CA2191079A1 - Stabilized homogenous suspensions as computed tomography contrast agents - Google Patents

Abstract

Novel stabilized suspensions of a gas as diagnostic contrast agents, including computed tomography (CT) and magnetic resonance (MR) contrast agents. In preferred embodiments, the suspensions comprise stabilized gas-filled microspheres. Also in some preferred embodiments, at least a portion of the gas is derived from a gaseous precursor.

Description

WO 95/3200~ 2 1 9 1 0 7 9 r~
.
.

STARTT T7:F.T) HOMOGENOUS SUSPENSIONS
AS COMPUTED TOMOGRAPHY CONTRAST AGENTS
Cross-Reference to F l~ed ~
This is a c~ in-part of copending U.S. application Serial No.
5 08/247,656, filed May 23, 1994, which is related to U.S. application Serial No.
08/116,982, filed September 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.
07/680,98i, filed April 5, 1991, now U.S. Patent 5,205,290, issued April 27, 1993.
The disclosures of each of the above ~ ;".. are iul~u~Julalcd herem by reference in their entirety.
Field of the Invention The present invention relates to r ' for computed IUIIIU~
More ~ " the present invention relates to stabilized, v~.b~L~Ul~ lly l.. .e,.. , , ~ as contrast agents for computed Lulllv~.
nA. 1~ ' 0f the In~ention Computed LUIIIV~ (CT) is a commonly used diagnostic technique for the diagnosis of various diseases and maladies of the body, mcluding abdominal and pelvic diseases. In 1978 alone, about 7.8 million body CT scans were l,~
CT imaging involves measuring the ~IJiudLl.. ,ily of matter.
;."~ ;ly is typically expressed in Tlou.. ,rl~ld Units (HU). TT.. fi 11 Units are

2 1 9 1 0 7 9 PCT/US95106491 ~

a measure of the relative absorption of computed lulllvgLa~LJh~ X-rays by matter and is directly ~lu~ul~iullal to electron density. Water has been arbitrarily assigned a value of 0 HU, air a value of -1000 HU, and dense cortical bone a value of 1000 EIU.
Various tissues in the body possess simila} densities. This has caused 5 difficulty in the generation of visual images by CT of tissues that possess similar densities and which are proximate each other. For exa~nple, it is difficult to generate separate CT images of the ~a~ L~ ' (GI) tract and adjacent structures, includmg, for example, the blood vessels and the Iymph nodes. Accordingly, contrast agentshave been developed in an attempt to change the relative densities of different tissues, 10 and thereby improve the diagnostic efficacy of CT. CT contrast agents a}e used in a majority of the CT imaging scans. For example, of the 7.B million body CT scans performed in lg78, 6.6 million involved the use of ill~ldv~,llUU:~ contrast agents for the .. ,l. --,.. l of vascular amd/or visceral images.
Traditional CT contrast agents for imaging the lsa~ ' ' '' 1 (GI) tract 15 are generally based on r~ of ipncp~ non-lhc~rh~hlp~ heavy metal materials. Such materials assist in imagmg the bowel by absorbing X-ray i which increases the ldLl;~d~ y of the bowel lumen. Common among such contrast agents are barium and iodinated, r ' includmg, for example, barium sulfate. Barium sulfate and iodinated: , ' have been used for itnaging the GI tract for the past 60 years and 20 are widely used today for enhancing CT images of the upper and lower Gl tract. They generally increase electron density in cer[aim regions of the body, and are therefore classified as a "positive contrast agents. n Despite their widespread use, barium and iodinated , ' suffer from various drawbacks. ~or example, they are generally i~ ~ ""IJ ' ;l .l.~ with other 25 and/or newer imaging i ' . . includimg vascular ilnaging techniques. This ' ~ J is observed, for example, in CT aul~iOg .' .y (CTA). In CTA, iodinated contrast agents are injected i.~La~.,llu~ly and images are obtained during the bolus phase of contrast. Highly detailed images of the ~a~ula~ul~ are generally obtained using CTA by ~ the axial images to yield a composite picture of 30 the vessels. During this lef ., . ~ the picture of the \ ' ~ is optimized based on the measured density in the vessels being visualized. To perform this imaging, various baseline image ' `.~a~liulL~ are performed. However, the vascular image ~ WO 9513200~i 2 1 9 ~ 0 7 ~ PcT/us9~06491 ,.
;.... becomes distorted and obscured in the presence in vhe bowel of radiodense contrast agents, for example, iodinated ~ . J. - -ly, difficulty is e.~.uulltclcd in ,u.,.rvlllliug CTA and CT in the GI tract ~ ly if any positive contrast agents, such as barnlm and iodinated: , ' are present.
Moreover, vhe viability of currently available CT contrast agents is generally extremely sensitive to If the c~ .,';.... is too low, little contrast is obsened. If the: is too high, beam hardening artifacts result and are obsened as streaks in the resulting CT images. In addition, difficulty is typically e.l,v...l~.c~ in visualizing the bowel mucosa with the currently available 10 contrast agents.
Accordingly, new and/or better contrast agents for CT are needed. The present invention is directed to this, as well as other, important ends.
Summo~ of the Invention The present mvention is directed to stabilized, sllhct~nti~lly 1..""~.~, ...".
, Specifically, in one aspect, there is provided a stabilized, ~ul~lall~ially 1O wspension of a gas. The suspension has a negative density of about ~0 T~lmc.ofiPI-I units (HU) or less. In preferred form, the suspension comprises stabilized 1~ iclu*~h_~c~. The suspension may addiliv lally comprise, as desired, a stabilr~ing material, ~ickening agent and/or a dispersing agent.
Anovher aspect of the invention relates to a stabilized, I
suspension of a gaseous precursor. In preferred form, the suspension comprises stabilized lli.,.v*,h~ .. The suspension may additionally comprise, as desired, a stabilizing material, thickening agent and/or a dispersing agent.
Yet another aspect of the invention relates to a contrast medi~m for5 computed Ivluv~Ela,ully. The contrast agent comprises a stabilr~ed, ! ' '- "~, suspension of a gas. At least a portion of the gas is derived from a gaseous precursor. In preferred form, the suspension comprises stabilized Still another aspect of the invention relates to a method for p~eparing a 30 stabilized, 'ly 1... --.~,.. ~ suspension of a gaseous precursor. The method wo 9s/32005 2 1 ~ ~ ~ 7 ~ /u~ J ~91 ~
comprises agitating an aqueous suspension of a stabilizing material in the presence of a gaseous precursor. In preferred form, the agitation comprises shaking or vortexing.
Yet another aspect of the invention relates to a method for preparing a stabilized, substantially l~ ,f ~ '` suspension of a gas for use as a computed 5 lulllv~la~lly contrast medium. The method comprises agitating an aqueous suspension of a stabilizing material in the presence of a gaseous precursor. The method further comprises activating the gaseous precursor. In preferred form, agitation of the aqueous suspension comprises shaking or vortexing. Also m preferred form, activation of the gaseous precursor involves a phase transition of a liquid gaseous precursor to a gas in lO vivo.
Still another aspect of the invention relates to a method of providing an rmage of an mternal region of a patient. The method comprises r ' ' ~ to the patient a suspension as described above. The metbod also comprises scanning the patient using computed lulll~la~lly to obtain visible images of the region.
Another aspect of the invention also relates to a method of providing an image of an internal region of a patient. The method comprises to the patient a stabilized, ~ suspension of a gaseous precursor. The gaseous precursor is allowed to undergo a phase transition from a liquid to a gas in vivo and the patient is scammed using computed lul~lu~la~lly to obtain visible images of 20 any diseased tissue in the patient.
The invention also relates to a method for diagnosing the presence of diseased tissue in a patient. The method comprises ,, to the patient a suspension as described above. The method furtber comprises scam~ing the patientusing computed Lu...O~Ialllly to obtain visible images of any diseased tissue in the 25 patient.
Another aspect of the mvention also relates to a method for diagnosing the presence of diseased tissue m a patient. The method comprises L- ' ' ' ' ,, to the patient a stabilized. ~ul~LallLi~lly l~.,.. c. ~ - suspension of a gaseous precursor.
The gaseous precursor is allowed to undergo a phase transition from a liquid to a gas 30 in vivo and. the patient is scanmed usmg computed Lu~ alJlly to obtain visible images of any diseased tissue in the patient.
.

~ wossl3200s 2 ~ 9 ~ 0 7 9 F~l/u~
Yet another aspect of the invention relates to a method for preparing in a patient a contrast medium for computed lu..lv~ ,vlly. The method comprises ~ to the patient a stabilized, substantially l~ n~ suspension of a gaseous precursor. The method further comprises allowing the gaseous precursor to undergo a phase transition from a liquid to a gas in vivo to provide the contrast medium.
These and other aspects of the invention will become more apparent from the present ~ and claims.
I~ ' ` ` Des~ri~tion of the Invention As employed above and tLlroughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
"Suspension" refers to a mixture, dispersion or emulsion of finely divided colloidal particles floating in a liquid. The particles may be solid, liquid or gaseous.
"Emulsion" refers to a mixture of two or more liquids and is generally im the form of a colloidal mixture.
"Stabilized" refers to ,~ which have been formulated as a mixture of finely divided colloidal particles floating in a liquid with minimal ~,5SlC~ iUII. Preferably, the , remain stabilized for a certain period of time.
20 As discussed in detail below, certain preferred ~ L of the present invention involve , of stabilized III;~IUD~UI~ CD~ In this context, the term "stabilized"
refers to ~IPi~,lu~l~h_lcD which are substantially resistant to ~ i.. that is caused, for example, by the loss of structural or ~ mtegrity im the walls of the llfil"lUD~ lCD and/or by the loss of any sigmficant portion of a gas or gaseous precursor, . ' within the uD~h~lc. As indicated above, the . of the present invention are stable for a certain period of time. Preferably, &e are stable for a period of time which permits use of the imcluding, for example, use af the suspension for generating visible images of a region of a patient by computed IU..IU~ ,UI'I1. Thus, the r ' are preferably stable for30 a period of time to at least permit their to a patient and subsequent scanning of the patient with computed lu..lo~ )h~

w0ss/3200s 2 1 9 1 07 ~ s ~.gl ~

"Stabilizing material" refers to a substance which is l,;.~ and which is capable of stabilizing the present ~ Thus, with respect to which comprise, for example, finely divided liquid droplets and/or gaseous bubbles, the stabilizing materials are capable of ,, aO6l,,6~lLiull of the droplets 5 and/orbubbles. With respect to ~ ~1 .a~ involving ~ of l~ ,IUD~/II.,lCD, the stabilizing materials are capable of promoting the formation of the ~ IUD~ , as well as enhancmg the resistance of the 1ll;.l~ ,' , once for~ned, to ~.gr- ' caused, for example, by the loss of structural or ~.. 1.. -:';.~.. ,.1 integrity in the walls of the II~;~,IUD~ and/or by the loss of any sigluficant portion of a gas or gaseous10 precursor , ' ' therein. In preferred ~ ~ ' , the stabilizing materials impart the aforesaid properties to the , for a certain period of time.
Preferably, the stabilrzing materials are capable of stabilizing ~ for a period of time which permits use of the Sll~r~n~;~.n mcluding, for example, in the generation of visible images of a region of a patient by computed lullluol~ll~. Thus, the 15 stabilizing materials are preferably capable of stabilizing the ~ ~1. .-: ...~ of the present mvention for a period of time to at least permit their p~ to a patient and subsequent scanning of the patient with computed O .' 1. In certam preferred 1 ~101; ~ , the stabilizing material comprises a surfactant. "Surfactant", as used herein, refers to a surface-active agent that is capable of altering (i) the surface tension 20 of an aqueous solution; (ii) the surface tension between two liquids; amd/or (iii) the surface tension between a liquid and a solid. Generally, alteration of the surface tension involves a reduction in surface tension. The surfactant may possess a net neutral, positive or negative charge. In certam preferred; ' ' , tbe stabilizingmaterial comprises a polymer. ~Polymer", as used herem, refers to molecules formed 25 from the chemical union of two or more repeating units. Accordingly, included within the term "polymer" are, for example, dimers, trimers and oligomers. The polymer rnay be synthetic, naturally-occurrmg or ~1.;1l.,1;.,. In preferred form, the term "polymer" refers to molecules which comprise 10 or more repeatmg units. In certain other preferred; ' the stabilizmg material comprises a non-polymeric 30 material, includmg, for example"....,.~... ;~ molecules.
"Dispersing agent" refers to a surface-active agent which, when added to a suspending medium of colloidal particles, including, for example, the ~

WO95132005 21 91 079 P~,l/u~

generally promotes uniform separation of particles. In certain preferred ....l,o.l; .,~, the dispersing agent comprises a polymeric siloxane compound.
"Lipid" refers to a synthetic, ~ liDylltll~L;~ or naturally-occurring ,', compound which comprises a llv.ll~ r~ ''' component and a l~ u~/llub;~.
5 c , Lipids include, for example, fatty acids, neutral fats, glycolipids, aliphatic alcohols and waxes, terpenes and steroids.
"Alkyl" refers to an aliphatic llydluLallJull group which may be straight or branched having 1 to about 30 carbon atoms. "Lower alkyl~ refers to an alkyl group having I to about 8 carbon atoms. "Higher alkyl" refers to an alkyl group 10 having about 10 to about 30 carbon atoms. The alkyl group may be optionally substituted with one or more alkyl group ~ il,`l;~. ..~ wluch may be the same ordifferent, where "alkyl group ~ubqL;iu~.llL" mcludes, for example, halo, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, carboxy alhu~yuaul,u.lyl, oxo and cycloalkyl. There may be optionally inserted along the alkyl group one or more 15 oxygen, sulphur or substituted nitrogen atoms, wherein the nitrogen substituent is lower allcyl. The alkyl group may be linear or branched. "Branched" refers to analkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. Exemplary alkyl groups include, for example; methyl, ethyl, i-propyl, n-butyl, t-butyl, n-pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, 20 tridecyl, tetradecyl, pentadecyl and hexadecyl. Preferred alkyl groups include the lower alkyl groups havmg 1 to about 4 carbon atoms and the higher alkyl groups havmg about 10 to about 16 carbon atoms.
"1\~ ,' " refers to a small spherical entity which is ~
by the presence of an internal void. Included among such entities are, for example, 25 liposomes, micelles, bubbles, ...i.,..' - ' . and the like. Preferred ~ u~h~ . are formulated from the stabilizing materials descdbed herem. As discussed im detailbelow, certain ~ of the invention mvolve , which comprise lipids. In ...;~.u~ll"lcq formulated from these , . the lipids may be in the form of a monolayer or bilayer, and the mono- or bilayer lipids may be used to form 30 one or more mono- or bilayers. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric. Thus, the lipids may be used to form a ull;l~ll,ll --;-, u~ ; (comprised of one monolayer or bilayer), an 171i~ q~f~11qr wo ss/3~00s 2 1 9 1 0 7 ~ - 8 - ~ ~ .c ~
lUD~ C (comprised of about two O} about three lIIUI~IDY~ID or bilayers) or a mllltil~Fll:~r ~ (comprised of more than about three IIIUIII)~ ID or bilayers). The internal void of the lll;~lo~h~lcD may contain a liquid, mcluding, for example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid or solute5 material, as desired.
"Gas filled IlI;I~lUD~h~ D~' refers tû III;~IUD~ D in which there is ' a gas. "Gaseous precursor filled Illi~lVD~ ," refers to I~ UD~ ICD m which there is i. , ' ' a gaseous precursor. The ll~ .' may be minimally, partially or substantially completely filled with the gas and/or gaseous precursor In 10 preferred .~1.o.~ . the III;~ h l~D are substantially completely filled with the gas and/or gaseous precursor.
"Thickening agent" refers to any of a variety of generally hydrophilic materials which, when i.l~ ' in the present ~ act as viscosity modifymg agents, ' C~h.~, and/or ' ' ' ~, agents, suspending agents, and 15 tonicity raising agents. It is ,' ' that the thickening agents are capable of aiding m ~ the stability of the `"~lJ "- '"'` due to Duch properties.
"Liposome" refers to a generally spherical cluster or aggregate of ,', ' , ' . including lipid c-~ro~n~ic typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as 20 lipid 111;~
"Semi-synthetic polymer" refers to a naturally-occurring polymer that has been chemically modified. Exemplary naturally-occurring polymers mclude, forexample, p~ly 1~
~i~.. ,l.-~il,l. " refers to materials which are generally not mjurious to 25 biological functions and which will not result in any degree of ~ F toxicity, including allergenic responses and disease states.
"Patient~ refers to anirnals, mcluding mammals, preferably humans.
The present inverltion is directed, m part, to stabiliiced, substantially c ~ In a preferred . ' ' t, there is provided a stabilized, 30 substantiallyl ,....~,. ,--suspensionofagas. The~ are,l ~ ~.;, lby having a negative density. This negative density makes the Dll;~ ' of the present invention pal~ lly suitable for use as contrast agents for computed IUIIl~, ,' ~

WO95131005 2 1 9 1 079 ~ 191 _ 9 _ (CT). Preferably, the ~ . - have a negative density of about -40 ~Trnmcf~
units (HU) or less. In more preferred ~ ' the ~ have a negative density of about -50 HU or less, such as about -60 HU, -70 HU, -80 ~T and -90 HU, with negative densities of about -100 HU or less, such as about -110 HU, -120 HU, -130 HU, -140 HU, -150 HU, -160 HU, -170 HU7 -180 HU and -190 HU, being even more preferred. Still more preferably, the ~'`l' .-: ~ - have a negative density of about -200 HU or less, such as about -210 HU, -220 HU, -230 HU, -240 HU, -250 HU, -260 HU, -270 HU, -280 HU and -290 HU, and even more preferably, about -300 HU or less, such as about -310 HU, -320 HU, -330 HU, -340 HU, -350 HU, -360 HU, -370 HU, -380 HU and -390 HU. .~llcr~nri~nc having a negative density of about -400 HU or less, such as about -410 HU, 420 HU, -430 HU, ~40 HU, -450 HU, -460 HU, -470 HU, 480 HU and -490 HU are still more preferred, with negativedensities of about -500 HU or less being yet more preferred.
The ~ of the present invention possess highly desirable stability properties. Indeed, a surprising and unexpected advantage of the plresent invention is that , can be rl ~ 1, as described herein, which ]remain stable for a certar~ period of time. As described above, the present ! ~ are preferably stable for at least a period of time which permits the generation of visible images, for example, CT images, of a region of a patient. Another surprising and20 ~ l advantage of the invention is that the , generally lack a measure of stability that is associated with various of the prior art contrast agents. For example, Quay, T...., ~;..---l Application No. WO 94/16739 discloses contr~st agents for ultrasound which are disclosed as having a stability of from about 5 days to about 1 year. Such high stability has certain a-lv ~ . includimg, for example, lollg shelf 25 lives, which help to elrminate the need to repeatedly prepare fresh contrast agent.
However, contrast agents which remain stable for such extended periods of time suffer from serious drawbacks in that difficulty can be l~ 1 in commection with their ,.. l-l...li~ . and excretion from the body. Such stable contrast agents can: ' in the body of the patient, ~ il,L O.II,~ in fatty tissue and the liver. This is of 30 particular concern inasmuch as the health effects from long term exposure to such contrast agents is unknown.

