CA2214855A1 - A method of magnetic resonance focused surgical and therapeutic ultrasound - Google Patents

A method of magnetic resonance focused surgical and therapeutic ultrasound Download PDF

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Publication number
CA2214855A1
CA2214855A1 CA002214855A CA2214855A CA2214855A1 CA 2214855 A1 CA2214855 A1 CA 2214855A1 CA 002214855 A CA002214855 A CA 002214855A CA 2214855 A CA2214855 A CA 2214855A CA 2214855 A1 CA2214855 A1 CA 2214855A1
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Canada
Prior art keywords
vesicles
gas
magnetic resonance
ultrasound
patient
Prior art date
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Abandoned
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CA002214855A
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French (fr)
Inventor
Evan C. Unger
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ImaRx Pharmaceutical Corp
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Individual
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Publication date
Priority claimed from US08/401,974 external-priority patent/US5922304A/en
Application filed by Individual filed Critical Individual
Publication of CA2214855A1 publication Critical patent/CA2214855A1/en
Abandoned legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01R—MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES
    • G01R33/00—Arrangements or instruments for measuring magnetic variables
    • G01R33/20—Arrangements or instruments for measuring magnetic variables involving magnetic resonance
    • G01R33/44—Arrangements or instruments for measuring magnetic variables involving magnetic resonance using nuclear magnetic resonance [NMR]
    • G01R33/48—NMR imaging systems
    • G01R33/54—Signal processing systems, e.g. using pulse sequences ; Generation or control of pulse sequences; Operator console
    • G01R33/56—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution
    • G01R33/5601—Image enhancement or correction, e.g. subtraction or averaging techniques, e.g. improvement of signal-to-noise ratio and resolution involving use of a contrast agent for contrast manipulation, e.g. a paramagnetic, super-paramagnetic, ferromagnetic or hyperpolarised contrast agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • A—HUMAN NECESSITIES
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    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
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    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
    • A61K49/103—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA
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    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A61K49/16—Antibodies; Immunoglobulins; Fragments thereof
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1806—Suspensions, emulsions, colloids, dispersions
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1833—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule
    • A61K49/1839—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with a small organic molecule the small organic molecule being a lipid, a fatty acid having 8 or more carbon atoms in the main chain, or a phospholipid
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    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1851—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule
    • A61K49/1863—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle having a (super)(para)magnetic core coated or functionalised with an organic macromolecular compound, i.e. oligomeric, polymeric, dendrimeric organic molecule the organic macromolecular compound being a polysaccharide or derivative thereof, e.g. chitosan, chitin, cellulose, pectin, starch
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    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
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    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1878—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles the nanoparticle having a magnetically inert core and a (super)(para)magnetic coating
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Abstract

A novel method of magnetic resonance focused surgical ultrasound by administering to a patient a magnetic resonance imaging (MRI) contrast medium comprising gas filled vesicles, then scanning the patient with MRI techniques, and then applying ultrasound to effect surgery. These methods may also use an MRI contrast medium comprising gaseous precursor filled vesicles which undergo a phase transition from a liquid to gas in vivo after administration. Additionally, the MRI contrast medium may comprise a therapeutic compound.

Description

CA 022148~ 1997-09-08 W096/28090 PCT~S96/03054 A h~-l'~U~ OF MAGNETIC RESONANCE FOCUSED
SURGICAL AND T~PEUTIC ULTRASOUND

REFERENCE TO COPENDING APPLICATIONS

This application is a continuation-in-part o~
copending application Serial No. 08/401,974, ~iled March 9, 1995, which is a continuation-in-part o~ copending application Serial No. 08/212,S53, ~iled March 11, 1994, the disclosures o~ which are hereby incorporated herein by reference, in their entirety, and priority to which is hereby claimed.
Copending application Serial No. 08/076,250, ~iled June 11, 1993, which is a continuation-in-part of U.S. Serial Nos. 716,899 and 717,084, each ~iled ~une 18, 1991, which in turn are continuations-in-part o~ U.S. Serial No. 569,828, ~iled August 20, 1990, which in turn is a continuation-in-part o~ U.S. Serial No. 455,707, ~iled December 22, 1989, discloses therapeutic drug delivery systems comprising gas ~illed microspheres containing a therapeutic agent, with particular emphasis on the use of ultrasound techni~ues to monitor and determine the presence o~ said microspheres in a patient's body, and then to rupture said microspheres in order to release said therapeutic agent in the region o~ the patient~s body where said microspheres are ~ound.
Copending application Serial No. 08/076,239, filed - June 11, 1993, which has the identical parentage o~ preceding applications as Serial No. 08/076,250 set out immediately above, discloses methods and apparatus ~or preparing gas ~illed microspheres suitable ~or use as contrast agents ~or ultrasonic CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 imaging or as drug delivery agents.
Copending applications Serial No. 08/307,305, filed September 16, 1994, and copending applications Serial No.
08/159,687 and Serial No. 08/160,232, both of which were filed November 30, 1993, which in turn are continuation-in-parts, respectively, of applications Serial No. 08/076,239 and U.S.
Serial No. 08/076,250, both of which were filed June 11, 1993, disclose novel therapeutic delivery systems and methods of preparing gas and gaseous precursor filled microspheres and multiphase lipid and gas compositions useful in diagnostic and therapeutic applications.
Benefit of the filing dates of applications Serial Nos. 08/307,305, 08/159,687, 08/160,232, 08/076,239 and 08/076,250, and their parentage, is hereby claimed, and they are incorporated herein by reference in their entirety.
Reference is also made to application Serial No.
07/507,125, filed April 10, 1990, which discloses the use of biocompatible polymers, either alone or in admixture with one or more contrast agents such as paramagnetic, superparamagnetic or proton density contrast agents. The polymers or polymer/contrast agent admixtures may optionally be admixed with one or more biocompatible gases to increase the relaxivity of the resultant preparation.

R~ROuND OF THE lNV~N-llON

Field of the Invention This invention relates to the field of magnetic resonance imaging, more specifically to the use of stabilized gas filled vesicles as contrast media for magnetic resonance imaging (MRI) directed ultrasound surgery.
There are a variety of imaging techniques that have been used to diagnose disease in hllm~n~ One of the ~irst imaging techniques employed was X-rays. In X-rays, the images produced of the patients' body reflect the different densities of body structures. To improve the diagnostic utility of this imaging technique, contrast agents are employed to increase the CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~

density of tissues of interest as compared to surrounding tissues to make the tissues of interest more visible on X-ray.
Barium and iodinated contrast media, for example, are used extensively for X-ray gastrointestinal studies to visualize the esophagus, stomach, intestines and rectum. Likewise, these contrast agents are used for X-ray computed tomographic studies (that is, computer assisted tomography or CAT) to improve visualization of the gastrointestinal tract and to provide, for example, a contrast between the tract and the structures adjacent to it, such as the vessels or lymph nodes. Such contrast agents permit one to increase the density inside the esophagus, stomach, intestines and rectum, and allow differentiation of the gastrointestinal system from surrounding structures.
Magnetic resonance imaging (MRI) is a relatively new imaging technique which, unlike X-rays, does not utilize ionizing radiation. Like computer assisted tomography (CAT), MRI can make cross-sectional images of the body, however MRI
has the additional advantage of being able to make images in any scan plane (i.e., axial, coronal, sagittal or orthogonal).
Unfortunately, the full utility of MRI as a diagnostic modality ~or the body is hampered by the need ~or new or better contrast agents. Without suitable agents, it is often difficult using MRI to differentiate the target tissue from adjacent tissues.
If better contrast agents were available, the overall usefulness of MRI as an imaging tool would improve, and the diagnostic accuracy of this modality would be greatly enhanced.
MRI employs a magnetic field, radio frequency energy and magnetic field gradients to make images of the body. The contrast or signal intensity di~ferences between tissues mainly re~lect the T1 (longitudinal) and T2 (transverse) relaxation values and the proton density (effectively, the free water content) o~ the tissues. In changing the signal intensity in a region of a patient by the use of a contrast medium, several ~ 35 possible approaches are available. For example, a contrast medium could be designed to change either the T1, the T2 or the proton density.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~4 Brief DescriPtion of the Prior Art In the past, attention has mainly been focused on paramagnetic contrast media for MRI. Paramagnetic contrast agents contain unpaired electrons which act as small local magnets within the main magnetic field to increase the rate of longitudinal (T1) and transverse (T2) relaxation. Most paramagnetic contrast agents are metal ions which in most cases are toxic. In order to decrease toxicity, these metal ions are generally chelated using ligands. The resultant paramagnetic metal ion complexes have decreased toxicity. Metal oxides, most notably iron oxides, have also been tested as MRI contrast agents. While small particles of iron oxide, e.g., under 20 nm diameter, may have paramagnetic relaxation properties, their predominant e~fect is through bulk susceptibility. Therefore magnetic particles have their predominant effect on T2 relaxation. Nitroxides are another class of MRI contrast agent which are also paramagnetic. These have relatively low relaxivity and are generally less effective than paramagnetic ions as MRI contrast agents. All of these contrast agents can suffer from some toxic e~fects in certain use contexts and none of them are ideal for use as perfusion contrast agents by themselves.
The existing MRI contrast agents suffer ~rom a number of limitations. For example, positive contrast agents are known to exhibit increased image noise arising ~rom intrinsic peristaltic motions and motions ~rom respiration or cardiovascular action. Positive contrast agents such as Gd-DTPA are subject to the further complication that the signal intensity depends upon the concentration of the agent as well as the pulse sequence used. Absorption of contrast agent from the gastrointestinal tract, for example, complicates interpretation of the images, particularly in the distal portion o~ the small intestine, unless su~iciently high concentrations of the paramagnetic species are used (Kornmesser et al., Maqn. Reson. Imaqinq, 6:124 (1988)). Negative contrast agents, by comparison, are less sensitive to variation in pulse sequence and provide more consistent contrast. However at high CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~4 concentrations, particulates such as ferrites can cause magnetic susceptibility artifacts which are particularly evident, for example, in the colon where the absorption of intestinal fluid occurs and the superparamagnetic material may be concentrated. Negative contrast agents typically exhibit superior contrast to fat, however on T1-weighted images, positive contrast agents exhibit superior contrast versus normal tissue. Since most pathological tissues exhibit longer T1 and T2 than normal tissue, they will appear dark on Tl-weighted and bright on T2-weighted images. This would indicate that an ideal contrast agent should appear bright on T1-weighted images and dark on T2-weighted images. Many of the currently available MRI contrast media fail to meet these dual criteria.
Toxicity is another problem with the existing contrast agents. With any drug there is some toxicity, the toxicity generally being dose related. With the ferrites there are often symptoms of nausea after oral administration, as well as flatulence and a transient rise in serum iron. The paramagnetic contrast agent Gd-DTPA is an organometallic complex of gadolinium coupled with the complexing agent diethylene triamine pentaacetic acid. Without coupling, the free gadolinium ion is highly toxic. Furthermore, the peculiarities of the gastrointestinal tract, for example, wherein the stomach secretes acids and the intestines release alkalines, raise the possibility of decoupling and separation of the free gadolinium or other paramagnetic agent from the complex as a result of these changes in pH during gastrointestinal use. Certainly, minimizing the dose of paramagnetic agents is important for minimizing any potential toxic effects.
New and/or better contrast agents useful in magnetic resonance imaging as well as improved imaging techniques are needed. The present invention is directed, inter alia, to these important ends.
In the work on MRI contrast agents described above for application Serial No. 07/507,125, filed April 10, 1990, CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 it has been disclosed how gas can be used in combination with polymer compositions and paramagnetic or superparamagnetic agents as MRI contrast agents. Therein it has been shown how the gas stabilized by said polymers would function as an effective susceptibility contrast agent to decrease signal intensity on T2 weighted images; and that such systems are particularly effective for use as gastrointestinal MRI contrast media.
Widder et al. published application EP-A-O 324 938 discloses stabilized microbubble-type ultrasonic imaging agents produced from heat-denaturable biocompatible protein, e . g., albumin, hemoglobin, and collagen.
There is also mentioned a presentation believed to have been made by Moseley et al., at a 1991 Napa, California meeting of the Society for Magnetic Resonance in Medicine, which is summarized in an abstract entitled "Microbubbles: A
Novel MR Susceptibility Contrast Agent." The microbubbles which are utilized comprise air coated with a shell of hl~m~n albumin. The stabilized gas filled vesicles of the present invention are not suggested.
For intravascular use, however, the inventors have found that ~or optimal results, it is advantageous that any gas be stabilized with flexible compounds. Proteins such as albumin may be used to stabilize the bubbles but the resulting bubble shells may be brittle and inflexible. This is undesirable for several reasons. Firstly, a brittle coating limits the capability of the bubble to expand and collapse.
As the bubble encounters different pressure regions within the body ( e . g., moving from the venous system into the arteries upon circulation through the heart) a brittle shell may break and the gas will be lost. This limits the e~fective period of time for which useful contrast can be obtained in vivo from the bubbles contrast agents. Also such brittle, broken fragments can be potentially toxic. Additionally the inflexible nature of brittle coatings such as albumin, and stiff resulting bubbles make it extremely difficult to measure pressure in vi vo .

CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 Quay published application WO 93/05819 discloses that gases with high Q numbers are ideal for forming stable gases, but the disclosure is limited to stable gases, rather than their stabilization and encapsulation, as in the present invention. In a preferred embodiment described on page 31, sorbitol is used to increase viscosity, which in turn is said to extend the li~e of a microbubble in solution. Also, it is not an essential requirement of the present invention that the gas involved have a certain Q number or diffusibility factor.
Lanza et al. published application WO 93/20802 discloses acoustically re~lective oligolamellar liposomes, which are multilamellar liposomes with increased aqueous space between bilayers or have liposomes nested within bilayers in a nonconcentric fashion, and thus contain internally separated bilayers. Their use as ultrasonic contrast agents to enhance ultrasonic imaging, and in monitoring a drug delivered in a liposome administered to a patient, is also described.
D'Arrigo U.S. Patents 4,684,479 and 5,215,680 disclose gas-in-liquidemulsions andlipid-coated microbubbles, respectively.
In accordance with the present invention it has been discovered that stabilized gas filled vesicles are extremely e~ective, non-toxic contrast agents ~or simultaneous magnetic resonance focused noninvasive ultrasound.

SUMMARY OF THE lN V~N-l lON

The present invention is directed to a method o~
magnetic resonance imaging focused surgical and therapeutic ultrasound comprising administering a contrast medium for magnetic resonance imaging comprising gas filled vesicles to a patient requiring surgery, using said contrast medium to scan the patient with magnetic resonance imaging to identi~y the region of the patient requiring surgery, and applying ~ ultrasound to the region to carry out surgery. The application of ultrasound may be ~ollowed by a second scanning step whereby the patient is scanned with magnetic resonance imaging. The CA 022148~ 1997-09-08 W096/28090 PCT~S96103054 ultrasound application may be ~simultaneous with magnetic resonance imaging. The sC~nn;ng and surgical ultrasound steps may be performed repeatedly until the desired effect is achieved. The gas filled vesicles may comprise a therapeutic which may be released to a localized region of a patient upon ultrasound.
In addition, the present invention comprises a method for the controlled delivery of a therapeutic to a region of a patient using magnetic resonance imaging focused therapeutic ultrasound comprising administering to the patient vesicles comprising gas-filled vesicles comprising a therapeutic compound; monitoring the vesicles using magnetic resonance imaging to determine the presence of the vesicles in the region; and rupturing the vesicles using ultrasound to release the therapeutic in the region.
The present invention is also directed to a method of magnetic resonance focused surgical ultrasound comprising administering a contrast medium for magnetic resonance imaging comprising gaseous precursor filled vesicles to a patient requiring surgery, allowing the gaseous precursor to undergo a phase transition from a liquid to a gas, scanning the patient with magnetic resonance imaging to identi~y the region o~ the patient requiring surgery, and applying surgical ultrasound to the region. The phase transition step and the magnetic resonance scanning step may be performed simultaneously.
The contrast medium comprises stabilized gas filled vesicles, wherein the gas is a biocompatible gas, e . g., nitrogen or perfluoro-propane, but may also be derived from a gaseous precursor, e.g., perfluorooctylbromide, and the vesicles are stabilized by being formed ~rom a stabilizing compound, e . g ., a biocompatible lipid or polymer. The present invention may be carried out, often with considerable attendant advantage, by using gaseous precursors to form the gas of the gas filled vesicles. These gaseous precursors may be activated by a number of factors, but preferably are temperature activated. Such a gaseous precursor is a compound which, at a selected activation or transition temperature, changes phases CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~4 from a liquid or solid to a gas.- Activation thus takes place by increasing the temperature of the compound from a point below, to a point above the activation or transition temperature. Where a lipid is used to ~orm the vesicle, the lipid may be in the form of a monolayer or bilayer, and the mono- or bilayer lipids may be used to form a series of concentric mono- or bilayers. Thus, the lipid may be used to ~orm a unilamellar liposome (comprised o~ one monolayer or bilayer lipid), an oligolamellar liposome (comprised of two or three monolayer or bilayer lipids) or a multilamellar liposome (comprised of more than three monolayer or bilayer lipids).
Pre~erably, the biocompatible lipid comprises a phospholipid.
Optionally, the contrast medium may include paramagnetic and/or superparamagnetic contrast agents, preferably encapsulated by the vesicles. Also, optionally, the contrast medium may further comprise a liquid ~luorocarbon compound, e.g., a perfluorocarbon, to ~urther stabilize the vesicles. Preferably the fluorocarbon liquid is encapsulated by the vesicles.
These and other aspects of the invention will become more apparent ~rom the following detailed description.

DET~TT.T~n DESC~IPTION OF THE lNV ~:N-llON

The present invention is directed, inter alla, to a method o~ magnetic resonance imaging focused surgical and therapeutic ultrasound comprising administering a contrast medium for magnetic resonance imaging comprising gas ~illed vesicles to a patient requiring surgery, using contrast medium to scan the patient with magnetic resonance imaging to identi~y the region o~ the patient requiring surgery, and applying ultrasound to the region to carry out surgery. The ultrasound step may be per~ormed simultaneously with magnetic resonance imaging. The application o~ ultrasound may be ~ollowed by a second s~nn;ng step whereby the patient is scanned with magnetic resonance imaging. The gas ~illed vesicles may comprise a therapeutic which may be released to a localized region o~ a patient upon ultrasound.

