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EP1001816A1 - Perfluorinated-ether compositions as diagnostic contrast agents - Google Patents

Perfluorinated-ether compositions as diagnostic contrast agents

Info

Publication number
EP1001816A1
EP1001816A1 EP97946447A EP97946447A EP1001816A1 EP 1001816 A1 EP1001816 A1 EP 1001816A1 EP 97946447 A EP97946447 A EP 97946447A EP 97946447 A EP97946447 A EP 97946447A EP 1001816 A1 EP1001816 A1 EP 1001816A1
Authority
EP
European Patent Office
Prior art keywords
contrast agent
agent according
perfluoroheterocyclyl
vesicles
perfluoroheteroaryl
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97946447A
Other languages
German (de)
French (fr)
Other versions
EP1001816A4 (en
Inventor
Evan C. Unger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ImaRx Pharmaceutical Corp
Original Assignee
ImaRx Pharmaceutical Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ImaRx Pharmaceutical Corp filed Critical ImaRx Pharmaceutical Corp
Publication of EP1001816A1 publication Critical patent/EP1001816A1/en
Publication of EP1001816A4 publication Critical patent/EP1001816A4/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/223—Microbubbles, hollow microspheres, free gas bubbles, gas microspheres

Definitions

  • the present invention relates to novel compositions of perfluorinated ethers. More particularly, the present invention relates to novel compositions of perfluorinated ethers and their use as contrast media for diagnostic imaging and in various other applications.
  • Ultrasound is a valuable diagnostic imaging technique for studying various areas of the body, for example, the vasculature, including tissue micro vasculature.
  • Ultrasound provides certain advantages over other diagnostic techniques. For example, diagnostic techniques involving nuclear medicine and X-rays generally results in exposure of the patient to ionizing electron radiation. Such radiation can cause damage to subcellular material, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Ultrasound does not involve such potentially damaging radiation. In addition, ultrasound is relatively inexpensive relative to other diagnostic techniques, including computed tomography (CT) and magnetic resonance imaging (MRI), which require elaborate and expensive equipment.
  • CT computed tomography
  • MRI magnetic resonance imaging
  • Ultrasound involves the exposure of a patient to sound waves. Generally, the sound waves dissipate due to abso ⁇ tion by body tissue, penetrate through the tissue or reflect off of the tissue. The reflection of sound waves off of tissue, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues. This differential reflection is due to various factors, including the constituents and the density of the particular tissue being observed. Ultrasound involves the detection of the differentially reflected waves, generally with a transducer that can detect sound waves having a frequency of 1 megahertz (MHZ) to 10 MHZ. The detected waves can be integrated into an image which is quantitated and the quantitated waves converted into an image of the tissue being studied.
  • MHZ megahertz
  • Contrast agents are used to improve the quality and usefulness of images which are obtained via ultrasound.
  • Exemplary contrast agents include, for example, suspensions of solid particles, emulsified liquid droplets, and gas-filled bubbles. See, e.g., Hilmann et al., U.S. Patent No. 4,466,442, and published International Patent Applications WO 92/17212 and WO 92/21382.
  • bubbles refers to vesicles which are generally characterized by the presence of one or more membranes or walls surrounding an internal void that is filled with a gas or precursor thereto.
  • Exemplary bubbles include, for example, vesicles which are surrounded by monolayers and/or bilayers to form, for example, unilamellar, oligolamellar and/or multilamellar vesicles, such as liposomes, micelles and the like.
  • the effectiveness of bubbles as contrast agents depends upon various factors, including, for example, the size and/or elasticity of the bubble.
  • the signal which is reflected off of a bubble is a function of the radius (r 6 ) of the bubble (Rayleigh Scatterer).
  • r 6 the radius of the bubble
  • ⁇ m the diameter of 4 micrometer ( ⁇ m)
  • ⁇ m the diameter of 4 micrometer
  • bubble size is limited by the diameter of capillaries through which the bubbles must pass.
  • contrast agents which comprise bubbles having a diameter of greater than 10 ⁇ m can be dangerous since microvessels may be occluded. Accordingly, it is preferred that greater than about 90% of the bubbles in a contrast agent have a diameter of less than about 10 ⁇ m, with greater than about 95% being more preferred, and greater than about 98% being even more preferred.
  • Mean bubble diameter is important also, and should be greater than 1 ⁇ m, with greater than 2 ⁇ m being preferred.
  • the volume weighted mean diameter of the bubbles should be about 7 to 10 micrometer.
  • the elasticity of bubbles is also important. This is because highly elastic bubbles can deform, as necessary, to "squeeze" through capillaries. This decreases the likelihood of occlusion.
  • the effectiveness of a contrast agent which comprises bubbles is also dependent on the bubble concentration. Generally, the higher the bubble concentration, the greater the reflectivity of the contrast agent.
  • bubble stability refers to the ability of bubbles to retain gas entrapped therein after exposure to a pressure greater than atmospheric pressure.
  • bubbles generally need to retain greater than 50% of entrapped gas after exposure to pressure of 300 millimeters (mm) of mercury (Hg) for about one minute.
  • Particularly effective bubbles retain 75% of the entrapped gas after being exposed for one minute to a pressure of 300 mm Hg, with an entrapped gas content of 90%) providing especially effective contrast agents.
  • bubble resilience is also highly desirable that, after release of the pressure, the bubbles return to their original size. This is referred to generally as "bubble resilience.”
  • Bubbles which lack desirable stability provide poor contrast agents. If, for example, bubbles release the gas entrapped therein in vivo, reflectivity is diminished. Similarly, the size of bubbles which possess poor resilience will be decreased in vivo, also resulting in diminished reflectivity.
  • bubbles including gas-filled liposomes, which comprise lipoidal walls or membranes. See, e.g., Ryan et al., U.S. Patent Nos. 4,900,540 and 4,544,545; Tickner et al., U.S. Patent No. 4,276,885; Klaveness et al., WO 93/13809 and Schneider et al., EPO 0 554 213 and WO 91/15244.
  • bubble stability has included, for example, the preparation of bubbles in which the membranes or walls thereof comprise materials that are apparently strengthened via crosslinking. See, e.g., Klaveness et al., WO 92/17212, in which there are disclosed bubbles which comprise proteins crosslinked with biodegradable crosslinking agents.
  • bubble membranes can comprise compounds which are not proteins but which are crosslinked also with biocompatible compounds. See, e.g., Klaveness et al., WO 92/17436, WO 93/17718 and WO 92/21382.
  • Such crosslinking may, in certain circumstances, impart rigidity to the membranes or walls of the bubbles. This results in bubbles having reduced elasticity and, therefore, a decreased ability to deform and pass through capillaries. Thus, there may be a greater likelihood of occlusion of vessels with prior art contrast agents that are stabilized via crosslinking.
  • liposomes may have certain advantageous properties, such as non-toxicity, biodegradability and non-immunogenicity. Nevertheless, prior art liposomes generally suffer from drawbacks in that they may be relatively unstable in vivo and/or may be cleared quickly from the body by the reticuloendothelial system. Accordingly, little success has been achieved to date in connection with the use of liposomes as delivery agents of materials, such as active agents.
  • CT computed tomography
  • HU Hounsefield Units
  • CT contrast agents have been developed in an attempt to change the relative densities of different tissues, and thereby improve the diagnostic efficacy of CT.
  • CT contrast agents are used in a majority of the CT imaging scans. For example, of the 7.8 million body CT scans performed in 1978, 6.6 million involved the use of intravenous contrast agents for the enhancement of vascular and/or visceral images.
  • CT contrast agents for imaging the gastrointestinal (GI) tract are generally based on radiodense, non-absorbable, heavy metal materials. Such materials assist in imaging the bowel by absorbing X-ray transmissions which increases the radiodensity of the bowel lumen.
  • Common among such contrast agents are barium and iodinated compounds, including, for example, barium sulfate. Barium sulfate and iodinated compounds have been used for imaging the GI tract for the past 60 years and are widely used today for enhancing CT images of the upper and lower GI tract. They generally increase electron density in certain regions of the body, and are therefore classified as a "positive contrast agents.” Despite their widespread use, barium and iodinated compounds suffer from various drawbacks.
  • CTA CT angiography
  • iodinated contrast agents are injected intravenously and images are obtained during the bolus phase of contrast.
  • Highly detailed images of the vasculature are generally obtained using CTA by reformatting the axial images to yield a composite picture of the vessels. During this reformatting, the picture of the vasculature is optimized based on the measured density in the vessels being visualized.
  • various baseline image subtractions are performed.
  • the vascular image optimization becomes distorted and obscured in the presence in the bowel of radiodense contrast agents, for example, iodinated compounds.
  • any positive contrast agents such as barium and iodinated compounds
  • the viability of currently available CT contrast agents is generally extremely sensitive to concentration. If the concentration is too low, little contrast is observed. If the concentration is too high, beam hardening artifacts result and are observed as streaks in the resulting CT images.
  • difficulty is typically encountered in visualizing the bowel mucosa with the currently available contrast agents. Accordingly, new and/or better stabilized contrast agents, as well as stabilized delivery means for active agents and methods for providing same, are needed. The present invention is directed to these, as well as other important ends.
  • a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
  • X is O or S;
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl;
  • R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • the contrast agents preferably also comprise a lipid, protein or polymer.
  • Another embodiment of the invention also relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
  • X is O or S
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • the contrast agents preferably also comprise a stabilizing compound.
  • a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
  • X is O or S;
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl;
  • R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • the contrast agents preferably also comprise a stabilizing compound which may be substantially non- crosslinked or substantially crosslinked, with substantially non-crosslinked stabilizing compounds being preferred.
  • Still another embodiment of the invention relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula R,-X-R 2
  • X is O or S
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
  • the contrast agents preferably also comprise a lipid which may be substantially non-crosslinked or substantially crosslinked, with substantially non-crosslinked lipids being preferred.
  • a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a perfluoroether compound, preferably a gaseous perfluoroether compound, selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers contain no more than one oxygen atom in the heterocyclyl ring system.
  • the contrast agents preferably also comprise a stabilizing compound selected from the group consisting of lipids, proteins and polymers.
  • a composition comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
  • X is O or S
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • the compositions preferably also comprise a lipid, protein or polymer.
  • Still another embodiment of the invention relates to a composition
  • a composition comprising, in an aqueous carrier, a perfluoroether compound, preferably a gaseous perfluoroether compound, selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers comprise no more than one oxygen atom in the heterocyclic ring system.
  • the compositions preferably also comprise a stabilizing compound selected from the group consisting of lipids, proteins and polymers.
  • Another embodiment of the invention relates to a method for preparing a composition which comprises a perfluoroether compound, preferably a gaseous perfluoroether compound, wherein the method comprises:
  • compositions prepared according to this method are preferably stabilized compositions, preferably by further including in the vessel in (a) a stabilizing compound.
  • a method for providing an image of a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a compound of the formula
  • X is O or S
  • Ri is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
  • R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of the region.
  • the contrast agents employed in the methods preferably also comprise a lipid, protein or polymer.
  • a contrast agent comprising, in an aqueous carrier, a compound of the formula
  • X is O or S;
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl;
  • R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of any diseased tissue in the patient.
  • the contrast agents employed in the methods preferably also comprise a lipid, protein or polymer.
  • Another embodiment of the invention relates to a method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a formulation which comprises, in combination with a bioactive agent, a composition of a compound of the formula
  • X is O or S
  • R ⁇ is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl;
  • R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • the compositions employed in the methods preferably also comprise a lipid, protein or polymer.
  • Figure 1 is a graphical representation of the acoustic activity, viewed at one minute intervals, of vesicle compositions containing (i) perfluoromethyl butyl ether and (ii) perfluoropropane.
  • Figure 2 is a graphical representation of the acoustic activity, upon exposure to increasing and decreasing pressure, of vesicle compositions containing (i) perfluoromethyl butyl ether and (ii) perfluoropropane.
  • Figure 3 is a graphical representation of the acoustic activity, viewed at one minute intervals, of a vesicle composition containing perfluoromethyl butyl ether formed and shaken at an initial temperature of -20°C.
  • Figure 4 is a graphical representation of the acoustic activity, upon exposure to increasing pressure, of a vesicle composition containing perfluoromethyl butyl ether formed at an initial temperature of 4°C and shaken at room temperature.
  • Figure 5 is a graphical representation of the acoustic activity, upon exposure to increasing and decreasing pressure, of a vesicle composition containing perfluoromethyl butyl ether shaken at room temperature.
  • Figure 6 is a graphical representation of the acoustic activity, upon exposure to increasing pressure, of a vesicle composition containing perfluoromethyl butyl ether shaken at -20°C.
  • Lipid refers to a naturally-occurring, synthetic or semisynthetic (also referred to as "modified natural") compound which is generally amphipathic.
  • the lipids typically contain a hydrophilic component and a hydrophobic component.
  • Exemplary lipids include, for example, fatty acids, neutral fats, phosphatides, oils, glycolipids, surface-active agents (surfactants), aliphatic alcohols, waxes, te ⁇ enes and steroids.
  • Protein refers to molecules comprising, and preferably consisting essentially of, ⁇ -amino acids in peptide linkages.
  • protein globular proteins, such as albumins, globulins and histones, and fibrous proteins such as collagens, elastins and keratins.
  • compound proteins wherein a protein molecule is united with a nonprotein molecule, such as nucleproteins, mucoproteins, lipoproteins, and metalloproteins.
  • the protein may be naturally-occurring, synthetic or semisynthetic.
  • Polymer or “polymeric” refers to molecules formed from the chemical union of two or more repeating units. Accordingly, included within the term “polymer” may be, for example, dimers, trimers and oligomers. The polymer may be synthetic, naturally-occurring or semisynthetic. In preferred form, the term “polymer” refers to molecules which comprise 10 or more repeating units.
  • Stabilizing compound refers to any material which is capable of improving the stability of compositions containing perfluoroether compounds as described herein including, for example, emulsions, suspensions, dipersions and vesicle compositions.
  • the improved stability involves, for example, the maintenance of a relatively balanced condition, and may be exemplified, for example, by increased resistance against destruction, decomposition, degradation and the like.
  • the stabilizing compounds may serve to improve the stability of the vesicles, for example, by minimizing or substantially (including completely) preventing the escape of gas entrapped within vesicles which may occur, for example, from rupture and/or coalescence of vesicles.
  • substantially as used in reference to the prevention of the escape of entrapped gas, means that greater than about 50%> of the gas is maintained entrapped.
  • greater than about 60% of the gas is maintained entrapped, with greater than about 70% being more preferred. Even more preferably, greater than about 80% of the gas is maintained entrapped, with greater than about 90%) being still more preferred.
  • the gas may be completely maintained entrapped (i.e., about 100%) of the gas is maintained entrapped).
  • the stabilizing compounds may comprise discrete, individual compounds (monomers), or may comprise polymers. Broadly speaking, the stabilizing compounds may comprise, for example, surfactants, film-forming materials, membranes and/or membrane forming materials. Exemplary stabilizing compounds which may be employed in the methods and compositions of the present invention include lipids, proteins and polymers. In certain embodiments, the stabilizing compounds may be substantially (including completely) crosslinked.
  • crosslink generally refers to the linking of two or more stabilizing compounds, including lipid, protein and polymer stabilizing compounds, by one or more bridges.
  • the bridges which may be composed of one or more elements, groups or compounds, generally serve to join an atom from a first stabilizing compound molecule to an atom of a second stabilizing molecule.
  • the crosslink bridges may involve covalent and/or non-covalent associations. Any of a variety of elements, groups and/or compounds may form the bridges in the crosslinks, and the stabilizing compounds may be crosslinked naturally or through synthetic means.
  • crosslinking may occur in nature in materials formulated from peptide chains which are joined by disulfide bonds of cystine residues, as in keratins, insulin, and other proteins.
  • crosslinking may be effected by suitable chemical modification, such as, for example, by combining a compound, such as a stabilizing material, and a chemical substance that may serve as a crosslinking agent, which are caused to react, for example, by exposure to heat, high- energy radiation, ultrasonic radiation, and the like.
  • crosslinked stabilizing compounds examples include, for example, crosslinking with sulfur which may be present, for example, as sulfhydryl groups in cysteine residues, to provide disulfide linkages, crosslinking with organic peroxides, crosslinking of unsaturated materials by means of high-energy radiation, crosslinking with dimethylol carbamate, and the like.
  • substantially as used in reference to crosslinked stabilizing compounds, means that greater than about 50%> of the stabilizing compounds contain crosslinking bridges. In certain embodiments, preferably greater than about 60%) of the crosslinked stabilizing compounds contain crosslinking bridges, with greater than about 70%) being more preferred.
  • the crosslinked stabilizing compounds may contain crosslinking bridges, with greater than about 90%> being still more preferred. In certain particularly preferred embodiments, greater than about 95%> of the crosslinked stabilizing compounds contain crosslinking bridges.
  • the substantially crosslinked stabilizing compounds may be completely crosslinked (i.e., about 100% of the crosslinked stabilizing compounds contain crosslinking bridges). In the most preferred embodiments, the stabilizing compounds may be substantially (including completely) non-crosslinked. The term "substantially”, as used in reference to non-crosslinked stabilizing compounds, means that greater than about 50%> of the stabilizing compounds are devoid of crosslinking bridges.
  • the stabilizing compounds are devoid of crosslinking bridges, with greater than about 70%> being more preferred. Even more preferably, greater than about 80%) of the stabilizing compounds are devoid of crosslinking bridges, with greater than about 90% > being still more preferred. In particularly preferred embodiments, greater than about 95% > of the stabilizing compounds are devoid of crosslinking bridges. If desired, the substantially non-crosslinked stabilizing compounds may be completely non-crosslinked (i.e., about 100% of the stabilizing compounds are devoid of crosslinking bridges).
  • Covalent association refers to intermolecular interaction among two or more separate molecules, which interaction generally involves the sharing of electrons by two atomic nuclei.
  • Non-covalent association refers to intermolecular interaction among two or more separate molecules, which interaction does not involve a covalent bond. Intermolecular interaction is dependent upon a variety of factors, including, for example, the polarity of the involved molecules, the charge (positive or negative), if any, of the involved molecules, and the like. Non-covalent associations are preferably selected from the group consisting of ionic interaction, dipole-dipole interaction and van der Waal's forces and combinations thereof.
  • Ionic interaction refers to intermolecular interaction among two or more molecules, each of which is positively or negatively charged.
  • ionic interaction refers to the attraction between a first, positively charged molecule and a second, negatively charged molecule.
  • exemplary ionic interactions include, for example, the attraction between a metal cation, for example, a calcium ion (Ca 2+ ), and a stabilizing compound that contains negatively charged groups and/or groups or atoms that contain unshaired pairs of electrons.
  • Dipole-dipole interaction refers generally to the attraction which can occur among two or more polar molecules.
  • dipole-dipole interaction refers to the attraction of the positive end of a first polar molecule to the negative end of a second polar molecule.
  • Dipole-dipole interactions are exemplified, for example, by the attraction between the electropositive head group, for example, the choline head group, of phosphatidylcholine, and an electronegative atom, for example, a heteroatom, such as oxygen, nitrogen or sulphur atoms, which may be present in another stabilizing compound.
  • Double-dipole interaction refers also to intermolecular hydrogen bonding in which a hydrogen atom serves as a bridge between electronegative atoms on separate molecules and in which a hydrogen atom is held to a first molecule by a covalent bond and to a second molecule by electrostatic forces.
  • “Van der Waal's forces” refers to the attractive forces between non-polar molecules that are accounted for by quantum mechanics. Van der Waal's forces are generally associated with momentary dipole moments which are induced by neighboring molecules and which involve changes in electron distribution.
  • “Stabilized composition” refers to a composition which contains one or more stabilizing compounds and preferably one or more perfluoroether compounds, typically in an aqueous medium. In preferred stabilized compositions, the stabilizing compounds are selected from lipids, proteins and polymers, with lipids being particularly preferred. Exemplary stabilized compositions include suspensions, emulsions and vesicle compositions, with vesicle compositions being preferred.
  • Lipid composition refers to compositions which comprise lipids, polymers and proteins, typically in an aqueous medium.
  • Lipid formulation refers to lipid, polymer and protein compositions which also comprise one or more bioactive agents.
  • Vesicle refers to an entity which is generally characterized by the presence of one or more walls or membranes which form one or more internal voids. Vesicles may be formulated, for example, from stabilizing compounds, such as a lipid, including the various lipids described herein, a polymer, including the various polymers described herein, or a protein, including the various proteins described herein.
  • the lipids, polymers, and or proteins may be natural, synthetic or semi-synthetic.
  • Preferred vesicles are those which comprise walls or membranes formulated from lipids. The walls or membranes may be concentric or otherwise.
  • the stabilizing compounds may be in the form of a monolayer or bilayer, and the mono- or bilayer stabilizing compounds 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 may be concentric, if desired. Stabilizing compounds may be used to form unilamellar vesicles (comprised of one monolayer or bilayer), oligolamellar vesicles (comprised of about two or about three monolayers or bilayers) or multilamellar vesicles (comprised of more than about three monolayers or bilayers).
  • the walls or membranes of vesicles prepared from lipids, polymers or proteins may be substantially solid (uniform), or they may be porous or semi- porous.
  • the vesicles described herein include such entities commonly referred to as, for example, liposomes, micelles, bubbles, microbubbles, microspheres, lipid-, protein-and/or polymer-coated bubbles, microbubbles and/or microspheres, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal H II phase structures, and the like.
  • the internal void of the vesicles may be filled with a liquid (including, for example, an aqueous liquid), a gas, a gaseous precursor, and/or a solid or solute material, including, for example, a vasodilator and/or bioactive agent, as desired.
  • a liquid including, for example, an aqueous liquid
  • gas including, for example, a gas, a gaseous precursor, and/or a solid or solute material, including, for example, a vasodilator and/or bioactive agent, as desired.
  • the internal void of the vesicle may also contain, alone or in combination with other materials or agents, a perfluorinated ether compound.
  • the vesicles may also comprise, if desired, a targeting ligand.
  • Vehicle composition refers to a composition, typically in an aqueous medium, which comprises vesicles.
  • Vehicle formulation refers to a vesicle composition which also comprises one or more bioactive agents. Suitable vesicles or vesicle species for use in vesicle formulations include, for example, gas and/or gaseous precursor filled vesicles.
  • Lipid vesicle refers respectively to vesicles formulated from one or more lipids, polymers and proteins.
  • Lipome refers to a generally spherical or spheroidal cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as lipid vesicles.
  • the liposomes may be formulated, for example, from ionic lipids and/or non-ionic lipids. Liposomes which are formulated from non-ionic lipids may also be referred to as "niosomes.”
  • “Micelle” refers to colloidal entities formulated from lipids.
  • the micelles comprise a monolayer or hexagonal H2 phase configuration.
  • the micelles may comprise a bilayer configuration.
  • “Aerogel” refers to generally spherical or spheroidal entities which are characterized by a plurality of small internal voids.
  • the aerogels may be formulated from synthetic materials (for example, a foam prepared from baking resorcinol and formaldehyde), as well as natural materials, such as polysaccharides or proteins.
  • “Clathrate” refers to a solid, semi-porous or porous particle which may be associated with vesicles.
  • the clathrates may form a cage-like structure containing cavities which comprise the vesicles.
  • One or more vesicles may be bound to the clathrate.
  • a stabilizing material may, if desired, be associated with the clathrate to promote the association of the vesicle with the clathrate.
  • Suitable materials from which clathrates may be formulated include, for example, porous apatites, such as calcium hydroxyapatite, and precipitates of polymers and metal ions, such as alginic acid precipitated with calcium salts.
  • Emmulsion refers to a mixture of two or more generally immiscible liquids and is generally in the form of a colloid.
  • the mixture may be of lipids, for example, which may be homogeneously or heterogeneously dispersed throughout the emulsion.
  • the lipids may be aggregated in the form of, for example, clusters or layers, including mono- or bilayers.
  • “Suspension” or “dispersion” refers to a mixture, preferably finely divided, of two or more phases (solid, liquid or gas), such as, for example, liquid in liquid, solid in liquid, gas in liquid, and the like, which can preferably remain stable for extended periods of time.
  • “Hexagonal H II phase structure” refers to a generally tubular aggregation of lipids in liquid media, for example, aqueous media, in which the hydrophilic portion(s) of the lipids generally face inwardly in association with an aqueous liquid environment inside the tube.
  • the hydrophobic portion(s) of the lipids generally radiate outwardly and the complex assumes the shape of a hexagonal tube.
  • a plurality of tubes is generally packed together in the hexagonal phase structure.
  • the vesicles of the present invention preferably contain a perfluorinated ether compound, typically in the form of a gas or gaseous precursor, and preferably in the form of a gas.
  • a perfluorinated ether compound typically in the form of a gas or gaseous precursor, and preferably in the form of a gas.
  • Gas filled vesicle refers to vesicles in which there is encapsulated a gas.
  • Gaseous precursor filled vesicle refers to vesicles in which there is encapsulated a gaseous precursor.
  • the vesicles may be substantially (including completely) filled with the gas and/or gaseous precursor.
  • substantially means that greater than about 50%) of the internal void volume of the vesicle consists of a gas and/or gaseous precursor.
  • greater than about 60% of the internal void of the substantially filled vesicles consists of a gas and/or gaseous precursor, with greater than about 70%) being more preferred.
  • greater than about 80% > of the internal void of the substantially filled vesicles consists of a gas and/or gaseous precursor, with greater than about 90%> being still more preferred.
  • the internal void of the vesicles consists of a gas and/or gaseous precursor.
  • the substantially filled vesicle may be completely filled (i.e., filled with about 100%) gas and/or gaseous precursor).
  • the vesicles may also contain, if desired, no or substantially no gas and or gaseous precursor.
  • compositions of the present invention may be administered to a patient.
  • patient refers to animals, including mammals, preferably humans.
  • internal region of a patient and “region of interest” refer to the entire patient or to a particular area or portion of the patient.
  • Internal regions of a patient and regions of interest may include, for example, areas being imaged with diagnostic imaging and/or areas being treated with a bioactive agent. Exemplary of such areas include, for example, the gastrointestinal region and, the cardiovascular region, including myocardial tissue, the renal region, as well as other bodily tissues and regions, including the vasculature and circulatory system, and diseased tissue, such as cancerous tissue.
  • cardiac region of a patient refers to the region of the patient defined by the heart (myocardium) and the vasculature leading directly to and from the heart.
  • vascular endothelial denotes the blood vessels (including arteries, veins and the like) in the body or in an organ or part of the body.
  • gastrointestinal region or “gastrointestinal tract” includes the region of a patient defined by the esophagus, stomach, small and large intestines and rectum.
  • renal region of a patient refers to the region of the patient defined by the kidney and the vaculature that leads directly to and from the kidney, and includes the abdominal aorta.
  • Bioactive agent refers to a substance which may be used in connection with an application that is therapeutic or diagnostic in nature, such as in methods for diagnosing the presence or absence of a disease in a patient and/or in methods for the treatment of disease in a patient.
  • bioactive agent refers also to substances which are capable of exerting a biological effect in vitro and/or in vivo.
  • the bioactive agents may be neutral or positively or negatively charged.
  • suitable bioactive agents include diagnostic and pharmaceutical agents, synthetic or natural organic or inorganic molecules, including, for example, proteins, peptides, vitamins, steroids, steroid analogs, antitumor agents, hormones, antiinflammatory agents, chemotherapeutic agents and genetic material, including nucleosides, nucleotides and polynucleotides.
  • Targeting ligand refers to any material or substance which may promote targeting of tissues and/or receptors in vitro or in vivo with the compositions of the present invention.
  • the targeting ligand may be synthetic, semi-synthetic, or naturally-occurring.
  • Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, such as saccharides, including mono- and polysaccharides, and carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs, and polynucleotides.
  • tissue refers generally to specialized cells which may perform a particular function.
  • tissue may refer to an individual cell or a plurality or aggregate of cells, for example, membranes or organs.
  • tissue also includes reference to an abnormal cell or a plurality of abnormal cells.
  • exemplary tissues include, for example, myocardial tissue (also referred to as heart tissue or myocardium), including myocardial cells and cardiomyocites, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, and tumors.
  • Receptor refers to a molecular structure within a cell or on the surface of the cell which is generally characterized by the selective binding of a specific substance.
  • exemplary receptors include, for example, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and cytoplasmic receptors for steroid hormones.
  • An exemplary receptor within the context of the present invention is the glycoprotein GPIIbllla, which is a platelet integrin.
  • Diagnostic agent refers to any agent which may be used in connection with methods for imaging an internal region of a patient and/or diagnosing the presence or absence of a disease in a patient.
  • diagnostic agents include, for example, contrast agents for use in connection with ultrasound imaging, magnetic resonance imaging or computed tomography imaging of a patient including. Diagnostic agents include, for example, the lipid, protein or polymer and/or vesicle compositions described herein.
  • “Pharmaceutical agent” or “drug” refers to any therapeutic or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury in a patient.
  • Therapeutically useful peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug.
  • “Genetic material” refers generally to nucleosides, nucleotides and polynucleotides and the like, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • the genetic material may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant or other technology, or by a combination thereof.
  • the DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded.
  • Genetic material refers also to ribozymes and RNA having a hammerhead motif (i.e., catalytic RNA), as well as sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
  • a hammerhead motif i.e., catalytic RNA
  • sense and anti-sense DNA and RNA that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
  • administered and “administration” refer generally to the administration to a patient of a biocompatible material, including, for example, lipid, polymer or protein and/or vesicle compositions described herein.
  • Biocompatible refers to materials which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic responses and disease states.
  • the compositions and components thereof (such as lipids, proteins, polymers, perfluoroethers, bioactive agents, etc.) employed in the present invention are typically biocompatible.
  • “In combination with” generally refers to the co-administration of a bioactive agent with a composition of the present invention, including the present lipid, polymer or protein, and/or vesicle compositions.
  • co-administration means that the bioactive agent may be administered before, during, or after the administration of the composition.
  • the bioactive agent in any of a variety of different ways.
  • the bioactive agent may be entrapped within the internal void of the vesicle.
  • the bioactive agent may be integrated within the layer(s) or wall(s) of the vesicle, for example, by being interspersed among the lipids, proteins or polymers that comprise the vesicle layer(s) or wall(s).
  • the bioactive agent may be entrapped between or among the lipid, polymer and/or protein components.
  • the bioactive agent may be located on the surface of the vesicle or the lipid, protein or polymer.
  • the bioactive agent may interact chemically with the surface (inner or outer) of the vesicle or the non- vesicular lipid, protein or polymer, and remain substantially adhered thereto. Such interaction may take the form of, for example, electrostatic interactions, hydrogen bonding, van der Waal's forces, covalent bonding or other interaction.
  • the bioactive agent may interact with the surface (inner or outer) of the vesicle or the non- vesicular lipid, protein or polymer in a limited manner.
  • Such limited interaction would permit migration of the bioactive agent, for example, from the surface of a first vesicle to the surface of a second vesicle, or from the surface of a first non- vesicular lipid, protein or polymer to the surface of a second non- vesicular lipid, protein or polymer.
  • Thickening agent refers to any of a variety of generally hydrophilic materials which, when inco ⁇ orated in the various compositions and formulations described herein, may act as viscosity modifying agents, emulsifying and/or solubilizing agents, suspending agents, and tonicity raising agents. It is contemplated that the thickening agents may be capable of aiding in maintaining the stability of the compositions and/or formulations due to such properties.
  • Dispersing agent refers to a surface-active agent which, when added to a suspending medium of colloidal particles, including, for example, certain of the compositions and formulations described herein, may promote uniform separation of particles.
  • the dispersing agent may comprise a polymeric siloxane compound such as, for example, polydimethylsiloxane (also referred to as "simethicone").
  • Perfluorinated ether compound refers to any and all compounds having at least one ether group and at least one aliphatic, alicyclic or aromatic group in which all of the hydrogen atoms that would otherwise be bonded to carbon atoms are replaced by fluorine atoms. Thus, perfluorinated ether compounds contain no carbon-hydrogen bonds. Accordingly, in the case, for example, of an alkyl group having a formula C n H 2n+1 , "perfluorinated” and/or “perfluoro” means that all of the hydrogen atoms are replaced with fluorine atoms to provide a group having a formula C n F 2n+1 .
  • the perfluorinated group(s) may be substituted with one or more chemical moieties that include hydrogen atoms bonded to atoms other than carbon atoms.
  • a perfluoroalkyl group may be substituted, for example, with an hydroxy (-OH) group.
  • halogen atoms i.e., chlorine, bromine or iodine atoms
  • ether group refers to an -O- or -S- atom, with -O- atoms being preferred (compounds with -S- atoms being sometimes referred to herein as thioethers, and compounds with -O- atoms being sometimes referred to herein as pure ethers).
  • the perfluorinated ether compounds employed in the methods and compositions described herein are preferably biocompatible, and may be employed in the gaseous or liquid (including gaseous precursor) form, with perfluoroethers which are employed in the gaseous form being preferred. If employed as a gaseous precursor, the perfluoroethers may be employed, for example, as a temperature sensitive gaseous precursor and/or a pressure sensitive gaseous precursor, with temperature sensitive gaseous precursors being generally preferred.
  • Halogen refers to fluorine, chlorine, bromine and iodine. In certain preferred aspects of the invention, the halogen is chlorine or bromine.
  • Aliphatic refers to hydrocarbon compounds that are generally characterized by straight- or branched-chain arrangements of the constituent carbon atoms. Aliphatic hydrocarbons generally comprise three subgroups: (1) paraffins (e.g., alkanes) which are typically saturated; (2) olefins (e.g., alkenes) which contain one or more carbon- carbon double bonds; and (3) acetylenes (e.g., alkynes) which contain one or more carbon- carbon triple bonds.
  • paraffins e.g., alkanes
  • olefins e.g., alkenes
  • acetylenes e.g., alkynes
  • Alicyclic refers to hydrocarbon compounds that are characterized by the arrangement of the constituent carbon atoms in closed ring structures. Alicyclic compounds generally comprise three subgroups: (1) cycloparaffins (e.g., cycloalkanes) which are typically saturated; (2) cycloolefins (e.g., cycloalkenes) which contain one or more carbon-carbon double bonds; and (3) cycloacetylenes (e.g., cycloalkynes) which contain one or more carbon-carbon triple bonds.
  • cycloparaffins e.g., cycloalkanes
  • cycloolefins e.g., cycloalkenes
  • cycloacetylenes e.g., cycloalkynes
  • alkyl groups preferably contain from 1 to about 7 carbon atoms in the chain, and all combinations and subcombinations of ranges of carbon atoms therein. Preferred alkyl groups contain from 1 to 6 carbon atoms. "Lower alkyl”, which refers to a short chain alkyl group, preferably containing from 1 to about 4 carbon atoms is more preferred.
  • alkyl group may be optionally substituted with one or more alkyl group substituents which may be the same or different, where "alkyl group substituent" includes hydroxy, thio, oxo, carboxy, perfluorinated aryl, perfluorinated alkoxy, perfluorinated aryloxy, perfluorinated alkyloxy, perfluorinated alkylthio, perfluorinated arylthio, perfluorinated aralkyloxy, perfluorinated aralkylthio, perfluorinated alkoxycarbonyl and perfluorinated cycloalkyl.
  • alkyl group substituent includes hydroxy, thio, oxo, carboxy, perfluorinated aryl, perfluorinated alkoxy, perfluorinated aryloxy, perfluorinated alkyloxy, perfluorinated alkylthio, perfluorinated arylthio, perfluorinated a
  • Branched refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. As noted above, preferred alkyl groups are perfluorinated.
  • Exemplary alkyl groups from which the perfluoroalkyl groups may be derived include, for example, methyl, ethyl, propyl (including rc-propyl and /-propyl), butyl (including w-butyl, t-butyl, sec-butyl and /-butyl), pentyl (including w-pentyl, f ' -pentyl and neopentyl) and hexyl (including w-hexyl and isohexyl).
  • Preferred perfluoroalkyl groups include, for example, perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluoropentyl and perfluorohexyl.
  • Alkenyl refers to an alkyl group containing at least one carbon-carbon double bond and preferably from 2 to about 7 carbons, and all combinations and subcombinations of ranges of carbon atoms therein. In certain preferred embodiments, the alkenyl groups contain from 2 to about 6 carbons, with lower alkenyl groups being more preferred. "Lower alkenyl” refers to a short chain alkenyl group, preferably containing from 2 to about 4 carbon atoms. The alkenyl group may be optionally substituted with one or more "alkyl group substituents".
  • alkenyl group There may be optionally inserted along the alkenyl group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent may be lower alkyl.
  • preferred alkenyl groups are perfluorinated.
  • Exemplary alkenyl groups from which prefluoroalkenyl groups may be derived include, for example, vinyl, allyl, propenyl, butenyl (including 1- and 2-butene and isobutylene), pentenyl, and hexenyl.
  • Preferred perfluoroalkenyl groups include perfluorovinyl, perfluoropropenyl, perfluorobutenyl, perfluoropententyl and perfluorohexenyl.
  • Alkynyl refers to an alkyl group containing at least one carbon-carbon triple bond and preferably from 2 to about 7 carbons, and all combinations and subcombinations of ranges of carbon atoms therein. In certain preferred embodiments, the alkynyl groups contain from 2 to about 6 carbons, with lower alkynyl groups being more preferred. "Lower alkynyl” refers to a short chain alkynyl group, preferably containing from 2 to about 4 carbon atoms. The alkynyl group may be optionally substituted with one or more "alkyl group substituents".
  • alkynyl groups are perfluoroalkynyl groups.
  • Exemplary alkynyl groups from which perfluoroalkynyl groups may be derived include, for example, ethynyl, propargyl, butynyl, pentynyl, hexynyl and heptynyl.
  • Preferred perfluoroalkynyl groups include perfluoroethynyl, perfluoropropargyl, perfluorobutynyl, perfluoropentynyl, perfluorohexynyl and perfluoroheptynyl .
  • Cycloalkyl refers to a non-aromatic mono- or multicyclic ring system containing from about 3 to about 7 carbon atoms, and all combinatinons and subcombinations of ranges of carbon atoms therein.
  • the cycloalkyl group may be optionally partially unsaturated, and may also be optionally substituted with one or more alkyl group substituents.
  • the cycloalkyl groups contain from about 3 to about 6 carbons, with cycloalkyl groups containing from about 4 to about 6 carbons being more preferred.
  • Preferred monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.
  • Preferred multicyclic cycloalkyl rings include cw-5-norbornene, 5-norbornene, norbornane and «t -3-oxo- tricyclo-[2.2.1.0 26 ]heptane.
  • Heterocyclyl refers to a ring system containing from about 3 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein, wherein at least one or more of the ring members is a heteroatom (that is, an element other than carbon).
  • Preferred heteroatoms include, for example, oxygen, sulfur and nitrogen, the latter of which may be optionally substituted with a lower alkyl group.
  • the heterocyclyl groups contain about 5 or 6 ring atoms. Also in preferred embodiments, there is a maximum of one oxygen atom in the heterocyclyl rings.
  • the heterocyclyl groups may be optionally substituted with one or more alkyl group substituents.
  • Preferred heterocyclyl groups are also perfluorinated. Exemplary perfluorinated heterocyclyl groups include perfluorotetrahydrofuranyl and perfluorotetrahydropyranyl.
  • Aryl refers to an aromatic carbocyclic radical containing from about 6 to about 10 carbons and all combinations and subcombinations of ranges of carbon atoms therein, with aryl groups containing about 6 carbons being preferred.
  • the aryl group may be optionally substituted with one or more aryl group substituents which may be the same or different, where "aryl group substituent” includes nitro, cyano, alkyl, hydroxy, carboxy, carbamoyl, optionally substituted amino, perfluoroalkenyl, perfluoroalkynyl, perfluoroaryl, perfluoroaralkyl, perfluoroalkoxy, perfluoroaryloxy, perfluoroaralkoxy, perfluoroaroyl, perfluoroalkoxycarbonyl, perfluoroaryloxycarbonyl, perfluoroaralkoxy carbonyl, perfluoroacyloxy, perfluoroacylamino, perfluoroaroylamino, perfluor
  • Heteroaryl refers to a ring system of from about 5 to about 7 ring members, wherein one or more of the ring members is a heteroatom.
  • Preferred heteroatoms in the heteroaryl groups include, for example, oxygen, nitrogen and sulfur atoms.
  • the heteroaryl group may be optionally substituted with one or more aryl group substituents.
  • the heteroaryl groups are preferably perfluorinated. Exemplary perfluoroheteroaryl groups include perfluorofuranyl and perfluorothienyl.
  • the present invention is directed, in part, to improved methods and compositions which may be used in diagnostic imaging, including, for example, improved methods and compositions for use in providing an image of an internal region of a patient.
  • Embodiments of the present invention may involve stabilized compositions comprising, in an aqueous carrier, a stabilizing compound and a perfluorinated ether compound.
  • Preferred embodiments of the present invention involve vesicle compositions comprising, in an aqueous carrier, vesicles and a perfluorinateed ether compound.
  • the aforementioned compositions may be administered to a patient who is scanned using diagnostic imaging, preferably ultrasound imaging, to obtain a visible image of the region.
  • diagnostic imaging preferably ultrasound imaging
  • compositions of the perfluoroether compounds of the present invention optionally (and preferably) comprise one or more stabilizing compounds.
