CA2219528A1

CA2219528A1 - Stabilized compositions of fluorinated amphiphiles as contrast agents for ultrasound

Info

Publication number: CA2219528A1
Application number: CA002219528A
Authority: CA
Inventors: Evan C. Unger
Original assignee: Individual
Current assignee: ImaRx Pharmaceutical Corp
Priority date: 1995-06-06
Filing date: 1996-05-28
Publication date: 1996-12-12
Also published as: EP0831927A1; AU715681B2; JP2001517192A; AU5936096A; CN1185744A; EP0831927A4; WO1996039197A1; US5997898A

Abstract

Stabilized compositions comprising, in combination with a gas, a fluorinated amphiphilic compound. The compositions are particularly suitable for use in diagnostic applications, including ultrasound. The compositions can take the form of vesicular compositions, such as micelles and liposomes.

Description

W O 96/39197 PCT/U'~GJ~7813 STABn ~7.~1) COMPOSIIIONS OF FLUORINATED
AMPHIPHILES AS CONTRAST AGENTS FOR ULTRASOUND
Field of the Il-v_..tic The present invention relates to novel compositions for ulLLdsoulld.
S More particularly, the present invention relates to novel compo~itif n~ of fluol..l~l~d ~mphirhi1ic colllpc,ul.ds for use as cr~ntr~et agents for ulL-dsoulld.

Back~round of the T v~ n UlL.dsou,ld is a valuable ~ nostic im~ging technique for studying various areas of the body including, for r~ .lc the v~cnl~tllre~ such as tissue 10 microv~cc11l~tl1re. Ultrasound provides certain advantages relative to other rli~gn~stic techniques. For example, ~ gnostic techniques involving nuclear m.or1icinr and X-rays generally results in ~A~.)O~ , of the patient to ionizing electron radiation. Such radiation can cause ~l~m~ge to subcell~ r m~t.Qri~l, inr.lnfling deoxyribnnllrleic acid (DNA), ribonucleic acid (RNA) and proteins. UlLla3ound does not involve such lS potentially ~l~m~ging r~ tion In ~1rlition, ulL-asou-ld is relatively i~ ive as coll.pa.~d, for example, to co.ll~ul~d tomography (CT) and m~gn.q.tic l~so~ r-e im~ging (MRI), which require elaborate and cA~"~l,si~e eq11irm~ nt Ultrasound involves the ~AI_IO~ of a patient to sound waves.
~ Generally, the sound waves ~ ir~te due to absorption by body tissue, penetrate 20 through the tissue or reflect off of the tissue. The reflection of sound waves off of tissue, generally referred to as b~L ec~ . . or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues. This di~e.~ ial reflection is due to various factors, CA 022l9528 l997-l0-27 W O 96/39197 PCT~US96/07813 including, for example, the cn.~ nt~ and the density of the particular tissue being observed. The dirrt;.~;l,Lally reflected waves are ~ietecte~1 typically with a trzm~ Pr that can detect sound waves having a frequency of one m~g~h~rtz (MHz) to ten MHz.
The ~-tectecl waves can be h~Leg.dl~d, ~ 1 and co~ve,l~;d into an image of the 5 tissue being st ~
UlllasOul~d im~ging techniques typically involve the use of contrast agents. Contrast agents are used to hll~lov~ the quality and usefulness of images which are obtained via ultrasound. Exemplary c(~ dsl agents include, for example, suspensions of solid particles, ~mlll~ifi~l liquid droplets, and gas-filled bubbles. See, e.g, E~ilm~nn et al., U.S. Patent No. 4,466,442, and published Tnt~?m~ti~)nal Patent Applications WO 92/17212 and WO 92/21382.
The quality of images produced from ultrasound has hll~ov~d significantly. Nevertheless, further h~ rovement is n~e-lefl patticularly with respect to images involving v~c~ tllre in tissues that are perfused with a vascular blood 15 supply. Accordingly, there is a need for h~lplov~d ultrasound techniques, inrl~l-iing improved co~ d~l agents, which are capable of providing m.~ lly useful images ofthe vasculature and vascular-related organs.
The reflection of sound from a liquid-gas interface is extremely efficient. Accordingly, bubbles, in~ hllling gas-filled bubbles, are useful as contrast 20 agents. The term "bubbles", as used herein, refers to vesicles which are generally ch~,..~ 1 by the presence of one or more membranes or walls surrounding an intlqm~l void that is filled with a gas or a ~lecL~ ol thereto. Exemplary bubbles include, for ~Y;.",ple, liposomes, micelles and the like. As ~ cllcse~l more fully hereinarl~, the erre~liveness of bubbles as col,l.~wl agents ~ep~on-l~ upon various 25 factors, including, for .oY~mple, the size, elasticity and/or stability of the bubble.
With respect to the effect of bubble size, the signal that is reflected off of a bubble is a filn~tion of the radius (r6) of the bubble (Rayleigh Scatterer). Thus, a bubble having a fii~m~ter of 4 rnicrometer (~m) possesses about 64 times the scattering ability of a bubble having a diameter of 2 ~m. Thus, generally spe~king, 30 the larger the bubble, the greater the reflected signal.
However, bubble size is limited by the ~ meter of capillaries through which the bubbles must pass. Contrast agents which comprise bubbles having a CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 met~r of greater than 10 ,um are generally dangerous since mic,ove,sels may be occluded. Accordingly, it is desired that greater than about 99% of the bubbles in a colllldsl agent have a ~ m~ter of less than 10 ~Lm. Mean bubble diameter is important also, and should be greater than 1 ,um, with greater than 2 ~Lm being S plef~ d. The volume weighted mean diameter of the bubbles should be about 7 to 10 ~m.
Bubble elasticity is also important because highly elastic bubbles can deform, as necessary, to "squeeze" through c~pill~ries. This decreases the likelihood of occlusion. In ~ liti~n, rescn~nce is more easily in~ ce-l in bubbles having 10 enh~n~e-l elasticity. This can be advantageous in that rPson~ting bubbles typically generate sound emissions at frequencies in the sllbh~rmcIlic regime (based on multiples of 0.5) or in the supra- or ultr~h~rmonic regime (based on multiples of 2).
The supraharmonic regime, inchltling second h~rm( nic im~gin~, is desirable in ultrasound since background noise is ~,ulr.,~ lly elimin~teA Elastic bubbles can15 t_el~role be used to produce desirable second h~rmonic images.
The effectiveness of a contrast agent involving bubbles is also dependent on the bubble concentration. Generally, the higher the bubble concentration, the greater the reflectivity of the contrast agent.
Another important characteristic which is related to the effectiveness of 20 bubbles as contrast agents is bubble stability. As used herein, particularly with reference to gas-filled bubbles, "bubble stability" refers to the ability of bubbles to retain gas el~L,d~ped therein after exposure to a ~ie~,~,u,c; greater than atmospheric 7'7111e. To be ~rr~ ivt as collLld~,L agents, bubbles generally need to retain an amount of the c;llLlal)ped gas in vivo. It is also highly desirable that, after release of 25 the ~ ''7:~iUle~ the bubbles return to their original size. This is referred to generally as "bubble resilience."
Bubbles which lack desirable stability provide poor contrast agents. If, for example, bubbles release the gas e,.L,d~ped therein in vivo, reflectivity is~limini~hP~l Similarly, the size of bubbles which possess poor resilience will be 30 decreased in vivo, also reslllting in tlimini~h~l reflectivity.
The stability of bubbles disclosed in the prior art is generally inadequate for use as contrast agents. For example, the prior art discloses bubbles, CA 02219~28 1997-10-27 WO 96/39197 PCTrUS96/07813 including gas-filled liposomes, which comprise lipoidal walls or membranes. See,- e.g, Ryan et al., U.S. Patent Nos. 4,900,540 and 4,544,545, Tickner et al., U.S. Patent No. 4,276,885, Klaveness et al., WO 93/13809 and Schn~ er et al., EPO 0 554 213 and WO 91/15244. The stability ofthe bubbles ~ closecl inthese references ispoorS in that as the solutions in which the bubbles are suspended become ~lihlte~l, for example, in vivo, the walls or membranes of the bubbles are thinn~-1 This results in a greater lik~lihood of rupture of the bubbles.
Various ~lL~"~ have been made to improve bubble stability. Such ~lLe",~l~ have included, for example, the ~ lion of bubbles in which the 10 membranes or walls thereof are a~ lllly stren~h~n~?~l via cro~linking See, e.g, Giddey etal., U.S. PatentNo. 5,310,540 andKlaveness etal., WO 92/17212, in which there are disclosed bubbles which comprise proteins cro~link~cl with cro~linking agents.
Prior art techniques for stabilizing bubbles, including the use of 15 cro~linke~l mAt~riAl~, suffer from various drawbacks. For example, the cros~link~
mAteriAl~ described, for example, in Giddey et al., U.S. Patent No. 5,310,540 and Klaveness et al., WO 92/17212, lack bioc-....l.A~;bility or possess unknown metabolic fates. Added costs are also incurred with the use of additional mAtl~riAl~ and process steps n~ce~sAry for cro~linking In addition, cro~linking can impart rigidity to the 20 membranes or walls of the bubbles. This results in bubbles having reduced elasticity and, therefore, a decreased ability to deform and pass Llll~)u~ll capillaries. Thus, there is a greater likelihood of occlusion of vessels with prior art collL-~l agents that are stabilized via cro~linking Accol.ih~gly, new and/or better stabilized contr~t agents and methods 25 for providing same are n~e(le-1 The present invention is directed to this, as well as other, important ends.

Summarv of the Invention The present invention is directed, in part, to stabilized compositions of a gas and a sulfonated or phosphorylated fluorinated Amphirhi1ic compound.
30 Specifically, in one aspect, the present invention relates to a stabilized composition CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 comprising, in combination with a gas, a compound of the formula ~i Rll~C
[Rl--Xl]y--~z--Y--R3--Z
[Xi Rllz wherein~
each of x, y and z is independently 0 or 1;
each Xl is indep~n~l~ntly -O-, -S-, -SO-, -SO2-, -NR4-, -C(=X2)-, S -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-;
X2 is O or S;
Y is a direct bond or ~X3~M(=O)(ORs)q~O~~ where q is 1 or 2;
X3 is a direct bond or -O-;
M is P or S;
Z is hydrogen, the residue of a hydrophilic polymer, a sqc~hqride residue or~T(R6)r, ~ here r is 2 o}3;
each R1 is independently alkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons;
R2 is a direct bond or an alkylene linking group of 1 to about 10 1 5 carbons;
R3 is a direct bond or an alkylene diradical of 1 to about 10 carbons;
each of R4 and R5 is indep~.n-lently hydrogen or alkyl of 1 to about 8 carbons; and each R6 is indep~-n~ntly hydrogen, alkyl of 1 to about 8 carbons or a residue of a hydlol)hilic polyrner;
provided that at least one of x, y and z is 1, at least one of Rl is fluorinated alkyl of 1 to about 30 carbons and when R2 is a direct bond, at least two of x, y and z are 0. In certain embo-liment~, the composition may further comprise, as desired, a gaseous ~ 25 plciCul~Ol. Also, if desired, the composition may further comprise a bioactive agent.
Another aspect of the invention relates to compositions comprising, in combination with a gas, a compound of the formula W O 96/39197 PCTrUS96/07813 CnP2n+l--(CH2)m C(O)~
CnF2n+l--(CH2)m--C(O)O--OP(O2)O-(CH2)~ N (CH3)3 wherem:
m is 0 to about 18;
n is 1 to about 12; and w is 1 to about 8.
Yet another aspect of the invention relates to a method for the .~dlion of a stabilized con.~o:jiLion of a flllorin~t~d ~mphirhilic compound and a gas. The m~tho-1 compri~çs ~git~ting an aqueous llli~lUle of a flnorin~tçcl ~mphirhilic compound in the presence of a gas.
Still another aspect of the invention relates to a method of providing an image of an int~rn~l region of a patient. The method c~-mpri.~çs ~lmini~tçrin~ to the patient a contrast me~ m comprising a stabilized composition comprising a gas and a fluorinated ~mphirhilic compound. The method further comprises sc~nnin~ the patient using ultrasound to obtain visible images of the region.
Another aspect of the invention relates also to a method of providing an image of an int~rn~l region of a patient. This method colll~lises ~1minictçring to the patient a vesicular composition cnmpri~ing, in an aqueous carrier, vesicles comprising a fluorinated ~mphirhilic compound and a gas or gaseous precursor. The method also involves sc~nning the patient using ultrasound to obtain a visible image of any ç:~e~l tissue in the patient.
Yet another aspect of the invention relates to a method for diagnosing the presence of ~ e~cefl tissue in a patient. The method involves ~tlmini~ttoring to the patient a contrast medium comprising a stabili~d composition comprising a gas and a CA 02219~28 1997-10-27 fluorinated ~ ,hil)hilic compound. The method further involves sc~nning the patient using ultrasound to obtain visible images of any ~lice~ce~l tissue in the patient.
Still another aspect of the invention relates to a method for the therapeutic delivery in vivo of a bioactive agent. The method compricec S ~lmini.ctering to a patient a therapeutically t;rL-;liv~ amount of a form~ tion which comprises, in combination with a bioactive agent, a stabilized composition of a fluorinated ~mphirhilic compound and a gas.
These and other aspects of the invention will become more a~ e.lt from the present specification and claims.

10 Detailed De~ n of the Invention As employed above and throughout the disclosure, the following terms, unless other~-vise indicated, shall be understood to have the following m~ning.c"~mphirhile" or "~mphirhilic compound" refers to a synthetic or naturally-occurring compound having a water-soluble, hycllo~hilic portion and a 15 water-insoluble, hydlol)hobic portion. Pl~L.led il...l,hil-hilic compounds are characterized by a polar head group, for example, a phosphatidylcholine group, and one or more nonpolar, aliphatic chains, for example, palmitoyl groups. "Fluorinated ~..,l,hiphile" refers to an ~mphirhilic compound in which at least one hydrogen atom of the a ll~h~llilic compound is replaced with a fluorine atom. In p,~L,l~id form, the 20 fluorinated ~...l,hil)hiliC compounds are polyfluorinated. "Polyfl~lorin~t~cl", as used herein, refers to ~mphirhilic compounds which contain two or more fluorine atoms.
In certain preferred embo~liment~ of the present invention, the ~...l~h;l.l~ilic compounds comprise a lipid. "Lipid", as used herein, refers to m~t~ri~l~, including fats and fat-derived m~t~ri~lc, which are relatively insoluble in water but relatively soluble in 25 organic solvents, such as b~n7~ne, chloroform, acetone and ether. Lipids include, for example, fatty acids, fatty acid esters, neutral fats, phosphatides (phospholipids), glycolipids, fatty alcohols, sterols, waxes, terpenes and steroids. As discussed in detail below, certain plef~ ed lipid compounds are phosphorylated and contain phosphate groups, for example, P04 groups, and certain other p,efelied lipid 30 compounds are slllf~ted and contain sulfate groups, for exarnple, S04 groups.

CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 "Amrl~ hy" refers to the ~imlllt~n~ous attraction and repulsion in a single molecule or ion co..~ P one or more groups having an affinity for the phase or medium in which they are dissolved, emul~ified and/or suspended, together with one or more groups that tend to be expelled from the involved phase or me~ lm S "Stabilized" refers to colllpo~iLions which have been f~rmlll~te(l as a i~lule of finely divided colloidal particles floating in a liquid with minimsll aggregation. As discussed in detail below, certain ~ler~ d emborlim~nt~ of the present invention involve compositions of stabilized vesicles. In this context, the term "stabilized" refers to vesicles which are subst~nti~lly resistant to degradation that is caused, for exarnple, by the loss of structural or compositional integrity in the walls of the vesicles and/or by the loss of any ~i nific~nt portion of a gas or gaseous precursor enr~pslll~t~d within the vesicle.
"~mrhirhilic composition" refers to a composition which comrri~c an ~mphirhilic compound. Exemplary ~ )h;l~hilic compositions include suspensions, emulsions and vesicular compositi~)n~
"Suspension" refers to a llli~Lu~" dispersion or emulsion of finely divided colloidal particles flo~tin~ in a liquid. The particles may be solid, liquid or gaseous.
"Emulsion" refers to a llli~LLu~e of two or more liquids and is generally in the form of a colloidal llli~Lulc.
"Vesicle" refers to a spherical entity which is ch~~ e~ 1 by the presence of an int~rn~l void. Pl~;r~;lled vesicles are form~ t~l from ~...l,hil,hilic compounds, including lipids. In any given vesicle, the ~,..l,hil,hilic compounds may be in the form of a monolayer or bilayer, and the mono- or bilayer ;l...l~hil,hil~s may 25 be used to form one or more mono- or bilayers. In the case of more than one mono-or bilayer, the mono- or bilayers are generally concentric. The vesicles described herein include such entities commonly referred to as liposomes, micelles, bubbles, microbubbles, microspheres and the like. Thus, the ~mphirhilic compounds may be used to for n a nnil~mell~r vesicle (comprised of one monolayer or bilayer), an 30 oligol~m~ r vesicle (comprised of about two or about three monolayers or bilayers) or a multilamellar vesicle (comprised of more than about three monolayers or bilayers). The internal void of the vesicles may be filled with a liquid, including, for CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 example, an aqueous liquid, a gas, a gaseous ~ ,ul~ol, and/or a solid or solute m~teri~l, including, for example, a bioactive agent, as desired.
"Liposome" refers to a generally spherical cluster or ag~ g~le of ~mphirhilic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as vesicles or ~ )hil~hilic vesicles.
"Gas filled vesicles" refers to vesicles in which there is en~rs~ te~l a gas. "Gaseous precursor filled vesicles" refers to vesicles in which there is enc~rsi-l~te~l a ~,~seous pl~-;ul~ol-. The vesicles may be minim~lly, partially or subst~nti~lly completely filled with the gas and/or gaseous precursor. In ~,ef~ d embo-liment~, the vesicles are subst~nti~lly completely filled with the gas and/or gaseous precursor.
"Vesicular composition" refers to a composition of vesicles that is prepared from ~mphirhilic compounds.
".~mrhirhilic formulation" refers to a composition which comrri~es an ~mphirhilic compound and a bioactive agent.
"Vesicle formulation" refers to a composition which compri~es vesicles and a bioactive agent.
"Patient" refers to ~nim~le, including m~mm~l~, preferably hllm~n~
''Bioco.,.~ ihle~ refers to materials which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic responses and disease states.
"Bioactive agent" refers to a sl~hst~n~e which is used in connection with an application that is therapeutic or ~ nostic in nature, such as in methods for diagnosing the presence or absence of a disease in a patient and/or in methods for the tre~tment of disease in a patient. As used herein, "bioactive agent" refers also to substances which are capable of exerting a biological effect in vitro and/or in vivo.
The bioactive agents may be neutral or positively or negatively charged. Examples of suitable bioactive agents include diagnostic agents, ph~rm~ellticals, drugs, synthetic organic molecules, proteins, peptides, vitamins, steroids and genetic material, including nucleosides, nucleotides and polynucleotides.

CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 "Diagnostic agent" refers to any agent which is used in cnnnecti~n with methods for diagnosing the presence or absence of a disease in a patient. Exemplary tli~gnnstic agents include, for eY~mple, contrast agents for use in connection with ultrasound, m~gn~tic resonance im~ging or colll~uled tomography of a patient.
"Genetic m~tPri~l" refers generally to nucleotides and polynucleotides, including deoxyribonucleic acid (DNA) and ribonllr,leic acid (RNA). The genetic material may be made by ~ylllllclic ch~mic~l methodology known to one of c,l.lhl~.y skill in the art, or by the use of recombinant technology, or by a combination of the two. The DNA and RNA may optionally comprise ullll~lul~l nucleotides and may be single or double stranded. "Genetic m~t~ri~l" refers also to sense and anti-sense DNA
and RNA, that is, a nucleotide sequence which is cnmrlement~ry to a specific sequence of nucleotides in DNA and/or RNA.
"phz.. .~ rcuLical" or "drug" refers to any the.d~culic or prophylactic agent which is used in the tre~tment including the plcvclllion, diagnosis, alleviation, 15 or cure, of a malady, affliction, disease or injury in a patient. Thc,d~culically useful peptides, polypeptides and polynucleotides are included within the me~ning of the term ph~rm~relltir,~l or drug.
"Polymer", as used herein, refers to molecules formed from the chtomic~l union of two or more lc~Jc~ling units. Accordingly, included within the 20 term "polymer" are, for example, dimers, trimers and oligomers.
"Alirh~tic" refers to one of the major groups of organic compounds, char~cct~ri7~rl by straight- or branched-chain arrangement of the con~titl~nt carbon atoms. Aliphatic hydrocarbons c~mri~e three subgroups: (1) paldrruls~ including the ~lk~n~s; (2) olefins, inchlfling the ~lkene~, which contain carbon-carbon double bonds;
25 and (3) acetylenes, in~hl-1in~ the alkynes, which contain carbon-carbon triple bonds.
"Alkyl" refers to an aliphatic hydrocarbon group which may be straight or branched having l to about 30 carbon atoms. "Lower alkyl" refers to an alkyl group having l to about 8 carbon atoms. "Higher alkyl" refers to an alkyl group having about lO to about 30 carbon atoms. The alkyl group may be optionally 30 snbstitllt~-l with one or more alkyl group substituents which may be the same or different, where "alkyl group :jub~Liluent" includes, for example, halo, aryl, hydroxy, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, carboxy alkoxycarbonyl, oxo and CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 cycloalkyl. There may be optionally inserted along the alkyl group one or more oxygen, sulphur or sùb~LiluLed nitrogen atoms, w~llc~eill the nitrogen ~lb~ nt is lower alkyl. The alkyl group may be linear or br~n~.h~(l "Branched" refers to analkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is ~tt~-h~Cl 5 to a linear alkyl chain. The alkyl group can include one or more points of unsaturation including, for example, carbon-carbon double bonds and carbon-carbon triple bonds. Exemplary alkyl groups in-~.hlcle, for example, methyl, ethyl, i-propyl, n-butyl, t-butyl, n-pentyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pent~(lecyl and h~x~ cyl. Preferred alkyl groups include the lower alkyl 10 groups having 1 to about 4 carbon atoms and the higher alkyl groups having about 10 to about 16 carbon atoms. Fx~n pl~ry alkyl groups which contain alkyl group substituents include hydroxylated alkyl groups, such as alkyl groups derived from glycerol, including, for example, 2,3-dihyd.oxy~,.u~-1-yl.
"Alkylene", when used in conjunction with the term "diradical", refers 15 to a bivalent ~lirh~tic hydrocarbon group having from 1 to about 30 carbon atoms.
When used in conjunction with the term "linking group", "alkylene" refers to a bi- or trivalent ~liph~tic hydrocarbon group having from 1 to about 30 carbons. The alkylene group may be straight, branched or cyclic. The alkylene group may be also optionally unsalu dlt;d and/or sllbstit~lte~l with one or more "alkyl group ~ul~20 There may be optionally inserted along the alkylene group one or more oxygen,sulphur or sllbstilllte~l or ...~ nitrogen atoms, wherein the nitrogen substituent is alkyl as previously described. F.xempl~ry alkylene groups includemethylene (-CH2-), ethylene (-CH2CH2-), propylene (-(CH2)3-), prop-1,2,3-triyl (-CH2-CH(-)-CH2-), cyclohexylene (-C6HIo-),-CH=CH-CH=CH-, -CH=CH-CH2-, 25 -~C~n~CH2)m-, whc~ci~ n is an integer from about 1 to about 22 and m is an integer from 0 to about 22, -(CH2)n-N(R)-(CH2)m-, wherein each of m and n is independently an integer from 0 to about 30 and R is hydrogen or alkyl, methylenedioxy ~ (-O-CH2-O-) and ethylenedioxy (-O-(CH2)2-O-). It is ~crc~ d that the alkylene group has about 2 to about 3 carbon atoms.
The present invention is directed, in part, to stabilized compositions which are useful, for example, as cor.LI~l agents for diagnostic and/or therapeutic ulL.clsoulld. The compositions comprise, in combination with a gas and preferably in CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 an aqueous carrier, a flllorin~t~d ~mphirhilic compound. The flnc~rin~t~ mphirhilic compounds, which are described in detail below, impart highly desirable ~ ;.lies to the compositions of the present invention. For eY~mrle, it has been surprisingly and unexpectedly found that the fluorinated ~mphirhilic compounds are capable of 5 stabilizing the present compositions, inrl~l-ling ~cr~lcd compoeitinn~ which c~mpri~e vesicles. It has been found also that the present fl~lorin~t~ ,hiphilic compounds are capable of promoting the formation of vesicles, as well as hll~lvvillg the stability of the formed vesicles. In embo~lim~nt~ in which the vesicles comprise gas-filled and/or gaseous plc~iul~or-filled vesicles, the fl~lorin~te-l ~...pl)il)hilic compounds 10 enable the vesicles to ~ub~L~.Lially retain the gas and/or gaseous plc~ ol with minim~l loss or leakage. This is surprising and unexpected and generally rendersmn~cç~ ry the use of additional stabilizing m~t.?ri~l~, including, for example, surfactants, and stabilizing techniques, in-hlflinp, for example, cros~linking of the materials in the walls of the vesicles. As ~ cllc~e~l above, such techniques are15 generally nece~ in connection with collLld~L agents of the prior art. Moreover, the present fluorinated ~...l~hil~l~ilic compounds are generally bioc~ mp~tible and can be obtained with ...i..;,..~l effort and at minim~l c;~cl~e. Accordingly, the present invention is directed to simple and efficient methods for providing stabilized compositions, including vesicular compositions, for use as ultrasound contrast agents.
A variety of fl~lorin~terl ~mphirhilic compounds can be employed in the present compositions. Pl~,r~ d fluorinated ~ -hil-l-iliC compounds are thosewhich, when combined with a gas, tend to form stabilized compositions and/or vesicles. Preferred also are fluorinated ~...l~hil~hilic compounds which are capable of stabilizing the vesicles, once formed.
In preferred embo-limente the fluorinated ~mphirhilic compounds are based on ~mphirhilic compounds, including lipids, and especially phospholipids, which comprise a polar head group including, for example, a phosphorylated head group, such as a phosph~tidylcholine group, or a snlf~te(l head group, and at least one nonpolar aliphatic chain, such as a palmitoyl group. In such embo-1im~nt~, the fluorine atoms are preferably sll~stitnt~cl on the nonpolar aliphatic chain portions of the involved ~mphirhilic compounds. As noted above, among the preferred amphiphilic compounds are phosphorylated and/or sulfated lipid compounds. It is CA 02219~28 1997-10-27 WO 96/39197 PCTrUS96/07813 collle,lll)lated that the term "phosphorylate", as used herein, enco..,~ ;es rhosph~t~
groups with various valences, including, for example, PO3 and P04 groups. Similarly, ~ it is contemrl~ted that the term "s llf~tt-~l", as used herein, encomp~es sulfate groups with various v~lenre~, inelll~ing, for example, SO3 and S04. It is colllcll,~lated that 5 in these ~lcrel~cd lipid compounds, the phosphate group andlor the sulfate group are preferably located within the backbone portions of the lipid compounds. Thus, generally spe~king, the phosphate and/or sulfate groups in the plcrcll~,d phosphorylated andlor slllf~t~d lipid compounds are desirably spaced from the end-portions of the compounds with, for example, alkyl groups, and as such, are referred 10 to herein as "int.-rn~l phl sph~te (and/or sulfate) groups". These ~lcrcll~d fluorinated ~mphirhilic compounds can be cOllL d~led with prior art fluorinated compounds, including, for example, the class of compounds which are commercially available as ZONYL~ fluorosurf~ct~nt~ (DuPont Ch~mic~l Corp., Wilmington DE), including the ZONYLTU phosphate salts and the ZONYL~ sulfate salts, which have t~..,.l..~l 15 rhosph~te or sulfate groups. ReplcscllL~ es of these salts are disclosed, for example, in U.S. Patent No. 5,276,146, wherein the ZONYL~ phosphate salt has the formula [F(CF2CF2)3 8CH2CH20]l 2P(O)(O-NH4+)2, and the ZONYLDI sulfate salt has the formula F(CF2CF2)3 8CH2CH2SCH2CH2N+ (CH3)3 -OSO2OCH3. In contrast to the ~lcfellcd phosphorylated and sl-lf~te~1 lipid compounds involved in the present 20 invention, the ZONYL~ phosph~t~ and sulfate salts, as depicted above, includephosphate and sulfate moieties in the t~rmin~l portions of the disclosed compounds.
In certain ~lcrcllcd embo~ .k~ ;, the fluorinated ~mrhiphilic compounds are polyfluorinated. As noted above, this means that the amphiphilic compounds are preferably ~ with at least two or more fluorine atoms. In 25 even more plcrcllcd embo~1iment~, the fluorinated ~mrhiphilic compounds are perfluorinated.
As noted above, plcr~ d ~mrhiphilic compounds on which the fluorinated ~mphirhiles are based, generally comprise at least one nonpolar aliphatic chain. In certain ~,lef~ ;d embo-limentc, the ~mrhirhilic compounds comprise, at30 most, one nonpolar aliphatic chain. Such compounds are referred to herein as "monoch~in compounds." In certain other preferred embor1imentc, the amphiphilic compounds comprise more than one, and preferably at least two or three, nonpolar CA 02219~28 1997-10-27 ~lirh~tic chains. Such compounds are referred to herein as "polychain compounds."
Whether monochain or polychain, it is prerGllGd that the fluorine atoms are s~lbsti1~1te~ on the chain portion(s) of the ~.,.pl.il~hilic compounds. The extent of the fluorination of the chain(s), and the location of the fluorine atoms on the chain(s), can 5 be selected, as desired, in the flllorin~te~ ~mrhirhilic compounds. For example, only a certain portion of the nonpolar chain group, such as the termin~l methyl group(CH3-) or an intt-rn~l methylene group (-CH2-), can be fluorin~teA with the remz-inA.~r of the nonpolar chain group being, for ex~mrle, lln~b~ ed ~lirh~tic, such as un~ liLulGd alkyl. AlLGlll~LivGly, it is collLGlll~lated that a sllbst~nti~l majority of the 10 nonpolar chain group can be fluorinated. The nonpolar chain group can includesubst~nti~lly equivalent portions which are fluorinated and nonfluorin~teA as ~let~rmin~A, for t-x~mrle7 by the number of carbon atoms in the fluorinated and nonfluorinated portions. In pLGrGllGd form, the nonpolar chain group contains both a nonfluorinated portion and a fl~lorin~tt-A portion.
With particular reference to polychain compounds, it is conlG...... ~lated that all of the chains can be fl~lo~ A or only certain of the chains can be fluorinated. In the latter case, the nonfl~lo.h~lGd chain(s) can be, for ~Y~mrle, m~..l.slil~e(l aliphatic, including unsubstituted alkyl. Such compounds are referred to herein as "asymmetric ~...l~h;l.hilic compounds", since both fluorinated and 20 nonfluorinated chains are present in the same molecule.
In cc,.llle~lion with the plGr~ ,d embo-liment~ of the present invention involving mono- and polychain ~mrhirhilic compounds, the nonpolar aliphatic chains can be referred to as having a ~roxilllal end portion and a distal end portion. The hllal end portion collG~ollds to that portion of the aliphatic chain which is 25 proximate the polar head group. Thus, in the case of phospholipids, including, for example, phosphatidylcholines, such as mono- and Air~llll;Lc~ylpllosphatidylcholines~
the ~loxil~lal end portions of the palmitoyl moieties correspond to the portions of the chains which are located near the carbonyloxy (-C(=O)-O-) groups and the glycerolphosphocholine moiety. Similarly, the distal end portions cc ll. ;,~o..d to the 30 portions of the chains which are located near the terrninal methyl groups of the alkyl chains of the palmitoyl moieties.

