition t~ of thegaseous precursor. Heating of the mixture to a ~clll~J.Ia~lllc above the liquid state to gas state phase transition L~ J..a~ulc of the gaseous precursor then causes the precursor to expand. Heating is then .l;~. .."i;,.,.. l and the tCI~ ld~UlC of the mixture is then be allowed to drop below the liquid state to gas state phase transition Lc.ll~J.,Ia~Ulc of the gaseous precursor. Shaking of the mixture may take place during 15 the heating step, or ~ ;ly after the mixture is allowed to cool.
The present invention also . l.. ~ the use of a method for preparing gaseous precursor-filled Illi~lU~ C~ comprising shaking an aqueous solution comprising a stabilizing compound such as a ~ c~ lipid in the presence of a gaseous precursor, and separating the resulting gaseous precursor-filled 20 l~ u~ c~ for computed ~UIllO~la~ y imaging use. Mi~lua~ c~ prepared by the foregoing methods are referred to herein as gaseous precursor filled llli~lua~
prepared by a gel state shaking gaseous precursor instillation method.
ConveMional, aqueous-filled liposomes of the prior art are routinely formed at a L~,1113J.la~UlC above the phase transition ~ J.Iaiulc of the lipids used to 25 make them, since they are more flexible and thus useful in biological systems in the liquid crystalline state. See, for example, Szoka and P~ ju~u llos~ Proc. Natl.
Acad. Sci. 1978, 75, 4194-4198. In contrast, the ...i..u,~l-.lc~ made according to preferred ~,.,I,o~ described herein are gaseous precursor filled, which imparts greater flexibility, since gaseous precursors after gas formation are more ~u~ lc:.~ible 30 and compliant than an aqueous solution. Thus, the gaseous precursor filled Illi~lU~ lC~ may be utilized in biological systems when formed at a t~ laiulc wos~32006 2 1 8 8557 ~ s~s~ ~

below the phase transition ~C~ L~lc of the lipid, even though the gel phase is more rigid.
The methods r--~~ r' by the present invention provide for shakmg an aqueous solution comprising a stabilizing compound, such as a biù~ ;l.lr lipid, S in the presence of a ~ r, activated gaseous precursor. Shaking, as used herein, is defined as a motion that agitates an aqueous solution such that gaseous precursor is introduced from the local ambient ~ lVilUlul~ into the aqueous solution. Any type of motion that agitates the aqueous solution and results in the ;IlLludu~Liul~ of gaseous precursor may be used for the shaking. The shaking must be of sufficient force to 10 allow the formation of foam after a period of time. Preferably, the shaking is of sufficient force such that foam is formed within a shon period of time, such as 30 minutes, and preferably within 20 minutes, and more preferably, within 10 minutes.
The shaking may be by microemulsifying, by ~ unuidiLillg, for example, swirling (such as by vonexing), side-to-side, or up and down motion. In the case of the 15 addition of gaseous precursor in the liquid state, sonication may be used in addition to the shaking methods set fonh above. Further, different types of motion may be combined. Also, the shaking may occur by shaking the container holding the aqueous lipid solution, or by sbaking the aqueous solution within the container without shaking the container itself. I~unher, the shaking may occur manually or by machine.
20 MPri~ ir:~l shakers that may be used include, for example, a shaker table, such as a VWR Scientific (cerritûs~ CA) shaker table, a llli~unuidi~cl, Wig-L-Bug~M (Crescent Dental MAIIIIr~ , Inc., Lyons, IL), which has been found to give ,ualLi~.ulally good results, and a mechanical paint mixer, as well as other known machines.
Another mean~i for producing shaking includes the action of gaseous precursor emitted 25 under high velocity or pressure. It will also be understood that preferably, with a larger volume of aqueous solution, the total amount of force will be ~ù~-c~uulldill~;ly increa~ied. Vigorous shaking is defined as at least about 60 shaking motions perminute, and is preferred. Vonexing at least 1000 revolutions per minute, an example of vigorous shaking, is more preferred. Vonexing at 1800 revolutions per minute is 30 most preferred.
The formation of gaseous precursor filled Illi~lO ~ C~ upon shaking can be detected by the presence of a foam on the top of the aqueous solution. This is w095/32006 2 1 88557 ,~I/L '~