  • stabilizing compounds may be employed in the compositions and methods described herein.
  • Preferred stabilizing compounds are compounds which are capable of enhancing the stability of the compositions of the present invention, including emulsions, suspensions and vesicle compositions.
  • the stabilizing compounds are preferably capable of minimizing aggregation of the bubbles.
  • the stabilizing compounds are preferably capable of promoting the formation of the vesicles, as well as enhancing the resistance of the vesicles, once formed, to degradation caused, for example, by the loss of structural or compositional integrity in the walls of the vesicles and/or by the loss of any significant portion of a gas encapsulated therein.
  • the stabilizing compounds impart the aforesaid properties to the present compositions for a minimum period of time.
  • the stabilizing compounds are capable of stabilizing the present compositions for a period of time which permits use of the composition, such as, for example, in the generation of visible images of a region of a patient by diagnostic imaging.
  • the stabilizing compounds are preferably capable of stabilizing the compositions of the present invention for a period of time to at least permit their administration to a patient and- subsequent scanning of the patient with diagnostic imaging including, for example, ultrasound imaging.
  • Stabilizing compounds which may be employed in the methods and compositions of the present invention include, for example, lipids, proteins or polymers, and such stabilizing compounds are preferred.
  • other stabilizing compounds in addition to the aforementioned classes of compounds, can of course also be utilized.
  • any of a wide variety of organic and inorganic materials may be employed as stabilizing materials in the methods and compositions of the present invention including, for example, solid materials, such as particulate materials including, for example, apatites, or liquid materials, such as oils, surfactants, and the like.
  • the stabilizing compounds employed preferably include surfactants, tensides, suspendng agents, emulsifying agents, tonicity modifying agents, viscosity modifying agents, anti- foaming agents, thickeners, dispersing agents, solvents, diluents, and the like.
  • the stabilizing compounds may comprise one or more lipids, proteins or polymers.
  • compositions which comprise one or more lipids are preferred.
  • This phase transition temperature is 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. Biol. Chem. 1974 249, 2512- 2521. It is generally believed that vesicles which are prepared from lipids that possess higher gel state to liquid crystalline state phase transition temperatures tend to have enhanced impermeability at any given temperature. See Arthur 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.
  • lipids may be inco ⁇ orating in the lipid compositions at least a minor amount, for example, about 1 to about 10 mole percent, based on the total amount of lipid employed, of a negatively charged lipid.
  • Suitable negatively charged lipids include, for example, phosphatidylserine, phosphatidic acid, and fatty acids. Without intending to be bound by any theory or theories of operation, it is contemplated that such negatively charged lipids may provide added stability by counteracting the tendency of vesicles to rupture by fusing together.
  • the negatively charged lipids may act 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 other vesicles which may be proximate thereto.
  • the vesicles may be less prone to come into touching proximity with each other, which may lead to a rupture of the membrane or skin of the respective vesicles and consolidation of 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.
  • the lipid materials used in certain of the compositions described herein, especially in connection with vesicle compositions based on lipids are also preferably flexible.
  • the vesicles can alter their shape, for example, to pass through an opening having a diameter that is smaller than the diameter of the vesicle.
  • a wide variety of lipids are believed to be suitable for inco ⁇ oration in the lipid and/or vesicle compositions.
  • vesicle compositions for example, micelles and/or liposomes, any of the materials or combinations thereof which are known to those skilled in the art as suitable for their preparation may be used.
  • the lipids used may be of natural, synthetic or semi-synthetic origin.
  • suitable lipids generally include, for example, fatty acids, neutral fats, phosphatides, glycolipids, aliphatic alcohols and waxes, oils, te ⁇ enes and steroids.
  • lipids which may be used to prepare lipid compositions include, for example, lysolipids; phosphocholines; phosphatidylcholine with both saturated and unsaturated lipids, including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine (
  • polymerizable lipids included among the lipids which may be suitable for use in the compositions of the present invention are polymerizable lipids.
  • examples of such polymerizable lipids include, for example, lipids which contain functionalities that may undergo polymerization reactions, including, for example, alkenyl moieties, such as oleyl, linoleyl, acryloyl and methacryloyl groups, with or without polar groups to enhance water solubility, and cyanoacrylate esters optionally carrying lipophilic esterifying groups.
  • alkenyl moieties such as oleyl, linoleyl, acryloyl and methacryloyl groups
  • cyanoacrylate esters optionally carrying lipophilic esterifying groups.
  • a cationic lipid may be used, such as, for example, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP); and l,2-dioleoyl-3-(4'-trimethylammonio)- butanoyl-sn-glycerol (DOTB).
  • DOTMA N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOTAP l,2-dioleoyloxy-3- (trimethylammonio)propane
  • DOTB l,2-dioleoyl-3-(4'-trimethylammonio)- butanoyl-sn-glycerol
  • the molar ratio of cationic lipid to non-cationic lipid may be from about 1 :2 to about 1 :10, with a ratio of from about 1 : 1 to about 1 :2.5 being preferred. Even more preferably, the molar ratio of cationic lipid to non-cationic lipid may be about 1 :1.
  • this non-cationic lipid comprises one or more of DPPC, DPPE and dioleoylphosphatidylethanolamine.
  • lipids bearing cationic polymers such as polylysine or polyarginine, as well as alkyl phosphonates, alkyl phosphinates and alkyl phosphites, may also be used in the lipid compositions.
  • the lipid compositions comprise phospholipids, particularly one or more of DPPC, DPPE, DPPA, DSPC, DSPE, and DAPC (20 carbons), with DPPC and DSPC being preferred, and DPPC being even more preferred.
  • Saturated and unsaturated fatty acids may also be employed in the lipid compositions described herein and may include molecules that preferably contain from about 12 carbons to about 22 carbons, in linear or branched form. Hydrocarbon groups which include isoprenoid units and/or prenyl groups may be used as well.
  • saturated fatty acids that are suitable include, for example, lauric, myristic, palmitic and stearic acids.
  • Suitable unsaturated fatty acids that may be used include, for example, lauroleic, physeteric, myristoleic, linoleic, linolenic, palmitoleic, petroselinic and oleic acids.
  • branched fatty acids that may be used include, for example, isolauric, isomyristic, isopalmitic and isostearic acids.
  • the lipid is inco ⁇ orated in a mixed solvent system in the present compositions in a ratio of about 8:1:1 or about 9:1 :1 normal saline : glycerol : propylene glycol.
  • the lipids employed in the present methods and compositions are phospholipids.
  • Preferred among the phospholipids are those having a dialkylated moiety in which each alkyl group contains from about 10 to about 24 carbons, with phospholipids having dialkylated moieties in which each alkyl group contains from about 14 to about 22 carbons being even more preferred.
  • Still more preferred are dialkylated phospholipids in which each of the alkyl groups contains from about 16 to about 20 carbons, with from about 16 to about 18 carbons being yet more preferred, and 16 carbons being especially preferred.
  • the phospholipids employed have a transition temperature from the liquid crystalline state to the gel state of over 40°C, and/or that the phospholipids be in the gel state at physiologic temperature as at the temperature at which the compositions of the present invention are prepared.
  • the phospholipids are formulated as a blend of two or more lipids.
  • the present invention also provides compositions, including vesicle compositions, formulated from proteins or derivatives thereof. Proteins which may be suitable for use in the present compositions include, for example, collagen,- fibrin and albumin. The proteins are preferably of human origin, with proteins prepared via recombinant technology being more preferred.
  • Vesicles which are formulated from proteins and which would be suitable for use in the methods and compositions of the present invention are described, for example, in Feinstein, U.S. Patent Nos. 4,572,203, 4,718,433 and 4,774,958, and Cerny et al., U.S. Patent No.
  • compositions including compositions which vesicles based on proteins, in addition to those described in the aforementioned patents, would be apparent to one of ordinary skill in the art, once armed with the present disclosure.
  • compositions formulated from lipids and/or proteins also provides compositions, including vesicle compositions, formulated from polymers.
  • polymers may be of natural, semi-synthetic (modified natural) or synthetic origin.
  • polymer denotes a compound comprised of two or more repeating monomeric units, and preferably 10 or more repeating monomeric units.
  • semi-synthetic polymer (or modified natural polymer), as employed herein denotes a natural polymer that has been chemically modified in some fashion.
  • Exemplary natural polymers suitable for use in the present invention include naturally occurring polysaccharides.
  • Such polysaccharides include, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextrin, dextrose, polydextrose, pustulan, chitin, agarose, keratan, chondroitan, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch, HETA-starch, saccharides of microbial origin, such as dextran, and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabi
  • Exemplary natural polymers also include polypeptides such as, for example, polyglutamic acid and polylysine. Suitable polymers may also include, for example, proteins as described above, such as albumin, fibrin and collagen. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropyl- methylcellulose, methylcellulose, and methoxycellulose.
  • Exemplary synthetic polymers suitable for use in the present invention include polyethylenes (such as, for example, polyethylene glycol, polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyoxyethylene and polyoxypropylene copolymers, including polyoxyethylene and polyoxypropylene block copolymers, polyoxyalkylene derivatives of polyethylene glycol (such as, for example, the class of compounds referred to as PluronicsTM, commercially available from BASF, Parsippany, NJ), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides (such as, for example, polyacrylamide and nylon), polystyrene, polylactic acids, fluorinated hydrocarbons (such as, for example, polytetrafluoroethylene), polyoxazolines (such as, for example, polyethyloxazoline) and polymethyl meth
  • biocompatible synthetic polymers or copolymers prepared from monomers, such as acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, methacrylates, including ethyl acrylate and methyl methacrylate, 2-hydroxyethyl methacrylate (HEM), lactic acid, glycolic acid, e- caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorohydrin, hydroxyalkyl-acrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkyl-methacrylates, N-substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4-pentadiene-l-ol, vinyl acetate, acrylonitrile, styrene, p-amino- styrene, p-
  • Preferable polymers include polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polyphosphazene, polysiloxane, polydimethylsiloxane, polylactic acid and copolymers thereof, such as polylactidecoglycolide, poly(e-caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), poly(vinyl alcohol) and polyamide polymers, such as nylon.
  • Preferable copolymers include polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl-methacrylate, polystyrene-polyacrylonitrile, polyethylene-poly(propylene glycol), and polylactide co-glycolide polymers.
  • a preferred copolymer is polyvinylidene-polyacrylonitrile.
  • Other suitable biocompatible monomers and polymers will be readily apparent to those skilled in the art, once armed with the present disclosure.
  • Vesicle derived from polymers for use in the methods of the present invention are preferably low density.
  • the term "low density" refers to vesicles which have an internal void (cavity) volume which is at least about 75%> of the total volume of the vesicle.
  • the vesicles have a void volume of at least about 80%>, more preferably at least about 85%), even more preferably at least about 90%, still more preferably at least about 95%), and yet more preferably about 100% of the total volume of the vesicles.
  • the stabilizing compounds employed in the present compositions may be substantially crosslinked or substantially non-crosslinked.
  • Synthetic methods which may be employed for crosslinking stabilizing compounds including stabilizing compounds in the form of lipids, proteins and polymers, would be apparent to one of ordinary skill in the art, once armed with the present disclosure.
  • the formation of covalent crosslinks may involve the use of coupling and/or activation agents which are well known to the skilled artisan. These agents, when activating, may desirably serve as electrophiles and thereby promote the formation of covalent bonds.
  • activating agents include, for example, carbonyldiimidazol (CDI), dicyclohexyl- carbodiimide (DCC), diisopropylcarbodiimide (DIC), methyl sulfonyl chloride, Castro's Reagent, and diphenyl phosphoryl chloride.
  • Covalent linkages which are exemplary of crosslinking bridges include, for example, esters, ethers, thioesters, sulfides, disulfides, carbamates, amides, thioamides, urethane and substituted imidate linkages.
  • Crosslinking may be effected, for example, by (1) adding a chemical substance (crosslinking agent) to a stabilizing compound and, if necessary and/or desired, exposing the resulting mixture to heat, or (2) subjecting a stabilizing compound to high energy radiation.
  • crosslinking agent a chemical substance
  • a variety of crosslinking agents of different lengths and/or functionalities are described, for example, in R.L. Lunbland, Techniques in Protein Modification, CRC Press, Inc., Ann Arbor, MI, pp. 249-68 (1995), the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • crosslinkers include, for example, 3,3'-dithiobis(succinimidyl- propionate), dimethyl suberimidate, and its variations thereof, based on hydrocarbon length, and bis-N-maleimido-l,8-octane.
  • crosslinking may be introduced by combining polyvalent counterions, for example, calcium ions, with one or more compounds that contain groups that are negatively charged and/or that contain unshared pairs of electrons, such as, for example, polyphosphazene and hyaluronic acid.
  • a perfluorinated ether compound there is further included in the present compositions a perfluorinated ether compound.
  • the perfluorinated ether compounds impart highly desirable and beneficial characteristics to the compositions of the present invention.
  • the perfluoroether compounds which may be inco ⁇ orated in the present compositions preferably have low densities.
  • the compositions of the present invention may be highly useful as contrast agents for computed tomography (CT).
  • CT computed tomography
  • the perfluoroether compounds employed in the present invention possess useful reflectivity characteristics, they may be extremely useful as contrast agents for ultrasound.
  • the perfluoroether compounds described herein significantly beneficially improve the stability of the compositions of the invention, including those based on vesicles.
  • the perfluorinated compounds which may be inco ⁇ orated in the compositions of the present invention preferably comprise, for example, perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers and perfluoroheteroaryl ethers, and combinations thereof.
  • the perfluoroether compound may be, for example, a perfluoroalicyclic aliphatic ether compound (that is, a perfluoroether compound which contains both alicyclic and aliphatic moieties), a perfluoroheterocyclyl aliphatic ether compound (that is, a perfluoroether compound which contains both heterocyclyl and aliphatic moieties), a perfluoroalicyclic heterocyclyl ether compound (that is, a perfluoroether compound which contains both alicyclic and heterocyclyl moieties), and the like.
  • the perfluorinated ether compounds may have the formula R[-X-R 2
  • X is O or S;
  • R is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl;
  • R 2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
  • R, and R 2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • X is O or S.
  • X is O.
  • R, in the above formula (I) is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
  • R is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl, with perfluoroalkyl being more preferred.
  • perfluoroalkyl groups are perfluoroalkyl of from 1 to about 7 carbons, and all combinations and subcombinations of ranges therein, with perfluoroalkyl of from about 1 to about 6 carbons being more preferred. Even more preferred are perfluoroalkyl groups of from 1 to about 4 carbons. Particularly preferred perfluoroalkyl groups are perfluoromethyl and perfluorobutyl.
  • Preferred among the perfluoroalkenyl groups are perfluoroalkenyl of from about 2 to about 7 carbons, with perfluoroalkenyl of from 2 to about 6 carbons being more preferred and perfluoroalkenyl of from 2 to about 4 carbons being even more preferred.
  • perfluoroalkynyl groups are perfluoroalkynyl of from 2 to about 7 carbons, with perfluoroalkynyl of from 2 to about 6 carbons being more preferred and perfluoroalkynyl of from 2 to about 4 carbons being even more preferred.
  • Preferred among the perfluorocycloalkyl groups are perfluorocycloalkyl of from about 3 to about 7 carbons, with perfluorocycloalkyl of from about 3 to about 6 carbons being more preferred and perfluorocycloalkyl of from about 4 to about 6 carbons being even more preferred.
  • perfluoroaryl groups are perfluoroaryl of from about 6 to about 10 carbons, with perfluoroaryl of about 6 carbons being more preferred.
  • R 2 in the above.formula (I) is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
  • R 2 is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl, with perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl being more preferred, and perfluoroalkyl being even more preferred.
  • Preferred among the perfluoroalkyl groups are perfluoroalkyl of from 1 to about 7 carbons, and all combinations and subcombinations of ranges therein, with perfluoroalkyl of from about 1 to about 6 carbons being more preferred. Even more preferred are perfluoroalkyl groups of from 1 to about 4 carbons.
  • Particularly preferred perfluoroalkyl groups are perfluoromethyl and perfluorobutyl.
  • Preferred among the perfluoroalkenyl groups are perfluoroalkenyl of from about 2 to about 7 carbons, with perfluoroalkenyl of from 2 to about 6 carbons being more preferred and perfluoroalkenyl of from 2 to about 4 carbons being even more preferred.
  • Preferred among the perfluoroalkynyl groups are perfluoroalkynyl of from 2 to about 7 carbons, with perfluoroalkynyl of from 2 to about 6 carbons being more preferred and perfluoroalkynyl of from 2 to about 4 carbons being even more preferred.
  • perfluorocycloalkyl groups are perfluorocycloalkyl of from about 3 to about 7 carbons, with perfluorocycloalkyl of from about 3 to about 6 carbons being more preferred and perfluorocycloalkyl of from about 4 to about 6 carbons being even more preferred.
  • perfluoroaryl groups are perfluoroaryl of from about 6 to about 10 carbons, with perfluoroaryl of about 6 carbons being more preferred.
  • R 2 may also be halo.
  • R 2 is preferably chloro or bromo.
  • R is preferably perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl, with perfluoroalkyl being more preferred.
  • the total number of carbons in R, and R 2 is preferably no greater than about 7.
  • R, and R 2 may be linked together to form (together with X) perfluoroheterocyclyl or perfluoroheteroaryl.
  • the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
  • R, and R 2 are linked together to form perfluoroheterocyclyl.
  • Preferred among the perfluoroheterocyclyl groups are perfluoroheterocyclyl of from about 3 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein, with perfluoroheterocyclyl of about 5 or about 6 ring members being more preferred.
  • the perfluoroheteroaryl groups are perfluoroheteroaryl of from about 5 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein.
  • the perfluorinated ethers comprise perfluoroaliphatic ether compounds.
  • Preferred among the perfluoroaliphatic ethers are compounds of the formula r 2n+ 1 n - - m r 2m+ 1
  • each of n and m is independently an integer of 1 to about 7 (and all combinations and subcombinations of ranges therein); and X is O or S.
  • the sum of m and n is no greater than about 7, with a sum of about 5 being more preferred. Even more preferably, one of m and n is about 1 and the other of m and n is about 4. Also in preferred embodiments, X is O.
  • Exemplary compounds of formula (II) include, for example, perfluorodimethyl ether (where m and n are 1); perfluoromethyl ethyl ether (where one of m and n is 1 and the other of m and n is 2); perfluoropropyl methyl ethers (where one of m and n is 3 and the other of m and n is 1) such as, for example, perfluoroisopropyl methyl ether and perfluoro- n-propyl methyl ether; perfluorodiethyl ether (where m and n are 2); perfluorobutyl methyl ethers (where one of m and n is 4 and the other of m and n is 1) such as, for example, perfluoro-t-butyl methyl ether, perfluoroisobutyl methyl ether and pefluoro-n-butyl methyl ether; perfluoropropyl ethyl ethers
  • the perfluoroether compounds may comprise perfluoroaliphatic alicyclic compounds.
  • Preferred among the perfluoroaliphatic alicyclic ether compounds are compounds of the formula
  • n is an integer of from about 2 to about 5 (and all combinations and subcombinations of ranges therein);
  • X is O or S.
  • the sum of m and n in formula III is no greater than about 7, with a sum of up to about 5 being more preferred. More preferably, n is about 2 or about 3 and m is 1 or about 2. Also in preferred embodiments, X is O.
  • Exemplary compounds of formula (III) include, for example, perfluorocyclopropyl ethers (where n is 2), such as, for example, perfluorocyclopropyl methyl ether, perfluorocyclopropyl ethyl ether, perfluorocyclopropyl propyl ether, perfluorocyclopropyl butyl ether, perfluorocyclopropyl pentyl ether and perfluorocyclopropyl hexyl ether.
  • perfluorocyclopropyl ethers such as, for example, perfluorocyclopropyl methyl ether, perfluorocyclopropyl ethyl ether, perfluorocyclopropyl propyl ether, perfluorocyclopropyl butyl ether, perfluorocyclopropyl pentyl ether and perfluorocyclopropyl hexyl ether.
  • Exemplary compounds of formula (III) also include, for example, perfluorocyclobutyl ethers (where n is 3) such as, for example, perfluorocyclobutyl methyl ether, perfluorocyclobutyl ethyl ether, perfluorocyclobutyl propyl ether, perfluorocyclobutyl butyl ether, perfluorocyclobutyl pentyl ether and perfluorocyclobutyl hexyl ether.
  • perfluorocyclobutyl ethers such as, for example, perfluorocyclobutyl methyl ether, perfluorocyclobutyl ethyl ether, perfluorocyclobutyl propyl ether, perfluorocyclobutyl butyl ether, perfluorocyclobutyl pentyl ether and perfluorocyclobutyl hexyl ether.
  • the preferred perfluoroether compounds may be represented by the formula
  • m is an integer of from 0 to about 3 (and all combinations and subcombinations of ranges therein); n is an integer from about 2 to about 6 (and all combinations and subcombinations of ranges therein);
  • X is O or S
  • R is perfluoroalkyl of 1 to about 4 carbons.
  • m is an integer of 0 or 1
  • n is an integer of about 4 or about 5
  • X is O
  • R is perfluoromethyl.
  • Exemplary compounds of formula (IV) include, for example, perfluorotetrahydropyran (where m is 0, n is 5 and X is O), perfluorotetrahydrofuran (where m is 0, n is 4 and X is O) and perfluoromethyltetrahydrofuran (where m is 1 , n is 4 and X is O).
  • compositions which contain them including, for example, compositions containing stabilizing compounds which may be used, for example, as contrast agents in connection with methods for medical imaging.
  • compositions which comprise perfluoroether compounds, especially vesicle compositions may demonstrate improved stability when exposed to increased pressures, including, for example, increased pressures which may occur in vivo.
  • compositions of the present invention may provide a desirable and beneficial improvement when used in connection with methods for ultrasound imaging.
  • compositions of perfluorinated ether compounds may provide increased and prolonged contrast during ultrasound imaging of an internal region of a patient.
  • the novel compositions of the present invention may su ⁇ risingly and unexpectedly possess improved biocompatibility and may advantageously comprise vesicles which have a desirably smaller size distribution.
  • the perfluoroether compounds may impart reduced densities to the compositions of the present invention.
  • the present compositions may also provide desirably dark regions in diagnostic images obtained using X-ray imaging including, for example, computed tomography (CT) imaging.
  • CT computed tomography
  • administration of the compositions of the present invention including oral administration, provides decreased density within the bowel.
  • Administration, for example, by intravascular injection may also decrease the attenuation within the blood vessels.
  • the present compositions may provide desirable Tl and T2 relaxation times, and thus, may be suitable for use in MRI.
  • the perfluoroether compounds be administered to a patient in a gaseous or substantially gaseous form.
  • substantially as used in the present disclosure in connection with the administration of a perfluoroether compound in gaseous form, means that greater than about 50%> of the perfluoroether compound is in the gas state at about the time of administration. In certain embodiments, preferably greater than about 60%> of the perfluoroether compound is in the gas state at about the time of administration, with greater than about 70%> being more preferred.
  • perfluoroether compound is in the gas state at about the time of administration, with greater than about 90%) being still more preferred. In certain particularly preferred embodiments, greater than about 95%) of the perfluoroether compound is in the gas state at about the time of administration. If desired, the perfluoroether compound may be completely gaseous at about the time of administration (i.e., about 100%) of the perfluoroether compound is in the gas state). In ultrasound imaging applications, in particular, the gaseous state, especially a completely gaseous state, is most preferred.
  • Perfluoroether- compounds which are administered to a patient in substantially gaseous form may be gases at conditions of ambient temperature and pressure.
  • Perfluoroether compounds which are administered to a patient in substantially gaseous form may also be, in the context of the present invention, gaseous precursors, i.e., generally liquid at conditions of ambient temperatures and pressures, that have been activated to form a gas prior to or at the time of administration.
  • activation means the partial, substantial or complete conversion of a gaseous precursor to a gas. Conversion of a gaseous precursor to a gas may involve, for example, heating the gaseous precursor to a temperature at or near the phase transition temperature of the gaseous precursor. Activation of the gaseous precursor may also involve, for example, exposure of the gaseous precursor to varying pressures, typically reduced pressures, so that the gaseous precursor undergoes a phase transition from a liquid to a gas.
  • the perfluoroether compound may be administered to a patient substantially as a liquid.
  • Perfluoroether compounds which may be administered to a patient substantially in liquid form include, for example, perfluoroether compounds which are gaseous precursors, i.e., liquids at conditions of ambient temperature and pressure, which are liquids that have not been activated to form a gas prior to or at the time of administration.
  • Perfluoroether compounds which may be administered to a patient substantially in liquid form also include, for example, perfluoroether compounds which are typically gases at conditions of ambient temperature and pressure, but which have been converted to a liquid prior to administration.
  • a perfluoroether compound in liquid form means that greater than about 50%) of the perfluoroether compound is in the liquid state at about the time of administration.
  • preferably greater than about 60%) of the perfluoroether compound is in the liquid state at about the time of administration, with greater than about 70%> being more preferred.
  • greater than about 80%) of the perfluoroether compound is in the liquid state at about the time of administration, with greater than about 90%> being still more preferred.
  • greater than about 95%> of the perfluoroether compound is in the liquid state at about the time of administration.
  • the perfluoroether compound may be completely in the liquid state at about the time of administration (i.e., about 100% of the perfluoroether compound is in the liquid state).
  • the liquid perfluoroether compound may also be substantially converted from a liquid to a gas in vivo. This conversion can occur by virtue of the phase transition temperature of the perfluoroether liquid in the particular composition, or through applied heat (including ultrasound energy, for example).
  • the perfluoroether compound whether administered to a patient as a gas or a gaseous precursor, may also, if desired, be formulated to substantially persist as a liquid in vivo.
  • a particular perfluorinated ether compound may exist in the liquid state when the compositions of the present invention are first made, and thereafter converted to a gas and thus, may be used as a gaseous precursor.
  • the perfluorinated ether compounds may exist in the gaseous state when the present compositions are made, and thus may be used directly as a gas. Whether the perfluorinated ether compound is used as a liquid or a gas may generally depend on the desired application and on the particular compound chosen and its liquid/gas phase transition temperature, or boiling point.
  • a preferred perfluorinated ether compound perfluorobutylmethyl ether (PFBME) has a liquid/gas phase transition temperature (boiling point) of 35.4°C.
  • PFBME perfluorobutylmethyl ether
  • PFBME is generally a liquid at room temperature (about 25 °C), but is converted to a gas, for example, within the human body, the normal temperature of which is about 37 °C, this temperature being above the phase transition temperature of PFBME.
  • PFBME is a gaseous precursor at room temperature.
  • PFDEE perfluorodiethyl ether
  • PFBEE perfluorobutylethyl ether
  • PFDEE liquid gas transition of PFDEE
  • PFBEE gaseous precursor because of its relatively high boiling point.
  • PV nRT
  • P pressure
  • V volume
  • n moles of substance
  • R the gas constant
  • T temperature
  • P and T may affect whether, for example, the perfluoroether compound is in the liquid or gaseous state. Temperatures and pressures can be ambient or applied.
  • Such pressures can include, for example, interfacial pressures, such as interactions-between surfaces and substances in vivo, including, for example, liquids, membranous tissues and the like, such as might exist in the bloodstream.
  • the perfluorinated ether compound is present in the compositions of the present invention as a gas or gaseous precursor, with gases being preferred.
  • the perfluorinated ether compound may be advantageously converted to a gas prior to administration to a patient. A novel and convenient method for accomplishing this gas activation has been discovered, which involves the use of mechanical agitation.
  • this mechanical agitation imparts energy to the liquid gaseous precursor which stimulates its conversion to a gas.
  • this gas activation through mechanical agitation may be accomplished by providing a sealed vessel, for example, a container, which contains the composition, including the perfluorinated ether compound, under reduced pressure.
  • Agitation can involve, for example, a prolonged shaking motion or a single abrupt motion, such as, for example, the withdrawal of the plunger of a syringe containing the composition, immediately prior to administration.
  • Agitation of the container desirably results in conversion of the perfluorinated ether compound to a gas, preferably in the form of microbubbles.
  • the mechanical agitation can be employed to substantially coat the microbubbles with one or more stabilizing compounds, including, for example, surfactants, film-forming materials, membranes and/or membrane forming materials, such as, for example, lipids, proteins or polymers, to provide stabilized gaseous perfluorinated ether bubbles.
  • stabilizing compounds including, for example, surfactants, film-forming materials, membranes and/or membrane forming materials, such as, for example, lipids, proteins or polymers, to provide stabilized gaseous perfluorinated ether bubbles.
  • perfluorinated ether compounds which may be inco ⁇ orated in the compositions of the present invention preferably have a boiling point of from about 0°C to about 100°C, and all combinations and subcombinations of ranges of temperatures therein.
  • the perfluorinated ether compound has a boiling point of from about 20°C to about 60°C, with a boiling point of from about 25°C to about 45°C being more preferred. Even more preferably, the perfluorinated ether compound has a boiling point of from about 30°C to about 40°C.
  • the perfluorinated ether compounds generally demonstrate substantially improved acoustic activity when inco ⁇ orated as gases in the stabilized compositions of the present invention, including lipid, protein and polymer compositions, and/or vesicle compositions, as compared to the acoustic activity of the perfluorinated ether compounds when inco ⁇ orated in the aforesaid compositions as liquids.
  • the perfluorinated ether compound may be a gas at room temperature, as well as at physiological temperature after administration to a patient.
  • the perfluorinated ether compound employed may be a liquid at room temperature which preferably undergoes phase transition to a gas upon being exposed to temperatures higher than room temperature, for example, physiological temperatures.
  • boiling points for exemplary perfluorinated ether compounds that may be inco ⁇ orated in the compositions of the present invention.
  • halogenation namely fluorination, especially perfluorination
  • the non-halogenated ether compound is identified in the table as the "parent" compound.
  • Octafluoropropyl ethyl ethers (boiling point of 99.5°C).
  • Nonafluoropropyl ethyl ethers (boiling points range from 76 to 110°C).
  • FLUOROETHERS Heptafluoroisopropyl methyl ether (boiling point of 72 ° C).
  • Perfluoroether compounds which may be employed in the methods and compositions of the present invention, including the perfluoroether compounds of formulas (I) to (IV) above, may be readily prepared using standard synthetic methodology well known to those of ordinary skill in the art. In addition, certain perfluoroether compounds are commercially available. With respect to synthetic methods, various compounds which may serve as starting materials for the preparation of the perfluoroether compounds described herein are commercially available, or may likewise be readily synthesized. For example, the perfluoroether compounds may be prepared from the fluorination reaction of an adduct formed by the free-radical addition of a fluoro-olefin and a hydrogen-containing ether.
  • the fluorinated ether may be partially or fully fluorinated during the fluorination reaction.
  • the hydrogen-containing ether is preferably of the formula R-O-R', wherein R and R' are independently hydrocarbon groups optionally substituted by chlorine or fluorine, or together form a single hydrocarbon group.
  • R and R' are independently alkyl, cycloalkyl, aralkyl and aryl.
  • the mole ratio of the fluoro-olefin and the hydrogen-containing ether in the adduct may be from about 6:1 to about 1:1, although higher or lower amounts of fluoro-olefin may be employed, depending, for example, on the particular fluoro-olefin and/or hydrogen-containing ethers employed.
  • the fluorination of the adduct may be effected by the use of a high valency metal fluoride as a fluorinating agent, preferably at elevated temperatures including, for example, above about 200 ° C.
  • a fluorination procedure of this type is described, for example, in "Advances in Fluorine Chemistry", Vol 1., Butterworth, p. 166 (1960), the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • Suitable fluorinating agents include, for example, cobalt trifluoride (CoF 3 ), alone or in combination with one or more alkali or alkaline earth metal fluorides, such as, for example, potassium fluoride (KF) and calcium fluoride (CaF 2 ).
  • the fluorination reaction may preferably be effected at elevated temperatures, preferably above about 200 ° C, with preferred temperature ranges being from about 300 ° C to about 600 ° C, and more preferably,f rom about 400 C to about 500 ° C.
  • higher temperatures may provide fluorinated ether compounds having higher degrees of fluorination, including perfluorination, whereas the use of lower temperatures may provide fluorinated ether compounds which are partially fluorinated.
  • Fluorination with cobalt trifluoride is a technique well known in the art, and is described in standard chemistry textbooks such as, for example, R. D. Chambers, "Fluorine in Organic Chemistry", see page 25, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • cobalt trifluoride can be regenerated by reacting elemental fluorine and the cobalt difluoride resulting from the organic fluorination reaction.
  • Exemplary fluorination reactions which may be used to prepare the present perfluoroether compounds are described, for example, in Drakesmith et al., Published International Patent Application No.
  • the perfluoroether compounds may also be synthesized with isotopes, for example, isotopes of oxygen, such as 17 O, to enhance the paramagnetic properties of the perfluoroether compounds. This may serve, for example, to enhance the utility of the present compositions as diagnostic agents for magnetic resonance imaging.
  • the perfluorinated ether compounds may be present in the stabilized compositions of the present invention as a gas or gaseous precursor, with gases being preferred.
  • the compositions of the present invention may further comprise additional gases and/or gaseous precursors.
  • additional gases the following discussion is provided.
  • gases are gases which are inert and which are biocompatible, that is, gases which are not injurious to biological function.
  • Preferred gases include those selected from the group consisting of air, noble gases, such as helium, rubidium hype ⁇ olarized xenon, hype ⁇ olarized argon, hype ⁇ olarized helium, neon, argon, xenon, carbon dioxide, nitrogen, fluorine, oxygen, sulfur-based gases, such as sulfur hexafluoride and sulfur tetrafluoride, fluorinated gases, including, for example, partially fluorinated gases or completely fluorinated gases.
  • Exemplary fluorinated gases include the fluorocarbon gases, such as the perfluorocarbon gases, and mixtures thereof.
  • Paramagnetic gases, such as I7 O 2 may also be used in the lipid and/or vesicle compositions.
  • the additional gases utilized in the compositions described herein may be fluorinated gas.
  • fluorinated gases include materials which contain at least one, or more than one, fluorine atom.
  • gases which contain more than one fluorine atom with perfluorocarbons (that is, fully fluorinated fluorocarbons) being more preferred.
  • the perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfiuoropentane, perfluorocyclobutane and mixtures thereof.
  • the perfluorocarbon gas is perfluoropropane or perfluorobutane, with perfluoropropane being particularly preferred.
  • Another preferable gas is sulfur hexafluoride.
  • Yet another preferable gas is heptafluoropropane, including 1,1,1,2,3,3,3- heptafluoropropane and its isomer, 1,1,2,2,3, 3, 3 -heptafluoropropane.
  • mixtures of different types of gases such as mixtures of a perfluorocarbon gas and another type of gas, such as air, can also be used in the compositions employed in the methods of the present invention.
  • gases including the gases exemplified above, would be readily apparent to one skilled in the art based on the present disclosure.
  • a gas for example, air or a perfluorocarbon gas
  • a liquid perfluorocarbon such as perfiuoropentane, perfluorohexane, perfluoroheptane, perfluorooctylbromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine and perfluorotributylamine.
  • the perfluoroether compounds described herein which may be in the form of a gaseous precursor, it may be desirable to inco ⁇ orate in the stabilized compositions of the present invention additional gaseous precursors.
  • Such precursors include materials that are capable of being converted to a gas in vivo.
  • the gaseous precursor is biocompatible, and the gas produced in vivo is biocompatible also.
  • agents which are sensitive to pH include materials that are capable of evolving gas, for example, upon being exposed to a pH that is neutral or acidic.
  • pH sensitive agents include salts of an acid which is selected from the group consisting of inorganic acids, organic acids and mixtures thereof.
  • Carbonic acid (H 2 CO 3 ) is an example of a suitable inorganic acid
  • aminomalonic acid is an example of a suitable organic acid.
  • Other acids, including inorganic and organic acids, would be readily apparent to one skilled in the art based on the present disclosure.
  • Gaseous precursors which are pH-sensitive and which are derived from salts are preferably selected from the group consisting of alkali metal salts, ammonium salts and mixtures thereof. More preferably, the salt is selected from the group consisting of carbonate, bicarbonate, sesquecarbonate, aminomalonate and mixtures thereof.
  • suitable gaseous precursor materials which are derived from salts include, for example, lithium carbonate, sodium carbonate, potassium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, magnesium carbonate, calcium carbonate, magnesium bicarbonate, ammonium carbonate, ammonium bicarbonate, ammonium sesquecarbonate, sodium sesquecarbonate, sodium aminomalonate and ammonium aminomalonate.
  • Aminomalonate is well known in the art, and its preparation is described, for example, in Thanassi, Biochemistry, Vol. 9, no. 3, pp. 525- 532 (1970); Fitzpatrick et al., Inorganic Chemistry, Vol. 13, no. 3 pp. 568-574 (1974); and Stelmashok et al., Koordinatsionnaya Khimiya, Vol. 3, no. 4, pp. 524-527 (1977). The disclosures of these publications are hereby inco ⁇ orated herein by reference, in their entireties.
  • the gaseous precursor materials may also comprise compounds which are sensitive to changes in temperature.
  • suitable gaseous precursors which are sensitive to changes in temperature are the perfluorocarbons.
  • a particular perfluorocarbon may exist in the liquid state when the stabilized compositions are first made, and thus may be used as a gaseous precursor.
  • the perfluorocarbon may exist in the gaseous state when the stabilized compositions are made, and thus may be used directly as a gas. Whether the perfluorocarbon is used as a liquid or a gas generally depends on its liquid/gas phase transition temperature, or boiling point.
  • a preferred perfluorocarbon, perfiuoropentane has a liquid/gas phase transition temperature (boiling point) of 29.5°C.
  • perfiuoropentane is generally a liquid at room temperature (about 25°C), but is converted to a gas within the human body, the normal temperature of which is about 37°C, this temperature being above the transition temperature of perfiuoropentane.
  • perfiuoropentane is a gaseous precursor.
  • there are the homologs of perfiuoropentane namely perfluorobutane and perfluorohexane.
  • perfluorobutane may be useful as a gaseous precursor, although more likely as a gas, whereas perfluorohexane may be useful as a gaseous precursor because of its relatively high boiling point.
  • perfluorobutane may be useful as a gaseous precursor, although more likely as a gas
  • perfluorohexane may be useful as a gaseous precursor because of its relatively high boiling point.
  • a wide variety of materials may be used as temperature-sensitive gaseous precursors in the compositions described herein. It is only required that the material be capable of undergoing a phase transition to the gas phase upon passing through the appropriate temperature (such as physiological temperatures or following direct application of biocompatible heat).
  • Suitable gaseous precursors include, for example, hexafluoroacetone, isopropyl acetylene, allene, tetrafluoroallene, boron trifluoride, 1,2- butadiene, 2,3-butadiene, 1,3-butadiene, 1, 2,3 -trichloro-2-fluoro- 1,3 -butadiene, 2-methyl- 1,3-butadiene, hexafluoro- 1,3-butadiene, butadiyne, 1-fluorobutane, 2-methylbutane, perfluorobutane, 1-butene, 2-butene, 2-methyl-l-butene, 3-methyl-l-butene, perfluoro-1- butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-l-butene-3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-
  • Pressure sensitive gaseous precursors may also be employed in the methods and compositions of the present invention.
  • pressure is directly proportional to temperature.
  • the ideal gas law may be used to predict, for example, that the boiling point of a substance increases upon exposure to increased pressures. Conversely, the boiling point of a substance is predicted to decrease upon exposure to reduced pressures.
  • a decrease in pressure may result, for example, from the abrupt withdrawal on the plunger of a syringe which contains the gaseous precursor.
  • the gaseous precursor may undergo a conversion to a gas, owing to the decrease in pressure.
  • Perfluorocarbons are both preferred as additional gases and gaseous precursors for use in connection with the compositions employed in the methods of the present invention. Included among such perfluorocarbons are saturated perfluorocarbons, unsaturated perfluorocarbons, and cyclic perfluorocarbons.
  • the saturated perfluorocarbons which are usually preferred, preferably have the formula C n F 2n+2 , where n ranges from 1 to about 12, and all combinations and subcombinations of ranges therein.
  • n in the foregoing formula for saturated perfluorocarbons is from about 2 to about 10, more preferably about 3 to about 8, and even more preferably about 3 to about 6.
  • Suitable perfluorocarbons include, for example, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluorocyclobutane, perfiuoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluorononane.
  • the saturated perfluorocarbon is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfiuoropentane, perfluorohexane and perfluorooctane, with perfluoropropane being particularly preferred.
  • cyclic perfluorocarbons which preferably have the formula C n F 2n , where n ranges from about 3 to 8, and all combinations and subcombinations of ranges therein.
  • n in the foregoing formula for cyclic perfluorocarbons is from about 3 to about 6.
  • Exemplary cyclic perfluorocarbons include, for example, hexafluorocyclopropane, octafluorocyclobutane, and decafluorocyclopentane.
  • Such fluorocarbons include heptafluoropropane, for example, 1,1, 1,2,3,3, 3 -heptafluoropropane and its isomer, 1 , 1 ,2,2,3,3,3-heptafluoropropane.
  • the gaseous precursor materials may be also photoactivated materials, such as diazonium ion and aminomalonate.
  • photoactivated materials refers to light sensitive compounds which are generally in solid or liquid form and which become a gas after exposure to light.