WO 96/39197 PCTrUS96/07813 In ~cÇ~.~cd embo~ , at least the distal end portion of the nonpolar chain(s) of the mono- and polychain ~...pl,;~ ilic compounds is fluorinated, with polyfluorinated distal ends being more p~c;r~ed. Even more preferably, the distal end portions of the nonpolar chains are perfluorin~tç-l Also in ~ciÇ~cd 5 embo-limPnt~, the ~ xhllal end portion of the nonpolar chain portion(s) are ~."~ h~ erl z~liph~tic, with ..,.~.~b~ cl alkyl being more ~.efe~ d. Thus, particularly ~.er~ d embo-lim~nt~ of the invention involve fluorinated ~mphirhilic compounds in which one or more nonpolar aliphatic chains contain both a perfluorinated distal end portion and an ul~ul~sliLul~d proximal end portion.
In certain particularly p-er~.ed embo-lim~nt~, the fluorinated ...phil)hilic compound has the formula ~iRll,x [Rl--Xl]y-~IR2--Y R3 Z
[Xi Rl]Z

wherein:
each of x, y and z is indep~nrl~ntly O or l;
each X, is in-1~p~nrl~ntly -O-, -S-, -SO-, -SO2-, -NR4-, -C(=X2)-, 15 -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-;
X2 is O or S, Y is a direct bond or ~X3~M(=O)(OR5)q~0~~ where q is 1 or 2, X3 is a direct bond or -0-;
M is P or S;
Z is hydrogen, the residue of a hydrophilic polymer, a ch~ricle residue or -N(R6)r, where r is 2 or 3;
each Rl is indepçn~1~ntly aLkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons;
R2 is a direct bond or an alkylene linking group of 1 to about 10 25 carbons;

CA 02219~28 1997-10-27 W O 96/39197 PCT~US~G~ 13 R3 is a direct bond or an alkylene diradical of 1 to about 10 S;
each of R4 and Rs is independently hydrogen or alkyl of 1 to about 8 carbons, and each R6 is intlepçntl~ntly hydrogen, alkyl of 1 to about 8 carbons or a residue of a hy~Lophilic polymer, provided that at least one of x, y and z is 1, at least one of Rl is fluorinated alkyl of 1 to about 30 carbons, and when R2 is a direct bond, two of x, y and z are each 0.In the above formula (I), each of x, y and z is independently 0 or 1, provided that at least one of x, y and z is 1. In certain plr r~ d embo~1iment~, two of x, y and z are each 0. In certain other ~-~f~lled embo~iment~, one of x, y and z is 0 or 1 and the other t~vo of x, y and z are each 1, with one of x, y and z being 0 and the other t~,vo of x, y and z being 1 being more plcr~ d. In certain other plere.l~id embo~liment~, each of x, y and z is 1.
Each Xl in formula (I) above is indep~n(1~ntly -O-, -S-, -SO-, -SO2-, -NR4-, -C(=X2)-, -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-.
Preferably, each X~ is indep~nr~ntly -O-, -S-, -C(=X2)-, -C(=X2)-O-, -O-C(=X2)-,-C(=X2)-NR4- or -NR4-C(=X2)-. More preferably, each Xl is independently -C(=X2)-O- or -O-C(=X2)-. Even more preferably, each Xl is -C(---X2)-O-.
In the above formula (I), each X2 is O or S. Preferably, each X2 is O.
In formula (I) above, Y is a direct bond or ~X3~M(=O)(ORs)q~O~~ where q is 1 or 2. Preferably, Y is -XrM(=O)(ORs)q-O-M in the above d~finition of Y is P or S. Preferably, M is P.
X3 in the above definition of Y is a direct bond or -O-. Preferably, X3 is a direct bond.
In formula (I) above, Z is hydrogen, the residue of a hydrophilic polymer, a saccharide residue or -N(R6)r, where r is 2 or 3. In l.lef~ d embo-liment~, Z is ~N(R6)r.
Each Rl in formula (I) is indep~-n~ -ntly alkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons, provided that at least one of Rl is fluorinated alkyl of 1 to about 30 carbons. Thus, when only one of x, y and z is 1, Rl is n~ocec~rily fluorinated alkyl of 1 to about 30 carbons. In certain preferred CA 02219~28 1997-10-27 WO 96/39197 PCTrUS96/07813 embo~1imlont~, where one or none of x, y and z is 0, and preferably where one of x, y and z is 0 and the other two of x, y and z are each 1, at least one of Rl is alkyl of 1 to about 30 carbons and at least one of R, is fluorinated alkyl of 1 to about 30carbons. In other ~ler~.~d embo~lim~nt~ each Rl is independently fluorinated alkyl S of 1 to about 30 carbons. When fluorinated alkyl of 1 to about 30 carbons, Rl is preferably polyfluorinated alkyl of 1 to about 30 carbons, with perfluorinated alkyl of 1 to about 30 carbons being more ~l~r~ d. When perfluorinated alkyl of 1 to about 30 carbons, Rl is preferably CnF2n+l-(CH2)m-, where n is 1 to about 12 and m is 0 to about 18. In these latter embo~liment~, n is preferably about 2 to about 10 and m is preferably about 2 to about 14. More preferably, n is about 4 to about 8 and m is about 4 to about 10.
In formula (I) above, R2 is a direct bond or an alkylene linking group of 1 to about 10 carbons, provided that when R2 is a direct bond, two of x, y and z are each 0. Preferably, R2 is a direct bond or an alkylene linking group of 1 to about 4 C~bOllS. More preferably, R2 is an alkylene linking group of about 3 carbons.
Even more preferably, R2 is -CH2-CH(-)-CH2-.
R3 in formula (I) above is a direct bond or an alkylene diradical of 1 to about 10 carbons. Preferably, R3 is a direct bond or an alkylene diradical of 1 to about 4 carbons. More ~.ere dl~ly, R3 is an alkylene diradical of about 2 carbons.
Even more preferably, R3 is -CH2CH2-.
In formula (I) above, each of R4 and R5 is indeprn-lçntly hydrogen or alkyl of 1 to about 8 carbons. Preferably, each of R4 and R5 is independently hydrogen or alkyl of 1 to about 4 carbons. More preferably, each of R4 and R5 ishydrogen.
R6 in formula (I) above is hydrogen, alkyl of 1 to about 8 carbons or a residue of a hydrophilic polymer. Preferably, R6 is hydrogen or alkyl of 1 to about 4 carbons. More preferably, R6 is hydrogen or methyl, with methyl being even more ~f~lled.
In formula (I) above, it is intrn-lçd that when any symbol appears more than once in a particular formula or substituent, its mç~ning in each instance is independent of the other. This indeFt?n~lçnre of me~ning is subject to any of the stated provisos.

CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 Also in formula (I), it is intf~n~ 1 that when each of two or more .ent symbols is defined as being "a direct bond" to provide mnltiple, $~c~jzcPntdirect bonds, the multiple and ~ çPnt direct bonds devolve into a single direct bond As noted above, Z and R6 in the definition of Z in forrmll~ (I), can be, S inter alia, the residue of a hydrophilic polymer Fxempl~ry polymers from which Z
and/or R~ can be derived include polymers in which the r~e~Li,lg units contain one or more hy~ xy groups (polyhy~o~y polymers), inrhl-ling, for P~mple, poly(vinyl alcohol), polymers in which the ,~e~lh~g units contain one or more amino groups (polyamine polymers), including, for çx~mrle~ peptides, polypeptides, proteins and 10 lipc~ Leills, such as albumin and natural li~o~,Jlt;i,~, polymers in which the ~ e~Li"g units contain one or more carboxy groups (polycarboxy polymers), including, for ç r~mple, carboxymethylcellulose, alginic acid and salts thereof, such as sodium and calcium ~l~in~te~ glycosaminoglycans and salts thereof, in~ 1ing salts of hyaluronic acid, phosphorylated and sulfonated derivatives of carbohydrates, genetic 15 material, such as interleukin-2 and hlL. .r~O~I, and phosphorothioate oligomers; and polymers in which the .~e~li..g units contain one or more ~crh~ride moieties (polysaccharide polymers), including, for e~mple, carbohyd,dles The molecular weight of the polymers from which Z and/or R6 are derived may vary, and is generally about 50 to about 5,000,000, with polymers having a molecular weight of 20 about 100 to about 50,000 being plc:r~ed. More pler~,ed polymers have a molecular weight of about 150 to about 10,000, with molecular weights of 200 to about 8,000 being even more ~ ,r~ d Pl~rt"ed polymers from which Z and/or R6 are derived include, for example, poly(ethyl~"e~ lycol) (PEG), poly(~/i..yl~.y-lolidine), polyoxomer~, 25 poly~o~baLe and poly(vinyl alcohol), with PEG polymers being particularly preferred P~fe~cd among the PEG polymers are PEG polymers having a molecular weight of from about 100 to about 10,000 More preferably, the PEG polymers have a molecular weight of from about 200 to about 8,000, with PEG 2,000, PEG 5,000 andPEG 8,000, which have molecular weights of 2,000, 5,000 and 8,000, respectively,30 being even more l..er~.-ed Other suitable hydrophilic polymers, in addition to those exemplified above, will be readily ap~alc;nt to one skilled in the art based on the present CA 02219~28 1997-10-27 WO 96/39197 PCTrUS9G~'~7~13 disclosure. Generally, polymers from which Z and/or R~ are derived include polymers that can be incol~u.dled in the fluorinated ~ )hil.hilic compounds via alkylation or acylation reactions.
As with the various polymers exemplified above, it is cùnl~ lated that ~ S the polymeric residues can contain functional groups in addition, for example, to those typically involved in linking the polymeric residues to the fluorinated ~"~î~hi~ hilic compounds. Such functi~n~liti~s include, for example, carboxyl, amine, hydroxy and thiol groups. These functional groups on the polymeric residues can be further reacted, if desired, with materials which are generally reactive with such 10 functional groups and which can assist in L~ g specific tissues in the body including, for e~mple, ~ e~e~l tissue. Exemplary materials which can be reacted with the additional functional groups include, for example, proteins, including antibodies, carbohydrates, peptides, glycopeptides, glycolipids, lectins and nucleosides.
In addition to residues of hydrophilic polymers, Z in formula (I) can also be a saccharide residue. Exemplary s~cch~ricles from which Z can be derivedinclude, for ~mple, monosaccharides or sugar alcohols, such as ery~rose, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, m~nnitol and sedoheptulose, with ~ler~..cd monl s~r(h~rid~c being fructose, m~nnose, xylose, arabinose, m~nnitol and 20 sorbitol, and ~ crh~ride~ such as lactose, sucrose, maltose and cellobiose. Other saccharides include, for example, inositol and ganglioside head groups.
Other suitable s~crh~ridrs, in addition to those exemplified above, will be readily a~ ;llL to one skilled in the art based on the present disclosure.Generally, s~rrh~rides from which Z is derived include saccharides that can be 25 incorporated in the fluorinated amphiphilic compounds via alkylation or acylation reactions.
A particularly l,.ert;..ed class of fluorinated amphiphilic compounds of the present invention, which are within the scope of the compound of forrnula (I), is r~.cst;l,l~d by the formula CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 Cn~2n+ 1--(CH2)m--C(O) ~
C~2~1--(cH2)m--C(O)O--OP(02)0-(CH2)~ N (CEO3 (II) wherem:
m is 0 to about 18, n is 1 to about 12, and w is 1 to about 8.
The integer m in formula (II) is 0 to about 18. Preferably, m is about 2 to about 14. More preferably, m is about 4 to about 10.
In formula (II) above, n is 1 to about 12. Preferably, n is about 2 to about 10, with about 4 to about 8 being more ~fellcd.
The integer w in formula (II) is 1 to about 8. Preferably, w is 1 to about 4. More preferably, w is about 2.
The fluorinated ~mphirhilic compounds of the compositions of the present invention, in~ lin~ the compounds of formulas (I) and (II), can be prepared readily using standard organic synthetic methodology well known to those of ordil~dly skill in the art. Suitable methods for ~lc~dlhlg fluorinated ~...ph;l,hilic compounds 15 are disclosed, for example, in C. ~nt~ et al., New Journal of Chemistry, 15, 685 (1991), the disclosures of which are hereby incorporated by reference, in their enlilcLy. Exemplary of the available methods for ~ ~hlg fluorinated ~nnphiphiliccompounds are synthetic methods based on the phosphorylation of 1,2-di-(F-alkylacyl)-3-glycerol dclivdLives. These methods can be utilized in the plcl,~dlion of 20 perfluoroaLkyl phosphatidylcholines, and particularly perfluoroalkyl phosphatidylethanol~min~, and are disclosed, for example, in the aforementioned S~nt~ publication. Such methods involve linear phosphorylating agents, includingthe Hirt and Berchtold reagent (2-bromoethyldichlorop~osphate (BEDP)), which is readily available in large quantities. Hirt et al., Pharm. ~cta. Helv., 33, 349 (1958).