CA 022l48~ 1997-09-08 W096/28090 PCT~S96/03054 The scanning and surgical ultrasound steps may be performed repeatedly until the desired effect is achieved. In accordance with the present invention, simultaneous refers to scanning with ultrasound and magnetic resonance concurrently or synchronously; sequentially or successively; such that visualization of the disruption of vesicles and tissues by ultrasound is observed. Thus, ultrasound and magnetic resonance may be performed at the same time, or one may be followed by the other. The use of magnetic resonance imaging together with ultrasound improves the accuracy of currently available imaging modalities. The precision of magnetic resonance imaging and ultrasound together confirm the location of the vesicles, as the entire body is able to be scanned by magnetic resonance imaging which provides a large field of view, and, once located, the vesicles may be ruptured by ultrasound in the given regions of the body.
The present invention is also directed to a method of magnetic resonance focused surgical ultrasound comprising administering a contrast medium for magnetic resonance imaging comprising gaseous precursor filled vesicles to a patient requiring surgery, allowing the gaseous precursor to undergo a phase transition from a liquid to a gas, using said contrast medium to scan the patient with magnetic resonance imaging to identi~y the region of the patient requiring surgery, and applying surgical ultrasound to the region.
In addition, the present invention comprises a method for the controlled delivery of a therapeutic to a region of a patient using magnetic resonance focused therapeutic ultrasound comprising administering to the patient contrast medium comprising gas-filled vesicles comprising a therapeutic compound; monitoring the vesicles using magnetic resonance imaging to determine the presence o~ the vesicles in the region; and rupturing the vesicles using ultrasound to release the therapeutic in the region.
As employed above and throughout the disclosure, the ~ollowing terms, unless otherwise indicated, shall be understood to have the ~ollowing meanings.

CA 022148~ 1997-09-08 W096/28090 PCT~S96/03054 "Magnetic resonance imaging" (MRI) uses a static main magnetic ~ield; pulsed radio~requency energy and pulsed magnetic gradients to create images, i.e. to visualize the vesicles. The radiofrequency and electrical gradients may be used to cause local energy deposition and activate the vesicles, however, ultrasound is the pre~erred energy ~or the purpose o~ activating the vesicles. In carrying out the magnetic resonance imaging method o~ the present invention, the contrast medium can be used alone, or in combination with other diagnostic, therapeutic or other agents. Such other agents include excipients such as ~lavoring or coloring materials.
The magnetic resonance imaging techniques which are employed are conventional and are described, ~or example, in D.M. Kean and M.A. Smith, Maqnetic Resonance Imaqinq: Princi~les and Ap~lications, (William and Wilkins, Baltimore 1986).
Contemplated MRI techniques include, but are not limited to, nuclear magnetic resonance (NMR) and electronic spin resonance (ESR), and magnetic resonance angioplasty (MRA). The pre~erred imaging modality is NMR. O~ course, in addition to MRI, magnetic imaging may also be used to detect vesicles within the scope o~ the present invention. Magnetic imaging uses a magnetic ~ield yet need not use pulsed gradients or radio~requency energy. Magnetic imaging may be used to detect magnetic vesicles, such as and not limited to ferromagnetic vesicles. Magnetic imaging may be per~ormed by a magnetometer superconducting quantum in~erometry device (SQUID). SQUID
permits rapid screening o~ all of the body tissues ~or the magnetic particles; the ultrasound may then be localized to those regions. In this application, magnetic resonance imaging includes magnetic imaging, while it is understood that magnetic imaging is the imaging o~ magnetic vesicles and does not include resonance of the nucleii thereo~.
"Ultrasound imaging" is per~ormed on the tissues o~
interest and ultrasound energy may be used to activate or rupture the vesicles once they reach their intended tissue destination. Focused or directed ultrasound re~ers to the application o~ ultrasound energy to a particular region o~ the CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 body, such that the ultrasound energy is concentrated to a selected area or target zone. In addition, focused refers to the magnetic resonance which guides the ultrasound by visualizing the vesicles and the target zone; and simultaneous with ultrasound visualizing the disruption of tissues thereby.
Noninvasive refers to the disruption or disturbance o~ internal body tissues without an incision in the skin. Ultrasound, as deflned in accordance with the present invention refers to surgery resulting in tissue necrosis, i.e. disruption or destruction of tissue; repair o~ apertures, openings, breaks, or tears in tissue (such as a hernia); alleviation of all or part of diseased tissue (such as tumors); and the activation or rupture of vesicles adjacent to tissue by ultrasonic energy.
Ultrasound is a diagnostic imaging technique which is unlike nuclear medicine and X-rays since it does not expose the patient to the harmful effects of ionizing radiation.
Moreover, unlike magnetic resonance imaging, ultrasound is relatively inexpensive and can be conducted as a portable ~m; n~tion. In using the ultrasound technique, sound is transmitted into a patient or animal via a transducer. When the sound waves propagate through the body, they encounter inter~aces ~rom tissues and fluids. Depending on the acoustic properties of the tissues and ~luids in the body, the ultrasound sound waves are partially or wholly reflected or absorbed. When sound waves are reflected by an inter~ace they are detected by the receiver in the transducer and processed to form an image. The acoustic properties of the tissues and fluids within the body determine the contrast which appears in the resultant image. Alternatively, ultrasound may be used to visualize the vesicles and magnetic resonance imaging may be used to activate the vesicles. In addition, the strength o~
ultrasound energy may be at an intensity to result in rupture or activation of vesicles. The activation o~ the vesicles in turn disrupts the adjacent tissue such that necrosis of the tissue results.
Any of the various types of diagnostic ultrasound imaging devices may be employed in the practice o~ the CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 invention, the particular type or model of the device not being critical to the method of the invention. Also suitable are devices designed for administering ultrasonic hyperthermia, such devices being described in U.S. Patent Nos. 4,620,546, 4,658,828, and 4,586,512, the disclosures of each of which are hereby incorporated herein by reference in their entirety.
Preferably, the device employs a resonant frequency (RF) spectral analyzer. The transducer probes may be applied externally or may be implanted. Ultrasound is generally initiated at lower intensity and duration, pre~erably at peak resonant frequency, and then intensity, time, and/or resonant frequency increased until the microsphere ruptures.
"Vesicle" re~ers to a spherical entity which is characterized by the presence of an internal void. Preferred vesicles are formulated ~rom lipids, including the various lipids described herein. In any given vesicle, the lipids may be in the form of a monolayer or bilayer, and the mono- or bilayer lipids may be used to form one or more mono- or bilayers. In the case of more than one mono- or bilayer, the mono- or bilayers are generally concentric. The vesicles described herein include such entities commonly referred to as liposomes, micelles, bubbles, microbubbles, aerogels, clathrate bound vesicles, and the like. Thus, the lipids may be used to ~orm a unilamellar vesicle (comprised of one monolayer or bilayer), an oligolamellar vesicle (comprised o~ 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 ~illed with a liquid, including, ~or example, an aqueous liquid, a gas, a gaseous precursor, and/or a solid or solute material, including, for example, a targeting ligand and/or a bioactive agent, as desired.
~ Liposome" re~ers to a generally spherical cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers. Most pre~erably the gas ~illed liposome is constructed of a single layer (i.e. unilamellar) or a single monolayer of CA 022l48~ l997-09-08 W096/~090 PCT~S96/030~

lipid. A wide variety of lipids may be used to fabricate the liposomes including phospholipids and non-ionic surfactants (e.g. niosomes). Most preferably the lipids comprising the gas filled liposomes are in the gel state at physiological temperature. The liposomes may be cross-linked or polymerized and may bear polymers such as polyethylene glycol on their surfaces. Targeting ligands directed to endothelial cells are bound to the surface of the gas filled liposomes. A targeting ligand is a substance which is bound to a vesicle and directs the vesicle to a particular cell type such as and not limited to endothelial tissue and/or cells. The targeting ligand may be bound to the vesicle by covalent or non-covalent bonds. The liposomes may also be referred to herein as lipid vesicles.
Most preferably the liposomes are substantially devoid of water in their interiors.
"Micelle" refers to colloidal entities which form from lipidic compounds when the concentration of the lipidic compounds, such as lauryl sulfate, is above a critical concentration. Since many of the compounds which form micelles also have surfactant properties (i.e. ability to lower surface tension and both water and fat loving - hydrophilic and lipophilic domains), these same materials may also be used to stabilize bubbles. In general these micellular materials prefer to adopt a monolayer or hexagonal H2 phase configuration, yet may also adopt a bilayer configuration.
When a micellular materlal is used to ~orm a gas filled vesicle, the compounds will generally adopt a radial configuration with the aliphatic (fat loving) moieties oriented toward the vesicle and the hydrophilic domains oriented away from the vesicle surface. For targeting to endothelial cells, the targeting ligands may be attached to the micellular compounds or to amphipathic materials admixed with the micellular compounds. Alternatively, targeting ligands may be adsorbed to the sur~ace o~ the micellular materials stabilizing the vesicles.
~ 'Aerogel" refers to structures which are similar to microspheres except that the internal structure o~ the aerogels CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 is generally comprised of multiple small voids rather than one void. Additionally the aerogels are pre~erably constructed o~
synthetic materials (e.g. a ~oam prepared ~rom baking resorcinol and ~ormaldehyde), however natural materials such as polysaccharides or proteins may also be used to prepare aerogels. Targeting ligands may be attached to the sur~ace of the aerogel.
~ Clathrates" are generally solid materials which bind the vesicles as a host rather than coating the sur~ace o~ the vesicle. A solid, semi-porous, or porous clathrate may serve as the agent stabilizing the vesicle, however the clathrate itsel~ does not coat the entire sur~ace o~ the vesicle.
Rather, the clathrate forms a structure known as a "cage"
having spaces into which the vesicles may ~it. One or more vesicles may be adsorbed by the clathrate. Similar to microspheres, one or more surfactants may be incorporated with the clathrate and these sur~actants will help to stabilize the vesicle. The sur~actants will generally coat the vesicle and help to maintain the association o~ the vesicle with the clathrate. Use~ul clathrate materials ~or stabilizing vesicles include porous apatites such as calcium hydroxyapatite and precipitates o~ polymers with metal ions such as alginic acid with calcium salts. Targeting ligands directed to endothelial cells may be incorporated into the clathrate itsel~ or into the sur~actant material used in association with the clathrate.
While not intending to be bound by any particular theory o~ operation, the present invention is believed to rely, at least in part, on the ~act that gas, liquid, and solid phases have di~erent magnetic susceptibilities. At the inter~ace o~ gas and water, ~or example, the magnetic domains are altered and this results in dephasing o~ the spins o~, e.g., the hydrogen nuclei. In imaging, this is seen as a decrease in signal intensity adjacent to the gas/water inter~ace. This e~fect is more marked on T2 weighted images = 35 and most prominent on gradient echo pulse sequences. The e~ect is increased by using narrow bandwidth extended read-out pulse sequences. The longer the echo time on a gradient echo CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 pulse sequence, the greater the effect (i. e., the greater the degree and size of signal loss).
The stabilized gas filled vesicles useful in the present invention are believed to rely on this phase magnetic susceptibility difference, as well as on the other characteristics described in more detail herein, to provide high per~ormance level magnetic resonance imaging contrast medium and effective rupture of vesicles of the contrast medium. The vesicles are formed from, i . e., created out of, a matrix of stabilizing compounds which permit the gas filled vesicles to be established and thereafter retain their size and shape for the period of time required to be useful in magnetic resonance imaging. The compounds also permit rupture of the vesicles at a certain energy level, which energy is preferably ultrasound energy. These stabilizing compounds are most typically those which have a hydrophobic/hydrophilic character which allows them to ~orm monolayers or bilayers, etc., and vesicles, in the presence o~ water. Thus, water, saline or some other water-based medium, often referred to hereafter as a diluent, is generally an aspect of the stabilized gas ~illed vesicle contrast medium of the present invention.
The stabilizing compound may, in ~act, be a mixture o~ compounds which contribute various desirable attributes to the stabilized vesicles. For example, compounds which assist in the dissolution or dispersion o~ the ~undamental stabilizing compound have been found advantageous. A ~urther element of the stabilized vesicles is a gas, which can be a gas at the time the vesicles are made, or can be a gaseous precursor which, responsive to an activating factor, such as temperature, 30 is transformed from the liquid or solid phase to the gas phase.
The various aspects of the stabilized gas filled contrast medium useful in the present invention will now be described.

CA 022l48~ l997-09-08 W096J28090 PCT~S96/03054 Method~ o~ U~e In accordance with the present invention there is provided a method of simultaneous magnetic resonance focused noninvasive ultrasound. The imaging process of the present invention may be carried out by administering a contrast medium for magnetic resonance imaging comprising gas filled vesicles to a patient requiring surgery, scanning the patient with magnetic resonance imaging to identify the region of the patient requiring surgery, and simultaneously applying ultrasound and magnetic resonance to the region. By region of a patient, it is meant the whole patient or a particular area or portion of the patient.
After administration to a patient, the vesicles, which are visible on magnetic resonance imaging, are visualized by MRI. When the location of the vesicles is determined to be in the desired region of the patient, as ascertained by MRI, then energy, preferably ultrasound energy, is applied to the region. The vesicles are activated by the energy, heating and direct and rapid coagulative necrosis of the surrounding tissue (i.e. surgical ultrasound) results. Simultaneously, the region may also be visualized by magnetic resonance imaging if desired. Pre~erably, the energy used ~or vesicle activation is high energy continuous wave ultrasound, preferably over 50 milliwatts/cm2, even more pre~erably over 100 milliwatts/cm2.
Depending upon the desired therapeutic effect, the energy may be even higher, up to about 10 watts/cm2. Most preferably, the energy is deposited into the tissues using a hand held magnetic resonance compatible ultrasound transducer. The ultrasound transducer is made out of non-ferrous and non-ferromagnetic material. The cables supplying energy to the ultrasound transducers may have Faraday shields to decrease the potential for artifacts which may be caused by the electrical energy passing through the cables to supply the transducers.
The amount of energy and pulse duration of ultrasound used for therapy will vary depending upon the therapeutic purpose. The ultrasonic energy is preferably focused and the ~ocal zone is chosen to target the desired regions of vesicles.

CA 022148~ 1997-09-08 W096/28090 PCT~S96/03054 Focused ultrasonic surgery may be performed at energies of about 2 watts/cm2. Focused ultrasonic surgery energy may be at least 2 watts/cm2 to about lO watts/cm2.
Direct and rapid coagulative necrosis of the tissue results.
Simultaneous MRI may be performed with the vesicles used to visualize the target zone or region. Then together with ultrasound, the vesicles potentiate the surgery in the target zone.
Energy range of from about 500 mW/cm2 to about lO
watts/cm2, preferably greater than about l watt is useful for cavitational tissue destruction. The vesicles lower the cavitation threshold such that cavitation will occur within the target tissues at a low energy threshold resulting in tissue destruction.
Rupture or activation of vesicles may take place at an energy range of from about 50 mW/cm2 to about 500 mW/cm2.
Vesicles may be ruptured by non-cavitational interaction. As the vesicle is pulsed rapidly and strongly enough by ultrasound energy, the vesicle membrane degenerates. While there is likely a transient microdomain o~ increased temperature associated with the vesicle rupture, this process may not damage the surrounding tissues when energy and pulsing is applied at an indicated energy range. This effect of vesicle rupture may be advantageously used for localized delivery of a therapeutic. Thus, a therapeutic may be released to a region of the body with this technique. Further, energy from vesicle rupture may be used to create shock waves so that the therapeutic is also deposited released to adjacent tissues.
This is particularly useful with gene therapy wherein the shock waves may be used to open transient pores in adjacent cell membranes and facilitate cellular uptake of genetic material.
An energy range o~ about 500 mW/cm2 to about 5 watts/cm2, with vesicles as nuclei, may be used to increase the conversion o~ high energy sound into localized tissue, thereby heating the tissue and inducing hyperthermia.
In the case of a gaseous precursor, as ultrasound energy is focused on the precursor, it causes the precursor to CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 convert to the gaseous state. The enlarging gaseous void creates a domain of increasing magnetic susceptibility and is readily monitored on the magnetic resonance images. Monitoring is particularly enhanced by selecting precursors with well de~ined liquid to gas conversion temperatures, such as perfluorohexane at 56~C. The invention thus may also be used for non-invasive temperature monitoring during MRI. As the vesicles ~orm from gaseous precursors, the materials surrounding the gaseous precursor may be ruptured. In addition, a therapeutic may be released locally into the adjacent tissue where a therapeutic is co-entrapped within the vesicle. As the vesicle forms, the absorption of energy by the vesicle interface increases. This may be used to increase heating for hyperthermia as well as to rupture the vesicle.
The contrast medium may be particularly useful in providing images of and permitting ultrasound mediated surgery and/or drug delivery in the cardiovascular region or the gastrointestinal region, but can also be employed more broadly such as in imaging the vasculature or in other ways as will be readily apparent to those skilled in the art. Cardiovascular region, as that phrase is used herein, denotes the region of the patient de~ined by the heart and the vasculature leading directly to and from the heart. The phrase gastrointestinal region or gastrointestinal tract, as used herein, includes the region of a patient de~ined by the esophagus, stomach, small and large intestines and rectum. The phrase vasculature, as used herein, denotes the blood vessels (arteries, veins, etc.) in the body or in an organ or part o~ the body. The patient can be any type of m~mm~l, but most preferably is a human.
The novel stabilized gas filled vesicles, useful as contrast medium in simultaneous magnetic resonance focused noninvasive ultrasound, will be ~ound to be suitable for use in all areas where MRI is employed.
As one skilled in the art would recognize, ~ 35 administration of the stabilized gas filled vesicles used in the present invention may be carried out in various fashions, such as intravascularly, orally, rectally, etc., using a CA 022148~ 1997-09-08 W096/28090 PCT~S96/030 variety of dosage forms. When the region to be scanned is the cardiovascular region, administration of the contrast medium of the invention is preferably carried out intravascularly.
When the region to be scanned is the gastrointestinal region, administration of the contrast medium of the invention is preferably carried out orally or rectally. The useful dosage to be administered and the particular mode of administration will vary depending upon the age, weight and the particular m~mm~l and region thereof to be scanned, and the particular contrast medium of the invention to be employed. Typically, dosage is initiated at lower levels and increased until the desired contrast enhancement is achieved. Various combinations of the stabilized gas filled vesicles may be used to modify the relaxation behavior of the medium or to alter properties such as the viscosity, osmolarity or palatability (in the case of orally administered materials). In carrying out the simultaneous magnetic resonance focused noninvasive ultrasound method of the present invention, the contrast medium can be used alone, or in combination with other diagnostic, therapeutic or other agents. Such other agents include excipients such as ~lavoring or coloring materials. The magnetic resonance imaging techniques which are employed are conventional and are described, for example, in D.M. Kean and M.A. Smith, Maqnetic Resonance Imaqinq: Principles and A~lications, (William and Wilkins, Baltimore 1986).
Contemplated MRI techniques include, but are not limited to, nuclear magnetic resonance (NMR) and electronic spin resonance (ESR). The preferred imaging modality is NMR.
As noted above, the routes of administration and areas of usefulness of the gas ~illed vesicles are not limited merely to the blood volume space, i.e., the vasculature.
Simultaneous magnetic resonance ~ocused noninvasive ultrasound can be achieved with the gas ~illed vesicles used in the present invention if the vesicles are ingested by mouth so as to image the gastrointestinal tract and rupture the vesicles therein. Alternatively, rectal administration of these stabilized gas vesicles can result in excellent imaging of the CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 lower gastrointestinal tract including the rectum, descending colon, transverse colon, and ascending colon as well as the appendix, and rupturing the vesicles therein. It may be possible to achieve imaging o~ the jejunum and conceivably the 5 ileum via this rectal route; and to rupture the vesicles in > these areas. As well, direct intraperitoneal administration may be achieved to visualize the peritoneum, and rupture the vesicles therein. It is also contemplated that the stabilized gas vesicles may be administered directly into the ear canals 10 such that one can visualize the canals as well as the Eustachian tubes and, if a perforation exists, the inner ear.
Further, activation or rupture of the vesicles in the ear may also take place. It is also contemplated that the stabilized gas vesicles may be administered intranasally to aid in the 15 visualization of the nasal septum as well as the nasal sinuses, and rupture of the vesicles therein. Interstitial administration is also possible.
Other routes of administration of the vesicle contrast agents of the present invention, and tissue areas 20 whose imaging and rupture of the vesicles is enhanced thereby include, but are not limited to 1) intranasally for imaging the nasal passages and sinuses including the nasal region and sinuses and sinusoidsi 2) intranasally and orally for imaging the remainder of the respiratory tract, including the trachea, 25 bronchus, bronchioles, and lungs; 3) intracochlearly ~or imaging the hearing passages and Eustachian tubes, tympanic membranes and outer and inner ear and ear canals; ~) intraocularly for imaging the regions associated with vision;
5) intraperitoneally to visualize the peritoneum; and 6) 30 intravesicularly, i.e., through the bladder, to image all regions of the genitourinary tract via the areas thereof, including, but not limited to, the urethra, bladder, ureters, kidneys and renal vasculature and beyond, e.g., to perform cystography or to confirm the presence of ureteral reflux. In ~ 35 addition, the brain, spine, pulmonay region, and soft tissues such as and not including adipose tissue, muscle, and organs may be similarly imaged and surgery of these areas may be CA 022148~ 1997-09-08 W096/28090 PCT~S96103054 achieved by ultrasound.
Use of the procedures of the invention permit ultrasound mediated surgery. By ultrasound mediated surgery it is meant surgery effectively causing tissue necrosis, i.e.
disruption, destruction, or repair of tissue, such as repair o~ small tears (apertures, openings, or breaks) in tissue membranes (such as a hernia); alleviation of all or part of diseased tissue (such as tumors); and the activation or rupture of vesicles adjacent to tissue by ultrasonic energy.