  • Exemplary photosensitive compounds include, for example, diazonium compounds, which may decompose to form nitrogen gas after exposure to ultraviolet light.
  • Another exemplary photosensitive compound is aminomalonate.
  • other gaseous precursors may be chosen which are capable of forming a gas after exposure to light. Depending upon the application, exposure to such light may be necessary prior to administration or, in some instances, can occur subsequent to administration.
  • Certain stabilized compositions may be formulated so that gas can be formed at the target tissue or by the action of sound on the composition.
  • gaseous precursors are described, for example, in U.S. Patent Nos. 5,088,499 and 5,149,319, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties.
  • Other gaseous precursors in addition to those exemplified above, will be apparent to one skilled in the art based on the present disclosure.
  • the gaseous substances and/or liquids (including gaseous precursors), including the perfluoroether compounds are preferably inco ⁇ orated in the stabilized compositions irrespective of the physical nature of the composition.
  • the gaseous substances and/or precursors thereto may be inco ⁇ orated, for example, in lipid compositions in which the lipids are aggregated randomly, as well as in vesicle compositions, including vesicle compositions which are formulated from lipids, such as micelles and liposomes.
  • Inco ⁇ oration of the gaseous substances and/or precursors thereto in the stabilized compositions may be achieved by using any of a number of methods.
  • the formation of gas filled vesicles can be achieved by shaking or otherwise agitating an aqueous mixture which comprises a gas or gaseous precursor and one or more lipids. This promotes the formation of stabilized vesicles within which the gas or gas precursor is encapsulated.
  • a gas may be bubbled directly into an aqueous mixture of stabilized compound, including lipids and/or vesicle-forming compounds.
  • a gas instillation method can be used as disclosed, for example, in U.S. Patent Nos. 5,352,435 and 5,228,446, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties. Suitable methods for inco ⁇ orating the gas or gas precursor in cationic lipid compositions are disclosed also in U.S. Patent No. 4,865,836, the disclosures of which are hereby inco ⁇ orated herein by reference. Other methods would be apparent to one skilled in the art based on the present disclosure.
  • the gas may be instilled in the stabilized compositions after or during the addition of the stabilizing material and/or during formation of vesicles.
  • the gaseous substances and/or gaseous precursor materials may be inco ⁇ orated in vesicle compositions, with micelles and liposomes being preferred.
  • vesicles in which a gas, gaseous precursor and/or perfluorinated ether compound are encapsulated are advantageous in that they provide improved reflectivity in vivo.
  • the stabilized compositions, and especially vesicle compositions be formulated from lipids and optional stabilizing compounds to promote the formation of stable vesicles.
  • the lipid and/or vesicle compositions comprise a highly stable gas as well.
  • the phrase "highly stable gas" refers to a gas which has limited solubility and diffusability in aqueous media.
  • Exemplary highly stable gases include the perfluoroethers described herein, as well as perfluorocarbons since they are generally less diffusible and relatively insoluble in aqueous media. Accordingly, their use may promote the formation of highly stable vesicles.
  • a fluorinated compound including a perfluorinated compound, which may be in the liquid state at the temperature of use of the stabilized compositions, including, for example, the in vivo temperature of the human body, to assist or enhance the stability of the stabilized compositions, and especially, the gas filled vesicles.
  • Suitable fluorinated compounds include, for example, fluorinated amphiphilic compounds. It is contemplated that these fluorinated compounds may be used in addition to, or instead of, the various stabilizing compounds described above. A variety of fluorinated amphiphilic compounds can be employed in the present compositions.
  • Preferred fluorinated amphiphilic compounds are those which, when combined with a gas, including the perfluoroether compounds described herein, as well as other stabilizing compounds, if desired, tend to form stabilized compositions and/or vesicles. Preferred also are fluorinated amphiphilic compounds which are capable of stabilizing the vesicles, once formed.
  • the fluorinated amphiphilic compounds are based on amphiphilic compounds, including lipids, and especially phospholipids.
  • the fluorine atoms are preferably substituted on the nonpolar aliphatic chain portions of the involved amphiphilic compounds.
  • Other fluorinated compounds which also may be suitable for use in the present compositions, include, for example, the class of compounds which are commercially available as ZONYLTM fluorosurfactants (DuPont Chemical Co ⁇ ., Wilmington DE), including the ZONYLTM phosphate salts and the ZONYLTM sulfate salts, which have terminal phosphate or sulfate groups. Representatives of these salts are disclosed, for example, in U.S. Patent No. 5,276,146, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • the ZONYLTM phosphate salt has the formula
  • Suitable ZONYLTM surfactants also include, for example, Telomer B fluorosurfactants, including Telomer B surfactants which are pegylated (i.e., PEG Telomer B surfactants).
  • PEG Telomer B surfactant which may be particularly suitable for use as an amphiphilic compound is the compound C 9 F, 9 CH 2 CH 2 (OCH 2 CH 2 ) 9 -OH.
  • the Telomer B surfactants are also commercially available from the DuPont Chemical Co ⁇ ., of Wilmington DE.
  • the fluorinated amphiphilic compounds are polyfluorinated. This means that the amphiphilic compounds are preferably substituted with at least two or more fluorine atoms. In even more preferred embodiments, the fluorinated amphiphilic compounds are perfluorinated.
  • the fluorinated amphiphilic compound has the formula
  • X 3 is a direct bond or -O-; M is P or S;
  • Z is hydrogen, the residue of a hydrophilic polymer, a saccharide residue or -N ⁇ ),, where r is 2 or 3; each R, is independently alkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons;
  • R 2 is a direct bond or an alkylene linking group of 1 to about 10 carbons
  • R 3 is a direct bond or an alkylene diradical of 1 to about 10 carbons; each of R 4 and R j is independently hydrogen or alkyl of 1 to about 8 carbons; and each Rg is. independently hydrogen, alkyl of 1 to about 8 carbons or a residue of a hydrophilic polymer; provided that at least one of x, y and z is 1, at least one of R, is fluorinated alkyl of 1 to about 30 carbons, and when R 2 is a direct bond, two of x, y and z are each 0.
  • fluorinated derivatives of common phospholipids diacylphosphatidyl serine, diacylphosphatidyl ethanolamine, diacylphosphatidyl glycerol, diacylphosphatidyl glycerol, etc.
  • fluorinated derivatives of fatty acyl esters and free fatty acids can be employed in the compositions of the present invention.
  • a particularly preferred class of fluorinated amphiphilic compounds which may be employed in the compositions of the present invention and which are within the scope of fluorinated amphiphilic compounds of formula (I), is represented by the formula (I).
  • fluorinated amphiphilic compounds which may be employed in the methods and compositions of the present invention, including the fluorinated amphiphilic compounds of formulas (I) and (II), can be prepared readily using standard organic synthetic methodology well known to those of ordinary skill in the art. Suitable methods for preparing fluorinated amphiphilic compounds are disclosed, for example, in C. Santaella et al., New Journal of Chemistry, 15, 685 (1991), the disclosures of which are hereby inco ⁇ orated by reference, in their entirety. Exemplary of the available methods for preparing fluorinated amphiphilic compounds are synthetic methods based on the phosphorylation of l,2-di-(F-alkylacyl)-3-glycerol derivatives.
  • BEDP phosphatidylethanolamine
  • BEDP is highly reactive and can be used to phosphorylate sterically hindered disubstituted glycerols. Hansen et al., Lipids, 17, 453 (1982).
  • the ammonium group can be introduced by reaction of an appropriate amine with the 1 ,2-diacylglycero- 3-(2-bromoethyl)phosphate intermediate to give the desired phosphatidylethanolamine derivatives. This synthetic methodology is depicted in the following reaction scheme.
  • the hydrogenolysis reaction can be monitored by ordinary- analytical techniques, including, for example, thin layer chromatography (TLC) and proton ( ⁇ ) nuclear magnetic resonance (NMR).
  • TLC thin layer chromatography
  • NMR proton ( ⁇ ) nuclear magnetic resonance
  • the reaction is generally complete in about one hour with little or no transesterification.
  • the hydrogenolysis reaction is preferably conducted in THF because both the starting material in the involved reaction (the compound of formula (III)) and the product (the compound of formula (IV)) tend to be highly soluble in THF.
  • THF is conveniently used as the solvent in the subsequent phosphorylation step.
  • the catalyst (Pd/C) can be removed by filtration.
  • the 1 ,2-diacylglycerols of formula (IV) can be reacted immediately with BEDP and an excess of triethylamine. Phosphorylation is typically completed in about 2 to about 4 hours, as measured by TLC. The remaining phosphochloride bond can be hydrolysed in aqueous base and generally requires about 22 hours for completion.
  • mineral bases or salts including, for example, Na ⁇ O ; ,, KC1 and EDTA sodium salt
  • the phosphate salts are highly insoluble in water or organic solvents. Fleischer, Methods Enzymol. 98, 263 (1983).
  • the brominated intermediate can be isolated as a stable and soluble hydrogenotriethylammonium salt if excess triethylamine is used. Acidification of the phosphate salts to form the corresponding acid is difficult because the glycerol ester bonds are hydrolyzed at the necessary pH (pH of 2 to 3). Product degradation occurs also during purification over silica gel. Accordingly, it is preferred to use the phosphate salt without further purification. Nucleophilic displacement of the bromide ion by a large excess of trimethylamine occurs in a solvent mixture of CHC1 3 /CH 3 CN at 45°C over a 12 hour period. The displaced bromide ion can be precipitated by the addition of silver carbonate.
  • Exemplary fluorinated amphiphilic compounds are described, for example, in Applicant's copending U.S. application Serial No. 08/465,868, filed June 6, 1995, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties. Additional fluorinated compounds which may be inco ⁇ orated in the present compositions are described for example, in Lohrmann, U.S. Patent No. 5,562,893, Reiss et al. U.S. Patent No. 5,344,930, Frezard, F., et al, Biochem Biophys Acta 1994, 1192:61-70, and Frezard, F., et al., Art. Cells Blood Subs and Immob Biotech.
  • fluorinated compounds which may be employed in the present compositions include liquid perfluorinated ether compounds, as well as liquid perfluorocarbons, such as for example, perfluorooctylbromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine, and perfluorotributylamine.
  • PFOB perfluorooctylbromide
  • perfluorodecalin perfluorododecalin
  • perfluorooctyliodide perfluorotripropylamine
  • perfluorotributylamine perfluorotributylamine
  • perfluorocarbons perfluorooctylbromide and perfluorohexane, which are liquids at room temperature, are preferred.
  • the gas which is present may be, for example, a perfluoroether compound, as well as other gases, such as nitrogen or perfluoropropane.
  • the liquid fluorinated compound may be situated at the interface between the gas and the membrane or wall surface of the vesicle.
  • a further stabilizing layer of liquid fluorinated compound on the internal surface of the stabilizing compound for example, a biocompatible lipid used to form the vesicle, and this perfluorocarbon layer may also prevent the gas from diffusing through the vesicle membrane.
  • a liquid fluorinated compound such as a perfluorocarbon
  • a perfluoroether compound for example, perfluorbutyl methyl ether
  • a perfluorocarbon which remains liquid after administration to a patient, that is, whose liquid to gas phase transition temperature is above the body temperature of the patient, for example, perfluorooctylbromide.
  • Perfluorinated surfactants such as ZONYL® fluorinated surfactants, may be used to improve the stability of the stabilized compositions, and to act, for example, as a coating for vesicles.
  • Preferred perfluorinated surfactants are the partially fluorinated phosphocholine surfactants.
  • the dual alkyl compounds may be fluorinated at the terminal alkyl chains and the proximal carbons may be hydrogenated.
  • a desired component of the compositions of the present invention is an aqueous environment, particularly with respect to compositions comprising vesicles.
  • Many of the stabilizing materials discussed above involve compounds which comprise both hydrophobic and hydrophilic properties. Accordingly, there can be a predisposition among the present to form vesicles, which are highly stable configurations 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 of dissolved salts which preferably will not interfere with the creation and maintenance of the stabilized compositions or their use, for example, as diagnostic agents, and normal saline and physiological saline.
  • buffers may also be employed the aqueous medium, including phosphate buffers or tris(hydroxymethyl)aminomethane buffers.
  • the size of the vesicles and/or particles in vesicle compositions, emulsions and/or suspensions, whether in aqueous media or as a lypholizate, can be adjusted for the particular intended end use including, for example, diagnostic and/or therapeutic use.
  • the vesicles and/or particles preferably have a mean diameter of less than about 100 microns, with a mean diameter of less than about 10 microns being more preferred. Even more preferred are mean diameters of from about 5 microns to about 10 microns. Also in preferred form, the diameter of over about 95%> of the particles and/or vesicles is less than about 10 microns.
  • the particles and/or vesicles in the compositions of the present invention have a diameter of less than about 10 microns.
  • the vesicles be no larger that about 30 ⁇ m in diameter, with smaller vesicles being preferred, for example, vesicles of no larger than about 12 ⁇ m in diameter.
  • the diameter of the vesicles may be about 7 ⁇ m or less, with vesicles having a mean diameter of about 5 ⁇ m or less being more preferred, and vesicles having a mean diameter of about 3 ⁇ m or less being even more preferred. It is contemplated that these smaller vesicles may perfuse small vascular channels, such as the microvasculature, while at the same time providing enough space or room within the vascular channel to permit red blood cells to slide past the vesicles.
  • compositions employed herein may also include, with respect to their preparation, formation and use, gaseous precursors that can be activated to change from a liquid or solid state into a gas by temperature (including energy, such as ultrasound), pH and light.
  • the gaseous precursors may be made into gas by storing the precursors at reduced pressure.
  • a vial stored under reduced pressure may create a headspace of perfiuoropentane or perfluorohexane gas, useful for creating a preformed gas prior to injection.
  • the gaseous precursors may be activated by temperature.
  • Set forth below is a table listing a series of gaseous precursors which undergo phase transitions from liquid to gaseous states at relatively close to normal body temperature (37°C) or below, and the size of the emulsified droplets that would be required to form a vesicle of a maximum size of 10 ⁇ m.
  • vesicle compositions formulated from lipids by using gases of limited solubility.
  • limited solubility refers to 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 may have a tendency to diffuse out of the vesicle.
  • a lesser solubility in the aqueous milieu may, on the other hand, decrease or eliminate the gradient between the vesicle and the interface such that diffusion of the gas out of the vesicle may be impeded.
  • the gas entrapped in the vesicle has a solubility less than that of oxygen, that is, about 1 part gas in about 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.
  • vesicles may be desirable, in certain embodiments, to formulate vesicles from substantially impermeable polymeric materials. In these embodiments, it may be unnecessary to employ a gas which is highly insoluble also.
  • stable vesicle compositions which comprise substantially impermeable polymeric materials may be formulated with gases having higher solubilities, for example, air or nitrogen.
  • the compositions described herein may comprise one or more stabilizing materials.
  • stabilizing materials are, for example, biocompatible polymers.
  • the stabilizing materials may be employed to desirably assist in the formation of vesicles and/or to assure substantial encapsulation of the gases or gaseous precursors. Even for relatively insoluble, non-diffusible gases, such as perfluoropropane or sulfur hexafluoride, improved vesicle compositions may be obtained when one or more stabilizing materials are utilized in the formation of the gas and gaseous precursor filled vesicles.
  • stable or “stabilized”, as used herein, means that the vesicles may be substantially resistant to degradation, including, for example, loss of vesicle structure or encapsulated gas or gaseous precursor, for a useful period of time.
  • the vesicles employed in the present invention have a desirable shelf life, often retaining at least about 90 %> by volume of its original structure for a period of at least about two to three weeks under normal ambient conditions.
  • the vesicles are desirably stable for a period of time of at least about 1 month, more preferably at least about 2 months, even more preferably at least about 6 months, still more preferably about eighteen months, and yet more preferably up to about 3 years.
  • the vesicles described herein, including gas and gaseous precursor filled vesicles may also be stable 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 described herein may be attributable, at least in part, to the materials from which the vesicles are made, including, for example, the lipids, polymers and/or proteins described above, and it is often not necessary to employ additional stabilizing materials, although it is optional and may be preferred to do so. Such additional stabilizing materials and their characteristics are described more fully hereinafter.
  • the materials from which the vesicles are constructed are preferably biocompatible lipid, protein or polymer materials, and of these, the biocompatible lipids are preferred.
  • these vesicles may be conveniently made on site.
  • vesicles which comprise three components: (1) a neutral lipid, for example, a nonionic or zwitterionic lipid, (2) a negatively charged lipid, and (3) a lipid bearing a stabilizing material, for example, a hydrophilic polymer.
  • a neutral lipid for example, a nonionic or zwitterionic lipid
  • a negatively charged lipid for example, a negatively charged lipid
  • a stabilizing material for example, a hydrophilic polymer.
  • the amount of the negatively charged lipid will be greater than about 1 mole percent of the total lipid present
  • the amount of lipid bearing a hydrophilic polymer will be greater than about 1 mole percent of the total lipid present.
  • Exemplary and preferred negatively charged lipids include phosphatidic acids.
  • the lipid bearing a hydrophilic polymer will desirably be a lipid covalently linked to the polymer, and the polymer will preferably have a weight average molecular weight of from about 400 to about 100,000, and all combinations and subcombinations of weight average molecular weights therein.
  • Suitable hydrophilic polymers are preferably selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, poly(vinyl alcohol), and poly(vinyl pyrrolidone) and copolymers thereof, with PEG polymers being preferred.
  • the PEG polymer has a molecular weight of from about 1000 to about 10000, and all combinations and subcombinations of molecular weight ranges therein, with molecular weights of from about 2000 to about 8000 being more preferred.
  • the PEG or other polymer may be bound to the lipid, for example, DPPE, through a covalent bond, such as an amide, carbamate or amine linkage.
  • the PEG or other polymer may be linked to a targeting ligand, or other phospholipids, with a covalent bond including, for example, amide, ester, ether, thioester, thioamide or disulfide bonds.
  • the hydrophilic polymer is PEG
  • a lipid bearing such a polymer will be said to be “pegylated.”
  • the lipid bearing a hydrophilic polymer may be DPPE-PEG, including, for example, DPPE-PEG5000, which refers to DPPE having a polyethylene glycol polymer of a mean weight average molecular weight of about 5000 attached thereto (DPPE-PEG5000).
  • DPPE-PEG5000 DPPE having a polyethylene glycol polymer of a mean weight average molecular weight of about 5000 attached thereto
  • Another suitable pegylated lipid is distearoylphosphatidylethanol- amine-polyethylene glycol 5000 (DSPE-PEG5000).
  • the lipid compositions may include about 77.5 mole % DPPC, 12.5 mole % of DPP A, and 10 mole % of DPPE-PEG5000. Also preferred are compositions which comprise about 80 to about 90 mole % DPPC, about 5 to about 15 mole % DPPA and about 5 to about 15 mole % DPPE-PEG5000. Especially preferred are compositions which comprise DPPC, DPPA and DPPE-PEG5000 in a mole % ratio of 82: 10:8, respectively. DPPC is substantially neutral, since the phosphatidyl portion is negatively charged and the choline portion is positively charged.
  • DPPA which is negatively charged
  • 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.
  • the DPPE-PEG may provide more vesicles of a smaller size which are safe and stable to pressure when combined with other lipids, such as DPPC and DPPA, in the given ratios.
  • the pegylated material because of its structural similarity to water, may be able to defeat 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 may function as diagnostic imaging contrast media.
  • the vesicle compositions may be prepared from other materials, in addition to the materials described above, provided that the vesicles so prepared meet the stability and other criteria set forth herein. These materials may be basic and fundamental, and form the primary basis for creating or establishing the stabilized gas and gaseous precursor filled vesicles. On the other hand, they may be auxiliary, and act as subsidiary or supplementary agents which can enhance the functioning of the basic stabilizing material or materials, or contribute some desired property in addition to that afforded by the basic stabilizing material.
  • a given material is a basic or an auxiliary agent, since the functioning of the material in question is determined empirically, for example, by the results produced with respect to producing stabilized vesicles.
  • these basic and auxiliary materials may function, it has been observed that the simple combination of a biocompatible lipid and water or saline when shaken will often give a cloudy solution subsequent to autoclaving for sterilization.
  • a cloudy solution may function as a contrast agent, but is aesthetically objectionable and may imply instability in the form of undissolved or undispersed lipid particles.
  • Cloudy solutions may be also undesirable where the undissolved particulate matter has a diameter of greater than about 7 ⁇ m, and especially greater than about 10 ⁇ m.
  • propylene glycol may be added to remove this cloudiness by facilitating dispersion or dissolution of the lipid particles.
  • the propylene glycol may also function as a wetting agent which can improve vesicle formation and stabilization by increasing the surface tension on the vesicle membrane or skin. It is possible that the propylene glycol can also function as an additional layer that may coat the membrane or skin of the vesicle, thus providing additional stabilization.
  • further basic or auxiliary stabilizing materials there are conventional surfactants which may be used; see D'Arrigo U.S. Patents Nos. 4,684,479 and 5,215,680, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties.
  • compositions of the present invention may include as an auxiliary and/or basic stabilizing material an oil including, for example, vegetable oils and other oils, such as peanut oil, avocado oil, mineral oil, canola oil, soybean oil, olive oil, palm oil, palm seed oil, safflower oil, sunflower oil, sesame oil, corn oil, castor oil, coconut oil, cotttonseed oil, or any other oil commonly known to be ingestible which is suitable for use as a stabilizing compound in accordance with the teachings herein.
  • vegetable oils and other oils such as peanut oil, avocado oil, mineral oil, canola oil, soybean oil, olive oil, palm oil, palm seed oil, safflower oil, sunflower oil, sesame oil, corn oil, castor oil, coconut oil, cotttonseed oil, or any other oil commonly known to be ingestible which is suitable for use as a stabilizing compound in accordance with the teachings herein.
  • Preferred oils include those which are hydrophobic aliphatic materials and which are generally liquids at room temperature.
  • compositions containing a perfluoroether compound and one or more oils, as well as additional stabilizing materials, if desired, may be formulated in a variety of ways, including, for example, as emulsions, suspensions, colloidal dispersions, and the like.
  • the perfluoroether compound may be combined with an oil in a weight ratio of perfluoroether compound:oil of about 0.001:1 to about 1 :1, and even up to about 10:1.
  • Preparation of compositions of the perfluoroether compound and oils may involve, for example, mixing by agitation or emulsification.
  • the oil-containing compositions may be stored as an oil in water emulsion or suspension.
  • one or more additional stabilizing materials may be included in the oil-containing compositions, including, for example, phospholipids, fluorinated lipids/surfactants, and glycerol.
  • compounds used to make mixed micelle systems may be suitable for use as basic or auxiliary stabilizing materials, and these include, for example, lauryltrimethylammonium bromide (dodecyl-), cetyltrimethylammonium bromide (hexadecyl-), myristyltrimethylammonium bromide (tetradecyl-), alkyldimethyl- benzylammonium chloride (where alkyl is C 12 , C 14 or C, 6 ,), benzyldimethyldodecyl- ammonium bromide/chloride, benzyldimethyl hexadecylammonium bromide/chloride, benzyldimethyl tetradecylammonium bromide/chloride, cetyldimethylethylammonium bromide/chloride, or cetylpyridinium bromide/chloride.
  • lauryltrimethylammonium bromide di
  • 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 materials described herein. These materials can affect these parameters of the vesicles, especially vesicles formulated from lipids, not only by their physical interaction with the membranes, but also by their ability to modify the viscosity and surface tension of the surface of the gas and gaseous precursor filled vesicle.
  • 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 modifiers, including, for example, carbohydrates and their phosphorylated and sulfonated derivatives; polyethers, preferably with molecular weight ranges between 400 and 100,000; and di- and trihydroxy alkanes and their polymers, preferably with molecular weight ranges between 200 and 50,000; (b) emulsifying and/or solubilizing agents including, for example, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, mono- and di-glycerides, mono- ethanolamine, oleic acid, oleyl alcohol, poloxamer, for example, poloxamer 188, poloxamer 184, and poloxamer 181, polyoxyethylene 50 stearate, polyoxyl 35 castor oil, poly
  • lipid and/or vesicle compositions such as micelles and/or liposomes. Included among these methods are, for example, shaking, drying, gas- installation, spray drying, and the like.
  • Spray drying may be used with a variety of stabilizing materials, including, for example, lipids, such as phospholipids, proteins, such as albumin, synthetic polymers, such as polylactide, and the like.
  • Spray drying may also involve the preparation of an emulsion or suspension of stabilizing agents which contain cores of a volatile blowing agent.
  • the volatile blowing agent desirably expands as the compositions are spray dried. This causes the creation of a central void space within the vesicles.
  • Dried samples of the resulting vesicles may be stored, for example, as a powder under a headspace of a suitable perfluoroether gas.
  • Volatile blowing agents which may be used in the cores in this spray drying technique include, for example, methylene chloride, isobutane, and various of the perfluoroether compounds described herein, including, for example, perfluoromethyl butyl ether.
  • Other suitable methods for preparing vesicle compositions from lipids are described, for example, in Unger et al., U.S. Patent No. 5,469,854, the disclosures of which are inco ⁇ orated herein by reference.
  • the vesicles are preferably prepared from lipids which remain in the gel state.
  • Micelles may be prepared using any one of a variety of conventional micellar preparatory methods which will be apparent to those skilled in the art. These methods typically involve suspension of one or more lipid compounds in an organic solvent, evaporation of the solvent, resuspension in an aqueous medium, sonication and centrifugation. The foregoing methods, as well as others, are discussed, for example, in Canfield et al., Methods in Enzymology, Vol. 189, pp. 418-422 (1990); El-Gorab et al, Biochem. Biophys. Acta, Vol. 306, pp. 58-66 (1973); Colloidal
  • the vesicle composition may comprise liposomes.
  • liposomes may be prepared using any one of a variety of conventional liposomal preparatory techniques which will be apparent to those skilled in the art. These techniques include, for example, solvent dialysis, French press, extrusion (with or without freeze-thaw), reverse phase evaporation, simple freeze-thaw, sonication, chelate dialysis, homogenization, solvent infusion, microemulsification, spontaneous formation, solvent vaporization, solvent dialysis, French pressure cell technique, controlled detergent dialysis, and others, each involving the preparation of the vesicles in various fashions.
  • Suitable freeze-thaw techniques are described, for example, in International Application Serial No. PCT/US89/05040, filed November 8, 1989, the disclosures of which are inco ⁇ orated herein by reference in their entirety. Methods which involve freeze-thaw techniques are preferred in connection with the preparation of liposomes. Preparation of the liposomes may be carried out in a solution, such as an aqueous saline solution, aqueous phosphate buffer solution, or sterile water.
  • a solution such as an aqueous saline solution, aqueous phosphate buffer solution, or sterile water.
  • the liposomes may also be prepared by various processes which involve shaking or vortexing. This may be achieved, for example, by the use of a mechanical shaking device, such as a Wig-L-BugTM (Crescent Dental, Lyons, IL), a Mixomat (Degussa AG, Frankfurt, Germany), a Capmix (Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany), a Silamat Plus (Vivadent, Lechtenstein), or a Vibros (Quayle Dental, England). Conventional microemulsification equipment, such as a MicrofluidizerTM (Microfluidics, Woburn, MA) may also be used.
  • a mechanical shaking device such as a Wig-L-BugTM (Crescent Dental, Lyons, IL), a Mixomat (Degussa AG, Frankfurt, Germany), a Capmix (Espe Fabrik Pharmazeutischer Praeparate GM
  • Spray drying may be also employed to prepare the gas-filled vesicles.
  • the lipids may be pre-mixed in an aqueous environment and then spray dried to produce gas-filled vesicles.
  • the vesicles may be stored under a headspace of a desired gas.
  • Stabilized compositions comprising a gas can be prepared by agitating an aqueous solution containing, if desired, a stabilizing material, in the presence of a gas and/or a gaseous precursor.
  • agitating means any shaking motion of an aqueous solution such that a gas which is proximate to the environment of the aqueous solution may be introduced into the aqueous solution.
  • agitating also means any shaking motion of an aqueous solution which results in the conversion of a gaseous precursor, including the perfluorinated ether compounds described herein, to a gas.
  • This agitation is preferably conducted at a temperature below the gel to liquid crystalline phase transition temperature of the lipid.
  • the agitation is also preferably conducted under reduced pressures within a sealed container. Preferred reduced pressures are from about 5 torr up to about 760 torr, and all combinations and subcombinations of ranges therein.
  • the agitation may be conducted at a pressure of from about 10 torr to about 400 torr, with pressures of from about 15 torr to about 300 torr being even more preferred.
  • the shaking involved in the agitation of the solutions is preferably of sufficient force to result in the formation of a stabilized composition, including lipid compositions, particularly vesicle compositions, and especially vesicle compositions comprising gas filled vesicles.
  • the shaking may be by swirling, such as by vortexing, side-to-side, or up and down motion. Different types of motion may be combined.
  • the shaking may occur by shaking the container containing the aqueous solution which contains the stabilizing compound, or by shaking the aqueous solution within the container without shaking the container itself.
  • 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, as well as any of the shaking devices described hereinbefore, with the Capmix (Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany) being preferred. It has been found that certain modes of shaking or vortexing can be used to make vesicles within a preferred size range. Shaking is preferred, and it is preferred that the shaking be carried out using the Espe Capmix mechanical shaker. In accordance with this preferred method, it is preferred that a reciprocating motion be utilized to generate the lipid compositions, and particularly vesicle compositions.
  • the motion be reciprocating in the form of an arc. It is contemplated that the rate of reciprocation, as well as the arc thereof, is particularly important in connection with the formation of vesicles.
  • the number of reciprocations or full cycle oscillations may be from about 1000 to about 20,000 per minute. More preferably, the number of reciprocations or oscillations may be from about 2500 to about 8000 per minute, with from about 3300 to about 5000 reciprocations or oscillations per minute being even more preferred.
  • the number of oscillations may be dependent upon the mass of the contents being agitated. Generally speaking, a larger mass may require fewer oscillations.
  • Another means for producing shaking includes the action of gas emitted under high velocity or pressure.
  • Vigorous shaking is defined as at least about 60 shaking motions per minute, and is preferred. Vortexing at about 60 to about 300 revolutions per minute is more preferred. Vortexing at about 300 to about 1800 revolutions per minute is even more preferred.
  • the vesicle compositions employed in the present invention are preferably prepared using a shaking technique.
  • the shaking technique involves agitation with a mechanical shaking apparatus, such as an Espe Capmix (Seefeld, Oberay Germany), using, for example, the techniques disclosed in copending U.S. application Serial No.
  • gas filled vesicles can be adjusted, if desired, by a variety of procedures, including, for example, microemulsification, vortexing, extrusion, filtration, sonication, homogenization, repeated freezing and thawing cycles, extrusion under pressure through pores of defined size, and similar methods.
  • Gas filled vesicles prepared in accordance with the methods described herein can range in size from less than about 1 ⁇ m to greater than about 100 ⁇ m.
  • agitation or shaking may provide vesicle compositions which can contain substantially no or minimal residual anhydrous lipid phase in the remainder of the solution.
  • the vesicles may be used as they are formed, without any attempt at further modification of the size thereof.
  • the vesicles preferably have diameters of less than about 30 ⁇ m, and more preferably, less than about 12 ⁇ m.
  • the vesicles may be significantly smaller, for example, less than about 100 nm in diameter.
  • the vesicles may be significantly larger, for example, up to a millimeter in size.
  • the vesicles may be sized to have diameters of from about 2 ⁇ m to about 100 ⁇ m.
  • the gas filled vesicles may be sized by a simple process of extrusion through filters wherein the filter pore sizes control the size distribution of the resulting gas filled vesicles.
  • the filter pore sizes control the size distribution of the resulting gas filled vesicles.
  • the gas filled vesicles can be selected to have a very narrow size distribution around 7 to 9 ⁇ m. After filtration, these gas filled vesicles can remain stable for over 24 hours.
  • the sizing or filtration step may be accomplished by the use, for example, of a filter assembly when the composition is removed from a sterile vial prior to use, or more preferably, the filter assembly may be inco ⁇ orated into a syringe 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 may be carried out by a step of extracting which comprises extruding the vesicles from the barrel through the filter fitted to the syringe between the barrel and the needle, thereby sizing the vesicles before they are administered to a patient.
  • the step of extracting may also comprise drawing the vesicles into the syringe, where the filter may 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 may function to ensure that only vesicles within the desired size range, or of the desired maximum size, are subsequently administered by extrusion from the syringe.
  • the vesicle compositions may be heat sterilized or filter sterilized and extruded through a filter prior to shaking.
  • vesicle compositions comprising a gas may be heat sterilized, and vesicle compositions comprising gaseous precursors may be filter sterilized. Once gas filled vesicles are formed, they may be filtered for sizing as described above. Performing these steps prior to the formation of gas and gaseous precursor filled vesicles provide sterile gas filled vesicles ready for administration to a patient.
  • a mixing vessel such as a vial or syringe may be filled with a filtered stabilized composition, including lipid and/or vesicle, compositions, and the composition may be sterilized within the mixing vessel, for example, by autoclaving.
  • Gas may be instilled into the composition to form gas filled vesicles by shaking the sterile vessel.
  • the sterile vessel is equipped with a filter positioned such that the gas filled vesicles pass through the filter before contacting a patient.
  • the step of extruding the solution of lipid compound through a filter decreases the amount of unhydrated material by breaking up any dried materials and exposing a greater surface area for hydration.
  • the filter has a pore size of about 0.1 to about 5 ⁇ m, more preferably, about 0.1 to about 4 ⁇ m, even more preferably, about 0.1 to about 2 ⁇ m, and still more preferably, about 1 ⁇ m. Unhydrated compound, which is generally undesirable, appears as amo ⁇ hous clumps of non- uniform size.
  • the sterilization step provides a composition that may be readily administered to a patient for diagnostic imaging including, for example, ultrasound or CT.
  • sterilization may be accomplished by heat sterilization, preferably, by autoclaving the solution at a temperature of at least about 100°C, and more preferably, by autoclaving at about 100°C to about 130°C, even more preferably, about 110°C to about 130°C, still more preferably, about 120°C to about 130°C, and even more preferably, about 130°C.
  • heating occurs for at least about 1 minute, more preferably, about 1 to about 30 minutes, even more preferably, about 10 to about 20 minutes, and still more preferably, about 15 minutes.
  • extrusion and heating steps may be reversed, or only one of the two steps can be used.
  • Other modes of sterilization may be used, including, for example, exposure to gamma radiation.
  • gaseous precursors contained in vesicles can be formulated which, upon activation, for example, by exposure to elevated temperature, varying pH, or light, may undergo a phase transition from, for example, a liquid, including a liquid entrapped in a vesicle, to a gas, expanding to create the gas filled vesicles described herein. This technique is described in detail in copending patent applications Serial Nos.
  • the preferred method of activating the gaseous precursor is by exposure to elevated temperature.
  • Activation or transition temperature refer to the boiling point of the gaseous precursor and is the temperature at which the liquid to gaseous phase transition of the gaseous precursor takes place.
  • Useful gaseous precursors are those materials which have boiling points in the range of about -100°C to about 70°C.
  • the activation temperature is particular to each gaseous precursor.
  • An activation temperature of about 37°C, or about human body temperature, is preferred for gaseous precursors in the context of the present invention.
  • a liquid gaseous precursor is activated to become a gas at about 37°C or below.
  • the gaseous precursor may be in liquid or gaseous phase for use in the methods of the present invention.
  • the methods of preparing the gaseous precursor filled vesicles may be carried out below the boiling point of the gaseous precursor such that a liquid is inco ⁇ orated, for example, into a vesicle.
  • the methods may be conducted at the boiling point of the gaseous precursor, such that a gas is inco ⁇ orated, for example, into a vesicle.
  • 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.
  • 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 situ in the targeted tissue or fluid, in 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.
  • the gaseous precursor may be 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.
  • the size of the vesicles upon attaining the gaseous state may be determined.
  • the gaseous precursors may be utilized to create stable gas filled vesicles which are pre-formed prior to use.
  • the gaseous precursor may be added to a container housing a lipid composition at a temperature below the liquid-gaseous phase transition temperature of the respective gaseous precursor. As the temperature is increased, and an emulsion is formed between the gaseous precursor and liquid solution, the gaseous precursor undergoes transition from the liquid to the gaseous state.
  • the gas displaces the air in the head space above the liquid mixture so as to form gas filled vesicles which may entrap the gas of the gaseous precursor, ambient gas (e.g. air), or coentrap gas state gaseous precursor and ambient air.
  • This phase transition can be used for optimal mixing and formation of the contrast agent.
  • the gaseous precursor, perfluorobutane can be entrapped in the lipid vesicles and as the temperature is raised beyond the boiling point of perfluorobutane (4°C), perfluorobutane gas is entrapped in the vesicles.
  • the gaseous precursors may be selected to form gas filled vesicles in vivo or may be designed to produce the gas filled vesicles in situ, during the manufacturing process, on storage, or at some time prior to use.
  • the maximum size of the vesicle may be estimated by using the ideal gas law, once the transition to the gaseous state is effectuated.
  • the gas phase may be assumed to form instantaneously and substantially no gas in the newly formed vesicle has been depleted due to diffusion into the liquid, which is generally aqueous in nature.
  • a mixture of a lipid compound and a gaseous precursor, containing liquid droplets of defined size may be formulated such that upon reaching a specific temperature, for example, the boiling point of the gaseous precursor, the droplets may expand into gas filled vesicles of defined size.
  • the defined size may represent an upper limit to the actual size because the ideal gas law generally cannot account for such factors as gas diffusion into solution, loss of gas to the atmosphere, and the effects of increased pressure.
  • the ideal gas law which can be used for calculating the increase in the volume of the gas bubbles upon transitioning from liquid to gaseous states, is as follows:
  • the amount, for example, in moles, and volume of liquid precursor may be calculated which, when converted to a gas, may expand into a vesicle of known volume.
  • the calculated volume may reflect an upper limit to the size of the gas filled vesicle, assuming instantaneous expansion into a gas filled vesicle and negligible diffusion of the gas over the time of the expansion.
  • r is the radius of the sphere.
  • the amount of liquid gaseous precursor in the droplet may be determined.
  • the following can be applied:
  • V gas 4/3 ⁇ 3 by the ideal gas law
  • V gas nRT/P gas or
  • a representative gaseous precursor may be used to form a vesicle of defined size, for example, 10 ⁇ m diameter.
  • the vesicle may be formed in the bloodstream of a human being, thus the typical temperature would be 37°C or 310 K.
  • 7.54 x 10 "17 moles of gaseous precursor may be required to fill the volume of a 10 ⁇ m diameter vesicle.
  • a mixture for example, an emulsion containing droplets with a radius of 0.0272 ⁇ m or a corresponding diameter of 0.0544 ⁇ m, may be formed to make a gaseous precursor filled vesicle with an upper limit of a 10 ⁇ m vesicle.
  • an emulsion of this particular size could be easily achieved by the use of an appropriately sized filter.
  • the size of the filter may also suffice to remove any possible bacterial contaminants and, hence, can be used as a sterile filtration as well.
  • This embodiment for preparing gas filled vesicles may be applied to all gaseous precursors activated by temperature.
  • depression of the freezing point of the solvent system allows the use of gaseous precursors which may undergo liquid-togas 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 buffered saline exhibits a freezing point depression well below the freezing point of water alone. By increasing the amount of propylene glycol or adding materials such as sodium chloride, the freezing point can be depressed even further.
  • solutes When substances, solid or liquid, herein referred to as solutes, are dissolved in a solvent, such as water based buffers, the freezing point may be lowered by an amount that is dependent upon the composition of the solution.
  • a solvent such as water based buffers
  • x a is the mole fraction of the solvent
  • x b is the mole fraction of the solute
  • ⁇ H fus is the heat of fusion of the solvent
  • T 0 is the normal freezing point of the solvent.
  • the normal freezing point of the solvent can be obtained by solving the equation. If x b is small relative to x a , then the above equation may be rewritten as follows.
  • x b ⁇ H ⁇ /R ⁇ - T 0 /T 0 T] « ⁇ H fus ⁇ T/RT 0 2
  • This equation assumes the change in temperature ⁇ T is small compared to T 2 .
  • This equation can be simplified further by expressing the concentration of the solute in terms of molality, m (moles of solute per thousand grams of solvent).
  • the equation can be rewritten as follows.
  • X b m/[m + 1000/m ⁇ mMa/1000 where Ma is the molecular weight of the solvent.
  • K f M a RT o 2 /1000 ⁇ H fus
  • K f is 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 solutions of gaseous-precursor filled vesicles. Accordingly, the above 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 and/or shaking an aqueous mixture of gaseous precursor and additional materials as desired, including, for example, stabilizing materials, thickening agents and/or dispersing agents.
  • Optional variations of this method include autoclaving before vortexing or shaking; heating an aqueous mixture of gaseous precursor; venting the vessel containing the mixture/suspension; shaking or permitting the gaseous precursor filled vesicle to form spontaneously and cooling down the suspension of gaseous precursor filled vesicles; and extruding an aqueous suspension of gaseous precursor through a filter of about 0.22 ⁇ m.