W O 96~9197 PCT~US96/07813 BEDP is highly reactive and can be used to phosphorylate st~ric~lly hindered disubstituted glycerols. Hansen et al., Lipids, 17, 453 (1982). The ammonium group can be introduced by reaction of an a~lop~;ate amine with the 1,2-diacylglycero-3-(2-bromoethyl)phosphate intermediate to give the desired phosphatidylethanolamine ~ 5 derivatives. This synthetic methodology is depicted in the following reaction scheme.
CnPm~(CH2)m--C(O)O H2, Pd/C, C~iF2~(cH2)m--C(O)O~
CnPm~(CH2)m--C(O)O~ CnP2n~(CH2)m--C(O)O--~I~ LCH2 ~ ~n~ -OH
1)B~H~OP(O)CI2,NE~3 2) H20 C~F2~(CH2)m--C(O)O-- 3) NMe3, A&C03 C~F2n~(CH2)m--C(O)O--- OP(Oi)O-(CH~2- N(CH3)3 (v) where m and n are as previously ~l~fine~l The 1,2-disubstituted-3-benzylglycerol derivatives of formula (III) are readily synth~ci7~-1 in high yields (85 to 90%) by acylation of l-benzylglycerol with the corresponding perfluoroalkanoyl h~ lçs Proc. Nafl. Acad. Sci. USA, 75, 4074 10 (1978). The benzyl protecting group can be removed by hydrogenolysis over a palladium on charcoal catalyst (Pd/C) in tetrahydrofuran (THF). Proc. Natl. Acad.
Sci. USA, 75, 4074 (1978). Short reaction times for the hydrogenolysis of the benzyl group are ~-efel.~d to avoid L~ e~le.;l~ication of the 1,2-diacylglycerol of formula (III) into the more thermodynamically stable 1,3-diacylglycerol isomer. The 15 hydrogenolysis reaction can be monitored by ordhl~ analytical techniques, including, for example, thin layer chlolllaLography (TLC) and proton (lH) nuclear m~gn~tic lesonallce (NMR). The reaction is generally complete in about one hour with little or no ~ ç~ cation. The hydrogenolysis reaction is preferably con~ ctecl in THF because both the starting material in the involved reaction (the 20 compound of formula (III)) and the product (the compound of formula (IV)) tend to be highly soluble in THF. In addition, THF is conveniently used as the solvent in the subsequent phosphQrylation step.
After hydrogenolysis, the catalyst (Pd/C) can be removed by filtration.
The 1,2-diacylglycerols of formula (IV) can be reacted immediately with BEDP and W O 96/39197 PCT~US96/07813 an excess of triethylamine. Phosphorylation is typically completed in about 2 toabout 4 hours, as measured by TLC. The r~ phosphochloride bond can be hydrolysed in aqueous base and generally ~ uil.~s about 22 hours for completion.When min.or~l bases or salts are used for hydrolysis including, for example, Na2CO3, 5 KCl and EDTA sodium salt, the rhosph~te salts are highly insoluble in water or organic solvents. Fleiccher, Methods Enzymol. 98, 263 (1983). The b~
intermetli~te can be isolated as a stable and soluble hydrogenotriethylammonium salt if excess triethylamine is used. Acidification of the phosph~te salts to form the corresponding acid is difficult becduse the glycerol ester bonds are hydrolyzed at the 10 n~ce~ix~.~, pH (pH of 2 to 3). Product degradation occurs also during purification over silica gel. Accordingly, it is pl~r~lled to use the phosphate salt without further purification. Nucleophilic displ~c~m~nt of the bromide ion by a large excess of trimethylamine occurs in a solvent l~ LLILe of CHCl3/CH3CN at 45~C over a 12 hour period. The ~lixpl~ce~l bromide ion can be ~recipildled by the addition of silver 15 carbonate.
The c~nc~-ntr~tion of flllorin~ted ~ )hiphilic compound in the present compositions can vary and depends upon various factors, in~hl-ling, for example, the particular ~ ,h;l,hilic compound(s) which are employed in the compositions.
Toxicity is ~ner~lly of limited concern since the present fl~lcrin~tecl ~mrhirhilir 20 conl~oullds are :iu~s~ y bioco...l,~l;hle. See C. ~nt~ et al., New Journal ofChemistry, 15, 685 (1991). In general, the col-r~ .,,l;on of fluorinated ~mrhirhilic compound in the present compositions is from about 0.001 mg/mL to about 200 mg/mL, with a concentration of about 0.01 mg/mL to 20 mg/mL being pl.r~ d.
More p~c;r~.dbly, the concPntration of fluorinated ~mrhiphilic co",~ou"d is about 0.05 25 mg/mL to about 10 mg/mL, with a conc~"L-dLion of about 0.1 mg/mL to about 5 mg/mL being even more ~lcr~ d.
The stabilized compositions of the present invention also comprise a gas, and preferably, an inert gas. The gases provide the compositions with enhanced reflectivity, particularly in vesicular composition in which the gas is entrapped within 30 the vesicles. This increases their effectiveness as co"L.~ agents.
P,ef~"ed gases are gases which are inere and which are biocompatible.
Preferable gases include those selected from the group concicting of air, noble gases, WO 96/39197 PCT~US96/07813 such as helium, neon, argon and xenon, carbon dioxide, nitrogen, fluorine, oxygen, sulfur hexafluoride, fluorocarbons, perfluorocarbons, and ~ Lules thereof.
Perfluorocarbons are ~,cfcll~d gases. Preferably, the perfluorocarbon is selected from the group c-ncictin~ of perfluornmeth~n~, perfluoroethane, perfluc.l~l~alle, ~ 5 perfluorobutane, perfluorocyclobutane and mi~Lu,~s thereof. More preferably, the perfluorocarbon gas is selected from the group concictin~ of perfluolu~lu,u~e and perfluorobutane, with perfluoru~r~le being particularly plefcll~d. Another preferable gas is sulfur tetrafluoride.
It is collLelllplated that l~ LulcS of dirr~ L types of gases, including llli~Lules of a perfluorocarbon gas and another type of gas, such as air, can also be used in the compositions of the present invention. The gases ~1iccllcce-1 in Quay, Tntt?rn~tional Application WO 93/05819, including the high "Q" factor gases described therein, may be used also. The disclosures of Quay, T..~ ional Application WO
93/05819 are incol~oldLcd herein by ,erelG"ce in their ellLhcLy~ In addition, par~m~gn~tic gases and gases of isotopes, such as 17O, may be used. Other gases,including the gases exemplified above, would be readily ~pa,ellt to one skilled in the art based on the present ~lieslos~lre. The gases can be selected, as desired, to provide compositions which are suitable for use as cOllLlasL agents in ulLldsou,ld, as well as other ~ gnostic techniques, such as cull,~uLcd tomography (CT) and m~gn~tic reeon~nrc im~ging (MRI).
In certain prer~l~cd embo~1imlonte~ a gas, for example, air or a perfluorocarbon gas, is combined with a liquid ~clfluolocarbon, such as perfluorohloY~n~, p~orflnoroheptane, perfluorooctylbromide (PFOB), perfluoro~l~ç~lin, perfluorododec~lin, perfluorooctyliodide, perfluoiotLip.o~ylamine and ~,lfluoruLlibutylamine.
It may also be desirable to il~ol~ e in the stabilized compositions a e~;u~.,or to a gaseous sl-hst~nl~e. Such precursors include materials that are capable of being converted to a gas in vivo. Preferably, the gaseous precursor is biocompalible, and the gas produced in vivo is biocomp~tible also.
A variety of gaseous ~lCCUl.7ClS are available for use in the present compositions including, for example, substances which are sensitive to changes to pH.
These snhst~n~es include materials that are capable of evolving gas, for example, CA 022l9528 l997-l0-27 W O 96/39197 PCT~US96/07813 upon exposure to a pH that is sllhst~nti~lly neutral or acidic. F.~mplçs of such pH
sellsilive substances include salts of an acid s~lecte-l from the group concictin~ of inorganic acids, organic acids and llli2~Lulcs thereof. Carbonic acid (H2C03) is an mrle of a suitable inorganic acid, and ~minnm~ nic acid is an example of a S suitable organic acid. Other acids, in~ fling inorganic and organic acids, would be readily d~ ll to one skilled in the art based on the present disclosure.
Preferably, the gaseous ~Ic;cul~or is a salt which is selected from the group concicting of an alkali metal salt, an ~mmonium salt and nli~Lulcs thereof.
More preferably, the salt is selected from the group c-)ncictin~ of c~lJoll-dt~,10 bicarbonate, sesquec~bu..d~ min~m~lon~t~ and llli~Lulcs thereof.
Exemplary of gaseous precursor m~tt-ri~lc for use in the compositionc of the present invention and which are salts include lithium carbonate, sodium carbonate, potassium carbonate, lithium bic~bolldL~, sodium bicarbonate, potassium bic~bolldLe, m~gn~cium c~ubolldl~;, calcium carbonate, m~gn~cillm bicarbonate, 15 ~mmnnium carbonate, ~mmonillm bic~hbolldl~, ammonium sesquecarbonate, sodium sesquec~l,olldLe, sodium aminomalonate and z~mmonium aminomz~l~n~te Aminom~lnn~t~ is well known in the art, and its ~ udLion is described, for example, in Th~n~cci, Biochemistry, Vol. 9, no. 3,pp. 525-532 (1970); Fit7I~trick et al., Inorganic Chemisfry, Vol. 13, no. 3 pp. 568-574 (1974), and Stelm~ch~k et al., Koordinatsionnaya Khimiya, Vol. 3, no. 4,pp. 524-527 (1977). The disclosures of these publications are hereby incol~uldLed herein by ler~.ence.
In addition to, or instead of, being s~l~iLivc to changes in pH, the gaseous precursor materials may also c-mrrice compounds which are sensitive to changes in temperature. Such telll~.dLule sensitive agents include, for example,m~teri~lc which have a boiling point of greater than about 37~C at atmospheric ples~ule. For example, perfluolu~ellL~le has a liquid/gas phase transition tenl~;ldLu (boiling point) of 29.5~C at ttmcsph~ric ~Jlei~ulc. This means that perfluoropentane will be a liquid at room ~em~ldL:lle (about 25~C), but will become a gas within the human body, the normal ttlllpcldLLllc of which is 37~C, which is above the transition t~lllpeldLIlre of perfluorupellL~e. Thus, under normal circ-lmct~nces, perfluoro~llt~le is a gaseous precursor. As a further example, there are the homologs of perfluolo~ e, namely perfluorobutane and perfluorohexane. The liquid/gas W O 96/39197 PCTrUS96107813 transition of perfluorobutane is 4~C and that of perfluor~-h~ne is 57~C. Thus, perfluorobutane is potentially useful as a gaseous plc.,ulsol, although more likely as a gas, wh~leas perfluorohexane would likely be useful as a gaseous precursor because of its relatively high boiling point.
S As discussed below, compositions co.. l;.;.. i~-g gaseous pl~ olS, such as perfluoropell~le and/or perfluor-)h~Y~n~, can be ~ d, for ç~mrle, by agitating in a suitable cont~in~r, such as a vial, an aqueous l~ Lule of a fluorinated z3mrhirhilic compound and a gaseous p.e~ or at standard telll~eldlule and pressure.
Under these conditions, the gaseous precursor would be in the form of a liquid.
10 Alternatively, compositions cn..~;1;.,;..~ gaseous pl~e~ ol~ can be pl'~ d at elevated t~ ;ldlule and ~tmospht~ric Pre~U1e in which case the gaseous plecul~ol can be vol~tili7~1 Thus, for example, when a composition of a fluorinated ~mrhirhile and perfluol~op~ e is ~ .~ed, for example, at a ten~eld~ule of about 35~C to about 40~C, the perfluol~l.cll~le is gaseous. The perfluolopt;ll~le would occupy the head 15 space of the collk~ in which the composition is ~le~ed.
In addition to elevated tt;lll~.,.d~ule, s...l)hil~hilic compositions CC~ g gaseous ~lC~ Ol:i can be ~l~a,ed at about room lelll~)~,.dlUIe but at a reduced ple~ e. As known to those of ordill~ skill in the art, the boiling point of various of the gaseous ple~ ols described herein, including the perfluorocarbons, 20 such as perfluol~o~ e and perfluoroh~n~ is lowered to about room lelll~.dlu.e or less under reduced pies~-l-e. Thus, gaseous ple~ ol~ can be hlcol~uldl~;d into the present s....ph;l.hilic compositions as a gas by ~ ~illg the compositions at a reduced plcs~ . The vol~tili7~1 gaseous p.~ or would then occupy the head space of the container in which the composition is prepared.
A wide variety of m~t~ri~l~ which are sensitive to changes in t~ .dLule can be used as gaseous precursors in the compositions of the present invention. Suitable gaseous precursors include, for example, hexafluoroacetone, isopropyl acetylene, allene, tetrafluoroallene, boron trifluoride, 1,2-butadiene, 2,3-bllt~ n.o, 1,3-butadiene, 1,2,3-trichloro-2-fluoro-1,3-butadiene, 2-methyl-1,3-butadiene, hexafluoro-1,3-butadiene, butadiyne, l-fluorobutane, 2-methylbutane, perfluorobutane, l-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1-butene, perfluoro-l-butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne, CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 butyl nitrate, l-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-hexafluorobutyne, 3-methyl-1-butyne, perfluoro-2-butyne, 2-bromo-butyraldehyde, c~lo.lyl sulfide, crotononitrile, cyclobutane, methyl-cyclobutane, octafluorocyclobutane, perfluorocyclobutene, 3-ch orocyclo~ lcne, perfluorocyclc,~c"l~,e, octafluorocyclo~enlt"e, cyclo~io~u,e, S perfluorocyclo~.u~alle, 1,2-dimethyl-cyclo~ol)a-le, 1,1-dimethylcyclop.u~ e, 1,2-di-methylcyclo~,u~ e, ethylcycloprù~le, methylcycloprù~le, diacetylene, 3-ethyl-3-methyl rli~7iritlinP, l,l,l-trifluorodiazoethane, dimethyl amine, hexafluorodimethyl-amine, dimethylethylamine, bis-~dimethylphosphine)amine, perfluorohex: nP, perfluoroheptane, perfluorooctane, 2,3-dimethyl-2-norbornane, perfluorodimethyl-10 amine, dimethyloxonium chl~ ride, 1,3-dioxolane-2-one, 4-methyl-1,1,1,2-tetrafluoro-ethane, 1,1,1-trifluoroethane, 1,1,2,2-tetrafluoroeth~nP; 1,1,2-trichloro-1,2,2-trifluoroethane, 1,1 -dichloroethane, 1,1 -dichloro- 1 ,2,2,2-tetrafluoroethane,1 ,2-difluoroethane, 1 -chloro- 1,1 ,2~2~2-pe~ uoroethane, 2-chloro- 1,1 -difluoroethane, 1,1-dichloro-2-fluoroethane, 1-chloro-1,1,2,2-tetrafluoroethane, 2-chloro-1,1-15 difluoroethane, chloroethane, chlor~e, ~ oroethane, dichloloL-;fluoroethane, fluoroethane, perfluoroethane, llillu~ n-lQroeth~nP~7 nitrosope..l~[luoroethane,perfluoroethylamine, ethyl vinyl ether, l,l-dichloroethane, l,l-dichloro-1,2-difluoroethane, 1,2-difluoroethane, methane, trifluorometh~npslllfonylchloride~