Gases and Gaseous Precursor~
The vesicles of the invention encapsulate a gas and/or gaseous precursor. The term "gas filled and/or gaseous precursor filled", as used herein, means that the vesicles to which the present invention is directed, have an interior volume that is comprised of at least about lO~ gas and gaseous precursor, preferably at least about 25~ gas and gaseous precursor, more preferably at least about 50~ gas and gaseous precursor, even more pre~erably at least about 75~ gas and gaseous precursor, and most preferably at least about 9O~ gas and gaseous precursor. In use, where the presence o~ gas is important, it is pre~erred that the interior vesicle volume comprise at least about lO~ gas, preferably at least about 25~, 50~, 75%, and most preferably at least about 90~ gas.
Any of the various biocompatible gases and gaseous precursors may be employed in the gas and gaseous precursor filled vesicles of the present invention. Such gases include, for example, air, nitrogen, carbon dioxide, oxygen, argon, fluorine, xenon, neon, helium, rubidium enhanced (hyperpolarized) xenon, rubidium enhanced argon, rubidium enhanced helium, and rubidium enhanced neon, or any and all combinations thereo~. O~ such gases, nitrogen and ~luorine are pre~erred. For example, the use o~ NMR together with l9F
provides more sensitive visualization than the use of a li~uid or a solid. Likewise, various fluorinated gaseous compounds, such as various perfluorocarbon, hydrofluorocarbon, and sulfur hexafluoride gases may be utilized in the preparation o~ the CA 022l48~ l997-09-08 W096128090 PCT~S96/03054 gas filled vesicles. Also, the gases discussed in Quay, published application WO 93/05819, including the high "Q"
factor gases described therein, the disclosures of which are hereby incorporated herein by reference in their entirety, may be employed. Further, paramagnetic gases or gases such as 170 may be used. The oxygen should be stabilized, since oxygen gas is soluble in blood. Stabilization may be accomplished by an impermeable shell, preferably of a polymerized or cross-linked liposome or a cyanoacrylate microsphere; or used together with a perfluorocarbon, such as perfluoropentane or perfluorobutane.
Of all of the gases, perfluorocarbons and sul~ur hexafluoride are preferred. Suitable perfluorocarbon gases include, ~or example, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane, perfluoropropane, and perfluoropentane, perfluorohexane, most preferably perfluoropropane. Also preferred are a mixture of different types of gases, such as a per~luorocarbon gas and another type of gas such as oxygen, etc. Indeed, it is believed that a combination of gases may be particularly useful in simultaneous magnetic resonance focused noninvasive ultrasound applications.
The gaseous precursors may also be in the form of a solid. Sodium bicarbonate crystals produce carbon dioxide gas upon activation o~ the solid precursor ~orm. Solid and liquid gaseous precursors are particularly useful in ultrasonic hyperthermia which activates the precursor into the gaseous state.
Notwithstanding the requirement that the gas and gaseous precursor ~illed vesicles be made ~rom stabilizing compounds, it is pre~erred that a rather highly stable gas be utilized as well. By highly stable gas is meant a gas selected from those gases which will have low (limited) solubility and dif~usability in aqueous media. Gases such as perfluorocarbons are less di~usible and relatively insoluble and as such are easier to stabilize into the form of bubbles in aqueous media.
~ 35 The use of gaseous precursors is an optional embodiment o~ the present invention. In particular, per~luorocarbons have been found to be suitable ~or use as CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 gaseous precursors. As the artisan will appreciate, a given perfluorocarbon may be used as a gaseous precursor, i.e., in the liquid or solid state when the vesicles of the present invention are first made, or may be used as a gas directly, i.e., in the gas state, to make the gas and gaseous precursor filled vesicles. Whether such a perfluorocarbon is a gas, liquid, or solid depends, of course, on its liquid/gas or solid/gas phase transition temperature, or boiling point. For example, one of the more preferred perfluorocarbons is perfluoropentane, which has a liquid/gas phase transition temperature or boiling point of 27~C, which means that it will be a liquid at ordinary room temperature, but will become a gas in the environment of the human body, where the temperature will be above its liquid/gas phase transition temperature or boiling point. Thus, under normal circumstance, perfluoropentane is a gaseous precursor. As further examples, there is perfluorobutane and perfluorohexane, the next closest homologs of perfluoropentane. The liquid/gas phase transition temperature o~ perfluorobutane is 4~C and that of perfluorohexane is 57~C, making the former potentially a gaseous precursor, but generally more useful as a gas, while the latter would generally be a gaseous precursor, except under unusual circumstances, because of its high boiling point.
Another aspect of the present invention is the use of a fluorinated compound, especially a perfluorocarbon compound, which will be in the liquid state at the temperature of use of the vesicles of the present invention, to assist or enhance the stability of said gas and gaseous precursor filled vesicles. Such ~luorinated compounds include various liquid fluorinated compounds, such as fluorinated surfactants manufactured by the DuPont Company (Wilmington, DE), e.g., ZONYL~, as well as liquid perfluorocarbons. Preferably the fluorinated compounds are perfluorocarbons. Suitable perfluorocarbons useful as additional stabilizing agents include perfluorooctylbromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyliodide, perfluoro-tripropylamine, and per~luorotributylamine. In general, CA 022l48~ l997-09-08 W096/28090 PCT~S9~3054 perfluorocarbons over six carbon atoms in length will not be gaseous, i. e., in the gas state, but rather will be liquids, i . e ., in the li~uid state, at normal human body temperature.
These compounds may, however, additionally be utilized in preparing the stabilized gas and gaseous precursor filled vesicles used in the present invention. Preferably this perfluorocarbon is perfluorooctylbromide or perfluorohexane, which is in the liquid state at room temperature. The gas which is present may be, e . g., nitrogen or perfluoropropane, or may be derived from a gaseous precursor, which may also be a perfluorocarbon, e . g ., perfluoropentane. In that case, the vesicles of the present invention would be prepared from a mixture of perfluorocarbons, which for the examples given, would be perfluoropropane (gas) or perfluoropentane (gaseous precursor) and perfluorooctylbromide (liquid). Although not intending to be bound by any theory, it is believed that the liquid fluorinated compound is situated at the interface between the gas and the membrane surface of the vesicle. There is thus formed a further stabilizing layer of liquid fluorinated compound on the internal surface of the stabilizing compound, e.g., a biocompatible lipid used to form the vesicle, and this perfluorocarbon layer also serves the purpose of preventing the gas from diffusing through the vesicle membrane.
A gaseous precursor, within the context of the present invention, is a liquid or a solid at the temperature of manufacture and/or storage, but becomes a gas at least at or during the time of use.
Thus, it has been discovered that a liquid fluorinated compound, such as a perfluorocarbon, when combined with a gas or gaseous precursor ordinarily used to make the vesicles of the present invention, may confer an added degree of stability not otherwise obtainable with the gas or gaseous precursor alone. Thus, it is within the scope of the present invention to utilize a gas or gaseous precursor, such as a ~ 35 perfluorocarbon gaseous precursor, e . g ., perfluoropentane, together with a perfluorocarbon which r~' n-~ liquid after administration to a patient, i.e., whose liquid to gas phase CA 022148~ 1997-09-08 transition temperature is above the body temperature o~ the patient, e . g., per~luoroctylbromide.
Any biocompatible gas or gaseous precursor may be used to form the stabilized gas and gaseous precursor filled vesicles. By "biocompatible" is meant a gas or gaseous precursor which, when introduced into the tissues o~ a hl7m,7n patient, will not result in any degree o~ unacceptable toxicity, including allergenic responses and disease states, and pre~erably are inert. Such a gas or gaseous precursor should also be suitable ~or making gas and gaseous precursor ~illed vesicles, as described herein.
The size o~ the gas or gaseous precursor filled vesicles becomes stabilized when the stabilizing compounds described herein are employed; and the size o~ the vesicles can then be adjusted for the particular intended MRI end use. For example, magnetic resonance imaging o~ the vasculature may require vesicles that are no larger that about 30~ in diameter, and that are pre~erably smaller, e.g., no larger than about 12~
in diameter. The size o~ the gas ~illed vesicles can be adjusted, if desired, by a variety o~ procedures including microemulsi~ication, vortexing, extrusion, ~iltration, sonication, homogenization, repeated ~reezing and thawing cycles, extrusion under pressure through pores o~ de~ined size, and similar methods.
For intravascular use the vesicles are generally under 30~ in mean diameter, and are pre~erably under about 12~
in mean diameter. For targeted intravascular use, e . g., to bind to a certain tissue such as a tumor, the vesicles can be appreciably under a micron, even under 100 nm diameter. For enteric, i. e., gastrointestinal use the vesicles can be much larger, e.g., up to a millimeter in size, but vesicles between 20~ and 100~ in mean diameter are pre~erred.
As noted above, the embodiments o~ the present invention may also include, with respect to their preparation, formation and use, gaseous precursors that can be activated by temperature. Further below is set out Table I listing a series o~ gaseous precursors which undergo phase transitions ~rom W096/28090 PCT~S96103054 liquid to gaseous states at relatively close to normal body temperature (37~ C) or below, and the size o~ the emulsi~ied droplets that would be re~uired to form a microbubble of a ~ maximum size o~ lO microns.
Table I
Physical Characteristics of ~aseous Precursors and Diameter of Emulsi~ied Droplet to Form a 10 ~m Vesicle~

Compound MolecularBoiling Point DensityDiameter (~m) of Weight (o C) em~ ifie~ droplet to make 10 micron vesicle perfluoro 288.04 28.5 1.7326 2.9 1 opentane 1- 76.11 32.5 6.7789 1.2 fluorobutane 2-methyl 72.15 27.8 0.6201 2.6 butane 15(is~e~ e) 2-methyl 1- 70.13 31.2 0.6504 2.5 butene 2-methyl-2- 70.13 38.6 0.6623 2.5 butene 2o1-butene-3- 66 10 34.0 0.6801 2.4 yne-2-methyl 3-methyl-1- 68.12 29.5 0.6660 2.5 butyne octafluoro 200.04 -5.8 1.48 2.8 2 5cyclobutane decafluoro 238.04 -2 1.517 3.0 butane hexafluoro 138.01 -78.1 1 607 2.7 ethane ~Source: Chemical Rubber Company Handbook of Chemistry and Physics, Robert C. Weast and David R. Lide, eds., CRC Press, Inc. Boca Raton, ~lorida (1989-1990).

SUBSTlTUTE SHEET (RULE 26) CA 022l48~ l997-09-08 Wo 96/28090 PCT/US96/03054 There is also set out below a list composed o:E
potential gaseous precursors that may be used to form vesicles o:E de~ined size. However, the list is not intended to be limiting, since it is possible to use other gaseous precursors 5 :Eor that purpose. In ~act, ~or a variety oi~ di~erent applications, virtually any liquid can be used to make gaseous precursors so long as it is capable o~ undergoing a phase transition to the gas phase upon passing through the appropriate temperature, so that at least at some point in use l O it provides a gas . Suitable gaseous precursors f or use in the present invention are the ~ollowing: hexa~luoro acetone, isopropyl acetylene, allene, tetra:Eluoro-allene, boron trifluoride, isobutane, 1, 2-butadiene, 2, 3-butadiene, 1, 3-butadiene, 1, 2, 3-trichloro-2-i~luoro-1, 3-butadiene, 2-methyl-15 1, 3-butadiene, hexa:Eluoro-1, 3-butadiene, butadiyne, 1-:Eluoro-butane, 2-methyl-butane, decai~luorobutane, 1-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1-butene, per~luoro-1-butene, peri~luoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-1-butene-3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-2O hexa:Eluoro-butyne, 3-methyl-1-butyne, peri~luoro-2-butyne, 2-bromo-butyraldehyde, carbonyl sul~ide, crotononitrile, cyc1obutane, methyl-cyclobutane, octai~luoro-cyclobutane, per~luoro - cyclobutene, 3 - chlorocyclopentene, octai~luorocyclopentene, cyclopropane, 1, 2 -dimethyl-25 cyclopropane, 1,1-dimethylcyclopropane, 1, 2-dimethyl-cyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene, 3-ethyl-3-methyl diaziridine, 1,1,1-tri~luorodiazoethane, dimethyl amine, hexa:Eluorodimethylamine, dimethylethylamine, bis- (dimethylphosphine) amine, 30 per~luorohexane, 2, 3 -dimethyl-2-norbornane, per~luorodimethylamine, dimethyloxonium chloride, 1, 3-dioxolane-2-one, 4-methyl-1, 1,1, 2-tetra~luoroethane, 1,1,1-trifluoroethane, 1,1, 2, 2-tetra~luoroethane, 1,1, 2-trichloro-1, 2, 2-tri~luoroethane, 1, 1-dichloroethane, 1, 1-dichloro-35 1, 2, 2, 2-tetra:~luoroethane, 1, 2-di~luoroethane, 1-chloro-1,1, 2, 2, 2-pental~luoroethane, 2-chloro-1, 1-di:Eluoroethane, 1,1-dichloro-2-~luoroethane, 1-chloro-1, 1, 2, 2-tetra:Eluoroethane, CA 022l48~ l997-09-08 Wo 96/28090 PCT/US96/030S4 2-chloro-1, 1-difluoroethane, chloroethane, chloropentafluoroethane, dichlorotrifluoroethane, fluoroethane, hexaf luoroethane, nitropenta:Eluoroethane, nitrosopentaf luoroethane, perf luoroethylamine, ethyl vinyl 5 ether, l~l-dichloroethane~ 1,1-dichloro-1,2-difluoroethane, 1,2-difluoroethane, methane, trifluoromethanesulfonylchloride, trifluoromethanesulfonyl:Eluoride ,bromodifluoronitrosomethane, bromof luoromethane, bromochlorof luoromethane, bromotrii~luoromethane, chlorodifluoronitromethane, 10 chlorodinitromethane, chlorof luoromethane, chlorotrif luoromethane, chlorodif luoromethane, dibromodifluoromethane, dichlorodifluoromethane, dichlorofluoromethane, difluoromethane, difluoroiodomethane, disilanomethane, fluoromethane, iodomethane, iodotrifluoromethane, nitrotri:Eluoromethane, nitrosotrif luoromethane, tetraf luoromethane, trichloro~luoromethane, trifluoromethane, 2-methylbutane, methyl ether, methyl isopropyl ether, methyllactate, methylnitrite, methylsulfide, methyl vinyl ether, neon, neopentane, nitrogen (N2), nitrous oxide, 1,2,3-nonadecane-tricarboxylic acid-2-hydroxytrimethylester, 1-nonene-3-yne, oxygen (~2) ~ 1~4-pentadiene~ n-pentane, per:Eluoropentane, 4-amino-4-methylpentan-2-one, 1-pentene, 2-pentene (cis), 2-pentene (trans) , 3-bromopent-1-ene, perfluoropent-1-ene, tetrachlorophthalic acid, 2,3,6-trimethylpiperidine, propane, 1, 1, 1, 2, 2, 3-hexai~luoropropane, 1, 2-epoxypropane, 2, 2-difluoropropane, 2-aminopropane, 2-chloropropane, hepta~luoro-1-nitropropane, heptafluoro-1-nitrosopropane, perfluoropropane, propene, hexa:Eluoropropane, 1, 1, 1, 2, 3, 3-hexafluoro-2, 3 dichloropropane, 1-chloropropane, chloropropane- (trans), 2-chloropropane, 3 - f luoropropane, propyne, 3, 3, 3 -trifluoropropyne, 3-fluorostyrene, sulfur hexafluoride, sulfur ( di ) - decaf luoride ( S2Flo), 2, 4 - diaminotoluene , trifluoroacetonitrile, trifluoromethyl peroxide, ~ 3 5 trif luoromethyl sulf ide, tungsten hexaf luoride, vinyl acetylene, vinyl ether, and xenon.
The perfluorocarbons, as already indicated, are CA 022l48~ l997-09-08 W096/28090 PCT~S96/03~54 preferred for use as the gas or gaseous precursors, as well as additional stabilizing components. Included in such perfluorocarbon compositions are saturated perfluorocarbons, unsaturated perfluorocarbons, and cyclic per~luorocarbons. The saturated perfluorocarbons, which are usually preferred, have the formula CnF2n+2, where n is from 1 to 12, preferably 2 to 10, t more preferably 4 to 8, and most preferably 5. Examples of suitable saturated perfluorocarbons are the following:
tetrafluoromethane, hexafluoroethane, octafluoropropane, deca~luorobutane, dodeca~luoropentane, perfluorohexane, and perfluoroheptane. Cyclic perfluorocarbons, which have the formula CnF2n, where n is from 3 to 8, preferably 3 to 6, may also be preferred, and include, e.g., hexafluorocyclopropane, octafluorocyclobutane, and decafluorocyclopentane.
It is part of the present invention to optimize the utility o~ the vesicles by using gases of limited solubility.
By limited solubility, is meant the ability of the gas to diffuse out of the vesicles by virtue of its solubility in the surrounding aqueous medium. A greater solubility in the aqueous medium imposes a gradient with the gas in the vesicle such that the gas will have a tendency to diffuse out o~ said vesicle. A lesser solubility in the aqueous milieu, will, on the other hand, decrease or eliminate the gradient between the vesicle and the interface such that the diffusion of the gas out o~ the vesicle will be impeded. Pre~erably, the gas entrapped in the vesicle has a solubility less than that o~
oxygen, i.e., 1 part gas in 32 parts water. See Matheson Gas Data Book, 1966, Matheson Company Inc. More preferably, the gas entrapped in the vesicle possesses a solubility in water less than that of air; and even more preferably, the gas entrapped in the vesicle possesses a solubility in water less than that of nitrogen.