  • filtering may be performed during in vivo administration of the vesicles such that a filter of about 0.22 ⁇ m is employed; (b) microemulsification, whereby an aqueous mixture of gaseous precursor is emulsified by agitation and heated to form, for example, vesicles prior to administration to a patient;
  • Preferred methods for preparing the temperature activated gaseous precursor filled vesicles comprise shaking an aqueous solution having a lipid compound in the presence of a gaseous precursor at a temperature below the liquid state to gas state phase transition temperature of the gaseous precursor. This is preferably conducted at a temperature below the gel state to liquid crystalline state phase transition temperature of the lipid.
  • the mixture may be then heated to a temperature above the liquid state to gas state phase transition temperature of the gaseous precursor which can cause the precursor to volatilize and expand. Heating may be then discontinued, and the temperature of the mixture may be allowed to drop below the liquid state to gas state phase transition temperature of the gaseous precursor. Shaking of the mixture may take place during the heating step, or subsequently after the mixture is allowed to cool.
  • aqueous-filled liposomes of the prior art are routinely formed at a temperature above the phase transition temperature of 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.
  • the vesicles made according to certain preferred embodiments described herein are gaseous precursor filled, which imparts greater flexibility, since gaseous precursors after gas formation are more compressible and compliant than an aqueous solution.
  • the preparatory methods may involve shaking an aqueous solution comprising a lipid, in the presence of a temperature activatable gaseous precursor.
  • the shaking is of sufficient force such that a foam is formed within a short period of time, such as about 30 minutes, and preferably within about 20 minutes, and more preferably, within about 10 minutes.
  • the shaking may involve microemulsifying, microfluidizing, swirling (such as by vortexing), side-to-side, or up and down motion.
  • sonication may be used in addition to the shaking methods set forth above.
  • different types of motion may be combined.
  • 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 machine.
  • Mechanical shakers that may be used include, for example, the mechanical shakers described hereinbefore, with an Espe Capmix (Seefeld, Oberay Germany) being preferred.
  • Another means for producing shaking includes the action of gaseous precursor emitted under high velocity or pressure.
  • a gas such as air
  • the local ambient atmosphere can include the atmosphere within a sealed container, as well as the external environment.
  • a gas may be injected into or otherwise added to the container having the aqueous lipid solution or into the aqueous lipid solution itself to provide a gas other than air.
  • Gases that are lighter than air are generally added to a sealed container, while gases heavier than air can be added to a sealed or an unsealed container. Accordingly, the present invention includes co-entrapment of air and/or other gases along with gaseous precursors.
  • the gaseous precursor filled vesicles can be used in substantially the same manner as the gas filled vesicles described herein, 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 the gaseous precursors undergo phase transitions from liquid to gaseous states at near the normal body temperature of the host, and are thereby activated, for example, by the in vivo temperature of the host so as to undergo transition to the gaseous phase therein. This can occur where, for example, the host tissue is human tissue having a normal temperature of about 37°C and the gaseous precursors undergo phase transitions from liquid to gaseous states near 37°C.
  • the lipid and/or vesicle compositions may be sterilized by autoclave or sterile filtration if these processes are performed before the instillation step or prior to temperature mediated conversion of the temperature sensitive gaseous precursors within the compositions.
  • one or more anti-bactericidal agents and/or preservatives may be included in the formulation of the compositions, such as sodium benzoate, 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.
  • sodium benzoate quaternary ammonium salts
  • sodium azide sodium azide
  • methyl paraben propyl paraben
  • sorbic acid ascorbylpalmitate
  • butylated hydroxyanisole butylated hydroxytoluene
  • chlorobutanol chlorobutanol
  • dehydroacetic acid ethylened
  • Such sterilization which may also be achieved by other conventional means, such as by irradiation, may be necessary where the stabilized vesicles are used for imaging under invasive circumstances, for example, intravascularly or intraperitonealy.
  • the appropriate means of sterilization will be apparent to the artisan based on the present disclosure.
  • Vesicle compositions which comprise vesicles formulated from proteins
  • albumin vesicles may be prepared by various processes, as will be readily apparent to those skilled in the art, once armed with the present disclosure. Suitable methods include those described, for example, in Feinstein, U.S. Patent Nos. 4,572,203, 4,718,433 and 4,774,958, and Cerny et al., U.S. Patent No. 4,957,656, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties. Included among the methods described in the aforementioned patents for the preparation of protein-based vesicles are methods which involve sonicating a solution pf a protein.
  • the starting material may be an aqueous solution of a heat-denaturable, water-soluble biocompatible protein.
  • the encapsulating protein is preferably heat-sensitive so that it can be partially insolubilized by heating during sonication.
  • Suitable heat- sensitive proteins include, for example, albumin, hemoglobin, collagen, and the like.
  • the protein is a human protein, with human serum albumin (HSA) being more preferred.
  • HSA is available commercially as a sterile 5% aqueous solution, which is suitable for use in the preparation of protein-based vesicles.
  • the concentration of HSA can vary and may range from about 0.1 to about 25%> by weight, and all combinations and subcombinations of ranges therein. It may be preferable, in connection with certain methods for the preparation of protein-based vesicles, to utilize the protein in the form of a dilute aqueous solution.
  • albumin it may be preferred to utilize an aqueous solution containing from about 0.5 to about 7.5%> by weight albumin, with concentrations of less than about 5% by weight being preferred, for example, from about 0.5 to about 3%> by weight.
  • the protein-based vesicles may be prepared using equipment which is commercially available. For example, in connection with a feed preparation operation as disclosed, for example, in Cerny, et al., U.S. Patent No. 4,957,656, stainless steel tanks which are commercially available from Walker Stainless Equipment Co. (New Lisbon, WI), and process filters which are commercially available from Millipore (Bedford, MA), may be utilized.
  • the sonication operation may utilize both a heat exchanger and a flow through sonicating vessel, in series.
  • Heat exchanger equipment of this type may be obtained from ITT Standard (Buffalo, NY).
  • the heat exchanger maintains operating temperature for the sonication process, with temperature controls ranging from about 65 °C to about 80°C, depending on the makeup of the media.
  • the vibration frequency of the sonication equipment may vary over a wide range, for example, from about 5 to about 40 kilohertz (kHz), with a majority of the commercially available sonicators operating at about 10 or 20 kHz.
  • Suitable sonicating equipment include, for example, a Sonics & Materials Vibra-Cell, equipped with a flat-tipped sonicator horn, commercially available from Sonics & Materials, Inc. (Danbury, CT).
  • the power applied to the sonicator horn can be varied over power settings scaled from 1 to 10 by the manufacturer, as with Sonics & Materials Vibra-Cell Model VL1500.
  • An intermediate power setting for example, from 5 to 9, can be used. It is preferred that the vibrational frequency and the power supplied be sufficient to produce cavitation in the liquid being sonicated. Feed flow rates may range from about 50 mL/min to about 1000 mL/min, and all combinations and subcombinations of ranges therein.
  • Residence times in the sonication vessel can range from about 1 second to about 4 minutes, and gaseous fluid addition rates may range from about 10 cubic centimeters (cc) per minute to about 100 cc/min, or 5%> to 25%) of the feed flow rate, and all combinations and subcombinations of ranges therein.
  • the power input to the sonicator horn may be increased, and the process may be operated under mild pressure, for example, about 1 to about 5 psi. Foaming may be easily detected by the cloudy appearance of the solution, and by the foam produced.
  • Suitable methods for the preparation of protein-based vesicles may also involve physically or chemically altering the protein or protein derivative in aqueous solution to denature or fix the material.
  • protein-based vesicles may be prepared from a 5%> aqueous solution of HSA by heating after formation or during formation of the contrast agent via sonication.
  • Chemical alteration may involve chemically denaturing or fixing by binding the protein with a difunctional aldehyde, such as gluteraldehyde.
  • the vesicles may be reacted with 0.25 grams of 50%) aqueous gluteradehyde per gram of protein at pH 4.5 for 6 hours. The unreacted gluteraldehyde may then be washed away from the protein.
  • Vesicle compositions which comprise vesicles formulated from polymers may be prepared by various processes, as will be readily apparent to those skilled in the art, once armed with the present disclosure. Exemplary processes include, for example, interfacial polymerization, phase separation and coacervation, multiorifice centrifugal preparation, and solvent evaporation. Suitable procedures which may be employed or modified in accordance with ihe present disclosure to prepare vesicles from polymers include those procedures disclosed in Garner et al, U.S. Patent No. 4,179,546, Garner, U.S. Patent No. 3,945,956, Cohrs et al., U.S. Patent No.
  • the vesicles may be prepared using a heat expansion process, such as, for example, the process described in Garner et al., U.S. Patent No. 4,179,546, Garner, U.S. Patent No. 3,945,956, Cohrs et al., U.S. Patent No. 4,108,806, British Patent No. 1,044,680, and Japan Kokai Tokkyo Koho 62 286534.
  • the heat expansion process may be carried out by preparing vesicles of an expandable polymer or copolymer which may contain in their void (cavity) a volatile liquid (gaseous precursor).
  • the vesicle is then heated, plasticising the vesicle and converting the volatile liquid into a gas, causing the vesicle to expand to up to about several times its original size.
  • the thermoplastic polymer retains at least some of its expanded shape.
  • Vesicles produced by this process tend to be of particularly low density, and are thus preferred.
  • the foregoing described process is well known in the art, and may be referred to as the heat expansion process for preparing low density vesicles.
  • Polymers useful in the heat expansion process will be readily apparent to those skilled in the art and include thermoplastic polymers or copolymers, including polymers or copolymers of many of the monomers described above.
  • Preferable of the polymers and copolymers described above include the following copolymers: polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate, and polystyrene-polyacrylonitrile.
  • a most preferred copolymer is polyvinylidene-polyacrylonitrile.
  • Volatile liquids useful in the heat expansion process will also be well known to those skilled in the art and include: aliphatic hydrocarbons such as ethane, ethylene, propane, propene, butane, isobutane, neopentane, acetylene, hexane, heptane; chlorofluorocarbons such as CC1 3 F, CC1 2 F 3 , CC1F 3 , CC1F 2 -CC1 2 F 2 , chloroheptafluoro- cyclobutane, and 1,2-dichlorohexafluorocyclobutane; tetraalkyl silanes, such as tetramethyl silane, trimethylethyl silane, trimethylisopropyl silane, and trimethyl n- propyl silane; as well as perfluorocarbons, including the perfluorocarbons described above.
  • aliphatic hydrocarbons such as ethane, ethylene
  • the volatile liquid not be a solvent for the polymer or copolymer being utilized. It is also preferred that the volatile liquid have a boiling point that is below the softening point of the involved polymer or co-polymer. Boiling points of various volatile liquids and softening points of various polymers and copolymers will be readily ascertainable to one skilled in the art, and suitable combinations of polymers or copolymers and volatile liquids will be easily apparent to the skilled artisan. By way of guidance, and as one skilled in the art would recognize, generally as the length of the carbon chain of the volatile liquid increases, the boiling point of that liquid increases also. Also, mildly preheating the vesicles in water in the presence of hydrogen peroxide prior to definitive heating and expansion may pre-soften the vesicle to allow expansion to occur more readily.
  • vinylidene and acrylonitrile may be copolymerized in a medium of isobutane liquid using one or more of the foregoing modified or unmodified literature procedures, such that isobutane becomes entrapped within the vesicles.
  • isobutane gas expands, which in turn expands the vesicles.
  • the expanded polyvinylidene and acrylo-nitrile copolymer vesicles remain substantially fixed in their expanded position.
  • the resulting low density vesicles are extremely stable both dry and suspended in an aqueous media.
  • Isobutane is utilized herein merely as an illustrative liquid, with the understanding that other liquids which undergo liquid/gas transitions at temperatures useful for the synthesis of these vesicles and formation of the very low density vesicles upon heating can be substituted for isobutane.
  • monomers other than vinylidene and acrylonitrile may be employed in preparing the vesicles.
  • the vesicles which are formulated from synthetic polymers and which may be employed in the methods of the present invention are commercially available from Expancel, Nobel Industries (Sundsvall, Sweden), including EXPANCEL 551 DETM microspheres.
  • the EXPANCEL 551 DETM microspheres are composed of a copolymer of vinylidene and acrylonitrile which have encapsulated therein isobutane liquid. Such microspheres are sold as a dry composition and are approximately 50 microns in size.
  • the EXPANCEL 551 DETM microspheres have a specific gravity of only 0.02 to 0.05, which is between one-fiftieth and one- twentieth the density of water.
  • lipid and/or vesicle formulations may be prepared from a mixture of lipid compounds, bioactive agent and gas or gaseous precursor.
  • lipid and/or vesicle compositions may be prepared as described above in which the compositions also comprise bioactive agent.
  • micelles can be prepared in the presence of a bioactive agent.
  • the preparation can involve, for example, bubbling a gas directly into a mixture of lipid compounds and one or more additional materials.
  • the lipid and/or vesicle compositions may be preformed from lipid compounds and gas or gaseous precursor.
  • the bioactive agent may be then added to the lipid and/or vesicle composition prior to use.
  • an aqueous mixture of liposomes and gas may be prepared to which the bioactive agent may be added and which is agitated to provide the liposome formulation.
  • the liposome formulation can be readily isolated since the gas and/or bioactive agent filled liposome vesicles generally float to the top of the aqueous solution. Excess bioactive agent can be recovered from the remaining aqueous solution.
  • various of the compositions containing stabilizing materials which are described herein, including, for example, lipid, polymer, protein and/or vesicle compositions and/or formulations, may be lyophilized for storage, and reconstituted, for example, with an aqueous medium (such as sterile water, phosphate buffered solution, or aqueous saline solution), with the aid of vigorous agitation.
  • an aqueous medium such as sterile water, phosphate buffered solution, or aqueous saline solution
  • Additives which may be useful include sorbitol, mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone and poly(ethylene glycol) (PEG), for example, PEG polymers having a molecular weight of from about 400 to about 10,000, with PEG polymers having molecular weights of about 1000, 3000 (such as PEG3350) and 5000 being preferred.
  • PEG poly(ethylene glycol)
  • Lyophilized preparations generally have the advantage of greater shelf life.
  • compositions of the present invention are useful as contrast agents for diagnostic imaging, including, for example, ultrasound (US) imaging, computed tomography (CT) imaging, including CT angiography (CTA) imaging, magnetic resonance (MR) imaging, magnetic resonance angiography (MRA), nuclear medicine, optical imaging and elastography.
  • diagnostic imaging including, for example, ultrasound (US) imaging, computed tomography (CT) imaging, including CT angiography (CTA) imaging, magnetic resonance (MR) imaging, magnetic resonance angiography (MRA), nuclear medicine, optical imaging and elastography.
  • the present invention provides methods for diagnosing the presence or absence of diseased tissue in a patient.
  • the methods of the present invention involve the administration of a contrast medium in the form, for example, a stabilized compositions, such as a lipid and/or vesicle composition, to a patient.
  • the patient is scanned using diagnostic imaging including, for example ultrasound imaging, to obtain visible images of an internal region of a patient.
  • diagnostic imaging including, for example ultrasound imaging, to obtain visible images of an internal region of a patient.
  • the methods are especially useful in providing images of the heart region, the gastrointestinal region or the lymphatic system, but can also be employed more broadly to image other internal regions of the patient including, for example, the vasculature.
  • the present methods can also be used in connection with the delivery of a bioactive agent to an internal region of a patient.
  • the stabilized compositions such as the lipid and/or vesicle compositions described herein may further comprise a targeting agent to promote targeting of tissues and/or receptors in vivo including, for example, myocardial tissue.
  • the targeting ligands may be associated with the lipid compounds, proteins, polymers and/or vesicles covalently or non-covalently.
  • the targeting ligand may be bound, for example, via a covalent or non-covalent bond, to at least one of the lipids inco ⁇ orated in the compositions.
  • the targeting ligand may be preferably bound covalently or non- covalently to one or more of the materials inco ⁇ orated in the vesicle walls. It is generally preferred that the targeting ligand be bound to the lipid and/or vesicles covalently.
  • the targeting ligand is bound to the cholesterol substantially only non-covalently, and/or that the targeting ligand is bound covalently to a component of the composition, for example, another lipid, such as a phospholipid, other than the cholesterol.
  • the targeting ligands may also be bound to other stabilizing materials, for example, biocompatible polymers, which may be present in the compositions.
  • the targeting ligands which are inco ⁇ orated in the compositions of the present invention are preferably substances which are capable of targeting receptors and/or tissues in vivo.
  • the targeting ligands are desirably capable of targeting heart tissue, including myocardial cells, and membranous tissues, including endothelial and epithelial cells.
  • the targeting ligands are desirably capable of targeting GPIIbllla receptors.
  • preferred targeting ligands for use in targeting tissues and/or receptors are selected from the group consisting of proteins, peptides, saccharides, steroids, steroid analogs, bioactive agents and genetic material, including, for example, antibodies, glycoproteins and lectins, with peptides being preferred.
  • An example of a protein which may be preferred for use as a targeting ligand is Protein A, which is protein that is produced by most strains of Staphylococcus aureus. Protein A is commercially available, for example, from Sigma Chemical Co. (St. Louis, MO). Protein A may then be used for binding a variety of IgG antibodies.
  • peptides which are particularly useful as targeting ligands include natural, modified natural, or synthetic peptides that inco ⁇ orate additional modes of resistance to degradation by vascularly circulating esterases, amidases, or peptidases.
  • One very useful method of stabilization of peptide moieties inco ⁇ orates the use of cyclization techniques.
  • the end-to-end cyclization whereby the carboxy terminus is covalently linked to the amine terminus via an amide bond may be useful to inhibit peptide degradation and increase circulating half-life.
  • a side chain-to-side chain cyclization is also particularly useful in inducing stability.
  • an end-to-side chain cyclization may be a useful modification as well.
  • an L-amino acid for a D-amino acid in a strategic region of the peptide may offer resistance to biological degradation.
  • Suitable targeting ligands, and methods for their preparation will be readily apparent to one skilled in the art, once armed with the disclosure herein. Exemplary targeting agents, methods for their inco ⁇ oration into stabilized compositions, and methods for the use of such targeted compositions, are described, for example, in copending U.S. application Serial No.08/660,032, filed June 6, 1996, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • parenteral administration includes administration by the following routes: intravenous; intramuscular; interstitially; intra-arterially; subcutaneous; intraocular; intrasynovial; transepithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular; rectal; and nasal inhalation via insufflation.
  • Intravenous administration is preferred among the routes of parenteral administration.
  • the useful dosage to be administered and the particular mode of administration will vary depending upon the age, weight and the particular mammal and region thereof to be scanned, and the particular contrast agent employed. Typically, dosage is initiated at lower levels and increased until the desired contrast enhancement is achieved.
  • Various combinations of the lipid compositions may be used to alter properties as desired, including viscosity, osmolarity or palatability.
  • the contrast medium can be used alone, or in combination with diagnostic, therapeutic or other agents. Such other agents include excipients such as flavoring or coloring materials.
  • CT imaging techniques which are employed are conventional and are described, for example, in Computed Body Tomography, Lee, J.K.T., Sagel, S.S., and Stanley, R.J., eds., 1983, Ravens Press, New York, N.Y., especially the first two chapters thereof entitled “Physical Principles and Instrumentation”, Ter-Pogossian, M.M., and “Techniques” , Aronberg, D.J., the disclosures of which are inco ⁇ orated by reference herein in their entirety.
  • ultrasonic energy may be applied to at least a portion of the patient to image the target tissue.
  • a visible image of an internal region of the patient may be then obtained, such that the presence or absence of diseased tissue can be ascertained.
  • ultrasonic imaging techniques including second harmonic imaging, and gated imaging, are well known in the art, and are described, for example, in Uhlendorf, "Physics of Ultrasound Contrast Imaging: Scattering in the Linear Range", IEEE Transactions on Ultrasonics, Ferroelectncs, and Frequency Control, Vol. 14(1), pp.
  • Ultrasound can be used for both diagnostic and therapeutic pu ⁇ oses.
  • diagnostic ultrasound ultrasound waves or a train of pulses of ultrasound may be applied with a transducer.
  • the ultrasound is generally pulsed rather than continuous, although it may be continuous, if desired.
  • diagnostic ultrasound generally involves the application of a pulse of echoes, after which, during a listening period, the ultrasound transducer receives reflected signals.
  • Harmonics, ultraharmonics or subharmonics may be used.
  • the second harmonic mode may be beneficially employed, in which the 2x frequency may be received, where x is the incidental frequency. This may serve to decrease the signal from the background material and enhance the signal from the transducer using the targeted contrast media of the present invention which may be targeted to the desired site.
  • Other harmonics signals such as odd harmonics signals, for example, 3x or 5x, may be similarly received using this method.
  • Subharmonic signals, for example, x/2 and x/3 may also be received and processed so as to form an image.
  • continuous wave ultrasound for example, Power Doppler
  • Power Doppler may be applied.
  • rigid vesicles for example, vesicles formulated from polymethyl methacrylate or cyanomethacrylate
  • the relatively higher energy of the Power Doppler may be made to resonate the vesicles and thereby promote their rupture.
  • This can create acoustic emissions which may be in the subharmonic or ultraharmonic range or, in some cases, in the same frequency as the applied ultrasound. It is contemplated that there may be a spectrum of acoustic signatures released in this process and the transducer so employed may receive the acoustic emissions to detect, for example, the presence of a clot.
  • the process of vesicle rupture may be employed to transfer kinetic energy to the surface, for example of a clot to promote clot lysis.
  • therapeutic thrombolysis may be achieved during a combination of diagnostic and therapeutic ultrasound.
  • Spectral Doppler may also be employed.
  • the levels of energy from diagnostic ultrasound are insufficient to promote the rupture of vesicles and to facilitate release and cellular uptake of the bioactive agents.
  • diagnostic ultrasound may involve the application of one or more pulses of sound. Pauses between pulses permits the reflected sonic signals to be received and analyzed. The limited number of pulses used in diagnostic ultrasound limits the effective energy which is delivered to the tissue that is being studied.
  • Higher energy ultrasound for example, ultrasound which is generated by therapeutic ultrasound equipment, is generally capable of causing rupture of the vesicle species.
  • devices for therapeutic ultrasound employ from about 10 to about 100%> duty cycles, depending on the area of tissue to be treated with the ultrasound. Areas of the body which are generally characterized by larger amounts of muscle mass, for example, backs and thighs, as well as highly vascularized tissues, such as heart tissue, may require a larger duty cycle, for example, up to about 100%).
  • continuous wave ultrasound is used to deliver higher energy levels.
  • continuous wave ultrasound is preferred, although the sound energy may be pulsed also.
  • the sound will generally be pulsed in echo train lengths of from about 8 to about 20 or more pulses at a time.
  • the echo train lengths are about 20 pulses at a time.
  • the frequency of the sound used may vary from about 0.025 to about 100 megahertz (MHz).
  • frequency for therapeutic ultrasound preferably ranges between about 0.75 and about 3 MHz, with from about 1 and about 2 MHz being more preferred.
  • energy levels may vary from about 0.5 Watt (W) per square centimeter (cm 2 ) to about 5.0 W/cm 2 , with energy levels of from about 0.5 to about 2.5 W/cm 2 being preferred.
  • Energy levels for therapeutic ultrasound involving hyperthermia are generally from about 5 W/cm 2 to about 50 W/cm 2 .
  • vesicles having a diameter of less than about 0.5 ⁇ m higher frequencies of sound are generally preferred. This is because smaller vesicles may be capable of absorbing sonic energy more effectively at higher frequencies of sound.
  • very high frequencies are used, for example, greater than about 10 MHz, the sonic energy may penetrate fluids and tissues to a limited depth only.
  • the ultrasonic energy may be suitable for skin and other superficial tissues. However, it is generally necessary for deep structures to focus the ultrasonic energy so that it is preferentially directed within a focal zone.
  • the ultrasonic energy may be applied via interstitial probes, intravascular ultrasound catheters or endoluminal catheters. Such probes or catheters may be used, for example, in the esophagus for the diagnosis and/or treatment of esophageal carcinoma.
  • the compositions described herein can be employed in connection with esophageal carcinoma or in the coronary arteries for the treatment of atherosclerosis, as well as the therapeutic uses described, for example, in U.S. Patent No. 5,149,319, U.S.
  • a therapeutic ultrasound device may be used which employs two frequencies of ultrasound.
  • the first frequency may be x
  • the second frequency may be 2x.
  • the device would be designed such that the focal zones of the first and second frequencies converge to a single focal zone.
  • the focal zone of the device may then be directed to the compositions, for example, vesicle compositions, within the tissue in the region of interest.
  • This ultrasound device may provide second harmonic therapy with simultaneous application of the x and 2x frequencies of ultrasound energy.
  • this second harmonic therapy may provide improved rupturing of vesicles as compared to ultrasound energy involving a single frequency.
  • the preferred frequency range may reside within the fundamental harmonic frequencies of the vesicles. Lower energy may also be used with this device.
  • An ultrasound device which may be employed in connection with the aforementioned second harmonic therapy is described, for example, in Kawabata, K. et al., Ultrasonics Sonochemistry, Vol. 3, pp. 1-5 (1996), the disclosures of which are hereby inco ⁇ orated herein by reference, in their entirety.
  • Other therapeutic ultrasound devices are described, for example, in U.S. Patent Nos. 4,620,546, 4,658,828 and 4,586,512, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties.
  • the perfluoroether compound may exist in a steady state equilibrium between liquid and gaseous (vapor) phases.
  • ultrasound is applied to a vesicle containing a perfluoroether compound containing, for example, 6 carbon atoms, such as perfluoromethyl butyl ether
  • the ultrasound energy may be reflected as well as absorbed by the vesicle.
  • the absorbed energy will generally increase the temperature within the vesicle, and ultrasound shock waves may also lower the pressure within the vesicle.
  • the fraction of the perfluoroether within the vesicle which is in the gaseous state may increase. This increase in the concentration of gas may also provide an increase in backscatter upon further application of ultrasound energy.
  • the concentration of lipid required to form a desired stabilized vesicle level may vary depending upon the type of lipid used, and may be readily determined by routine experimentation.
  • the concentration of 1 ,2- dipalmitoylphosphatidylcholine (DPPC) used to form stabilized vesicles according to the methods of the present invention may be from 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 even more preferably from about 1 mg/mL to about 10 mg/mL of saline solution.
  • DPPC dipalmitoylphosphatidylcholine
  • the concentration of distearoylphosphatidylcholine (DSPC) used in preferred embodiments may be from 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 even more preferably from about 1 mg/mL to about 10 mg/mL of saline solution.
  • the amount of composition which is administered to a patient can vary. Typically, the IV dose may be less than about 10 mL for a 70 Kg patient, with lower doses being preferred.
  • the stabilized compositions described herein, and especially the vesicle compositions are useful as contrast media in diagnostic imaging, and may also be suitable for use in all areas where diagnostic imaging is employed. However, the stabilized vesicles are particularly useful for perfusion imaging.
  • methods of imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient may be carried out by administering a contrast agent to a patient, and then scanning the patient using, for example, ultrasound, computed tomography, and/or magnetic resonance imaging, to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region.
  • region of a patient refers to the whole patient or a particular area or portion of the patient.
  • the contrast agent may be particularly useful in providing images of the gastrointestinal and cardiovascular regions, 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 denotes the region of the patient defined by the heart and the vasculature leading directly to and from the heart.
  • vasculature denotes the blood vessels (including arteries, veins and the like) in the body or in an organ or part of the body.
  • the patient can be any type of mammal, but most preferably is a human.
  • the present invention also provides methods of diagnosing the presence of diseased tissue in a patient.
  • Diseased tissue includes, for example, endothelial tissue which results from vasculature that supports diseased tissue.
  • endothelial tissue which results from vasculature that supports diseased tissue.
  • administration of the compositions described herein may be carried out in various fashions, such as intravascularly, orally, rectally, and the like, using a variety of dosage forms.
  • administration of the contrast medium is preferably carried out intravascularly.
  • administration of the contrast medium 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 mammal and region thereof to be scanned, and the particular contrast medium to be employed. Typically, dosage may be initiated at lower levels and increased until the desired contrast enhancement is achieved.
  • lipid and/or vesicle compositions 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).
  • the present invention may be performed with ultrasound or computed tomography according to conventional methods known by skilled artisans.
  • 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.
  • ultrasound is relatively inexpensive and can be conducted as a portable examination. 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 interfaces from tissues and fluids.
  • the ultrasound sound waves are partially or wholly reflected or absorbed.
  • sound waves are reflected by an interface 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.
  • Computed tomography imaging principles and techniques which are employed are conventional and are described, for example, in Computed Body Tomography, Lee, J.K.T., Sagel, S.S., and Stanley, R.J., eds., Ch. 1, pp. 1-7 (Raven Press, NY 1983).
  • the contrast agent can be used alone, or in combination with other diagnostic, therapeutic or other agents.
  • Such other agents include excipients such as flavoring 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, Magnetic Resonance Imaging: Principles and Applications, (William and Wilkins, Baltimore 1986).
  • Contemplated MRI techniques include, but are not limited to, nuclear magnetic resonance (NMR) and electronic spin resonance (ESR).
  • NMR nuclear magnetic resonance
  • ESR electronic spin resonance
  • the preferred imaging modality is NMR.
  • the lipid and/or vesicle compositions may be administered by syringe, that is, by intravenous (IV) injection.
  • IV intravenous
  • the location on the body of the patient at which the stabilized compositions, such as lipid and/or vesicle compositions, are injected may vary and depends upon a variety of factors, including, for example, the particular lipid and/or vesicle composition employed, the contemplated application, such as diagnostic or therapeutic application, and the particular region of interest.
  • the lipid and/or vesicle compositions may be injected intravenously, for example, in the arm of a patient.
  • the IV administration of the contrast agents described herein including, for example, the vesicle compositions may involve administration via syringe. This may be achieved, for example, by an appropriate medical technician who handles the syringe or syringes manually. Alternatively, administration by syringe may be achieved mechanically, for example, via a mechanical injector, such as a mechanical injector which operates using pneumatic or hydraulic pressure. Suitable mechanical injectors which may be used in the methods of the present invention include a Syringe Pump Model 351, commercially available from Sage Instruments (a division of Orion Research Inc., Boston, MA), a MedRadTM power injector, commercially available from Medrad, Inc.
  • compositions including lipid and/or vesicle compositions which are administered via injection
  • administration by injection generally involves injection of the compositions into a blood vessel.
  • the blood flow in many blood vessels, and especially smaller blood vessels may be limited. Due to this limited blood flow, the injected lipid and/or vesicle compositions may pool or accumulate at or near the site of injection.
  • a flush may be administered.
  • the flush may act, generally by mechanical action, to "push” or “wash” the injected compositions into the bloodstream.
  • a flush may be administered after injection of the lipid and/or vesicle composition to facilitate its movement through the circulatory system and delivery to the region of interest, for example, the heart region.
  • embodiments of the present invention may involve perfluoroether compounds which are gaseous precursors, that is, perfluoroether compounds which are liquids at conditions of ambient pressure and temperature.
  • perfluoroether compounds are preferably included in compositions in the form of emulsions and/or suspensions.
  • the perfluoromethyl butyl ether will generally exist in the liquid state. Pulling back on the syringe plunger, while maintaining the seal in the syringe, may produce a decrease in the pressure within the syringe. This may advantageously cause the perfluoromethyl butyl ether to expand, which increases the propensity of the perfluoromethyl butyl ether to vaporize to a gas within the syringe.
  • the syringe plunger may be rapidly released to promote rupture of the vesicles into smaller sized vesicles during the shock wave within the syringe.
  • the syringe may be fitted with a filter to promote the break up of any large vesicles prior to injection.
  • the echogenicity of vesicles, and especially, gas filled vesicles, and the ability to rupture vesicles at, for example, the peak resonant frequency using ultrasound permits the controlled delivery of bioactive agents to an internal region of a patient.
  • the vesicles may be monitored subsequent to their administration to a patient to determine the rate ajt which the vesicles arrive, for example, to a desired region.
  • the vesicles may be ruptured using ultrasound to release the bioactive agent in the region.
  • perfluoroethers of the present invention may also be employed in a variety of other ways, in addition to those ways described hereinbefore.
  • Such additional applications will be apparent to one of ordinary skill in the art, once placed in possession of the present disclosure, and include, for example, their use as blood substitutes. See, e.g., Long, Jr., U.S. Patent Nos. 4,865,836, 4,987,154 and 4,927,623, the disclosures of which are hereby inco ⁇ orated herein by reference, in their entireties.
  • Such additional uses are intended to be within the scope of the present invention.
  • Example 1 to 9 are actual examples, while Example 10 is a prophetic example.
  • Example 1 This example is directed to the preparation of perfluoroether gas filled vesicles within the scope of the present invention.
  • l,2-Dipalmitoyl-sn-glycero-3-phosphocholine 50 mg; MW 734.05; powder, Lot No. 160pc-183 (Avanti-Polar Lipids, Alabaster, AL) was weighed and hydrated with saline solution (5 mL; 0.9%> NaCl) or phosphate buffered saline (5 mL; 0.8%> sodium chloride, 0.02%> potassium chloride, 0.115%) dibasic sodium phosphate and 0.02%) monobasic potassium phosphate, pH adjusted to 7.4) in a centrifuge tube.
  • saline solution 5 mL; 0.9%> NaCl
  • phosphate buffered saline 5 mL; 0.8%> sodium chloride, 0.02%> potassium chloride, 0.115%) dibasic sodium phosphate and 0.02%
  • the resulting perfluoromethyl butyl ether (PFMBE) filled vesicles were sized by optical microscopy. It was observed that the vesicles ranged in size from about 0.5 microns to about 50 to about 60 microns. The average size of the vesicles ranged from about 8 to about 12 microns. The PFMBE filled vesicles were then filtered through a 10 or 12 micron
  • the survival rate of the PFMBE filled vesicles which corresponds to the percentage of vesicles which remained after the extrusion process described above, was about 83 to about 92%>.
  • the volume of foam was about 12 mL and the volume of aqueous solution was about 4 mL.
  • the volume of foam was about 10 to 11 mL and the volume of aqueous solution was about 4 mL.
  • This example includes a description of experiments which were conducted to determine the effect of varying the temperature and the particular gaseous perfluorinated compound on the production of gas-filled vesicles.
  • samples of perfluoromethyl butyl ether (PFMBE) and perfiuoropentane (PFP), with and without a stabilizing compound were mixed on a Wig-L-Bug® shaking device at about 3000 to 3400 ⁇ m or a Capmix® shaking device at about 4500 to 5000 ⁇ m after incubation at temperatures of -20 ° C, 4 ° C or room temperature. Heating produced by the Capmix® device increased the sample temperature by about 20 to about 25 ° C.
  • DPPC dipalmitoylphosphatidylcholine
  • DOPC dioleoylphosphatidylcholine
  • nwm refers to number weighted mean o
  • the nwm values for PFMBE filled vesicles prepared at room temperature range from 2.2 (Sample 2E) to 2.95 (Sample 2F), as compared to the nwm values for PFMBE filled vesicles prepared at -20 ° C, which range from 2.9 (Sample 2H) to 5.3 (Sample 21).
  • the nwm values for PFP filled vesicles prepared at room temperature range from 3.88 (Sample 2N) to 4.65 (Sample 2V), as compared to the nwm values for PFP filled vesicles prepared at -20 ° C, which range from 4.5 (Sample 2Q) to 7.72 (Sample 2R).
  • samples prepared at room temperature generally possess a more uniform distribution of vesicles.
  • samples prepared at room temperature generally comprise a greater percentage of vesicles sized below lO ⁇ . Inspection of the data in Table 2 also indicates that, if the shaking process induces a phase-transition in the perfluoroether compound, as may occur with an ESPE Capmix® shaking device, vesicles may be obtained with a more uniform size distribution, independent of the initial incubation sample temperature.
  • Example 3A a composition of the type prepared in Example 1 above, referred to herein as Example 3A, and a composition containing vesicles prepared from DPPC, DPPA, DPPE- PEG5000 and perfluoropropane, referred to herein as Example 3B.
  • the acoustic analysis involved a study of the duration of vesicle activity in an ultrasonic field. Also studied were the effects of increasing pressure, followed by a rapid decrease in pressure. These measurements provided information respecting the stability of the vesicles upon exposure to conditions of associated with ultrasound in a diagnostic setting.
  • the results of these studies are depicted graphically in Figures 1 and 2 which show comparative histograms of time-dependent attenuation of vesicles subjected to varying frequencies of ultrasound (Figure 1) and pressure-dependent attenuation at varying frequencies (Figure 2). Inspection of the Figures 1 and 2 reveals that vesicles containing PFMBE (Example 3A) generally demonstrated improved attenuation effects (db) over time, as well as improved stability to increasing pressure, followed by a rapid decrease in pressure.
  • An anesthetized mongrel dog was imaged using an Acoustic Imaging 5200S ultrasound machine (Phoenix, AZ). Physiological parameters monitored included systemic arterial pressure, pulmonary arterial pressure, heart rate and oxygen saturation. Vesicle compositions as in Examples 1 and 3B were prepared by shaking at room temperature on an ESPE Capmix®. A dose of 0.005 cc/kg of each composition was administered via the cephalic vein. Cardiac imaging was carried out using a 7.5Mhz tightly curved linear array (TCLA) transducer. After injection of the Example 3B composition, a distinct attenuation artifact was observed followed by marked ventricular opacification and visible myocardial opacification.
  • TCLA tightly curved linear array
  • Example 3B composition began to clear after 2-3 minutes of continuous imaging.
  • the Example 1 composition showed reduced attenuation artfacts.
  • the ventricular contrast obtained with the Example 1 composition was brilliant and long-lasting, and myocardial opacification was easily visualized.
  • Ventricular contrast with the Example 1 composition remained visible after 3-4 minutes of continuous imaging. No changes in physiological parameters were observed with either contrast agent.
  • Example 5 This example includes an analysis of the effect of varying temperature on the stability, as measured by the attenuation reflectivity, of PFMBE filled vesicles.
  • Figure 3 shows the attenuation reflectivity from 0 to 5 minutes, at 1 minute intervals, of a composition of PFBME filled vesicles when formed and shaken at an initial sample temperature of about -20°C.
  • This shaking protocol involved warming the compositions to beyond the phase transition temperature of PFMBE (35.4 ° C), so that at time 0, the temperature was relatively warmer than as in subsequent minutes 1 to 5. This experiment was repeated, except that the vesicles were formed at 4°C, but shaken at ambient temperatures.
  • This example is directed to the preparation of albumin coated vesicles filled with PFMBE gas.
  • Vesicles were prepared by dissolving 5%> bovine serum albumin (Sigma,
  • This example is directed to the preparation of PFMBE gas vesicles stabilized with a surfactant material (Pluronic).
  • Vesicles were prepared by dissolving 1% Pluronic F68 (Spectrum, Gardena, CA) in 8:1 :1 saline/propylene glycol/glycerol. This solution was then aliquotted into two 2 mL serum vials. One vial sample was shaken immediately while the headspace gas for the other vial was removed and PFMBE was added. Both samples were sized using an Accusizer 770. The sample with a headspace of air had no visible foam while the sample containing PFMBE contained a large quantity of visible foam. Neither sample was sufficiently stable to size.
  • This example is directed to a comparison of the toxicity of PFMBE and perfiuoropentane (PFP) in mice.
  • Lipid vesicles were prepared as in Example 1 using either PFMBE or PFP. The samples were cooled to -20 ° C before the addition of the perfluorinated material. The headspace was evacuated briefly with a vacuum after which the samples were warmed to room temperature with shaking for 60 seconds on an ESPE Capmix ® .
  • mice Groups of outbred mice were immobilized and the vesicle compositions (50 ⁇ L) were injected into the mice. No effects were seen in the mice receiving the PFMBE vesicles at a dosage of approximately 2 mL/kg. The group receiving the PFP filled vesicles experienced 2 immediate mortalities, and the third animal was sluggish and in respiratory distress.
  • a mixture of 82 mole percent DOPC, 10 mole percent DOPA and 8 mole percent DOPE-PEG 5000 was prepared and lyophilized.
  • the phase transition of liquid crystalline to gel state of these lipids is about 4°C. Accordingly, the shaking/gas instillation step described below occurred while the lipids were in the liquid crystalline state.
  • the lipid mix was hydrated in a solution of 8:1 :1 saline/propylene glycol/glycerol at a lipid concentration of 1 mg/mL in a 2 mL vial.
  • PFMBE (20 ⁇ L) was added to the vial, the contents were chilled to 4°C and the headspace of air was evacuated under vacuum.
  • the vial was shaken with a ESPE Capmix® at 4500 ⁇ m for 60 seconds.
  • the temperature of the solution after shaking was about 26°C, which is well above the phase transition temperature of the lipid.
  • the concentration of vesicles was 6.73 X 10 7 vesicles/mL as compared to 1.90 X 10 9 vesicles/mL when gel state lipids were used. (See Sample 2T in data tabulated above.)
  • the vesicle composition was examined for acoustic activity at 37°C, and was found to be substantially inactive as compared to the vesicle compositions which were prepared with gel state lipids as shown in Figures 4 and 6.
  • This example is directed to the preparation of a vesicle composition containing a perfluoroether and a bioactive agent.
  • Polylactic acid (4 g; m.w. 15,000-25,000) and dichloromethane (20 mL) are combined in a 30 mL serum bottle.
  • PFMBE (20-50 ⁇ L) is also added.