trifluoromP.th~n~slllro"ymuoride, bromodifluorûl il~usometh:~np~ bromofluoromP.thslne, 20 bromochlorofluoromPth~nP, bromotrifluor~ mPth~n~, chlorodifluorol,illul..ethane, chlorodh~ o~Pth~nP~ chlorofluoromethane, chlolollinuoromPth~n~7 chlorodifluoro-methane, dibromodifluor~mP~th~nP, dichlorodifluor )mP,thzmP dichlorofluor~ meth~nP, difluor-methzmP, difluoroiodompthzmp~ n~methane~ fluor~ mP,th~ne, iot10mP,thsm~7 iodotrifluoromPth~nP, ~uL-oL-ifluoromethane, nitrosotrifluoromPth~ne, tetrafluoro-25 mPth~n~, trichlorofluoromethane, trifluoromethane, 2-methylbutane, methyl ether, methyl iso~lu~yl ether, methyll~t~t~tP, melhylllil.ile~ methyl~nlfifle, methyl vinyl ether, neo~.~"l;."~, nitrous oxide, 1~2~3-non~dec~ne-tricarboxylic acid-2-hyd.cxyL~i...ethyl-ester, l-nonene-3-yne, 1,4-pentadiene, n-pentane, perfluoro~c--~le, 4-amino-4-l--clhyl~entan-2-one, l-pentene, 2-pentene (cis), 2-pentene (trans), 3-bromopent-1-ene, 30 ~ .nuù~u~ellt-l-ene, tetrachlorophthalic acid, 2,3,6-trimethylpiperidine, propane, 1,1,1,2,2,3-hexafluo.Oplu~ le~ 1,2-epoxypropane, 2,2-difluolOplu~ane~ 2-amino-WO 96/39197 PCTAUS9C~7~13 propane, 2-chloru~lu~ e~ heptafluoro-l-niL.u~lopalle, heptafluoro-l-nitrosc,~,lu~e, perfluoru~lo~le, propene, h~-Y~fll~olu~lupdlle, 1,1,1,2,3,3-hexafluoro-2,3-dichloro-propane, 1-chlol~ro~ e, chloro~luy~le-(trans), 2-chlolo~lu~le, 3-fluorc,~lùp~e, plo,uylle~ 3,3,3-trifluorù~,lo~ylle, 3-fluorostyrene, sulfur (di)--lec~flll-)ride (S2Flo), 5 2,4-~ minotoluene, trifluoro~cetonitrile7 trifluoromethyl peroxide, trifluoromethyl sulfide, tlln_.eten hexafluoride, vinyl acetylene and vinyl ether. FY~mples of gaseous ,Ul~iUl;:i are generally described, for ex~mrle, in U.S. Patent Nos. 5,088,499 and 5,149,319, the disclosures of which are hereby incorporated herein by reference in their ellLil~,ly.
Perfluorocarbons are pler~ d g~seous ~lle~,Ul:iOl:j for use in the compositions of the present invention. Exemplary of perfluorocarbon gaseous ,Ule~;Ul:jOl~ are perfluorocarbons selected from the group coneietinp of perfluolopt;llL~le, perfluorohPY~n~, perfluoroheptane, perfluorooctane and perfluorononane. Preferably, the perfluorocarbon gaseous precursor is selected from 15 the group coneieting of perfluoro~cilll~le, perfluoroh~Y~ne and perfluorooctane, with perfluoropentane being particularly pl~er~ d.
The gaseous ple~ul~or m~t~ri~le may be also photoactivated materials, such as dia~ollium ion and ~minnm~lonate. As ~ieclleeed more fully hel~i~r~
certain stabilized compositions, and particularly vesicular compositions, may be20 form~ te-1 so that gas is formed at the target tissue or by the action of sound on the particle. Other gaseous precnrsor.s, in ~cl-liti~ n to those exemplified above, will be a~pa~c;llL to one skilled in the art based on the present disclosure.
As noted above, certain ~l~;rt;.l.,d embo-limente of the present invention involve vesicular compositions. The si~ of the vesicles can be adjusted, if desired, 25 by a variety of procedures including, for example, micro~mllleification, vortexing, extrusion, filtration, sonic~tion~ homoge i~lion, repeated cycles of freezing and thawing, extrusion under ~ie~ through pores of defined size, and similar methods.
For intravascular use, the vesicles preferably have diameters of less than about 30 ~m, and more preferably, less than about 12 ~Lm. For targeted 30 intravascular use including, for example, binding to certain tissue, such as cancerous tissue, the vesicles can be significantly smaller, for example, less than 100 nm in metcr. For enteric or g~L-oi~ use, the vesicles can be significantly larger, W O 96/39197 PCTrU~GJ~/~13 for example, up to a millim(?ter in size. Preferably, the vesicles are sized to have diameters between about 20 ~Lm and 100 ~m.
Tabulated below is a listing of a series of gaseous precursors which undergo phase tr~n~iti-n~ from liquid to gas at relatively close to normal human body S temperature (37~C) or below. Also listed in the table are the sizes, in diameter, of emulsified droplets that would be required to form a vesicle of a ..li1x;lllllll, size of about lO ,um.
TABLE I
Physical Characl~l;.Aics of Gaseous I~e~ 2j and D ~ le~ of F.. l~;r-tl Droplet to Form a 10 ~m Vesicle Diameter (~m) of Pm~ ifiP~l droplet MolecularBoiling Point to make 10 micron Co~ uulld Weight (~C) Density vesicle perfluoro- 288.04 29.5 1.7326 2.9 pt;ll~ e 1- 76.11 32.5 6.7789 1.2 15fluorobutane 2-methyl- ~72.15 27.8 0.6201 2.6 butane (isopentane) 2-methyl-1- 70.13 31.2 0.6504 2.5 20butene 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 253-methyl-1- 68.12 29.5 0.6660 2.5 butyne oct~ oro- 200.04 -5.8 1.48 2.8 cyclobutane ~e~ ~fl~oro- 238.04 -2 1.517 3.0 30butane hP~c~fllloro- 138.01 -78.1 1.607 2.7 ethane CA 02219~28 1997-10-27 WO 96/39197 PCT~US96/07813 *Source: Ch~m~ Rubber C~ ally Handbook of Chemistry and Physics Robert C.
Weast and David R. Lide, eds. CRC Press, Inc. Boca Raton, Florida. (1989 - l99O).
The utility of the compo~itionc of the present invention can be op~ cl, for example, by using gases of limited solubility. T.imh~-l solubility, as 5 used herein, refers to the ability of the gas to diffuse, for example, out of vesicles by virtue of its solubility in the sullou,lding aqueous m~ m A greater solubility in the aqueous mto~ lm imposes a gradient with the gas in the vesicle such that the gas will have a tçntl~on~y to diffuse out of the vesicle. A lesser solubility in the aqueous m~r~ m will decrease the gra~ nt b~;Lwt:ell the vesicle and the int~ re such that the 10 diffusion of the gas out of the vesicle will be impe~1ç~1 Preferably, the gas e"Ll~ped in the vesicle has a solubility less than that of oxygen, namely, 1 part gas in 32 parts water. See Matheson Gas Data Book, Matheson Co~ ally, Inc. (1966). More preferably, the gas enLld~ed in the vesicle possesses a solubility in water less than that of air; and even more preferably, the gas e"L~ped in the vesicle poc~.~sses a solubility 15 in water less than that of nitrogen.
The gases and/or gaseous ~ ,Ul;!iOl~ are preferably incorporated in the stabilized collll,o~iLions of the present invention irrespective of the physical nature of the composition. Thus, it is contemplated that the gases and/or precursors thereto are incorporated in compositions in which the fluorinated amphiphilic compounds are ag~ lcgaled, for example, sllbst~nti~lly r~n~ mly, as well as compositions in which the fluoli"a~t:d amphirhilie co",l uul,ds form vesicles, in~ ing mi~çlles and liposomes.
Incc,~olalion of the gases and/or gaseous pl'e~;Ul:iOl:!i in the present compositions may be achieved by using any of a number of methods. For example, the formation of gas-filled vesicles can be achieved by .~h~king or otherwise ~git~ting an aqueous llli~t 25 which colll~lises a gas or gaseous ~lc~;ul~ol and the fluorinated ~mphirhili~coul~oullds~ This promotes the form~tion of stabilized vesicles within which the gas or gaseous ple-;ul~ol is enc~ tt--l In addition, a gas may be bubbled directly into an aqueous llli~lUle of the fluorinated amphiphilic compounds. ~lt~orn~tively, a gas in~till~tion method can be 30 used as disclosed, for example, in U.S. Patent Nos. 5,352,435 and 5,228,446, the disclosures of each of which are hereby incorporated herein by l~fele,lce in their entirety. Suitable methods for incorporating the gas or gaseous precursor in the present compositions are disclosed also in U.S. Patent No. 4,865,836, the disclosures of which are hereby incul~uldLcd herein by lcrclcnce. Other mPfhn~lc would be a~al~ to one skilled in the art based on the present disclosure.
In pler~.lcd embo~1imP-ntc, the gases and/or gaseous ~lC~;Ul~or ~ lc 5 are incc,l~uldkd in vesir~ r compositions, with mirelles and liposomes being pl~er~,~lcd. As ~ e(l in detail below, vesicles in which a gas or gas ~lec~ul~or or bûth are el-c~ ted are advantageous in that they provide hll~lovcd reflectivity in vivo.
In certain embo-l;...~ , the stabilized compositions of the present 10 invention can further colll~lise one or more additional ~mrhirhilic materials. Due to the ~ )h;l.,.lhy of these additional alll~h;l.hilir m~tPri~lc, they are generally capable, in ~lefellcd form, of e..l1~l)ri.~g or otherwise illl~ Vill~ the stability of the present colll~o~ilions. It has been found that hlcol~uldling one or more additional amphiphilic materials is particularly advantageous in connection with compositions involving15 asymmPtric fluorinated amphiphilic colll~.~ullds. Particularly ~lcr~ ,d additional amphiphilic m~tPri~lc include compounds which are biocn~ a~ le and which are capable of promoting the formation of vesicles, and Pspeci~lly stabilized vesicles, in the present stabilized compositions.
In certain ~lcfellcd embo~ , the additional amphirhilir- materials 20 colll~lise a lipid compound. Suitable lipids include, for example, phospholipids, such as phnsph~ti-1ylcholine with both ~ rd and ~ ed fatty acids, inrhl~ling dioleoylphosphatidylcholine, diulyli~l~yl~hosphatidylcholine, ~lir~h~ oyl~hosphatidyl-choline and di~Lal~yl~hosphatidylrllnlinP; rhnsph~tillylethanol~minPs, such as ~iirz~h~iloyll~hnsph~ti~lylethanolamine~ dioleoylrhncph~ti~;ylethanolamine~ N-succinyl-25 dioleoylphosphatidylethanolamine and 1-hPx~d~Pcyl-2-palmitoylgly-;cluphosphoethanol-amine; phosphatidylserine; phosphatidylglycerol; sphingolipids; glycolipids, such as ganglioside GMl; glucolipids; s~llf~ti-le~ glycosphingolipids; phosphatidic acids, such as ~lir~lmitQlylphosph~ti-lic acid; p~lmitic acid; stearic acid; ar~rhi~lonic acid; oleic acid; lipids bearing polymers, such as polyethyleneglycol or polyvinylpyrrolidone;
30 cholesterol and cholesterol hPmi~l~ccinate; 12-(((7'-diethylaminocoumarin-3-yl)-carbonyl)methylamino)oct~-lec~nnic acid; N-tl2-(((7'-diethylaminocoumarin-3-yl)-carbonyl)methylamino)oct~lec~noyl]-2-aminop~lmitir acid; cholesteryl-(4'-trimethyl-CA 02219~28 1997-10-27 amino)b~ ..n~r; 1,2-dioleoyl-sn-glycerol; 1,2--1ir~lmitoyl-sn-3-succinylglycerol; 1,3-ir~lmitoyl-2-succinylglycerol; and palmitoylhomo-;y~ltille.
Suitable additional amphirhilir m~t~ri~l~ include also lipid compounds used to make mixed micelle systems, such as laulyLlilllethylammonium bromide; cetyl-S trimethyl~mm-)nil-m bromide; lllylisLylLlillletheylammonium bromide; alkyl-l;...~ll.yl-benzylammonium chloride (where alkyl is, for example, Cl2, Cl4 or Cls);
benzyl-lim~thyldodecyl~mmonillm bromide/chloride; benzyl~lim~thylh~Y~ cyl-~llllWlli~llll bromide/chloride; benzyl~limr!l-yll~Lladecylammonium bromide/chloride;
cetyldilllcLllylethylammonium bromide/chloride; and c~Lyl~yl;~linillm bromide/
1 0 chloride.
Suitable additional amphiphilic m~t~ri~l~ for use in the present compositions include also lipid compounds carrying a net charge, for example, anionic and/or cationic lipids. Exemplary cationic lipids include, for example, N-tl-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride) (DOTMA), dioleoyloxy-3-15 (trimethylammonium)~l~alle) (DOTPA) and 1,2,-dioleoyloxy-e-(4'-Llhll~Lllyl-ammonium)butanoyl-sn-glycerol .
In ~ ition to, or instead of, the lipid c ~ )ollllds described above, the additional amphirhilir m~trri~l~ may comprise aliphatic carboxylic acids, for example, fatty acids. Preferred fatty acids include those which contain about 5 to about 22 20 carbon atoms in the ~lirh~tir group. The ~lirh~tir group can be either linear or branched. Exemplary saLul~Led fatty acids include, for example, (iso)lauric, (iso)lllyli~Lic, (iSo)F~lmitir and (iso)stearic acids. Exemplary u~aLulàL~d fatty acids include, for example, lauroleic, phy~c;L~lic, lllyli~Loleic, palmitoleic, petroselinic, and oleic acid. Suitable fatty acids include also, for example, fatty acids in which the 25 ~lirh~tir group is an isupl~l-oid or prenyl group.
The present compositions can also colll~lise, if desired, one or more neutral or positively or ne~alively charged m~trri~l~. Exemplary neutral materials include, for example, oils, such as peanut oil, canola oil, olive oil, safflower oil and corn oil; lecithin; sphingomyelin; cholesterol and derivatives thereof; squalene;
30 terpenes and terpenoid compounds; triglycerides; gums, such as x~nth~n, tr~g~r~nth, locust bean, guar and carrageenan gums; methoxylated pectin; starch; agarose;
cellulose and semi-~yllLh~LiC cellulose, for example, methyl cellulose, hyd~o~yt:Lllyl -W O 96/39197 PCT~US96/07813 cellulose, mPthnxy cellulose and hydlu~yL)lo~yl cellulose; nonionic lual~ials, inrlllfling, for example, polyu~-yclllylene fatty acid esters, polyu~ydllylene fatty alcohols, polyu~Lycl~lylene fatty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol O~y..Lcalalc, glycerol polyethylene glycol S ricinoleate, ethoxylated sc~ylJcd-~ sterols, ethoxylated castor oil, poly~ yelhylene-polyo~y~lu~ylene polymers and polyoxyethylene fatty acid ~r~ rs; acacia; agar;
bell~ol ilcs, including purified be~Q~ c; m~gm~; carbomer 934P; dextrin; gelatin; di-and trihydlo~y su~ ilulcd aLkanes and their polymers, inrlnrling polyvinylalcohol;
mono-, di- and triglycerides; amino alcohols; mon- c~c ch~ri.1Pc or sugar alcohols, such 10 as elylluuse, threose, ribose, arabinose, xylose, lyxose, fructose, sorbitol, ...A....ilol and sedoheptulose, with pier.,~l~,d monoc~cch~ri-lPc being fructose, m~nn~sP, xylose, arabinose, .."...~ ol and sorbitol; and ~1ic~rh,~ Ps~ such as lactose, sucrose, m~ltose and cellobiose.
Suitable positively charged m~tPri~lc include culll~uullds co~ ;..;..g, for 15 example, plo~ dled or ~ y amino groups, including polymers in which the lc~eatill~, units contain one or more amino groups, such as peptides, polypeptides, plotcills and li~u~lolculs, such as albumin and natural li~rolciuls. Exemplary positively charged m~teri~lc include, for example, chitin; alkanol~minPs, such as monoethanolamine, rlieth~nolamine and triPth~nnlamine, and llli~lulcs thereof, including, for example, trolamine; polylysine; poly~gil c; polyethylelleilllille;
chitos~n; and peptides, inrlll-1in~ mPl~nin c~...re~ o. holululle and dynorphin.Suitable llegdlivcly charged m~tPri~lc are cullll.oullds CO..~;1;..i.~g, for example, carboxy (CO2-) groups, including poly~;~bu~y polymers. Exemplary negatively cl~cd m~tPri~lc include, for c.~ lc, carboxymethylrelllllose; salts of alginic acid, such as sodium and c~lrillm ~lgin~te; salts of glycos,....;.~-glycans, inrl~ ing salts of hyaluronic acid; phosphorylated and sulfonated delivdlives of carboliy-lldLcs; genetic material, such as intPrlellkin-2 and iul~lf~lvu; phosphorothioate oligomers; and negatively charged peptides, such as deltorphin. In addition, carbolly-lldLes bearing polymers may be used in the present compositions. Carbohydrate bearing lipids are described, for 30 example, in U.S. Patent No. 4,310,505, the disclosures of which are hereby incorporated by l~felcllce herein, in their entirey.