WO95/32005 2 1 9 1 0 7 q .~ ,gl ~

In addition to their excellent efficacy as contMst agents for computed Lu~uo~Lrlly, the present ~ geneMlly remain stable throughout their useful life, and not geneMlly _ ~ 'y beyond that time fMme, typically for a period of time which at least permits their: ' to, and scanning of, a patient to 5 geneMte visible images, for example, by CT. Thus, the , of the present invention are stable for at least about several minutes. If desired, the ~- q, ,- - ,.. . can be formulated to be stable for periods longer than several minutes. For example,~...l.. . -:, ~ which are stable for seveMI hours or about a day can be formulated according to the methods described herein. If desired, ~"`L' l ` can be formulated 10 which are stable for up to several days. As is apparent to one of ordinary skill in the art, based on the present disclosure, the length of time that any particular suspension is stable depends on various factors, including, for example, the c""~ of the ~crr~ncinn the method used to prepare the . the C~lvi UlUll~ to which the suspension is exposed after r."". ~ ;"" and the like. Dependimg on these various15 factors, the ~ of the present invention will begim to sepaMte and lose their ll"..",o.... ::y after about seveMI minutes to about seveMI days. Thus, the ~
remain stable for a period of time which permits their: ' to a patient and the patient to be scanned with, for example, CT. It is , ' ' that the of the present invention geneMlly do not remain stable ~ , after 20 the use thereof. For example, it is , ' ' that they generally do not remain stable for a period of time substantially beyond that required for their - ' to, and scanning of, a patient. Thus, the ~ of the present invention carl be readily .... .: .I .l,, 1 by a patient, without danger of _ in fat tissue or the organs.
Moreover, it has been ~UI~UIi~ Oly and u--- ~ h ~ y found that l..",..,~,. ::y of the - , can be restored cu..v~ lLly with minimal effort.
T~l."",~,.... :y is important for uniformly accuMte computed ~u~uuOIALr~ scanning and can be easily RstoRd by merely agitating the Cllcr^~ n Such agitation can take the form of, for example, shaking the CIICr~nQi-^n Techniques for agitating the 30 l~ --- is described in detail below in cormection with methods for the IJl~Al~A~iull of the prcsent c~ After agitation and IC r ' , the suspension will remain stable for a period of time of from seveMI minutes to several days, as described ~, W0 9513~iO05 2 1 9 1 0 7 q h. .~ .. F~c, Accordingly, the ~ l. . of the present invention possess potentially indefinite shelf lives, since .c 'l" ~ of a separated and/or otherwise nor~-1..,.,.~,~,. .--- mixture is readily achieved.
~.sc~c G r ~S
In one aspect of the present invention, the , comprise a gas.
Preferred gases are gases which are extremely stable. The term stable gas, as used herein, refers to gases which are substantially inert and which are ~
Preferred also are gases which have low solubility and/or dirru~ib;li~y in aqueous media. In addition, preferred gases are those which provide the present 10 with a desirable negative density, including a negative density of about 10 HU or less, as described above. Gases, such as ~."auulul allJul~, are especially preferred since they are less diffusible and are relatively insoluble in aqueous media.
Exemplary gases which may be i..cu.~" ' in the present ~
include those selected from the group consisting of air, noble gases, such as hdium, 15 neon, argon and xenon, carbon dioxide, nitrogen, fluorine, oxygen, sulfur-based gases, such as sulfur l.~ . ;.1. and sulfur ~ U~llbUI~l pl - Ul,/~ll gases, and mixtures thereof. As noted above, y lr~uu~u~ are preferred gases.
Exemplary ~. '' u~bu.. gases include, for example, p, ~ , , ~.,lauulu.,;l~lc, p.,llluulu}Jlu,uall." ~u~ .,lnuulu~l ~(' and mixtures thereof. It is ~- . ' ' that mixtures of different types of gases, such as mixtures of a p~,lnuuluc~ ull gas and another type of gas, such as air, can also be used in the of the present invention. The gases discussed in Quay, ln~rr~
Application WO 93/05819, including the high "Q" factor gases described tbe~rein, may be used also. The disclosures of Quay, T" . -~;,.---l Application WO 93/05819 are 2iS iU.,Ul,UI ' ' herein by reference in their entirety In addition, l~ ~, l---~,.l ;;~ gases and gases of isotopes, such as 170, may be used. It is ,' ' that ~ c which comprise these latter gases may also be used as contrast agents in connection with diagnostic techniques in addition to computed t~ ,, . ' .y, such as magnetic resonance imaging (M~I).
Other gases, including the gases ~ l above, would be readily apparent to one skilled in the art based on the present disclosure.

W095/32005 21 q 1 0 79 r~ ) C ~

In ~l~iuulally preferred ~ ' ' of the present invention, the comprise lII;~IUD~ D~ The lII;UIUD~ L~D are essentially bubbles of very small diamehr comprising a "skin" or "envelope" that surrounds or encloses a cavity or void filled with liquid or a gas. Mi.,l~ . ' are ~ ly preferred in 5 corlnection with gases that are derived from gaseous precursors, which are also preferred in the context of the present invention.
Gaseous precursors irlclude materials that are capable of being converted in vivo to a gas. Exemplary precursors are materials which are liquids at room t~ ,ldLulc and which, after being ~ ' ~,1 to a patient, umdergo a phase lû trarlsition to a gas in vivo. Preferably, the gaseous precursor is ~ , and the gas produced in vivo is bi~ also. In addition, preferred gaseous precursors are materials which, when converted to a gas, provide the ~ with a desirable negative density, including a negative density of about ~0 HU or less, as described above in cormection with the gases. Exemplary of suitable gaseous precursors are the 15 ~.llluvlu~,~lbùl~D. As the artisan will appreciah, a particular ~,lnuuluualbu~ rnay exist in the liquid state when the . .l. ,-: ~-- - are first made, amd are thus used as a gaseous precursor. ~ ,ly, the p~,llluulu~l~ may be used directly as a gas.
Whether the I ~ u~ bùll is used as a liquid or a gas generally depends on its liquid/gas phase transition t.ll~ , or boiling point. For example, a preferred 20 y~lnuulu~lbull, ~lnuul~ r ' , has a liquid/gas phase transition h.u~ .c (boilmg point) of 29.5C. This means that l ~ , . will be a liquid at room : (about 25C), but will become a gas withm the human body, the normal h...~ L~ of which is 37C, which is above the transition L . of ~.,.nuUlu~ ~l.,. Thus, under normal ~ ,lnuulu~..ll~.~ is a gaseous 25 precursor. As a further example, there are the homologs of P~ namely ~l~Jl~Jbu~ and p, r~ , ~ The liquid/gas transition of ~ nuul~ ' is 4C and that of ~ n~ ull~,A~.., is 57C. Thus, ~.,.lluul~ is potentially useful as a gaseous precursor, although more likely as a gas, whereas }J~ uUl~ ~ would likely be useful as a gaseous precursor only because of its relatively high boilmg point.
A wide variety of materials can be used as gaseous precursors in the present ~ It is only required that the material be capable of, ~, ~ a phase transition to the gas phase upon passmg tbrough the C~ UIUl ' ' i ~ ' wossl3200s ~ l 9 0 7 ~ r~l~O~ Sl Suitable gaseous precursors include, for example, I~Adlluulu~ ,tull~" isopropyl acetylene, allene, r~ udllene~ boron trifluoride, 1,2-butadiene, 2,3-butadiene, 1,3-butadiene, 1 ,2,3-trichloro-2-fluoro-1 ,3-butadiene, 2-methyl-1 ,3-butddiene, hexafluoro-1,3-butadiene, butadiyne, l-lluul-~ ' , 2-1~ ,lllu~lubuuul~, 5 1-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1-butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne, butyl nitrate, l-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-~ IIC, 3-methyl-1-butyne, perfluoro-2-butyne, 2-bromo-bu~y ' ' ' ~ " carbonyl sulfide, .,lu~ , ;lr ~,l~u allc~ methyl-~-,l~uullc" ' '' ucyululJulalle, ~lnuulu~l~ut~

3--,Llulu~ lnuulu~ u~y,~ lU~lul)lulc~
p~,ln~lulU~IU~)lUlJall~ 2-dimethyl~ ."ul~ y~.lù~Jlu~lal~
1~2~1;1ll~,al~1cy~,1u~lu~ " c~ ,lu~ulu~alll" Ill~,LllylL,~,lu~!lu~l~, diacetylene, 3-ethyl-3-methyl diaziridme, l ,1,1-1~illuulu.1;~ ." dimethyl amine, L,Aalluululliul~' ' , dilll"l~/lcl.~lcull'ul~" bis-(lu.l.,a.~ ' )amme, 15 ~.,ln~)luL~i~u~, y~,llluOIull~tallc~ ~"llluu~uu~ , 2,3-dimethyl-2-1-.,.1"".- -, U~ ' ' ylU~.UIIiUIII chloride, 1,3-dioxolane-2-one, 4-methyl-1,1~1~2-kllal;luulul,llculc, 1,1,1-L~illuulu"~lau~" 1,1,2,2;
1,1,2-trichloro-1,2,2-LIilluulu.,tl.~ule, l,1-dicl~lulu~ ul~" 1,1-dichloro-1,2,2,2-~lanuu~u~ ul~" 1,2-~''' U~ UI~" 1-chloro-1,1,2,2,2y.,ll~alluu.. ' , 2-chloro-l,1-dinuulu~lllallc, 1,1-dichloro-2-r' , ' , 1-chloro-1,1,2,2 tcuanuul~ ' , 2-chloro-1,1-,lilluu-, ' , CLIU~U~ ulC~ diCLIUlULlillUUlU-ethane, nuUI, , I r~ u~
UUlU~lh,r- , ethyl Yinyl ether, 1,1-di~ '' , 1,1-dichloro-1,2-difluoro-ethane, 1,2~'r' , ' , methane, L~inuu., r Jluh!ù~idc, trifluoro-J Ulll~ bromo-~LIUIUIIU~JIUIII~ UI~ u~l~u~nrl~.JIul.~;l ul." ~ lulu~linuull '' u~ u~" cl~loro-' u..l~GI~ul~ ..n."".", Ih--,,~I.luluLlin~.Jl~ ' ,clllu.u~lin,,~,l, ~ luluodir.l,ul~ ' , di~,llluludill~.ululll~llaulc~ di~l-lu-~ " , difluoro-methane, dilluul~ , d;C;IA.""". 11 - ~, nuululll~,Lhallc, i."1~. :I ---c, iodo-30 l.inuv.u..l"a.~u.c, I i~-u~linlluluuu.,~llallc, lui~ c~lanuululll~,lllàlL, lli~lulunuul~ lilluululll.,ii~ll." 2-~ y-' ' , methyl ether, mefhyl wo ss/3200~ 2 1 9 1 0 7 9 r~ . c - ~s~

isopropyl ether, ~ Llly" , Il.,lly' lll~,Ll~ylDulrldc~ methyl vinyl ether, rl~rlrPr~q-lP, nitrous oxide, 1,2,3~ ./-LIi~ lbvAyliu acid-2-llydluAyLli ester, 1-nonene-3-yne, 1,4-pentadiene, n-pentane, ~.,.n Jlu~.,,lLul~, 4-amino4-Il,~.llyl~."lL lll-2-one, 1-pentene, 2-pentene (cis), 2-pentene (trans), 3-blulllu~.,l.. 1-ene, s l ~,.nuvl, . 1-ene, t~ ,hlvlu~llLl~lic acid, 2,3,6-LIiul~.,dlyl~ , propane, 1,1,1,2,2,3 LA~auuluL~lu~...r ,1,2-~,L/UAYAUIU~J~.. " 2,2~1inhvlu~lu"~ " 2-ammo-propane, 2-cLhJlu~Jlu,ua".,, heptafluoro-1- U~)IU~ heptafluoro-1- UDU~IUL/~
I~ '' U~llU~la~lC, propene, 1 r~ U~llU~làllc, 1,1,1~2,3,3 h.,A.Inuulu-2,3-dichloro-propane, 1-~ 1ulu~lu~àlle, chlulu~,lu~ -(trans), 2-UIllUlUUlU~J~lC~ 3-llUVlU~
10 propyne, 3,3,3-i ~ U~lU~)yllc, 3-lluuluDLylcllc, sulfur (di)~ ,11 n,;,l.~ (S2FIo), 2,4~ , Ll r' .~ lr, Llilluulull-~,LIlyl peroxide, Llin. Jlu."~,Lhyl sulfide, tungsten lI~-AI~ II;~Ir, vinyl acetylene and vinyl ether.
rl r~ Ul ~ are both preferred gases and preferred gaseous precursors for use in the . of the present invention. Preferably, the 15 ~ -vlu~,albul~ has from about 1 carbon atom (4 fluorine atoms) to about 9 carbon atoms ~20 fluorine atoms). Exemplary of ~.lluuluc~l of 1 to about 9 carbons are u~ul,u.~ selected from the group consisting of ~.,.I1UU~U~ LI~
p~.n~ .n~,v.uy.ul,lu.c, ~ u~,y~
nuul~r . ~ ull~tallc, ~ uuluu~,L~ulc amd 20 ~.,.lluul~ . Preferably, the I '' u,all/ull is selectcd from the group consistmg of l r~ UII~A~UIC and I '' uo~,L~ulc, with ~ [IUUIU~ IL~IIC being Au~ll 'Y preferred In certam preferred ~ ' " , a gas, for example, air or a Ruu~u~lbu~ gaD, is combined with a liquid l,~.,lluulu.,~ such as 25 ~J~,In~_Jluu~,Lylhlull~ic (PFOB), l ~ uJ~alm, u, r~ .,rln~l~ r ,1;"
iluuluu~,lyliull;~, y~.~lluuluLIi~lu,uy' , and I '' uLIib..,y' As noted above, certain preferred ~ " of the present invention imvolve , which comprise llliCl~ ,' The size of the lllicl~ can be adjusted, if desired, by a variety of procedures including, for example, 30 lUiClU' ~ vortexing, extrusion, filtration, sonication, l..."...,~
repeated cycles of freezimg and thawing, extrusion under pressure through pores of defined size, and similar methods.