coupled with a decrease in the volume of the aqueous solution upon the formation of foam. Preferably, the final volume of the foam is at least about two times the initial volume of the aqueous lipid solution; more preferably, the final volume of the foam is at least about three times the initial volume of the aqueous solution; even more5 preferably, the final volume of the foam is at least about four times the initial volume of the aqueous solution; and most preferably, all of the aqueous lipid solution is converted to foam.
The required duration of shaking time may be determined by detection of the formation of foam. For example, 10 ml of lipid solution in a 50 ml centrifuge 10 tube may be vortexed for ~L~ 15-20 mmutes or until the viscosity of the gaseous precursor-filled ~ ua,ull~lca becomes sufficiently thick so that it no longer clings to the side walls as it is swir~ed. At this time, the foam may cause the solution containing the gaseous precursor-filled Illi~lua~ to raise to a level of 30 to 35 ml.
The c~ i.... of stabilizing compound, especially lipid required to 15 form a preferred foam level will vary depending upon the type of stabilizing compound such as l,:.. ~.. ~ - ;l.l~ lipid used, and may be readily determined by one skilled in the art, once armed with the present disclosure. For example, in preferred rl.ll.ù.l;....:~, the fu. ~ - of 1,2-dipal...;~uyl~ yl~l~oli-~ (DPPC) used to form gaseous precursor-filled Ill;~,lu~ ca according to methods ~ . ' ' by the present 20 invention is about 20 mg/ml to about 30 mg/ml saline solution. The c~ .. of Lliai~ luyll.il~ ylcllulillc- (DSPC) used in preferred ~...1..~.1;..,...1~ is about 5 mg/ml to about 10 mg/ml saline solution.
Specifically, DPPC in a cnnffnrr~rinn of 20 mg/ml to 30 mg/ml, upon shaking, yields a total suspension and entrapped gaseous precursor volume four times 25 greater than the suspension volume alone. DSPC in a c.. l,,.li..l. of 10 mg/ml, upon shaking, yields a total volume completely devoid of any liquid suspension volume and contains entirely foam.
It will be understood by one skilled in the art, once armed with the present disclosure, that the lipids and other stabilizing compounds used as startmg 30 materials, or the Illi~lualJh.,lc final products, may be 111~ ' ' prior and subsequent to being subjected to the methods ~ . ' ' by the present invention. For example,the stabilizing compound such as a bif.c~ ,lf lipid may be hydrated and then wo ss/32006 Z 1 8 8 5 5 7 P~

Iyophilized, processed through freeze and thaw cycles, or simply hydrated. In preferred . ."l.~ the lipid is hydrated and then Iyophilized, or hydrated, then processed through freeze and thaw cycles and then Iyophilized, prior to the formation of gaseous precursor-filled Illil lU*lll~,lC~
According to the methods ,' ' by the present invention, the presence of gas, such as and not limited to air, may also be provided by the local ambient dllllu*~ . The local ambient dlulu~ull.,l~ may be the atmosphere within a sealed container, or in an unsealed container, may be the external ~IlVilU.l,ll.,.ll.
Alternatively, for example, a gas may be injected into or otherwise added to thecontainer having the aqueous lipid solution or iMo the aqueous lipid solution itself in order to provide a gas other than air. Gases that are not heavier than air may be added to a sealed container while gases heavier than air may be added to a sealed or an unsealed container. Accordingly, the present invention includes co e~ d,u~ ll of air and/or other gases along with gaseous precursors.
As already described above in the section dealing with the stabilizing compound, the preferred methods ~ , ' ' by the preseM invention are carried out at a t~ ,ldiUI~ below the gel state to liquid crystalline state phase transitiont lll~ldlUlC of the lipid employed. By "gel state to liquid crystalline state phase transition t~,lll~.,ld~Ul~n, it is meant the t~ ldiUl~ at which a lipid bilayer will convert 20 from a gel state to a liquid crystalline state. See, for example, Chapman et al.. J.
Biol. Chem. 1974, 249, 2512-2521.
Hence, the stabilized Illiwu~ precursors described above, can be used in the same manner as the other stabilized Illil,lU~,Ull lC~ used in the present invention, once activated by application to the tissues of a host, where such factors as 25 I~ ldlUI~ or pH may be used to cause generation of the gas. It is preferred that this l~lllbOllUII~ is one wherein the gaseous precursors undergo phase transitions from liquid to gaseous states at near the normal body ~ ),ld~UlC~ of said host, and are thereby activated by the i~lll,U~ld~ of said host tissues so as to undergo transition to the gaseous phase therein. More preferably still, this method is one wherein the host 30 tissue is human tissue having a normal ~IlI,U~,ldiUlC; of about 37C, and wherein the gaseous precursors undergo phase transitions from liquid to gaseous states near 37C.