Stabilizinq C~...~o.~ ds One or more stabilizing compounds are employed to form the vesicles, and to assure continued encapsulation of the gases or gaseous precursors. Even for relatively insoluble, CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 non-di~fusible gases such as perfluoropropane or sulfur hexafluoride, improved vesicle preparations are obtained when one or more stabilizing compounds are utilized in the formation of the gas and gaseous precursor filled vesicles. These compounds maintain the stability and the integrity of the vesicles with regard to their size, shape and/or other attributes.
The terms "stable" or "stabilized", as used herein, means that the vesicles are substantially resistant to degradation, i. e., are resistant to the loss of vesicle structure or encapsulated gas or gaseous precursor for a useful period of time. Typically, the vesicles of the invention have a good shelf life, often retaining at least about 9O percent by volume of its original structure for a period of at least about two or three weeks under normal ambient conditions, although it is preferred that this period be at least a month, more at least preferably two months, even more preferably at least six months, still more preferably eighteen months, and most preferably three years. Thus, the gas and gaseous precursor filled vesicles typically have a good shelf life, sometimes even under adverse conditions, such as temperatures and pressures which are above or below those experienced under normal ambient conditions.
The stability of the vesicles of the present invention is attributable, at least in part, to the materials from which said vesicles are made, and it is often not necessary to employ additional stabilizing additives, although it is optional and often preferred to do so; and such additional stabilizing agents and their characteristics are explained in more detail herein. The materials from which the vesicles used in the present invention are constructed are preferably biocompatible lipid or polymer materials, and of these, the biocompatible lipids are especially preferred. In addition, because of the ease o~ formulation, i. e., the ability to produce the vesicles just prior to administration, these vesicles may be conveniently made on site.
The lipids and polymers employed in preparing the ' CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 vesicles of the invention _are biocompatible. By "biocompatible" is meant a lipid or polymer which, when introduced into the tissues of a human patient, will not result in any degree of unacceptable toxicity, including allergenic responses and disease states. Preferably the lipids or polymers are inert.

-Biocompatible Lipids For the biocompatible lipid materials, it is preferred that such lipid materials be what is often referred to as "amphiphilic" in nature (i . e, polar lipid), by which is meant any composition o~ matter which has, on the one hand, lipophilic, i . e ., hydrophobic properties, while on the other hand, and at the same time, having lipophobic, i. e., hydrophilic properties.
~ydrophilic groups may be charged moieties or other groups having an affinity for water. Natural and synthetic phospholipids are examples of lipids useful in preparing the stabilized vesicles used in the present invention. They contain charged phosphate "head" groups which are hydrophilic, attached to long hydrocarbon tails, which are hydrophobic.
This structure allows the phospholipids to achieve a single bilayer (unilamellar) arrangement in which all o~ the water-insoluble hydrocarbon tails are in contact with one another, leaving the highly charged phosphate head regions ~ree to interact with a polar aqueous environment. It will be appreciated that a series of concentric bilayers are possible, i . e., oligolamellar and multilamellar, and such arrangements are also contemplated to be an aspect of the present invention.
The ability to form such bilayer arrangements is one feature of the lipid materials use~ul in the present invention.
The lipid may alternatively be in the ~orm of a monolayer, and the monolayer lipids may be used to form a single monolayer (unilamellar) arrangement. Alternatively, the monolayer lipid may be used to form a series o~ concentric monolayers, i. e., oligolamellar or multilamellar, and such arrangements are also considered to be within the scope of the CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 invention.
It has also been found advantageous to achieving the stabilized vesicles of the present invention that they be prepared at a temperature below the gel to liquid crystalline phase transition temperature of a lipid used as the stabilizing compound. This phase transition temperature is the temperature at which a lipid bilayer will convert ~rom a gel state to a liquid crystalline state. See, for example, Chapman et al., J. Biol. Chem. 1974 249, 2512-2521.
It is believed that, generally, the higher the gel state to li~uid crystalline state phase transition temperature, the more impermeable the gas and gaseous precursor filled vesicles are at any given temperature. See Derek Marsh, CRC
Handbook of Lipid Bilayers (CRC Press, Boca Raton, FL 1990), at p. 139 for main chain melting transitions of saturated diacyl-sn-glycero-3-phosphocholines. The gel state to liquid crystalline state phase transition temperatures of various lipids will be readily apparent to those skilled in the art and are described, for example, in Gregoriadis, ed., Liposome Technology, Vol. I, 1-18 (CRC Press, 1984). Table 2, below, lists some of the representative lipids and their phase transition temperatures:

CA 022l48~ l997-09-08 W096/28090 PCT~S96tO3054 Table 2 Saturated Diacyl sn-Glycero(3)Phosphocholines:
Main Chain Phase Transition Temperatures Carbons in Acyl Main Phase ~h~; n~ Transition Temperature ~C
1,2-(12:0) -1.0 1,2-(13:0) 13.7 1,2-(14:0) 23.5 1,2-(15:0) 34.5 1,2-(16:0) 41.4 1,2-(17:0) 48.2 1,2-(18:0) 55.1 1,2-(19:0) 61.8 1,2-(20:0) 64.5 1,2-(21:0) 71.1 1,2-(22:0) 74.0 1,2-(23:0) 79.5 1,2-(24:0) 80.1 ~Derek Marsh, "CRC Handbook of Lipid Bilayers", CRC Press, Boca Raton, Florida (1990), page 139.
It has been found possible to enhance the stability of the vesicles used in the present invention by incorporating at least a small amount, i.e., about 1 to about 10 mole percent of the total lipid, o~ a negatively charged lipid into the lipid from which the gas and gaseous precursor ~illed vesicles are to be formed. Suitable negatively charged lipids include, e.g., phosphatidylserine, phosphatidic acid, and fatty acids.
Such negatively charged lipids provide added stability by counteracting the tendency of the vesicles to rupture by ~using together, i.e., the negatively charged lipids tend to establish a uniform negatively charged layer on the outer surface of the vesicle, which will be repulsed by a similarly charged outer layer on the other vesicles. In this way, the vesicles will tend to be prevented ~rom coming into touching proximity with each other, which would o~ten lead to a rupture of the membrane or skin of the respective vesicles and consolidation o~ the contacting vesicles into a single, larger vesicle. A
continuation of this process of consolidation will, of course, lead to significant degradation of the vesicles.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 The lipid material or other stabilizing compound used to form the vesicles is also preferably flexible, by which is meant, in the context of gas and gaseous precursor filled vesicles, the ability of a structure to alter its shape, for example, in order to pass through an opening having a size smaller than the vesicle.
In selecting a lipid for preparing the stabilized vesicles used in the present invention, a wide variety of lipids will be found to be suitable for their construction.
Particularly useful are any of the materials or combinations thereof known to those skilled in the art as suitable for liposome preparation. The lipids used may be of either natural, synthetic, or semi-synthetic origin.
Lipids which may be used to prepare the gas and gaseous precursor filled vesicles used in the present invention include but are not limited to: lipids such as fatty acids, lysolipids, phosphatidylcholine with both saturated and unsaturated lipids including dioleoylphosphatidylcholine;
dimyristoylphosphatidylcholine; dipentadecanoyl-phosphatidylcholinei dilauroylphosphatidylcholine;dipalmitoylphosphatidylcholine (DPPC); distearoyl-phosphatidylcholine (DSPC); phosphatidylethanolamines such as dioleoylphosphatidylethanolamine and dipalmitoyl-phosphatidylethanolamine (DPPE); phosphatidylserine;
phosphatidylglycerol; phosphatidylinositol; sphingolipids such as sphingomyelin; glycolipids such as ganglioside GM1 and GM2i glucolipids;sulfatidesiglycosphingolipidsiphosphatidicacids such as dipalymitoylphosphatidic acid (DPPA); palmitic acid;
stearic acid; arachidonic acid; oleic acid; lipids bearing polymers such as polyethylene glycol, i.e., PEGylated lipids, chitin, hyaluronic acid or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, oligo- or polysaccharides; cholesterol, cholesterol sulfate and cholesterol hemisuccinate; tocopherol hemisuccinate; lipids with ether and ester-linked fatty acids;
polymerized lipids (a wide variety of which are well known in the art); diacetyl phosphate; dicetyl phosphate; stearylamine;
cardiolipin; phospholipids with short chain fatty acids of 6-8 CA 022148~ 1997-09-08 WO 96/28090 PCTfUS96/03054 carbons in lengthi synthetic phospholipids with asymmetric acyl rh~ ; n.~ (e.g., with one acyl chain o~ 6 carbons and another acyl chain o~ 12 carbons)i ceramides; non-ionic liposomes including niosomes such as polyoxyethylene fatty acid esters, polyoxyethylene ~atty alcohols, polyoxyethylene ~atty alcohol ethers, polyoxyethylated sorbitan fatty acid esters, glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, polyoxyethylene-polyoxypropylene polymers, and polyoxyethylene ~atty acid stearates; sterol aliphatic acid esters including cholesterol sul~ate, cholesterol butyrate, cholesterol iso-butyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate, and phytosterol n-butyrate; sterol esters of sugar acids including cholesterol glucuroneide, lanosterol glucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate; esters o~ sugar acids and alcohols including lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoyl gluconate, and stearoyl gluconate;
esters o~ sugars and aliphatic acids including sucrose laurate, ~ructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, accharic acid, and polyuronic acid; saponins including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, and digitoxigenin; glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate, glycerol and glycerol esters including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate; longchain alcohols including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and n-octadecyl alcohol; 6-(5-cholesten-3~-yloxy)-1-thio-~-D-galactopyranoside;digalactosyldiglyceride;6-(5-cholesten-3~-yloxy)hexyl-6-amino-6-deoxy-1-thio-~-D-galactopyranoside; 6-(5-cholesten-3~-yloxy)hexyl-6-amino-6-deoxyl-1-thio-~-D-mannopyranoside; 12-(((7~-diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoic acid; N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methyl-amino) octadecanoyl]-CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 2-aminopalmitic acid; cholesteryl)4~-trimethyl-~mmo~i o) butanoatei N-succinyldioleoylphosphatidylethanol-amine;
1~2-dioleoyl-sn-glycerolil~2-dipalmitoyl-sn-3-succinylglycer 1~3-dipalmitoyl-2-succinylslycerolil-hexadecyl-2-palmitoyl-glycerophosphoethanolamine and palmitoylhomocysteine, and/orcombinations thereof.
If desired, a variety of cationic lipids such as DOTMA, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammoium chloride; DOTAP, 1~2-dioleoyloxy-3-(trimethylammonio)propanei and DOTB, 1,2-dioleoyl-3-(~'-trimethyl-ammonio)butanoyl-sn-glycerol may be used. In general the molar ratio of cationic lipid to non-cationic lipid in the liposome may be, for example, 1:1000, 1:100, preferably, between 2:1 to 1:10, more preferably in the range between 1:1 to 1:2.5 and most preferably 1:1 (ratio of mole amount cationic lipid to mole amount non-cationic lipid, e.g., DPPC). A wide variety of lipids may comprise the non-cationic lipid when cationic lipid is used to construct the vesicle. Preferably, this non-cationic lipid is dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine or dioleoylphosphatidyl-ethanolamine. In lieu o~ cationic lipids as described above, lipids bearing cationic polymers such as polylysine or polyarginine, as well as alkyl phosphonates, alkyl phosphinates, and alkyl phosphites, may also be used to construct the vesicles.
The most preferred lipids are phospholipids, preferably DPPC, DPPE, DPPA and DSPC, and most preferably DPPC.
In addition, examples of saturated and unsaturated ~atty acids that may be used to prepare the stabilized vesicles used in the present invention, in the form o~ gas and gaseous precursor filled mixed micelles, may include molecules that may contain pre~erably between 12 carbon atoms and 22 carbon atoms in either linear or branched form. Hydrocarbon groups consisting o~ isoprenoid units and/or prenyl groups can be used as well. Examples of saturated fatty acids that are suitable include, but are not limited to, lauric, myristic, palmitic, and stearic acids; examples o~ unsaturated ~atty acids that may CA 022148~ 1997-09-08 W096l28090 PCT~S96/03054 be used are, but are not limited to, lauroleic, physeteric, myristoleic, palmitoleic, petroselinic, and oleic acids;
examples of branched fatty acids that may be used are, but are not limited to, isolauric, isomyristic, isopalmitic, and isostearic acids. In addition, to the saturated and unsaturated groups, gas and gaseous precursor filled mixed micelles can also be composed of 5 carbon isoprenoid and prenyl groups. In addition, partially fluorinated phospholipids can be used as stabilizing compounds for coating the vesicles.

- Biocom~atible PolYmers The biocompatible polymers useful as stabilizing compounds for preparing the gas and gaseous precursor ~illed vesicles used in the present invention can be of either natural, semi-synthetic (modified natural) or synthetic origin.
15 As used herein, the term polymer denotes a compound comprised of two or more repeating monomeric units, and preferably 10 or more repeating monomeric units. The phrase semi-synthetic polymer (or modi~ied natural polymer), as employed herein, denotes a natural polymer that has been chemically modi~ied in some fashion. Exemplary natural polymers suitable ~or use in the present invention include naturally occurring polysaccharides. Such polysaccharides include, for example, arabinans, ~ructans, fucans, galactans, galacturonans, glucans, m~nn~n.~, xylans (such as, ~or example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectin, amylose, pullulan, glycogen, amylopectin, cellulose, dextran, pustulan, chitin, agarose, keratan, chondroitan, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch and various other natural homopolymer or heteropolymers such as those containing one or more o~ the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof. Exemplary 5 semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers suitable ~or use in the present invention include polyethylenes (such as, ~or example, polyethylene glycol, 10 polyoxyethylene,andpolyethyleneterephthlate), polypropylenes (such as, ~or example, polypropylene glycol), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides including nylon, polystyrene, polylactic acids, fluorinated 15 hydrocarbons, fluorinated carbons (such as, for example, polytetrafluoroethylene), and polymethylmethacrylate, and derivatives thereof. Methods for the preparation of such polymer-based vesicles will be readily apparent to those skilled in the art, once armed with the present disclosure, 20 when the present disclosure is coupled with information known in the art, such as that described and re~erred to in Unger, U.S. Patent No. 5,205,290, the disclosures of which are hereby incorporated herein by re~erence, in their entirety.
Pre~erably, when intended to be used in the 25 gastrointestinal tract, the polymer employed is one which has a relatively high water binding capacity. When used, ~or example, in the gastrointestinal region, a polymer with a high water binding capacity binds a large amount of free water, enabling the polymer to carry a large volume o~ liquid through 30 the gastrointestinal tract, thereby filling and distending the tract. The filled and distended gastrointestinal tract permits a clearer picture o~ the region. In addition, where imaging rof the gastrointestinal region is desired, pre~erably the polymer employed is also one which is not substantially 35 degraded within and absorbed from the gastrointestinal region.
Minimization o~ metabolism and absorption within the gastrointestinal tract is preferable, so as to avoid the -CA 022148~ 1997-09-08 W096/28090 PCT~Sg6/030 removal of the contrast agent from the tract as well as avoid the formation of gas within the tract as a result of this degradation. Moreover, particularly where gastrointestinal usage is contemplated, polymers are preferably such that they are capable of displacing air and minimizing the formation of large air bubbles within the polymer composition.
Particularly pre~erred embodiments of the present invention include vesicles wherein the stabilizing compound from which the stabilized gas and gaseous precursor filled vesicles are formed comprises three components: (1) a neutral (e.g., nonionic or zwitterionic) lipid, (2) a negatively charged lipid, and (3) a lipid bearing a hydrophilic polymer.
Preferably, the amount of said negatively charged lipid will be greater than 1 mole percent of total lipid present, and the amount of lipid bearing a hydrophilic polymer will be greater than 1 mole percent of total lipid present. It is also preferred that said negatively charged lipid be a phosphatidic acid. The lipid bearing a hydrophilic polymer will desirably be a lipid covalently bound to said polymer, and said polymer will preferably have a weight average molecular weight of from about 400 to about lOO,O00. Said hydrophilic polymer is preferably selected from the group consisting of polyethyleneglycol, polypropyleneglycol, polyvinylalcohol, and polyvinylpyrrolidone and copolymers thereof. The PEG or other polymer may be bound to the DPPE or other lipid through a covalent linkage, such as through an amide, carbamate or amine linkage. Alternatively, ester, ether, thioester, thioamide or disulfide (thioester) linkages may be used with the PEG or other polymer to bind the polymer to, for example, cholesterol or other phospholipids. Where the hydrophilic polymer is polyethyleneglycol, a lipid bearing such a polymer will be said to be "PEGylated," which has reference to the abbreviation for polyethyleneglycol: "PEG." Said lipid bearing a hydrophilic polymer is preferably dipalmitoylphosphatidylethanolamine-p o 1 y e t h y 1 e n e g 1 y c o 1 5 0 O 0 , i . e . , adipalmitoylphosphatidylethanolamine lipid having a polyethyleneglycol polymer of a mean weight average molecular CA 022l48~ l997-09-08 W096/28090 PCT~S~5'~3054 weight of about 5000 attached thereto (DPPE-PEG5000); or distearoyl-phosphatidylethanolamine-polyethyleneglycol 5000.
Preferred embodiments of the vesicle contemplated by the present invention would include, e.g., 77.5 mole percent dipalmitoylphophatidylcholine (DPPC), with 12.5 mole percent o~ dipalmitoylphosphatidic acid (DPPA), and with 10 mole percent of dipalmitoylphosphatidylethanolamine-polyethyleneglycol-5000 (DPPE/PEG5000). These compositions in a 82/10/8 ratio of mole percentages, respectively, is also preferred. The DPPC component is effectively neutral, since the phosphtidyl portion is negatively charged and the choline portion is positively charged. Consequently, the DPPA
component, which is negatively charged, is added to enhance stabilization in accordance with the me~h~n;sm described further above regarding negatively charged lipids as an additional agent. The third component, DPPE/PEG, provides a PEGylated material bound to the lipid membrane or skin of the vesicle by the DPPE moiety, with the PEG moiety free to surround the vesicle membrane or skin, and thereby form a physical barrier to various enzymatic and other endogenous agents in the body whose function is to degrade such foreign materials It is also theorized that the PEGylated material, because of its structural similarity to water, is able to de~eat the action of the macrophages of the human immune system, which would otherwise tend to surround and remove the foreign object. The result is an increase in the time during which the stabilized vesicles can function as contrast media.