W O 96/39197 PCTrUS96/07813 In certain ci~ res, it may be desirable to i~lcol~ulate one or more charged species into the present ~mphirhilic compositions. It is believed that such charged species can contribute to the stability of the present amphiphilir- compositions.
Examples of suitable charged species include, for example, cations, such as metal ions, S or anions. Exemplary cations include, for example, calcium, ...~-g;~i-rse, m~gnPeillm, copper, gadolinium or dysprosium cations, or any other cation which is c-.~.p~lihle for use in connrction with ph~. ",~ lir~l applications. Suitable anions include, forexample, sulphur, peroxides or superoxides. The anionic species may be cl~rl~t.o~l with chelating agents, for example, ethylenrrli,....;..~ r~l;r acid (EDTA) and 10 diethyl~ --i--fyc--lh~r~lir acid (DTPA).
Other materials which can be incorporated in the present compositions and which are contemplated as being capable of u~ ovillg the stability of the compositions include polymeric m~teri~lc. Exemplary of such polymeric materials include, for example, the polymers from which Z and/or R6 in formula (I) are derived, 15 as ~lieclle~e~d hclcull~cr~lG.
It may be desirable to include in the amphiphilic compositions of the present invention anti-bactericidal agents and/or pl'~Cl valiveS. Examples of these . . .~te, ;~le include, for example, sodium bel.~o~ y ~mmonillm salts, sodium azide, me~yl paraben, propyl paraben, sorbic acid, ascoll,yl, p~lmit~t~, butylated 20 hydlu~y~:iole, butylated hydroxytoluene, chlorobutanol, dehydluacetic acid, ethyler~ , monothioglycerol, pot~eeillm ben~o~f~, pot~eeillm metabisulfite,pot~e~ m sorbate, sodium bisulfite, sulphur dioxide and organic mc~-;uliâl salts.
Other ~ litir,n~l materials which can be hlcol~ld~ed in the present compo~eiti~ne would be ~alcll~ to one skilled in the art based on the present 25 disclosure. Preferably, the ~ tion~l materials are selecte~l to opLillli~c certain desirable ~ pcllies of the compositions, inrll--ling serum stability and plasma half-life.
This can be achieved without undue c~cl;...~nt~tion, based on the present disclosure.
The collcellL.àlion of the additional materials in the present compositions can vary and depends, for example, upon the particular fluorinated amphiphilir 30 colllpùullds which are employed. In plerell~d embo~lim~ntc, the c~llcenL~dtion of additional materials is from about 0.01 mg/mT~ to about 200 mg/mT . More preferably, the conren~tion of additional materials is from about 0.05 mg/mL to about 5 mg/mL, CA 022l9528 l997-l0-27 W O 96/39197 PCT~J~,GI~7~13 with co.~rc~ ns of about 15 mg/mT~ to about 2.5 mg/mL being even more - pl.,rtllcd.
In certain plcfellcd embo-iimlo-nt~ of the invention, the stabilized compositions comprise a vesir~ r composition. The ve~ir~ r cc.ll.~o~ilions may 5 col..~lise mirçl1~s and/or liposomes. A wide variety of methods are available for the plc~alalion of vç~irlll~r compositions, including, for example, .~h~king, drying, gas-in~t~ tion, spray drying, and the like. Suitable mlqth~ for pie~ing the ve~iclll~r compositions are described, for example, in U.S. applir~ti~ n Serial No. 307,305, filed September 16, 1994, the disclosures of which are incorporated herein by lcçclc~ce.
In ~lcrcllcd embo~1im~ont~, fluorinated ~mphirhilir col.~ ds are used in the plcpalalion of the present compositions, inrlnlling vecirlll~r compositions, w_ich are in the gel state at physiological l~ cl, ~ . As known to one of oldil~y skill in the art, the main phase transition Iclll~clalulc (Tc) of nonfluorinated a,ll~ hilir compounds, such as lipids, including, for example, phosphatidylcholines, is based 15 gen~r~lly on the length of the carbon chains which are linked to the glycerol moiety.
Exemplary lcp~s~ livc lipids and their phase transition ~lll~clalulcS are set forth in the following table.
TABLE II
Salul~led Diacyl-sn-Glycero-3-Phosphocholines:
Main Chain Meltin~ Transition Temp~.d~u~cs Number of Carbons in Main Phase Acyl Chains Tr~n~iti- n Tclll~clalulc (~C) ,2-(12:0) -1.0 1,2-(13:0) 13.7 251,2-(14:0) 23.5 1,2-(15:0) 34.5 1,2-(16:0) 41.4 CA 022l9528 l997-l0-27 TABLE II
Saturated Diacyl-sn-Glycero-3-Phospho- holin-~c-Main Chain M~l~in~ Transition T~~ alul~s Number of Carbons in Main Phase Acyl Chains Transition Te~u~.dLur~ (~C) 1,2-(17:0) 48.2 1,2-(18:0) 55.1 1,2-(19:0) 61.8 1,2-(20:0) 64.5 1,2-(21:0) 71.1 1,2-(22:0) 74.0 1,2-(23:0) 79.5 1,2-(24:0) 80.1 See, for example, Derek Marsh, CRC Handbook of Lipid Bilayers, p. 139 (CRC Press, lO Boca Raton, FL 1990).
With respect to the fluorinated amphiphiles involved in the present invention, it is believed that the number of carbon atoms which are fluorinated has a greater effect on Tc, as ~osed to the length of the chain(s), generally. In general, the greater the number of carbons fluorinated in one or more of the chains, the higher ~ 15 the Tc~ Main chain transition L~ c~aLules of the compound of formula (V), which is the product in the fo-~goillg reaction scheme and which is depicted below, are set forth W O 96~9197 PCT~US96/07813 in the following table.