wo s~i2006 r~

All of the above , I.r.~l;.,. 1~ involving ~ of the stabili_ed gas and gaseous precursor filled IlliLIu*~ ca used in the present invention, may be sterili_ed by autoclave or sterile filtration if these processes are performed before either the gas instillation step or prior to LLIII~l~,ldiUI~ mediated gas conversion of the 5 ~rlll~ .lr sensitive gaseous precursors within the CllcrPncir,n Alternatively, one or more anti-b~trriri~ agents and/or ,UIC~ may be included in the rulluulr~iu of the contrast medium, such as sodium ben7oate, all quaoernary ~m~r,nillnn salts, sodium a_ide, methyl paraben, propyl paraben. sorbic acid, ascorbylpalmitate, butylated ll~llu~ y....;~ulc, butylated hydroxytoluene, ~IIlulubuLdllol, d~ llu~ iu acid, 10 LLIIy~ .,;"~ lyc~lul~ potassium ben_oate, potassium ..,.; ~ ri ., potassium sorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts. Such crrrjli7Arirln which may also be achieved by other uu~ ,llLiulldl means, such as by irradiation, will be necessary where the stabili_ed Illi~lua,ull~ are used for imaging under invasive .,il, ., ., .~ e.g., illL~d~ ,uLuly or illLld~ y . The 5 d,U~III . ' ' means of ~r ;1;~ "~ will be apparent to the artisan instructed by the present description of the stabili_ed gas and gaseous precursor filled Illil,l~ ,' La and their use. The contrast medium is generally stored as an aqueous suspension but in the case of dried Illiclualull.,lca or dried lipidic spheres the contrast medium may be stored as a dried powder ready to be l~ prior to use.
20 Methods o~ use The novel stabili_ed gas and gaseous precursor filled III;~lua~ .lLa, useful as contrast media in CT imaging, will be found to be suitable for use in all areas where computed ~UlllU~ld~JIly imaging is employed.
In accordance with the present invention there is provided a method of 25 imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient. The imaging process of the present invention may be carried out by ,~.1...;..;~.. ;.-~ a contrast medium of the invention tû a patient, and then scanning the patient using computed Lulllo~ld~ y imaging to obtain visible images of an internal region of a patient andlor of any diseased tissue in that region. By region of a patient, 30 it is meaM the whole patient or a particular area or portion of the patient. The contrast medium is pdlLiuulduly useful in providing images of the ~daLIuillt~,~Lilldl region, but wo 95132006 ~ c can also be employed more broadly such as in imaging the vasculature or in other ways as wili be readily apparent to those skilled in the art. The phrase K~i~L1u;~
region or ~ciaLiuilllc~ 1 tract, as used herein, includes the region of a patient defined by the esophagus, stomach, small and large intestines and rectum. The phrase 5 vasculature, as used herein, denotes the blood vessels in the body or in an organ or part of the body. The patient can be any type of mammal, but most preferably is a human.
As one skilled in the art would recognize, s~ , - of the stabilized gas and gaseous precursor filled IlliL~ua,ull~,lca used in the present inveMion 10 may be carried out in various fashions, such as intravascularly, orally, intrarectally, intravaginally, illLld~ ul~illy, i"l",l,.. ;l.. AIIy, illildCo.lll.. ,lly, ~ 'ly, etc., using a variey of dosage forms. When the region to be scanned is the ~L1uil1Lca~ l region, ^II .;" ~ of the contrast medium of the invention is preferably carried out orally or rectally. The useful dosage to be dd11~ ia~1cd and the 15 particular mode of ^~ will vary depending upon the age, weight and the particular mammal and region thereof to be scanmed, and the particular contrast medium of the invention to be employed. Typically, dosage is initiated at lower levels and increased until the desired contrast . .,~ . ., .11 is achieved. Various 1 ~"ll1,;., 1;.,ll~ of the stabilized gas and gaseous precursor filled ~ ,lua~ll.,lc~ may be 20 used to alter properties such as the viscosity, osmolarity or palatability, in the case of orally a-'11.i..;~.1cd materials. In carryin~ out the CT imaging method of the present invention, the contrast medium can be used alone, or in . .",.I,;"-~;.", with diagnostic, therapeutic or other agents. Such other agents include excipients such as flavoring or coloring materials. The CT imaging techniques which are employed are ~ullv~ Liul1~il 25 and are described, for example, in Computed Body Tomography, i~ ee, J.K.T., Sagel, S.S., and Staniey, R.J., eds., 1983. Ravens Press, New York, N.Y., especially the first two chapters thereof entitled "Physical Principles and In~ ion", Ter-Pogossian, M.M., and "Techni~ues", Aronberg, D.J.
The routes of dL1llill;a~ Liu11 and areas of uâefulness of the gas and 30 gaseous precursor filled Ill;Llua,ul--l~a are not limited merely to the blood volume space, i.e., the vasculature. CT imaging can be achieved with the gas and gaseous precursor filled 111iLlUa~)l1C.ca used in the present invention if the I11iL1uaLJl~ a are , _ _ _ _ .