  • the vial is attached to a Heavy Duty #6 Wig-L-Bug and shaken for 2 minutes. After the sample is cooled, it is added to a solution of 0.5%) polyvinyl alcohol m.w. 86,000 to 145,000 (Aldrich).
  • This solution is agitated at 7500 ⁇ m using a Silverson mixer (Waterside, England). The sample is stirred for an additional 2 minutes and then transferred to a hotplate at 30°C and mixed for 2 hours with a Talboy stirrer (South Montrose, PA). Particles are then observed using a Nikon Diaphot Microscope (Tokyo, Japan) and sized on the Accusizer 770.

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Abstract

Compositions and methods for providing an image of an internal region of a patient. Embodiments of the invention involve vesicle compositions comprising, in an aqueous carrier, a perfluorinated ether compound and vesicles comprising one or more stabilizing compounds. The compositions may be administered to a patient, and the patient may be scanned using diagnostic imaging, such as ultrasound, to obtain a visible image of an internal region.

Description

PERFLUORINATED-ETHER COMPOSITIONS AS DIAGNOSTIC CONTRAST AGENTS
Cross-Reference to Related Application This application claims the benefit of U.S. Provisional Application Serial
No. 60/031,903, filed November 25, 1996, the disclosures of which are hereby incorporated herein by reference, in their entirety.
Field of the Invention
The present invention relates to novel compositions of perfluorinated ethers. More particularly, the present invention relates to novel compositions of perfluorinated ethers and their use as contrast media for diagnostic imaging and in various other applications.
Background of the Invention
Ultrasound is a valuable diagnostic imaging technique for studying various areas of the body, for example, the vasculature, including tissue micro vasculature.
Ultrasound provides certain advantages over other diagnostic techniques. For example, diagnostic techniques involving nuclear medicine and X-rays generally results in exposure of the patient to ionizing electron radiation. Such radiation can cause damage to subcellular material, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins. Ultrasound does not involve such potentially damaging radiation. In addition, ultrasound is relatively inexpensive relative to other diagnostic techniques, including computed tomography (CT) and magnetic resonance imaging (MRI), which require elaborate and expensive equipment.
Ultrasound involves the exposure of a patient to sound waves. Generally, the sound waves dissipate due to absoφtion by body tissue, penetrate through the tissue or reflect off of the tissue. The reflection of sound waves off of tissue, generally referred to as backscatter or reflectivity, forms the basis for developing an ultrasound image. In this connection, sound waves reflect differentially from different body tissues. This differential reflection is due to various factors, including the constituents and the density of the particular tissue being observed. Ultrasound involves the detection of the differentially reflected waves, generally with a transducer that can detect sound waves having a frequency of 1 megahertz (MHZ) to 10 MHZ. The detected waves can be integrated into an image which is quantitated and the quantitated waves converted into an image of the tissue being studied. Ultrasound imaging techniques generally also involve the use of contrast agents. Contrast agents are used to improve the quality and usefulness of images which are obtained via ultrasound. Exemplary contrast agents include, for example, suspensions of solid particles, emulsified liquid droplets, and gas-filled bubbles. See, e.g., Hilmann et al., U.S. Patent No. 4,466,442, and published International Patent Applications WO 92/17212 and WO 92/21382.
The quality of images produced from ultrasound has improved significantly. Nevertheless, further improvement is needed, particularly with respect to images involving vasculature in tissues that are perfused with a vascular blood supply. Accordingly, there is a need for improved ultrasound techniques, including improved contrast agents which are capable of providing medically useful images, especially of the vasculature and vascular-related organs.
The reflection of sound from a liquid-gas interface is extremely efficient. Accordingly, bubbles, including gas-filled bubbles, are useful as contrast agents. The term "bubbles", as used herein, refers to vesicles which are generally characterized by the presence of one or more membranes or walls surrounding an internal void that is filled with a gas or precursor thereto. Exemplary bubbles include, for example, vesicles which are surrounded by monolayers and/or bilayers to form, for example, unilamellar, oligolamellar and/or multilamellar vesicles, such as liposomes, micelles and the like. As discussed more fully hereinafter, the effectiveness of bubbles as contrast agents depends upon various factors, including, for example, the size and/or elasticity of the bubble. With respect to the effect of bubble size, the following discussion is provided. As known to the skilled artisan, the signal which is reflected off of a bubble is a function of the radius (r6) of the bubble (Rayleigh Scatterer). Thus, a bubble having a diameter of 4 micrometer (μm) possesses about 64 times the scattering ability of a bubble having a diameter of 2 μm. Thus, generally speaking, the larger the bubble, the greater the reflected signal. However, bubble size is limited by the diameter of capillaries through which the bubbles must pass. Generally, contrast agents which comprise bubbles having a diameter of greater than 10 μm can be dangerous since microvessels may be occluded. Accordingly, it is preferred that greater than about 90% of the bubbles in a contrast agent have a diameter of less than about 10 μm, with greater than about 95% being more preferred, and greater than about 98% being even more preferred. Mean bubble diameter is important also, and should be greater than 1 μm, with greater than 2 μm being preferred. The volume weighted mean diameter of the bubbles should be about 7 to 10 micrometer. As noted above, the elasticity of bubbles is also important. This is because highly elastic bubbles can deform, as necessary, to "squeeze" through capillaries. This decreases the likelihood of occlusion. The effectiveness of a contrast agent which comprises bubbles is also dependent on the bubble concentration. Generally, the higher the bubble concentration, the greater the reflectivity of the contrast agent.
Another important characteristic which is related to the effectiveness of bubbles as contrast agents is bubble stability. As used herein, particularly with reference to gas-filled bubbles, "bubble stability" refers to the ability of bubbles to retain gas entrapped therein after exposure to a pressure greater than atmospheric pressure. To be effective as contrast agents, bubbles generally need to retain greater than 50% of entrapped gas after exposure to pressure of 300 millimeters (mm) of mercury (Hg) for about one minute. Particularly effective bubbles retain 75% of the entrapped gas after being exposed for one minute to a pressure of 300 mm Hg, with an entrapped gas content of 90%) providing especially effective contrast agents. It is also highly desirable that, after release of the pressure, the bubbles return to their original size. This is referred to generally as "bubble resilience."
Bubbles which lack desirable stability provide poor contrast agents. If, for example, bubbles release the gas entrapped therein in vivo, reflectivity is diminished. Similarly, the size of bubbles which possess poor resilience will be decreased in vivo, also resulting in diminished reflectivity.
The stability of bubbles disclosed in the prior art has generally been inadequate for use as contrast agents. For example, the prior art discloses bubbles, including gas-filled liposomes, which comprise lipoidal walls or membranes. See, e.g., Ryan et al., U.S. Patent Nos. 4,900,540 and 4,544,545; Tickner et al., U.S. Patent No. 4,276,885; Klaveness et al., WO 93/13809 and Schneider et al., EPO 0 554 213 and WO 91/15244. The stability of the bubbles disclosed in the aforementioned references is poor in that as the solutions in which the bubbles are suspended become diluted, for example, in vivo, the walls or membranes of the bubbles are thinned. This results in a greater likelihood of rupture.
Various studies have been conducted in an attempt to improve bubble stability. Such studies have included, for example, the preparation of bubbles in which the membranes or walls thereof comprise materials that are apparently strengthened via crosslinking. See, e.g., Klaveness et al., WO 92/17212, in which there are disclosed bubbles which comprise proteins crosslinked with biodegradable crosslinking agents. Alternatively, bubble membranes can comprise compounds which are not proteins but which are crosslinked also with biocompatible compounds. See, e.g., Klaveness et al., WO 92/17436, WO 93/17718 and WO 92/21382. Such crosslinking may, in certain circumstances, impart rigidity to the membranes or walls of the bubbles. This results in bubbles having reduced elasticity and, therefore, a decreased ability to deform and pass through capillaries. Thus, there may be a greater likelihood of occlusion of vessels with prior art contrast agents that are stabilized via crosslinking.
Since the early 1970's, bubbles, especially liposomes, have also been investigated repeatedly and intensively for their use as a delivery means for a wide variety of active agents including, for example, cosmetics, drugs, vitamins and biopolymers. Liposomes may have certain advantageous properties, such as non-toxicity, biodegradability and non-immunogenicity. Nevertheless, prior art liposomes generally suffer from drawbacks in that they may be relatively unstable in vivo and/or may be cleared quickly from the body by the reticuloendothelial system. Accordingly, little success has been achieved to date in connection with the use of liposomes as delivery agents of materials, such as active agents. In addition to ultrasound, computed tomography (CT) is a commonly used diagnostic technique for the diagnosis of various diseases and maladies of the body, including abdominal and pelvic diseases. In 1978 alone, about 7.8 million body CT scans were performed. CT imaging involves measuring the radiodensity of matter. Radiodensity is typically expressed in Hounsefield Units (HU). Hounsefield Units are a measure of the relative absoφtion of computed tomography X-rays by matter and is directly proportional to electron density. Water has been arbitrarily assigned a value of 0 HU, air a value of -1000 HU, and dense cortical bone a value of 1000 HU.
Various tissues in the body possess similar densities. This has caused difficulty in the generation of visual images by CT of tissues that possess similar densities and which are proximate each other. For example, it is difficult to generate separate CT images of the gastrointestinal (GI) tract and adjacent structures, including, for example, the blood vessels and the lymph nodes. Accordingly, contrast agents have been developed in an attempt to change the relative densities of different tissues, and thereby improve the diagnostic efficacy of CT. CT contrast agents are used in a majority of the CT imaging scans. For example, of the 7.8 million body CT scans performed in 1978, 6.6 million involved the use of intravenous contrast agents for the enhancement of vascular and/or visceral images.
Traditional CT contrast agents for imaging the gastrointestinal (GI) tract are generally based on radiodense, non-absorbable, heavy metal materials. Such materials assist in imaging the bowel by absorbing X-ray transmissions which increases the radiodensity of the bowel lumen. Common among such contrast agents are barium and iodinated compounds, including, for example, barium sulfate. Barium sulfate and iodinated compounds have been used for imaging the GI tract for the past 60 years and are widely used today for enhancing CT images of the upper and lower GI tract. They generally increase electron density in certain regions of the body, and are therefore classified as a "positive contrast agents." Despite their widespread use, barium and iodinated compounds suffer from various drawbacks. For example, they are generally incompatible with other and/or newer imaging techniques, including vascular imaging techniques. This incompatibility is observed, for example, in CT angiography (CTA). In CTA, iodinated contrast agents are injected intravenously and images are obtained during the bolus phase of contrast. Highly detailed images of the vasculature are generally obtained using CTA by reformatting the axial images to yield a composite picture of the vessels. During this reformatting, the picture of the vasculature is optimized based on the measured density in the vessels being visualized. To perform this imaging, various baseline image subtractions are performed. However, the vascular image optimization becomes distorted and obscured in the presence in the bowel of radiodense contrast agents, for example, iodinated compounds. Consequently, difficulty is encountered in performing CTA and CT in the GI tract concurrently if any positive contrast agents, such as barium and iodinated compounds, are present. Moreover, the viability of currently available CT contrast agents is generally extremely sensitive to concentration. If the concentration is too low, little contrast is observed. If the concentration is too high, beam hardening artifacts result and are observed as streaks in the resulting CT images. In addition, difficulty is typically encountered in visualizing the bowel mucosa with the currently available contrast agents. Accordingly, new and/or better stabilized contrast agents, as well as stabilized delivery means for active agents and methods for providing same, are needed. The present invention is directed to these, as well as other important ends.
Summary of the Invention
The present invention, is directed, at least in part, to contrast agents which may be useful for diagnostic imaging. Specifically, in one embodiment, there is provided a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
R]-X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The contrast agents preferably also comprise a lipid, protein or polymer. Another embodiment of the invention also relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
R|-X-R
'(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The contrast agents preferably also comprise a stabilizing compound.
Yet another embodiment of the invention relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
R,-X-R2 (I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The contrast agents preferably also comprise a stabilizing compound which may be substantially non- crosslinked or substantially crosslinked, with substantially non-crosslinked stabilizing compounds being preferred.
Still another embodiment of the invention relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula R,-X-R2
(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The contrast agents preferably also comprise a lipid which may be substantially non-crosslinked or substantially crosslinked, with substantially non-crosslinked lipids being preferred. Another embodiment of the invention relates to a contrast agent for diagnostic imaging comprising, in an aqueous carrier, a perfluoroether compound, preferably a gaseous perfluoroether compound, selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers contain no more than one oxygen atom in the heterocyclyl ring system. The contrast agents preferably also comprise a stabilizing compound selected from the group consisting of lipids, proteins and polymers. Yet another embodiment of the invention relates to a composition comprising, in an aqueous carrier, a compound, preferably a gaseous compound, of the formula
R|-X-R2
(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The compositions preferably also comprise a lipid, protein or polymer.
Still another embodiment of the invention relates to a composition comprising, in an aqueous carrier, a perfluoroether compound, preferably a gaseous perfluoroether compound, selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers comprise no more than one oxygen atom in the heterocyclic ring system. The compositions preferably also comprise a stabilizing compound selected from the group consisting of lipids, proteins and polymers.
Another embodiment of the invention relates to a method for preparing a composition which comprises a perfluoroether compound, preferably a gaseous perfluoroether compound, wherein the method comprises:
(a) providing a vessel containing a perfluoroether compound; and (b) agitating said vessel to provide the composition. The compositions prepared according to this method are preferably stabilized compositions, preferably by further including in the vessel in (a) a stabilizing compound.
In yet another embodiment of the invention, there are provided methods for providing an image of a patient. Exemplary of the methods of this embodiment is a method for providing an image of an internal region of a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a compound of the formula
RrX-R2 (I) wherein:
X is O or S;
Ri is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of the region. The contrast agents employed in the methods preferably also comprise a lipid, protein or polymer.
In still another embodiment of the invention, there are provided methods for diagnosing the presence of diseased tissue in a patient using the contrast agents of the invention. Exemplary of the methods of this embodiment are methods for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a compound of the formula
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of any diseased tissue in the patient. The contrast agents employed in the methods preferably also comprise a lipid, protein or polymer.
Another embodiment of the invention relates to a method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a formulation which comprises, in combination with a bioactive agent, a composition of a compound of the formula
(I) wherein:
X is O or S; R{ is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. The compositions employed in the methods preferably also comprise a lipid, protein or polymer.
These and other aspects of the invention will become more apparent from the present specification and claims. Brief Description of the Drawings
For the puφose of illustrating embodiments of the invention, there is shown in the drawings forms which are presently preferred. It should be understood, however, that this invention is not limited to the precise arrangements and instrumentalities shown. Figure 1 is a graphical representation of the acoustic activity, viewed at one minute intervals, of vesicle compositions containing (i) perfluoromethyl butyl ether and (ii) perfluoropropane.
Figure 2 is a graphical representation of the acoustic activity, upon exposure to increasing and decreasing pressure, of vesicle compositions containing (i) perfluoromethyl butyl ether and (ii) perfluoropropane.
Figure 3 is a graphical representation of the acoustic activity, viewed at one minute intervals, of a vesicle composition containing perfluoromethyl butyl ether formed and shaken at an initial temperature of -20°C.
Figure 4 is a graphical representation of the acoustic activity, upon exposure to increasing pressure, of a vesicle composition containing perfluoromethyl butyl ether formed at an initial temperature of 4°C and shaken at room temperature.
Figure 5 is a graphical representation of the acoustic activity, upon exposure to increasing and decreasing pressure, of a vesicle composition containing perfluoromethyl butyl ether shaken at room temperature. Figure 6 is a graphical representation of the acoustic activity, upon exposure to increasing pressure, of a vesicle composition containing perfluoromethyl butyl ether shaken at -20°C.
Detailed Description of the Invention
As employed above and throughout the disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings.
"Lipid" refers to a naturally-occurring, synthetic or semisynthetic (also referred to as "modified natural") compound which is generally amphipathic. The lipids typically contain a hydrophilic component and a hydrophobic component. Exemplary lipids include, for example, fatty acids, neutral fats, phosphatides, oils, glycolipids, surface-active agents (surfactants), aliphatic alcohols, waxes, teφenes and steroids. "Protein", as used herein, refers to molecules comprising, and preferably consisting essentially of, α-amino acids in peptide linkages. Included within the term "protein" are globular proteins, such as albumins, globulins and histones, and fibrous proteins such as collagens, elastins and keratins. Also included are "compound proteins", wherein a protein molecule is united with a nonprotein molecule, such as nucleproteins, mucoproteins, lipoproteins, and metalloproteins. The protein may be naturally-occurring, synthetic or semisynthetic.
"Polymer" or "polymeric" refers to molecules formed from the chemical union of two or more repeating units. Accordingly, included within the term "polymer" may be, for example, dimers, trimers and oligomers. The polymer may be synthetic, naturally-occurring or semisynthetic. In preferred form, the term "polymer" refers to molecules which comprise 10 or more repeating units.
"Stabilizing compound" refers to any material which is capable of improving the stability of compositions containing perfluoroether compounds as described herein including, for example, emulsions, suspensions, dipersions and vesicle compositions. The improved stability involves, for example, the maintenance of a relatively balanced condition, and may be exemplified, for example, by increased resistance against destruction, decomposition, degradation and the like. In the case of preferred embodiments involving vesicles, especially gas filled vesicles, the stabilizing compounds may serve to improve the stability of the vesicles, for example, by minimizing or substantially (including completely) preventing the escape of gas entrapped within vesicles which may occur, for example, from rupture and/or coalescence of vesicles. The term "substantially", as used in reference to the prevention of the escape of entrapped gas, means that greater than about 50%> of the gas is maintained entrapped. Preferably, greater than about 60% of the gas is maintained entrapped, with greater than about 70% being more preferred. Even more preferably, greater than about 80% of the gas is maintained entrapped, with greater than about 90%) being still more preferred. In particularly preferred embodiments, greater than about 95% of the gas is maintained entrapped. If desired, the gas may be completely maintained entrapped (i.e., about 100%) of the gas is maintained entrapped). The stabilizing compounds may comprise discrete, individual compounds (monomers), or may comprise polymers. Broadly speaking, the stabilizing compounds may comprise, for example, surfactants, film-forming materials, membranes and/or membrane forming materials. Exemplary stabilizing compounds which may be employed in the methods and compositions of the present invention include lipids, proteins and polymers. In certain embodiments, the stabilizing compounds may be substantially (including completely) crosslinked. The terms "crosslink", crosslinked" and "crosslinking", as used herein, generally refers to the linking of two or more stabilizing compounds, including lipid, protein and polymer stabilizing compounds, by one or more bridges. The bridges, which may be composed of one or more elements, groups or compounds, generally serve to join an atom from a first stabilizing compound molecule to an atom of a second stabilizing molecule. The crosslink bridges may involve covalent and/or non-covalent associations. Any of a variety of elements, groups and/or compounds may form the bridges in the crosslinks, and the stabilizing compounds may be crosslinked naturally or through synthetic means. For example, crosslinking may occur in nature in materials formulated from peptide chains which are joined by disulfide bonds of cystine residues, as in keratins, insulin, and other proteins. Alternatively, crosslinking may be effected by suitable chemical modification, such as, for example, by combining a compound, such as a stabilizing material, and a chemical substance that may serve as a crosslinking agent, which are caused to react, for example, by exposure to heat, high- energy radiation, ultrasonic radiation, and the like. Examples include, for example, crosslinking with sulfur which may be present, for example, as sulfhydryl groups in cysteine residues, to provide disulfide linkages, crosslinking with organic peroxides, crosslinking of unsaturated materials by means of high-energy radiation, crosslinking with dimethylol carbamate, and the like. The term "substantially", as used in reference to crosslinked stabilizing compounds, means that greater than about 50%> of the stabilizing compounds contain crosslinking bridges. In certain embodiments, preferably greater than about 60%) of the crosslinked stabilizing compounds contain crosslinking bridges, with greater than about 70%) being more preferred. Even more preferably, greater than about 80%) of the crosslinked stabilizing compounds contain crosslinking bridges, with greater than about 90%> being still more preferred. In certain particularly preferred embodiments, greater than about 95%> of the crosslinked stabilizing compounds contain crosslinking bridges. If desired, the substantially crosslinked stabilizing compounds may be completely crosslinked (i.e., about 100% of the crosslinked stabilizing compounds contain crosslinking bridges). In the most preferred embodiments, the stabilizing compounds may be substantially (including completely) non-crosslinked. The term "substantially", as used in reference to non-crosslinked stabilizing compounds, means that greater than about 50%> of the stabilizing compounds are devoid of crosslinking bridges. Preferably, greater than about 60%) of the stabilizing compounds are devoid of crosslinking bridges, with greater than about 70%> being more preferred. Even more preferably, greater than about 80%) of the stabilizing compounds are devoid of crosslinking bridges, with greater than about 90%> being still more preferred. In particularly preferred embodiments, greater than about 95%> of the stabilizing compounds are devoid of crosslinking bridges. If desired, the substantially non-crosslinked stabilizing compounds may be completely non-crosslinked (i.e., about 100% of the stabilizing compounds are devoid of crosslinking bridges).
"Covalent association" refers to intermolecular interaction among two or more separate molecules, which interaction generally involves the sharing of electrons by two atomic nuclei. "Non-covalent association" refers to intermolecular interaction among two or more separate molecules, which interaction does not involve a covalent bond. Intermolecular interaction is dependent upon a variety of factors, including, for example, the polarity of the involved molecules, the charge (positive or negative), if any, of the involved molecules, and the like. Non-covalent associations are preferably selected from the group consisting of ionic interaction, dipole-dipole interaction and van der Waal's forces and combinations thereof. "Ionic interaction" refers to intermolecular interaction among two or more molecules, each of which is positively or negatively charged. Thus, for example, "ionic interaction" refers to the attraction between a first, positively charged molecule and a second, negatively charged molecule. Exemplary ionic interactions include, for example, the attraction between a metal cation, for example, a calcium ion (Ca2+), and a stabilizing compound that contains negatively charged groups and/or groups or atoms that contain unshaired pairs of electrons.
"Dipole-dipole interaction" refers generally to the attraction which can occur among two or more polar molecules. Thus, "dipole-dipole interaction" refers to the attraction of the positive end of a first polar molecule to the negative end of a second polar molecule. Dipole-dipole interactions are exemplified, for example, by the attraction between the electropositive head group, for example, the choline head group, of phosphatidylcholine, and an electronegative atom, for example, a heteroatom, such as oxygen, nitrogen or sulphur atoms, which may be present in another stabilizing compound. "Dipole-dipole interaction" refers also to intermolecular hydrogen bonding in which a hydrogen atom serves as a bridge between electronegative atoms on separate molecules and in which a hydrogen atom is held to a first molecule by a covalent bond and to a second molecule by electrostatic forces.
"Van der Waal's forces" refers to the attractive forces between non-polar molecules that are accounted for by quantum mechanics. Van der Waal's forces are generally associated with momentary dipole moments which are induced by neighboring molecules and which involve changes in electron distribution. "Stabilized composition" refers to a composition which contains one or more stabilizing compounds and preferably one or more perfluoroether compounds, typically in an aqueous medium. In preferred stabilized compositions, the stabilizing compounds are selected from lipids, proteins and polymers, with lipids being particularly preferred. Exemplary stabilized compositions include suspensions, emulsions and vesicle compositions, with vesicle compositions being preferred.
"Lipid composition," "polymer composition" and "protein composition" refer respectively to compositions which comprise lipids, polymers and proteins, typically in an aqueous medium.
"Lipid formulation," "polymer formulation" and "protein formulation" refer respectively to lipid, polymer and protein compositions which also comprise one or more bioactive agents.
"Vesicle" refers to an entity which is generally characterized by the presence of one or more walls or membranes which form one or more internal voids. Vesicles may be formulated, for example, from stabilizing compounds, such as a lipid, including the various lipids described herein, a polymer, including the various polymers described herein, or a protein, including the various proteins described herein. The lipids, polymers, and or proteins may be natural, synthetic or semi-synthetic. Preferred vesicles are those which comprise walls or membranes formulated from lipids. The walls or membranes may be concentric or otherwise. In the preferred vesicles, the stabilizing compounds may be in the form of a monolayer or bilayer, and the mono- or bilayer stabilizing compounds 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 may be concentric, if desired. Stabilizing compounds may be used to form unilamellar vesicles (comprised of one monolayer or bilayer), oligolamellar vesicles (comprised of about two or about three monolayers or bilayers) or multilamellar vesicles (comprised of more than about three monolayers or bilayers). The walls or membranes of vesicles prepared from lipids, polymers or proteins may be substantially solid (uniform), or they may be porous or semi- porous. The vesicles described herein include such entities commonly referred to as, for example, liposomes, micelles, bubbles, microbubbles, microspheres, lipid-, protein-and/or polymer-coated bubbles, microbubbles and/or microspheres, microballoons, microcapsules, aerogels, clathrate bound vesicles, hexagonal H II phase structures, and the like. The internal void of the vesicles may be filled with a liquid (including, for example, an aqueous liquid), a gas, a gaseous precursor, and/or a solid or solute material, including, for example, a vasodilator and/or bioactive agent, as desired. The internal void of the vesicle may also contain, alone or in combination with other materials or agents, a perfluorinated ether compound. The vesicles may also comprise, if desired, a targeting ligand.
"Vesicle composition" refers to a composition, typically in an aqueous medium, which comprises vesicles.
"Vesicle formulation" refers to a vesicle composition which also comprises one or more bioactive agents. Suitable vesicles or vesicle species for use in vesicle formulations include, for example, gas and/or gaseous precursor filled vesicles.
"Lipid vesicle", "polymer vesicle" and "protein vesicle" refer respectively to vesicles formulated from one or more lipids, polymers and proteins.
"Liposome" refers to a generally spherical or spheroidal cluster or aggregate of amphipathic compounds, including lipid compounds, typically in the form of one or more concentric layers, for example, bilayers. They may also be referred to herein as lipid vesicles. The liposomes may be formulated, for example, from ionic lipids and/or non-ionic lipids. Liposomes which are formulated from non-ionic lipids may also be referred to as "niosomes."
"Micelle" refers to colloidal entities formulated from lipids. In certain preferred embodiments, the micelles comprise a monolayer or hexagonal H2 phase configuration. In other preferred embodiments, the micelles may comprise a bilayer configuration. "Aerogel" refers to generally spherical or spheroidal entities which are characterized by a plurality of small internal voids. The aerogels may be formulated from synthetic materials (for example, a foam prepared from baking resorcinol and formaldehyde), as well as natural materials, such as polysaccharides or proteins. "Clathrate" refers to a solid, semi-porous or porous particle which may be associated with vesicles. In preferred form, the clathrates may form a cage-like structure containing cavities which comprise the vesicles. One or more vesicles may be bound to the clathrate. A stabilizing material may, if desired, be associated with the clathrate to promote the association of the vesicle with the clathrate. Suitable materials from which clathrates may be formulated include, for example, porous apatites, such as calcium hydroxyapatite, and precipitates of polymers and metal ions, such as alginic acid precipitated with calcium salts.
"Emulsion" refers to a mixture of two or more generally immiscible liquids and is generally in the form of a colloid. The mixture may be of lipids, for example, which may be homogeneously or heterogeneously dispersed throughout the emulsion.
Alternatively, the lipids may be aggregated in the form of, for example, clusters or layers, including mono- or bilayers.
"Suspension" or "dispersion" refers to a mixture, preferably finely divided, of two or more phases (solid, liquid or gas), such as, for example, liquid in liquid, solid in liquid, gas in liquid, and the like, which can preferably remain stable for extended periods of time.
"Hexagonal H II phase structure" refers to a generally tubular aggregation of lipids in liquid media, for example, aqueous media, in which the hydrophilic portion(s) of the lipids generally face inwardly in association with an aqueous liquid environment inside the tube. The hydrophobic portion(s) of the lipids generally radiate outwardly and the complex assumes the shape of a hexagonal tube. A plurality of tubes is generally packed together in the hexagonal phase structure.
The vesicles of the present invention preferably contain a perfluorinated ether compound, typically in the form of a gas or gaseous precursor, and preferably in the form of a gas. "Gas filled vesicle" refers to vesicles in which there is encapsulated a gas. "Gaseous precursor filled vesicle" refers to vesicles in which there is encapsulated a gaseous precursor. In certain preferred embodiments, the vesicles may be substantially (including completely) filled with the gas and/or gaseous precursor. The term "substantially", as used in reference to the gas and/or gaseous precursor filled vesicles, means that greater than about 50%) of the internal void volume of the vesicle consists of a gas and/or gaseous precursor. Preferably, greater than about 60% of the internal void of the substantially filled vesicles consists of a gas and/or gaseous precursor, with greater than about 70%) being more preferred. Even more preferably, greater than about 80%> of the internal void of the substantially filled vesicles consists of a gas and/or gaseous precursor, with greater than about 90%> being still more preferred. In particularly preferred embodiments, greater than about 95%> of the internal void of the vesicles consists of a gas and/or gaseous precursor. If desired, the substantially filled vesicle may be completely filled (i.e., filled with about 100%) gas and/or gaseous precursor). Although not considered a preferred embodiment of the present invention, the vesicles may also contain, if desired, no or substantially no gas and or gaseous precursor.
As discussed in detail below, the compositions of the present invention may be administered to a patient. As used herein, "patient" refers to animals, including mammals, preferably humans.
The phrases "internal region of a patient" and "region of interest" refer to the entire patient or to a particular area or portion of the patient. Internal regions of a patient and regions of interest may include, for example, areas being imaged with diagnostic imaging and/or areas being treated with a bioactive agent. Exemplary of such areas include, for example, the gastrointestinal region and, the cardiovascular region, including myocardial tissue, the renal region, as well as other bodily tissues and regions, including the vasculature and circulatory system, and diseased tissue, such as cancerous tissue. The phrase "cardiovascular region of a patient" refers to the region of the patient defined by the heart (myocardium) and the vasculature leading directly to and from the heart. The phrase "vasculature" denotes the blood vessels (including arteries, veins and the like) in the body or in an organ or part of the body. The phrase "gastrointestinal region" or "gastrointestinal tract" includes the region of a patient defined by the esophagus, stomach, small and large intestines and rectum. The phrase "renal region of a patient" refers to the region of the patient defined by the kidney and the vaculature that leads directly to and from the kidney, and includes the abdominal aorta. "Bioactive agent" refers to a substance which may be used in connection with an application that is therapeutic or diagnostic in nature, such as in methods for diagnosing the presence or absence of a disease in a patient and/or in methods for the treatment of disease in a patient. As used herein, "bioactive agent" refers also to substances which are capable of exerting a biological effect in vitro and/or in vivo. The bioactive agents may be neutral or positively or negatively charged. Examples of suitable bioactive agents include diagnostic and pharmaceutical agents, synthetic or natural organic or inorganic molecules, including, for example, proteins, peptides, vitamins, steroids, steroid analogs, antitumor agents, hormones, antiinflammatory agents, chemotherapeutic agents and genetic material, including nucleosides, nucleotides and polynucleotides.
"Targeting ligand" refers to any material or substance which may promote targeting of tissues and/or receptors in vitro or in vivo with the compositions of the present invention. The targeting ligand may be synthetic, semi-synthetic, or naturally-occurring. Materials or substances which may serve as targeting ligands include, for example, proteins, including antibodies, antibody fragments, hormones, hormone analogues, glycoproteins and lectins, peptides, polypeptides, amino acids, sugars, such as saccharides, including mono- and polysaccharides, and carbohydrates, vitamins, steroids, steroid analogs, hormones, cofactors, bioactive agents, and genetic material, including nucleosides, nucleotides, nucleotide acid constructs, and polynucleotides. "Tissue" refers generally to specialized cells which may perform a particular function. It should be understood that the term "tissue," as used herein, may refer to an individual cell or a plurality or aggregate of cells, for example, membranes or organs. The term "tissue" also includes reference to an abnormal cell or a plurality of abnormal cells. Exemplary tissues include, for example, myocardial tissue (also referred to as heart tissue or myocardium), including myocardial cells and cardiomyocites, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, and tumors.
"Receptor" refers to a molecular structure within a cell or on the surface of the cell which is generally characterized by the selective binding of a specific substance. Exemplary receptors include, for example, cell-surface receptors for peptide hormones, neurotransmitters, antigens, complement fragments, and immunoglobulins and cytoplasmic receptors for steroid hormones. An exemplary receptor within the context of the present invention is the glycoprotein GPIIbllla, which is a platelet integrin.
"Diagnostic agent" refers to any agent which may be used in connection with methods for imaging an internal region of a patient and/or diagnosing the presence or absence of a disease in a patient. Exemplary diagnostic agents include, for example, contrast agents for use in connection with ultrasound imaging, magnetic resonance imaging or computed tomography imaging of a patient including. Diagnostic agents include, for example, the lipid, protein or polymer and/or vesicle compositions described herein. "Pharmaceutical agent" or "drug" refers to any therapeutic or prophylactic agent which may be used in the treatment (including the prevention, diagnosis, alleviation, or cure) of a malady, affliction, disease or injury in a patient. Therapeutically useful peptides, polypeptides and polynucleotides may be included within the meaning of the term pharmaceutical or drug. "Genetic material" refers generally to nucleosides, nucleotides and polynucleotides and the like, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genetic material may be made by synthetic chemical methodology known to one of ordinary skill in the art, or by the use of recombinant or other technology, or by a combination thereof. The DNA and RNA may optionally comprise unnatural nucleotides and may be single or double stranded. "Genetic material" refers also to ribozymes and RNA having a hammerhead motif (i.e., catalytic RNA), as well as sense and anti-sense DNA and RNA, that is, a nucleotide sequence which is complementary to a specific sequence of nucleotides in DNA and/or RNA.
The terms "administered" and "administration" refer generally to the administration to a patient of a biocompatible material, including, for example, lipid, polymer or protein and/or vesicle compositions described herein.
"Biocompatible" refers to materials which are generally not injurious to biological functions and which will not result in any degree of unacceptable toxicity, including allergenic responses and disease states. The compositions and components thereof (such as lipids, proteins, polymers, perfluoroethers, bioactive agents, etc.) employed in the present invention are typically biocompatible. "In combination with" generally refers to the co-administration of a bioactive agent with a composition of the present invention, including the present lipid, polymer or protein, and/or vesicle compositions. The term "co-administration" means that the bioactive agent may be administered before, during, or after the administration of the composition. In embodiments in which the bioactive agent is included in the composition as in a formulation, the bioactive agent with the composition in any of a variety of different ways. For example, in the case of vesicle compositions, the bioactive agent may be entrapped within the internal void of the vesicle. In addition, the bioactive agent may be integrated within the layer(s) or wall(s) of the vesicle, for example, by being interspersed among the lipids, proteins or polymers that comprise the vesicle layer(s) or wall(s). In the case of non- vesicular lipid, polymer and/or protein compositions, the bioactive agent may be entrapped between or among the lipid, polymer and/or protein components. It is also contemplated that the bioactive agent may be located on the surface of the vesicle or the lipid, protein or polymer. In this case, the bioactive agent may interact chemically with the surface (inner or outer) of the vesicle or the non- vesicular lipid, protein or polymer, and remain substantially adhered thereto. Such interaction may take the form of, for example, electrostatic interactions, hydrogen bonding, van der Waal's forces, covalent bonding or other interaction. Also, the bioactive agent may interact with the surface (inner or outer) of the vesicle or the non- vesicular lipid, protein or polymer in a limited manner. Such limited interaction would permit migration of the bioactive agent, for example, from the surface of a first vesicle to the surface of a second vesicle, or from the surface of a first non- vesicular lipid, protein or polymer to the surface of a second non- vesicular lipid, protein or polymer.
"Thickening agent" refers to any of a variety of generally hydrophilic materials which, when incoφorated in the various compositions and formulations described herein, may act as viscosity modifying agents, emulsifying and/or solubilizing agents, suspending agents, and tonicity raising agents. It is contemplated that the thickening agents may be capable of aiding in maintaining the stability of the compositions and/or formulations due to such properties. "Dispersing agent" refers to a surface-active agent which, when added to a suspending medium of colloidal particles, including, for example, certain of the compositions and formulations described herein, may promote uniform separation of particles. In certain preferred embodiments, the dispersing agent may comprise a polymeric siloxane compound such as, for example, polydimethylsiloxane (also referred to as "simethicone").
"Perfluorinated ether compound," "perfluoroethers", and "perfluoroether compound" refer to any and all compounds having at least one ether group and at least one aliphatic, alicyclic or aromatic group in which all of the hydrogen atoms that would otherwise be bonded to carbon atoms are replaced by fluorine atoms. Thus, perfluorinated ether compounds contain no carbon-hydrogen bonds. Accordingly, in the case, for example, of an alkyl group having a formula CnH2n+1, "perfluorinated" and/or "perfluoro" means that all of the hydrogen atoms are replaced with fluorine atoms to provide a group having a formula CnF2n+1. It should be understood that, in accordance with the present invention, the perfluorinated group(s) may be substituted with one or more chemical moieties that include hydrogen atoms bonded to atoms other than carbon atoms. For example, a perfluoroalkyl group may be substituted, for example, with an hydroxy (-OH) group. In preferred form, other than fluorine atoms, there are no halogen atoms (i.e., chlorine, bromine or iodine atoms) bonded to carbon atoms in the perfluorinated ether compounds. In addition, the term "ether group," as used herein, alone or in combination with, for example, perfluoroether compounds, and the like, refers to an -O- or -S- atom, with -O- atoms being preferred (compounds with -S- atoms being sometimes referred to herein as thioethers, and compounds with -O- atoms being sometimes referred to herein as pure ethers). The perfluorinated ether compounds employed in the methods and compositions described herein are preferably biocompatible, and may be employed in the gaseous or liquid (including gaseous precursor) form, with perfluoroethers which are employed in the gaseous form being preferred. If employed as a gaseous precursor, the perfluoroethers may be employed, for example, as a temperature sensitive gaseous precursor and/or a pressure sensitive gaseous precursor, with temperature sensitive gaseous precursors being generally preferred.
"Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine. In certain preferred aspects of the invention, the halogen is chlorine or bromine. "Aliphatic" refers to hydrocarbon compounds that are generally characterized by straight- or branched-chain arrangements of the constituent carbon atoms. Aliphatic hydrocarbons generally comprise three subgroups: (1) paraffins (e.g., alkanes) which are typically saturated; (2) olefins (e.g., alkenes) which contain one or more carbon- carbon double bonds; and (3) acetylenes (e.g., alkynes) which contain one or more carbon- carbon triple bonds.
"Alicyclic" refers to hydrocarbon compounds that are characterized by the arrangement of the constituent carbon atoms in closed ring structures. Alicyclic compounds generally comprise three subgroups: (1) cycloparaffins (e.g., cycloalkanes) which are typically saturated; (2) cycloolefins (e.g., cycloalkenes) which contain one or more carbon-carbon double bonds; and (3) cycloacetylenes (e.g., cycloalkynes) which contain one or more carbon-carbon triple bonds. "Alkyl" refers to an aliphatic hydrocarbon group which may be straight or branched. In connection with the perfluorinated ether compounds, alkyl groups preferably contain from 1 to about 7 carbon atoms in the chain, and all combinations and subcombinations of ranges of carbon atoms therein. Preferred alkyl groups contain from 1 to 6 carbon atoms. "Lower alkyl", which refers to a short chain alkyl group, preferably containing from 1 to about 4 carbon atoms is more preferred. The alkyl group may be optionally substituted with one or more alkyl group substituents which may be the same or different, where "alkyl group substituent" includes hydroxy, thio, oxo, carboxy, perfluorinated aryl, perfluorinated alkoxy, perfluorinated aryloxy, perfluorinated alkyloxy, perfluorinated alkylthio, perfluorinated arylthio, perfluorinated aralkyloxy, perfluorinated aralkylthio, perfluorinated alkoxycarbonyl and perfluorinated cycloalkyl. There may be optionally inserted along the alkyl group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent may be lower alkyl. "Branched" refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is attached to a linear alkyl chain. As noted above, preferred alkyl groups are perfluorinated. Exemplary alkyl groups from which the perfluoroalkyl groups may be derived include, for example, methyl, ethyl, propyl (including rc-propyl and /-propyl), butyl (including w-butyl, t-butyl, sec-butyl and /-butyl), pentyl (including w-pentyl, f'-pentyl and neopentyl) and hexyl (including w-hexyl and isohexyl). Preferred perfluoroalkyl groups include, for example, perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluorobutyl, perfluoropentyl and perfluorohexyl.
"Alkenyl" refers to an alkyl group containing at least one carbon-carbon double bond and preferably from 2 to about 7 carbons, and all combinations and subcombinations of ranges of carbon atoms therein. In certain preferred embodiments, the alkenyl groups contain from 2 to about 6 carbons, with lower alkenyl groups being more preferred. "Lower alkenyl" refers to a short chain alkenyl group, preferably containing from 2 to about 4 carbon atoms. The alkenyl group may be optionally substituted with one or more "alkyl group substituents". There may be optionally inserted along the alkenyl group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent may be lower alkyl. As with the alkyl groups discussed above, preferred alkenyl groups are perfluorinated. Exemplary alkenyl groups from which prefluoroalkenyl groups may be derived include, for example, vinyl, allyl, propenyl, butenyl (including 1- and 2-butene and isobutylene), pentenyl, and hexenyl. Preferred perfluoroalkenyl groups include perfluorovinyl, perfluoropropenyl, perfluorobutenyl, perfluoropententyl and perfluorohexenyl.