~ WO 95/32005 2 1 9 1 0 7 q r~l~u~ sl For hlLId~ use, the UD~ D preferably have diameters of less than about 30 ,um, and more preferably, less than about 12 ~Lm. For targeted illLId~da~uLll use including, for example, binding to certain tissue, such as cancerous tissue, the , ~' can be a;}~.fir~ ly smaller, for example, less than lO0 rlm m 5 diameter. For enteric or ~aaLI~ ' I use, the ~ ,lua~ lca can be a;~. irl.~lLIylarger, for example, up to a millimeter in size. Preferably, the Ill;.lua~ll.l~,D are sized to have diameters between about 20 ~m and 100 ~m.
Tabulated belo~v is a listing of a series of gaseous precursors ~vhich undergo phase transitions from liquid to gas at relatively close to normal hur.~an body 10 L l~ ulc (37C) or below. Also listed in the table are the sizes, in diameter, of emulsified droplets that ~vould be required to form a Ill;-lu~ lG of a maximum size of about 10 ~m.
TABLE I
Physical Cl~ of Gaseous ~ a and Diameter of ~ ' '~ ' Droplet to Form a 10 ILm l\I;~, ' c Diameter (llm) of emulsified droplet Molecular Boilmg Point to make 10 micron Compound Weight ( C) Density ua~h~,.c perfluoro- 288.04 29.5 1.7326 2.9 pentane 1- 76.11 32.5 6.7789 1.,!
20 ul,.
2-methyl- 72.15 27.8 0.6201 2.
butane (jcnp~.nt~
2-methyl-1- 70.13 31.2 0.6504 2.5 25 butene 2-methyl-2- 70.13 38.6 0.6623 2.. 5 butene 1-butene-3- 66.10 34.0 0.6801 2.4 yne-2-methyl 30 3-methyl-1- 68.12 29.5 0.6660 2.5 butyne w09s/3200s 2191~7~

T~BLE 1 Physical Cll~ t~;aliu~ oF Gaseous Precursors and Diameter of r ~ Droplet to Form a 10 llm Mi.~
Diameter (~um) of emulsified droplet Molecular Boiling Point to make 10 micron Compound Weight ( C) Density lll;~,l~ ,' octanuoro- 200.04 -5.8 1.48 2.8 decafluoro- 238.04 -2 1.517 3.0 butane Shexafluoro- 138.01 -78.1 1.607 2.7 ethane ~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 an aspect of the present invention to optimize the utility of the 10 `".1" ~ including ~ which comprise ll~;clu~ .,lc~, by using gases of limited solubility. Limited solubiliy, as used herein, refers to the ability of the gas to diffuse, for example, out of IlI;~,lva~Ll~ i~ by virtue of its solubility in the :~UII~ " ~
aqueous medium. A greater solubility in the aqueous medium imposes a gradient with the gas in the lll;.,l~ .' c such that the gas will have a tendency to diffuse out of the 1~ lui.l~, c. A lesser solubility in the aqueous medium will decrease the gradient between the ~ ,lu~ul~,.c and the mterface such that the diffusion of the gas out of tbe lul,uh_~c will be impeded. Preferably, the gas entrapped in the lUiCl~ has a solubility less than that of oxygen, namely, 1 patt gas in 32 parts water. See Matheson Gas Data Book, Matheson Company, Inc. (1966). More preferably, the gas entrapped20 in the ~ , possesses a solubility in water less than that of air; and even more preferably, the gas entrapped in the u*~ c possesses a solubility m water less ~m that of nitrogen.
The amount of gas and/or gaseous precursor which is i... u.~, ' in the present ~ can vary, and depends upon various factors, including, for 2~ example, the particular stabilizing materials, thickening agents, dispersing agents, and the like, which, if desired, are employed m the ! . ' It is preferred that the ~ W09513200S 2 l q 1 0 7 9 II~,I/U~ _:'CS~Sl rl;ll of gas or gaseous precursor is at least sufficient to impart desirable properties to the . including, for example, a desirable negative density when used as contrast agents. In preferred ' ' , which involve a gaseous precursor, as well as a: ' of gaseous precursors, the total of g~seous 5 precursor is from about 0.1 wt. % to about 5 wt. % . More preferably, the total C I ~ l rl ;1 l of gaseous precursor is from about 0.5 wt. % to about 2 wt. %, with a total of about 1 wt.% being even more preferred.
Stabilizine Materials In certain preferred 1 --~l~o~ of the present invention, the 0 ~ r ' further comprise a stabilizing material. It has been ~U~ ly and 1y found that the stabilizing materials, as defined herein, are capable of promoting the formation of substantially 1~ It has also been ~ul~ ;llgly and ... 1~ ly found that the stabilizing materials, as defined herem, are capable of promoting the formation of u i. .~, ' , as well as enh~mcing the 15 resistance of Illil l~ .' once formed, to d~ caused by, for example, the Ioss of structural or 1 , ' integrity in the walls of the 1ll;.,ll, ' amd/or by the loss of any significant portion of a gas or gaseous precursor which may be ' within the 1ll;.,l~ ,' A wide variety of substances are available which can be used as 20 stabilizing materials in the ~ of the present invention. Preferred stabilizing materials are substances tbat are ' , -- ' . Preferred also are substances which are capable of raismg the viscosity of the ! I It has been found that surface-active agents, mcluding, for example, r , are U..ll;~,ul.llly suitable for use as stabilizing materials in the r Of the present mvention. In preferred 25 ~ ~ '- . the surfactmts are selected from the group consisting of anionic, cationic, ~,w amd nonionic r Preferred among these surfactmts are tbe nonionic r~-f~rtq-ltc Exemplary nonionic surfactants include, for example, PC~IY~ .YI~
pvlyw y,ulu~ylu.~ glycol block UUIJUIY~ sorbitan fatty acid esters and fluorine-30 containing ~ rfS~rt~ntr~ Preferred among the POIYVA~.dIYIUII~ VIYU~YI~IU~I~ glycolblock cu~ulyl~ are ~-hydroxy ~ hJLu}.y,uuly(u~.~.,lllyh,.l~;)-poly(uAy,ulu~yh,..~)-woss/3200s 21 91 07~ r~l,u.. r~

poly(u,~ lullc) block cuyvl~ ,la. These latter block cuyvlyl.A,la are generally referred to as poloxamer uuyûlylll~la. Examples of poloxamer cuyvlyl~ a which are y~ ,ulally suitable for use in tbe present . include, for example, poloxamer F68, poloxamer L61 and poloxamer L64. These poloxamer CUYUIYIII~,ID are 5 crm~r~r~ y available from Spectrum 1100 (Houston, TX).
Preferred among the sorbitan faKy acid esters are, for example, poly(oxy-1,2-ethanediyl) derivatives of higher alkyl esters of sorbitan. Examples of such esters of sorbitan include, for example, sorbitan ' , sorbitan ~' , sorbitan l r ~ ' and sorbitan . ~ ' These, as well as 10 other derivatives of sorbitan, are typically referred to as puly , including, for example, polysorbate 20, pOlyavli 40, poly ' 60 and pûly ' 80. Various of the yulyavlb...~,D are ~ly available from Spectrum 1100 (Houston, TX).
The fluorine-containing surfactants include surfactants containing one or more fluorine atoms. l~xemplary of such surfactants include those ~ ..., . . , :- l'y 15 available from DuPont Chemicals (Wilmington, DE) and which are sold under ~e tradename Zonyl'U, including, for example, Zonyln' FSN-100 and Zonyl'Y FSO-100.
As noted above, ionic ~ fqrtqntc for example, anionic and cationic cnrflrt~ntc may be used as stabilizing materials in the present , Exemplary of suitable anionic surfactants is sodium lauryl sulfate, lly available from 20 Witco Corp. (New York, NY). Suitable cationic surfactants include q.n~rni~m salts, y~. Li,,ul~.ly ammonium salts substituted with higher alkyl groups. Included among such ammonium salts are l~.-.ly' ' J' ' bromide, c~ylL~iul.~llly bromide, l..J~ialy' ' ~' bromide, all ylJ ..~ YIU~IILY- ' chloride (where alkyl is, for example, C,2, C,~ or C,~), b.,llLy ' ' Jl~vd~,.,yl-25 : I,.vlll;J ~-lllvli~c, I~ yl~ aly'- ' Y- I,.v liJ~ .l.lv I,~,.ILyl~ a~ J~.,y' I~lu..l;dc~,lluliJ~ .ty'- jl~,aly blUII~ ,IIIUIiJC:~ and ul.lylyyliJil..~. bromidelchloride. In addition, the cationic surfactant can comprise a cationic lipid, including for example, N-[1-(2,3-Jiul~.vylul~y)yluyyl]-N,N,N-llilll~,aly- chloride (DOTMA), 1,2~1iulc~)ylu~.y-30 3-(LIi..l~a.y' -)propane (DOTAP), 1,2-dioleoyl-e-(4'-i y- 3-butanoyl-sn-glycerol (DOTB) and lipids bearing cationic polymers, such as polylysine and yvlyal~, ~ WO95l32005 2 1 9 1 07q J~ Vv ~ 9l ~" - 19 -Other ~ f~rt~ c, including those .' ~ ' aboYe, would be apparent to one of ordinary skill in the art based on the present disclosure.
As indicated above, ~ m which there is ill,UI~)~ ' ' a stabilizing material represent a preferred e ~ of the present invention. It is , ' ' that the ~ can comprise a mixture of stabilrzmg mate,rials.
The . of each stabilizing material, when present m the ,...,....~;....~ of the present invention, can vary and depends upon various factors, includimg, for example, the particular stabilizing material(s), gases, gaseous precursors, and the like, employed. It is preferred that the total of stabilizimg 10 material is at least sufficient to cause `~ ,, of the l If the iuspension comprises .II;~IU~I~ D~ it is preferred that the total c~". 1.,-1;...1 of stabilizmg material is at least sufficient to promote formation of the llli~luDI,h~,cD as wcll as subsequent ' thereof. In preferred 1 .~l o(l; -~, the c~- of each stabilizing material is from about 10 ppm to about 1,000 ppm. More preferably, the c, ~ ;. .,. of each stabilizing material is from about 50 ppm to about 750 ppm, with of about 100 ppm to about 500 ppm bemg even more preferred.
A~ents In certain preferred L.,~l.o~l;--.. ,l~ of the present invention, the further comprise a thickening agent. If desired, two or more thickening 20 agents can be employed in the present .~ Suitable tbickening agel1ts for use m the present , include starches, gums, pectin, casein, gelatin and ~hY~U~OIIU;~D~ including ~ -. algm and agar; semi-syntbetic cellulose d~ ati~D, polyvinyl alcohol and ~ubu~yvilly' , and bentonite, silicates and colloidal silica. Exemplary of the foregoing materials are, for example, .,c.l~ ' yl-25 and the l' ,' y' ' and sulfonated derivatives thereof; agarose; polyethers,including polyethers havmg a molecular weight of, for example, from about 400 to about 100,000; di- and trihydroxy alkames and their polymers having a molecular weight of, for example, about 200 to about 50,000; acacia; ' ' ' , glycerol , lanolin alcohols; lecithin; mono- and d; ;ly~cli~D~
30 oleic acid; oleyl alcohol; polyu~.llyl.,l.. 50 stearate; polyoxyl 35 castor oil; polyoxyl 10 oleyl ether; polyoxyl 20 cetostearyl ether; polyoxyl 40 stearate; propylere glycol W095/32005 2 1 9 1 0 79 ~ J 5~9l ~

diacetate; propylene glycol , sodium stearate; stearic acid; trolamme;
c~ ;ry;ll~; wax; agar; alginic acid; aluminum ll~ f' ~ , bentonite; magma;
carbomer 934P; hJLu~ JI starch; ~ubu~ylll~lllJl~,-,llulose; calcium amd sodium and sodium 12; 1 " cellulose; dextran; gelatin; guar gum; locust bean gum;
S veegum; ~l~dlu~ l cellulose; llyllu~yluyyllll.,llyl-,ellulose; ~ aluminum-silicate; ...~.I.yl.ellu ~ , pectin; yvl~ lullc oxide; povidone; propylene glycol alginate; silicon dioxide; sodium alginate; tragacanth; xanthan gum; ~-d-L~ ~ , glycerol; mannitol; pol~ h.-l~~ ,ol (PEG); puly~;..JlAu~llulidu (PVP); p~ly~ ' ' ' ' (PVA); pOIyyluyyl~ ; glycol; pol~ , sorbitol;
10 ylvyyl~ l; and glycerol.
Preferred amorlg the foregoing thickening agents are gums, including xanthan gum, cellulose derivatives, including methyl cellulose and c~hw-ylll.,ll~l cellulose, and . ~....c,.. .~, r...~i~ul~lly preferred among the thickening agents is methyl cellulose.
The ~ rl;......... of thickening agent, when present in the ~
of the present invention, can vary and depends upon various factors, imcluding, for example, the particular thickening agent(s), gases, gaseous precursors, stabili_ir.g materials, amd tLe like, employed. It is preferred that the total r.. ...ti,.l;~" of thickening agent is at least sufficient to impart desirable properties to the 20 including, for example, cfsllili7~tirn of the , In preferred, ' " , the r.. ,~.,-1;l.. of thickening agent is from about 0.1 wt.% to about 10 wt.%. More preferably, the ~ of thickening agent is from about 0.2 wt. % to about 7.5 wt. %, with ~ of about 0.25 wt. % to about 5 wt. % bemg even more preferred. Still more preferably, the, of thickening agent is from about 25 0.3 wt. % to about 3 wt. % .
~isDersin~ A~ents The ~ of the present invention also preferably comprise one or more dispersmg agents. It is ~ that the dispersmg agents are also capable of (....,l-;l- -;;-~ to the stability of the A In certain preferred ~
30 the dispersing agent comprises a polymeric siloxane compound. Preferably, thepolymeric siloxane compound is substantially or completely alkylated with alkyl ~ W09513200~i 2 1 9 1 0 79 ~ u ~s~l groups, with lower alkyl groups being preferred. More preferably, the polymeric siloxane compound is 3ub3L~llLidlly or completely Ill.,.lly' ' A ~ i.UlDlly suitable polymeric siloxane compound for use as a dispersing agent in the , of the present invention is ~-(i ylDilyl)~ ,Ll~yllJùly[Oxy( " JIDilylul~)], which is 5 also referred to as , amd which is Cullllll~,ll,idlly available from Dow Corrling (Midland, MI).
The amount of dispersing agent which is included m the , can vary, and depends upon various factors, including, for example, the particular .- ~(c), stabilizing materials, thickening agents, gases, gaseous l and 10 the like, which, as desired, are employed in the ~ It is preferred that the -- of dispersing agent is at least sufficient to impart desirable prûperties to the ~ . including, for example, promoting desirable Sr~ i7~-in- of the In preferred . . . ~ , the ~ ; -l . of dispersmg agent is from about 10 ppm to about 1,000 ppm. More preferably, the . of dispersing agent is from about 50 ppm to about 750 ppm, with of from about 100 ppm to about 500 ppm being even more preferred.
Aiuxili~iy r- -In addition to the stabili_ing materials, thickeners and dispersing agents discussed above, there exists a wide variety of auxiliary materials which, if desired, 20 can be h~.u~l ' in the . of the present invention. Dependmg upon, for example, tbe physical and/or chemical properties of the auxiliary materials, they may impart desirable properties to the , For example, it is , ' ' that certain of the auxiliary materials are capable of - _ to the formatiol1 and/ûr 31_l,;1;,~1;~ of the present . In involving llliUl~ ,- the 25 auxiliary materials may promote formation and/or ~.l, l;,~l;---- of the llliuluD~Jh_~.D. In addition, the auxiliary materials may er~hamce the r ~ of the stabilizing materials of the present invention, or may contribute some desired property in addition to that afforded by the present stabilizing material. For example, it is l , ' that certarn of the auxiliary materials can enhance the ~ ;r ~ i properties of the WO95132005 2 1 9 1 079 P~ ,9l ~

As would be apparent to one of ordinary skill in the art, based on the teachings in the present disclosure, it is not always apparent whether a particular material, when iII~UI,UI ' ' in the present ~ acts as a stabilizing material, thickening agent or dispersing agent as described above, or whether it is acting as an S auxiliary material, smce the ~ ,, of the substance in question is generally determined empirically, or by the results produced with resrect to the r~ . of aparticular stabilized ~rrn~inn For example, the simple . ' of a l.:n~ -';l,lr lipid, which, as described more fully hereinafter, is generally anauxiliary material within the context of tbe present invention, and water or saline, 10 when shaken, will often give a cloudy solution subsequent to: ' Vh.6 for .~ .;li,~l;.", Such a cloudy solution may function as a contrast agent, but is aesthetically ubj.,.,Liul~l,le and may imply instability in the form of Ulld;~ UI~AI or Iipid particles. Propylene glycol, which is identified herein as a thickenmg agent, may be added to remove this cloudiness by facilitating dispersion or dissolution of the lipid particles. However, it is . ' ' that the propylene glycol may also function to improve formation of ~ lua~h_lca and ' ' by increasmg the surface tension on the l~ u*~h~lc membrane or skin. It is possible also that thepropylene glycol functions as an additiorlal layer that coats the membrane or skin of a lll;~l~ ,' , thus providing additional ! ' ' ''' "
Materials which function as stabilizing materials and/or as auxiliary materials for use in the ~ of stabilized, ~u ~ ~ would be apparent to one skilled in the art based on the present disclosure. Such materials include cu..~.
surfactants which are disclosed, for example, in D'Arrigo, U.S. Patents Nos.

4,684,479 and 5,215,680, the disclosures of which are 'u~w~,ul ' ' herein by reference, in their entirety.
Additional auxiliary and/or stabilhzing materials mclude, for example, oils, such as peanut oil, canola oil, olive oil, safflower oil, corn oil, or any other Oh which is commonly known to be ingestible. Another ! ' ' "' ' ,,/~ h~y material is trehalose.
As noted above, I.;-~c--~- ~u ~ lr lipids may be included in the of the present invention. Exemplary of such lipids include, for example, Iysolipids, , such as l~llu~lulk~liu~h~ with both saturated and, ' lipids, .. . . ~ . .