wo ss/32006 2 1 8 8 5 5 7 . ~

ingested by mouth so as to image the ~.lalluill~caLiu~l tract. Alternatively, rectal " of these stabilized gas Illil,lUa~ can result in excellent imaging of the lower ~;~aLluiul,a,alil~l tract including the rectum, descending colon, transverse colon, and ascending colon, as well as the appendix. It may be possible to achieve 5 imaging of the ileum, and Cull.C;~.IiJly the jejunum, by way of this rectal route. As well, direct ;.,~ may be achieved to visuali_e tile ~,, It is also c~ ' ' that the stabilized gas and gaseous precursor filled Illil,lVal)~ ,a may be a illlil..,~lc i directly into the ear canals such that one can visuali_e the canais as well as the Eustachian tubes and, if a perforation exists, the inner ear. It is also 0 I,UII r' ' ~ that the stabili7ed gas and gaseous precursor filled Illil,lUa~ ,lCa may be a i.l.iuliv~,,cd intranasally to aid in the viC~li7:~ri- n of the nasal septum as well as the nasal sinuses by computed tulllO~;Ia~Jlly imaging.
Other routes of ~l",;";~ ,- of the IlliUlU~ C contrast agents of the present invention, and tissue areas whose imaging is enhanced thereby include, but are 15 not limited to 1) intranasally for imaging the nasal passages and sinuses including the nasal region and sinuses and sinusoids; 2) mtranasally and orally for imaging the remainder of the respiratory tract, including the trachea, bronchus, blull~,llivlca, and lungs; 3) ;~ O ~ y for imaging the hearing passages and Eustachian tubes, tympanic "-. .,.l""". c and outer and inner ear and ear canals; 4) illllA~ y for20 imaging the regions associated with vision; 5) ;.,l-"l,;l,. Ally to visualize the p~ UII~ lll; and 6) intravesicularly, i e., through the bladder, to image all regions of the ~ lI l ;l IA ly tract via the areas thereof, including, but not limited to, the urethra, bladder, ureters, kidneys and renal i~al~UI~lLUlC and beyond, e.g., to perform 1yaiU~;l~lly or to confum the presence of ureteral reflux.
The invention is further described in the following examples. Ail of the examples are actual examples. These examples are for illustrative purposes oniy, and are not to be construed as limiting the appended claims.
ExamPles Various of the materiais used in the following examples are commercially available. All of the lipids were purchased from Avanti Polar Lipids wo 95/32006 ` 2 l 8 8 5 5 7 ~ ~"~ c~e ~ ~

(Alabaster, AL). r~,.lluulu~ L~ and p~lluu~vl~ dl~c we}e purchased from PCR
Chemicals, Inc. (Gainesville, FL).
In the following examples, "DPPE" refers to ~', ' ,y~ yl-r~ ";"., "DPPA" refers to di~ uyll~ acid; and "DPPC" refers to 5 dipal~ uyll.l...~l.l.,.~;~lyl~llvliulc. "PEG 50Q0" refers to poly(e~hylene glycol) polymer having a molecular weight of about 5000. "DPPE-PEG-5000" refers to DPPE which is covalently bound to PEG 5000.
F~y~ lDle 1 This example describes the ult~ ,liu.. of gas and gas precursor filled Illi~,lua~ .lca within the scope of the invention.
DPPC (77.5 mole %), DPPA (12.5 mole %), and DPPE-PEG 5000 (10 mole %) were introduced into a carrier solution of normal saline with glycerol (10 wt.%) and propylene glycol (10 wt.%). To this mixture was added u~"nuu.u~l,l.ia.,~
and a portion of the suspension (6 mL) was placed in a 18 mL glass vial and autoclaved for 15 minutes at 121C. The resulting translucent suspension was allowed to cool to room t~ Lul~. No ~ .,i~le foam could be seen was observed, but gentle shaking produced a few small bubbles at the top of v"e SllcrPncinn Shaking on a Wig-L-Bug~ (Crescent DeMal Mfg. Corp., Lyons, IL) for 2 minutes resulted in a dense foam that substantially filled the entire volume of the vial.
Samples of the lipid/lJ~lrlllvlu~ liallc (PFP) CIICrl~nCinn with and without shaking, were scanned by computed lUIIIV~ i)IIY using a Siemens DRH
Somatom Ill (Siemens, Iselin, NJ), at 125 peak kilovolts with 410 milli ,,~ and an 8 millimeter slice thickness and a zoom factor of 1.4. The images processed wivh a window width of 380 l:~mlncl~fi~ Units (HU) and a center of 30 HU showed fluid densiy in the unshaken sample and complete blackness in the shaken sample. When examined with a window width of 1,500 HU and a center of -600 ~U, which ~vll~alJulld~ to window senings of the type used for lung scamning, the unshakensample appeared bright white and the shaken sample was only faintly visible The density of the samples was measured and the unshaken sample measured 84.2 HU
(S.D. 38.02) and the shaken sample measured -548.3 HU i~ 5.92 HU.