"Alkynyl" refers to an alkyl group containing at least one carbon-carbon triple bond and preferably from 2 to about 7 carbons, and all combinations and subcombinations of ranges of carbon atoms therein. In certain preferred embodiments, the alkynyl groups contain from 2 to about 6 carbons, with lower alkynyl groups being more preferred. "Lower alkynyl" refers to a short chain alkynyl group, preferably containing from 2 to about 4 carbon atoms. The alkynyl group may be optionally substituted with one or more "alkyl group substituents". There may be optionally inserted along the alkynyl group one or more oxygen, sulphur or substituted or unsubstituted nitrogen atoms, wherein the nitrogen substituent may be lower alkyl. Preferred alkynyl groups are perfluoroalkynyl groups. Exemplary alkynyl groups from which perfluoroalkynyl groups may be derived include, for example, ethynyl, propargyl, butynyl, pentynyl, hexynyl and heptynyl. Preferred perfluoroalkynyl groups include perfluoroethynyl, perfluoropropargyl, perfluorobutynyl, perfluoropentynyl, perfluorohexynyl and perfluoroheptynyl .
"Cycloalkyl" refers to a non-aromatic mono- or multicyclic ring system containing from about 3 to about 7 carbon atoms, and all combinatinons and subcombinations of ranges of carbon atoms therein. The cycloalkyl group may be optionally partially unsaturated, and may also be optionally substituted with one or more alkyl group substituents. In certain preferred embodiments, the cycloalkyl groups contain from about 3 to about 6 carbons, with cycloalkyl groups containing from about 4 to about 6 carbons being more preferred. Preferred monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl. Preferred multicyclic cycloalkyl rings include cw-5-norbornene, 5-norbornene, norbornane and «t -3-oxo- tricyclo-[2.2.1.026]heptane. "Heterocyclyl" refers to a ring system containing from about 3 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein, wherein at least one or more of the ring members is a heteroatom (that is, an element other than carbon). Preferred heteroatoms include, for example, oxygen, sulfur and nitrogen, the latter of which may be optionally substituted with a lower alkyl group. In preferred embodiments, the heterocyclyl groups contain about 5 or 6 ring atoms. Also in preferred embodiments, there is a maximum of one oxygen atom in the heterocyclyl rings. The heterocyclyl groups may be optionally substituted with one or more alkyl group substituents. Preferred heterocyclyl groups are also perfluorinated. Exemplary perfluorinated heterocyclyl groups include perfluorotetrahydrofuranyl and perfluorotetrahydropyranyl.
"Aryl" refers to an aromatic carbocyclic radical containing from about 6 to about 10 carbons and all combinations and subcombinations of ranges of carbon atoms therein, with aryl groups containing about 6 carbons being preferred. The aryl group may be optionally substituted with one or more aryl group substituents which may be the same or different, where "aryl group substituent" includes nitro, cyano, alkyl, hydroxy, carboxy, carbamoyl, optionally substituted amino, perfluoroalkenyl, perfluoroalkynyl, perfluoroaryl, perfluoroaralkyl, perfluoroalkoxy, perfluoroaryloxy, perfluoroaralkoxy, perfluoroaroyl, perfluoroalkoxycarbonyl, perfluoroaryloxycarbonyl, perfluoroaralkoxy carbonyl, perfluoroacyloxy, perfluoroacylamino, perfluoroaroylamino, perfluoroalkyl carbamoyl, perfluorodialkylcarbamoyl, perfluoroarylthio, perfluoroalkylthio and perfluoroalkylene An exemplary perfluoroaryl group is perfluorophenyl.
"Heteroaryl" refers to a ring system of from about 5 to about 7 ring members, wherein one or more of the ring members is a heteroatom. Preferred heteroatoms in the heteroaryl groups include, for example, oxygen, nitrogen and sulfur atoms. The heteroaryl group may be optionally substituted with one or more aryl group substituents. The heteroaryl groups are preferably perfluorinated. Exemplary perfluoroheteroaryl groups include perfluorofuranyl and perfluorothienyl. The present invention is directed, in part, to improved methods and compositions which may be used in diagnostic imaging, including, for example, improved methods and compositions for use in providing an image of an internal region of a patient. Embodiments of the present invention may involve stabilized compositions comprising, in an aqueous carrier, a stabilizing compound and a perfluorinated ether compound.
Preferred embodiments of the present invention involve vesicle compositions comprising, in an aqueous carrier, vesicles and a perfluorinateed ether compound. In embodiments of the present invention which involve methods for diagnostic imaging, the aforementioned compositions may be administered to a patient who is scanned using diagnostic imaging, preferably ultrasound imaging, to obtain a visible image of the region. The compositions and methods provided herein are particularly useful for diagnosing the presence of diseased tissue in a patient.
Compositions of the perfluoroether compounds of the present invention optionally (and preferably) comprise one or more stabilizing compounds. A wide variety of stabilizing compounds may be employed in the compositions and methods described herein. Preferred stabilizing compounds are compounds which are capable of enhancing the stability of the compositions of the present invention, including emulsions, suspensions and vesicle compositions. Thus, with respect to compositions which comprise, for example, suspensions of finely divided gaseous bubbles, the stabilizing compounds are preferably capable of minimizing aggregation of the bubbles. With respect to embodiments involving vesicle compositions, the stabilizing compounds are preferably capable of promoting the formation of the vesicles, as well as enhancing the resistance of the vesicles, once formed, to degradation caused, for example, by the loss of structural or compositional integrity in the walls of the vesicles and/or by the loss of any significant portion of a gas encapsulated therein. In preferred embodiments, the stabilizing compounds impart the aforesaid properties to the present compositions for a minimum period of time. Preferably, the stabilizing compounds are capable of stabilizing the present compositions for a period of time which permits use of the composition, such as, for example, in the generation of visible images of a region of a patient by diagnostic imaging. Thus, the stabilizing compounds are preferably capable of stabilizing the compositions of the present invention for a period of time to at least permit their administration to a patient and- subsequent scanning of the patient with diagnostic imaging including, for example, ultrasound imaging.
Stabilizing compounds which may be employed in the methods and compositions of the present invention include, for example, lipids, proteins or polymers, and such stabilizing compounds are preferred. Of course, as would be apparent to one of ordinary skill in the art, once armed with the teachings of the present disclosure, other stabilizing compounds, in addition to the aforementioned classes of compounds, can of course also be utilized. Broadly speaking, any of a wide variety of organic and inorganic materials may be employed as stabilizing materials in the methods and compositions of the present invention including, for example, solid materials, such as particulate materials including, for example, apatites, or liquid materials, such as oils, surfactants, and the like. The stabilizing compounds employed preferably include surfactants, tensides, suspendng agents, emulsifying agents, tonicity modifying agents, viscosity modifying agents, anti- foaming agents, thickeners, dispersing agents, solvents, diluents, and the like. In preferred embodiments, the stabilizing compounds may comprise one or more lipids, proteins or polymers. In these embodiments, compositions which comprise one or more lipids are preferred. In embodiments involving lipid compositions, particularly in lipid compositions which are in the form of vesicle compositions, it may be advantageous to prepare the lipid compositions at a temperature below the gel to liquid crystalline phase transition temperature of the involved lipids. This phase transition temperature is 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. Biol. Chem. 1974 249, 2512- 2521. It is generally believed that vesicles which are prepared from lipids that possess higher gel state to liquid crystalline state phase transition temperatures tend to have enhanced impermeability 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). The following table lists some of the representative lipids and their phase transition temperatures. TABLE 1
Saturated Diacyl-sn-Glycero-3-Phosphocholines: Main Chain Melting Transition Temperatures
See, for example, Derek Marsh, CRC Handbook of Lipid Bilayers, p. 139 (CRC Press, Boca Raton, FL 1990). It may be possible to enhance the stability of vesicles formulated from lipids by incoφorating in the lipid compositions at least a minor amount, for example, about 1 to about 10 mole percent, based on the total amount of lipid employed, of a negatively charged lipid. Suitable negatively charged lipids include, for example, phosphatidylserine, phosphatidic acid, and fatty acids. Without intending to be bound by any theory or theories of operation, it is contemplated that such negatively charged lipids may provide added stability by counteracting the tendency of vesicles to rupture by fusing together. Thus, the negatively charged lipids may act 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 other vesicles which may be proximate thereto. In this way, the vesicles may be less prone to come into touching proximity with each other, which may lead to a rupture of the membrane or skin of the respective vesicles and consolidation of 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. The lipid materials used in certain of the compositions described herein, especially in connection with vesicle compositions based on lipids, are also preferably flexible. This means that, for example, in the case of vesicle compositions based on lipids, the vesicles can alter their shape, for example, to pass through an opening having a diameter that is smaller than the diameter of the vesicle. A wide variety of lipids are believed to be suitable for incoφoration in the lipid and/or vesicle compositions. With particular reference to vesicle compositions, for example, micelles and/or liposomes, any of the materials or combinations thereof which are known to those skilled in the art as suitable for their preparation may be used. The lipids used may be of natural, synthetic or semi-synthetic origin. As noted above, suitable lipids generally include, for example, fatty acids, neutral fats, phosphatides, glycolipids, aliphatic alcohols and waxes, oils, teφenes and steroids. Exemplary of these lipids which may be used to prepare lipid compositions include, for example, lysolipids; phosphocholines; phosphatidylcholine with both saturated and unsaturated lipids, including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC); and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE); phosphatidylserine; phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG); phosphatidylinositol; sphingolipids, such as sphingomyelin; glycolipids, such as ganglioside GM1 and GM2; glucolipids; sulfatides; glycosphingohpids; phosphatidic acids, such as dipalmitoylphosphatidic acid (DPP A) and distearoylphosphatidic acid (DSPA); palmitic acid; stearic acid; arachidonic acid; oleic acid; lipids bearing biocompatible polymers, including chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG), such as PEG2000, PEG5000 and PEG8000, which refer respectively to PEG polymers having mean average molecular weights of about 2,000, 5,000 and 8,000 (lipids bearing PEG polymers are also referred to herein as "pegylated lipids", with preferred lipids bearing polymers including DPPE-PEG, which refers to the lipid DPPE having a PEG polymer attached thereto, including, for example, DPPE-PEG5000, which refers to DPPE having attached thereto a PEG polymer having a mean average molecular weight of about 5000); 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); polymerizable lipids; diacetyl phosphate; dicetyl phosphate; stearylamine; cardiolipin; phospholipids with short chain fatty acids of about 6 to about 8 carbons in length; synthetic phospholipids with asymmetric acyl chains, such as, for example, one acyl chain of about 6 carbons and another acyl chain of about 12 carbons; ceramides; non-ionic liposomes including niosomes such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohols, polyoxyethylene fatty alcohol ethers, polyoxyalkylene sorbitan fatty acid esters (such as, for example, the class of compounds referred to as TWEEN™, commercially available from ICI Americas, Inc., Wilmington, DE), glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, ethoxylated soybean sterols, ethoxylated castor oil, polyoxyethylene-polyoxypropylene polymers and polyoxyethylene fatty acid stearates; sterol aliphatic acid esters, including cholesterol sulfate, cholesterol butyrate, cholesterol iso-butyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate and phytosterol n-butyrate; sterol esters of sugar acids including cholesterol glucuronide, lanosterol glucuronide, 7-dehydrocholesterol glucuronide, ergosterol glucuronide, cholesterol gluconate, lanosterol gluconate and ergosterol gluconate; esters of -sugar acids and alcohols including lauryl glucuronide, stearoyl glucuronide, myristoyl glucuronide, lauryl gluconate, myristoyl gluconate and stearoyl gluconate; esters of sugars and aliphatic acids, including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid and polyuronic acid; saponins, including sarsasapogenin, smilagenin, hederagenin, oleanolic acid and digitoxigenin; glycerols, including glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate, glycerol and glycerol esters, such as glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate and glycerol trimyristate; long chain alcohols, including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol and n-octadecyl alcohol; 6-(5-cholesten-3β-yloxy)-l-thio-β-D-galactopyranoside; digalactosyldiglyceride; 6-(5-cholesten-3β-yloxy)hexyl-6-amino-6-deoxy-l-thio-β-D-galactopyranoside; 6-(5- cholesten-3β-yloxy)hexyl-6-amino-6-deoxyl-l-thio-α-D-mannopyranoside; 12-(((7'- diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoic acid; N-[ 12-(((7'- diethylaminocoumarin-3-yl)carbonyl)methylamino)-octadecanoyl]-2-aminopalmitic acid; cholesteryl)4'-trimethylammonio)butanoate; N-succinyldioleoylphosphatidylethanol- amine; 1 ,2-dioleoyl-sn-glycerol; l,2-dipalmitoyl-sn-3-succinylglycerol; l,3-dipalmitoyl-2- succinylglycerol; 1 -hexadecyl-2-palmitoyl-glycerophosphoethanolamine and palmitoylhomocysteine, and/or combinations thereof.
As noted above, included among the lipids which may be suitable for use in the compositions of the present invention are polymerizable lipids. Examples of such polymerizable lipids include, for example, lipids which contain functionalities that may undergo polymerization reactions, including, for example, alkenyl moieties, such as oleyl, linoleyl, acryloyl and methacryloyl groups, with or without polar groups to enhance water solubility, and cyanoacrylate esters optionally carrying lipophilic esterifying groups. Exemplary polymerizable lipid compounds which may be utilized in the compositions of the present invention are illustrated below. 3
H3
O SH II I CH2— O-C— CH— (CH2)13-CH3
O SH CH2-CH2-O-C— (CH2)8-C≡C-C≡C— (CH2)12— CH3 II I CH— O-C-CH— (CH2)13-CH3 CH3— N O γH3 CH2_CH2_0_C__(CH2)8_C≡C_C≡C_(CH2)I2_CH3
CH2— O-P— CH2-CH2- N-CH3
^e CH3
O — (CH2)9 CsC-C≡C— (CH2)9-CH3 CH3s@/(CH2)ιβ-S HO/ X0— (CH2)9-C≡C-C≡C— (CH2)9-CH3 CH3 (CH2)ιβ_S O
II
O O CH2=CH-CH2 CH2-CH2-0-C-CH2-(CF2)7-CF3
H0-P-0-(CH2)9-CsC-C≡C— (CH2)β -P-OH ' i °
OH OH CH3 CH2-CH2-O-C-CH2-(CF2)7-CF3 n-CιβHι7 NCO(CH2)2- ) Xs N-C n-CιβHι
CH3-(CH2)i2-C≡C-CsC-(CH2)8-COO-(CH2)2 *.\©<-v.H Q, CH3-(CH2)ι -CH2 CH3
V X
CH3-(CH2)i2-C≡C-CsC-(CH2)8-COO-(CH2)2 CH3 CH2=(H3C)C-COO-(CH2)ιo-CH2 κ CH3
H
CH2=CH(CH2)8-COO-(CH2)2. © .CH3
N Br CH2=CH(CH2)8-COO-(CH2)2 NCH2-CH2OH
If desired, a cationic lipid may be used, such as, for example, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), l,2-dioleoyloxy-3- (trimethylammonio)propane (DOTAP); and l,2-dioleoyl-3-(4'-trimethylammonio)- butanoyl-sn-glycerol (DOTB). If a cationic lipid is employed in the lipid compositions, the molar ratio of cationic lipid to non-cationic lipid may be, for example, from about 1 : 1000 to about 1:100. Preferably, the molar ratio of cationic lipid to non-cationic lipid may be from about 1 :2 to about 1 :10, with a ratio of from about 1 : 1 to about 1 :2.5 being preferred. Even more preferably, the molar ratio of cationic lipid to non-cationic lipid may be about 1 :1. In the case of lipid compositions which contain both cationic and non- cationic lipids, a wide variety of lipids may be employed as the non-cationic lipid. Preferably, this non-cationic lipid comprises one or more of DPPC, DPPE and dioleoylphosphatidylethanolamine. In lieu of the cationic lipids listed above, lipids bearing cationic polymers, such as polylysine or polyarginine, as well as alkyl phosphonates, alkyl phosphinates and alkyl phosphites, may also be used in the lipid compositions. In preferred embodiments, the lipid compositions comprise phospholipids, particularly one or more of DPPC, DPPE, DPPA, DSPC, DSPE, and DAPC (20 carbons), with DPPC and DSPC being preferred, and DPPC being even more preferred.
Saturated and unsaturated fatty acids may also be employed in the lipid compositions described herein and may include molecules that preferably contain from about 12 carbons to about 22 carbons, in linear or branched form. Hydrocarbon groups which include isoprenoid units and/or prenyl groups may be used as well. Examples of saturated fatty acids that are suitable include, for example, lauric, myristic, palmitic and stearic acids. Suitable unsaturated fatty acids that may be used include, for example, lauroleic, physeteric, myristoleic, linoleic, linolenic, palmitoleic, petroselinic and oleic acids. Examples of branched fatty acids that may be used include, for example, isolauric, isomyristic, isopalmitic and isostearic acids.
In preferred form, the lipid is incoφorated in a mixed solvent system in the present compositions in a ratio of about 8:1:1 or about 9:1 :1 normal saline : glycerol : propylene glycol.
In certain preferred embodiments, the lipids employed in the present methods and compositions are phospholipids. Preferred among the phospholipids are those having a dialkylated moiety in which each alkyl group contains from about 10 to about 24 carbons, with phospholipids having dialkylated moieties in which each alkyl group contains from about 14 to about 22 carbons being even more preferred. Still more preferred are dialkylated phospholipids in which each of the alkyl groups contains from about 16 to about 20 carbons, with from about 16 to about 18 carbons being yet more preferred, and 16 carbons being especially preferred. Preferred also are phospholipids in which the alkyl groups are saturated. It is also preferred, in connection with certain embodiments of the invention, that the phospholipids employed have a transition temperature from the liquid crystalline state to the gel state of over 40°C, and/or that the phospholipids be in the gel state at physiologic temperature as at the temperature at which the compositions of the present invention are prepared. Preferably the phospholipids are formulated as a blend of two or more lipids. In addition to compositions formulated from lipids, the present invention also provides compositions, including vesicle compositions, formulated from proteins or derivatives thereof. Proteins which may be suitable for use in the present compositions include, for example, collagen,- fibrin and albumin. The proteins are preferably of human origin, with proteins prepared via recombinant technology being more preferred. Vesicles which are formulated from proteins and which would be suitable for use in the methods and compositions of the present invention are described, for example, in Feinstein, U.S. Patent Nos. 4,572,203, 4,718,433 and 4,774,958, and Cerny et al., U.S. Patent No.
4,957,656. Other compositions, including compositions which vesicles based on proteins, in addition to those described in the aforementioned patents, would be apparent to one of ordinary skill in the art, once armed with the present disclosure.
In addition to compositions formulated from lipids and/or proteins, the present invention also provides compositions, including vesicle compositions, formulated from polymers. Such polymers may be of natural, semi-synthetic (modified natural) or synthetic origin. 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 modified natural polymer), as employed herein, denotes a natural polymer that has been chemically modified in some fashion. Exemplary natural polymers suitable for use in the present invention include naturally occurring polysaccharides. Such polysaccharides include, for example, arabinans, fructans, fucans, galactans, galacturonans, glucans, mannans, xylans (such as, for example, inulin), levan, fucoidan, carrageenan, galatocarolose, pectic acid, pectins, including amylose, pullulan, glycogen, amylopectin, cellulose, dextrin, dextrose, polydextrose, pustulan, chitin, agarose, keratan, chondroitan, dermatan, hyaluronic acid, alginic acid, xanthan gum, starch, HETA-starch, saccharides of microbial origin, such as dextran, and various other natural homopolymer or heteropolymers, such as those containing one or more of the following aldoses, ketoses, acids or amines: erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, mannitol, sorbitol, lactose, sucrose, trehalose, maltose, cellobiose, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, glucuronic acid, gluconic acid, glucaric acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid, and naturally occurring derivatives thereof.
Exemplary natural polymers also include polypeptides such as, for example, polyglutamic acid and polylysine. Suitable polymers may also include, for example, proteins as described above, such as albumin, fibrin and collagen. Exemplary semi-synthetic polymers include carboxymethylcellulose, hydroxymethylcellulose, hydroxypropyl- methylcellulose, methylcellulose, and methoxycellulose. Exemplary synthetic polymers suitable for use in the present invention include polyethylenes (such as, for example, polyethylene glycol, polyoxyethylene, and polyethylene terephthlate), polypropylenes (such as, for example, polypropylene glycol), polyoxyethylene and polyoxypropylene copolymers, including polyoxyethylene and polyoxypropylene block copolymers, polyoxyalkylene derivatives of polyethylene glycol (such as, for example, the class of compounds referred to as Pluronics™, commercially available from BASF, Parsippany, NJ), polyurethanes (such as, for example, polyvinyl alcohol (PVA), polyvinylchloride and polyvinylpyrrolidone), polyamides (such as, for example, polyacrylamide and nylon), polystyrene, polylactic acids, fluorinated hydrocarbons (such as, for example, polytetrafluoroethylene), polyoxazolines (such as, for example, polyethyloxazoline) and polymethyl methacrylate, and derivatives thereof. Preferred are biocompatible synthetic polymers or copolymers prepared from monomers, such as acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, methacrylates, including ethyl acrylate and methyl methacrylate, 2-hydroxyethyl methacrylate (HEM), lactic acid, glycolic acid, e- caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorohydrin, hydroxyalkyl-acrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkyl-methacrylates, N-substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4-pentadiene-l-ol, vinyl acetate, acrylonitrile, styrene, p-amino- styrene, p-amino-benzyl-styrene, sodium styrene sulfonate, sodium 2- sulfoxyethylmethacrylate, vinyl pyridine, aminoethyl methacrylates, 2- methacryloyloxytrimethyl ammonium chloride, and polyvinylidene, as well polyfunctional crosslinking monomers such as N,N'-methylene-bisacrylamide, ethylene glycol dimethacrylates, 2,2'-(p-phenylenedioxy)-diethyl dimethacrylate, divinylbenzene, triallylamine and methylenebis-(4-phenyl-isocyanate), including combinations thereof. Preferable polymers include polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polyphosphazene, polysiloxane, polydimethylsiloxane, polylactic acid and copolymers thereof, such as polylactidecoglycolide, poly(e-caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), poly(vinyl alcohol) and polyamide polymers, such as nylon. Preferable copolymers include polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl-methacrylate, polystyrene-polyacrylonitrile, polyethylene-poly(propylene glycol), and polylactide co-glycolide polymers. A preferred copolymer is polyvinylidene-polyacrylonitrile. Other suitable biocompatible monomers and polymers will be readily apparent to those skilled in the art, once armed with the present disclosure.
Methods for the preparation of vesicles comprising polymers will be readily apparent to those skilled in the art, once armed with the present disclosure, when the present disclosure is coupled with information known in the art, such as that described and referred to in Unger, U.S. Patent No. 5,205,290, the disclosures of which are hereby incoφorated herein by reference in their entirety.
Vesicle derived from polymers for use in the methods of the present invention are preferably low density. The term "low density" refers to vesicles which have an internal void (cavity) volume which is at least about 75%> of the total volume of the vesicle. Preferably, the vesicles have a void volume of at least about 80%>, more preferably at least about 85%), even more preferably at least about 90%, still more preferably at least about 95%), and yet more preferably about 100% of the total volume of the vesicles.
As noted above, the stabilizing compounds employed in the present compositions may be substantially crosslinked or substantially non-crosslinked. Synthetic methods which may be employed for crosslinking stabilizing compounds, including stabilizing compounds in the form of lipids, proteins and polymers, would be apparent to one of ordinary skill in the art, once armed with the present disclosure. For example, the formation of covalent crosslinks may involve the use of coupling and/or activation agents which are well known to the skilled artisan. These agents, when activating, may desirably serve as electrophiles and thereby promote the formation of covalent bonds. Examples of such activating agents include, for example, carbonyldiimidazol (CDI), dicyclohexyl- carbodiimide (DCC), diisopropylcarbodiimide (DIC), methyl sulfonyl chloride, Castro's Reagent, and diphenyl phosphoryl chloride. Covalent linkages which are exemplary of crosslinking bridges include, for example, esters, ethers, thioesters, sulfides, disulfides, carbamates, amides, thioamides, urethane and substituted imidate linkages. Crosslinking may be effected, for example, by (1) adding a chemical substance (crosslinking agent) to a stabilizing compound and, if necessary and/or desired, exposing the resulting mixture to heat, or (2) subjecting a stabilizing compound to high energy radiation. A variety of crosslinking agents of different lengths and/or functionalities are described, for example, in R.L. Lunbland, Techniques in Protein Modification, CRC Press, Inc., Ann Arbor, MI, pp. 249-68 (1995), the disclosures of which are hereby incoφorated herein by reference, in their entirety. Exemplary crosslinkers include, for example, 3,3'-dithiobis(succinimidyl- propionate), dimethyl suberimidate, and its variations thereof, based on hydrocarbon length, and bis-N-maleimido-l,8-octane. In addition, crosslinking may be introduced by combining polyvalent counterions, for example, calcium ions, with one or more compounds that contain groups that are negatively charged and/or that contain unshared pairs of electrons, such as, for example, polyphosphazene and hyaluronic acid.
In accordance with the present invention, there is further included in the present compositions a perfluorinated ether compound. The perfluorinated ether compounds impart highly desirable and beneficial characteristics to the compositions of the present invention. For example, the perfluoroether compounds which may be incoφorated in the present compositions preferably have low densities. Accordingly, the compositions of the present invention may be highly useful as contrast agents for computed tomography (CT). Also, because the perfluoroether compounds employed in the present invention possess useful reflectivity characteristics, they may be extremely useful as contrast agents for ultrasound. In addition, it is believed that the perfluoroether compounds described herein significantly beneficially improve the stability of the compositions of the invention, including those based on vesicles. The perfluorinated compounds which may be incoφorated in the compositions of the present invention preferably comprise, for example, perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers and perfluoroheteroaryl ethers, and combinations thereof. Thus, the perfluoroether compound may be, for example, a perfluoroalicyclic aliphatic ether compound (that is, a perfluoroether compound which contains both alicyclic and aliphatic moieties), a perfluoroheterocyclyl aliphatic ether compound (that is, a perfluoroether compound which contains both heterocyclyl and aliphatic moieties), a perfluoroalicyclic heterocyclyl ether compound (that is, a perfluoroether compound which contains both alicyclic and heterocyclyl moieties), and the like. In certain preferred embodiments, the perfluorinated ether compounds may have the formula R[-X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
In the above formula (I), X is O or S. Preferably, X is O.
R, in the above formula (I) is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl. Preferably, R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl, with perfluoroalkyl being more preferred.
Preferred among the perfluoroalkyl groups are perfluoroalkyl of from 1 to about 7 carbons, and all combinations and subcombinations of ranges therein, with perfluoroalkyl of from about 1 to about 6 carbons being more preferred. Even more preferred are perfluoroalkyl groups of from 1 to about 4 carbons. Particularly preferred perfluoroalkyl groups are perfluoromethyl and perfluorobutyl. Preferred among the perfluoroalkenyl groups are perfluoroalkenyl of from about 2 to about 7 carbons, with perfluoroalkenyl of from 2 to about 6 carbons being more preferred and perfluoroalkenyl of from 2 to about 4 carbons being even more preferred. Preferred among the perfluoroalkynyl groups are perfluoroalkynyl of from 2 to about 7 carbons, with perfluoroalkynyl of from 2 to about 6 carbons being more preferred and perfluoroalkynyl of from 2 to about 4 carbons being even more preferred. Preferred among the perfluorocycloalkyl groups are perfluorocycloalkyl of from about 3 to about 7 carbons, with perfluorocycloalkyl of from about 3 to about 6 carbons being more preferred and perfluorocycloalkyl of from about 4 to about 6 carbons being even more preferred. Preferred among the perfluoroaryl groups are perfluoroaryl of from about 6 to about 10 carbons, with perfluoroaryl of about 6 carbons being more preferred. R2 in the above.formula (I) is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl. In certain preferred embodiments, R2 is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl, with perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl being more preferred, and perfluoroalkyl being even more preferred. Preferred among the perfluoroalkyl groups are perfluoroalkyl of from 1 to about 7 carbons, and all combinations and subcombinations of ranges therein, with perfluoroalkyl of from about 1 to about 6 carbons being more preferred. Even more preferred are perfluoroalkyl groups of from 1 to about 4 carbons. Particularly preferred perfluoroalkyl groups are perfluoromethyl and perfluorobutyl. Preferred among the perfluoroalkenyl groups are perfluoroalkenyl of from about 2 to about 7 carbons, with perfluoroalkenyl of from 2 to about 6 carbons being more preferred and perfluoroalkenyl of from 2 to about 4 carbons being even more preferred. Preferred among the perfluoroalkynyl groups are perfluoroalkynyl of from 2 to about 7 carbons, with perfluoroalkynyl of from 2 to about 6 carbons being more preferred and perfluoroalkynyl of from 2 to about 4 carbons being even more preferred. Preferred among the perfluorocycloalkyl groups are perfluorocycloalkyl of from about 3 to about 7 carbons, with perfluorocycloalkyl of from about 3 to about 6 carbons being more preferred and perfluorocycloalkyl of from about 4 to about 6 carbons being even more preferred. Preferred among the perfluoroaryl groups are perfluoroaryl of from about 6 to about 10 carbons, with perfluoroaryl of about 6 carbons being more preferred.
As noted above, R2 may also be halo. In these embodiments, R2 is preferably chloro or bromo. Also in these embodiments, R, is preferably perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl, with perfluoroalkyl being more preferred. In certain embodiments of the present invention, the total number of carbons in R, and R2 is preferably no greater than about 7.
Also in the compounds of formula (I) above, R, and R2 may be linked together to form (together with X) perfluoroheterocyclyl or perfluoroheteroaryl. Preferably, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom. Preferably, R, and R2 are linked together to form perfluoroheterocyclyl. Preferred among the perfluoroheterocyclyl groups are perfluoroheterocyclyl of from about 3 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein, with perfluoroheterocyclyl of about 5 or about 6 ring members being more preferred. Preferred among the perfluoroheteroaryl groups are perfluoroheteroaryl of from about 5 to about 7 ring members, and all combinations and subcombinations of ranges of ring members therein. In certain particularly preferred embodiments of the present invention, the perfluorinated ethers comprise perfluoroaliphatic ether compounds. Preferred among the perfluoroaliphatic ethers are compounds of the formula r 2n+ 1 n- - mr 2m+ 1
(II) wherein: each of n and m is independently an integer of 1 to about 7 (and all combinations and subcombinations of ranges therein); and X is O or S. In preferred embodiments, the sum of m and n is no greater than about 7, with a sum of about 5 being more preferred. Even more preferably, one of m and n is about 1 and the other of m and n is about 4. Also in preferred embodiments, X is O. Exemplary compounds of formula (II) include, for example, perfluorodimethyl ether (where m and n are 1); perfluoromethyl ethyl ether (where one of m and n is 1 and the other of m and n is 2); perfluoropropyl methyl ethers (where one of m and n is 3 and the other of m and n is 1) such as, for example, perfluoroisopropyl methyl ether and perfluoro- n-propyl methyl ether; perfluorodiethyl ether (where m and n are 2); perfluorobutyl methyl ethers (where one of m and n is 4 and the other of m and n is 1) such as, for example, perfluoro-t-butyl methyl ether, perfluoroisobutyl methyl ether and pefluoro-n-butyl methyl ether; perfluoropropyl ethyl ethers (where one of m and n is 3 and the other of m and n is 2) such as, for example, perfluoroisopropyl ethyl ether and perfluoro-n-propyl ethyl ether; perfluorobutyl ethyl ether (where one of m and n is 4 and the other of m and n is 2); and perfluorodipropyl ethers (where m and n are 3) such as, for example, bis(perfluoro- isopropyl) ether.
In alternate preferred embodiments, the perfluoroether compounds may comprise perfluoroaliphatic alicyclic compounds. Preferred among the perfluoroaliphatic alicyclic ether compounds are compounds of the formula
wherein: m is an integer of from 1 to about 6 (and all combinations and subcombinations of ranges therein); n is an integer of from about 2 to about 5 (and all combinations and subcombinations of ranges therein); and
X is O or S.
In preferred embodiments, the sum of m and n in formula III is no greater than about 7, with a sum of up to about 5 being more preferred. More preferably, n is about 2 or about 3 and m is 1 or about 2. Also in preferred embodiments, X is O. Exemplary compounds of formula (III) include, for example, perfluorocyclopropyl ethers (where n is 2), such as, for example, perfluorocyclopropyl methyl ether, perfluorocyclopropyl ethyl ether, perfluorocyclopropyl propyl ether, perfluorocyclopropyl butyl ether, perfluorocyclopropyl pentyl ether and perfluorocyclopropyl hexyl ether. Exemplary compounds of formula (III) also include, for example, perfluorocyclobutyl ethers (where n is 3) such as, for example, perfluorocyclobutyl methyl ether, perfluorocyclobutyl ethyl ether, perfluorocyclobutyl propyl ether, perfluorocyclobutyl butyl ether, perfluorocyclobutyl pentyl ether and perfluorocyclobutyl hexyl ether.
In yet another alternate embodiment, the preferred perfluoroether compounds may be represented by the formula
(IV)
wherein: m is an integer of from 0 to about 3 (and all combinations and subcombinations of ranges therein); n is an integer from about 2 to about 6 (and all combinations and subcombinations of ranges therein);
X is O or S; and
R is perfluoroalkyl of 1 to about 4 carbons. Preferably, in formula (IV) above, m is an integer of 0 or 1, n is an integer of about 4 or about 5, X is O and R is perfluoromethyl. Exemplary compounds of formula (IV) include, for example, perfluorotetrahydropyran (where m is 0, n is 5 and X is O), perfluorotetrahydrofuran (where m is 0, n is 4 and X is O) and perfluoromethyltetrahydrofuran (where m is 1 , n is 4 and X is O).
Other perfluorinated ether compounds which would be suitable for use in the methods and compositions of the present invention, in addition to those exemplified above, would be readily apparent to one of ordinary skill in the art once placed in possession of the present disclosure.
In accordance with the present invention, it has been found unexpectedly that the presence of at least one element other than carbon, such as, for example, oxygen or sulfur, imparts suφrising and beneficial properties to the perfluorinated ether compound, as well as to the compositions which contain them including, for example, compositions containing stabilizing compounds which may be used, for example, as contrast agents in connection with methods for medical imaging. For example, as compared to certain prior art contrast agents, compositions which comprise perfluoroether compounds, especially vesicle compositions, may demonstrate improved stability when exposed to increased pressures, including, for example, increased pressures which may occur in vivo. In addition, compositions of the present invention may provide a desirable and beneficial improvement when used in connection with methods for ultrasound imaging. Specifically, compositions of perfluorinated ether compounds may provide increased and prolonged contrast during ultrasound imaging of an internal region of a patient. The novel compositions of the present invention may suφrisingly and unexpectedly possess improved biocompatibility and may advantageously comprise vesicles which have a desirably smaller size distribution. As noted above, the perfluoroether compounds may impart reduced densities to the compositions of the present invention. Accordingly, the present compositions may also provide desirably dark regions in diagnostic images obtained using X-ray imaging including, for example, computed tomography (CT) imaging. Thus, administration of the compositions of the present invention, including oral administration, provides decreased density within the bowel. Administration, for example, by intravascular injection, may also decrease the attenuation within the blood vessels. In addition, the present compositions may provide desirable Tl and T2 relaxation times, and thus, may be suitable for use in MRI. As noted above, for certain uses, it is preferred that the perfluoroether compounds be administered to a patient in a gaseous or substantially gaseous form. The term "substantially", as used in the present disclosure in connection with the administration of a perfluoroether compound in gaseous form, means that greater than about 50%> of the perfluoroether compound is in the gas state at about the time of administration. In certain embodiments, preferably greater than about 60%> of the perfluoroether compound is in the gas state at about the time of administration, with greater than about 70%> being more preferred. Even more preferably, greater than about 80%) of the perfluoroether compound is in the gas state at about the time of administration, with greater than about 90%) being still more preferred. In certain particularly preferred embodiments, greater than about 95%) of the perfluoroether compound is in the gas state at about the time of administration. If desired, the perfluoroether compound may be completely gaseous at about the time of administration (i.e., about 100%) of the perfluoroether compound is in the gas state). In ultrasound imaging applications, in particular, the gaseous state, especially a completely gaseous state, is most preferred. Perfluoroether- compounds which are administered to a patient in substantially gaseous form may be gases at conditions of ambient temperature and pressure. Perfluoroether compounds which are administered to a patient in substantially gaseous form may also be, in the context of the present invention, gaseous precursors, i.e., generally liquid at conditions of ambient temperatures and pressures, that have been activated to form a gas prior to or at the time of administration. As used herein, the term "activation" means the partial, substantial or complete conversion of a gaseous precursor to a gas. Conversion of a gaseous precursor to a gas may involve, for example, heating the gaseous precursor to a temperature at or near the phase transition temperature of the gaseous precursor. Activation of the gaseous precursor may also involve, for example, exposure of the gaseous precursor to varying pressures, typically reduced pressures, so that the gaseous precursor undergoes a phase transition from a liquid to a gas.
It is also contemplated that, in certain other preferred embodiments, the perfluoroether compound may be administered to a patient substantially as a liquid. Perfluoroether compounds which may be administered to a patient substantially in liquid form include, for example, perfluoroether compounds which are gaseous precursors, i.e., liquids at conditions of ambient temperature and pressure, which are liquids that have not been activated to form a gas prior to or at the time of administration. Perfluoroether compounds which may be administered to a patient substantially in liquid form also include, for example, perfluoroether compounds which are typically gases at conditions of ambient temperature and pressure, but which have been converted to a liquid prior to administration. By substantially, as used in the present disclosure in connection with the administration of a perfluoroether compound in liquid form, means that greater than about 50%) of the perfluoroether compound is in the liquid state at about the time of administration. In certain embodiments, preferably greater than about 60%) of the perfluoroether compound is in the liquid state at about the time of administration, with greater than about 70%> being more preferred. Even more preferably, greater than about 80%) of the perfluoroether compound is in the liquid state at about the time of administration, with greater than about 90%> being still more preferred. In certain particularly preferred embodiments, greater than about 95%> of the perfluoroether compound is in the liquid state at about the time of administration. If desired, the perfluoroether compound may be completely in the liquid state at about the time of administration (i.e., about 100% of the perfluoroether compound is in the liquid state).
Depending on the particular perfluoroether compound(s) and/or stabilizing material(s) employed in a given composition and the desired application, the liquid perfluoroether compound may also be substantially converted from a liquid to a gas in vivo. This conversion can occur by virtue of the phase transition temperature of the perfluoroether liquid in the particular composition, or through applied heat (including ultrasound energy, for example). The perfluoroether compound, whether administered to a patient as a gas or a gaseous precursor, may also, if desired, be formulated to substantially persist as a liquid in vivo.