~ W0 95f32005 2 1 9 1 0 7 9 P~,lf~ 9i ~t ~-- 23 --including dl~lLvy~l' ,' " '~,I.,Ilulil.. " dill~y~ia~uy~ lylLllùlif yl~u~ lylLllvl;~ lila~lluyll~ ; yll,l-ulil-~, ir pllua~ idyl~ uli.. _ (DPPC) and di~ Laluylllll~ lylullolil.~ (DSPC);
rjhJayllalidy- - ' ' such as d;~l~vy~ ,' '' ' ' , distearoyl-~Ja"haliJy' ' ' ' and ~ir-~ xy~ l, " l- ; (DPPE);
~llua~lialidyla.,li.~, l' , ' '!flbly~lul~ llua~JhaLidy' ' . ~ " .l;~.;.l~. such as !,' ~ f~,l;ll; glycolipids, such as _ ~' ' GM1 and GM2; ,.l.. ,1;~
sulfatides; ,;ly. ,' ' ~ ~1;'1;' acid, such as ~, ' ' CY1II~ acid ~DPPA); palmitic acid; stearic acid; ~ ;1- - acid; oleic acid; lipids bealing 10 polymers, includmg l~ydlu~Jhili~, polymers, such as ~Jul~f~llylLIl~ f~,ul~ chitin, hyaluronic acid or pGlyvi..fl~yllulidonc, with preferred lipids bearing polymersincludmg dilJalllliLuyllll~ ;-lyl~ -PEG 5000 ~DPPE-PEG 5000), which means a 'i, ' ' Yll~ ; IY' ~' ' lipid having a PEG polymer of a mean average molecular weight of about 5000 attached thereto; lipids bearing sulfonated 15 mono-, di-, oligo- or p~ly~ - . l, -- ;,1~ ~. such as arabinans, fructans, fucans, galactans, ~I~.,Lu., ., glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, -. ,..~,. . ,. gala~,Lu~,alulùSC~ pectic acid, amylose, pullulan, glycogen, alllylu~ ,Lill, cellulose, dextran, pustulan, chitin, agarose, keratm, UIIUII~
dermatan, hyaluronic acid, alginic acid, xanthan gum, starch and various ol:her natural 20 hull~olyl.~. or h., L~u~JOIylll~l~, including those contaming one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, Iyxose, allose, altrose, glucose, malmose, gulose, idose, galactose, talose, ~lyLh ulva~, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, r,ysteme, 25 tyrosine, acr~inP, glutamme, aspartic acid, glutamic acid, Iysme, arginine, histidme, glucuronic acid, gluconic acid, glucaric acid, ~ala~,Lul~ acid, u.~ acid, c~ , L ~( . and neuraminic acid, and naturally occurrmg derivatives thereof; chulLat~,luls and cholesterol I ' ' ', i , uls and tocopherol , lipids with ether and ester-linked fatty acids; pOIylll.,li~ lipids;
30 diaceyl phosphate; dicetyl phosphate; aLLalylalllill~ ,al~'' ", ' . r~ with short chain fatty acids (C6 to C8~,; synthetic r~ with aay acyl chains, for example, a first acyl cham of C6 and a second acyl cham of Cl2; ceramides;

w09s/3200s 2191 079 r~l,.a.. .o~lsi ~

p~lyu.~y~ yl~, fatty acid esters, polyu7~.,LIIyl-,l.., fatty alcohols, polyu~y~ ylull~. fatty alcohol ethers, glycerol poly.,l-yl.ll., glycol u~y ', glycerol ~ulJ~.llyl~ , glycol ricinoleate, sterols, cLIIO~y' ' ' soybean sterols, CLIIu~y' ' ' castor oil, andyulyu~.y~llyl~.ll., fatty acid stearates; sterol aliphatic acid esters, includmg cholesterol S sulfate, cholesterol butyrate, cholesterol ' ' ~J , cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate, and phytosterol n-butyrate; sterol esters of sugar acids, including cholesterol ~ ' ullh~c, lanosterol Elu~ulu~ " 7-d~hyJIul,lwlu~lul Eluuulu.uJu, ergosterol ~,' UlliJI" cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate; esters of sugar acids and alcohols, 10 including lauryl ~IUI~UIUIIid~ stearoyl Eh.~ ul~;ic, myristoyl ~lù~u-~ ' ', lauryl gluconate, myristoyl gluconate, and stearoyl gluconate; esters of sugars and aliphatic acids, includmg sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid, and polyuronic acid; saponins, including ~al~ ly~ ' oleanolic acid, and ,'i,,' ',, ' glycerol 15 dilaurate, glycerol trilaurate, glycerol 1il ' ' , glycerol and glycerol esters, including glycerol i 'I ' " ', glycerol distearate, glycerol tristearate, glycerol dilll.y ' ', glycerol lLillly ' ' ', long cham 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 1' .' ' and alkyl 20 1 ' . ' ' , 6-(5-cholesten-3,B-yloxy)-l-thio-,~-D-E,..l,l.,luyy..l..u~;J~, digalactosyl-~;r~lY~lidc~ 6-(S-cholesten-3~-yloxy)hexyl-6-amino-6-deoxy-l-thio-~-D-galacto-yyl~llu~;J~, 6-(5-cholesten-3,~-yloxy)hexyl-6-amino-6-deoxy-1-thio-~-D-malmo-~y ' ', 12-(((T-~ y' ' ' 3-yl)carbonyl` ~ yl~l,ll"10~ ~
acid; N-[12-(((7'-d;~,lhy' 3-yl)carbonyl` y )~ rr ~----yl]-2-25 ' ~ ' ' ' acid; ~,11ul~i~u,lyl(4'-i ' ' J' ' 3butanoate; N-~u~.~.;uyldivl~ yl-~o,yl~iiJyl~ ,1,2-dioleoyl ~ gly~lul; 1,2-dipalmitoyl-sn-3-succmyl-glycerol; 1 ,3~ipalmitoyl-2-~u..i lylEly~,.,lul; l-hexadecyl-2-1 ' " yl~,ly~.~l, l ' . ' -" ' ' , yaLIliLuylllulllO~,y~.t~ c, saturated fatty acids, including, for example,lauric, myristic, palmitic and stearic acids; and I ' ' ' fatty acids, includmg, for 30 example, isolauric, isomyristic, isopalmitic and isostearic acids; and/or, ' ' ' thereof.

~W095132005 2191079 I~ "I

In certain rl~ t~, the auxiliary materials preferably comprise a mixture of two or more lipids. An example of a preferred mixture of lipids is a - mixture of DPPC, DPPA and DPPE-PEG-5000.
As discussed in detail below, a wide variety of methods are a~ailable for

5 the l l of the present , including , comprismg U~ ,.I. Included among these methods are, for example, shaking, drying, gas-otif-n spray drying, and the like. In I ' which involve the use of a lipid as, for example, an auxiliary material, the ~ are preferably plrepared from lipids which remain in the gel state, tnis being the t ~ at whicll a lipid 10 bilayer converts from the gel state to the liquid crystalline state. See, e.g., Cbapman et al., J. Biol. Chem. 1974 249, 2512-2521, the disclosures of which are hereby ill~,VI~ by reference herein, in their entirety. The following table lists CIJIC.~ iY~ lipids and their phase transition ~ r~

Saturated Diacyl-sn-Glycelro-3-Ph~
Main Chain Phase Transition T~
Carbons in Acyl Main Phase Transition Chains Temperature C
1 ,2-(12:û) -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-(1~: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 See, e.g., Derek Marsh, CRC Handbook of Lipid Bilayers, p. 139 (CRC Press, Boca Raton, FL 1990).

woss/3200~ 21 9 1 07~ r~ s. ~I9l ~

Preferably, the ~ of the present invention are formulated with one or more materials which possess relatively high water bindmg capacities Whenused, for example, in the Gl region, such materials can bind large amounts of free water. This enables the materials to carry a large volume of liquid through the GI
5 tract, thereby filling and distendmg the tract. The filled and distended GI tract permits enhanced CT imaging of the region.
In addition, where imagmg of the GI region is desired, the materials from which the ~ are formulated are preferably not substantially degraded rn, and absorbed from, the GI region. Thus" ~ and absorption within the GI
10 tract is preferably minimized to avoid removal of the contrast agent. This also avoids the possible formation of gas within the GI tract from such l ~ ;.." For imagingthe GI region, preferred materials are capable of displacing air and ~ the formation of large air bubbles withm the contrast medium.
As noted above, it is often desirable to ;..~.UI~J~ ' one or more auxiliary 15 materials which are capable of enhancing the ~ ol.~, properties of the It is ~ ,' ' that various of these materials are capable also of acting as a stabilrEing material, thickener andlor dispersant. Included among auxiliary materials which can improve the . .. ~ properties of the ~ are sweetening agents, for example, sucrose, fructose, lactose, saccharin, or aspartame, 2û and flavoring agents, for example, ~ . oil of ~ t~ ;l~ll or cherry flavormg.
Preferably, a sweetening agent is ill,UII~ ' ' in the present ~ . with fructose being a preferred sweetening agent.
The amount of . ~ enhancing agent which is included in the .. , can vary, and depends upon various factors, mcluding, for example, the 25 particular stabilizing materials, thickening agents, gases, gaseous precursors, dispersing agents, and the like, which, as desired, are employed in the ~ It is preferred that the ~." ~ , of ",~ erlhancing agent is at least sufficient to rmpart desirable properties to the , , mcluding, for example, improved taste.
In preferred ~ ...1.,~.1; -- .~, the ~ ; -, of ul~ GI~lil, enhancing agent is from 30 about 0.1 wt. % to about 10 wt. %. More preferably, the . of ~
enhmcing agent is from about 0.5 wt.% to about 5 wt.%, with ~.. ,1.,.1;.~.. ~ of about 2 wt. % being even more preferred.

~woss/3200s ; 9~a79 P~ gl A~ueous Diluents A desired component of the stabilized , of the present invention is an aqueous ~ v;lul~ y~Li~ul~lll.y with respect to !
,, l~ IVDYII~ICD~ Many of the stabilizing materials discussed above involve 5 . , which comprise both h.~lluyllub;u and I~J~uyll;liu properties.
~A~ , there can be a Yl~ L~ among the present ~ to form ul;uluayll.,l~,D, which are highly stable .,~ ~, in such an ".lv;lu~ull.,.l~. The diluents which can be employed to create such an aqueous cllvilul"l._.lL include, but are not limited to, water, either deioni2ed or containmg any number of dissolved salts 10 which will not interfere with the creation and ~ -;.,' . c of the stabilized or their use as CT agents, and normal saline and pl,~ ' ~, I saline.
Methods of I' y... ~;
The stabilized ~ .,. - - -,..~ of the present invention may be pr~,pared by a number of suitable methods. These are described below separately for ~ r 15 which comprise a gas, a gaseous precursor, and ~ Culllyliaill~ botll a gas and a gaseous precursor.
~thQIIs. of I~..,,l.. '- Usin~ a Gas ru~ncif.nc comprising a gas can be prepared by agitating an aqueous solution containing, if desired, a stabilizing material, preferably a surfactant, in the 20 presence of a gas. If a lipid is i.l.,ull ' in the , the agitating is preferably conducted at a i , - below the gel to liquid crystalline phase transition t~,llly~ l of the lipid. The term agitating, and variations thereof, as used herein, means any motion that shakes an aqueous solution such that gas is introduced from the local ambient c..vi,, into the aqueous solution. The shaking must be of5 sufficient force to result m the formation of a stabilized ! , , mcluding stabilized of uliuluayh_~cD~ and y.,.; ' '~, gas filled lll;.,l~ ,' The shaking may be by swirling, such as by vortexmg, side-to-side, or up and down motion.
Different types of motion may be combincd. Also, the shaking may occur by shaking the container holding the aqueous lipid solution, or by shaking the aqueous solution 30 within the container without shaking the container itself.

Further, the shaking may occur manually or by machine. ~
shakers that may be used include, for example, a shaker table such as a VWR
Scientific (Cerritos, CA) shaker table, or a Wig-L-Bug0 Shaker from Dental Mfg. Ltd.
(Lyons, IL), which has been found to give excellent results. It has been found tbat 5 certain modes of shaking or vortexing can be used to make stable ~ c, within a preferred size range. Shaking is preferred, and it is preferred that the shaking be carried out using the Wig-L-Bug~ mechanical shaker. In accordance with this preferred method, it is preferred that a ~ UWi;..6 motion be utilized to generate the stabilr~ed . , and ,U~ iCULIII,~ stabilized ~ comprising llliCll .' 10 It is even mûre preferred that the motion be IC~ lUL.lLill6 in the form of an arc. It is still more preferred that the motion be ~cc;,uluwlill6 in the form of an arc between about 2 and about 20, and yet further preferred that the arc be between about 5 and about 8. It is most preferred that the motion is lc~ lu~ l6 between about 6 and about 7, most l,~ui ' ~!~ about 6.5. It is .~ . ' ' that the rate of 15 l~u;~ulu..~ll;ull, as well as the arc thereof, is palli~uLIlly important in connection with the formation Illk,l~ .' Preferably, the number of Ic~ uwliull~ or full cycle nc~ innc is from about lOOû to about 20,000 per minute. More preferably, the number of ~.,;,u-uc.,liu.~ or oscillations is from about 5000 to about 8000. The Wig-L-Bug~, referred to above, is a mechanical shaker which provides 2000 pestle strikes 20 eYery 10 seconds, i.e., 6000 oscillations every mimute. Of course, the number of oscillations is dependent upon the mass of the contents being agitated, with tne larger the mass, the fewer the number of nc~ innc Another means for producing shaking includes the action of gas emitted under bigh velocity or pressure.
It will also be understood that preferably, with a larger volume of 25 aqueous solution, the total amount of force will be co..c r " _'~, increased.Vigorous shaking is defined as at least about 60 shaking motions per mmute, and is preFerred. Vortexing at about 60 to 300 revolutions per minute is more preferred.
Vortexing at about 300 to 1800 revolutions per mimute is even more preferred.
The formation of gas filled lUiCl~ upon shaking can be detected 30 visually. In addition to the simple shaking methods described above, more elaborate methods can also be employed. Such elaborate methods include, for example, liquid crystalline shaking gas instillation processes and vacuum drying gas instillation ~ WO 95/32005 2 ~ 9 ~ 0 7 9 r~
~ - 29 -processes, such as those described in copending U.S. application Serial No.
08/076,250, filed June 11, 1993, which is illcUlyl ' herein by reference, in itsentirety. When such processes are used, the stabilized ~ ,lu~yll~,lc~ which are to be gas filled, may be prepared prior to gas installation using any one of a variety of 5 UUII~I '' ' ylC, ' y techniques, including Cull~l I liposome yl,. y techniques in connectiûn with ~ r containing lipids, and which will be apparent to those skilled in the art. Among the yl, . y techniques are freeze-thaw, as well as techniques such as sonication, chelate dialysis, l~ ;..., solvent infusion, ~iCl~ v~ formation, solvent ~ i.. French pressure cell 10 technique, controlled detergent dialysis, and others, each involving preparing the Ill;clu~yll~lc:, in various fashions. See, e.g., Madden et al., Chemist~y and ,~hysics of Lipids, lg90 53, 3746, the disclûsures of which are hereby ;llCUlyl ' herein by reference im their entirety.
Gas filled llliCl~ .' prepared in accordance with the metbûds 15 described herein can range in size from below a micron to over 100 ~m. In addition, after extrusion and - ~ ;l ,. . procedures, agitation or shaking provides ~
of u*~ll.,.c~ which, if prepared from . comprising lipid, prov;des ;A11Y no or minimal residual anhydrous lipid phase in the remainder of the solution. (Bangham, A.D., Standish, M.M, & Watkins, J.C. (1965) J. Mol. Biol. Vol.
20 13, pp. 238-252 (1965).
The size of gas filled l..k.l~ . ' c~ can be adjusted, if desired, by a variety of procedures, including IlliCl'J' .. I~;r~ vortexing, extrusion, filtration, sonication, l~..--.. t.. ,-;;.... repeated freezing and thawing cycles, extrusion under pressure through pores of defmed size, and similar methods. It may also be desirable 25 to use the ui~u ~yll.,lc~ of the present invention as they are fûrmed, without any attempt at further . ~ of the size thereof.
The gas filled 1ll;~ ,;, may be sized by a simple process of extrusion through filters; the filter pore sizes control the size ,l:~l.il...li.... of the resulting gas filled Illi.,lU*.~ C~. By using two or more cascaded ûr stacked set of 30 filters, for example, a 10 ~um filter followed by an 8 ~m filter, the gas fillecl uli~lu*lL,c~ can be selected to have a very narrow size ~1ictnhllti~n a~ound 7 to 9 ~Lm.

wo ss/3200s 2 1 9 1 0 7 9 F~~ 191 ~

After filtration, these stabilized gas filled I~ ua~ll..lc~ remain stable for over 24 hours.
The sr~img or filtration step may be ~. ~..,..~,1;~1~. J by the use of a filter assembly when the suspension is removed from a sterile vial prior to use, or more S preferably, the filter assembly may be ill~ ' into the syringe itself during use.
The method of sizing the Ill;l~luD~ lci~ will then comprise usimg a syringe comprising a barrel, at least one filter, and a needle; and will be carried ûut by a step of extracting which comprises extruding said 1ll;.l~ ,- ca from said barrel through said filter fitted to said syringe between said barrel and said needle, thereby sizing said L~ ualJh_l~
10 before they are - ' cd to a patient in the course of using the I~I;LIU~ D as CT
contrast agents in accordance with the present invention. The step of extracting may also comprise drawing said Illi~luaulll.lca into said syringe, where the filter will function in the same way to size the Ill;~lua~ll.,lca upon entrance into the syrmge.
Anotber alternative is to fill such a syringe with Ill;~lu~.t~ " which have already been 15 sized by some other means, in which case the filter now functions to ensure that only ua~ll.,l.,D within the desired size range, or of the desired maximum size, are .~". .~ ly: ~ cd by extrusion from the syringe.
In preferred l the solution or suspension of Illi~lUa~ ,lCa is extruded through a filter and is heat sterilized prior to shaking. Once gas filled 20 Illi~l~ ,' ca are formed, they may be filtered for sizimg as described above. These steps prior to the formation of gas and gaseous precursor filled Illi.l~ . ' c~ provide the ~Iv_ ~ , for example, of reducing the amount of unhydrated stabilizimg compound, amd thus providing a ~;bllir~ ly higher yield of gas filled ~ .lu*~h_lc~, as weD as and providing sterile gas filled Ill;~lua,uL~.lc~ ready for ~' to a 25 patient. For example, a mixing vessel such as a vial or syringe may be filled with a filtered stabilizing material, and the suspension may then be sterilized within the mixmg vessel, for example, by _ ' Y;~ Gas may be instilled into the suspension to form gas filled ~ lua~ .lca by shaking the sterile vessel. Preferably, the sterile vessel is equipped with a filter positioned such that the gas filled 111;~ ' pass 30 through the filter before contacting a patient.
The first step of this preferred method, extruding the solution of stabilizing compound through a filter, decreases the amount of IlllL~y~ ' ~ material by . . .