Other and AuxiliarY Stabilizinq Com~ounds It is also contemplated to be a part of the present invention to prepare stabilized gas and gaseous precursor filled vesicles using compositions of matter in addition to the biocompatible lipids and poly~mers described above, provided that the vesicles so prepared meet the stability and other criteria set forth herein. These compositions may be basic and flln~m~ntal , i . e ., form the primary basis for creating or establishing the stabilized gas and gaseous precursor filled CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 vesicles. On the other hand, they may be auxiliary, i.e., act as subsidiary or supplementary agents which either ~nh~nce the functioning of the basic stabilizing compound or compounds, or else contribute some desired property in addition to that afforded by the basic stabilizing compound.
However, it is not always possible to determine whether a given compound is a basic or an auxiliary agent, since the functioning o~ the compound in question is determined empirically, i.e., by the results produced with respect to producing stabilized vesicles. As examples of how these basic and auxiliary compounds may ~unction, it has been observed that the simple combination o~ a biocompatible lipid and water or saline when shaken will o~ten give a cloudy solution subsequent to autoclaving for sterilization. Such a cloudy solution may ~unction as a contrast agent, but is aesthetically objectionable and may imply instability in the form of undissolved or undispersed lipid particles. Thus, propylene glycol may be added to remove this cloudiness by facilitating dispersion or dissolution o~ the lipid particles. The propylene glycol may also function as a thickening agent which improves vesicle formation and stabilization by increasing the sur~ace tension on the vesicle membrane or skin. It is possible that the propylene glycol further functions as an additional layer that coats the membrane or skin of the vesicle, thus providing additional stabilization. As examples of such further basic or auxiliary stabilizing compounds, there are conventional sur~actants which may be used; see D'Arrigo U.S. Patents Nos. 4,684,479 and 5,215,680.
Additional auxiliary and basic stabilizing compounds include such agents as peanut oil, canola oil, olive oil, sa~flower oil, corn oil, or any other oil commonly known to be ingestible which is suitable for use as a stabilizing compound in accordance with the requirements and instructions set ~orth in the instant speci~ication.
In addition, compounds used to make mixed micelle systems may be suitable ~or use as basic or auxiliary stabilizing compounds, and these include, but are not limited CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 to: lauryltrimethylammonium bromide (dodecyl-), cetyltrimethylammonium bromide (hexadecyl-), myristyltrimethyl~mmon;um bromide (tetradecyl-), alkyldimethyl-benzylammonium chloride (alkyl=Cl2,Cl4,Cl6,), benzyldimethyl-dodecylammonium bromide/chloride, benzyldimethylhexadecylammonium bromide/ chloride, benzyldimethyl tetradecylammonium bromide/chloride, cetyldimethylethyl~mmon;um bromide/chloride, or cetylpyridinium bromide/chloride.
It has been ~ound that the gas and gaseous precursor filled vesicles used in the present invention may be controlled according to size, solubility and heat stability by choosing from among the various additional or auxiliary stabilizing agents described herein. These agents can af~ect these parameters of the vesicles not only by their physical interaction with the lipid coatings, but also by their ability to modify the viscosity and surface tension of the surface of the gas and gaseous precursor filled vesicle. Accordingly, the gas and gaseous precursor filled vesicles used in the present invention may be favorably modified and further stabilized, for example, by the addition of one or more of a wide variety of (a) viscositY modi~iers, including, but not limited to carbohydrates and their phosphorylated and sul~onated derivatives; and polyethers, preferably with molecular weight ranges between 400 and 100,000; di- and trihydroxy alkanes and their polymers, preferably with molecular weight ranges between 200 and 50,000; (b) emulsifyinq and/or solubilizinq aqents may also be used in conjunction with the lipids to achieve desired modi~ications and ~urther stabilization; such agents include, but are not limited to, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, mono- and di-glycerides, mono-ethanolamine, oleic acid, oleyl alcohol, poloxamer (e.g., poloxamer 188, poloxamer 184, and poloxamer 181), polyoxyethylene 50 stearate, polyoxyl 35 castor oil, polyoxyl 10 oleyl ether, polyoxyl 20 cetostearyl ether, polyoxyl 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, propylene glycol diacetate, propylene glycol monostearate, sodium lauryl sul~ate, sodium CA 022l48~ l997-09-08 W096~28090 PCT~S96/03054 stearate, sorbitan mono-laurate, sorbitan mono-oleate, sorbitan mono-palmitate, sorbitan monostearate, stearic acid, trolamine, and emulsi~ying wax; (c) suspendina and/or viscosity-increasinq aqents that may be used with the lipids include, but are not limited to, acacia, agar, alginic acid, all7m;nl7m mono-stearate, bentonite, magma, carbomer 934P, carboxymethylcellulose, calcium and sodium and sodium 12, carrageenan, cellulose, dextran, gelatin, guar gum, locust bean gum, veegum, hydroxyethyl cellulose, hydroxypropyl methylcellulose, magnesium-aluminum-silicate, methylcellulose, pectin, polyethylene oxide, povidone, propylene glycol alginate, silicon dioxide, sodium alginate, tragacanth, xanthum gum, ~-d-gluconolactone, glycerol and mannitol; (d) synthetic suspendinq aqents may also be utilized such as polyethyleneglycol (PEG), polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), polypropylene glycol, and polysorbate; and (e) tonicity raisinq aqents may be included; such agents include but are not limited to sorbitol, propyleneglycol and glycerol.

Aoueous Diluents As mentioned earlier, where the vesicles are lipid in nature, a particularly desired component of the stabilized vesicles is an aqueous environment o~ some kind, which induces the lipid, because o~ its hydrophobic/hydrophilic nature, to ~orm vesicles, the most stable con~iguration which it can achieve in such an environment. The diluents which can be employed to create such an aqueous environment include, but are not limited to water, either deionized or containing any number o~ dissolved salts, etc., which will not inter~ere with creation and maintenance o~ the stabilized vesicles or their use as MRI contrast agents; and normal saline and physiological saline.

Par~m7~n~tic and Superparamaqnetic Contrast Aqents In a ~urther embodiment o~ the present invention, the stabilized gas ~illed vesicle based contrast medium o~ the invention may ~urther comprise additional contrast agents such CA 022l48~ l997-09-08 W096/28090 PCT~S96/03~4 as conventional contrast agents, which may serve to increase the e~ficacy o~ the contrast medium ~or simultaneous magnetic resonance focused noninvasive ultrasound. Many such contrast agents are well known to those skilled in the art and include paramagnetic and superparamagnetic contrast agents.
Exemplary paramagnetic contrast agents suitable ~or use in the subject invention include stable ~ree radicals (such as, ~or example, stable nitroxides), as well as compounds comprising transition, lanthanide and actinide elements, which may, i~ desired, be in the ~orm o~ a salt or may be covalently or noncovalently bound to complexing agents (including lipophilic derivatives thereo~) or to proteinaceous macromolecules.
Preferable transition, lanthanide and actinide elements include Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III). More pre~erably, the elements include Gd(III), Mn(II), Cu(II), Fe(II), Fe(III), Eu(III) and Dy(III), especially Mn(II) and Gd(III).
These elements may, i~ desired, be in the ~orm o~ a salt, such as a manganese salt, e . g., manganese chloride, manganese carbonate, manganese acetate, and organic salts o~
manganese such as manganese gluconate and manganese hydroxylapatite; and such as an iron salt, e . g., iron sul~ides and ~erric salts such as ~erric chloride.
These elements may also, if desired, be bound, e.g., covalently or noncovalently, to complexing agents (including lipophilic derivatives thereo~) or to proteinaceous macromolecules. Pre~erable complexing agents include, ~or example, diethylenetriamine-pentaacetic acid (DTPA), ethylene-diaminetetraacetic acid (EDTA), 1,4,7,10-tetraazacyclododecane-N,N',N',N'''-tetraacetic acid (DOTA), 1,4,7,10-tetraazacyclododecane-N,N',N''-triacetic acid (DO3A), 3,6,9-triaza-12-oxa-3,6,9-tricarboxymethylene-10-carboxy-13-phenyl-~ 35 tridecanoic acid (B-19036), hydroxybenzylethylene-diamine diacetic acid (HBED), N,N'-bis(pyridoxyl-5-phosphate)ethylene diamine, N,N'-diacetate (DPDP), 1,4,7-triazacyclononane-CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 N,N',N''-triacetic acid (NOTA), 1,4,8,11-tetraazacyclotetradecane-N,N'N'',N'''-tetraaceticacid (TETA), kryptands (that is, macrocyclic complexes), and des~erriox~lne. More pre~erably, the complexing agents are EDTA, DTPA, DOTA, DO3A and kryptands, most pre~erably DTPA.
Pre~erable lipophilic complexes thereo~ include alkylated derivatives o~ the complexing agents EDTA, DOTA, etc., ~or example, EDTA-DDP, thatis, N,N'-bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N'-diacetate; EDTA-ODP, that is N,N'-bis-(carboxy-octadecylamido-methyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N'-diacetate; EDTA-~DP N,N'-Bis-(carboxy-laurylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N'-diacetate; etc.; such as those described in U.S. Serial No. 887,290, filed May 22, 1992, the disclosures o~ which are hereby incorporated herein by re~erence in its entirety. Pre~erable proteinaceous macromolecules include albumin, collagen, polyarginine, polylysine, polyhistidine, ~-globulin and ~-globulin. More pre~erably, the proteinaceous macromolecules comprise albumin, polyarginine, polylysine, and polyhistidine.
Suitable complexes thus include Mn(II)-DTPA, Mn(II)-EDTA,Mn(II)-DOTA, Mn(II)-DO3A, Mn(II)-kryptands, Gd(III)-DTPA, Gd(III)-DOTA, Gd(III)-DO3A, Gd(III)-kryptands, Cr(III)-EDTA, Cu(II)-EDTA, or iron-des~errioxamine, especially Mn(II)-DTPA
or Gd(III)-DTPA.
Paramagnetic chelates, such as alkylated chelates o~
paramagnetic ions, as disclosed in U.S. Patent No. 5,312,617, the disclosure o~ which is incorporated herein by re~erence in its entirety, paramagnetic copolymeric chelates as in U.S.
Patent No. 5,385,719 use~ul ~or attaching to gas ~illed liposomes and to the sur~ace o~ gas ~illed polymeric liposomes, nitroxide stable ~ree radicals (NSFRs) use~ul ~or attaching to lipids in gas ~illed liposomes as well as to polymers ~or construction gas ~illed liposomes and hybrid complexes comprised of chelate moieties containing one or more paramagnetic ions in close proximity with one or more NSFRs as outlined in U.S. Patent No. 5,407,657, may be used ~or CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 constructing paramagnetic gas filled liposomes. These hybrid complexes have greatly increased relaxivity and therefore increase the sensitivity to the vesicle on magnetic resonance.
Nitroxides are paramagnetic contrast agents which increase both T1 and T2 relaxation rates by virtue of one unpaired electron in the nitroxide molecule. The paramagnetic effectiveness of a given compound as an MRI contrast agent is at least partly related to the number of unpaired electrons in the paramagnetic nucleus or molecule, specifically to the square of the number of unpaired electrons. For example, gadolinium has seven unpaired electrons and a nitroxide molecule has only one unpaired electron; thus gadolinium is generally a much stronger MRI contrast agent than a nitroxide.
However, effective correlation time, another important parameter ~or assessing the e~fectiveness of contrast agents, confers potential increased relaxivity to the nitroxides. When the effective correlation time is very close to the proton Larmour frequency, the relaxation rate may increase dramatically. When the tumbling rate is slowed, e . g., by attaching the paramagnetic contrast agent to a large structure, it will tumble more slowly and thereby more effectively transfer energy to hasten relaxation o~ the water protons. In gadolinium, however, the electron spin relaxation time is rapid and will limit the extent to which slow rotational correlation times can increase relaxivity For nitroxides, however, the electron spin correlation times are more favorable and tremendous increases in relaxivity may be attained by slowing the rotational correlation time of these molecules. The gas filled vesicles o~ the present invention are ideal ~or attaining the goals o~ slowed rotational correlation times and resultant improvement in relaxivity. Although not intending to be bound by any particular theory of operation, it is contemplated that since the nitroxides may be designed to coat the perimeters of the gas filled vesicles, e . g., by making alkyl derivatives thereof, that the resulting correlation times can be optimized. Moreover, the resulting contrast medium of the present invention may be viewed as a magnetic sphere, a CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 geometric configuration which maximizes relaxivity.
If desired, the nitroxides may be alkylated or otherwise derivitized, such as the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, free radical, and 2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (TMPO).
Exemplary superparamagnetic contrast agents suitable for use in the subject invention include metal oxides and sul~ides which experience a magnetic domain, ferro- or ferrimagnetic compounds, such as pure iron, magnetic iron oxide (such as magnetite), ~-Fe2O3, Fe3O4, iron sulfides,manganese ferrite, cobalt, ferrite, nickel ferrite, and ferritin filled with magnetite or other magnetically active materials such as ferromagnetic and superparamagnetic materials.
The contrast agents, such as the paramagnetic and superparamagnetic contrast agents described above, may be employed as a component within the vesicles or in the contrast medium comprising the vesicles. They may be entrapped within the internal space of the vesicles, administered as a solution with the vesicles or incorporated into the stabilizing compound forming the vesicle wall.
Superparamagnetic agents may be used as clathrates to adsorb and stabilize vesicles For example, emulsions o~
various perfluorocarbons, such as perfluoroh~ne or perfluorochlorocarbons mixed with irregular shaped iron oxide compounds. The hydrophobic cle~ts in the iron oxides cause nano-droplets of the liquid gaseous precursor to adhere to the surface o~ the solid material.
For example, if desired, the paramagnetic or superparamagnetic agents may be delivered as alkylated or other derivatives incorporated into the stabilizing compound, especially the lipidic walls of the vesicles. In particular, the nitroxides 2,2,5,5-tetramethyl-1-pyrrolidinyloxy, ~ree radical and2,2,6,6-tetramethyl-1-piperidinyloxy, ~reeradical, can form adducts with long chain ~atty acids at the positions of the ring which are not occupied by the methyl groups, via a number o~ different linkages, e.g., an acetyloxy group. Such adducts are very amenable to incorporation into the stabilizing CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 compounds, especially those o~ a liPidic nature, which ~orm the walls o~ the vesicles o~ the present invention.
Mixtures o~ any one or more o~ the paramagnetic agents and/or superparamagnetic agents in the contrast media may similarly be used.
The paramagnetic and superparamagnetic agents described above may also be coadministered separately, i~
desired.
The gas filled vesicles used in the present invention may not only serve as e~ective carriers of the superparamagnetic agents, e . g., iron oxides, but also appear to magni~y the e~ect o~ the susceptibility contrast agents.
Superparamagnetic contrast agents include metal oxides, particularly iron oxides but including manganese oxides, and as iron oxides, containing varying amounts o~ manganese, cobalt and nickel which experience a magnetic domain. These agents are nano or microparticles and have very high bulk susceptibilities and transverse relaxation rates. The larger particles, e . g., lO0 nm diameter, have much higher R2 relaxivities than R1 relaxivities but the smaller particles, e . g., lO to 15 nm diameter have somewhat lower R2 relaxivities, but much more balanced Rl nd R2 values. The smallest particles, e.g., monocrystalline iron oxide particles, 3 to 5 nm in diameter, have lower R2 relaxivities, but probably the most balanced R1 and R2 relaxation rates. Ferritin can also be ~ormulated to encapsulate a core o~ very high relaxation rate superparamagnetic iron. It has been discovered that stabilized gas ~illed vesicles used in the present invention can increase the ef~icacy and sa~ety o~ these conventional iron oxide based MRI contrast agents.
The iron oxides may simply be incorporated into the stabilizing compounds ~rom which the vesicles are made.
Particularly, the iron oxides may be incorporated into the walls o~ the lipid based vesicles, e . g., adsorbed onto the sur~aces o~ the vesicles, or entrapped within the interior o~
the vesicles as described in U.S. Patent 5,088,499, issued February 18, 1992.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 Although there is no intention to limit the present invention to any particular theory as to its mode of action, it is believed that the vesicles increase the efficacy of the superparamagnetic contrast agents by several mechanisms.
First, it is believed that the vesicles function so as to increase the apparent magnetic concentration of the iron oxide particles. Second, it is believed that the vesicles increase the apparent rotational correlation time of the MRI contrast agents, both paramagnetic and superparamagnetic agents, so that relaxation rates are increased. Finally, the vesicles appear to operate by way of a novel mechanism which increases the apparent magnetic domain of the contrast medium and is believed to operate in the manner described immediately below.
The vesicles may be thought of as flexible spherical domains of differing susceptibility from the suspending medium, i.e., the aqueous suspension of the contrast medium and the gastrointestinal fluids in the case of gastrointestinal administration, and blood or other body fluids in the cases of intravascular injection or injection into another body space.
When considering ferrites or iron oxide particles, it should be noted that these agents have a particle size dependent e~ect on contrast, i.e., it depends on the particle diameter of the iron oxide particle. This phenomenon is very common and is o~ten re~erred to as the "secular" relaxation of the water molecules. Described in more physical terms, this relaxation mechanism is dependent upon the effective size of the molecular complex in which a paramagnetic atom, or paramagnetic molecule, or molecules, may reside. One physical explanation may be described in the ~ollowing Solomon-Bloembergen e~uations which define the paramagnetic contributions to the Tl and T2 relaxation times o~ a spin l/2 nucleus with gyromagnetic ratio g perturbed by a paramagnetic ion:

1/T,M =(2/15) S(S + 1) y2g2~B2/r6 [ 3T / (1 + C~2T2) +
7TC / (1 + C-1s2TC2) ] + (2/3) S(S+ 1)A2 /h2 [ Te I (1 + ~s2Te2)]

CA 022l48~ l997-09-08 W096128090 PCT~S96/030 and ~ -1/T2M = (1/15) S(S + 1) y2g2~2/r6 [ 4Tc + 3Tc/ (1 +~,2TC2) +
13TC / (1 + WS2TC2) ] + (1/3) S(S+ 1)A2 /h2 [ Te I (1 + CIJs2Te2)]

where:
S = electron spin quantum number;
g = electronic g factor;
,a = Bohr magneton;
c~JI and ~5 (= 657 w~) = Larmor angular precession frequencies for the nuclear spins and electron spins;
r = ion-nucleus distancei A = hyperfine coupling constant;
Tc and Te = correlation times for the dipolar and scalar interactions, respectivelyi and h = Planck's constant.
15 See, e.g., Solomon, I. Phys. Rev. 99, 559 (1955) and Bloembergen, N. J. Chem. Phys. 27, 572, 595 (1957), the disclosures of which are hereby incorporated by reference in their entirety.
A few large particles will generally have a much 20 greater effect than a larger number of much smaller particles, primarily due to a larger correlation time. I~
one were to make the iron oxide particles very large however, they might be toxic and embolize the lungs or activate the complement cascade system. Furthermore, it is 25 not the total size o~ the particle that matters, but particularly the diameter o~ the particle at its edge or outer surface The domain of magnetization or susceptibility effect falls off exponentially from the surface o~ the particle. Generally speaking, in the case of dipolar (through space) relaxation mechanisms, this exponential fall of~ exhibits an r6 dependence. Literally interpreted, a water molecule that is 4 angstroms away from a paramagnetic sur~ace will be in~luenced 64 times less than a water molecule that is 2 angstroms away from the same 35 paramagnetic surface. The ideal situation in terms of maximizing the contrast effect would be to make the iron oxide particles hollow, flexible and as large as possible.