w09~/32006 21 88557 r~ rJ~

ExamDle 2 This example is directed to an analysis the effect of manual amd m~rh~lnir~l shaking on ~ ,lva~ c size.
A lipid/PFP suspension was prepared aâ described in Example 1. A
5 sample of the suspension was shaken at room ~ IC manually (much less vigorously than with the Wig-L-Bug m~orh~nir~l shaker utilized in Example 1).
S~hc~nti~lly no foam was produced, only a few bubbles at the top of the liquid layer.
However, when the sample was warmed to body t~ ..~lC, 37C, i.e., above the 27.5C boiling point of the p.,lnuulup~.lidll~, and shaken manually, foam readily appeared and filled the entire Yial. When the foam produced by the Wig-L-Bug m- rh~nir:ll shaker at room L,llly.ldLu,c was compared to the foam produced manually at body t~ J.ld~UlC, it was noted that the Illi~lUD,Ull~lCD produced by manual shaking were somewhat larger than the Illi~luD~ lca produced by the Wig-L-Bug m-~rh~nir~l shaker. The Illi~lUD~JIl.lCD produced by manual shaking rose to the surface morequickly than the Illiulua~ll-lca produced by the Wig-L-Bug mechanical shaker, a further indication that the Illi-lUDl)ll.lCD produced by mechanical shaking were smaller tharl the Illi,luD,ull.les produced by manual shaking, since larger Illi.lUD~);I.lcD will rise more quickly.
Examl~le 3 This example is direcoed to the formation of stabilized gas-filled ua,u~..lcS comprising lipid bilayers with polyvinyl alcohol.
The effect of a polymer, namely, polyvinylalcohol, on the size of i~lua~Jll.lcS containing ~.lnuulu...ll,ul" is illustrated in this example. Gas-filled luD~ll.lcs comprising a lipid were prepared by the addition of 5 mg/mL of a suspension of DPPC ~ n~ acid, and DPPE-PEG 500û in a molar weight ratio of 82:8:10 in a vehicle containing 5% by weight of polyvinylalcohol (weight average - M.W. 5û,000, 99~ % hydrolyzed) in normal saline. To this mixture was added 50 ~LL
of ~..i[lUolu~ dllc. An identical suspension to the above described suspension was also prepared except that the vehicle was normal saline, glycerol, and propylene glycol 30 in a ratio of 8:1:1, v:v:v (Spectrum Chemical Co., Gardena, Calif.). The ~..~1....i;..,.
were then autoclaved at 121C for 21 minutes in a Barnstead/Thermolyne autoclave . _, _ . _ . ... .. ... . . . .. . . . .. _ _ _ .. _ . . _ . . .

~1 88557 (Barnstead/Thermolyne, Rancho Dominguez, Calif) The Ll,~ ulca of the resultant products were then ~ i to 30 C in a VWR Model 2500 incubator (VWR
M~,.,.r,. ~ Corp., ~l . , , New Mexico). The slightly opaque ~
were then shaken on a Wig-kBug shaker (Crescent Dental Mfg. Corp., Lyons, Il.) for 5 two minutes. This led to the production of foams. The subsequent foam samples were then sized on a Particle Sizing Systems Model 770 light vi ~ sizer. The instrument was calibrated with standard sized latex beads ranging in size from 2.02 ~m to 41.55 ILm (Coulter Electronics, Inc., Hialeah, Fla.). The sampling vehicle was deionized water. The size .l;~ of the PVA-containing sample vs. the normal 10 saline, glycerol, propylene glycol sample were as follows:

Sizing of Gas-Filled Mi.~ a Comprising Lipid Bilayers with and without Poly~inyl Alcohol (PVA) 5% PVA Sample Normal Saline, Glycerol, Propylene Glycol Sample Average Size 5.51 ~m 5,82 ~m 95% less than 14.45 ~ml 19.1 ~Lm 99.9% less than 72.2 ~m 75.6 ~m ~xample 4 This example describes the use of p".lluu-u~ dl.~ in the ~ ldliU~l of llli.lua~ .ca comprising lipid bilayers.
In an 18 mL vial, 6 mL of a suspension of 5 mglmL lipid consisting of 77.5 mole % 1,2 dipalmitoyl-3-sn gly~ lyl-llolill~ (DPPC), 12.5 mole %
;. acid, and 10 mole % 1,2 dipalmitoyl-3-sn-~ullu~u~ idyl~
pol~ yl~ ly.ul 5000 (DPPE-PEG 5000) was added followed by the addition of 50 IlL of pl,llluvlu~..lLdl~e. injected into the solution at room Lc~ diUlc. The head PCTIUS'~5/0649~
21 88557 ~P~51 7 Jl~J 1g98 space in the vial was filled with air at ambient pressure and the Yial was sealed with a teflon stopper and aluminum seal (VWR, Albuquerque, New Mexico). The vial was then autoclaved at 121C for lS minutes (Barnstead Thermolyne, Dubuque, lowa). Atranslucent hull~vg.ll~,vu~ suspension resulted. The vial was then removed from the S autoclave, allowed to cool to room t~ U~ liulG~ and then shaken for two minutes on a Wig-L-Bug shaker (Crescent Dental ~,..", rA~ ;llg Corp., Lyons, Ill.). The entire vial was then found to be filled with foam. The vial was thereafter placed in a ~rli~ tvl at 4C and the foam persisted. By ~ foam prepared with air or nitrogen gas alone in the same mixture of lipids, i.e., without the addition of y~lluu~uy~ ldl~c-, did not persist as long as the foam produced using y.,llluvlùy.,l-Ld--~. The duration of the foam prepared with the mixture of y~,lnuuluy~ dll~ and air was surprising; ~dl~il,ulally so when it is considered that the boiling pomt of y~,lnuuluy"liL~ is dy,ul~ 'y 27.5C. Room ~lUy~ld~ulc under the conditions of this ~ i" ,l was about 20C, and thus at 4C it would have expected that the foam would collapse. This ~Ay~,li thus l1rl~ the surprising discovery that the presence of y~,lnuulul,dubul~, despite being in the liquid state, can contribute to ~Ldbili~dlivl~ of the foam.
Examle 5 This example describes the use of perfluorohexane in the yl~ydldliv~l ûf ,lu~yll~,lu~ UUIllyli~iulg lipid bilayers.
To further ~' that a liquid-state ~,llluuluc~uboll can contribute to ~ " of a gas-filled Illi~,~u~yll~l~ foam, the result obtained using perfluorohexane (b.p. 56C) was evaluated. A suspension of lipids was prepared as described above in Example 4, except that 50 ~L of y~,lnUULUl~ dLl~ was added to the vial in lieu of the y~llluvlvy~,llLdl~t;. The suspension was autoclaved yielding a translucent-to-cloudy suspension of lipids. The material was shaken on the Wig-L-Bug (Crescent Dental Mfg. Corp., Lyons, Ill.) for two minutes and a foam again resulted.
The volume of foam was greater than the control sample, which utilized air alone as the ambient gas. Once again, the foam remained stable and persisted longer than the air sample. This clearly d~,lllvlL~lldL~s that the presence of the perfluorohexane, which 3û is liquid at human physiological ~Illp~,ldLUl~, functions to stabilize a gas-filled i~,lU~yllClc; foam.
~MC~ û SH~ET