~ woss/3200s 21 91 079 r~llL~ ~l9l breaking up any dried materials and exposing a greater surface area for hydration.
Preferably, the filter has a pore size of about 0.1 to about 5 ~Lm, more preferably, about 0.1 to about 4 ~m, even more preferably, about 0.1 to about 2 ~Lm, and still more preferably, about 1 I-m. Unhydrated compound, appears as amorphous clumps 5 of non-uniform si7e and is ....~
The second step, ~. .;l;, l;~,.. provides a , that may be readily ~ c~ to a patient for CT imaging. Preferably, ~ l ;. . is tl by heat ~ ;-. preferably, by: ' v;..~, the solution at a C of at least about 100C, and more preferably, by ' v;..~, at about 10 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 1 to about 30 minutes, even more preferably, about 10 to about 20 minutes, and still more preferably, about 15 minutes.
If desired, dlt~ , the first and second steps, as outlined above, may be reversed, or only one of the two steps can be used.
Where ~ I occurs by a process other than heat ~ro~ili7Afit~ af. a ~ ltlU.I~ which would cause rupture of the gas filled III;CIU:~UIl~ I may occur subsequent to the formation of the gas filled III;~IU~ .IC~, and is preferred. For 20 example, gamma radiation may be used before and/or after gas filled III;CIU~ .lC~ are formed.
Methods of r ~ Usin~ a Gdseous Precursor In addition to the r " i ~ _ '' ', gaseous precllrsors contamed in Ill;~,lua~.ci~ can be formulated which, upon activation, for example, by 25 exposurc to elevated tCl~ d~Ul~ varying pH, or light, undergo a pbase tra~sition from, for example, a liquid, including a liquid entrapped in a Ill;clu~ c, to a gaseous state, expanding to create the stabili_ed, l~ s ~ of the present invention. In preferred ~,.~ ' - activation of the gaseous precursor converts gaseous precursor filled l..;~lu~ c~ to gas-filled lll;cll ,' This 30 technique is described in detail in copending patent A~ Serial Nos.

wo ss/3zoo5 2 1 9 ~ 0 7 ~ r~ c ~

08/160,232, filed November 30, 1993 and 08/159,687, filed November 30, 1993, thedisclosures of each of which are i~ ' herein by reference in their entirety.
The preferred method of activating the gaseous precursor is by exposure to elevated t~ UlC Activation or transition . c, and like terms, refer to 5 the boiling point of the gaseous precursor which is the ~t , c at which the liquid to gaseous phase 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 i , c is particular to each gaseous precursor. An activation i , of about 37C, or about human body tc~ ldlu.c, is preferred 10 for gaseous precursors of the present invention. Thus, in preferred form, a liquid gaseous precursor is activated to become a gas at 37C. However, the gaseous precursor may be in liquid or gaseous phase for use in the methods of the present mvention.
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 iul.,ull l, for example, into a ",;",, . ' In addition, the methods may be performed at the boiling point of the gaseous precursor such tbat a gas is 1, for example, into a u~lJh_lc. For gaseous precursors havmg low c boiling points, liquid precursors may be emulsified using a ~llic~unuidi 20 device chilled to a low h r 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 i r ' C 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 b~e 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 r ' , The methods of producing the i activated gaseous precursor-filled U~ IC~ may be carried out at a t -T c below the boiling point of the gaseous precursor. In this t~ ' t~ the gaseous precursor is entrapped within a lll;c~u~ lc such that the phase transition does not 30 occur durirlg ~ ll.lrd,,lu,c. Instead, the gaseous precursor-filled l~iu~u~h_lc~ are ~ in the liquid phase of the gaseous precursor. Activation of the phase transition may take place at any time as the i~ U~C is allowed to exceed the ,~ WO95/32005 2 1 9 1 079 PCT/US95~06491 boilmg pomt of the precursor. Also, knowing the amount of liquid in a droplet ofliquid gaseous precursor, the size of the IlliUlU~lUIl.,lC~ upon attaining the gascous state may be tl~tf~ n~
Alternatively, the gaseous prccursors may be utilized to create stable S gas-filled III;LIU~,UII..lC~ which are pre-formed prior to use. In this ~ lu~ ' t, the gaseous precursor is added to a container housing a suspending and/or stabilrzing medium at a ~ .-IIIIG below the liquid-gaseous phase transition i ~ of the respective gaseous precursor. As the t~,UlU~ c is then increased, and an emulsion is formed between the gaseous precursor and liquid solution, the gaseous precursor 10 undergoes transition from the liquid to the gaseous state. As a result of this heating and gas formation, the gas displaces the air m the head space above the liquid suspension so as to form 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 ~ . of the CT
15 imaging contrast medium. For example, the gaseous precursor, Iu~"nuulubuLulc~ can be entrapped in the 1- ~ stabilizing compoumd, and as the i . is raised, beyond 4C, which is the boilmg pomt of p.,~nuululJu~u~, ~ '' uluukulL gas is entrapped in llli~ . ' As an additional example, the gaseous precursor lluu~ ' can be suspended in an aqueous suspension containing, for example, 20 ~ ul~;ryill~ and/or thickening agents, such as glycerol or propylene glycol, and vortexed on a c.. , :_l vortexer. Vortexmg is c~. ,--.. ~d at a ~ "l" .,.l.,.c low enough that the gaseous precursor is liquid amd is continued as the A ' C of thesample is raised past the phase transition i . from the liquid to gaseous state.In so doing, the precursor converts to the gaseous state during the IU;~
25 process. In the presence of the ~llu,ulul materials, including, for examplc, stabilrzing materials, thickenmg agents and/or dispersing agents, stable gas-filled u~l~h_lc~ are provided.
Accordingly, the gaseous precursors may be selected to form a gas-filled u~L,h.,.c in vivo or may be designed to produoe the gas-filled Ill;~lU*~ IC in situ, 30 during the ..~.... r~, 1.,. ;,~ process, on storage, or at some time prior to use.
As a further b~ ' of this mvention, by pre-forming the gaseous precursor in the liquid state mto an aqueous emulsion, the maximum size of the wogsr32oo~ 21 91 079 r~ '0~131 ~
ll~;,lu~ ,.c may be estimated by using the ideal gas law, once the transition to the gaseous state is ~1~ ' For the purpose of making gas-filled Illiclu~ll.,lc~ fromgaseous precursors, the gas phase is assumed to form '~, and ~llh~t~nti~liy no gas in the newly formed Ill;Cl~ . has been depleted due to diffusion into the 5 liquid, which is generally aqueous in nature. Hence, from a known liquid volume in tbe emulsion, one would be able to predict an upper limit to the size of the gas-filled 1111~ . C.
Pursuant to the present invention, a mixture of a stabilizing material and a gaseous precursor, containing liquid droplets of defined size, may be formulated such 10 that upon reaching a specific t~ .,ld~UIc, the boiling point of the gaseous precursor, the droplets will expand into gas-filled Ill;,,lU~JII.,IC~ of defined 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 .' c, and the effects of increased pressure are factors for which the ideal gas law car~not 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 is pressure in: ' .' c~ (atm);
V is volume in liters (L);
n is moles of gas;
T is t~ 'I"'G in degrees Kelvin (K); and R is the ideal gas constant (22.4 ~atm/K-mole).
With knowledge of volume, density, and i . c of the liquid in the 25 rnixture of liquids, the amount (e.g. number of moles) of liquid precursor as well as the volume of liquid precursor may be calculated which, when converted to a gas, will expand into a I~P~lu~ .,lc of known volume. The calculated volume will reflect an upper limit to the sr~e of the gas-filled "~i-,,, ,' , assuming i ~ exparlsion into a gas-filled l~ ,lu~LIc and negligible diffusion of the gas over the time of the 3û expansion.

woss/3200s 2 1 9 1 0 7 9 P~,~/u~ 51 .- 35 -Thus, for ~ of the precursor in the liquid state in a mixhure wherein the precursor droplet is spherical, the volume of the precursor droplet may be determined by the equation:
Volume (sphere) = 4/3 ~rr3 5 where r is the radius of the spherc.
Thus, once the volume is predicted, and knowing the density of the liquid at the desired t ..~1.. .,-h..c, the amount of liquid (gaseous precursor) i~ the droplet may be APtl~rrninP~i In more descriptive terms, the followmg can be applied:
V~ps = 413 ~r(rl,S)3 by the ideal gas law, PV=nRT
reveals, V", = nRT/P~2s or, (A) n = 4/3 [~rr",3] P/RT
a}nount n = 413 [1rrG~3 P/RT]-MWn Converting back to a liquid volume (B) Vljq = [4/3 [~r~3] P/RT]-MWn/D]
20 where D is the density of the precursor.
Solving for the diameter of the liquid droplet, (C) dia~neter/2 = [3147r [4/3-[~rr"~3] P/RT] MWn/D]"3 which reduces to Diameter = 2[[r",3] P/RT [MWn/D]]I'3.
As a further means of preparmg ~ u~ of the desired size for use as CT imaging conh-ast agents in accordance with the present invention, andl with a knowledge of the volume and especially the radius of the stabilizing compound/precursor liquid droplets, one can use .~ sized filters ;n order to size the gaseous precursor droplets to the ~ . diameter sphere.
A ~c~ gaseous precursor may be used to form a Illi............... l~ .-of defined size, for example, 10 ~m diameter. In this example, the ~ u~lll.,lc is woss/3200s 2 1 9 1 0 7 9 Pv~ 9i formed in the blo~ ll of a human being, thus the typical ~ c would be 37C or 310 K. At a pressure of 1 ~Illlu~ c and using the equation in (A), 7.54 x 10-~ moles of gaseous precursor would be required to fill the volume of a 10 ~m diameter Ill;~,lu*~ .lc.
S Using the above calculated amount of gaseous precursor, and l-n~.,l.' , which possesses a molecular weight of 76.11, a boiling pomt of 32.5C and a density of 0.7789 g/mL at 20C, further . All "I.t,"..~ predict that 5.74 x 10-~5 grams of this precursor would be required for a 10 ~m ~ ,lu~ ,lc.
~.Yt~rnl further, amd with the knowledge of the density, equation (B) further 10 predicts that 8.47 x 10-16 mL of liquid precursor is necessary to form a lll;clu~h_lc with an upper limit of 10 ~Lm.
Finally, using equation (C), a mixture, for example, an emulsion containing droplets with a radius of 0.0272 llm or a COIl~ r ~' v diameter of 0.0544 /~m, is formed to make a gaseous precursor filled Ill;,,l~ ,.c with an upper limit of a 15 10 ~Lm lll;~
An emulsion of this particular size could be easily achieved by the use of an _~I)IU~ I 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 f~lter would also suffice to remove any possible bacterial ~.,- ~- .... --.l~ and, hence, can be used as a sterile 20 filtration as well.
This; ' ' for preparing gas-filled lll;l,ll . ' used as CT
imaging contrast agents in the methods of the present invention may be applied to all gaseous precursors actiYated by ~....l...AII. ~ In fact, depression of the freezing pomt of the solvent system allows the use of gaseous precursors which would undergo 25 liquid-to-gas phase transitions at i . below 0C. The solvent system can be selected to provide a medium for suspension of the gaseous precursor. For example, 20% propylene glycol miscible in buffered saline exhibits a freezing pomt depression well below the freezmg point of water alone. By increasing the amount of propylene glycol or addmg materials such as sodium chloride, the freezing point can be depressed 30 even further.
The selection of cl~l~llU~/li..~ solvent systems may be determined by physical methods as well. Wh~n substances, solid or liquid, herein referred to as ~ W095/32005 2 ~ 9 ~ ~ 7~ P~
':
solutes, are dissolYed in a solvent, such as water based buffers, the freezing point is lowered by an amount that is dependent upon the . of the solution. Thus, as defined by Wall, one can express the freezing point depression of the sol~ent by the following equation:
Inx, = In (1- xb) = AH",,/R(I/To- 1/T) where x, is the mole fraction of the solvent;
xb is the mole fraction of the solute;
~Hh, is the heat of fusion of the solvent; and To is the normal freezing point of the solvent.
The normal freezing point of the solvent can be obtained by solving the equation. If xb is small relative to x~, then the above equation may be rewritten as follows.
Xb = aH""/R[T- To/ToT] ~ ~Hh~T/RT~Z
15 The above equation assumes the change in i r ' AT is small compared to T2.
This equation can be simplifled further by expressing the ~,~ of the solute in terms of molality, m (moles of solute per thousand grams of solvent). Thus, the equation can be rewritten as follows.
XO =m/[m + 1000/m,] ~ mMa/1000 20 where Ma is the molecular weight of the solvent.
Thus, ' ~ for the fraction Xb:
~T = [M~RTo~lloonA~
or AT = K,m, where K,--M~RTo~llooo~
K, is referred to as the molal freezing point and is equal to 1.86 degrees per unit of molal ~ .. a.. l;.". for water at one ~ pressure. The above woss~3~005 2 1 9 1 0 79 ~ t~

equation may be used to accurately determine the molal freezing point of gaseous-precursor filled I~ uD~h-lc solutions used in the present invention.
Hence, the above equation can be applied to estimate freezing point d-,~lCDDiUlla and to determine the a~ . ~ of liquid or solid solute S necessary to depress the solvent freezing i r c to an a~JIu~J value.
Methods of preparing the L..l.~, c activated gaseous precursor-filled . i.,.uDL,h.l,,;, include:
(a) vortexing and/or shaking an aqueûus mixture of gaseous precursor and additional materials as desired, including, for example, stabilizing materials, 10 thickening agents andtor dispersing agents. Optional varjations of this method include au~u~,klvill~ before vortexing or shaking; heating an aqueous mixture of gaseousprecursor; venting the vessel containing the mixturel r ' , shaking or permitting the gaseous precursor l--;,, ua~,L.,IGD to form -r ' ~y and cooling down the suspension of gaseous precursor filled Illi.-~ ,' , and extrudmg an aqueous 15 suspension of gaseous precursor through a filter of about 0.22 fLm. A ~ ,ly, filtering may be performed during in vivo A~i of the IllicluD~ll.,lcD such that a filter of about o.æ ~m is employed;
(b) a ...ic.u . ~ i., method whereby an aqueous mixture of gaseous precursor is emulsified by agitation and heated to form, for example, 20 1ll;.,l~ . ' CD prior to - to a patient;
(c) heating a gaseous precursor in a mixture, with or without agitation, whereby the less dense gaseous precursor-filled u.;u., .' float to the top of the solution by expamding and displacing other 1..;._., ,- in the vessel and ventmg the vessel to release air; and (d) utilizing m any of the above methods a sealed vessel to hold the aqueous suspension of gaseous precursor and 7 the suspension at a L~ IaLulc below the phase transition i . c of the gaseous precursor, followed by ~u.u~ vi--g to raise the ~ At G above the phase transition i I c, optionally with shaking, or permitting the gaseous precursor ~ .' to form ~ y~ whereby the expanded gaseous precursor in the sealed vessel mcreases the pressure in the vessel, amd cooling down the gas-filled l- ic-~ .' c after which shaking may also take place.