As the artisan will appreciate, a particular perfluorinated ether compound may exist in the liquid state when the compositions of the present invention are first made, and thereafter converted to a gas and thus, may be used as a gaseous precursor. Alternatively, the perfluorinated ether compounds may exist in the gaseous state when the present compositions are made, and thus may be used directly as a gas. Whether the perfluorinated ether compound is used as a liquid or a gas may generally depend on the desired application and on the particular compound chosen and its liquid/gas phase transition temperature, or boiling point. For example, a preferred perfluorinated ether compound, perfluorobutylmethyl ether (PFBME), has a liquid/gas phase transition temperature (boiling point) of 35.4°C. This means that PFBME is generally a liquid at room temperature (about 25 °C), but is converted to a gas, for example, within the human body, the normal temperature of which is about 37 °C, this temperature being above the phase transition temperature of PFBME. Thus, under normal circumstances, PFBME is a gaseous precursor at room temperature. As further examples, there are perfluorodiethyl ether (PFDEE) and perfluorobutylethyl ether (PFBEE). The liquid gas transition of PFDEE is 3 to 4.5°C and that of PFBEE is 60°C. Thus, PFDEE may be useful as a gas, whereas PFBEE may be useful as a gaseous precursor because of its relatively high boiling point. As one skilled in the art would recognize, in accordance with the ideal gas law, PV = nRT, where P is pressure, V is volume, n is moles of substance, R is the gas constant, and T is temperature, parameters such as P and T may affect whether, for example, the perfluoroether compound is in the liquid or gaseous state. Temperatures and pressures can be ambient or applied. Such pressures can include, for example, interfacial pressures, such as interactions-between surfaces and substances in vivo, including, for example, liquids, membranous tissues and the like, such as might exist in the bloodstream. In preferred embodiments, particularly where diagnostic imaging is desired, the perfluorinated ether compound is present in the compositions of the present invention as a gas or gaseous precursor, with gases being preferred. In the case of compositions which comprise the perfluorinated ether compound as a gaseous precursor, the perfluorinated ether compound may be advantageously converted to a gas prior to administration to a patient. A novel and convenient method for accomplishing this gas activation has been discovered, which involves the use of mechanical agitation. It is contemplated that this mechanical agitation imparts energy to the liquid gaseous precursor which stimulates its conversion to a gas. Preferably, this gas activation through mechanical agitation may be accomplished by providing a sealed vessel, for example, a container, which contains the composition, including the perfluorinated ether compound, under reduced pressure. Agitation can involve, for example, a prolonged shaking motion or a single abrupt motion, such as, for example, the withdrawal of the plunger of a syringe containing the composition, immediately prior to administration. These, as well as other forms and mechanisms of agitation will be well within the ambit of one skilled in the art in view of the present disclosure. Agitation of the container desirably results in conversion of the perfluorinated ether compound to a gas, preferably in the form of microbubbles. At the same time, the mechanical agitation can be employed to substantially coat the microbubbles with one or more stabilizing compounds, including, for example, surfactants, film-forming materials, membranes and/or membrane forming materials, such as, for example, lipids, proteins or polymers, to provide stabilized gaseous perfluorinated ether bubbles. Generally speaking, perfluorinated ether compounds which may be incoφorated in the compositions of the present invention preferably have a boiling point of from about 0°C to about 100°C, and all combinations and subcombinations of ranges of temperatures therein. Preferably, the perfluorinated ether compound has a boiling point of from about 20°C to about 60°C, with a boiling point of from about 25°C to about 45°C being more preferred. Even more preferably, the perfluorinated ether compound has a boiling point of from about 30°C to about 40°C. The perfluorinated ether compounds generally demonstrate substantially improved acoustic activity when incoφorated as gases in the stabilized compositions of the present invention, including lipid, protein and polymer compositions, and/or vesicle compositions, as compared to the acoustic activity of the perfluorinated ether compounds when incoφorated in the aforesaid compositions as liquids. Thus, it may be desirable to employ for example, as ultrasound contrast media, the lipid, polymer or protein compositions, and/or vesicle compositions of the present invention, in which the perfluorinated ether compound is a gas under the conditions of use. Thus, in the case of ultrasound contrast media, the perfluorinated ether compounds may be a gas at room temperature, as well as at physiological temperature after administration to a patient. Also, if desired, the perfluorinated ether compound employed may be a liquid at room temperature which preferably undergoes phase transition to a gas upon being exposed to temperatures higher than room temperature, for example, physiological temperatures. In the table below, there are provided boiling points for exemplary perfluorinated ether compounds that may be incoφorated in the compositions of the present invention. As can be seen in the chart, halogenation, namely fluorination, especially perfluorination, generally lowers the boiling point of the compound as compared to the corresponding non-halogenated ether compound. The non-halogenated ether compound is identified in the table as the "parent" compound. Without being intended to be bound by any theory or theories of operation, it is contemplated that as the degree of halogenation of a compound is increased, there may be an associated reduction in boiling point owing to the reduced availability of hydrogen atoms to engage in intermolecular hydrogen bonding. This effect appears to be more pronounced for smaller, less symmetrical ethers. BP (°C) Parent BP CO
C6 A. PERFLUORO
1. Perfluorobutylethyl ether 60 92
2. Bisφerfluoroisopropyl) ether 54 69 BP CO Parent BP CC)
C5
A. PERFLUOROETHERS
1. Perfluorotetrahydropyran 34 88 2. Perfluoromethyl tetrahydrofuran 27 80
3. Perfluoro-n-butyl methyl ether 35.4 71
B. FLUOROETHERS
1. Heptafluoropropyl ethyl ethers (boiling points range from 88 to 110°C).
2. Octafluoropropyl ethyl ethers (boiling point of 99.5°C). 3. Nonafluoropropyl ethyl ethers (boiling points range from 76 to 110°C).
4. Decafluoropropyl ethyl ethers (boiling point of 115"C).
5. Undecafluoropropyl ethyl ethers (boiling point of 65°C)
6. Nonafluoroisopropyl ethyl ether (boiling point of 60°C).
BP Parent BP CO
C4
A. PERFLUOROETHERS 1. Perfluorodiethyl ether 3 to 4.5 34.6
B. FLUOROETHERS 1. Heptafluoroisopropyl methyl ether (boiling point of 72°C).
2. Nonafluoropropyl methyl ether (53°C)
BP (°α Parent BP CO
C3 A. PERFLUOROETHERS 1. Perfluoromethyl ethyl ether 11
B. FLUOROETHERS
1. Pentafluoromethyl ethyl ether (boiling points range from 43 to 53°C) C2
B. FLUOROETHERS
1. Trifluorodimethyl ether (boiling point of 33°C)
Perfluoroether compounds which may be employed in the methods and compositions of the present invention, including the perfluoroether compounds of formulas (I) to (IV) above, may be readily prepared using standard synthetic methodology well known to those of ordinary skill in the art. In addition, certain perfluoroether compounds are commercially available. With respect to synthetic methods, various compounds which may serve as starting materials for the preparation of the perfluoroether compounds described herein are commercially available, or may likewise be readily synthesized. For example, the perfluoroether compounds may be prepared from the fluorination reaction of an adduct formed by the free-radical addition of a fluoro-olefin and a hydrogen-containing ether. The fluorinated ether may be partially or fully fluorinated during the fluorination reaction. The hydrogen-containing ether is preferably of the formula R-O-R', wherein R and R' are independently hydrocarbon groups optionally substituted by chlorine or fluorine, or together form a single hydrocarbon group. In preferred form, R and R' are independently alkyl, cycloalkyl, aralkyl and aryl. Included among the preferred hydrogen-containing ethers which may be used to prepare the perfluoroethers described herein are, for example, dimethyl ether, diethyl ether, dipropyl ether, tetrahydrofuran, dioxane, tetrahydropyran, trimethylene oxide, and ethylene glycol dimethyl ether. Included among the preferred fluoro-olefins which may be used to prepare the perfluoroethers described herein include, for example, difluoroethylene, chlorotrifluoroethylene, perfluorocyclobutene, trifluoroethylene and hexafluoropropene. The mole ratio of the fluoro-olefin and the hydrogen-containing ether in the adduct may be from about 6:1 to about 1:1, although higher or lower amounts of fluoro-olefin may be employed, depending, for example, on the particular fluoro-olefin and/or hydrogen-containing ethers employed.
Generally speaking, the fluorination of the adduct may be effected by the use of a high valency metal fluoride as a fluorinating agent, preferably at elevated temperatures including, for example, above about 200°C. A fluorination procedure of this type is described, for example, in "Advances in Fluorine Chemistry", Vol 1., Butterworth, p. 166 (1960), the disclosures of which are hereby incoφorated herein by reference, in their entirety. Suitable fluorinating agents include, for example, cobalt trifluoride (CoF3), alone or in combination with one or more alkali or alkaline earth metal fluorides, such as, for example, potassium fluoride (KF) and calcium fluoride (CaF2). As noted above, the fluorination reaction may preferably be effected at elevated temperatures, preferably above about 200°C, with preferred temperature ranges being from about 300°C to about 600° C, and more preferably,f rom about 400 C to about 500°C. Generally speaking, the use of higher temperatures may provide fluorinated ether compounds having higher degrees of fluorination, including perfluorination, whereas the use of lower temperatures may provide fluorinated ether compounds which are partially fluorinated.
Fluorination with cobalt trifluoride is a technique well known in the art, and is described in standard chemistry textbooks such as, for example, R. D. Chambers, "Fluorine in Organic Chemistry", see page 25, the disclosures of which are hereby incoφorated herein by reference, in their entirety. As known to the skilled artisan, cobalt trifluoride can be regenerated by reacting elemental fluorine and the cobalt difluoride resulting from the organic fluorination reaction. Exemplary fluorination reactions which may be used to prepare the present perfluoroether compounds are described, for example, in Drakesmith et al., Published International Patent Application No. WO 84/02909, the disclosures of which are hereby incoφorated by reference herein, in their entirety. As would be apparent to the skilled artisan, once armed with the present disclosure, the perfluoroether compounds may also be synthesized with isotopes, for example, isotopes of oxygen, such as 17O, to enhance the paramagnetic properties of the perfluoroether compounds. This may serve, for example, to enhance the utility of the present compositions as diagnostic agents for magnetic resonance imaging.
As discussed above, the perfluorinated ether compounds may be present in the stabilized compositions of the present invention as a gas or gaseous precursor, with gases being preferred. In addition to the gases and/or gaseous precursors represented by the perfluorinated ether compounds, the compositions of the present invention may further comprise additional gases and/or gaseous precursors. With respect to additional gases, the following discussion is provided. Preferred gases are gases which are inert and which are biocompatible, that is, gases which are not injurious to biological function. Preferred gases include those selected from the group consisting of air, noble gases, such as helium, rubidium hypeφolarized xenon, hypeφolarized argon, hypeφolarized helium, neon, argon, xenon, carbon dioxide, nitrogen, fluorine, oxygen, sulfur-based gases, such as sulfur hexafluoride and sulfur tetrafluoride, fluorinated gases, including, for example, partially fluorinated gases or completely fluorinated gases. Exemplary fluorinated gases include the fluorocarbon gases, such as the perfluorocarbon gases, and mixtures thereof. Paramagnetic gases, such as I7O2, may also be used in the lipid and/or vesicle compositions.
In preferred embodiments, the additional gases utilized in the compositions described herein may be fluorinated gas. Such fluorinated gases include materials which contain at least one, or more than one, fluorine atom. Preferred are gases which contain more than one fluorine atom, with perfluorocarbons (that is, fully fluorinated fluorocarbons) being more preferred. Preferably, the perfluorocarbon gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfiuoropentane, perfluorocyclobutane and mixtures thereof. More preferably, the perfluorocarbon gas is perfluoropropane or perfluorobutane, with perfluoropropane being particularly preferred. Another preferable gas is sulfur hexafluoride. Yet another preferable gas is heptafluoropropane, including 1,1,1,2,3,3,3- heptafluoropropane and its isomer, 1,1,2,2,3, 3, 3 -heptafluoropropane. It is contemplated that mixtures of different types of gases, such as mixtures of a perfluorocarbon gas and another type of gas, such as air, can also be used in the compositions employed in the methods of the present invention. Other gases, including the gases exemplified above, would be readily apparent to one skilled in the art based on the present disclosure.
In certain preferred embodiments, a gas, for example, air or a perfluorocarbon gas, may be combined with a liquid perfluorocarbon, such as perfiuoropentane, perfluorohexane, perfluoroheptane, perfluorooctylbromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine and perfluorotributylamine. As noted above, in addition to certain of the perfluoroether compounds described herein which may be in the form of a gaseous precursor, it may be desirable to incoφorate in the stabilized compositions of the present invention additional gaseous precursors. Such precursors include materials that are capable of being converted to a gas in vivo. Preferably, the gaseous precursor is biocompatible, and the gas produced in vivo is biocompatible also.
Among the gaseous precursors which are suitable for use in the stabilized compositions described herein are agents which are sensitive to pH. These agents include materials that are capable of evolving gas, for example, upon being exposed to a pH that is neutral or acidic. Examples of such pH sensitive agents include salts of an acid which is selected from the group consisting of inorganic acids, organic acids and mixtures thereof. Carbonic acid (H2CO3) is an example of a suitable inorganic acid, and aminomalonic acid is an example of a suitable organic acid. Other acids, including inorganic and organic acids, would be readily apparent to one skilled in the art based on the present disclosure.
Gaseous precursors which are pH-sensitive and which are derived from salts are preferably selected from the group consisting of alkali metal salts, ammonium salts and mixtures thereof. More preferably, the salt is selected from the group consisting of carbonate, bicarbonate, sesquecarbonate, aminomalonate and mixtures thereof. Examples of suitable gaseous precursor materials which are derived from salts include, for example, lithium carbonate, sodium carbonate, potassium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, magnesium carbonate, calcium carbonate, magnesium bicarbonate, ammonium carbonate, ammonium bicarbonate, ammonium sesquecarbonate, sodium sesquecarbonate, sodium aminomalonate and ammonium aminomalonate. Aminomalonate is well known in the art, and its preparation is described, for example, in Thanassi, Biochemistry, Vol. 9, no. 3, pp. 525- 532 (1970); Fitzpatrick et al., Inorganic Chemistry, Vol. 13, no. 3 pp. 568-574 (1974); and Stelmashok et al., Koordinatsionnaya Khimiya, Vol. 3, no. 4, pp. 524-527 (1977). The disclosures of these publications are hereby incoφorated herein by reference, in their entireties.
In addition to, or instead of being sensitive to changes in pH, the gaseous precursor materials may also comprise compounds which are sensitive to changes in temperature. In addition to many of the perfluoroethers themselves, exemplary of suitable gaseous precursors which are sensitive to changes in temperature are the perfluorocarbons. As with the perfluoroether compounds described above, a particular perfluorocarbon may exist in the liquid state when the stabilized compositions are first made, and thus may be used as a gaseous precursor. Alternatively, the perfluorocarbon may exist in the gaseous state when the stabilized compositions are made, and thus may be used directly as a gas. Whether the perfluorocarbon is used as a liquid or a gas generally depends on its liquid/gas phase transition temperature, or boiling point. For example, a preferred perfluorocarbon, perfiuoropentane, has a liquid/gas phase transition temperature (boiling point) of 29.5°C. This means that perfiuoropentane is generally a liquid at room temperature (about 25°C), but is converted to a gas within the human body, the normal temperature of which is about 37°C, this temperature being above the transition temperature of perfiuoropentane. Thus, under normal circumstances, perfiuoropentane is a gaseous precursor. As a further example, there are the homologs of perfiuoropentane, namely perfluorobutane and perfluorohexane. The liquid/gas transition of perfluorobutane is 4°C and that of perfluorohexane is 57°C. Thus, perfluorobutane may be useful as a gaseous precursor, although more likely as a gas, whereas perfluorohexane may be useful as a gaseous precursor because of its relatively high boiling point. A wide variety of materials may be used as temperature-sensitive gaseous precursors in the compositions described herein. It is only required that the material be capable of undergoing a phase transition to the gas phase upon passing through the appropriate temperature (such as physiological temperatures or following direct application of biocompatible heat). Suitable gaseous precursors include, for example, hexafluoroacetone, isopropyl acetylene, allene, tetrafluoroallene, boron trifluoride, 1,2- butadiene, 2,3-butadiene, 1,3-butadiene, 1, 2,3 -trichloro-2-fluoro- 1,3 -butadiene, 2-methyl- 1,3-butadiene, hexafluoro- 1,3-butadiene, butadiyne, 1-fluorobutane, 2-methylbutane, perfluorobutane, 1-butene, 2-butene, 2-methyl-l-butene, 3-methyl-l-butene, perfluoro-1- butene, perfluoro-2-butene, 4-phenyl-3-butene-2-one, 2-methyl-l-butene-3-yne, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-l,l,l,4,4,4-hexafluorobutyne, 3 -methyl- 1-butyne, perfluoro-2-butyne, 2-bromobutyraldehyde, carbonyl sulfide, crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane, perfluorocyclobutene, 3-chlorocyclopentene, perfluorocyclopentane, octafluorocyclopentene, cyclopropane, perfluorocyclopropane, 1 ,2-dimethyl-cyclopropane, 1 , 1 -dimethylcyclopropane, 1 ,2-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene, 3-ethyl-3-methyl diaziridine, 1 , 1 , 1 -trifluorodiazoethane, dimethyl amine, hexafluorodimethylamine, dimethylethyl- amine, bis(dimethylphosphine)amine, perfluorohexane, perfluoroheptane, perfluoro- octane, 2,3-dimethyl-2-norbornane, perfluorodimethylamine, dimethyloxonium chloride, l,3-dioxolane-2-one, 4-methyl-l,l,l,2-tetrafluoroethane, 1,1,1-trifluoroethane, 1,1,2,2- tetrafluoroethane, l,l,2-trichloro-l,2,2-trifluoroethane, 1,1-dichloroethane, 1,1-dichloro- 1 ,2,2,2-tetrafluoroethane, 1 ,2-difluoroethane, 1 -chloro- 1 , 1 ,2,2,2-pentafluoroethane, 2-chloro-l,l-difluoroethane, l,l-dichloro-2-fluoroethane, 1 -chloro- 1,1,2, 2-tetrafluoro- ethane, 2-chloro-l,l-difluoroethane, chloroethane, chloropentafluoroethane, dichloro- trifluoroethane, fluoroethane, perfluoroethane, nitropentafluoroethane, nitrosopenta- fluoroethane, perfluoroethylamine, ethyl vinyl ether, 1,1-dichloroethane, 1,1-dichloro- 1 ,2-difluoroethane, 1 ,2-difluoroethane, methane, trifluoromethanesulfonyl chloride, trifluoromethanesulfonylfluoride, bromodifluoronitrosomethane, bromofluoromethane, bromochlorofluoromethane, bromotrifluoromethane, chlorodifluoronitromethane, chlorodinitromethane, chlorofluoromethane, chlorotrifluoromethane, chlorodifluoro- methane, dibromodifluoromethane, dichlorodifluoromethane, dichlorofluoromethane, difluoromethane, difluoroiodomethane, disilanomethane, fluoromethane, iodomethane, iodotrifluoromethane, nitrotrifluoromethane, nitrosotrifluoromethane, tetrafluoromethane, trichlorofluoromethane, trifluoromethane, 2-methylbutane, methyl ether, methyl isopropyl ether, methyllactate, methylnitrite, methylsulfide, methyl vinyl ether, neopentane, nitrous oxide, 1,2,3-nonadecanetricarboxylic acid 2-hydroxytrimethyl ester, l-nonene-3-yne, 1 ,4-pentadiene, n-pentane, perfiuoropentane, 4-amino-4-methylpentan- 2-one, 1-pentene, 2-pentene (cis and trans), 3-bromopent-l-ene, perfluoropent-1-ene, tetrachlorophthalic acid, 2,3,6-trimethyl-piperidine, propane, 1,1, 1,2,2,3 -hexafluoro- propane, 1 ,2-epoxypropane, 2,2-difluoropropane, 2-aminopropane, 2-chloropropane, heptafluoro-1-nitropropane, heptafluoro-1-nitrosopropane, perfluoropropane, propene, hexafluoropropane, 1,1,1 ,2,3 ,3-hexafluoro-2,3-dichloropropane, 1 -chloropropane, chloropropane-(trans), 2-chloropropane, 3-fluoropropane, propyne, 3,3,3-trifluoro- propyne, 3-fluorostyrene, sulfur (di)-decafluoride (S2F,0), 2,4-diaminotoluene, trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene and vinyl ether.
Pressure sensitive gaseous precursors may also be employed in the methods and compositions of the present invention. By "pressure sensitive," it is meant, in accordance with the ideal gas law, PV=nRT, where P is pressure, V is volume, n is the amount (generally in moles) of the gaseous precursor, R is the ideal gas law constant, and T is temperature, that many of the aforementioned temperature sensitive gaseous precursors, including the perfluoroethers, may be subjected in a desirable fashion to pressure changes to promote a change in physical state, for example, from a liquid to a gas. In this connection, as known to one of ordinary skill in the art, according to the ideal gas law, pressure is directly proportional to temperature. Thus, the ideal gas law may be used to predict, for example, that the boiling point of a substance increases upon exposure to increased pressures. Conversely, the boiling point of a substance is predicted to decrease upon exposure to reduced pressures. Thus, in accordance with the discussion above respecting the conversion of a liquid gaseous precursor to a gas, it is contemplated that a decrease in pressure may result, for example, from the abrupt withdrawal on the plunger of a syringe which contains the gaseous precursor. Depending, for example, on the rate at which the plunger is withdrawn, as well as the particular gaseous precursor involved, the gaseous precursor may undergo a conversion to a gas, owing to the decrease in pressure.
Perfluorocarbons are both preferred as additional gases and gaseous precursors for use in connection with the compositions employed in the methods of the present invention. Included among such perfluorocarbons are saturated perfluorocarbons, unsaturated perfluorocarbons, and cyclic perfluorocarbons. The saturated perfluorocarbons, which are usually preferred, preferably have the formula CnF2n+2, where n ranges from 1 to about 12, and all combinations and subcombinations of ranges therein. Preferably, n in the foregoing formula for saturated perfluorocarbons is from about 2 to about 10, more preferably about 3 to about 8, and even more preferably about 3 to about 6. Suitable perfluorocarbons include, for example, perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, perfluorocyclobutane, perfiuoropentane, perfluorohexane, perfluoroheptane, perfluorooctane and perfluorononane. Preferably, the saturated perfluorocarbon is selected from the group consisting of perfluoropropane, perfluorobutane, perfluorocyclobutane, perfiuoropentane, perfluorohexane and perfluorooctane, with perfluoropropane being particularly preferred. Also preferred are cyclic perfluorocarbons, which preferably have the formula CnF2n, where n ranges from about 3 to 8, and all combinations and subcombinations of ranges therein. Preferably, n in the foregoing formula for cyclic perfluorocarbons is from about 3 to about 6. Exemplary cyclic perfluorocarbons include, for example, hexafluorocyclopropane, octafluorocyclobutane, and decafluorocyclopentane. In addition to the perfluorocarbons, it may be desirable to utilize stable fluorocarbons which are not completely fluorinated. Such fluorocarbons include heptafluoropropane, for example, 1,1, 1,2,3,3, 3 -heptafluoropropane and its isomer, 1 , 1 ,2,2,3,3,3-heptafluoropropane.
The gaseous precursor materials may be also photoactivated materials, such as diazonium ion and aminomalonate. The term "photoactivated materials", as used herein, refers to light sensitive compounds which are generally in solid or liquid form and which become a gas after exposure to light. Exemplary photosensitive compounds include, for example, diazonium compounds, which may decompose to form nitrogen gas after exposure to ultraviolet light. Another exemplary photosensitive compound is aminomalonate. As one skilled in the art would recognize, other gaseous precursors may be chosen which are capable of forming a gas after exposure to light. Depending upon the application, exposure to such light may be necessary prior to administration or, in some instances, can occur subsequent to administration. Certain stabilized compositions, and particularly vesicle compositions, may be formulated so that gas can be formed at the target tissue or by the action of sound on the composition. Examples of gaseous precursors are described, for example, in U.S. Patent Nos. 5,088,499 and 5,149,319, the disclosures of which are hereby incoφorated herein by reference, in their entireties. Other gaseous precursors, in addition to those exemplified above, will be apparent to one skilled in the art based on the present disclosure. The gaseous substances and/or liquids (including gaseous precursors), including the perfluoroether compounds, are preferably incoφorated in the stabilized compositions irrespective of the physical nature of the composition. Thus, it is contemplated that the gaseous substances and/or precursors thereto may be incoφorated, for example, in lipid compositions in which the lipids are aggregated randomly, as well as in vesicle compositions, including vesicle compositions which are formulated from lipids, such as micelles and liposomes. Incoφoration of the gaseous substances and/or precursors thereto in the stabilized compositions may be achieved by using any of a number of methods. For example, in the case of stabilized compositions which comprise vesicles formulated from lipids, the formation of gas filled vesicles can be achieved by shaking or otherwise agitating an aqueous mixture which comprises a gas or gaseous precursor and one or more lipids. This promotes the formation of stabilized vesicles within which the gas or gas precursor is encapsulated.
In addition, a gas may be bubbled directly into an aqueous mixture of stabilized compound, including lipids and/or vesicle-forming compounds. Alternatively, a gas instillation method can be used as disclosed, for example, in U.S. Patent Nos. 5,352,435 and 5,228,446, the disclosures of which are hereby incoφorated herein by reference, in their entireties. Suitable methods for incoφorating the gas or gas precursor in cationic lipid compositions are disclosed also in U.S. Patent No. 4,865,836, the disclosures of which are hereby incoφorated herein by reference. Other methods would be apparent to one skilled in the art based on the present disclosure. Preferably, the gas may be instilled in the stabilized compositions after or during the addition of the stabilizing material and/or during formation of vesicles.
In preferred embodiments, the gaseous substances and/or gaseous precursor materials, including the perfluoroether compounds, may be incoφorated in vesicle compositions, with micelles and liposomes being preferred. As discussed in detail below, vesicles in which a gas, gaseous precursor and/or perfluorinated ether compound are encapsulated are advantageous in that they provide improved reflectivity in vivo.
As discussed more fully hereinafter, it is preferred that the stabilized compositions, and especially vesicle compositions, be formulated from lipids and optional stabilizing compounds to promote the formation of stable vesicles. In addition, it is also preferred that the lipid and/or vesicle compositions comprise a highly stable gas as well. The phrase "highly stable gas" refers to a gas which has limited solubility and diffusability in aqueous media. Exemplary highly stable gases include the perfluoroethers described herein, as well as perfluorocarbons since they are generally less diffusible and relatively insoluble in aqueous media. Accordingly, their use may promote the formation of highly stable vesicles.
In certain embodiments, it may be desirable to use a fluorinated compound, including a perfluorinated compound, which may be in the liquid state at the temperature of use of the stabilized compositions, including, for example, the in vivo temperature of the human body, to assist or enhance the stability of the stabilized compositions, and especially, the gas filled vesicles. Suitable fluorinated compounds include, for example, fluorinated amphiphilic compounds. It is contemplated that these fluorinated compounds may be used in addition to, or instead of, the various stabilizing compounds described above. A variety of fluorinated amphiphilic compounds can be employed in the present compositions. Preferred fluorinated amphiphilic compounds are those which, when combined with a gas, including the perfluoroether compounds described herein, as well as other stabilizing compounds, if desired, tend to form stabilized compositions and/or vesicles. Preferred also are fluorinated amphiphilic compounds which are capable of stabilizing the vesicles, once formed.
In preferred embodiments, the fluorinated amphiphilic compounds are based on amphiphilic compounds, including lipids, and especially phospholipids. In such embodiments, the fluorine atoms are preferably substituted on the nonpolar aliphatic chain portions of the involved amphiphilic compounds. Other fluorinated compounds which also may be suitable for use in the present compositions, include, for example, the class of compounds which are commercially available as ZONYL™ fluorosurfactants (DuPont Chemical Coφ., Wilmington DE), including the ZONYL™ phosphate salts and the ZONYL™ sulfate salts, which have terminal phosphate or sulfate groups. Representatives of these salts are disclosed, for example, in U.S. Patent No. 5,276,146, the disclosures of which are hereby incoφorated herein by reference, in their entirety. The ZONYL™ phosphate salt has the formula
[F(CF2CF2)3.8CH2CH2O]1 2P(O)(O-NH4 +)2,, and the ZONYL™ sulfate salt has the formula F(CF2CF2)3.8CH2CH2SCH2CH2N+(CH3)3 OSO2OCH3. Suitable ZONYL™ surfactants also include, for example, Telomer B fluorosurfactants, including Telomer B surfactants which are pegylated (i.e., PEG Telomer B surfactants). An example of a PEG Telomer B surfactant which may be particularly suitable for use as an amphiphilic compound is the compound C9F,9CH2CH2(OCH2CH2)9-OH. The Telomer B surfactants are also commercially available from the DuPont Chemical Coφ., of Wilmington DE.
In certain preferred embodiments, the fluorinated amphiphilic compounds are polyfluorinated. This means that the amphiphilic compounds are preferably substituted with at least two or more fluorine atoms. In even more preferred embodiments, the fluorinated amphiphilic compounds are perfluorinated.
In certain particularly preferred embodiments, the fluorinated amphiphilic compound has the formula
[Rr-X^ Ra-Z (I) wherein: each of x, y and z is independently 0 or 1 ; each X, is independently -O-, -S-, -SO-, -SQ -, -NR^ -, -C(=X. )-, -C(=X2)-O-, -O-C(=X2)-, -C(=X2)-NR4- or -NR4-C(=X2)-; X2 is O or S; Y is a direct bond or -X3-M(=O)(OR5)q-O-, where q is 1 or 2;
X3 is a direct bond or -O-; M is P or S;
Z is hydrogen, the residue of a hydrophilic polymer, a saccharide residue or -N^),, where r is 2 or 3; each R, is independently alkyl of 1 to about 30 carbons or fluorinated alkyl of 1 to about 30 carbons;
R2 is a direct bond or an alkylene linking group of 1 to about 10 carbons;
R3 is a direct bond or an alkylene diradical of 1 to about 10 carbons; each of R4 and Rj is independently hydrogen or alkyl of 1 to about 8 carbons; and each Rg is. independently hydrogen, alkyl of 1 to about 8 carbons or a residue of a hydrophilic polymer; provided that at least one of x, y and z is 1, at least one of R, is fluorinated alkyl of 1 to about 30 carbons, and when R2 is a direct bond, two of x, y and z are each 0. Those skilled in the art will appreciate that fluorinated derivatives of common phospholipids (diacylphosphatidyl serine, diacylphosphatidyl ethanolamine, diacylphosphatidyl glycerol, diacylphosphatidyl glycerol, etc.) as well as fluorinated derivatives of fatty acyl esters and free fatty acids can be employed in the compositions of the present invention. A particularly preferred class of fluorinated amphiphilic compounds which may be employed in the compositions of the present invention and which are within the scope of fluorinated amphiphilic compounds of formula (I), is represented by the formula
Crfrri ι— (Crtøm— C(0)O— ι Crf 2nn— (Cr m— C(0)0—
— OP(Q>)O- (Cr w- ft (Cr+
(II) wherein: m is 0 to about 18; n is 1 to about 12; and w is 1 to about 8.
The fluorinated amphiphilic compounds which may be employed in the methods and compositions of the present invention, including the fluorinated amphiphilic compounds of formulas (I) and (II), can be prepared readily using standard organic synthetic methodology well known to those of ordinary skill in the art. Suitable methods for preparing fluorinated amphiphilic compounds are disclosed, for example, in C. Santaella et al., New Journal of Chemistry, 15, 685 (1991), the disclosures of which are hereby incoφorated by reference, in their entirety. Exemplary of the available methods for preparing fluorinated amphiphilic compounds are synthetic methods based on the phosphorylation of l,2-di-(F-alkylacyl)-3-glycerol derivatives. These methods can be utilized in the preparation of perfluoroalkyl phosphatidylcholines, and particularly perfluoroalkyl phosphatidylethanolamines, and are disclosed, for example, in the aforementioned Santaella publication. Such methods involve linear phosphorylating agents, including the Hirt and Berchtold reagent (2-bromoethyldichlorophosphate
(BEDP)), which is readily available in large quantities. Hirt et al., Pharm. Ada. Helv., 33, 349 (1958). BEDP is highly reactive and can be used to phosphorylate sterically hindered disubstituted glycerols. Hansen et al., Lipids, 17, 453 (1982). The ammonium group can be introduced by reaction of an appropriate amine with the 1 ,2-diacylglycero- 3-(2-bromoethyl)phosphate intermediate to give the desired phosphatidylethanolamine derivatives. This synthetic methodology is depicted in the following reaction scheme.
2) H20
CnF2ιvι— (CH2)m— C(0)0— a) NMe3, Ag2Cθ3 CnF2n 1— (CH2)m-C(0)0—
-OP(C2)0-(CH2)2- N (CH3)3
(V) where m and n are as previously defined in connection with the fluorinated amphiphilic compounds of formula II. The l,2-disubstituted-3-benzylglycerol derivatives of formula (III) are readily synthesized in high yields (85 to 90%>) by acylation of 1 -benzylglycerol with the corresponding perfluoroalkanoyl halides. Proc. Natl. Acad. Sci. USA, 75, 4074 (1978). The benzyl protecting group can be removed by hydrogenolysis over a palladium on charcoal catalyst (Pd/C) in tetrahydrofuran (THF). Proc. Natl. Acad. Sci. USA, 75, 4074 (1978). Short reaction times for the hydrogenolysis of the benzyl group are preferred to avoid transesterification of the 1,2-diacylglycerol of formula (III) into the more thermodynamically stable 1,3-diacylglycerol isomer. The hydrogenolysis reaction can be monitored by ordinary- analytical techniques, including, for example, thin layer chromatography (TLC) and proton (Η) nuclear magnetic resonance (NMR). The reaction is generally complete in about one hour with little or no transesterification. The hydrogenolysis reaction is preferably conducted in THF because both the starting material in the involved reaction (the compound of formula (III)) and the product (the compound of formula (IV)) tend to be highly soluble in THF. In addition, THF is conveniently used as the solvent in the subsequent phosphorylation step.
After hydrogenolysis, the catalyst (Pd/C) can be removed by filtration. The 1 ,2-diacylglycerols of formula (IV) can be reacted immediately with BEDP and an excess of triethylamine. Phosphorylation is typically completed in about 2 to about 4 hours, as measured by TLC. The remaining phosphochloride bond can be hydrolysed in aqueous base and generally requires about 22 hours for completion. When mineral bases or salts are used for hydrolysis including, for example, Na^O;,, KC1 and EDTA sodium salt, the phosphate salts are highly insoluble in water or organic solvents. Fleischer, Methods Enzymol. 98, 263 (1983). The brominated intermediate can be isolated as a stable and soluble hydrogenotriethylammonium salt if excess triethylamine is used. Acidification of the phosphate salts to form the corresponding acid is difficult because the glycerol ester bonds are hydrolyzed at the necessary pH (pH of 2 to 3). Product degradation occurs also during purification over silica gel. Accordingly, it is preferred to use the phosphate salt without further purification. Nucleophilic displacement of the bromide ion by a large excess of trimethylamine occurs in a solvent mixture of CHC13/CH3CN at 45°C over a 12 hour period. The displaced bromide ion can be precipitated by the addition of silver carbonate.
Exemplary fluorinated amphiphilic compounds are described, for example, in Applicant's copending U.S. application Serial No. 08/465,868, filed June 6, 1995, the disclosures of which are hereby incoφorated herein by reference, in their entireties. Additional fluorinated compounds which may be incoφorated in the present compositions are described for example, in Lohrmann, U.S. Patent No. 5,562,893, Reiss et al. U.S. Patent No. 5,344,930, Frezard, F., et al, Biochem Biophys Acta 1994, 1192:61-70, and Frezard, F., et al., Art. Cells Blood Subs and Immob Biotech. 1994, 22:1403-1408, the disclosures of each of which are also incoφorated herein by reference, in their entireties. Other fluorinated compounds which may be employed in the present compositions include liquid perfluorinated ether compounds, as well as liquid perfluorocarbons, such as for example, perfluorooctylbromide (PFOB), perfluorodecalin, perfluorododecalin, perfluorooctyliodide, perfluorotripropylamine, and perfluorotributylamine. In general, perfluorocarbons comprising about six or more carbon atoms will be liquids at normal human body temperature. Among these perfluorocarbons, perfluorooctylbromide and perfluorohexane, which are liquids at room temperature, are preferred. The gas which is present may be, for example, a perfluoroether compound, as well as other gases, such as nitrogen or perfluoropropane. Although not intending to be bound by any theory or theories of operation, it is believed that, in the case of vesicle compositions, the liquid fluorinated compound may be situated at the interface between the gas and the membrane or wall surface of the vesicle. There may be thus formed a further stabilizing layer of liquid fluorinated compound on the internal surface of the stabilizing compound, for example, a biocompatible lipid used to form the vesicle, and this perfluorocarbon layer may also prevent the gas from diffusing through the vesicle membrane.
Thus, a liquid fluorinated compound, such as a perfluorocarbon, when combined with a perfluorinated ether compound as described herein, may confer an added degree of stability. Thus, it is within the scope of the present invention to utilize a perfluoroether compound, for example, perfluorbutyl methyl ether, together with a perfluorocarbon which remains liquid after administration to a patient, that is, whose liquid to gas phase transition temperature is above the body temperature of the patient, for example, perfluorooctylbromide. Perfluorinated surfactants, such as ZONYL® fluorinated surfactants, may be used to improve the stability of the stabilized compositions, and to act, for example, as a coating for vesicles. Preferred perfluorinated surfactants are the partially fluorinated phosphocholine surfactants. In these preferred fluorinated surfactants, the dual alkyl compounds may be fluorinated at the terminal alkyl chains and the proximal carbons may be hydrogenated.
A desired component of the compositions of the present invention is an aqueous environment, particularly with respect to compositions comprising vesicles. Many of the stabilizing materials discussed above involve compounds which comprise both hydrophobic and hydrophilic properties. Accordingly, there can be a predisposition among the present to form vesicles, which are highly stable configurations 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 of dissolved salts which preferably will not interfere with the creation and maintenance of the stabilized compositions or their use, for example, as diagnostic agents, and normal saline and physiological saline. If desired, buffers may also be employed the aqueous medium, including phosphate buffers or tris(hydroxymethyl)aminomethane buffers.
The size of the vesicles and/or particles in vesicle compositions, emulsions and/or suspensions, whether in aqueous media or as a lypholizate, can be adjusted for the particular intended end use including, for example, diagnostic and/or therapeutic use. The vesicles and/or particles preferably have a mean diameter of less than about 100 microns, with a mean diameter of less than about 10 microns being more preferred. Even more preferred are mean diameters of from about 5 microns to about 10 microns. Also in preferred form, the diameter of over about 95%> of the particles and/or vesicles is less than about 10 microns. Indeed, it has been surprisingly discovered that over 98%> of the particles and/or vesicles in the compositions of the present invention have a diameter of less than about 10 microns. In connection with particular uses, for example, intravascular use, including magnetic resonance imaging of the vasculature, it may be preferred that the vesicles be no larger that about 30 μm in diameter, with smaller vesicles being preferred, for example, vesicles of no larger than about 12 μm in diameter. In certain preferred embodiments, the diameter of the vesicles may be about 7 μm or less, with vesicles having a mean diameter of about 5 μm or less being more preferred, and vesicles having a mean diameter of about 3 μm or less being even more preferred. It is contemplated that these smaller vesicles may perfuse small vascular channels, such as the microvasculature, while at the same time providing enough space or room within the vascular channel to permit red blood cells to slide past the vesicles.
The size of the gas filled vesicles can be adjusted, if desired, by a variety of procedures including, for example, shaking, microemulsification, vortexing, extrusion, filtration, sonication, homogenization, repeated freezing and thawing cycles, extrusion under pressure through pores of defined size, and similar methods. As noted above, compositions employed herein may also include, with respect to their preparation, formation and use, gaseous precursors that can be activated to change from a liquid or solid state into a gas by temperature (including energy, such as ultrasound), pH and light. The gaseous precursors may be made into gas by storing the precursors at reduced pressure. For example, a vial stored under reduced pressure may create a headspace of perfiuoropentane or perfluorohexane gas, useful for creating a preformed gas prior to injection. Preferably, the gaseous precursors may be activated by temperature. Set forth below is a table listing a series of gaseous precursors which undergo phase transitions from liquid to gaseous states at relatively close to normal body temperature (37°C) or below, and the size of the emulsified droplets that would be required to form a vesicle of a maximum size of 10 μm.
TABLE
Physical Characteristics of Gaseous Precursors and Diameter of Emulsified Droplet to Form a 10 μm Vesicle*
TABLE
Physical Characteristics of Gaseous Precursors and Diameter of Emulsified Droplet to Form a 10 μm Vesicle*
'Source: Chemical Rubber Company Handbook of Chemistry and Physics, Robert C. Weast and David R. Lide, eds., CRC Press, Inc. Boca Raton, Florida (1989-1990). As noted above, it is preferred to optimize the utility of the stabilized compositions, especially vesicle compositions formulated from lipids, by using gases of limited solubility. The phrase "limited solubility" refers to 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 may have a tendency to diffuse out of the vesicle. A lesser solubility in the aqueous milieu, may, on the other hand, decrease or eliminate the gradient between the vesicle and the interface such that diffusion of the gas out of the vesicle may be impeded. Preferably, the gas entrapped in the vesicle has a solubility less than that of oxygen, that is, about 1 part gas in about 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.
It may be desirable, in certain embodiments, to formulate vesicles from substantially impermeable polymeric materials. In these embodiments, it may be unnecessary to employ a gas which is highly insoluble also. For example, stable vesicle compositions which comprise substantially impermeable polymeric materials may be formulated with gases having higher solubilities, for example, air or nitrogen.
In addition to, or instead of, the lipid, proteinaceous and/or polymeric compounds discussed above, the compositions described herein may comprise one or more stabilizing materials. Exemplary of such stabilizing materials are, for example, biocompatible polymers. The stabilizing materials may be employed to desirably assist in the formation of vesicles and/or to assure substantial encapsulation of the gases or gaseous precursors. Even for relatively insoluble, non-diffusible gases, such as perfluoropropane or sulfur hexafluoride, improved vesicle compositions may be obtained when one or more stabilizing materials are utilized in the formation of the gas and gaseous precursor filled vesicles. These compounds may help improve 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 may be substantially resistant to degradation, including, for example, loss of vesicle structure or encapsulated gas or gaseous precursor, for a useful period of time.
Typically, the vesicles employed in the present invention have a desirable shelf life, often retaining at least about 90 %> by volume of its original structure for a period of at least about two to three weeks under normal ambient conditions. In preferred form, the vesicles are desirably stable for a period of time of at least about 1 month, more preferably at least about 2 months, even more preferably at least about 6 months, still more preferably about eighteen months, and yet more preferably up to about 3 years. The vesicles described herein, including gas and gaseous precursor filled vesicles, may also be stable 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 described herein may be attributable, at least in part, to the materials from which the vesicles are made, including, for example, the lipids, polymers and/or proteins described above, and it is often not necessary to employ additional stabilizing materials, although it is optional and may be preferred to do so. Such additional stabilizing materials and their characteristics are described more fully hereinafter.
The materials from which the vesicles are constructed are preferably biocompatible lipid, protein or polymer materials, and of these, the biocompatible lipids are preferred. In addition, because of the ease of formulation, including the capability of preparing vesicles immediately prior to administration, these vesicles may be conveniently made on site.