2 1 885~7 wo ~s/32006 ~ . 6 t~
5~
Example 6 This example describes trehalose ~Ldbili~liu.l of gas-filled IlliLlu~pllLlc~
comprising lipid bilayers.
A gas-filled lll;LIU~yl~,~ foam was prepared from a stabilizing 5 compound vehicle comprising normal saline:glycerol:propylene glycol (8:1:1, v:v:v) with the lipids set forth in Example 4 above, and shaken as described therein on a Wig-L-Bug for two minutes, yielding d~ / 6 mL of foam at room .. r After fûur days, it was discovered that the foam was no longer present.
When the above experiment was repeated with the same lipids, e~cept that trehalose, 10 D-~lu~u~,yldllù~, a ~ I, was added in a 1:1 molar ratio of trehalose to lipid, the foam was found to persist longer than the control. Repeating the experiment yielded simiiar results. This experiment clearly (' that trehalose can function as an auxiliary stabilizing compound to lengthen the time duration of gas-filled IlliLlU~)h_lC~ of the present invention.
The disclosures of each patent, patent application and publication cited or described in this document are hereby illLUl~JI ' ' by reference, in their entirety.
Various ~.n.lilirA~ of the inYention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing ~ipsrrirtinn Such ~ i;r; Al~ . are also intended to fall within the scope of the appended claims.

Claims (51)

What is claimed is:

1. A contrast medium for computed tomography imaging comprising gas-filled microspheres.

2. A contrast medium according to Claim 1 wherein said gas is selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, fluorine, helium argon, xenon and neon.

3. A contrast medium according to Claim 1 wherein said gas comprises a fluorinated gas.

4. A contrast medium according to Claim 1 wherein said fluorinated gas is selected from the group consisting of perfluorocarbons and sulfur hexafluoride.

5. A contrast medium according to Claim 3 wherein said perfluorocarbon gas is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane and perfluoropentane.

6. A contrast medium according to Claim 1 wherein said microspheres are prepared from a biocompatible lipid.

7. A contrast medium according to Claim 6 wherein said lipid is selected from the group consisting of fatty acids, lysolipids, phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylglycerols, phosphatidylinositols, sphingolipids, glycolipids, glucolipids, sulfatides;
glycosphingolipids, phosphatidic acids, palmitic acids, stearic acids, arachidonic acids, oleic acids, lipids bearing polymers. lipids bearing sulfonated monosaccharides, lipids bearing sulfonated disaccharides, lipids bearing sulfonated oligosaccharides, lipids bearing sulfonated polysaccharides, cholesterols, tocopherols, lipids with ether-linked fatty acids, lipids with ester-linked fatty acids, polymerized lipids, diacetyl phosphates, dicetyl phosphates, stearylamines, cardiolipin, phospholipids with fatty acids of 6-8 carbons in length, synthetic phospholipids with asymmetric acyl chains, ceramides, non-ionic lipids, sterol aliphatic acid esters, sterol esters of sugar acids, esters of sugar acids, esters of sugar alcohols, esters of sugars, esters of aliphatic acids, saponins, glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate, glycerol, glycerol esters, alcohols of 10 to 30 carbons in length, 6-(5-cholesten-3.beta.-yloxy)-1-thio-.beta.-D-galacto-pyranoside, digalactosyldiglyceride, 6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxy-1-thio-.beta.-D-galactopyranoside, 6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxy-1-thio-.alpha.-D-mannopyranoside, 12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoic acid, N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanouyl]-2-aminopalmitic acid, cholestery(4'-trimethylammonio)butanoate, N-succinyldioleoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol, 1,2-dipalmitoyl-sn-3-succinylglycerol, 1,3-dipalmitoyl-2-succinylglycerol, 1-hexadecyl-2-palmkitoylglycero-phosphoethanolamine, palmitoylhomocysteine, cationic lipids, N-[1-(2,3-dioleoyloxy)-propyl]-N,N,N-trimethylammonium chloride, 1,2-dioleoyloxy-3-(trimethylammonio)-propane, 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol, lipids bearing cationic polymers, alkyl phosphonates, alkyl phosphinates, and alkyl phosphites.