woss/3200s 2 ~ 9 ~ (~ 79 r~ 9l Freeze drying is useful to remove water and organic materials from the stabilr~ing materials prio} to tbe shaking installation method. Drying installation methods may be used to remove water from ~l ic~u~ll.,.c~. By pre-entrapping the gaseous precursor in the dried ...;.,., ,' c~ (i.e. prior to drying) after warming, the 5 gaseous precursor may expand to fill the ~ .' Gaseous precursors can also be used to fill dried l~ ,' c~ after they have been subjected to vacuum. As the dried lUil,l~ ,' C:l are kept at a i r ' I below their gel state to liquid crystallinet,lll~ldLulC~ the drying chamber can be slowly filled with the gaseous precursor rn its gaseous state, e.g. ~ ~UIdl~ can be used to fill dried u ic~u~ . at 10 ~lll~,ld~UlC;~ above 4C (the boiling point of ~..lluul~ ' ).
Preferred methods for preparing the Lc.l.~.ld~ulc activated gaseous precursor filled ...i.,.u~..,.c~ comprise shaking an aqueous solution havrng a stabilr~ing material rn the presence of a gaseous precursor at a i l c below the liquid state to gas state phase transition tClll~ ..C of the gaseous precursor. In the case of 15 aqueous solutions which also contain lipid, this is preferably conducted at at~,ll.,)~,ld~UlC below the gel state tb liquid crystalline state phase transition i of the lipid. The mr~ture is then heated to a i , above the liquid state to gas state phase transition , c of the gaseous precursor which causes the precursor to volatilize and expand. Heating is then ' ~, and the t~ of the0 mixture is then allowed to drop below the liquid state to gas state phase transition of the gaseous precursor. Shaking of the mixture may take place during the heating step, or ~ .-- -lly after the mixture is allowed to cool.
Other methods for preparing gaseous precursor-filled lUiClU*)}'I.,lC;
involve shaking an aqueous solution of, for example, a stabilizing material and a 25 gaseous precursor, and separating the resulting gaseous precursor-filled ui~..~ .' for use in computed Lu...~.d~l.J imaging.
Cu..~, l, aqueous-filled liposomes of the prior art are roultinely formed at a t `l" ...II..G above the phase transition tUl.l~ c of the lipids used to make them, since they are more flexible and thus useful in biological systems in the 30 liquid crysta!line state. See, for example, Szoka and r~ Proc. Na~l.
Acad. Sci. 1978, 75, 4194-4198. In contrast, the Illi~.lU:>,UIl.,l~,;. made according to preferred , l,o.l; '~ described herein are gaseous precursor filled, which imparts w095132005 2 1 ~ 1 07~ ",~ . 191 greater flexibility, since gaseous precursors after gas formation are more Culll,u~ C
and compliant than an aqueous solution.
The methods . ' ' by the present invention provide for shaking an aqueous solution comprising a stabilizing material, in the presence of a 1~ . c activatable gaseous prccursor. Shaking, as used herein, is defined as a motion that agitates an aqueous solution such that gaseous precursor is introduccd from the local ambient C~;IUIIII~ .. into the aqueous solution. Any type of motion that agitates the aqueous solution and results m tbe illlluducliùll of gaseous precursor may be used for the shaking. Preferably, the shaking is of sufficient force such that a foam is formed 10 within a short period of time, such as 30 minutes, and preferably within 20 minutes, and more preferably, witbin 10 minutes. The shaking may involve ~ lu.lll.~l~;ryillg, 1..;.,11 '' ' ~, swirling (such as by vortexing), side-to-side, or up and down motion.
In the case of the addition of gaseous precursor in the liquid state, sonication may be used irl addition to the shaking methods set forth above. Further, different types of 15 motion may be combmed. Also, the shaking may occur by shaking the contamer holding the aqueous lipid solution, or by shaking the aqueous sûlution within the container without shaking the container itself. Further, the shaking may occur manually or by machme. MPr~ r:~l shakers that may be uscd mclude, for example, ashaker table, such as a VWR Scientific (Cerritos, CA) shaker table, a ~ll;.,lulL.;d~l, 20 Wig-L,Bug~ (Crescent Dental r~ Inc., Lyons, IL), which has been foumd to give ~u~uLi-,ul~uly good results, and a mechanical pamt mixer, as well as other known and available equipment. Another means for producing shaking mcludes the action of gasc~ous precursor emitted under high velocity or pressure. It will also be understood that preferably, with a la~ger volume of aqueous solution, the total amount of force 25 will be ~.ullcD,uulldiL~ mcreased. Vigorous shaking is defmed as at least about 60 shaking motions per minute, and is preferred. Vortexmg at about 1000 revolutions per mmute is more preferred. Vortexing at 1800 revolutions per minute is even more preferred.
The formation of gaseous precursor filled Illi.,lU~llCl.~ upon shakmg 30 can be detected by the presence of a foam on the top of the aqueous solution. This is coupled with a decrease in the volume of the aqueous solution upon the formation of foam. Preferably, the fmal volume of the foam is at least about two times the initial ~ W0 95~3200S 2 1 9 ~ 0 7 9 r~

volume of the aqueous solution. More preferably, the final volume of the f~am is at least about three times the initial volume of the aqueous solution, with a fmal volume of the foam of at least about four times the initial volume of the aqueous so}ution being even more preferred. Still more preferred, all of the aqueous solution is co~verted to 5 foam.
The required duration of shaking time may be determined by detection of the formation of foam. For example, 10 mL of solution in a 50 mL centrifuge tube may be vortexed for ~ , 15 to 20 minutes or until the viscosity of the gaseous precursor-filled l~ U~I I.L.c~ becomes rr ' '~/ thick so that it no longer 10 clings to the side walls as it is swirled. At this time, the foam may cause the solution containing the gaseous precursor-filled III;~IU~ to raise to a level of 30 to 35 mL.
According to the methods c, .' ' by the present invention, the presence of gas, such as air, may also be provided by the local ambient dilll~
The local ambient ,' can include the dLlllU~L~II.,I~ within a sealed container, or 15 in an unsealed conhiner, the external .,..v;., Alternatively, for example, a gas may be injected into or otherwise added to the container having the aqueous lipid solution or into the aqueous lipid solution itself in order to provide a gas other than air.
Gases that are lighter than air are generally added to a sealed container while gases heavier than air can be added to a sealed or an unsealed container. Accordi~gly, the 20 present imvention includes co, . of air and/or other gases along witll gaseous precursors.
Hence, the stabilized , ,' ~ precursors described above~ can be used im the same manner as the other stabilized ~ u~ used in the present mvention, once activated by application to the tissues of a host, where such factors as 25 ~ ; or pH may be used to cause generation of the gas. It is preferred that the gaseous precursors undergo phase transitions from liquid to gaseous states at near the normal body i , ~ of said host, and are thereby activated, for example, by the in vivo . of the host so as to undergo transition to the gaseous phase therem.
This can occur where, for example, the host tissue is human tissue havmg a normal 30 ~...~ of about 37C and the gaseous precursors undergo phase transitions from liquid to gaseous states near 37C.

wo ss/3200s 2 1 9 1 0 7 ~ c~

All of the above ~ ù l;. ~ involving IJlcL)~ua~ioll~ of the stabilized , including .~ of gas and/or gaseous precursor filled llli,lU >~ .,lC~., may be sterilized by autoclave or sterile filtration if these processes are performed before the installation step or prior to tclll~.,ld~lllc mediated conversion of the 5 . c sensitive gaseous precursors within the Cl~cp^~ n Alternatively, one or more anti-ltr^r~riri~ agents and/or l~ clvaLi~ may be included in the r.,.. ~
of the contrast medium, such as sodium benzoate, quaternary salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, a~,ulby~i ' , butylated l.~.l,u~.llu.,olc, butylated ll,~-llu~ ,IIu~u~lol, .Ic~.,d~u~ ..i., acid, 10 ctl~ ul~ potassium benzoate, potassium ", ~ r;~., potassium sorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts. Such ~ .;l;,_l;...~ which may also be achieved by other cull~ iundl means, such as byirradiation, will be necessary where the stabilized Illi~ .' are used for imagirlg umder invasive ~,ill , e.g., intravascularly or illLL~l 'y. The 15 a~JIul means of ! ' ''' " will be apparent to the artisan based on the present disclosure.
The contrast medium of the present invention is generally stored as an aqueous , but the contrast medium can be stored as a dried powder ready to be ,~ ' prior to use in the case of dried III;.,IU~III.,lC~l.
20 Methods of Use As discussed above, the stabilized ~ . of the present invention, irlcluding ~ of gas and/or gaseous precursor filled ~ .lu~ .;" are usefulas contrast agents for computed lulllu6.~h~ (CT) imaging, including CT ~ O ,' y (CTA) imaging. It is c, .' ' that the present stabilized . are useful as 25 contrast agents in connection with other diagnostic -~ inlr~ , rncluding, for example, magnetic resonance (MR) imaging and magnetic resonance (MRA).
In accordance with the present invention, there is provided a method of imaging one or more regions of a patient. The present invention provide also imagrng 30 methods which involve diagnosing the presence or absence of diseased tissue in a patient. The imaging methods of the present invention can be carried out by ~ W0 9S132005 2 1 9 ~ 0 7 9 r~
-~43 -~ ~' ,, a contrast medium, in the form of a stabilized Cl.cr,-r~;.,n to a patient.
The patient is scanned using computed Lulllù~ Jlly imaging, or other imaging 1~. il.~..l..l~:l~, to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region. By region of a patient, it is meant the whole l~atient or a particular area or portion of the patient. The contrast medium is ~ uæful in providing images of the ~ LI~ ' ' I region, but can also be employed more broadly such as in imaging the Y~ul~tlllc or in other ways as will be readily apparent to those skilled in the art. The phrase ~s~-DLI~ region or ~ ,LIu;.~alil~l tract, as usedherein, includes the region of a patient defined by the esophagus, stomach, small and 10 large intestines and rectum. The phrase 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, A l~..;.. ~l.Al;.". of the stabilized ,~ - of the present invention can be carried out in various ~ashions,15 such as illLl~y~ uLuly~ orally, intrarectally, illLl~ly~ illally~ illLl~y~a;~ul~l~y~
y~ . l,l - ly~ Iy, and the like, using a variety of dosage forms. When the region to be scanned is the g~laLl~ ' 1 region, A.1...;..;~1.,.1;.~.. of the ! ' of the present invention is preferably carried out orally or rectally. The useful dosage to be a l.lli. ~ and the particular mode of 20 ' will vary depending upon the age, weight and the particular nnammal and region thereof to be scanned, and the particular contrast mediurn of the invention to be employed. Typically, dosage is mitiated at lower levels and increased until the desired contrast . ' is achieved. Various ~ ofthe stabilized may be used to alter properties as desired, nncluding viscosity, osmolarity 25 or ~llaLa~ y~ In carryying out the imaging methods of the present invenltioll, the contrast medium can be used alone, or in ' with diagnostic, theralpeutic or other agents. Such other agents include excipients such as flavoring or coloringmaterials. CT imaging techniques which are employed are ;u...~ iu~l and are described, for example, in Compl~ted Body 'romography, Lee, J.K.T., Sagel, S.S., and 30 Stanley, R.J., eds., 1983, Ravens Press, New York, N.Y., especially the first two chapters thereof entitled "Physlcal Principles and 11~l ", Ter-Po~ossian, WO 95/32005 ` 2 ~ 9 1 0 7 9 P~ C D ' .91 ~

M.M., and ~Techniques", Aronberg, D.J., the disclosures of wi~iich are i...,u.
by reference herein in their entirety.
The routes of ' and areas of usefulness of the present are not limited merely to the blood volume space, mcluding the vasculature. CT imaging can be achieved with the ~ of the present invention if ingested orally to image the ~ u;ll~c~ l (Gl) tract. Alterriatively, rectal of these stabili7ed ~ can result m excellent imaging of the lower Gl tract, including the rectum, descending colon, transverse colon, and ascending colon, as well as the appendix. The , of the present invention, 10 and especially gas filled and gaseous precursor filled l-liu-u~ .c~, are pi~Lil~ul~lly well suited for imaging the GI tract. For example, ~ - comprising gas filled or gaseous precursor filled ~ic~u*)l~ c~ can be ~ cd to the patient orally or rectally. It has been found that, ~l' . ...- of gaseous precurso} filled Illi~.lU*~I'l_lC:~
are generally more palatable than ~ of gas filled l~ ,lu~yh~lc~. Accordingly, 15 in the case of oral a~ of gaseous precursor filled l~ u~
are preferred. In t~ie case of ! , ' of llliulu*~ lca fi'iled with a gaseous precursor having a transition t~ ..alulc at near body hl.ll.. , t~ie gaseous prccursor fililed lliiC~ .' are desirably and cu..._....,..ily converted to gas filled Illi-,iua,ull~ withm the Gl tract of the patient.
In addition to the areas of the Gl tract which can be imaged with the of the present invention, including the rectum, descending colon, transverse colon, ascending colon, and appendix, it is l ,' ' that the ileum, and CUIIU~ ~IY the jejunum, can be imaged by the methods described herein by way of rectal ~ ' In addition, direct i~ ' may be achievcd to visua'ii_e the ~. It is also , ' ' that the stabilized - -r ' may be a~' directly into 'ihe ear canals such that one can visua'ii_e the cana'is as well as the Eustachian tribes and, if a perforation exists, the ilmer ear. It is also ,' ' that the stabilized , may be ~ l intranasa'ily to aid in the vi~ li7~ti-n of the nasal septum as well as the nasal sinuses by CT
Other rouhs of _' of the ~ - of the present invention, and tissue areas whose imaging is enhanced thereby include, for example, (i) intranasa'ily for imaging the nasal passages and sinuses, including the nasai region and _ _ _ , _ _ _ _ _ _ _ _ _ , . _ . . _ _ _ _ _ _ _ _ _ _ ~ W095/32005 21 91 079 r~ c~
~ 45 -sinuses and sinusoids; (ii) intranasally and orally for imaging the remainder of the respiratory tract, includmg the trachea, bronchus, blul~uh ~ , and lungs; (iii) ly for imaging the hearing passages and Eustachian tubes, tympanic ", , l"~ " ~ and outer and inner ear and ear canals; (iv) ;.~l.,.... --1~ 1~ for r~naging the 5 regions associated with vision; (v) , '~/ to visualr~e the p ~ . --..; and (Vi) illL~ ,uL~ , i.e., through the bladder, to rmage all regions of the g .y tract rncluding, for example, the uretbra, bladder, ureters, kidneys and renal ~lal,ul~iulc and beyond, for example, to perform cy~u~l~lly or to confr~m the presence of ureteral reflux.
The rnvention is further described in the following examples. Examples 1 to 168 are actual examples. Examples 169 to 170 are prophetic examples. These examples are for illustrative purposes only, and are not to be construed as lirniting the appended clarins.
The following examples (Examples 1 to 10) describe the l)lC~ iUII of 15 various stabilr~ed ~ within the scope of the present invention. Various of these examples (Examples 1 to 6) also describe methods which are within the scope of the present invention and which involve imaging mternal regions of mammals usingCT. Example 7 describes methods which are witbin the scope of tbe present invention and which involve imaging internal regiorls of marnmals using MR.
20 Exsunple 1 To purified water (730 mL) were added 0.5 wt%
(Spectrum, Houston TX), Poloxamer F68 (500 ppm) (Spectrum, Houston TX), Polysorbate 40 (200 ppm) (Spectrum, Houston, TX), ~ . (50û ppm~ (Dow Corning, Midland, Ml) and 0.1% potassium sorbate (Spectrum, Houston TX). The 25 mixture was mixed for 5 mrnutes with a Silverson L4RT mixer (Silverson Machine LTD, Waterside, Englamd) with the general assembly and the square hole high shear screen set at 5000 rpm. The resulting mixture was cooled to 7C in an ice bath.
r~ ~ , r ' (5 mL) (PCR Chemicals Inc., Gainsville, FL) was added to the cooled mixture. The mixture was then agitated for 1 minute with the same Imixer and 30 screen as before except the speed was adjusted to a higher setting (10,300 rpm). The resulting stabilrzed suspension Of,u~,lnuulu~ cul~ had a viscosity of 30.8 cps as .