CA 022l48~ l997-09-08 W096128090 PCT~S96/03054 Up until now it has not been possible to do this;
furthermore, these benefits have probably been unrecognized until now. By coating the inner or outer sur~aces of the vesicles with the contrast agents, even though the individual contrast agents, e. g., iron oxide nanoparticles or paramagnetic ions, are relatively small structures, the effectiveness of the contrast agents may be greatly enhanced. In so doing, the contrast agents may function as an effectively much larger sphere wherein the effective domain of magnetization is determined by the diameter of the vesicle and is maximal at the sur~ace of the vesicle. These agents afford the advantage of flexibility, i. e., compliance. While rigid vesicles might lodge in the lungs or other organs and cause toxic reactions, these flexible vesicles slide through the capillaries much more easily.

Methods o~ Preparation The stabilized gas filled vesicles used in the present invention may be prepared by a number of suitable methods. These are described below separately for the case where the vesicles are gas ~illed, and where they are gaseous precursor ~illed, although vesicles having both a gas and gaseous precursor are part of the present invention.

-Utilizinq a Ga~
A preferred embodiment comprises the steps of agitating an a~ueous solution comprising a stabilizing compound, preferably a lipid, in the presence of a gas at a temperature below the gel to liquid crystalline phase transition temperature o~ the lipid to ~orm gas ~illed vesicles. The term agitating, and variations thereo~, as used herein, means any motion that shakes an aqueous solution such that gas is introduced from the local ambient environment into the aqueous solution. The shaking must be of su~icient force to result in the formation o~ vesicles, particularly stabilized vesicles. The shaking may be by swirling, such as by vortexing, side-to-side, or up-and-down CA 022148~ 1997-09-08 motion. Different types o~ motion may be combined. Also, the shaking may occur by shaking the container holding the aqueous lipid solution, or by shaking the aqueous solution within the container without shaking the container itself.
Further, the shaking may occur manually or by t machine. Mechanical shakers that may be used include, ~or example, a shaker table such as a VWR Scientific (Cerritos, CA) shaker table, or a Wig-L-Bug Shaker from Crescent Dental Mfg. Ltd., Lyons, Ill., which has been found to give excellent results. It is a preferred embodiment of the present invention that certain modes of shaking or vortexing be used to make stable vesicles within a preferred size range. Shaking is preferred, and it is preferred that this shaking be carried out using the Wig-L-Bug mechanical shaker. In accordance with this preferred method, it is preferred that a reciprocating motion be utilized to generate the gas filled vesicles. It is even more preferred that the motion be reciprocating in the form of an arc. It is still more preferred that the motion be reciprocating in the form of an arc between about 2~ and about 20~, and yet further preferred that the arc be between about 5~ and about 8~. It is most preferred that the motion is reciprocating between about 6~ and about 7~, most particularly about 6.5~.
It is contemplated that the rate o~ reciprocation, as well as the arc thereof, is critical to determining the amount and size of the gas filled vesicles formed. It is a preferred embodiment of the present invention that the number of reciprocations, i.e., ~ull cycle oscillations, be within the range of about 1000 and about 20,000 per minute.
More preferably, the number of reciprocations or oscillations will be between 2500 and 8000. The Wig-L-Bug~, referred to above, is a mechanical shaker which provides 2000 pestle strikes every 10 seconds, i.e., 6000 oscillations every minute. Of course, the number o~
oscillations is dependent upon the mass of the contents being agitated, with the larger the mass, the ~ewer the number of oscillations). Another means for producing CA 022l48~ l997-09-08 W096/28090 PCT~S96103054 shaking includes the action of gas emitted under high velocity or pressure.
It will also be understood that preferably, with a larger volume of aqueous solution, the total amount of force will be correspondingly increased. Vigorous shaking is defined as at least about 60 shaking motions per minute, and is preferred. Vortexing at least 60-300 revolutions per minute is more preferred. Vortexing at 300-1800 revolutions per minute is most preferred. The formation of gas filled vesicles upon shaking can be detected visually. The concentration of lipid required to form a desired stabilized vesicle level will vary depending upon the type of lipid used, and may be readily determined by routine experimentation. For example, in preferred embodiments, the concentration of 1,2-dipalimitoyl-phosphatidylcholine (DPPC) used to form stabilized vesicles according to the methods of the present invention is about 0.1 mg/ml to about 30 mg/ml of saline solution, more preferably from about 0.5 mg/ml to about 20 mg/ml o~ saline solution, and most preferably from about 1 mg/ml to about 10 mg/ml of saline solution. The concentration of distearoylphosphatidylcholine (DSPC) used in preferred embodiments is about 0.1 mg/ml to about 30 mg/ml of saline solution, more preferably from about 0.5 mg/ml to about 20 mg/ml of saline solution, and most preferably from about 1 mg/ml to about 10 mg/ml of saline solution.
In addition to the simple shaking methods described above, more elaborate, but for that reason less preferred, methods can also be employed, e.g., liquid crystalline shaking gas instillation processes, and vacuum drying gas instillation processes, such as those described in U.S. Serial No. 076,250, filed June 11, 1993, which is incorporated herein by reference, in its entirety. When such processes are used, the stabilized vesicles which are to be gas filled, may be prepared prior to gas installation using any one of a variety of conventional liposome preparatory techniques which will be apparent to those CA 022l48~ l997-09-08 W096l28090 PCT~S96103054 skilled in the art. These techniques include freeze-thaw, as well as techniques such as sonication, chelate dialysis, homogenization, solvent infusion, microemulsification, spontaneous formation, solvent vaporization, French pressure cell technique, controlled detergent dialysis, and others, each involving preparing the vesicles in various fashions in a solution containing the desired active ingredient so that the therapeutic, cosmetic or other agent is encapsulated in, enmeshed in, or attached the resultant polar-lipid based vesicle. See, e . g., Madden et al., Chemistry and Physics of Lipids, 1990 53, 37-46, the disclosure of which is hereby incorporated herein by re~erence in its entirety.
The gas filled vesicles prepared in accordance with the methods described above range in size ~rom below a micron to over lOO~ in size. In addition, it will be noted that after the extrusion and sterilization procedures, the agitation or shaking step yields gas filled vesicles with little to no residual anhydrous lipid phase (Bangham, A.D., Standish, M.M, & Watkins, J.C. (1965) J. Mol. Biol. 13, 238 - 252) present in the remainder o~ the solution. The resulting gas filled vesicles remain stable on storage at room temperature ~or a year or even longer.
The size o~ gas filled vesicles can be adjusted, i~ desired, by a variety o~ procedures including microemulsi~ication, vortexing, extrusion, ~iltration, sonication, homogenization, repeated ~reezing and thawing cycles, extrusion under pressure through pores of defined size, and similar methods. It may also be desirable to use the vesicles o~ the present invention as they are ~ormed, without any attempt at further modi~ication of the size thereo~.
The gas ~illed vesicles may be sized by a simple process of extrusion through ~ilters; the ~ilter pore sizes control the size distribution o~ the resulting gas filled ~ 35 vesicles. By using two or more cascaded, i. e., a stacked set o~ ~ilters, e.g., lO~ ~ollowed by 8~, the gas filled vesicles have a very narrow size distribution centered CA 022l48~ l997-09-08 W096/28090 PCT~S96/~3054 around 7 - 9 ~m. After filtration, these stabilized gas filled vesicles remain stable for over 24 hours.
The sizing or filtration step may be accomplished by the use of a filter assembly when the suspension is removed from a sterile vial prior to use, or even more preferably, the filter assembly may be incorporated into the syringe itself during use. The method of sizing the vesicles will then comprise using a syringe comprising a barrel, at least one filter, and a needle; and will be carried out by a step of extracting which comprises extruding said vesicles from said barrel through said filter fitted to said syringe between said barrel and said needle, thereby sizing said vesicles before they are administered to a patient in the course of using the vesicles as MRI
contrast agents in accordance with the present invention.
The step of extracting may also comprise drawing said vesicles into said syringe, where the filter will function in the same way to size the vesicles upon entrance into the syringe. Another alternative is to fill such a syringe with vesicles which have already been sized by some other means, in which case the filter now functions to ensure that only vesicles within the desired size range, or of the desired maximum size, are subsequently administered by extrusion from the syringe.
Typical of the devices which can be used for carrying out the sizing or filtration step, is the syringe and filter combination shown in Figure 2 of U.S. application Serial No. 08/401,974, filed March 9, 1995, the disclosure of which is incorporated by reference in its entirety.
In preferred embodiments, the stabilizing compound solution or suspension is extruded through a filter and the said solution or suspension is heat sterilized prior to shaking. Once gas filled vesicles are formed, they may be filtered for sizing as described above. These steps prior to the formation of gas filled vesicles provide the advantages, for example, of reducing the amount of unhydrated stabilizing compound, and thus providing a CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 significantly higher yield of gas filled vesicles, as well as and providing sterile gas filled vesicles ready for administration to a patient. For example, a mixing vessel such as a vial or syringe may be filled with a filtered stabilizing compound, especially lipid suspension, and the suspension may then be sterilized within the mixing vessel, for example, by autoclaving. Gas may be instilled into the lipid suspension to form gas filled vesicles by shaking the sterile vessel. Preferably, the sterile vessel is equipped with a filter positioned such that the gas filled vesicles pass through the filter before contacting a patient.
The first step of this preferred method, extruding the stabilizing, especially lipid, solution through a filter, decreases the amount of unhydrated compound by breaking up the dried compound and exposing a greater surface area ~or hydration. Pre~erably, the filter has a pore size of about 0.1 to about 5 ~m, more pre~erably, about 0.1 to about 4 ~m, even more pre~erably, about 0.1 to about 2 ~m, and most preferably, about 1 ~m. Unhydrated compound, especially lipid, appears as amorphous clumps of non-uniform size and is undesirable.
The second step, sterilization, provides a composition that may be readily administered to a patient ~or MRI imaging. Pre~erably, sterilization is accomplished by heat sterilization, preferably, by autoclaving the solution at a temperature o~ at least about 100~C, and more preferably, by autoclaving at about 100~C to about 130~C, even more pre~erably, about 110~C to about 130~C, even more preferably, about 120~C to about 130~C, and most pre~erably, about 130~C. Preferably, heating occurs for at least about 1 minute, more preferably, about 1 to about 30 minutes, even more preferably, about 10 to about 20 minutes, and most pre~erably, about 15 minutes.
If desired, alternatively the first and second steps, as outlined above, may be reversed, or only one of the two steps employed.
Where sterilization occurs by a process other than CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 heat sterilization at a temperature which would cause rupture of the gas filled vesicles, sterilization may occur subsequent to the formation of the gas filled vesicles, and is preferred. For example, gamma radiation may be used before and/or after gas filled vesicles are formed.

-Utilizin~ a Gaseous Precursor In addition to the a~orementioned embodiments, one can also use gaseous precursors contained in the lipid-based vesicles that can, upon activation by temperature, light, or pH, or other properties of the tissues of a host to which it is administered, undergo a phase transition from a liquid or solid entrapped in the lipid-based vesicles, to a gaseous state, expanding to create the stabilized, gas filled vesicles used in the present invention. This technique is described in detail in patent applications Serial Nos.
08/160,232, filed November 30, 1993, and 08/159,687, ~iled November 30, 1993, both of which are incorporated herein by re~erence in their entirety. The techniques ~or preparing gaseous precursor filled vesicles are generally similar to those described for the preparation of gas filled vesicles herein, except that a gaseous precursor is substituted ~or the gas.
The preferred method of activating the gaseous precursor is by temperature. Activation or transition temperature, and like terms, re~er to the boiling point of the gaseous precursor, the temperature at which the liquid to gaseous phase transition of the gaseous precursor takes place. Useful gaseous precursors are those gases which have boiling points in the range o~ about -100~ C to 70~ C. The activation temperature is particular to each gaseous precursor. An activation temperature o~ about 37~ C, or human body temperature, is pre~erred for gaseous precursors of the present invention. Thus, a liquid gaseous precursor is activated to become a gas at 37~ C. However, the gaseous precursor may be in liquid or gaseous phase ~or use in the methods of the present invention. The methods o~ preparing CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 the MRI contrast agents used in t~he present invention may be carried out below the boiling point of the gaseous precursor such that a liquid is incorporated into a vesicle. In addition, the said methods may be performed at the boiling point of the gaseous precursor such that a gas is incorporated into a vesicle. For gaseous precursors having low temperature boiling points, liquid precursors may be emulsified using a microfluidizer device chilled to a low temperature. The boiling points may also be depressed using solvents in liquid media to utilize a precursor in liquid form. Further, the methods may be performed where the temperature is increased throughout the process, whereby the process starts with a gaseous precursor as a liquid and ends with a gas.
The gaseous precursor may be selected so as to form the gas in sl tu in the targeted tissue or fluid, ln vivo upon entering the patient or animal, prior to use, during storage, or during manufacture. The methods of producing the temperature-activated gaseous precursor-filled vesicles may be carried out at a temperature below the boiling point of the gaseous precursor. In this embodiment, the gaseous precursor is entrapped within a vesicle such that the phase transition does not occur during manufacture.
Instead, the gaseous precursor-filled vesicles are manufactured in the liquid phase of the gaseous precursor.
Activation of the phase transition may take place at any time as the temperature is allowed to exceed the boiling point of the precursor. Also, knowing the amount of liquid in a droplet of liquid gaseous precursor, the size of the vesicles upon attaining the gaseous state may be determined.
Alternatively, the gaseous precursors may be utilized to create stable gas filled vesicles which are pre-formed prior to use. In this embodiment, the gaseous precursor is added to a container housing a suspending and/or stabilizing medium at a temperature below the liquid-gaseous phase transition temperature of the respective gaseous precursor. As the temperature is then exceeded, and CA 022l48~ l997-09-08 W096/28090 PCT~S96103054 an emulsion is ~ormed between the gaseous precursor and liquid solution, the gaseous precursor undergoes transition from the liquid to the gaseous state. As a result of this heating and gas formation, the gas displaces the air in the 5 head space above the liquid suspension so as to form gas filled lipid spheres which entrap the gas of the gaseous r precursor, ambient gas (e.g., air) or coentrap gas state gaseous precursor and ambient air. This phase transition can be used for optimal mixing and stabilization of the MRI
lO contrast medium. For example, the gaseous precursor, perfluorobutane, can be entrapped in the biocompatible lipid or other stabilizing compound, and as the temperature is raised, beyond 4~ C (boiling point of perfluorobutane) stabilizing compound entrapped fluorobutane gas results. As 15 an additional example, the gaseous precursor fluorobutane, can be suspended in an aqueous suspension containing emulsifying and stabilizing agents such as glycerol or propylene glycol and vortexed on a commercial vortexer.
Vortexing is commenced at a temperature low enough that the 20 gaseous precursor is liquid and is continued as the temperature o~ the sample is raised past the phase transition temperature from the liquid to gaseous state. In so doing, the precursor converts to the gaseous state during the microemulsification process. In the presence of the 25 appropriate stabilizing agents, surprisingly stable gas filled vesicles result.
Accordingly, the gaseous precursors may be selected to form a gas filled vesicle in vivo or may be designed to produce the gas filled vesicle in situ, during 30 the manufacturing process, on storage, or at some time prior to use.
As a further embodiment of this invention, by pre-forming the liquid state of the gaseous precursor into an aqueous emulsion and maintaining a known size, the maximum 35 size of the microbubble may be estimated by using the ideal gas law, once the transition to the gaseous state is effectuated. For the purpose o~ making gas filled vesicles CA 022148~ 1997-09-08 W096128090 PCT~S96103054 from gaseous precursors, the gas phase is assumed to form instantaneously and no gas in the newly formed vesicle has been depleted due to dif~usion into the liquid (generally aqueous in nature). Hence, from a known liquid volume in the emulsion, one actually would be able to predict an upper limit to the size of the gaseous vesicle.
Pursuant to the present invention, an emulsion of a stabilizing compound such as a lipid, and a gaseous precursor, containing liquid droplets of defined size may be formulated, such that upon reaching a speci~ic temperature, the boiling point of the gaseous precursor, the droplets will expand into gas filled vesicles of defined size. The defined size represents an upper limit to the actual size because factors such as gas diffusion into solution, loss of gas to the atmosphere, and the effects of increased pressure are factors for which the ideal gas law cannot account.
The ideal gas law and the equation for calculating the increase in volume of the gas bubbles on transition from the liquid to gaseous states is as follows:
PV = nRT

where P = pressure in atmospheres V = volume in liters n = moles of gas T = temperature in ~ K
R = ideal gas constant = 22.4 L atmospheres deg~l mole~l With knowledge of volume, density, and temperature of the liquid in the emulsion of liquids, the amount (e.g., number of moles) of liquid precursor as well as the volume o~ liquid precursor, a priorl, may be calculated, which when converted to a gas, will expand into a vesicle of known volume. The calculated volume will reflect an upper limit to the size of the gas filled vesicle, assuming instantaneous expansion into a gas filled vesicle and - 35 negligible diffusion of the gas over the time of the expansion.
Thus, for stabilization of the precursor in the liquid state in an emulsion wherein the precursor droplet is CA 022l48~ l997-09-08 W096l28090 PCT~S96/03054 spherical, the volume o~ the precursor droplet may be determined by the equation:
Volume (sphere) = 4/3 ~r3 where r = radius of the sphere Thus, once the volume is predicted, and knowing the density of the liquid at the desired temperature, the amount o~ liquid (gaseous precursor) in the droplet may be determined. In more descriptive terms, the following can be applied:
Vgas = 4/3 ~(rgas) by the ideal gas law, PV=nRT
substituting reveals, VgaS = nRT/Pgas or, (A) n = 4/3 [~rgaS3] P/RT
amount n = 4/3 [~rgaS3 P/RT] * MWn Converting back to a liquid volume (B) Vli~ = [4/3 [~rga53] P/RT] * MWn/D]
where D = the density o~ the precursor Solving ~or the diameter o~ the liquid droplet, (C) diameter/2 = [3/4~ [4/3 * [~rgaS3] P/RT] MWn/D] 1/3 which reduces to Diameter = 2[[rgaS3] P/RT [MWn/D] ] 1/3 As a further means o~ preparing vesicles o~ the desired size for use as MRI contrast agents in the present invention, and with a knowledge o~ the volume and especially the radius o~ the stabilizing compound/precursor liquid droplets, one can use appropriately sized filters in order to size the gaseous precursor droplets to the appropriate diameter sphere.
A representative gaseous precursor may be used to form a vesicle o~ de~ined size, ~or example, 10~ diameter.
In this example, the vesicle is formed in the bloodstream of a human being, thus the typical temperature would be 37O C
or 310~ K. At a pressure of 1 atmosphere and using the CA 022148~ 1997-09-08 W096/28090 PCT~S961030 equation in (A), 7.54 x lO-l7 moles of gaseous precursor would be required to fill the volume of a lO~ diameter vesicle.
Using the above calculated amount of gaseous precursor, and l-fluorobutane, which possesses a molecular weight of 76.ll, a boiling point of 32.5~ C and a density of 0.7789 grams/mL~l at 20~ C, further calculations predict that 5.74 x lo-l5 grams o~ this precursor would be required for a lO~ vesicle. Extrapolating further, and armed with the knowledge of the density, equation (B) further predicts that 8.47 x lo-l6 mLs of liquid precursor are necessary to form a vesicle with an upper limit o~ lO~.
Finally, using equation (C), an emulsion of lipid droplets with a radius of 0.0272~ or a corresponding diameter of 0.0544~ need be formed to make a gaseous precursor ~illed vesicle with an upper limit of a lO~
vesicle.
An emulsion of this particular size could be easily achieved by the use of an appropriately sized filter.
In addition, as seen by the size of the filter necessary to form gaseous precursor droplets o~ defined size, the size o~
the filter would also su~fice to remove any possible bacterial cont~m;n~ntS and, hence, can be used as a sterile filtration as well.
This embodiment for preparing gas filled vesicles used as simultaneous magnetic resonance ~ocused noninvasive ultrasound contrast agents in the methods o~ the present invention may be applied to all gaseous precursors activated by temperature. In fact, depression o~ the ~reezing point of the solvent system allows the use gaseous precursors which would undergo liquid-to-gas phase transitions at temperatures below 0~ C. The solvent system can be selected to provide a medium for suspension of the gaseous precursor.
For example, 20~ propylene glycol miscible in buf~ered saline exhibits a ~reezing point depression well below the freezing point of water alone. By increasing the amount o~
propylene glycol or adding materials such as sodium chloride, the freezing point can be depressed even ~urther.