C~;~+l--(CH2)m--C(O)O--CnF2~l--(CEI2)m--C(O)O--OP(02)0-(CH2)2--N (cE~3)3 TABLE m Perflou~ aled Diacyl-sn-Glycero-3-Phosphoel~r~
Main Chain Melting Transition Te~dLules Main Phase Transition Tc~ claLulc S Compound (V) m n (~C) (A) 4 4 a~
~) 4 6 a~
(C) 4 8 69.3 ~) lO 4 18.6 0 ~) lO 6 56.4 ~) lO 8 > 95 An ~cteri~lr (*) means that no transition was ~let~cte~l between tPmper~ res of 4~C and 95~C.
With particular lcrcl~,lce to the plcpalation of micelle compositions, the 15 following ~liccllccion is provided. Micelles may be prepared using any one of a variety of conventional mirell~r ~lc~alatOly mlotho~c which will be ~e..L to those skilled in the art. T_ese methods typically involve suspension of the fluorinated amphiphilir cc,.l.poul.d and additional amphiphilic compound, as desired, in an organic solvent, evaporation of the solvent, resllcpencion in an aqueous m~o~lillm, sonication and 20 ce~L.irugation. The foregoing methods, as well as others, are ~liccucce(l for example, CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 in ~nfiPI~l et al., Methods in Enzymology, Vol. 189, pp. 418422 (1990); El-Gorab et al, Biochem. Biophys. Acta, Vol. 306, pp. 58-66 (1973); Colloidal Surfactant, ~ Shinoda, K., N~k~g~n~, T~l.. , .. --~il.i and Isejura, ~r~ omir. Press, NY (1963) (especially "The Formation of Micelles", Shinoda, Chapter 1, pp. 1-88); Catalysis in S Micellar and Macromolecular Systems, Fendler and Fendler, ~r~flrrnir Press, NY(1975). The disclosures of each of the folcgoillg publications are incorporated by crclcllce herein, in their elllilcLy.
The vesic~ r culllposiLion may also culllylise liposomes. In any given liposome, the involved ~llyh;l~hilin compound(s), including fluorinated and 10 nonfluorinated amphiphilic collly~ulld~ may be in the form of a monolayer or bilayer, and the mono- or bilayers may be used to form one or more mono- or bilayers. In the case of more than one mono- or bilayer, the mono- or bilayers are generally collccllLlic. Thus, the amphiphilic compounds may be used to form lmil~m.oll~r liposomes (colllylised of one mr)nnl~yer or bilayer), oligol~mPll~r liposomes 15 (cclllplised of two or three monolayers or bilayers) or mnltil~mPll~r liposomes (comprised of more than three monolayers or bilayers).
A wide variety of m~thn-lc are available in connection with the ~le~dldLion of liposome compositions. Accoldingly, the liposomes may be ylc~alcdusing any one of a variety of conventional liposomal yrcy~toly terhniqll~os which will 20 be aypar~ to those skilled in the art. These techniques include solvent dialysis, French press, extrusion (with or without freeze-thaw), reverse phase evaporation, microeTmll~ifir~tir~n and simple freeze-thawing. The liposomes may also be plcydlcd by various processes which involve ~h~king or ~/ollcx~illg. This may be achieved, for example, by the use of a mrch~nir.~l ~h~king device, such as a Wig-L-Bug~ (Crescent 25 Dental, Lyons, IL). Conventional micrormlll~ifir~tion e(luiylllcllL~ such as a Microflni~1i7sr~ (Microfluidics, Woburn, MA) may be used also.
Additional mPthnti~ for the ylcpdldLion of liposome compositions include, for example, sonir~ti-)nJ chelate dialysis, homogcl.i~Lion, solvent infusion, ~y.llL;~leous formation, solvent vaporization, controlled dclcigcllL dialysis, and others, 30 each involving the preparation of liposomes in various fashions. Methods which involve freeze-thaw techniques are ylcfell~d in connection with the ylcpaldLion of liposomes. Suitable freeze-thaw techniques are described, for example, in copending CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 U.S. application Serial N o. 07/838,504, filed February 19, 1992, the disclosures of which are incorporated herein by leçe.cllce in their ellLi~ly. E~lc~ on of the liposomes may be carried out in a solution, such as an aqueous saline solution, aqueous phosphate buffer solution, or sterile water.
As noted above, the size of the vesicles, inrhlAing liposomes, can be adjusted, if desired, by a variety of t~chniqnes, inr~ ing extrusion, filtr~tion, sonication and homo~ n In addition, the size of the liposomes can be adjusted by the introduction of a l~min~r stream of a core of liquid into an immiscible sheath of liquid. Other methods for adju~ the size of the liposomes and for mor~ ting the 10 reslllt~nt liposomal biodi~LlibuLion and cl~,re of the liposomes would be ~ alcllL to one skilled in the art based on the present ~ clc)sllre. Preferably, the size of the liposomes is adjusted by extrusion under prei,~uic through pores of a ~l.ofinl-~ size.
Although liposomes employed in the subject invention may be of any one of a variety of sizes, the liposomes are preferably small, that is, less than about 100 ..~....".
(nm) in outside ~ m~ter.
Many of the r~,l~Oillg liposomal pr~ o. y t.orhniql~es, as well as others, are ~ .s~ , for example, in U.S. Patent No. 4,728,578; U.K. Patent Application GB 2193095 A; U.S. Patent N o. 4,728,575; U.S. Patent N o. 4,737,323;
T.~ nal Application Serial N o. P C T/U S85/01161; Mayer et al., Biochimica et Biophysica Acta, V ol. 858, pp. 161-168 (1986); Hope et al., Biochimica et Biophysica Acta, V ol. 812, pp.55-65 (1985); U.S. Patent No. 4,533,254; Mayhew et al., Method~ in En2ymology, V ol. 149, pp.64-77 (1987); Mayhew et al., Biochimica et Biophysica Acta, V ol 755, pp. 169-74 (1984); Cheng et al, Investigative Rcu1iology, V ol. 22, pp. 47-55 (1987); Tl.lr. .~ n~l Application Serial No. P C T/U S89/05040;
U.S. Patent No. 4,162,282; U.S. Patent No. 4,310,505; U.S. Patent N o. 4,921,706;
and Liposome Technology, G~cgc,liadis, G., ed., Vol. I, pp. 29-31,51-67 and 79-108 (CRC Press Inc., Boca Raton, FL 1984), the disclosures of each of which are hereby incorporated by ~crelcllce herein, in their clllilcly.
Although any of a number of varying t~ hniq~-es can be used, it is contemplated that the vecit~ r compositions of the present invention are preferably ~cpalcd using a .ch~king technique. Preferably, the ~h~king technique involves agitation with a m~rh~ni~ h~king ap~alaLus, such as a Wig-L-BugTY (Crescent CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96/07813 Dental, Lyons, IL), such as those disclosed in copending U.S. application Serial No.
160,232, filed November 30, 1993, the ~licclQs ~reS of which are hereby inco,~oldLcd herein by ,cre,cll~e in their ellLileLy.
In certain embo-l;...k..l~ of the present invention, the colll~os;~i- nc S further colll~,ise a bioactive agent. These compositions are lcr~,~led to herein as "amphiphilir f~ tionc", and can be used for the thcld~cuLic delivery in vivo of - bioactive agents. Preferably, the amphiphilir form~ tions co~ ,ise vesirlll~r formlll~tions. In veciclll~r formulations, circulation and delivery of the vesicles to the targeted tissue can be observed via a non-invasive procedure. In conn~octi- n with gas-lO filled or gaseous precursor-filled vesicles, the applir~tinn of high energy ultrasound, radio frequency, optical energy, for example, laser light, and/or heat, to produce areas of lly~cl~ , can be used, if desired, to rupture the vesicles in vivo and thereby promote release of the e~lLl~ed gas and/or gaseous precursor and bioactive agent.
Thus, veciclll~r formlll~tions permit the controlled release of a bioactive agent in vivo.
As those skilled in the art will recognize, any of the present stabilized compositions and/or formlll~ti~mc may be lyophili7r~1 for storage, and lcc~ l, for example, with an aqueous m~1illm (such as sterile water or phosph~t~ burr~,,ed solution, or aqueous saline solution), with the aid of vigorous ~git~tion To ~)lCVClll ag~l--li--i lion or fusion of the fluorinated ~mphirhilir compounds and/or ~lr1itinn~l 20 amp_ip_ilic compounds as a result of lyophili7~tion, it may be useful to include additives which pl~vcllL such fusion or ~g~ll.li..~lion from occ~...;..g. Additives which may be useful include sorbitol, ...~....ilol, sodium chloride, glucose, tre_alose, polyvillyl~yll.lidone and poly(ethylene glycol), for example, PEG 400. These andother additives are described in the literature, such as in the U.S. Pl.~....~copeia, USP
25 X~I, NF XVII, The United States Ph~rm~ropeia, The National Formulary, United States ph~rm~ropeial Convention Inc., 12601 Twinbrook Pdlh~ay, Rockville, MD
20852, t_e disclosures of w_ich are hereby incorporated herein by rercrcl~ce in their entirety. Lyophilized ~lep~alions generally have the advantage of greater shelf life.
In the case of veciclll~r compositions, inrltltling mirell~oc and liposomes, 30 it is colllellll,lated that the bioactive agent is preferably ellLI~ped within a void of the vesicles. In certain cases, the bioactive agent can be incorporated also into the membrane walls of the vesicle. In the case of amphiphilic compositions in which the WO 96/39197 PCTrUS96/07813 ~mphirhilir c(jlllpuullds are subst~nti~lly agg,cgalcd randomly, or ~ub~li~..li~lly not ag~,c~ ~It;d, it is contemplated that the bioactive agent is generally di~ ed homogeneously throughout the cù",~osiLion.
The bioactive agent which is hlcc~l~ulalcd in the present compositi-)n~ is S preferably a sl-bst~nre which is capable of exerting a t~a~culiC biological effect in vitro and/or in vivo. rl~ rr~.lir~l.e, drugs and genetic m~t~ri~l are examples of suitable bioactive agents. Examples of genetic m~t~ri~l~ include, for example, genes carried on e~,cssion vectors, such as plasmids, ph~g~mill~, cosmids, yeast artificial chromosomes (YACs) and dere~livc- or "helper" viruses; anti-sense and sense 10 oligonucleotides; phosphorothioate oligodeoxynucleotides; antigene nucleic acids; and single and double str~n~led RNA and DNA, including DNA which encodes at least a portion of a gene, for e~mple, DNA w_ich encodes for human leukocyte antigen (HLA), dystrophin, cystic fibrosis tran.cm~...hla"e l~,ct:~lol (CFTR), inf~rlel-kin-2 (IL-2), tumor necrosis factor (TNF) and granulocyte-",acrophage colony stim~ ting factor 15 (GMCSF). The DNA can also encode certain ~Lolt:ills which may be used in the tre~tmPnt of various types of pathologies or conditions, inrlll-ling those which are associated with the loss or dclt:,iulalion of immnnP c~...pcl~re. Such pathologies or con-liti~ n~ involving ill~ P co~ C~ re include, for example, acquired immnnP
deficiency ~y~dlu~e (AIDS), cancer, chronic viral infections, and ~uloi.. ~ disease.
Specifically, DNA may be sel~octecl w_ich c~lc~es adenosine ~le.. i.. ~e (ADA) for the ll~.~t..~..l of ADA ~erir;~ y; growth hormone for the l~ ..l of growth deficiency or to aid in the healing of tissues; insulin for the trP~tmPnt of hetrs; luL~il~iulg hormone rclP~ei~-~ hormone (LHRH) antagonist as a birth control agent; LHRH for the tre~tmpnt of ~lus~lt; or breast cancer; tumor necrosis factor 25 and/or int~rlrllkin-2 for the llc~ ll of advanced c~n~rs; high-density lipoprol~;iu, (HDL) lcCc:~lol for the L~ of liver disease; thymidine k~nase for the tre~tm~ t of ovarian cancer, brain tumors, or human immnnodeficiency virus (HIV) infection;
HLA-B7 for the treatment of m~ n~nt m.ol~nnm~; IL-2 for the L,c~t...-.~l of neurobl~tom~, m~lign~nt melanoma or kidney cancer; interleukin~ (IL~) for the 30 tre~tm~nt ûf cancer; HIV env for the L~ t---f---l of HIV infection; antisense ras/pS3 for the Llc~ of lung cancer; and Factor VIII for the tre~tmPnt of Hemophilia B. Such CA 02219~28 1997-10-27 WO 96/39197 PCTAJS~G~'~7&13 therapies are described, for example, in Science, Vol. 258, pp. 744-746 (1992), the disclosure of which is incc,l~oldlcd herein by l,fclcllce in its ellLil~y.
In certain embo.l;...~ ;, it is desirable to incc,l~uldLe in the amphiphilic compositions mdLclials which colll~liOe, for example, a polymer, such as an alginic S acid polymer, that is covalently linked to a bioactive agent. An çx~mr)le of such a m~teri~l is n~lirlixic acid ~lgin~t~q in which a polymer (alginic acid) is covalently linked to a bioactive "~ l (n"li~lixir acid). Such materials are desirable in that it is believed they are capable of illl~JlUVill~3, and/or elllldllcillg the stability of the amphiphilir compositions, as well as providing a source of bioactive agent. It is 10 contemplated also that after ~ L~dLion, the materials are hydrolyzed in vivo, to bioactive agent (n~lirlixit~ acid) and polymer (alginic acid).
As with the stabilized ~ll~hi~hilic compositions, a wide variety of techniques also exist for the pl~d~dLion of stabilized ~mphiI~hilir formulations. For example, the formulations may be plc~dlcd from a llli~Lulc of ~mphirhilir compounds, 15 bioactive agent and gas or gaseuus precursor. In this case, stabilized compositions are pr~dlcd as described above in the ~l~,sellce of a bioactive agent. Thus, for example, micelles and liposomes can be plc~dled in the ~l~,se.lce of a bioactive agent. The c~llation can involve, for example, bubbling a gas dilccLly into an aqueous llli~lUlC
of the fluolilla~ed amphirhilir colllpuullds and additional ~ll~hil~hilir m,ltrri~l~, as 20 desired, and bioactive agent. ~llr-ll,livcly, the ~mphirhilir compositions may be ~lcfolllled from fluorinated amphip_ilic cc~l~uullds and gas or gaseous precursor. In tbe latter case, the bioaclivc agent is then added to the ~mphirhilir colll~osilion prior to use. For example, an aqueous ll~i~lul~, of liposomes and gas may be plc~aled to which the bioactive agent is added and which is ~gif"t~fl to provide the liposome formulation.
25 The liposome formulation is readily isolated also in that the gas- and/or bioactive agent-filled liposome vesicles generally float to the top of the aqueous solution. Excess bioactive agent can be recovered from the rem~ining aqueous solution.
The present amphiphilic compositions and/or formulations are suitable for diagnostic and/or therapeutic applications. In the case of diagnostic applications, 30 such as ultrasound, the amphiphilic compositions, which may further comprise a gaseous ~l~,culOOl, are ~1mini~tered to a patient. Energy, preferably in the form of ultrasonic energy, is applied to at least a portion of the patient to image a region, which may include targeted tissue. A visible image of an infrrn~l region of the patient is then obtained, such that the p.~ se,.ce or ~hspnre of ~ ç~ tissue can be ascertained.
Ultrasonic im~gin~ teehni~ s, inr.lll~ling second h~rmonir. im~in~, are S well known in the art, and are described, for example, in Uhl~n-lorf, "Physics of Ulllasuul~d Contrast Tm~ging: .~c~lle~ in the Linear Range", IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, Vol. 14(1), pp. 70-79 (1994) and .Cnth~o.rl~ntl, et al., " Color Doppler Myocardial Tm~ging: A New Technique for t-h-e ~e~m~nt of Myocardial Function", Journal of the American Society of Echocardiography, Vol. 7(5), pp. 441-458 (1994), the rli~c.losnres of which are hereby incorporated herein by reference in their ell~ ly.
With respect to th~_~apculic applications, the present ~mphirhilir formnl~tiQns can be used in either in vitro or in vivo appli~ .c. In the case of in vitro applications, including cell culture applications, the amphirhilir f~rmnl~ti~ns can 15 be added to the cells in cultures and then inr.~lb~trcl If desired, where li~osollles are employed, energy, such as sonic energy, may be applied to the culture media to burst the liposollles and release any Ih..L,~l~c--lir. agents.
With respect to in vivo applir~ti~mc~ the f )rm~ tinn~ of the present invention can be ~.h..;..i~ -cd to a patient in a variety of forms adapted to the chosen 20 route of ~fl...;..;.~i~aLion, inrln-ling, for example, pdlcll~ àl, oral, or hllLa~tlilollcâl.
rdicl~ dl ~ ;x~ on~ which is ~lcfell~d, inr.hl-:les ~ -dlion by the following routes: hlLIavclluus; i"~ r; ;..I~ lly; intra-arterially; sl~hL;~ .uu:~;
intraocular; intrasynovial; L~dlls~;lh~ l, inrln-ling tr~n~lerm~l; pulmonary viainh~l~tion; ophth~lmir.; sublingual and buccal; topically, inrln-ling ophth~lmir; ~e-rm~l;
25 ocular; rectal; and nasal inh~l~tinn via in~lffl~tion. Illlldv~l~us ~h~ dlion is ~fe~l~d among the routes of ~alc;ll~lal ~h..;,.;!i~.dlion.
After ve.cic -l~r forml~l~tiQns which cc,lll~lise a gas and bioactive agent and, optionally, a ~ dse.~us ~ ;ul~ol, have been ~ d to a patient, energ,v, preferably in the form of ultrasonic energy, can be applied to the target tissue to 30 identify the location of the vesicles. The applied energ,v may also be employed to effect release of the bioactive agent and to facilitate cellular uptake of the bioactive agent. As one skilled in the art would recognize, based on the present disclosure, this CA 02219~28 1997-10-27 W O 96/39197 PCTrUS96~7813 mf th~ rl of mP~ ting cellular uptake of bioactive agents with ultrasonic energy is preferably effected with tissues whose acoustic window pc,~ the ~ ion of ultrasonic energy. This is the case for most tissues in the body, inrln-ling, for example, muscle and organ tissues, such as the heart and liver, as well as most other 5 vital ~l~u~;Lu-~:s. With respect to brain tissue, it may be ~f'.Cf'~ y to create a "surgical window" by removing part of the skull, in~cmllrh as ullldsollic energy generally does not llal~lllil through bone. Illllavas~;ular and/or endoll-rnin~l ulllasuulld tr~nc~lllcers may be used to apply the ultrasound energy to selected tissues and/or sites in the body, for example, the aorta and the esophagus.
Fluorinated ~mphirhilic ft~rmnl~tions can be formnl~tf d to be sllffirif ntly stable in the v~cc -l~t--re such that they circulate throughout the body and provide blood pool equilibration. As one skilled in the art would recognize, based on the present disclosure, the formnl~tions~ inr~ ling those which colll~lise ~u~e~ions, em--lcic)ns and/or vesicles, such as liposomes and micelles, may be coated with certain materials 15 to ~ o uptake by the reticuloendothelial system. Suitable co~tingc include, for example, g~ngliosi(les and glycolipids w_ich bind c~rrh,.. ;-le moieties, such as glu~;ulullalt:, g~ ollale~ gululul~ , poly(ethylene glycol), poly(propylene glycol), polyvhlyl~yllolidone, poly(vinyl alcohol), dextran, starch, pho~hc,lylated and sulrol~td mono-, di-, tri-, oligo- and poly~àccllalides and albumin. Provided that the 20 circulation half-life of the formnl~tions is of a snmri~ont period of time, they will generally pass through the target tissue while passing through the body. In the case of form~ tions which colll~-ise a bioactive agent, energy, for example, sonic energy, may be focused on the tissue to be treated, for example, ~licf~cf~cl tissue. The bioactive agent will then be ~~lcased locally in the target tissue. The illVt;lllOl~i have found also 25 that antibodies, carbol-yd-al~;s, peptides, glyco~t~lides, glycolipids and lectins also assist in the ~ ~li~ of tissue. Accordingly, these m~tf ri~l~ may be incorporated into the fluorinated ~ .l.;philir f~rmnl~tions also.
DirÇf;.cll~ levels of energy are generally associated with diagnostic and thf ~ ir ultrasound. For example, the levels of energy associaL~d with diagnostic 30 UlLIasOulld are generally in~nffirient to cause rupture of vesicles and/or to facilitate release and cellular uptake of the bioactive agents. Moreover, ~i~gnnstir ultrasound involves the application of one or more pulses of sound. Pauses between pulses CA 02219~28 1997-10-27 W O 96/39197 PCT~US96/07813 ~""il~ the reflrctr~l sonic signals to be received and analyzed. The limited number of pulses used in ~1iAgnnsti~ ultrasound limits the effective energy which is delivered to the tissue that is being s~ d On the other hand, higher energy ultrasound, for example, nltrAcol~ntl S which is gelle,d~ed by Illcld~t;u~ic ultrasound eql~ipm~ont, is genrrAlly capable of ~
rupture of the vesicles. In ge~rAl, thc.d~cu~ic ultrasound mArhin~s use from about 10 to about 100% duty cycles, depending on the area of tissue to be treated with the ulL~souLd. Areas of the body which are generally çh~ c(~ l by larger amounts of muscle mass, for example, backs and thighs, as well as highly vA~c -l~ri7~o~1 tissues, 10 such as heart tissue, may require a larger duty cycle, for example, up to about 100%.
In ~ e~ r ultrasound, continuous wave ultrasound is used to deliver higher energy levels. For the rupture of vesicles, continuous wave ultrasound isprcfc,,cd, although the sound energy may be pulsed also. If pulsed sound energy is used, the sound will gen~rAlly be pulsed in echo train lengths of about 8 to about 20 or 15 more pulses at a time. Preferably, the echo train lengths are about 20 pulses at a time.
In addition, the frequency of the sound used may vary from about 0.25 to about 100 m.-g~hrrtz (~IHz). In general, frequency for Ih~ ~dl~ ultrasound ranges between about 0.75 and about 3 MHz are ~lcrcllcd with about 1 and about 2 MHz being morep~crcLlcd. In addition, energy levels may vary from about 0.5 Watt (W) per square 20 centimrt~r (cm2) to about 5.0 W/cm2, with energy levels of about 0.5 to about 2.5 W/cm2 being ~lcre~,d. Energy levels for theld~cu~ic ultrasound involving hy~ iA are genrrAlly from about S W/cm2 to about 50 W/cm2. For very small vesicles, for example, vesicles having a t~ -lrl of less than about 0.5 micron, higher frequencies of sound are generally ~lcrcllcd. This is becduse smaller vesicles are 25 capable of abso,l,i"g sonic energy more erÇc~lively at higher frequencies of sound.
When very high frequencies are used, for example, greater than about 10 MHz, thesonic energy will generally p~ fluids and tissues to a limited depth only. Thus, e~ctern~l application of the sonic energy may be suitable for skin and other superficial tissues. However, for deep ~Lluc~ulcS it is generally nPcessAry to focus the ultrasonic 30 energy so that it is p,cre,c~-lially directed within a focal zone. ~ltlornAtively, the ultrasonic energy may be applied via illLc,~LiLial probes, intravascular ultrasound c~thrters or endoluminal c~thrter.c. Such probes or r~th~ters may be used, for W O 96~9197 PCTrUS96/07813 example, in the esophagus for the ~ nosi~ and/or tr~tm~nt of esophageal cal.;illollla.
In ~d~lition to the theld~c.l~ic uses ~iig~ ed above, the present compo~iti- n~ can be employed in co.-.-P~-~ion with esophageal ca,-;i lullla or in the corollaly arteries for the tre~tm~nt of alllclosclerosis~ as well as the th~ ldl.e~l;r uses ~lescrihe<l, for example, in S U.S. Patent No. 5,149,319, the disclosures of which are hereby illcw~o~dted herein by .erc-c.lce in their ell~ile~y.
The useful dosage to be ~ ed and the particular mode of d~ion will vary depending upon such factors as the age, weight and the particular animal and region thereof to be treated, the particular amphiphilic compound 10 or cc,lll~uullds used, the plcsellce or absence of a bioactive agent, the ~ gnnstir or thc.d~cu~ic use coll~elllplated, and the form of the involved compositions, for example, micelle or liposome, as will be readily a~cll~ to those skilled in the art. Typically, dosage is ~ lcd at lower levels and illc-eased until the desirable therapeutic effect is achieved. The amount of ~mrhirhilir co.ll~oulld that is ~-lmi.~ lcd can vary lS and generally depends upon the amount of particular fluorinated ~mrhirhili~ compound and ~Mition~l amphirhilir- material, as desired, ~ cd.
The present invention is further described in the following examples.
Examples 1, 2 and 4 to 7 describe the ~lcp,..~inn of vesir~ r colll~o~iLions within the scope of the present invention. Example 1 also describes an evaluation of the ~JlC:~:iUl'~, 20 stability of a veciclll~r composition within the scope of the present invention and a composition ~ dlcd from a nonflllorin~trtl ~llpl~i~hilic compound. F.Y~mrle 3 describes the freeze-fracture analysis of ve~ir7ll~r compositions within the scope of the present invention. All of the examples are pn~hclic examples. These examples arefor ill~ ive purposes only, and are not to be construed as limiting the appended25 claims.