8. A contrast medium according to Claim 7 wherein said phosphatidylcholine is selected from dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine; wherein the phosphatidylethanolamine is selected from dioleoylphosphatidylethanolamine, distearoylphosphatidylethanolamine and dipalmitoylphosphatidylethanolamine; wherein the sphingolipid is sphingomyelin;
wherein the glycolipid is selected from the group consisting of ganglioside GM1 and ganglioside GM2; wherein in the lipids bearing polymers, the polymer is selected from the group consisting of polyethyleneglycol, chitin, hyaluronic acid and polyvinylpyrrolidone; wherein the sterol aliphatic acid esters are selected from the group consisting of cholesterol sulfate, cholesterol butyrate, cholesterol isobutyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate, and phytosterol n-butyrate; wherein the sterol esters of sugar acids are selected from the group consisting of cholesterol glucuronide, lanosterol glucuronide, 7-dehydro-cholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate; wherein the esters of sugar acids and the esters of sugar alcohols are selected from the group consisting of lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoyl gluconate, and stearoyl gluconate; wherein the esters of sugars and the esters of aliphatic acids are selected from the group consisting of sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid, and polyuronic acid;
wherein the saponins are selected from the group consisting of sarsasapogenin, smilagenin, hederagenin, oleanolic acid, and digitoxigenin; wherein the glycerol esters are selected from the group consisting of glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol and trimyristate; wherein the alcohols of 10-30 carbon atoms are selected from the group consisting of n-decylalcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and n-octadecyl alcohol;
wherein in the lipids bearing cationic polymers, the cationic polymers are selected from the group consisting of polylysine and polyarginine

9. A contrast medium according to Claim 6 wherein said lipid is selected from the group consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine and dipalmitoylphosphatidic acid

10. A contrast medium according to Claim 9 wherein polyethylene glycol is bound to said dipalmitoylphosphatidylethanolamine.

11. A contrast medium according to Claim 6 wherein said lipid comprises dipalmitoylphosphatidylethanolamine and phosphatidic acid in a combined amount of from about 0.5 to about 30 mole percent

12. A contrast medium according to Claim 11 wherein said lipid is selected from the group consisting of dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, in an amount of from about 70 to about 100 mole percent.

13. A contrast medium according to Claim 6 wherein said lipid comprises: (i) a neutral lipid, (ii) a negatively charged lipid, and (iii) a lipid bearing a hydrophilic polymer; wherein the amount of said negatively charged lipid is greater than 1 mole percent of the total lipid present and the amount of lipid bearing ahydrophilic polymer is greater than 1 mole percent of the total lipid present.

14. A contrast medium according to Claim 13 wherein said negatively charged lipid is phosphatidic acid and wherein the polymer in said lipid bearing a hydrophilic polymer has a weight average molecular weight of from about 400 to about 100,000 and is covalently bound to said lipid.

15. A contrast medium according to Claim 14 wherein said hydrophilic polymer is selected from the group consisting of polyethyleneglycol, polypropyleneglycol, polyvinylalcohol and polyvinylpyrrolidone and copolymers thereof, and wherein said lipid of said lipid bearing a hydrophilic polymer is selected from the group consisting of dipalmitoylphosphatidylethanolamine and distearoylphosphatidylethanolamine.

16. A contrast medium according to Claim 6 wherein said lipid comprises about 77.5 mole percent of dipalmitoylphosphatidylcholine, about 12.5 mole percent of dipalmitoylphosphatidic acid, and about 10 mole percent of dipalmitoylphosphatidylethanolamine-polyethyleneglycol 5000.

17. A contrast medium according to Claim 6 wherein said lipid comprises about 82 mole percent of dipalmitoylphosphatidylcholine, about 10 molepercent of dipalmitoylphosphatidic acid, and about 8 mole percent of dipalmitoylphosphatidylethanolamine-polyethyleneglycol 5000.

18. A contrast medium according to Claim 1 wherein said microspheres are prepared from a biocompatible polymer selected from the group consisting of a polysaccharide, a semisynthetic polymer and a synthetic polymer.

19. A contrast medium according to Claim 18 wherein said polysaccharide is selected from the group consisting of arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans, levan, fucoidan, carrageenan, galactocarolose, pectic acid, pectin, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratan, chondroitan, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch, natural homopolymers and heteropolymers containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic, acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof.

20. A contrast medium according to Claim 18 wherein said semisynthetic polymer is selected from the group consisting of carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose.