CA 022148~ 1997-09-08 WO 96/28090 PCT/USg6/03054 The selection of appropriate solvent systems may be explained by physical methods as well. When substances, solid or liquid, herein referred to as solutes, are dissolved in a solvent, such as water based buffers for example, the freezing point is lowered by an amount that is dependent upon the composition of the solution. Thus, as defined by Wall, one can express the freezing point depression of the solvent by the following equation:
Inxa = In (1 - Xb) = ~HfUs/R(l/To - 1/T) where:
xa = mole fraction of the solvent xb = mole fraction of the solute aHfUs = heat of fusion of the solvent To = Normal freezing point of the solvent The normal ~reezing point o~ the solvent results from solving the equation. I~ xb is small relative to xa, then the above equation may be rewritten:
xb = aHfus/R[T - To/ToT] ~ aHfusaT/RTo The above equation assumes the change in temperature aT is small compared to T2. The above equation can be simplified further assuming the concentration of the solute (in moles per thousand grams of solvent) can be expressed in terms of the molality, m Thus, Xb =m/[m + 1000/ma] ~ mMa/1000 where:
Ma = Molecular weight of the solvent, and m = molality o~ the solute in moles per 1000 grams.
Thus, substituting for the fraction Xb:
aT = [MaRTo2/loooaHfus]m or aT = Kfm, where Kf =MaRTo2/1000 aHfU5 Kf iS referred to as the molal freezing point and is equal to 1. 86 degrees per unit of molal concentration for water at one atmosphere pressure. The above equation may be used to accurately determine the molal freezing point of gaseous-precursor filled vesicle solutions used in the present invention.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/030S4 Hence, the a~ove equation can be applied to estimate freezing point depressions and to determine the appropriate concentrations of liquid or solid solute necessary to depress the solvent freezing temperature to an appropriate value.
Methods of preparing the temperature activated gaseous precursor-filled vesicles include:
(a) vortexing an aqueous suspension of gaseous precursor-filled vesicles used in the present invention;
variations on this method include optionally autoclaving before shaking, optionally heating an aqueous suspension of gaseous precursor and lipid, optionally venting the vessel containing the suspension, optionally shaking or permitting the gaseous precursor vesicles to form spontaneously and cooling down the gaseous precursor filled vesicle suspension, and optionally extruding an aqueous suspension o~ gaseous precursor and lipid through a filter o~ about 0.22 ~, alternatively, filtering may be performed during in vivo administration of the resulting vesicles such that a ~ilter of about 0.22 ~ is employed;
(b) a microemulsification method whereby an aqueous suspension o~ gaseous precursor-~illed vesicles of the present invention are emulsified by agitation and heated to ~orm vesicles prior to administration to a patient; and (c) forming a gaseous precursor in lipid suspension by heating, and/or agitation, whereby the less dense gaseous precursor-~illed vesicles ~loat to the top of the solution by expanding and displacing other vesicles in the vessel and venting the vessel to release air; and (d) in any of the above methods, utilizing a sealed vessel to hold the aqueous suspension of gaseous precursor and stabilizing compound such as biocompatible lipid, said suspension being maintained at a temperature below the phase transition temperature of the gaseous precursor, followed by autoclaving to move the temperature above the phase transition temperature, optionally with shaking, or permitting the gaseous precursor vesicles to CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 ~orm spontaneously, whereby the expanded gaseous precursor in the sealed vessel increases the pressure in said vessel, and cooling down the gas filled vesicle suspension.
Freeze drying is use~ul to remove water and organic materials ~rom the stabilizing compounds prior to the shaking gas instillation method. Drying-gas instillation methods may be used to remove water ~rom vesicles. By pre-entrapping the gaseous precursor in the dried vesicles (i . e., prior to drying) a~ter warming, the gaseous precursor may expand to fill the vesicle. Gaseous precursors can also be used to ~ill dried vesicles a~ter they have been subjected to vacuum. As the dried vesicles are kept at a temperature below their gel state to liquid crystalline temperature, the drying chamber can be slowly ~illed with the gaseous precursor in its gaseous state, e. g., per~luorobutane can be used to ~ill dried vesicles composed of dipalmitoylphosphatidylcholine (DPPC) at temperatures between 4~ C (the boiling point o~
per~luorobutane) and below 40~ C, the phase transition temperature o~ the biocompatible lipid. In this case, it would be most preferred to fill the vesicles at a temperature about 4~ C to about 5O C.
Pre~erred methods for preparing the temperature activated gaseous precursor-~illed vesicles comprise shaking an aqueous solution having a stabilizing compound such as a biocompatible lipid in the presence o~ a gaseous precursor at a temperature below the gel state to liquid crystalline state phase transition temperature o~ the lipid. The present invention also contemplates the use o~ a method ~or preparing gaseous precursor-~illed vesicles comprising shaking an aqueous solution comprising a stabilizing compound such as a biocompatible lipid in the presence of a gaseous precursor, and separating the resulting gaseous precursor-~illed vesicles ~or MRI imaging use. Vesicles prepared by the ~oregoing methods are re~erred to herein as gaseous precursor-~illed vesicles prepared by a gel state shaking gaseous precursor instillation method.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 Conventional, aqueous-~illed liposomes of the prior art are routinely formed at a temperature above the phase transition temperature o~ the lipids used to make them, since they are more flexible and thus useful in biological systems in the liquid crystalline state. See, for example, Szoka and Papahadjopoulos, Proc . Natl . Acad .
Sci. 1978, 75, 4194-4198. In contrast, the vesicles made according to preferred embodiments described herein are gaseous precursor-filled, which imparts greater ~lexibility, since gaseous precursors after gas formation are more compressible and compliant than an aqueous solution. Thus, the gaseous precursor-filled vesicles may be utilized in biological systems when formed at a temperature below the phase transition temperature of the lipid, even though the gel phase is more rigid.
The methods contemplated by the present invention provide ~or shaking an aqueous solution comprising a stabilizing compound such as a biocompatible lipid in the presence of a temperature activated gaseous precursor.
Shaking, as used herein, is defined as a motion that agitates an aqueous solution such that gaseous precursor is introduced ~rom the local ambient environment into the aqueous solution. Any type of motion that agitates the aqueous solution and results in the introduction of gaseous precursor may be used ~or the shaking. The shaking must be of suf~icient force to allow the ~ormation o~ a suitable number of vesicles after a period o~ time. Pre~erably, the shaking is of sufficient force such that vesicles are formed within a short period o~ time, such as 30 minutes, and preferably within 20 minutes, and more preferably, within 10 minutes. The shaking may be by microemulsifying, by micro~luidizing, for example, swirling (such as by vortexing), side-to-side, or up and down motion. In the case o~ the addition o~ gaseous precursor in the liquid state, sonication may be used in addition to the shaking methods set forth above. Further, different types of motion may be combined. Also, the shaking may occur by shaking the CA 022l48~ l997-09-08 W096/28090 PCT~S96/030 container holding the aqueous lipid solution, or by shaking the aqueous solution within the container without shaking the container itself. Further, the shaking may occur manually or by machine. Mechanical shakers that may be used include, for example, a shaker table, such as a VWR
Scientific (Cerritos, CA) shaker table, a microfluidizer, Wig-L-Bug~ (Crescent Dental Manufacturing, Inc., Lyons, IL), which has been found to give particularly good results, and a mechanical paint mixer, as well as other known machines.
Another means for producing shaking includes the action of gaseous precursor emitted under high velocity or pressure.
It will also be understood that preferably, with a larger volume of aqueous solution, the total amount of force will be correspondingly increased. Vigorous shaking is defined as at least about 60 shaking motions per minute, and is preferred. Vortexing at least 1000 revolutions per minute, an example o~ vigorous shaking, is more pre~erred.
Vortexing at 1800 revolutions per minute is most preferred.
The ~ormation of gaseous precursor-filled vesicles upon shaking can be detected by the presence of a foam on the top of the aqueous solution. This is coupled with a decrease in the volume o~ the aqueous solution upon the formation of foam. Preferably, the final volume of the foam is at least about two times the initial volume of the 2s aqueous lipid solution; more pre~erably, the ~inal volume o~
the foam is at least about three times the initial volume of the aqueous solutioni even more preferably, the final volume o~ the ~oam is at least about ~our times the initial volume of the aqueous solution; and most preferably, all o~ the aqueous lipid solution is converted to foam.
The required duration of shaking time may be determined by detection o~ the ~ormation of foam. For example, 10 ml of lipid solution in a 50 ml centri~uge tube may be vortexed for approximately 15-20 minutes or until the viscosity of the gaseous precursor-filled vesicles becomes su~iciently thick so that it no longer clings to the side walls as it is swirled. At this time, the ~oam may cause CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 the solution containing the gaseous precursor-filled vesicles to raise to a level of 30 to 35 ml.
The concentration of stabilizing compound, especially lipid required to form a preferred foam level will vary depending upon the type of stabilizing compound such as biocompatible lipid used, and may be readily determined by one skilled in the art, once armed with the present disclosure. For example, in preferred embodiments, the concentration of 1,2-dipalimitoylphosphatidylcholine (DPPC) used to ~orm gaseous precursor-filled vesicles according to methods contemplated by the present invention is about 20 mg/ml to about 30 mg/ml saline solution. The concentration o~ distearoylphosphatidylcholine (DSPC) used in preferred embodiments is about 5 mg/ml to about 10 mg/ml saline solution.
Specifically, DPPC in a concentration of 20 mg/ml to 30 mg/ml, upon shaking, yields a total suspension and entrapped gaseous precursor volume four times greater than the suspension volume alone. DSPC in a concentration of 10 mg/ml, upon shaking, yields a total volume completely devoid of any liquid suspension volume and contains entirely foam.
It will be understood by one skilled in the art, once armed with the present disclosure, that the lipids and other stabilizing compounds used as starting materials, or the vesicle ~inal products, may be manipulated prior and subsequent to being subjected to the methods contemplated by the present invention. For example, the stabilizing compound such as a biocompatible lipid may be hydrated and then lyophilized, processed through ~reeze and thaw cycles, or simply hydrated. In preferred embodiments, the lipid is hydrated and then lyophilized, or hydrated, then processed through freeze and thaw cycles and then lyophilized, prior to the ~ormation o~ gaseous precursor-filled vesicles.
According to the methods contemplated by the present invention, the presence of gas, such as and not limited to air, may also be provided by the local ambient atmosphere. The local ambient atmosphere may be the CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 atmosphere within a sealed container, or in an unsealed container, may be the external environment. Alternatively, for example, a gas may be injected into or otherwise added to the container having the aqueous lipid solution or into the aqueous lipid solution itself in order to provide a gas other than air. Gases that are not heavier than air may be added to a sealed container while gases heavier than air may be added to a sealed or an unsealed container. Accordingly, the present invention includes co-entrapment of air and/or other gases along with gaseous precursors.
As already described above in the section dealing with the stabilizing compound, the pre~erred methods contemplated by the present invention are carried out at a temperature below the gel state to liquid crystalline state phase transition temperature o~ the lipid employed. By 'rgel state to liquid crystalline state phase transition temperature", it is meant the temperature at which a lipid bilayer will convert from a gel state to a liquid crystalline state. See, for example, Chapman et al., J.
20 Biol. Chem. 1974, 249, 2512-2521.
Hence, the stabilized vesicle precursors described above, can be used in the same manner as the other stabilized vesicles used in the present invention, once activated by application to the tissues of a host, where such factors as temperature or pH may be used to cause generation of the gas. It is preferred that this embodiment is one wherein the gaseous precursors undergo phase transitions from liquid to gaseous states at near the normal body temperature of said host, and are thereby activated by the temperature of said host tissues so as to undergo transition to the gaseous phase therein. More preferably still, this method is one wherein the host tissue is human tissue having a normal temperature o~ about 37~C, and wherein the gaseous precursors undergo phase transitions from liquid to gaseous states near 37~C.
All of the above embodiments involving preparations o~ the stabilized gas ~illed vesicles used in CA 022148~ 1997-09-08 W096/28090 PCT~S96103054 the present invention, may be sterilized by autoclave or sterile filtration if these processes are per~ormed before either the gas instillation step or prior to temperature mediated gas conversion of the temperature sensitive gaseous precursors within the suspension. Alternatively, one or more anti-bactericidal agents and/or preservatives may be included in the formulation of the contrast medium, such as sodium benzoate, all quaternary ammonium salts, sodium azide, methyl paraben, propyl paraben, sorbic acid, ascorbylpalmitate, butylated hydroxyanisole, butylated hydroxytoluene, chlorobutanol, dehydroacetic acid, ethylenediamine, monothioglycerol, potassium benzoate, potassium metabisulfite, potassium sorbate, sodium bisulfite, sulfur dioxide, and organic mercurial salts.
Such sterilization, which may also be achieved by other conventional means, such as by irradiation, will be necessary where the stabilized microspheres are used for imaging under invasive circumstances, e.g., intravascularly or intraperitonealy. The appropriate means of sterilization will be apparent to the artisan instructed by the present description of the stabilized gas filled vesicles and their use. The contrast medium is generally stored as an aqueous suspension but in the case of dried vesicles or dried lipidic spheres the contrast medium may be stored as a dried powder ready to be reconstituted prior to use.
The invention is further demonstrated in the following prophetic Examples 1-11. The examples, however, are not intended to in any way limit the scope o~ the present invention.

EXAMPLES
Example 1 Trans~errin is coupled to dextran and this is added to a solution of iron salts. The solution of superparamagnetic iron oxides is prepared by dissolving a mixture o~ ~errous and ~erric iron salts in water and HCl at pH l.o in an anaerobic environment in the chamber of a Heat CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 Systems Probe (Heat Systems, Farmingdale, N.Y.) sonicator equipped with an atmosphere and pressure chamber. The sonicator is activated using the standard sized horn on medium/high power and as oxygen gas is bubbled through the solution the pH is raised suddenly to pH = 12. The result is iron oxide nanoparticles composed of magnetite, Fe~O4.
The nanoparticles are washed in normal saline and differential centrifugation is used to harvest the nanoparticles with diameter of 20 nm and less. These nanoparticles are suspended in n-hexane at a concentration of 10 mg per ml nanoparticles with 10 mg per ml dipalmitoylphospatidylcholine. The n-hexane is evaporated and the lipid coated iron oxide nanoparticles are lyophilized. Accordingly, iron oxide nanoparticles are prepared which are coated with dextran bearing transferrin.
The superparamagnetic iron oxide nanoparticles at a concentration of lO mg per ml are mixed with 2 mg per ml perfluoropentane and 20 mg per ml pluronic F-68 with lO mg per ml dioleoylphosphatidylcholine in sterile water with 5.5 ~ by weight mannitol. This is microfluidized and results in a colloidal suspension of perfluoropentane coated with trans~errin labeled magnetite particles. This is administered i.v. (dose = 5 ml) to a 25 year old female patient with suspected ectopic pregnancy. The magnetically labeled vesicles localize in the ectopic pregnancy as the transferrin binds to the fetal tissue and this is visualized by MRI. High energy continuous wave ultrasound, 2 MHz, 2.5 Watts/cm is applied to the ectopic fetal tissue. By virtue of increased absorption i~ sound energy caused by the vesicles the ectopic fetal tissue is then destroyed by the ultrasound energy. This avoids an open procedure such as laparotomy or a more invasive procedure such as laparoscopy as the therapeutic ultrasound under magnetic resonance guidance can generally be performed transcutaneously without having to gain surgical access.

CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~4 ExamPle 2 A colloidal suspension of perfluorohexane (0.2 mg per ml) and perfluorpentane (0.2 mg per ml) is prepared in 10 mg per ml phospholipid (82 mole percent DPPC, 7 mole percent DPPE-PEG 5000 and 10 mole percent DPPA and 1 mole DPPE-PEG 5000-anti fibrin antibody) with 20 mg per ml pluronic F-68 and 5.5 ~ by weight mannitol. To this is added 5 mg per ml of iron oxide nanoparticles and this material is microfluidized as disclosed in preceding examples. This material is administered i.v. to a patient with suspected vascular thromobosis. A magnetometer superconducting quantum inferometry device (SQUID), a type of magnetic imaging, is scanned over the patient~s body, localizing an apparent region of increased magnetic susceptibility to the patient's ilial veins. The presence of clot is confirmed via ultrasound imaging. High energy ultrasound, 500 milliwatts per cm2 is then applied to the regions of clot to which the vesicles are bound. Sonication is performed under the guidance of the SQUID or magnetometer. The ultrasound transducer might be equipped with a SQUID as a part of the transducer. As the sonication occurs, a change in the magnetic susceptibility is detected by the magnetometer. The microvesicles cause increased absorption of sonic energy; the liquid to gaseous phase conversion of the perflourohexane in the emulsion is readily visible on either ultrasound or by magnetic imaging as the vesicles expand during the heat expansion process and this results in local lysis of the clot and noninvasive surgical alleviation of the thrombosis.