w0 9sl3200s 2 1 9 1 0 7 9 . ~ C ~ I91 ~

measured by a Gilmont I~ lu..~ size No. 2 falling ball vi~u~i--l.,L., model GV-2200 (Gilmont Instr., R~rrin,,fon, IL). The number weighted mean particle size was 4.58 ~m as measured using an AccuSizer 770 optical pa~ticle sizer (Particle Sizing Systems Inc., Santa Barbara, CA).
S A~ , 500 mL of the stabilized suspension was a~h~ clal to a dog (17.25 kg) which had been kept NPO (none per oral) for 24 hours. This uullc r ' to a dosage oF 30 mL/kg. The suspension was dispensed to the dog in a dish over a two hour period. Prior to CT imaging, the dog was ~ .l with a dose oF 0.25 cc/kg sodium pentothal and kept under anesthesia witb isoflurane. CT
imaging was performed with a Toshiba 900S CT scanner (Toshiba Medical Systems, Nasu, Japan) and tbe i~nages were recorded on X-ray film. Superior images of thebowel were obtained. The entire bowel was visualized as lumena filled with O. ~ v~ black contrast. The mucosal surfaces were readily discerned throughout the entire bowel.
CT Angiography (CTA) was performed on a Picker 2000 CT scanner (Picker Medical Systems, Cleveland, OH) with a dose of 0.5 mL/kg iohexol (SanofiWintbrop, N.Y., NY) to create positive contrast in the abdominal ~ cuL~ c. CTA
was acquired with a slice thickness of 5 mm with a pitch of 1.25 and power ættings of 130 kV and 100 ma on a modified spiral pancreas setting. Superior images of the abdominal vasculature were obtamed. No ' c~,c with ~,;----I;, Ii---- of the blood vessels was caused by the black bowel. By ~ CTA performed after iohexol (I.V.) in subjects _' c~ oral barium sulfate was markedly degraded by density m the bowel u~ L~ ;A.o and obscuring tbe abdominal Exarnple 2 Poloxamer F68 (0.25 g), - - lI.:r............ ~ (0.25 g), ~u;y ' 40 (0.1 g), sodium benzoate (0.5g), fructose (10 g), l.~ hyl~cl~ulv~c (5 g) and purified water (484 g) were combined and the resulting mixture was mixed umtil 1~ - using a Silverson L4RT mixer equipped with the 1" tubular assembly and the square hole high-shear screen set at 8000 rpm~ The l".. O. ,.. ~ mixture was cooled to 4C in an 30 ice bath and ~IIUUIU~ LU~ (3 mL) was added. The mixture was then mixed for 1 minute with high shearing using the same mixer as above and with the assembly set at ~ W095/3:~005 2191G79 P~ o~

a higher speed (12,000 rpm). The resulting stabilr~ed suspension had a viscosity of 103 cps as measured by a Gilmont l.~.lu.l.~ L, size No. 2 falling ball V;:~O:~UI~
model GV-2200 (Gilmont Instr., P~nin~tnn, IL). The number weighted mean particlesr~e was 4.07 llm as measured using an AccuSizer 770 optical particle sizer (Particle 5 Sizing Systems Inc., Santa Barbara, CA).
A Sprague-Dawley rat (250 g) that had been NPO for 24 hr was gavaged with the mixture (4 mL) and imaged at 1 and 1.5 hours with a Toshiba 900S CT
macbine which was set at 100 milliamps, 140 kilovolts and 4 mm slice thickness.
TTl,. ,.. " . v~ ~ negative contrast of the Gl tract was observed.
10 ExamDle 3 Poloxamer F68 (0.125 g), (0.125 g), pvly~ 40 (0.05 g), sodium benzoate (0.25 g), fructose (5 g), VeegumTY (5 g) (Spectlum, Houston, TX) and purified water (239 g) were combined and the resulting mixture was mi1xed until o v~ ~ using a Silverson L4RT mixer equipped with the 1 " tubular assembly 15 and the square hole high-shear screen set at 8000 rpm. The l~ O ~ mixture was cooled to 4C in an ice bath and ~,.llluulu~ ~ (1.6 mL) was added. The mixture was then mixed for 1 minute with high shearing using the same mixer as above andwith the assembly set at a higher speed (12,000 rpm). The resulting stabilized suspension had a viscosity of 5.6 cps as measured by a Gilmont l.~LI~,ll~ size No. 2 20 falling ball vi~.u~ l model GV-2200 (Gihmont Instr., r ,, IL). The number weighted mean particle size was 2.67 ~m as measured using an AccuSr~er 770 optical particle srzer (Particle Sizing Systems Inc., Santa Barbara, CA).
A Sprague-Dawley rat (250 g) that had been NPO for 24 hrs was gavaged with the suspension (4 mL) and imaged at 1 and 1.5 hours with a Toshiba 25 900S CT machine (Toshiba Medical Systems, Nasu, Japan) set at 100 milliamps, 140 kilovolts and 4 mm slice thickness. Tl".. ~. v ~ negative contrast of the GI tract was observed.
~Ex~nple 4 Poloxamer F68 (0.125 g), ~ - (0.125 g), p~ 40 (0.05 30 g), sodium beate (0.25 g), fructose (5 g), xanthan gum (2.5g) (Kelco, San Diego, woss/3200s 2 1 9 1 079 Y~ cs~" ~

CA) and purified water (242 g) were combined until ll.-, ..,C. ~ usmg a Silverson L4RT mixer equipped with the 1" tubular assembly and the square hole highshear screen set at 8000 rpm. The l.. ~,.. - mixture was cooled to 4C m an ice bath and ~ n..J.~ ,l., (1.6 mL) was added. The mixture was then mixed for 1 minute 5 with high shearing using the same mixer as above with the assembly set at a higher speed (12,000 rpm). The viscosity of the resulting stabilized suspension was too thick to measure with a Gilmont T~ size No. 2 falling ball lda~ l model GV-2200 (Gilmont Instr., R ~-rin~tr n, IL). The number weighted mean particle size was 4.06 ~m as measured using an AccuSizer 770 optical particle sizer ( Particle10 Sizing Systems Inc., Santa Barbara, CA).
A Sprague-Dawley rat (250 g) that had been NPO for 24 hr was gavaged with the suspension (4 mL) and imaged at 1 and 1.5 hours with a Toshiba 900S CT
machine (Toshiba Medical Systems, Nasu, Japan) which was set at 100 milliamps, 140 kilovolts and 4 mm slice thickness. Extremely 1~ ,. u - negative contrast, at a 15 density ~ that of air, was observed in the Gl tract.
Exam~le 5 Poloxamer F68 (0.3125 g), ~ h~ (0.3125 g), poly;~ullJ~t~ 40 (0.125 g), sodium benzoate (0.625 g), fructose (18 g) and purified water (500 g) were combmed and the resulting mixture was mixed until l~ ,. v using a Silverson 20 L4RT mixer equipped with the 1" tubular assembly and the square hole high-shear screen set at 8000 rpm. The pH of the 11 ''""D' ''''''- mixture was adjusted to pH 4 with 1 N EICI (Fisher Chemical Supply, Fair Lawn, NJ) and the mixture was cooled to 4C
in an ice bath. The cooled mixture was placed m a M-110TM;~,~onuilli~l (Mircofluidics Corp., Newton, MA) equipped with a cooling chamber through which a 25 ~ llyl alcohol solution was cycled to cool the fluidized mixture to 10C while operating at 15,000 psi. The cooled mixture was cycled ~ , 2 passes and then p~lnuv..r (3.9 mL) was added to the cycling mixture. The M-1 lOT
Mi~,~unuidi~ was then allowed to cycle 500 times. A 2.5% solution of methylcellulose was prepared by the addition of methylcellulose (12.5 g) to purified 30 water (487 g) which was mixed using a Silverson L4RT mixer (Silverson MachimeLTD, Waterside, England) with the general assembly and the square hole high shear .. .. .. . . . .. .. . . _ _ . _ _ _ _ _ _ WO95132005 2 1 9 1 0 7 9 . ~ fl~l screen set at 5000 rpm. The cooled mixture prepared above (200 mL) was added to the 2.5% .l..,~l,,.,:hll~e solution (50 mL) and blended by stirring. The resulting stabilized suspension had a viscosity of 15.5 cps as measured by a Gilmont IllDUh...~
sr~e No. 2 falling ball Vi~l..U~ ,t~,l model GV-2200 (Gilmont Instr., ~rin~rnn IL).
5 The number weighted mean particle size was 1.76 ~m as measured using an AccuSizer 770 optical particle sizer ( Particle Sizing Systems Inc., Santa Barbara, CA).
Two SpMgue-Dawley rats (250 g each) which were NPO for 24 hr were each gavaged with the blended suspension (4 mL) and imaged at 1 and 1.5 hours with a Toshiba 900S CT machine (Toshiba Medical Systems, Nasu, Japan) which was set at 10 100 milliamps, 140 kilovolts and 4 mm slice thickness. Extremely ~ ,. u ..
negative contrast, at a density ~ ul~ ' ~ that of air, was observed in the GI tract.
ExamDle 6 Poloxamer F68 (0.195 g), ~ (0.195 g) and potassium sorbate (0.432 g) were combined and brought to 300 g with purified water. The resulting 15 mixture was mixed umtil l--- ---~ using a Braun M~ irr~rrir Hand Blender (Braun Inc, Lynnfield, MA). The pH of the l.. -- .~,_.. mixture was adjusted to pH

6 with 1 N HCI (Fisher Chemical Supply, Fair Lawn, Nn and the mixture was cooledto 4C m an ice bath. A portion of the cooled h....... ,,. .,.~ mixture (250 mL) was placed in a M-llOT ~ (M;.~.~n..'-l:~ ~ Corp., Newton, MA) equipped with 20 a cooling chamber through which a w /lll.,tllyl alcohol solution was cycled to cool the fluidized mixture to 10C while operating at 15,000 psi. The l~ .. ~,. -,.. ~ mrxture was cycled .~ , 2 passes and then ~lluul~ r ' (1.9 mL) was added to the cyclmg mixture. The M-llOT Mi.,.~lnuidi~l was then allowed to cycle 250 times.
A portion of this mixture (187.5 mL) was added to a 1% xanthan gum solution (62.5 25 mL~ and the resultmg mixture was blended by stirring to provide a stabilized pn~inn The suspension had a viscosity of 16.1 cps as measured by a Gilmont I~L.~...,~..Ib size No. 2 &llmg ball ~ model GV-2200 (Gilmûnt Irlstr., R~rr~'ngtnn IL). The number weighted mean particle size was 6.50 ~m as measured using an AccuSizer 770 optical particle sizer (Particle Sizing Systems Inc., Santa 30 Barbara, CA?.

woss/3200s 21 9 l O79 ~ r~

A Sprague-Dawley rat (250 g) that had been NPO for 24 hr was gavaged with the suspension (4 mL) and the rat was imaged at 1.5 and 2 hours with a Toshiba 900S CT machine (Toshiba Medical Systems, Nasu, Japan) wbich was set at 100 milliamps, 140 kilovolts and 4 mm slice thickness. Extremely i..,.,..,~ ... v - negative 5 contrast, at a density d~ that of air, was observed in the GI tract.
Example 7 Into a 15 mL ~ly~JIu~yl~ . screw cap tube (VWR, West Chester, PA) were introduced water (7.3 mL), a 1% solution of xanthan gum (1 mL), soy oil (l mL), a 1% aqueous solution of sodium lauryl sulfate (300 ~L) (Duponol C, Witco 10 Corp., N.Y., NY), a 1% aqueous solution of P~IJ~OI 40 (200 ~LL), a 1% aqueoussolution of potassium sorbate (100 ~LL), ~.", l,; ,--- (5 ~L), and p~nuulu~ luul~; (100 ~L). The mixture was agitated using a Vortex-Genie 2 (Scientific Industries Inc., Frar~lin Lakes, NJ) on full speed for 1 minute, thereby creating a stabilized .

A Sprague-Dawley rat (250 g) that had been NPO for 24 br was gavaged with the suspension (4 mL). After one hour, the rat was ~ ,. Ih t;~ 1 with 1.3 cc/lcg of a 10:1 mixture of ketamine HCI (Ketaset, Aveco Co. Inc., Fort Dodge, IA) and d~ maleate (PromAce, Aveco Co. Inc., Fort Dodge, IA). The rat was imaged using multiple protocols in a GE Signa 1.5 Tesla MR scamner (GE Signa, 20 Milwaukee, WI) using the extremity coil. Four protocols were used: MEMP, VEMP, GRASS and Fast GRASS. Settings for MEMP imaging were as follows: 250 TR, 14 ms TE (Auto), 11 cm FOV, 4 mm slice thickness, 1 mm gap, 4 NEX and a 256 x 192 matrix. Settings for VEMP imaging were as follows: 2500 ms TR, 19/80 ms TE, 11 cm FOV, 4 mm slice thickness, 1 mm gap, 1 NEX and a 256 x 192 matrix. Settings 25 for GRASS imaging were as follows: 13.1 ms TR, 4.2 ms TE, 20 degree flip angle, 15 cm FOV, 4 mm slice thickness, 1 mm gap, 2 NEX and a 256 x 192 matrix.
Settings for Fast GRASS imaging were as follows: 100 ms TR, 6 ms TE, 30 degree flip angle, 11 cm FOV, 4 mm slice thickness, I mm gap, I NEX and a 256 x 192 matrix. The MR images showed l"~ low signal intensity (signal voids) 30 within the entire GI tract. The mucosal detail was excellent in the MEMP and VEMP
images and all of the images were free of, ~ ;ly artifacts. The ~

~ W095132005 21 91 079 r~/u~s~rl/l with MR imagmg verify that the ~ .v~ of the present invention function as an effective negative (black) contrast agent for MR.
E~nDle 8 Poloxamer F68 (0.25 g), ! ' ~ ' (0.25 g), pvly~uli 4~ (0.05 g), 5 sodium benzoate (0.5 g), fructose (10 g), methyl cellulose (2.5 g) and purifi~d water (486 g) were combined and the resulting mixture was mixed until l~ ,. (10 minutes) using a Silverson L4RT mixer equipped with a 1 inch tubular asserflbly and a square hole high-shear screen set at 500û rpm. A portiûn of this ~ 6~ mixture (250 mL) was cooled to 4C in an ice bath to which ~,."nuv-~ r ' (0.16 mL) was 10 added. The resulting mixture was mixed for 1 minute with high shearing using the same mixer and assembly as above at a higher speed setting (12,900 rpm). A firstportion of this sheared suspension was sized for particles using an AccuSizer 770 optical particle sizer (Particle Sizing Systems Inc., Santa Barbara, CA). A second portion of the sheared suspension was also similarly sized. After 30 days, the sheared 15 l . which had become non~ - was IG' -~ f d by agitation and sized again. Comparison of the various sizings showed little change in the particle ~' ' ~LI
Examlde 9 Poloxamer F68 (0.25 g), ! ' '- (0.25 g), puly ' ' 40 (0.05 g), sodium benzoate (0.5 g), fructose (10 g), Viscarin GP-209, ,, (1.25 g) (FMC
Corp., pl,.1 ~f ~ -~, PA) and purified water (487 g) were combined and the resulting nuxture was mixed until l'~ 's ~J"` (10 minutes) using a Silverson L4RT mixer equipped with the 1 inch tubular assembly and the square hole highshear screen set at 5000 rpm. The pH of the l~ .. ~,. --. - mixture was adjusted to pH 4 with 1 N HCI.
25 A portion of this mixture (250 mL) was cooled to 4C in an ice bath and p~,lnuulu~ t~l~ (0.16 mL) was added. The resulting mixture was then mixed for 1 minute with high shearing usmg the same mixer and assembly at high speed (12,900rpm). The resulting stabilized suspension had a viscosity of 15.1 cps as measured by a Gilmorlt T - size No. 2 falling ball \d~,u~iu~,t~" model GV-2200 (Gilmont 30 Instr., r ~ IL). The number weighted mean particle size was 4.16 llm as woss/3200s 2 1 9 1 0 7 9 r~l,uv E~
measured using an AccuSizer 770 optical particle sizer (Particle Sizing Systems Inc., Santa Barbara, CA).
Examl~le 10 Poloxamer F68 (0.25 g), ~ (0.25 g), polysorbate 40 (0.05 g), 5 sodium benzoate (0.5 g), fructose (10 g), Kelgum (1.25 g) (Kelco, San Diego, CA) and purified water (487 g) were combined and the resulting mixture was mixed until l..."..,,.... v - (10 minutes) using the Silverson L4RT mixer equipped with the 1 inch tubular assembly and the square hole high-shear screen at ~000 rpm. The pH of the l.(.".~. ."~ mixture was adjusted to pH 4 with 1 N HCI. A portion of this mixture 10 (250 mL) was cooled to 4C in an ice bath and ~c.nu~vlu~ ul~ (0.16 mL) was added.
The resulting mixture was mixed for 1 minute with high shearing using the sarne mixer and assembly as above, set at a higher speed (12,900 rpm). The resulting stabilized suspension had a viscosity of 8.6 cps as measured by a Gilmont T size No. 2 falling ball ~ ll.,L. model GV-2200 (Gilmont Instr., r V IL). The number 15 weighted mean particle size was 3.62 ~m as measured using an AccuSizer 770 optical particle sizer (Particle Sizing Systems Inc., Santa Barbara, CA).
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W095/3200~ 2 ~ 9 ~ 0 7 9 r~ r~ 9l ExamPIe 168 This example describes ,UIII~ iVc imaging studies involving the stabilized . ~ :.,"~ of the present invention and prior art contrast agents.
Methylcellulose (2.5 g), poloxamer F68 (0.25 g), `;~ (0.25 g), S pol,~:.ulb_:c 40 (0.1 g), fructose (10 g), sodium benzoate (0.5 g) (Spectrum Houston 17~) and purified wate} (486 mL) were combined and the resulting mixture was mixed until 1,~...,-.~,.... ~ using a Silverson L4RT mixer equipped with the 1" tubular assembly and the square hole high-shear screen set at 8000 rpm. The Ill....h~.,~.....
mixture was cooled to 4C in an ice bath and ~ .n,.vlu~ (3 mL) was added.
10 The resulting mixture was 1,-~ by mixing for I minute using the same mixer and assembly as above, set at a higher speed (12,000 rpm).
Four 250 g Sprague-Dawley rats (rats (A), (B), (C) and (D)) were ad-~ .cd the stabilized suspension prepared above, contrast agent of the prior art, amd mixtures of the above suspension and contrast agent of the prior art. Rat (A), 15 which had been NPO for 24 hr, was gavaged with the stabilized suspension prepared above. Rat (B) was gavaged with Scan c~u (4 mL) (Smith and Nephew Diagnostics, Memphis, TN) which is a prior art, barium sulfate contrast agent. Rat (C) ~1vas gavaged with a mixture (4 mL) of the stabilized suspension prepared above to which 0.2% by weight barium sulfate had been added. Rat (D) was gavaged with Scan C'Y
20 (2 mL) followed by a second gavage one hour later with the stabilized suspension prepared above. All of the rats were imaged at 1.25 and 2.25 hours after ~.1.,.:.,:~1".1..", on a Somotom DRH (Iselin, NJ) with settings of Tl 3, KVp 125, AS
0.28, Slice thickness 4 mm, and a Gantly tilt of 0. The images obtained of rat (A) were l.. h~.. v ~ amd black throughout the Gl tract. The images obtairled of rat (B) 25 showed ~ of the barium sulfate contrast. The images obtained for rat (C) showed ~c.~u~..cd barium sulfate (positive contrast) mixed with areas of negative contrast (i.e. a two phase contrast). The images obtained of rat (D) were ulldi~ from those of rat (A) at 1.25 hours; however, the barium sulfate a~ .stclc~ in the lower bowel at ~.25 hours.