EXAMPLES
In the following examples, lcrelcllce to the compounds of formulas (V)(A), (V)(E) and (V)(F) are to the coll~ ollding compounds set forth in Table m above. In addition, "DPPC" in Example 1 refers to rlir~lmitoylphosphatidylcholine.

W O 96/39197 PCT~US96/07813 F,Y~rnp'~ 1 The cun~uu.ld of formula (V)(A) will be ~ ~cd using the procedures described hereinbefore. The cc~ uulld will be suspended, at a co~cel.lldLion of 1 mg/mL, in a solution of saline:glycerol:propylene glycol (8:1:1 wlwlw). The reslllting S mixture (1.5 mL) will be placed into a sterile 3 mL glass vial. The head space in the vial will be replaced with p~rfl-lo~,~a.le gas and the vial will be sealed. The vial will be shaken for 5 ...;....lrs at 4,200 r.p.m. on a Wig-L-Bug~ (Crescent Dental, Lyons, IL) .ch~king al~pdlalus to provide perfluo,ù~u~ e gas-filled vesicles (liposomes) having a mean ~ m~ter of about 3 ,um. The gas-filled vesicles will be 10 stable for several weeks at room Lclll~cldlulc. This will be repeated except that the compound of formula (V)(A) is replaced with DPPC. The ~,cs~u,c stability of the veciclll~r composition Cûlll~ illg the fluorinated ~mrhirhile of formula (V)(A) and the composition cont~ining DPPC will be col,l~alcd in an acuu~Lic laboratory. The colll~o~ilions will be exposed to elevated pleS:!iUlC (300 mm Hg), and the loss in 15 ~ ion and b~r~ l between the compositions will be cOlll~ d. The vesi~ r composition colll~lish~g the flnorin~te(l ~mrhirhilir compound of formula (V)(A) will exhibit hll~lc,ved ~ UlC stability.

nr'F 2 The compound of formula (V)(F) will be ~lc~ucd as descrihed above.
20 A vesirlll~r composition COlll~ illg this cc,lll~uu~d will then be ~l~a~cd using the procedure described in Example 1 except that the collcclllldlion of the saline:glycerol:propylene glycol ~u~cnsion will be 1.25 mg/mL and the head space of the vial will be replaced with perfluurul~uL~le gas. The resllltin~ perfluorobutane gas-filled ve~irlll~r composition will exhibit improved ~lu~c~ies,in~ ing ~lC~UlC
25 stability.

~rnrl~ 3 Freeze-fracture electron miclusco~y (FFEM), using standard techniques, will be ~clrolllled on the vesicular compositions within the scope of the present invention and which were ~lc~aled in Examples 1 and 2. FFEM will show that the CA 02219~28 1997-10-27 WO 96/39197 PCTrUS96/07813 gas-filled vesicles of the present invention possess smooth walls having a thirl~-n~c.c of about a single bilayer.

Example 4 The compound of formula (V)(E) will be plc~alcd using the procedures 5 described above. A l~ Lul~ of the compound of formula (V)(E) (10 mg/mL) in normal saline with trehalose (20 mg/mL) and poloxamer F68 (polyoxyethylene-polyoxy~lo~ylene glycol block copolymer) (25 mg/mL) will be heated to 60~C and passed through a Microfl~ i7er (Mircofluidics Corp., Newton, MA) 20 times at 16,000 pSi. The reslllting veci~ r composition will be lyophili_ed and the head space 10 of the container in which the culll~o~ilion is stored will be in~tille-l gradually with nitrogen gas and reeqnilihrated to ambient ~)lCS:!iUlC over a period of 48 hours. The resllltin~ vesiclll~r composition will colll~lise gas (nitrogen) filled vesicles (liposomes) having a mean .li,..l.rl~ l of 200 nm. The vesicles can be stored dry until use, as desired, and can be rehydrated with sterile water and shaken prior to injection.
Example 5 The compound of formula (V)(E) will be ~lcpalcd as described above and suspended, at a cullce~ ion of 1 mg/mL, in a solution of saline:glycerol:
propylene glycol (8:1:1 wlwlw). The resnlting ll~ u~e (1.5 mL) will be placed into a sterile 3 mL glass vial at 35~C. The head space in the vial will be replaced with perfluorvpcu~le gas and the vial will be sealed. The vial will be shaken for S minlltes at 4,200 r.p.m. on a Wig-L-Bug~ (Crescent Dental, Lyons, IL) ~h~king a~ alus while the l~lll~la~ul~ of the vial is ".~;"~ at 35~C. Perfluolv~ ~le gas-filled vesicles (liposomes) will be obtained.

FY~nple 6 Example 5 will be repeated except that the perfluoropentane gas is replaced with a ll~ul~ of perfluoiope~ gas and nitrogen gas (1:1 v/v). Cooling the reslllting liposomes to below 30~C may result in the condensation of the perfluolupell~le gas. However, the con~len~e~l perfluolupe~ ne will exist as a nanodroplet within the vesicles and can be vol~tili7~d by W~llPillg to provide the gas-W O 96/39197 PCTrUS9G1~7~13 filled vesicles. This W~ llillg can be caused by alLirlcial means ~c vivo or by the body IC111~C1~U1C of a patient after ~ iQn of the v~ r composition.

Example 7 The colll~lulld of formula (V)(F) will be pl~,~arcd as described above 5 and snspçn~ at a co.~ liQn of 1 mg/mL, in a solution of saline:glycerol:propylene glycol (7:1.5:1.5 wlwlw). The rçsll1ting llli~Lulc (1.5 mL) will be placed into a sterile 3 mL glass vial at 60~C. The head space in the vial will be replaced with perfluol.)h~-x 1i~~ gas at 60~C. The vial will be sealed and shaken for 5 ...i....~s at 4,400 r.p.m. on a Wig-L-BuglU (Crescent Dental, Lyons, IL) ~h~king 10 a~aldLus while the Lelll~cl~lulc of the vial is ...~ ;.i..Pd at 60~C. Perfluoroh~ n~-gas-filled vesicles (liposomes) will be obtained.
The disclosures of each patent, patent application and publication cited or described in this docllm~-nt are hereby incc,l~ol~-lcd by lcrclcllce, in their enLilcLy.
Various mf~flifir~tions of the invention, in addition to those described 15 herein, will be a~ lL to those skilled in the art from the foregoing descli~Lion.
Such mo~ tiQns are also intf nflf d to fall within the scope of the appended claims.

Claims

What is claimed is:

1. A stabilized composition comprising, in combination with a gas, a compound of the formula wherein:
each of x, y and z is independently 0 or 1;
each X1 is independently -O-, -S-, -SO-, -SO2-, -NR4-, -C(=X2)-, -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-;
X2 is O or S;
Y is a direct bond or -X3-M(=O)(OR5)q-O-, where q is 1 or 2;
X3 is a direct bond or -O-;
M is P or S;
Z is hydrogen, the residue of a hydrophilic polymer, a saccharide residue or -N(R6)r, where r is 2 or 3;
each R1 is independently alkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons;
R2 is a direct bond or an alkylene linking group of 1 to about 10 carbon;
R3 is a direct bond or an alkylene diradical of 1 to about 10 carbons; and each of R4 and R5 is independently hydrogen or alkyl of 1 to about 8 carbons; and each R6 is independently hydrogen, alkyl of 1 to about 8 carbons or a residue of a hydrophilic polymer;
provided that at least one of x, y and z is 1, at least one of R1 is fluorinated alkyl of 1 to about 30 carbons, and when R2 is a direct bond, two of x, y and z are 0.

2. A composition according to Claim 1 wherein:
two of x, y and z are each 0.

3. A composition according to Claim 1 wherein:
one of x, y and z is 0 or 1; and the other two of x, y and z are each 1.

4. A composition according to Claim 3 wherein:
one of x, y and z is 0, and the other two of x, y and z are each 1.

5. A composition according to Claim 3 wherein:
each of x, y and z is 1.

6. A composition according to Claim 1 wherein:
X2 is O.

7. A composition according to Claim 6 wherein:
each X1 is independently -O-, -S-, -C(=X2)-, -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-.

8. A composition according to Claim 7 wherein:
each X1 is independently -C(=X2)-O- or -O-C(=X2)-.

9. A composition according, to Claim 8 wherein:
X1 is-C(=X2)-O-.

10. A composition according to Claim 9 wherein:
Y is -X3-M(=O)(OR5)q-

11. A composition according to Claim 10 wherein:
q is 1, X3 is a direct bond, and M is P.

12. A composition according to Claim 11 wherein:
Z is -N(R6)r.

13. A composition according to Claim 3 wherein:
at least one of R1 is alkyl of 1 to about 30 carbons and at least one of R1 is fluorinated alkyl of 1 to about 30 carbons.

14. A composition according to Claim 3 wherein:
each R1 is independently fluorinated alkyl of 1 to about 30 carbons.

15. A composition according to Claim 3 wherein:
each R1 is independently polyfluorinated alkyl of 1 to about 30 carbons.

16. A composition according to Claim 15 wherein:
each R1 is independently perfluorinated alkyl of 1 to about 30 carbons.

17. A composition according to Claim 16 wherein:
R1 is CnF2n+1-(CH2)m-, where n is 1 to about 12 and m is 0 to about 18.

18. A composition according to Claim 17 wherein:
n is about 2 to about 10 and m is about 2 to about 14.

19. A composition according to Claim 18 wherein:
n is about 4 to about 8 and m is about 4 to about 10.

20. A composition according to Claim 1 wherein:
- R2 is a direct bond or an alkylene linking group of 1 to about 4 carbons;
R3 is a direct bond or an alkylene diradical of 1 to about 4 carbons; and each of R4, R5 and R6 is independently hydrogen or alkyl of 1 to about 4 carbons.

21. A composition according, to Claim 20 wherein:
R2 is an alkylene linking group of about 3 carbons;
R3 is an alkylene diradical of about 2 carbons;
each of R4 and R5 is hydrogen; and R6 is hydrogen or methyl.

22. A composition according to Claim 21 wherein:
R2 is -CH2-CH-CH2-;

R3 is -CH2CH2-; and R6 is methyl.

23. A composition according to Claim 1 which is a stabilized vesicular composition.

24. A composition according to Claim 23 wherein said vesicles are selected from the group consisting of micelles and liposomes.

25. A composition according to Claim 23 wherein said vesicles comprise gas-filled vesicles.

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

27. A composition according to Claim 1 wherein said gas comprises a fluorinated gas.

28. A composition according to Claim 27 wherein said fluorinated gas is selected from the group consisting of a perfluorocarbon gas and sulfur hexafluoride.

29. A composition according to Claim 28 wherein said perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane and perfluorocyclobutane.

30. A composition according to Claim 29 wherein said perfluorocarbon gas is selected from the group consisting of perfluoropropane and perfluorobutane .

31. A composition according to Claim 30 wherein said perfluorocarbon gas comprises perfluoropropane.

32. A composition according to Claim 1 wherein said gas is derived, at least in part, from a gaseous precursor.

33. A composition according to Claim 1 further comprising a gaseous precursor.

34. A composition according to Claim 33 wherein said gaseous precursor comprises a perfluorocarbon having a boiling point of greater than about room temperature.

35. A composition according to Claim 34 wherein said gaseous precursor is selected from the group consisting of perfluoropentane, perfluorohexane and perfluorooctane.

36. A composition according to Claim 35 wherein said gaseous precursor comprises perfluoropentane.

37. A composition according to Claim 1 further comprising a bioactive agent.

38. A composition according to Claim 1 which is for diagnostic imaging.

39. A composition according to Claim 38 wherein said imaging comprises ultrasound.

40. A composition comprising, in combination with a gas, a compound of the formula wherein:
m is 0 to about 18;
n is 1 to about 12; and w is 1 to about 8.

41. A composition according to Claim 40 wherein m is about 2 to about 14;
n is about 2 to about 10; and w is 1 to about 4.

42 A composition according to Claim 41 wherein m is about 4 to about 10;
n is about 4 to about 8; and w is about 2.

43. A composition according to Claim 40 which is a stabilized vesicular composition.

44. A composition according to Claim 43 wherein said vesicles are selected from the group consisting of micelles and liposomes.

45 A composition according to Claim 43 wherein said vesicles comprises gas-filled vesicles.

46. A composition according to Claim 40 wherein said gas is selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, fluorine, helium, argon, xenon and neon.

47. A composition according, to Claim 40 wherein 1 said gas comprises a fluorinated gas.

48. A composition according to Claim 47 wherein said fluorinated gas is selected from the group consisting of a perfluorocarbon gas and sulfur hexafluoride.

49. A composition according to Claim 48 wherein said perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane and perfluorocyclobutane.

50. A composition according to Claim 49 wherein said perfluorocarbon gas is selected from the group consisting of perfluoropropane and perfluorobutane .

51. A composition according to Claim 50 wherein said perfluorocarbon gas comprises perfluoropropane.

52. A composition according to Claim 40 wherein said gas is derived, at least in part, from a gaseous precursor.

53. A composition according to Claim 40 further comprising a gaseous precursor.

54. A composition according to Claim 53 wherein said gaseous precursor comprises a perfluorocarbon having a boiling point of greater than about room temperature.

55. A composition according to Claim 54 wherein said gaseous precursor is selected from the group consisting of perfluoropentane, perfluorohexane and perfluorooctane.

56. A composition according to Claim 55 wherein said gaseous precursor comprises perfluoropentane.

57. A composition according to Claim 40 further comprising a bioactive agent.

58. A stabilized composition comprising a gas and a sulfonated or phosphorylated fluorinated amphiphilic compound.

59. A composition according to Claim 58 which is a stabilized vesicular composition.

60. A composition according to Claim 59 wherein said vesicles are selected from the group consisting of micelles and liposomes.

61. A composition according to Claim 59 wherein said vesicles comprise gas-filled vesicles.

62. A composition according to Claim 58 wherein said gas is selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, fluorine, helium, argon, xenon and neon.

63. A composition according to Claim 58 wherein said gas comprises a fluorinated gas.

64. A composition according to Claim 63 wherein said fluorinated gas is selected from the group consisting of a perfluorocarbon gas and sulfur hexafluoride.

65. A composition according to Claim 64 wherein said perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane and perfluorocyclobutane.

66. A composition according to Claim 65 wherein said gas is selected from the group consisting of perfluoropropane and perfluorobutane.

67. A composition according to Claim 66 wherein said gas comprises perfluoropropane.

68. A composition according to Claim 58 wherein said fluorinated amphiphilic compound comprises a fluorinated lipid.

69. A composition according to Claim 68 wherein said fluorinated lipid comprises a fluorinated phospholipid.

70. A composition according to Claim 69 wherein an organic chain of said phospholipid is fluorinated.

71. A composition according to Claim 70 wherein said fluorinated phospholipid comprises a fluorinated monochain phospholipid.

72. A composition according to Claim 70 wherein said fluorinated phospholipid comprises a fluorinated polychain phospholipid.

73. A composition according to Claim 72 wherein at least one of said chains is nonfluorinated.

74. A composition according to Claim 58 further comprising a gaseous precursor.

75. A composition according to Claim 74 wherein said gaseous precursor comprises a perfluorocarbon having a boiling point of greater than about room temperature.

76. A composition according to Claim 75 wherein said gaseous precursor is selected from the group consisting of perfluoropentane, perfluorohexane and perfluorooctane.

77. A composition according to Claim 76 wherein said gaseous precursor comprises perfluoropentane.

78. A composition according to Claim 59 further comprising a bioactive agent.

79. A composition according to Claim 78 wherein said bioactive agent is substantially entrapped in said vesicles.

80. A stabilized composition consisting essentially of a gas and a fluorinated amphiphilic compound.

81. A stabilized composition consisting of a gas and a fluorinated amphiphilic compound.

82. A method for the preparation of a stabilized composition of a fluorinated amphiphilic compound and a gas comprising agitating an aqueous mixture of a fluorinated amphiphilic compound in the presence of a gas.

83. A method of providing an image of an internal region of a patient comprising (i) administering to the patient a contrast medium comprising a stabilized composition comprising a gas and a fluorinated amphiphilic compound, and (ii) scanning the patient using ultrasound to obtain visible images of the region.

84. A method of providing an image of an internal region of a patient comprising(i) administering to the patient a vesicular composition comprising, in an aqueous carrier, vesicles comprising a fluorinated amphiphilic compound and a gas or gaseous precursor; and (ii) scanning the patient using ultrasound to obtain a visible image of any diseased tissue in the patient.

85. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast medium comprising a stabilized composition comprising a gas and a fluorinated amphiphilic compound, and (ii) scanning the patient using ultrasound to obtain visible images of any diseased tissue in the patient.

86. A method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a formulation which comprises, in combination with a bioactive agent, a stabilizedcomposition of a fluorinated amphiphilic compound and a gas.