Example 3 Gas filled microvesicles impregnated with paramagnetic iron oxide particles in the microvesicle membrane are injected into the antecubital ~ossa. The vesicles are pre~erential taken up by the lymphatic vessels which could be identified on magnetic resonance with a superconducting quantum in~erometry device (SQUID) as clear CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 outlines of tumor burdened nodes. Upon identification of the nodes, a focused ultrasound transducer is then aimed at the site of the identified tumor burden and a continuous wave train of ultrasound is applied under SQUID guidance.
The microvesicles are made to resonate and release energy in the form o~ thermal energy, thereby heating and subsequently destroying the tumor. Upon re-imaging by magnetic imaging using the SQUID device, the image of the tumor burden in the lymphatic is no longer identifiable, indicating destruction of the tumor, thereby providing a noninvasive surgical technique.

Ex~ple 4 Gas filled microvesicles prepared with manganese N,N'-Bis-(carboxy-decylamidomethyl-N-2,3-dihydroxypropyl)-ethylenediamine-N,N'-diacetate, (MN-EDTA-DDP), and Mn-EDTA-ODP, a paramagnetic complex disclosed in U.S. Patent No.
5,312,617, the disclosure of which is incorporated herein by reference in its entirety, is injected into a patient with malignant melanoma. The exact size and location o~ the tumor in the lymph chain is identi~ied on MRI and treated with real time simultaneous magnetic resonance imaging and sonication using a magnetic resonance compatible transducer operating at 1.5 Watts per cm2 and a ~requency of 0.75 MHz.
The presence o~ the bubbles results in increased deposition of energy, heating of the tissue as well as local cavitation. The degree of tissue necrosis as well as the temperature of the tissue is monitored non-invasively by simultaneous real time magnetic resonance imaging on a machine equipped with echoplanar imaging gradients, thus ef~ecting noninvasive surgery.

Example 5 In a patient with a cerebral arteriovenous malformation (AVM), a skull ~lap is created and the dura is surgically exposed. The patient is placed in an MRT-0.5 Tesla, interventional magnetic resonance imaging system, (GE

CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 Medical Systems, Milwaukee, WI). This magnetic resonance system allows access to the patient during surgical procedures while simultaneous magnetic resonance imaging.
r The patient is injected with 0.2 ml per kg of Aerosomes~
5 composed of 2 mg per ml of lipid containing 75 mole~ DPPC, 8 mole ~ DPPA and 8 mole ~ DPPE-PEG 5000 with 9 mole ~
Platelet-Activation Factor (PAF), Avanti Polar Lipids, Alabaster Alabama entrapping a mixture o~ air and per~luorobutane gas. The purpose of the PAF is to activate 10 platelets after release o~ the PAF ~rom the Aerosomes~ to stimulate thrombosis of the AVM. During magnetic resonance imaging a high energy magnetic resonance compatible ultrasound transducer equipped with imaging and therapy functions is positioned over the AVM. After the Aerosomes~
15 are injected I.V. they pass through the large vessels and microcirculation supplying the AVM. The microbubbles are readily visualized by both magnetic resonance and ultrasound. On magnetic resonance the bubbles are portrayed as signal voids on gradient echo bright blood images 20 obtained during transit through the AVM and on ultrasound as a snow-storm of specular reflections. The ultrasound transducer is focussed onto the AVM such that the focal zone of the ultrasound corresponds to the vascular nidus.
Simultaneous magnetic resonance imaging and ultrasound are 25 per~ormed as the power level on ultrasound is increased (e.g. up to several watts). While emissions from cavitation obscure visualization of much of the anatomy on ultrasound, magnetic resonance still shows the surrounding tissues with exquisite detail. The surgeon operating the high energy 30 ultrasound is there~ore much better able to control the aiming, firing and energy levels of the high energy transducer and avoid damaging critical surrounding cerebral vascular structures. The procedure results in ablation of the AVM, regions of coagulative necrosis as well as 35 thrombosis o~ the vascular nidus. At the end of the procedure much of the anatomy is obscured on ultrasound due to the coagulative necrosis and collections of microbubbles CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 within the tissue, but magnetic resonance shows the entire surgical ~ield as well as the surgical e~ects on the treated and surrounding tissues.

Example 6 A trauma victim with suspected hemorrhage is scanned by MRI. The scan shows a hemorrhage from the spleen. Aerosomes as in the example above except that the Aerosomes also entrap 1 mg per ml o~ thrombin are injected I.V. and simultaneous magnetic resonance and ultrasound imaging are per~ormed. As the Aerosomes pass through the splenic artery the ultrasound power on the magnetic resonance compatible ultrasound transducer is increased by the surgeon to about 1.0 Watt per cm2 and the Aerosomes pop releasing PAF and thrombin. Thrombosis is achieved and the hemorrhage is stopped. Simultaneous magnetic resonance angiography con~irms the thrombosis of the splenic artery.
This m;n;m~l ly invasive procedure is cheaper and caused less morbidity than conventional open surgery.

Example 7 100 ml o~ per~luoropentane vesicles coated with phospholipid, 82 mole ~ DPPC, 8 mole ~ DPPE-PEG 2000 and 10 mole ~ DPPA (mean diameter = about 1 micron) with 10 mole percent alkylated complexes o~ manganese (Mn-EDTA-ODP) bearing antibodies, Monoclonal Antibody to Breast Cancer (human) CA-15-3, IgG1, (SIGNET LABS, Dedham, MA) to human breast carcinoma is administered intravenously to a patient with breast carcinoma. The vesicles also contain 10 mole percent alkylated derivative o~ doxorubicin bound to the vesicle membranes. Four hours later the patient is scanned via MRI. Enhancing lymph nodes are identi~ied in the axilla indicating metastatic disease. An magnetic resonance compatible 1 MHz ultrasound probe is positioned over the lymph nodes and high energy continuous wave ultrasound at 200 mW/cm2 is applied to the lymph nodes. Simultaneous real time magnetic resonance imaging is per~ormed on a CA 022l48~ l997-09-08 W096/28090 PCT~S961030S4 commercially available magnet, e.g. 1.5 Tesla (GE Signa, Milwaukee, WI), using rapid pulse sequences, e.g. Spoiled GRASS, TR = 30 msec and TE = 5 msec with a 30~ flip angle.
r As the vesicles expand during heating they are 5 seen as an increased region of low signal intensity on the magnetic resonance images corresponding to the zone of magnetic susceptibility caused by the vesicles. As the vesicles "pop" this is seen as a transient region of even more demonstrable hypointensity. Thereafter the vesicles lO disappear a~ter they have "popped" and cleared. As the vesicles pop, the doxorubicin pro-drug is released and activated in the neoplastic lymph nodes.

Ex~m~le 8 Superparamagnetic iron oxides are prepared by 15 dissolving a mixture of ferrous and ferric iron salts in water and HCl at pH 1.0 in an anaerobic environment in the chamber of a Heat Systems Probe (Heat Systems, Farmingdale, N.Y.) sonicator equipped with an atmosphere and pressure chamber. The sonicator is activated using the standard 20 sized horn on medium/high power and as oxygen gas is bubbled through the solution the pH is raised suddenly to pH = 12.
The result is iron oxide nanoparticles composed of magnetite, Fe3O4. The nanoparticles are washed in normal saline and differential centri~ugation is used to harvest 25 the nanoparticles with diameter of 20 nm and less. These nanoparticles are suspended in n-hexane at a concentration of lO mg per ml nanoparticles with 10 mg per ml dipalmitoylphospatidylcholine. The n-hexane is evaporated and the lipid coated iron oxide nanoparticles are 30 lyophilized. The lipid coated iron oxide nanoparticles are then added 10 ~ by weight to 1 mg per ml of phospholipid composed of 82 mole ~ dipalmitoylphosphatidylcholine (DPPC) with 8 mole percent dipalmitylphosphatidylethanolamine-PEG
5,000 (DPPE-PEG 500) and 8 mole percent 35 dipalmitoylphosphatidic acid (DPPA) with 2 mole percent palmitoylated cis-platinum derivative. This mixture o~

-CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 lipids, lipid coated iron oxides and palmitoylated pro-drug is suspended at a final lipid concentration of 1 mg per ml in normal saline in a sealed sterile container with a head space of perfluorobutane gas. The material is shaken for 2 minutes on a Wig-L-Bug~ at 4,200 r.p.m and results in lipid coated pro-drug bearing vesicles, the surfaces o~ which are studded with iron oxide nanoparticles. A dose of 20 ml of these vesicles (mean diameter = about 2 microns, bubble conc. = 1 x 109 per ml) is injected intravenously into a patient and magnetic resonance imaging is performed obtaining rapid GRASS se~uences. The iron oxide labeled vesicles are easily seen on magnetic resonance as susceptibility agents causing regions of hypointensity. The pro-drug vesicle based contrast agent is shown to accumulate in regions of vascularized tumors involving the lymph nodes and other tissues. Ultrasound 1 MHz is applied as above to ~popll the vesicles under magnetic resonance guidance and achieve local drug delivery.

ExamPle 9 Anti-myosin antibody, anti-myosin antibody, Chicken Muscle, Catalogue No. 476123, (CAL BIOCHEM, La Jolla, CA) is coupled to dipalmitolyphosphatidyl-ethanolamine. This is added 0.1 mg per ml to a concentration o~ 0.1 mg per ml perfluoropentane and 5 mg per ml of phospholipid (90 mole ~ DPPC with 10 mole ~ DPPA) in 5.5 ~ by weight mannitol in sterile water. This mixture is bubbled with oxygen and oxygen-17 and then microemulsi~ied using a micro~luidizer (Micro~luidics, Newton, MA) at 16,000 psi for 20 passes resulting in emulsi~ied antibody bearing colloids o~ per~luoropentane. Ten cc of this material is injected into a patient with suspected myocardial in~arction and magnetic resonance imaging is performed. A region o~
enhanced susceptibility is shown in the area of in~arcted myocardium on the rapid GRASS magnetic resonance images. An magnetic resonance compatible continuous wave ultrasound transducer is positioned-over the myocardium and the CA 022l48~ l997-09-08 W096/28090 PCT~S96/03054 myocardium is treated with 0.100 watts per cm2 ultrasound energy. This causes the bubbles to pop and release oxygen locally into the ischemic tissue.

Example 10 A vesicle is prepared as in Example 9, except that the cationic lipid DOTMA, N-[l(-2,3-dioleoyloxy)propyl]N,N-trimethylammonium chloride is substituted for DPPA and the DPPC is substituted with DPPE. The antibody bearing colloidal particles of per~luoropentane are then prepared as above and loaded with Oxygen-17 gas within the perfluoropentane. Then 10 micrograms per ml of DNA encoding the gene ~or vascular endothelial growth ~actor (VE&F) is added and the suspension is vortexed at low power setting for 2 minutes at 4~C. Magnetic resonance imaging is performed in a patient with suspected myocardial infarction.
A region of low signal intensity is identi~ied in the myocardium about 30 minutes after administration of the contrast agent. An magnetic resonance compatible ultra-sound transducer is ~ocused on the region o~
ischemic/infarcted myocardium and the perfluoropentane microbubbles are ~popped" under real time simultaneous magnetic resonance imaging using a gradient echo echoplanar imaging technique in a magnetic resonance system equipped with resonating gradients, (Advanced NMR, Woburn, MA), 2S retrofit 1.5 Tesla, (GE Signa System, Milwaukee, WI). This results in local integration o~ the gene for VEGF within the infarcted/ischemic myocardium and local gene expression.
New blood vessels proli~erate in response to the VEGF and there is increased growth of new blood vessels. This results in healthier regional myocardium.

Example 11 Cationic vesicles are prepared by shaking a mixture o~ lipid 82 mole ~ DPPC with 7 mole ~ DPPE-PEG 5,000 with 5 mole ~ DPPA and 6 mole ~ DOTMA with a head space of gas prepared ~rom a mixture of per~luorobutane and oxygen-CA 022l48~ l997-09-08 W096/28090 PCT~S96/030~4 16. After shaking, as disclosed in preceding examples with the Wig-L-Bug~, 100 micrograms per ml of DNA for the VEGF
gene is added and the mixture is gently vortexed for 1 minute. This results in absorption of the DNA onto the t 5 surface of the microvesicles. A somewhat higher amount of vesicles is necessary for visualization under magnetic resonance than the magnetically labeled vesicles but the vesicles are still detectable on T2 weighted spin echo or fast spin echo or gradient echo pulse sequences. Magnetic 10 resonance angiography (MRA), a type of magnetic resonance imaging, using a 2D Time of Flight pulse sequence is performed an a diabetic patient with peripheral vascular disease involving the lower extremities. A significant stenosis is shown in the popliteal artery. The 15 microvesicles bearing DNA are administered i.v. and on the basis of the vesicle signals from MRI and ultrasound, high energy ultrasound is applied under magnetic resonance imaging guidance from the skin surface to focus energy on the region of stenosis in the popliteal artery. This 20 results in localized bubble rupture and gene release at the site of arterial stenosis. Localized expression o~ VEGF
encourages collateral and new vessel ~ormation improving blood flow to the distal leg.

The disclosures of each patent, patent application 25 and publication cited or described in this document are hereby incorporated herein by reference, in their entirety.
Various modification o~ the invention, in addition to those described herein, will be apparent to those skilled in the art from the ~oregoing description. Such 30 modi~ications are also intended to ~all within the scope of the appended claims.

_

Claims (31)

What is claimed is:
1. A method of magnetic resonance focused surgical ultrasound comprising:
administering a contrast medium for magnetic resonance imaging comprising gas filled vesicles to a patient requiring surgery, scanning said patient with magnetic resonance imaging using said contrast medium to identify the region of the patient requiring surgery, and applying ultrasound to said region to carry out said surgery.
2. The method of claim 1 wherein said application of ultrasound is simultaneous with a second scanning step whereby said patient is scanned with magnetic resonance imaging.
3. The method of claim 1 wherein said application of ultrasound is followed by a second scanning step whereby said patient is scanned with magnetic resonance imaging.
4. The method of claim 1 wherein said surgery is carried out in one of the following regions: vascular;
cardiovascular; gastrointestinal; intranasal tract; auditory canal; intraocular region; intraperitoneal region; kidneys;
urethra; genitourinary tract, brain, spine, pulmonary region, and soft tissues.
5. A method of claim 1 wherein said vesicles comprise a targeting agent.
6. The method of claim 1 wherein said gas filled vesicles further comprise a therapeutic which is released upon application of ultrasound.
7. The method of claim 6 wherein said therapeutic is selected from the group consisting of an oligonucleotide sequence, an antisense sequence, an antibody, and a chemotherapeutic agent.
8. The method of claim 1 wherein said ultrasound repairs an aperture in said region of said patient.
9. The method of claim 8 wherein said aperture is in the vasculature of said patient.
10. The method of claim 1 wherein said gas filled vesicles are administered intravenously.
11. The method of claim 1 wherein the gas is selected from the group consisting of air, nitrogen, carbon dioxide, oxygen, fluorine, helium, argon, xenon, and neon.
12. The method of claim 1 wherein the gas is a fluorinated gas.
13. The method of claim 12 wherein the fluorinated gas is selected from the group consisting of perfluorocarbons and sulfur hexafluoride.
14. The method of claim 13 wherein the perfluorocarbon gas is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfluoromethane, perfluoroethane, perfluorohexane, and perfluoropentane.
15. The method of claim 1 wherein said gas is 170,
16. The method of claim 1 wherein said contrast medium further comprises a paramagnetic agent or a superparamagnetic agent.
17. The method of claim 16 wherein the contrast agent is a paramagnetic agent.
18. The method of claim 17 wherein the paramagnetic agent comprises a paramagnetic ion selected from the group consisting of transition, lanthanide and actinide elements.
19. The method of claim 18 wherein the paramagnetic ion is selected from the group consisting of Gd(III), Mn(II), Cu(II), Cr(III), Fe(II), Fe(III), Co(II), Er(II), Ni(II), Eu(III) and Dy(III).
20. The method of claim 19 wherein the paramagnetic ion is Mn(II).
21. The method of claim 17 wherein the paramagnetic agent comprises a nitroxide.
22. The method of claim 16 wherein the contrast agent is a superparamagnetic agent.
23. The method of claim 22 wherein the superparamagnetic agent comprises a metal oxide or metal sulfide.
24. The method of claim 23 wherein the superparamagnetic agent comprises a metal oxide wherein the metal is iron.
25. The method of claim 22 wherein the superparamagnetic agent is ferritin, iron, magnetic iron oxide, .gamma.-Fe2O3, manganese ferrite, cobalt ferrite and nickel ferrite.
26. A method of magnetic resonance focused surgical ultrasound comprising:
administering a contrast medium for magnetic resonance imaging comprising gaseous precursor filled vesicles to a patient requiring surgery, allowing the gaseous precursor to undergo a phase transition from a liquid to a gas, scanning said patient with magnetic resonance imaging using said contrast medium to identify the region of the patient requiring surgery, and applying ultrasound to said region to carry out said surgery.
27. The method of claims 26 wherein said gaseous precursor phase transition from a liquid to a gas and said imaging with magnetic resonance take place simultaneously.
28. A method for the controlled delivery of therapeutic compounds to a region of a patient using magnetic resonance focused therapeutic ultrasound comprising:
administering a contrast medium for magnetic resonance imaging comprising gas filled vesicles and a therapeutic compound to a patient;
scanning said patient with magnetic resonance imaging using said contrast medium to determine the presence of the vesicles in the region; and applying ultrasound to said region to rupture said vesicles to release the therapeutic compound in the region.
29. The method of claim 1 wherein said gas filled vesicle is filled with 19F and said magnetic resonance imaging is nuclear magnetic resonance.
30. The method of claim 1 wherein said gas filled vesicles are administered interstitially.
31. The method of claim 1 wherein said gas is selected from the group consisting of rubidium enhanced xenon, rubidium enhanced argon, rubidium enhanced helium, and rubidium enhanced neon.
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US08/401,974 US5922304A (en) 1989-12-22 1995-03-09 Gaseous precursor filled microspheres as magnetic resonance imaging contrast agents
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US08/476,317 US6088613A (en) 1989-12-22 1995-06-07 Method of magnetic resonance focused surgical and therapeutic ultrasound
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EP0852477A4 (en) 1998-09-23
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AU5184496A (en) 1996-10-02
US6088613A (en) 2000-07-11
US6461586B1 (en) 2002-10-08
US20030088176A1 (en) 2003-05-08
EP0852477A1 (en) 1998-07-15
JPH11501839A (en) 1999-02-16

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