w095/3200~ 2 1 9 1 079 - 68 - ~ 3i ~
' 169 = _ ~ ~
This example describes a ~cli~ cuy toxicity study which were conducted on stabilized ~ within the scope of the present invention. The studies involved comparing the toxicity of the present ~ -"c with the toxicity of 5 a control (mixtures prepared without a gas or gaseous precursor).
Pl~, . " of Stabilized ~
Into a 15 mL pol~ yl, .,~, screw cap tube (vwr~, West Chester, PA) were introduced water (7.3 mL), 1% xanthan gum (I mL), soy oil (I mL), a 1%
aqueous solution of sodium lauryl sulfate (300 IlL) (Duponol C, Witco Corp., N.Y., 10 NY), a 1% aqueous solution of roly~."l,..~c 40 (200 IlL), a 1% aqueous solution of potassium sorbate (100 IlL), ~....~.1.- -~e (5 IlL), and ~.,lnuul~ (100 IlL).
This mixture was mixed for I minute using a Vortex-Genie 2 (Scientific Industries Inc., Franklin Lal~es, NJ) on full speed to produce a hnmf ~-n-~oll~ mixture.
P, ""... ' of Control Mixture _ _ A mixture was prepared as described above except there was no p~,lluvlvl,.,..kulf. This is referred to herein as "the control mixture".
Tox~citv Studv Twelve Sprague-Dawley rats (250 to 300 g) were used in the toxicity study and were divided into three groups (Groups (A), (B) and (C)) of four rats each.
:ZO The rats of Group (A) were dosed with the control mixture (4 mL) once a day for seven days. The rats of Group (B) were dosed witb the stabilized suspension prepared above (4 mL) once a day for seven days. The rats of Group (C) were dosed v~/ith the stabilized suspension prepared above (3 mL) twice a day. After four days, the second daily dose which was being adl..i..iatc.cd to the Group (C) rates was25 ~ d due to excessive irritation of the esophagi from the gavage tubes. The Group (C) rats were then dosed with a single daily dose (4 mL) for the remainingthree days. No substantial differences were observed in the food (....~ ., or the weights of the rats of Groups (A), (B) and (C). The rats were sacrificed after the seven day treatment period and blood and tissue samples were collected. Blood 30 chemistry analysis showed no significant differences among the rats of Groups (A), (B) and (C). In addition, tissue samples of multiple organs showed no significant differences or toxicity indications among the rats of Groups (A), (B) and (C). A

wossl3200~ 2 ~ 9 1 o 79 PCT/US9S106491 possible renal tubular epithelium Ir~ in the collecting ducts of the rats of Group C was observed. However, it is believed that this was due to the advanced age of the rats studied in Group C.
The following examples, (Examples 170 to ~77) are lly,uulll..i~ol 5 examples and describe the various clinical ~l.l.li- ~';....~ of the stabilized of the present invention.
ExamPle 171 This example describes the use of the present stabilizing ~ in magnetic resonance all~;O~la~lly (MRA).
In a patient sufferin~ from abdominal disease, 10 mLlkg of the stabilized suspension prepared in Example I will be ~d~ cl orally. Magnetic resonance (MR) imaging will be performed obtaining axial Tl arld T2 weigllted images. The bowel will appear l.~ ,. u ~l~ with low signal intensity as a signalvoid. MR .~ l.y (MRA) of the abdominal vaaculdl~c will be perforrned also 15 using a 2D Time of Flight pulse sequence, axial arT~icition The MRA image will be of high quality, with no discernible artifacts caused by the stabilized suspension of the present invention.
Two cu..lualaLivc MRA studies will be conducted to evaluate the efficacy of the stabilized ~ of the present invention. In the first 20 cull.Au~alivc study, patients will be r ' ' ' ' cd orally a positive Gl MR contrast agent, such as dilute (4 millimolar) gadolirlium di~ 1.. pentaacetic acid (Gd-DTPA). MRA images will be degraded by ghosting artifacts from the high signal intensity in the bowel lumen. In the second Cu~llJ~alivc study, a patient will be r ' ' ' ' cd iron oxide particles as a GI contrast medium. MRA images will be5 degraded by ellerPrtihili~y artifacts. These studies will show that the stabilized of the present invention provide excellent Gl contrast and are also effective as adjuncts for MRA in the abdomen or pelvis.

wo ss/3200s 2 1 ~ ~ ~ 7 9 ~ 9~ ~

F~ ple 172 =
This example describes the use of tbe stabilized ~ . of the present invention for improving the CT imaging ..l, -- ,.... ;~l;. ~ of prior art positive contrast agents.
60% Hypaque~ iodinated contrast media (300 mL) will be combined with a stabilized suspension of l~"nuu~u~ ~lc (600 mL) within the scope of the present invention. The suspension will preferably comprise ~.clnuulu~ ul~ filledllu~,lua~h_lca. The ... .. ,l l "l;.", of Hypaque~ in the resulting "l ' " contrast medium will be 20% by volume and the ~ ." of ~ .,.nuulv~ lt~le filled 10 I~ ,lualJh_l~a will be 1% by volume. The contrast medium will be r I
orally to a patient at a dose of 15 mL/kg. After ingestion, the ,u~lnuu.uu~l.la..., will be converted to a gas and the overall volume of the contrast medium will expand.This expansion will result in controlled and uniform distension of the GI tract with ll",.,-.~.. ,... high density. Improved definition of the mucosal surfaces in the GI
15 tract will be observed with the ,~ ;.", contrast medium, as compared to HypaquelU alone. This improved definition is due to distension of tbe GI tract caused by the stabilized suspension of the present invention.
li. ' 173 Tbis example describes the use of the stabilized ~ of the 20 present invention for improving the CT and X-ray imaging ~ of prior art contrast agents.
A patient will be ad~ull;at~,lcli a 0.4% by weight ~ , of barium sulfate (4ûO mL) that is designed to adhere to the mucosal surfaces of the bowel.
Preparation of the barium sulfate ...,..l.-~ l..,.. will involve grinding barium sulfate in 25 a colloid mill with a, ~ powder. The resulting micron-sized particles of barium sulfate coated with ,, are designed to have an affinity for the mucosal surfaces of the bowel. Four hours after ~.l",; ~1".1;.~.., the patient will be cd a stabilized suspension of the present invention (15 mL/kg). The A ~ ;"" of both barium sulfate and a stabilized suspension of the present 30 invention will provide a double contrast . - .,;,.- ;~.., of the bowel for computed ~ WO 951320W 2 1 q 1 0 7 9 r~

IUIIIU~;I~IIY or lluulu~,V,uy. The mucosal surfaces of the bowel will be hi~hli~ht.
with a f~ne, tbin layer of high density and the lumen will be black.
EXamPIe 174 This example describes the use of the stabilized ~ of the 5 present invention for improving the MR imaging ~ of prior art contrast agents.
A patient will be adl.l;-l;atc.cd of l.".,....TI- .- eTV (400 mL) (Bracco Diagnostics, Princeton, NJ) which contains manganese chloride (MgCI2) in admixture with pOly~;aLI~Luuulll~ acid. Four hours afler ~ 1;."., the patient will be 10 d~ln~ LIcd a stabilized suspension of the present invention (15 mL/kg). MR
imaging will be performed on a cullllll. l.,;cl MR imager with Tl weighted spin echo technique, for example, where TR is 300 msec and TE is 11 msec. The resulting images will show a two phase contrast. Due to the ~ ;..,. of the stabilized suspension of the present invention, the lumen of the bowel will be 1~ ly 15 dark and will appear as a signal void. The mucosal surface will appear as a thin, bright rim. Mucosal surfaces are thereby hi~hli~ht, ~ with a dual contrast MR
technique.
EXamPIe 175 T_is example describes the use of the stabilized ~ of the 20 present invention for improving the MR imaging ~ of prior art contrast agents alLIl..,l~Llcd by l.V.
A stabilized suspension of the present invention is av~',l~v (20 mL/kg) over a two hour period to a patient suffering from polyps in the bowel. Ten n~inutes prior to scamning with MR, 500 millimolar solution of Gd-DTPA (Schering25 A.G., Berlin, Germany), providing a dose of 0.1 mL/kg, will be a,l.~..;.t~.c,i l.V. Tl weighted MR images will be obtained with parameters as described above i:rl Example 170 except that fat saturatiorl will be used also with imaging on a 1.5 Tesla magnet.
The resultmg MR images will show the polyps as high signal, contrast enhancing structures witbin a black background. Accordingly, improved c ul~J;cuily of the 30 polyps will be obtained. The resulting pattern of contrast will be useful for detectmg wogs/3~00~ 2191079 ~ .c-~gl ~

a variety of neoplasms arising throughout the GI tract. With genetic screening, this will be a useful surveillance technique for patients with oncogenes at risk for d~,loy~ ll of GI tract neoplasms.
F 176 _ _ A study similar to that conducted in Exarnple 175 will be repeated except that the patient will be ddllli~ d 0.010 millimoles/kg of a compound containing m~g?nr~r, for example, manganese dipyri-l- Y-' ~ (Nycomed, Oslo, Norway) instead of the gadolinium compoumd. As with the results obtained in Example 171, areas of abnormai mucosa throughout the GI tract will be readily 10 apparent as contrast enhancing lesions on MR.
F `-1 7 7 . _ ~
This example describes the use of the stabiliæd ~ of the present invention in connection with targeted, positive contrast agents.
A contrast agent comprising a targeted yalallla~ tic or radiodense 15 agent, for example, MnCI2 nanogels (100 mg) labelled with an antibody for ~;llO~ lyull;~ antigen, or barium sulfate particles (I g) labelled with endothelial growth factor, wili be adll~..l.~ d to a patient suffering from cancer of the Gl tract.
Twenty-four hours after ~ m of the aforesaid targeted contrast agent, the patient will be a illlihl...~ d 20 mL/kg of a stabilized suspension of the present 20 invention. CT or i~R imagimg will be perforrned. The bowel will appear black and an area of carcinoma will be detected within the bowel as a bright spot ~llll ;",l,.,~l upon the black background.
Tile disclosures of each patent, patent application and publication cited or described in this document are hereby ill~,Olyu~ i by reference, in their entirety.
Various ,.. ,.1;11. ~.;".. ~ of the invention, in addition to those describedherein, will be apparent to those skilled in the art from the foregoing ~Ir~rrirti~n Such ~ ".c are aiso intended to fall within the scope of the appended claims.

Claims

What is Claimed:
1. A stabilized, substantially homogenous suspension of a gas, wherein the suspension has a negative density of about -40 HU or less.
2. A suspension according to Claim 1 which has a negative density of about -50 HU or less.
3. A suspension according to Claim 2 which has a negative density of about -100 HU or less.
4. A suspension according to Claim 3 which has a negative density of about -500 HU or less.
5. A suspension according to Claim 1 which is stabilized with a stabilizing material.
6. A suspension according to Claim 5 wherein said stabilizing material comprises a surfactant.
7. A suspension according to Claim 6 wherein said surfactant is selected from the group consisting of anionic, cationic, zwitterionic and nonionic surfactants.

8. A suspension according to Claim 7 wherein said surfactant comprises a nonionic surfactant.
9. A suspension according to Claim 8 wherein said nonionic surfactant is selected from the group consisting of a polyoxyethylene-polyoxypropylene glycol block copolymer and a sorbitan fatty acid ester.

10. A suspension according to Claim 1 wherein said gas comprises a volatilized perfluorocarbon.
11. A suspension according to Claim 10 wherein said perfluorocarbon is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoropentane, perfluorohexane and perfluorooctane.
12. A suspension according to Claim 11 wherein said perfluorocarbon is selected from the group consisting of perfluoropentane, perfluorohexane and perfluorooctane.
13. A suspension according to Claim 12 wherein said perfluorocarbon comprises perfluoropentane.
14. A suspension according to Claim 1 further comprising a thickening agent.
15. A suspension according to Claim 14 wherein said thickening agent is selected from the group consisting of starches, gums, pectin, casein, agarose, gelatin, carrageenan and cellulose derivatives.
16. A suspension according to Claim 15 wherein said thickening agent is selected from the group consisting of gums, carrageenan and cellulose additives.
17. A suspension according to Claim 16 wherein said gum comprises xanthan gum.
18. A suspension according to Claim 16 wherein said cellulose derivative is selected from the group consisting of methyl cellulose and carboxymethyl cellulose.

19. A suspension according to Claim 18 wherein said cellulose derivative comprises methyl cellulose.
20. A suspension according to Claim 1 further comprising a dispersing agent.
21. A suspension according to Claim 20 wherein said dispersing agent comprises a polymeric siloxane compound.
22. A suspension according to Claim 21 wherein said polymeric siloxane compound is substantially completely alkylated with alkyl groups.
23. A suspension according to Claim 22 wherein said alkyl groups comprise lower alkyl groups.
24. A suspension according to Claim 23 wherein said lower alkyl groups are methyl groups.
25. A suspension according to Claim 21 wherein said polymeric siloxane compound is.alpha.-(trimethylsilyl)-.omega.-methylpoly[oxy(dimethylsilyleene)].
26. CANCELLED.
27. CANCELLED.
28. A suspension according to Claim 1 further comprising a compound selected from the group consisting of lipids, ingestible oils, viscosity modifiers, emulsifying and/or solubilizing agents, suspending or viscosity-increasing agents, synthetic suspending agents, and tonicity-raising agents.

29. A suspension according to Claim 1 wherein at least a portion of said gas is derived from a gaseous precursor.
30. A stabilized, substantially homogenous suspension of a gaseous precursor.
31. CANCELLED.
32. CANCELLED.
33. CANCELLED.
34. A suspension according to Claim 30 for use as a contrast medium for computed tomography or magnetic resonance.
35. A suspension according to Claim 34 for use as a contrast medium for computed tomography.
36. A contrast medium for computed tomography comprising a stabilized, substantially homogenous suspension of a gas.
37. A contrast medium according to Claim 36 wherein at least a portion of said gas is derived from a gaseous precursor.
38. CANCELLED.
39. CANCELLED.

40. CANCELLED.
41. A method for preparing a stabilized, substantially homogenous suspension of a gaseous precursor comprising agitating an aqueous mixture of a stabilizing material in the presence of the gaseous precursor.
42. CANCELLED.
43. CANCELLED.
44. A method according to Claim 41 further comprising agitating said aqueous mixture in the presence of a thickener.
45. A method according to Claim 41 further comprising agitating said aqueous mixture in the presence of a dispersing agent.
46. A method according to Claim 41 wherein said agitating comprises shaking or vortexing.
47. A method according to Claim 41 further comprising agitating said aqueous mixture in the presence of a gas.
48. A method for preparing a stabilized, substantially homogenous suspension of a gas for use as a computed tomography contrast agent comprising agitating an aqueous mixture of a stabilizing material in the presence of a gaseous precursor; and activating said gaseous precursor.

49. CANCELLED.
50. CANCELLED.
51. CANCELLED.
52. A method according to Claim 48 wherein said activating step comprises causing said gaseous precursor to undergo a phase transition from a liquid to a gas in vivo.
53. A method of providing an image of an internal region of a patient comprising (i) administering to the patient a contrast medium comprising a suspension according to Claim 1, and (ii) scanning the patient using computed tomography to obtain visible images of the region.
54. A method according to Claim 53 wherein the region comprises the vasculature region.
55. A method according to Claim 53 wherein the region comprises the cardiovascular region.
56. A method according to Claim 53 wherein the region comprises the gastrointestinal region.
57. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast medium comprising a suspension according to Claim 1, and (ii) scanning the patient using computed tomography to obtain visible images of any diseased tissue in the patient.

65. A method for preparing in a patient a contrast medium for computed tomography comprising (i) administering to the patient a stabilized, substantially homogenous suspension of a gaseous precursor, and (ii) allowing said gaseous precursor to undergo a phase transition from a liquid to a gas in vivo to provide the contrast medium.
66. A method according to Claim 41 wherein said agitating comprises homogenization.
67. A method according to Claim 48 wherein said agitating comprises homogenization.