21. A contrast medium according to Claim 18 wherein said synthetic polymer is selected from the group consisting of polyethylenes, polypropylenes, polyurethanes, polyamides, polystyrene, polylactic acids, fluorinated hydrocarbons, fluorocarbons and polymethylmethacrylate.

22. A contrast medium according to Claim 21 wherein said polyethylene is selected from the group consisting of polyethylene glycol, polyoxyethylene and polyethylene terephthalate: wherein said polypropylene is polypropylene glycol; wherein said polyurethane is selected from the group consisting of polyvinyl alcohol, polyvinylchloride and polyvinylpyrrolidone; wherein said polyamide is nylon; and wherein said fluorocarbon is polytetrafluoroethylene.

23. A contrast medium according to Claim 1 further comprising compounds selected from the group consisting of ingestible oils; mixed micelle systems compounds; viscosity modifiers; emulsifying and/or solubilizing agents; suspending or viscosity-increasing agents; synthetic suspending agents; and tonicity-raising agents.

24. A contrast medium according to Claim 1 wherein said gas is derived at least in part from a gaseous precursor.

25. A contrast medium according to Claim 1 wherein said microspheres a are prepared from a composition comprising dipalmitoylphosphatidylcholine, glycerol and propylene glycol.

26. A method for preparing gas-filled microspheres for use as a computed tomography contrast medium comprising agitating an aqueous suspension of a biocompatible lipid in the presence of a gas.

27. A method according to Claim 26 wherein said agitating comprises shaking or vortexing.

28. A method according to Claim 27 wherein said shaking comprises a reciprocating motion in the form of an arc.

29. A method according to Claim 28 wherein said arc is between about 2° and 20°, and the number of reciprocations per minute is between about 1000 and about 20,000

30. A method according to Claim 29 wherein said arc is between about 6° and about 7°, and the number of reciprocations per minute is between about 5000 and about 8000.

31. A method according to Claim 26 wherein said agitating is carried out at a temperature below the gel to liquid crystalline phase transition temperature of said biocompatible lipid.

32. A method for preparing gas-filled microspheres for use as a computed tomography contrast medium comprising agitating an aqueous suspension of a biocompatible lipid in the presence of a gaseous precursor to provide gaseous precursor-filled microspheres; and activating said gaseous precursor to provide the gas-filled microspheres.

33. A method according to Claim 32 wherein said agitating comprises shaking or vortexing.

34. A method according to Claim 33 wherein said shaking comprises a reciprocating motion in the form of an arc.

35. A method according to Claim 34 wherein said arc is between about 2° and about 20°, and the number of reciprocations per minute is between about 1000 and about 20,000.

36. A method according to Claim 35 wherein said arc is between about 6° and about 7°, and the number of reciprocations per minute is between about 5000 and about 8000.

37. A method according to Claim 32 wherein said agitating is carried out at a temperature below the gel to liquid crystalline phase transition temperature of said biocompatible lipid.

38. A method according to Claim 32 further comprising agitating said suspension in the presence of a gas.

39. A method of providing an image of an internal region of a patient comprising (i) administering to the patient a contrast medium according to Claim 1, and (ii) scanning the patient using computed tomography to obtain visible images of the region.

40. A method according to Claim 39 wherein the region comprises the vasculature.

41. A method according to Claim 39 wherein the region comprises the cardiovascular region.

42. A method according to Claim 39 wherein the region comprises the gastrointestinal region.

43. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast medium according to Claim 1, and (ii) scanning the patient using computed tomography to obtain visible images of any diseased tissue in the patient.

44. A method according to Claim 43 wherein the region comprises the vasculature.

45. A method according to Claim 43 wherein the region comprises the cardiovascular region.

46. A method according to Claim 43 wherein the region comprises the gastrointestinal region.

47. A method according to Claim 43 wherein said scanning is of a region of a patient selected from the group consisting of the intranasal tract, the auditory canal, the introcular region, the intraperitoneal region, the kidneys, the urethra and the genitourinary tract.

48. A method of providing an image of an internal region of a patient comprising (i) administering to the patient gaseous precursor-filled microspheres, (ii) allowing said gaseous precursor to undergo a phase transition from a liquid to a gas, and (iii) scanning the patient using computed tomography to obtain visible images of said region.

49. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient gaseous precursor-filled microspheres, (ii) allowing said gaseous precursor to undergo a phase transition from a liquid to a gas, and (iii) scanning the patient using computed tomography to obtain visible images of any diseased tissue in the patient.

50. A method for preparing in situ in the tissue of a patient a contrast medium for computed tomography, the contrast medium comprising gas-filled microspheres, comprising (i) administering to the patient gaseous precursor-filled microspheres, and (ii) allowing the gaseous precursor to undergo a phase transition from a liquid to a gas to provide the gas-filled microspheres.

51. A method according to Claim 48 wherein said gaseous precursor undergoes a phase transition from liquid to gaseous states at near the normal body temperature of the patient.