Particularly preferred embodiments of the present invention involve vesicles which comprise three components: (1) a neutral lipid, for example, a nonionic or zwitterionic lipid, (2) a negatively charged lipid, and (3) a lipid bearing a stabilizing material, for example, a hydrophilic polymer. Preferably, the amount of the negatively charged lipid will be greater than about 1 mole percent of the total lipid present, and the amount of lipid bearing a hydrophilic polymer will be greater than about 1 mole percent of the total lipid present. Exemplary and preferred negatively charged lipids include phosphatidic acids. The lipid bearing a hydrophilic polymer will desirably be a lipid covalently linked to the polymer, and the polymer will preferably have a weight average molecular weight of from about 400 to about 100,000, and all combinations and subcombinations of weight average molecular weights therein. Suitable hydrophilic polymers are preferably selected from the group consisting of polyethylene glycol (PEG), polypropylene glycol, poly(vinyl alcohol), and poly(vinyl pyrrolidone) and copolymers thereof, with PEG polymers being preferred. Preferably, the PEG polymer has a molecular weight of from about 1000 to about 10000, and all combinations and subcombinations of molecular weight ranges therein, with molecular weights of from about 2000 to about 8000 being more preferred. The PEG or other polymer may be bound to the lipid, for example, DPPE, through a covalent bond, such as an amide, carbamate or amine linkage. In addition, the PEG or other polymer may be linked to a targeting ligand, or other phospholipids, with a covalent bond including, for example, amide, ester, ether, thioester, thioamide or disulfide bonds. Where the hydrophilic polymer is PEG, a lipid bearing such a polymer will be said to be "pegylated." In preferred form, the lipid bearing a hydrophilic polymer may be DPPE-PEG, including, for example, DPPE-PEG5000, which refers to DPPE having a polyethylene glycol polymer of a mean weight average molecular weight of about 5000 attached thereto (DPPE-PEG5000). Another suitable pegylated lipid is distearoylphosphatidylethanol- amine-polyethylene glycol 5000 (DSPE-PEG5000).
In certain preferred embodiments of the present invention, the lipid compositions may include about 77.5 mole % DPPC, 12.5 mole % of DPP A, and 10 mole % of DPPE-PEG5000. Also preferred are compositions which comprise about 80 to about 90 mole % DPPC, about 5 to about 15 mole % DPPA and about 5 to about 15 mole % DPPE-PEG5000. Especially preferred are compositions which comprise DPPC, DPPA and DPPE-PEG5000 in a mole % ratio of 82: 10:8, respectively. DPPC is substantially neutral, since the phosphatidyl portion is negatively charged and the choline portion is positively charged. Consequently, DPPA, which is negatively charged, may be added to enhance stabilization in accordance with the mechanism described above. 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. The DPPE-PEG may provide more vesicles of a smaller size which are safe and stable to pressure when combined with other lipids, such as DPPC and DPPA, in the given ratios. It is also theorized that the pegylated material, because of its structural similarity to water, may be able to defeat 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 may function as diagnostic imaging contrast media.
The vesicle compositions may be prepared from other materials, in addition to the materials described above, provided that the vesicles so prepared meet the stability and other criteria set forth herein. These materials may be basic and fundamental, and form the primary basis for creating or establishing the stabilized gas and gaseous precursor filled vesicles. On the other hand, they may be auxiliary, and act as subsidiary or supplementary agents which can enhance the functioning of the basic stabilizing material or materials, or contribute some desired property in addition to that afforded by the basic stabilizing material.
However, it is not always possible to determine whether a given material is a basic or an auxiliary agent, since the functioning of the material in question is determined empirically, for example, by the results produced with respect to producing stabilized vesicles. As examples of how these basic and auxiliary materials may function, it has been observed that the simple combination of a biocompatible lipid and water or saline when shaken will often give a cloudy solution subsequent to autoclaving for sterilization. Such a cloudy solution may function as a contrast agent, but is aesthetically objectionable and may imply instability in the form of undissolved or undispersed lipid particles. Cloudy solutions may be also undesirable where the undissolved particulate matter has a diameter of greater than about 7 μm, and especially greater than about 10 μm. Manufacturing steps, such as sterile filtration, may also be problematic with solutions which contain undissolved particulate matter. Thus, propylene glycol may be added to remove this cloudiness by facilitating dispersion or dissolution of the lipid particles. The propylene glycol may also function as a wetting agent which can improve vesicle formation and stabilization by increasing the surface tension on the vesicle membrane or skin. It is possible that the propylene glycol can also function as an additional layer that may coat the membrane or skin of the vesicle, thus providing additional stabilization. As examples of such further basic or auxiliary stabilizing materials, there are conventional surfactants which may be used; see D'Arrigo U.S. Patents Nos. 4,684,479 and 5,215,680, the disclosures of which are hereby incoφorated herein by reference, in their entireties.
The compositions of the present invention may include as an auxiliary and/or basic stabilizing material an oil including, for example, vegetable oils and other oils, such as peanut oil, avocado oil, mineral oil, canola oil, soybean oil, olive oil, palm oil, palm seed oil, safflower oil, sunflower oil, sesame oil, corn oil, castor oil, coconut oil, cotttonseed oil, or any other oil commonly known to be ingestible which is suitable for use as a stabilizing compound in accordance with the teachings herein. Preferred oils include those which are hydrophobic aliphatic materials and which are generally liquids at room temperature. Compositions containing a perfluoroether compound and one or more oils, as well as additional stabilizing materials, if desired, may be formulated in a variety of ways, including, for example, as emulsions, suspensions, colloidal dispersions, and the like. In general, the perfluoroether compound may be combined with an oil in a weight ratio of perfluoroether compound:oil of about 0.001:1 to about 1 :1, and even up to about 10:1. Preparation of compositions of the perfluoroether compound and oils may involve, for example, mixing by agitation or emulsification. The oil-containing compositions may be stored as an oil in water emulsion or suspension. As indicated above, one or more additional stabilizing materials may be included in the oil-containing compositions, including, for example, phospholipids, fluorinated lipids/surfactants, and glycerol.
Various other auxiliary and basic stabilizing materials are disclosed, for example, in U.S. application Serial No. 08/444,574, filed May 19, 1995, the disclosures of which are incoφorated herein by reference, in their entirety.
In addition, compounds used to make mixed micelle systems may be suitable for use as basic or auxiliary stabilizing materials, and these include, for example, lauryltrimethylammonium bromide (dodecyl-), cetyltrimethylammonium bromide (hexadecyl-), myristyltrimethylammonium bromide (tetradecyl-), alkyldimethyl- benzylammonium chloride (where alkyl is C12, C14 or C,6,), benzyldimethyldodecyl- ammonium bromide/chloride, benzyldimethyl hexadecylammonium bromide/chloride, benzyldimethyl tetradecylammonium bromide/chloride, cetyldimethylethylammonium bromide/chloride, or cetylpyridinium bromide/chloride.
It has also been found 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 materials described herein. These materials can affect these parameters of the vesicles, especially vesicles formulated from lipids, not only by their physical interaction with the membranes, 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 modifiers, including, for example, carbohydrates and their phosphorylated and sulfonated derivatives; polyethers, preferably with molecular weight ranges between 400 and 100,000; and di- and trihydroxy alkanes and their polymers, preferably with molecular weight ranges between 200 and 50,000; (b) emulsifying and/or solubilizing agents including, for example, acacia, cholesterol, diethanolamine, glyceryl monostearate, lanolin alcohols, lecithin, mono- and di-glycerides, mono- ethanolamine, oleic acid, oleyl alcohol, poloxamer, for example, 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 sulfate, sodium stearate, sorbitan and sorbitan derivatives, including, for example, sorbitan mono-laurate, sorbitan mono- oleate, sorbitan mono-palmitate, and sorbitan monostearate, stearic acid, trolamine, and emulsifying wax; (c) suspending and/or viscosity-increasing agents, including, for example, acacia, agar, alginic acid, aluminum 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, Zeolites®, methylcellulose, pectin, polyethylene oxide, povidone, propylene glycol alginate, silicon dioxide, sodium alginate, tragacanth, xanthan gum, α-d-gluconolactone, glycerol and mannitol; (d) synthetic suspending agents, such as polyethylene glycol (PEG), poly(vinyl pyrrolidone) (PVP), poly(vinyl alcohol) (PVA), poly(propylene glycol) (PPG), and polysorbate; and (e) tonicity raising agents which stabilize and add tonicity, including, for example, sorbitol, mannitol, trehalose, sucrose, propylene glycol and glycerol.
A wide variety of methods are available for the preparation of stabilized compositions, including lipid and/or vesicle compositions, such as micelles and/or liposomes. Included among these methods are, for example, shaking, drying, gas- installation, spray drying, and the like. Spray drying may be used with a variety of stabilizing materials, including, for example, lipids, such as phospholipids, proteins, such as albumin, synthetic polymers, such as polylactide, and the like. Spray drying may also involve the preparation of an emulsion or suspension of stabilizing agents which contain cores of a volatile blowing agent. In this technique, the volatile blowing agent desirably expands as the compositions are spray dried. This causes the creation of a central void space within the vesicles. Dried samples of the resulting vesicles may be stored, for example, as a powder under a headspace of a suitable perfluoroether gas. Volatile blowing agents which may be used in the cores in this spray drying technique include, for example, methylene chloride, isobutane, and various of the perfluoroether compounds described herein, including, for example, perfluoromethyl butyl ether. Other suitable methods for preparing vesicle compositions from lipids are described, for example, in Unger et al., U.S. Patent No. 5,469,854, the disclosures of which are incoφorated herein by reference. As noted above, the vesicles are preferably prepared from lipids which remain in the gel state.
With particular reference to the preparation of micelle compositions, the following discussion is provided. Micelles may be prepared using any one of a variety of conventional micellar preparatory methods which will be apparent to those skilled in the art. These methods typically involve suspension of one or more lipid compounds in an organic solvent, evaporation of the solvent, resuspension in an aqueous medium, sonication and centrifugation. The foregoing methods, as well as others, are discussed, for example, in Canfield et al., Methods in Enzymology, Vol. 189, pp. 418-422 (1990); El-Gorab et al, Biochem. Biophys. Acta, Vol. 306, pp. 58-66 (1973); Colloidal
Surfactant, Shinoda, K., Nakagana, Tamamushi and Isejura, Academic Press, NY (1963) (especially "The Formation of Micelles", Shinoda, Chapter 1, pp. 1-88); Catalysis in Micellar and Macromolecular Systems, Fendler and Fendler, Academic Press, NY (1975). The disclosures of each of the foregoing publications are incoφorated by reference herein, in their entirety.
As noted above, the vesicle composition may comprise liposomes. A wide variety of methods are available in connection with the preparation of liposome compositions. Accordingly, the liposomes may be prepared using any one of a variety of conventional liposomal preparatory techniques which will be apparent to those skilled in the art. These techniques include, for example, solvent dialysis, French press, extrusion (with or without freeze-thaw), reverse phase evaporation, simple freeze-thaw, sonication, chelate dialysis, homogenization, solvent infusion, microemulsification, spontaneous formation, solvent vaporization, solvent dialysis, French pressure cell technique, controlled detergent dialysis, and others, each involving the preparation of the vesicles in various fashions. See, e.g., Madden et al., Chemistry and Physics of Lipids, 1990 53, 37-46, the disclosures of which are hereby incoφorated herein by reference in their entirety. Suitable freeze-thaw techniques are described, for example, in International Application Serial No. PCT/US89/05040, filed November 8, 1989, the disclosures of which are incoφorated herein by reference in their entirety. Methods which involve freeze-thaw techniques are preferred in connection with the preparation of liposomes. Preparation of the liposomes may be carried out in a solution, such as an aqueous saline solution, aqueous phosphate buffer solution, or sterile water. The liposomes may also be prepared by various processes which involve shaking or vortexing. This may be achieved, for example, by the use of a mechanical shaking device, such as a Wig-L-Bug™ (Crescent Dental, Lyons, IL), a Mixomat (Degussa AG, Frankfurt, Germany), a Capmix (Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany), a Silamat Plus (Vivadent, Lechtenstein), or a Vibros (Quayle Dental, Sussex, England). Conventional microemulsification equipment, such as a Microfluidizer™ (Microfluidics, Woburn, MA) may also be used.
Spray drying may be also employed to prepare the gas-filled vesicles. Utilizing this procedure, the lipids may be pre-mixed in an aqueous environment and then spray dried to produce gas-filled vesicles. The vesicles may be stored under a headspace of a desired gas.
Many liposomal preparatory techniques which may be adapted for use in the preparation of vesicle compositions are discussed, for example, in U.S. Patent No. 4,728,578; U.K. Patent Application GB 2193095 A; U.S. Patent No. 4,728,575; U.S. Patent No. 4,737,323; International Application Serial No. PCT/US85/01161; Mayer et al., Biochimica et Biophysica Acta, Vol. 858, pp. 161-168 (1986); Hope et al., Biochimica et Biophysica Acta, Vol. 812, pp. 55-65 (1985); U.S. Patent No. 4,533,254; Mayhew et al., Methods in Enzymology, Vol. 149, pp. 64-77 (1987); Mayhew et al, Biochimica et Biophysica Acta, Vol 755, pp. 169-74 (1984); Cheng et al, Investigative Radiology, Vol. 22, pp. 47-55 (1987); International Application Serial No.
PCT/US89/05040; U.S. Patent No. 4,162,282; U.S. Patent No. 4,310,505; U.S. Patent No. 4,921,706; and Liposome Technology, Gregoriadis, G., ed., Vol. I, pp. 29-31, 51-67 and 79-108 (CRC Press Inc., Boca Raton, FL 1984), the disclosures of each of which are hereby incoφorated by reference herein, in their entirety. Stabilized compositions comprising a gas can be prepared by agitating an aqueous solution containing, if desired, a stabilizing material, in the presence of a gas and/or a gaseous precursor. The term "agitating," as used throughout the present disclosure, means any shaking motion of an aqueous solution such that a gas which is proximate to the environment of the aqueous solution may be introduced into the aqueous solution. The term "agitating", as used herein, also means any shaking motion of an aqueous solution which results in the conversion of a gaseous precursor, including the perfluorinated ether compounds described herein, to a gas. This agitation is preferably conducted at a temperature below the gel to liquid crystalline phase transition temperature of the lipid. The agitation is also preferably conducted under reduced pressures within a sealed container. Preferred reduced pressures are from about 5 torr up to about 760 torr, and all combinations and subcombinations of ranges therein. More preferably, the agitation may be conducted at a pressure of from about 10 torr to about 400 torr, with pressures of from about 15 torr to about 300 torr being even more preferred. The shaking involved in the agitation of the solutions is preferably of sufficient force to result in the formation of a stabilized composition, including lipid compositions, particularly vesicle compositions, and especially vesicle compositions comprising gas filled vesicles. The shaking may be by swirling, such as by vortexing, side-to-side, or up and down motion. Different types of motion may be combined. Also, the shaking may occur by shaking the container containing the aqueous solution which contains the stabilizing compound, or by shaking the aqueous solution within the container without shaking the container itself. 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, as well as any of the shaking devices described hereinbefore, with the Capmix (Espe Fabrik Pharmazeutischer Praeparate GMBH & Co., Seefeld, Oberay Germany) being preferred. It has been found that certain modes of shaking or vortexing can be used to make vesicles within a preferred size range. Shaking is preferred, and it is preferred that the shaking be carried out using the Espe Capmix mechanical shaker. In accordance with this preferred method, it is preferred that a reciprocating motion be utilized to generate the lipid compositions, and particularly vesicle compositions. It is even more preferred that the motion be reciprocating in the form of an arc. It is contemplated that the rate of reciprocation, as well as the arc thereof, is particularly important in connection with the formation of vesicles. Preferably, the number of reciprocations or full cycle oscillations may be from about 1000 to about 20,000 per minute. More preferably, the number of reciprocations or oscillations may be from about 2500 to about 8000 per minute, with from about 3300 to about 5000 reciprocations or oscillations per minute being even more preferred. Of course, the number of oscillations may be dependent upon the mass of the contents being agitated. Generally speaking, a larger mass may require fewer oscillations. Another means for producing 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 may be correspondingly increased. Vigorous shaking is defined as at least about 60 shaking motions per minute, and is preferred. Vortexing at about 60 to about 300 revolutions per minute is more preferred. Vortexing at about 300 to about 1800 revolutions per minute is even more preferred.
In addition to the simple shaking methods described above, more elaborate methods can also be employed. Such elaborate methods include, for example, liquid crystalline shaking gas instillation processes and vacuum drying gas instillation processes, such as those described in copending U.S. application Serial No. 08/076,250, filed June 11, 1993, the disclosures of which are incoφorated herein by reference, in their entirety. Although any of a number of varying techniques can be used, the vesicle compositions employed in the present invention are preferably prepared using a shaking technique. Preferably, the shaking technique involves agitation with a mechanical shaking apparatus, such as an Espe Capmix (Seefeld, Oberay Germany), using, for example, the techniques disclosed in copending U.S. application Serial No. 160,232, filed November 30, 1993, the disclosures of which are hereby incoφorated herein by reference in their entirety. The size of gas filled vesicles can be adjusted, if desired, by a variety of procedures, including, for example, microemulsification, vortexing, extrusion, filtration, sonication, homogenization, repeated freezing and thawing cycles, extrusion under pressure through pores of defined size, and similar methods. Gas filled vesicles prepared in accordance with the methods described herein can range in size from less than about 1 μm to greater than about 100 μm. In addition, after extrusion and sterilization procedures, which are discussed in detail below, agitation or shaking may provide vesicle compositions which can contain substantially no or minimal residual anhydrous lipid phase in the remainder of the solution. (Bangham, A.D., Standish, M.M, & Watkins, J.C., J. Mol. Biol. Vol. 13, pp. 238-252 (1965). If desired, the vesicles may be used as they are formed, without any attempt at further modification of the size thereof. For intravascular use, the vesicles preferably have diameters of less than about 30 μm, and more preferably, less than about 12 μm. For targeted intravascular use including, for example, binding to certain tissue, such as cancerous tissue, the vesicles may be significantly smaller, for example, less than about 100 nm in diameter. For enteric or gastrointestinal use, the vesicles may be significantly larger, for example, up to a millimeter in size. Preferably, the vesicles may be sized to have diameters of from about 2 μm to about 100 μm.
The gas filled vesicles may be sized by a simple process of extrusion through filters wherein the filter pore sizes control the size distribution of the resulting gas filled vesicles. By using two or more cascaded or stacked set of filters, for example, a 10 μm filter followed by an 8 μm filter, the gas filled vesicles can be selected to have a very narrow size distribution around 7 to 9 μm. After filtration, these gas filled vesicles can remain stable for over 24 hours.
The sizing or filtration step may be accomplished by the use, for example, of a filter assembly when the composition is removed from a sterile vial prior to use, or more preferably, the filter assembly may be incoφorated into a syringe 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 may be carried out by a step of extracting which comprises extruding the vesicles from the barrel through the filter fitted to the syringe between the barrel and the needle, thereby sizing the vesicles before they are administered to a patient. The step of extracting may also comprise drawing the vesicles into the syringe, where the filter may 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 may function to ensure that only vesicles within the desired size range, or of the desired maximum size, are subsequently administered by extrusion from the syringe. In certain preferred embodiments, the vesicle compositions may be heat sterilized or filter sterilized and extruded through a filter prior to shaking. Generally speaking, vesicle compositions comprising a gas may be heat sterilized, and vesicle compositions comprising gaseous precursors may be filter sterilized. Once gas filled vesicles are formed, they may be filtered for sizing as described above. Performing these steps prior to the formation of gas and gaseous precursor filled vesicles provide 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 stabilized composition, including lipid and/or vesicle, compositions, and the composition may be sterilized within the mixing vessel, for example, by autoclaving. Gas may be instilled into the composition 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 step of extruding the solution of lipid compound through a filter decreases the amount of unhydrated material by breaking up any dried materials and exposing a greater surface area for hydration. Preferably, the filter has a pore size of about 0.1 to about 5 μm, more preferably, about 0.1 to about 4 μm, even more preferably, about 0.1 to about 2 μm, and still more preferably, about 1 μm. Unhydrated compound, which is generally undesirable, appears as amoφhous clumps of non- uniform size.
The sterilization step provides a composition that may be readily administered to a patient for diagnostic imaging including, for example, ultrasound or CT. In certain preferred embodiments, sterilization may be accomplished by heat sterilization, preferably, by autoclaving the solution at a temperature of at least about 100°C, and more preferably, by autoclaving at about 100°C to about 130°C, even more preferably, about 110°C to about 130°C, still more preferably, about 120°C to about 130°C, and even more preferably, 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 still more preferably, about 15 minutes.
If desired, the extrusion and heating steps, as outlined above, may be reversed, or only one of the two steps can be used. Other modes of sterilization may be used, including, for example, exposure to gamma radiation. In addition to the aforementioned embodiments, gaseous precursors contained in vesicles can be formulated which, upon activation, for example, by exposure to elevated temperature, varying pH, or light, may undergo a phase transition from, for example, a liquid, including a liquid entrapped in a vesicle, to a gas, expanding to create the gas filled vesicles described herein. This technique is described in detail in copending patent applications Serial Nos. 08/160,232, filed November 30, 1993 and 08/159,687, filed November 30, 1993, the disclosures of which are incoφorated herein by reference, in their entireties. The preferred method of activating the gaseous precursor is by exposure to elevated temperature. Activation or transition temperature, and like terms, refer to the boiling point of the gaseous precursor and is the temperature at which the liquid to gaseous phase transition of the gaseous precursor takes place. Useful gaseous precursors are those materials which have boiling points in the range of about -100°C to about 70°C. The activation temperature is particular to each gaseous precursor. An activation temperature of about 37°C, or about human body temperature, is preferred for gaseous precursors in the context of the present invention. Thus, in preferred form, a liquid gaseous precursor is activated to become a gas at about 37°C or below. The gaseous precursor may be in liquid or gaseous phase for use in the methods of the present invention.
The methods of preparing the gaseous precursor filled vesicles may be carried out below the boiling point of the gaseous precursor such that a liquid is incoφorated, for example, into a vesicle. In addition, the methods may be conducted at the boiling point of the gaseous precursor, such that a gas is incoφorated, for example, 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 situ in the targeted tissue or fluid, in 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 may be 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 may be added to a container housing a lipid composition at a temperature below the liquid-gaseous phase transition temperature of the respective gaseous precursor. As the temperature is increased, and an emulsion is formed 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 head space above the liquid mixture so as to form gas filled vesicles which may entrap the gas of the gaseous precursor, ambient gas (e.g. air), or coentrap gas state gaseous precursor and ambient air. This phase transition can be used for optimal mixing and formation of the contrast agent. For example, the gaseous precursor, perfluorobutane, can be entrapped in the lipid vesicles and as the temperature is raised beyond the boiling point of perfluorobutane (4°C), perfluorobutane gas is entrapped in the vesicles. Accordingly, the gaseous precursors may be selected to form gas filled vesicles in vivo or may be designed to produce the gas filled vesicles in situ, during the manufacturing process, on storage, or at some time prior to use.
As a further embodiment of this invention, by pre-forming the gaseous precursor in the liquid state into an aqueous emulsion, the maximum size of the vesicle may be estimated by using the ideal gas law, once the transition to the gaseous state is effectuated. For the puφose of making gas filled vesicles from gaseous precursors, the gas phase may be assumed to form instantaneously and substantially no gas in the newly formed vesicle has been depleted due to diffusion into the liquid, which is generally aqueous in nature. Hence, from a known liquid volume in the emulsion, one may predict an upper limit to the size of the gas filled vesicle.
In embodiments of the present invention, a mixture of a lipid compound and a gaseous precursor, containing liquid droplets of defined size, may be formulated such that upon reaching a specific temperature, for example, the boiling point of the gaseous precursor, the droplets may expand into gas filled vesicles of defined size. The defined size may represent an upper limit to the actual size because the ideal gas law generally cannot account for such factors as gas diffusion into solution, loss of gas to the atmosphere, and the effects of increased pressure. The ideal gas law, which can be used for calculating the increase in the volume of the gas bubbles upon transitioning from liquid to gaseous states, is as follows:
PV = nRT where
P is pressure in atmospheres (atm); V is volume in liters (L); n is moles of gas;
T is temperature in degrees Kelvin (K); and R is the ideal gas constant (22.4 L-atm/K-mole).
With knowledge of volume, density, and temperature of the liquid in the mixture of liquids, the amount, for example, in moles, and volume of liquid precursor may be calculated which, when converted to a gas, may expand into a vesicle of known volume. The calculated volume may reflect an upper limit to the size of the gas filled vesicle, assuming instantaneous expansion into a gas filled vesicle and negligible diffusion of the gas over the time of the expansion.
Thus, for stabilization of the precursor in the liquid state in a mixture wherein the precursor droplet is spherical, the volume of the precursor droplet may be determined by the equation: Volume (spherical vesicle) = 4/3 πr3 where
r is the radius of the sphere.
Thus, once the volume is predicted, and knowing the density of the liquid at the desired temperature, the amount of liquid gaseous precursor in the droplet may be determined. In more descriptive terms, the following can be applied:
Vgas = 4/3 π^ 3 by the ideal gas law,
PV=nRT substituting reveals, Vgas = nRT/Pgas or,
(A) n = 4/3 [πrgas 3] P/RT amount n = 4/3 [πrgas 3 P/RT]«MWn Converting back to a liquid volume (B) Vlιq = [4/3 [πrgas 3] P/RT].MWn D] where D is the density of the precursor. Solving for the diameter of the liquid droplet, (C) diameter/2 = [3/4π [4/3»[πrgas 3] P/RT] MWn/D]1/3 which reduces to Diameter = 2[[rgas 3] P/RT [MWn/D]],/3.
As a further means of preparing vesicles of the desired size for use in the methods of the present invention, and with a knowledge of the volume and especially the radius of the liquid droplets, one can use appropriately sized filters to size the gaseous precursor droplets to the appropriate diameter sphere. A representative gaseous precursor may be used to form a vesicle of defined size, for example, 10 μm diameter. In this example, the vesicle may be formed in the bloodstream of a human being, thus the typical temperature would be 37°C or 310 K. At a pressure of 1 atmosphere and using the equation in (A), 7.54 x 10"17 moles of gaseous precursor may be required to fill the volume of a 10 μm diameter vesicle. Using the above calculated amount of gaseous precursor and
1 -fluorobutane, which possesses a molecular weight of 76.11, a boiling point of 32.5°C and a density of 0.7789 g/mL at 20°C, further calculations predict that 5.74 x 10"15 grams of this precursor may be required for a 10 μm vesicle. Extrapolating further, and with the knowledge of the density, equation (B) further predicts that 8.47 x 10"16 mL of liquid precursor may be necessary to form a vesicle with an upper limit of 10 μm.
Finally, using equation (C), a mixture, for example, an emulsion containing droplets with a radius of 0.0272 μm or a corresponding diameter of 0.0544 μm, may be formed to make a gaseous precursor filled vesicle with an upper limit of a 10 μm 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 of defined size, the size of the filter may also suffice to remove any possible bacterial contaminants and, hence, can be used as a sterile filtration as well.
This embodiment for preparing gas filled vesicles may be applied to all gaseous precursors activated by temperature. In fact, depression of the freezing point of the solvent system allows the use of gaseous precursors which may undergo liquid-togas 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 buffered saline exhibits a freezing point depression well below the freezing point of water alone. By increasing the amount of propylene glycol or adding materials such as sodium chloride, the freezing point can be depressed even further.
The selection of appropriate solvent systems may be determined by physical methods as well. When substances, solid or liquid, herein referred to as solutes, are dissolved in a solvent, such as water based buffers, the freezing point may be 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:
Inxβ = In (l - ) = ΔHflB /R(l/TJ - 1/T) where xa is the mole fraction of the solvent; xb is the mole fraction of the solute; ΔHfus is the heat of fusion of the solvent; and T0 is the normal freezing point of the solvent. The normal freezing point of the solvent can be obtained by solving the equation. If xb is small relative to xa, then the above equation may be rewritten as follows. xb = ΔH^/RΓ - T0/T0T] « ΔHfusΔT/RT0 2 The above equation assumes the change in temperature ΔT is small compared to T2. This equation can be simplified further by expressing the concentration of the solute in terms of molality, m (moles of solute per thousand grams of solvent). Thus, the equation can be rewritten as follows.
Xb =m/[m + 1000/m ~ mMa/1000 where Ma is the molecular weight of the solvent. Thus, substituting for the fraction xb: ΔT = [MaRTo 2/1000ΔHfus]m or ΔT = K,m, where
Kf=MaRTo 2/1000ΔHfus Kf is 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 solutions of gaseous-precursor filled vesicles. Accordingly, the above 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 and/or shaking an aqueous mixture of gaseous precursor and additional materials as desired, including, for example, stabilizing materials, thickening agents and/or dispersing agents. Optional variations of this method include autoclaving before vortexing or shaking; heating an aqueous mixture of gaseous precursor; venting the vessel containing the mixture/suspension; shaking or permitting the gaseous precursor filled vesicle to form spontaneously and cooling down the suspension of gaseous precursor filled vesicles; and extruding an aqueous suspension of gaseous precursor through a filter of about 0.22 μm. Alternatively, filtering may be performed during in vivo administration of the vesicles such that a filter of about 0.22 μm is employed; (b) microemulsification, whereby an aqueous mixture of gaseous precursor is emulsified by agitation and heated to form, for example, vesicles prior to administration to a patient;
(c) heating a gaseous precursor in a mixture, with or without agitation, whereby the less dense gaseous precursor filled vesicles may float to the top of the solution by expanding and displacing other vesicles in the vessel and venting the vessel to release air; (d) utilizing in .any of the above methods a sealed vessel to hold the aqueous suspension of gaseous precursor and maintaining the suspension at a temperature below the phase transition temperature of the gaseous precursor, followed by autoclaving to raise the temperature above the phase transition temperature, optionally with shaking, or permitting the gaseous precursor vesicle to form spontaneously, whereby the expanded gaseous precursor in the sealed vessel increases the pressure in the vessel, and cooling down the gas filled vesicle suspension, after which shaking may also take place; and
(e) spray drying as described above. Freeze drying may be useful to remove water and organic materials prior to the shaking instillation method. Drying instillation methods may be used to remove water from vesicles. By pre-entrapping the gaseous precursor in the dried vesicles (i.e. prior to drying) after warming, the gaseous precursor may expand to fill the vesicle. Gaseous precursors can also be used to fill dried vesicles after 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 filled with the gaseous precursor in its gaseous state. For example, perfluorobutane can be used to fill dried vesicles at temperatures above 4°C (the boiling point of perfluorobutane).
Preferred methods for preparing the temperature activated gaseous precursor filled vesicles comprise shaking an aqueous solution having a lipid compound in the presence of a gaseous precursor at a temperature below the liquid state to gas state phase transition temperature of the gaseous precursor. This is preferably conducted at a temperature below the gel state to liquid crystalline state phase transition temperature of the lipid. The mixture may be then heated to a temperature above the liquid state to gas state phase transition temperature of the gaseous precursor which can cause the precursor to volatilize and expand. Heating may be then discontinued, and the temperature of the mixture may be allowed to drop below the liquid state to gas state phase transition temperature of the gaseous precursor. Shaking of the mixture may take place during the heating step, or subsequently after the mixture is allowed to cool. Other methods for preparing gaseous precursor filled vesicles can involve shaking an aqueous solution of, for example, a lipid and a gaseous precursor, and separating the resulting gaseous precursor filled vesicles. Conventional, aqueous-filled liposomes of the prior art are routinely formed at a temperature above the phase transition temperature of 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 certain preferred embodiments described herein are gaseous precursor filled, which imparts greater flexibility, since gaseous precursors after gas formation are more compressible and compliant than an aqueous solution.
The preparatory methods may involve shaking an aqueous solution comprising a lipid, in the presence of a temperature activatable gaseous precursor.
Preferably, the shaking is of sufficient force such that a foam is formed within a short period of time, such as about 30 minutes, and preferably within about 20 minutes, and more preferably, within about 10 minutes. The shaking may involve microemulsifying, microfluidizing, swirling (such as by vortexing), side-to-side, or up and down motion. In the case of the addition of gaseous precursor in the liquid state, sonication may be used 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 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, the mechanical shakers described hereinbefore, with an Espe Capmix (Seefeld, Oberay Germany) being preferred. Another means for producing shaking includes the action of gaseous precursor emitted under high velocity or pressure.
According to the methods described herein, a gas, such as air, may also be provided by the local ambient atmosphere. The local ambient atmosphere can include the atmosphere within a sealed container, as well as 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 to provide a gas other than air. Gases that are lighter than air are generally added to a sealed container, while gases heavier than air can be added to a sealed or an unsealed container. Accordingly, the present invention includes co-entrapment of air and/or other gases along with gaseous precursors. Hence, the gaseous precursor filled vesicles can be used in substantially the same manner as the gas filled vesicles described herein, 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 the gaseous precursors undergo phase transitions from liquid to gaseous states at near the normal body temperature of the host, and are thereby activated, for example, by the in vivo temperature of the host so as to undergo transition to the gaseous phase therein. This can occur where, for example, the host tissue is human tissue having a normal temperature of about 37°C and the gaseous precursors undergo phase transitions from liquid to gaseous states near 37°C.
As noted above, the lipid and/or vesicle compositions may be sterilized by autoclave or sterile filtration if these processes are performed before the instillation step or prior to temperature mediated conversion of the temperature sensitive gaseous precursors within the compositions. Alternatively, one or more anti-bactericidal agents and/or preservatives may be included in the formulation of the compositions, such as sodium benzoate, 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, may be necessary where the stabilized vesicles are used for imaging under invasive circumstances, for example, intravascularly or intraperitonealy. The appropriate means of sterilization will be apparent to the artisan based on the present disclosure. Vesicle compositions which comprise vesicles formulated from proteins
(also referred to as protein encapsulated microbubbles), such as albumin vesicles, may be prepared by various processes, as will be readily apparent to those skilled in the art, once armed with the present disclosure. Suitable methods include those described, for example, in Feinstein, U.S. Patent Nos. 4,572,203, 4,718,433 and 4,774,958, and Cerny et al., U.S. Patent No. 4,957,656, the disclosures of which are hereby incoφorated herein by reference, in their entireties. Included among the methods described in the aforementioned patents for the preparation of protein-based vesicles are methods which involve sonicating a solution pf a protein. In preferred form, the starting material may be an aqueous solution of a heat-denaturable, water-soluble biocompatible protein. The encapsulating protein is preferably heat-sensitive so that it can be partially insolubilized by heating during sonication. Suitable heat- sensitive proteins include, for example, albumin, hemoglobin, collagen, and the like. Preferably, the protein is a human protein, with human serum albumin (HSA) being more preferred. HSA is available commercially as a sterile 5% aqueous solution, which is suitable for use in the preparation of protein-based vesicles. Of course, as would be apparent to one of ordinary skill in the art, other concentrations of albumin, as well as other proteins which are heat-denaturable, can be used to prepare the vesicles. Generally speaking, the concentration of HSA can vary and may range from about 0.1 to about 25%> by weight, and all combinations and subcombinations of ranges therein. It may be preferable, in connection with certain methods for the preparation of protein-based vesicles, to utilize the protein in the form of a dilute aqueous solution. For albumin, it may be preferred to utilize an aqueous solution containing from about 0.5 to about 7.5%> by weight albumin, with concentrations of less than about 5% by weight being preferred, for example, from about 0.5 to about 3%> by weight.
The protein-based vesicles may be prepared using equipment which is commercially available. For example, in connection with a feed preparation operation as disclosed, for example, in Cerny, et al., U.S. Patent No. 4,957,656, stainless steel tanks which are commercially available from Walker Stainless Equipment Co. (New Lisbon, WI), and process filters which are commercially available from Millipore (Bedford, MA), may be utilized.
The sonication operation may utilize both a heat exchanger and a flow through sonicating vessel, in series. Heat exchanger equipment of this type may be obtained from ITT Standard (Buffalo, NY). The heat exchanger maintains operating temperature for the sonication process, with temperature controls ranging from about 65 °C to about 80°C, depending on the makeup of the media. The vibration frequency of the sonication equipment may vary over a wide range, for example, from about 5 to about 40 kilohertz (kHz), with a majority of the commercially available sonicators operating at about 10 or 20 kHz. Suitable sonicating equipment include, for example, a Sonics & Materials Vibra-Cell, equipped with a flat-tipped sonicator horn, commercially available from Sonics & Materials, Inc. (Danbury, CT). The power applied to the sonicator horn can be varied over power settings scaled from 1 to 10 by the manufacturer, as with Sonics & Materials Vibra-Cell Model VL1500. An intermediate power setting, for example, from 5 to 9, can be used. It is preferred that the vibrational frequency and the power supplied be sufficient to produce cavitation in the liquid being sonicated. Feed flow rates may range from about 50 mL/min to about 1000 mL/min, and all combinations and subcombinations of ranges therein. Residence times in the sonication vessel can range from about 1 second to about 4 minutes, and gaseous fluid addition rates may range from about 10 cubic centimeters (cc) per minute to about 100 cc/min, or 5%> to 25%) of the feed flow rate, and all combinations and subcombinations of ranges therein.
It may be preferable to carry out the sonication in such a manner to produce foaming, and especially intense foaming, of the solution. Generally speaking, intense foaming and aerosolating are important for obtaining a contrast agent having enhanced concentration and stability. To promote foaming, the power input to the sonicator horn may be increased, and the process may be operated under mild pressure, for example, about 1 to about 5 psi. Foaming may be easily detected by the cloudy appearance of the solution, and by the foam produced.
Suitable methods for the preparation of protein-based vesicles may also involve physically or chemically altering the protein or protein derivative in aqueous solution to denature or fix the material. For example, protein-based vesicles may be prepared from a 5%> aqueous solution of HSA by heating after formation or during formation of the contrast agent via sonication. Chemical alteration may involve chemically denaturing or fixing by binding the protein with a difunctional aldehyde, such as gluteraldehyde. For example, the vesicles may be reacted with 0.25 grams of 50%) aqueous gluteradehyde per gram of protein at pH 4.5 for 6 hours. The unreacted gluteraldehyde may then be washed away from the protein.
Vesicle compositions which comprise vesicles formulated from polymers may be prepared by various processes, as will be readily apparent to those skilled in the art, once armed with the present disclosure. Exemplary processes include, for example, interfacial polymerization, phase separation and coacervation, multiorifice centrifugal preparation, and solvent evaporation. Suitable procedures which may be employed or modified in accordance with ihe present disclosure to prepare vesicles from polymers include those procedures disclosed in Garner et al, U.S. Patent No. 4,179,546, Garner, U.S. Patent No. 3,945,956, Cohrs et al., U.S. Patent No. 4,108,806, Japan Kokai Tokkyo Koho 62 286534, British Patent No. 1,044,680, Kenaga et al., U.S. Patent No. 3,293,114, Morehouse et al., U.S. Patent No. 3,401,475, Walters, U.S. Patent No.
3,479,811, Walters et al., U.S. Patent No. 3,488,714, Morehouse et al., U.S. Patent No. 3,615,972, Baker et al., U.S. Patent No. 4,549,892, Sands et al., U.S. Patent No. 4,540,629, Sands et al., U.S. Patent No. 4,421,562, Sands, U.S. Patent No. 4,420,442, Mathiowitz et al., U.S. Patent No. 4,898,734, Lencki et al., U.S. Patent No. 4,822,534, Herbig et al., U.S. Patent No. 3,732,172, Himmel et al., U.S. Patent No. 3,594,326, Sommerville et al., U.S. Patent No. 3,015,128, Deasy, Microencapsulation and Related Drug Processes, Vol. 20, Chs. 9 and 10, pp. 195-240 (Marcel Dekker, Inc., N.Y., 1984), Chang et al., Canadian J. of Physiology and Pharmacology, Vol 44, pp. 115-129 (1966), and Chang, Science, Vol. 146, pp. 524-525 (1964), the disclosures of each of which are incoφorated herein by reference in their entireties.
In accordance with a preferred synthesis protocol, the vesicles may be prepared using a heat expansion process, such as, for example, the process described in Garner et al., U.S. Patent No. 4,179,546, Garner, U.S. Patent No. 3,945,956, Cohrs et al., U.S. Patent No. 4,108,806, British Patent No. 1,044,680, and Japan Kokai Tokkyo Koho 62 286534. In general terms, the heat expansion process may be carried out by preparing vesicles of an expandable polymer or copolymer which may contain in their void (cavity) a volatile liquid (gaseous precursor). The vesicle is then heated, plasticising the vesicle and converting the volatile liquid into a gas, causing the vesicle to expand to up to about several times its original size. When the heat is removed, the thermoplastic polymer retains at least some of its expanded shape. Vesicles produced by this process tend to be of particularly low density, and are thus preferred. The foregoing described process is well known in the art, and may be referred to as the heat expansion process for preparing low density vesicles.
Polymers useful in the heat expansion process will be readily apparent to those skilled in the art and include thermoplastic polymers or copolymers, including polymers or copolymers of many of the monomers described above. Preferable of the polymers and copolymers described above include the following copolymers: polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate, and polystyrene-polyacrylonitrile. A most preferred copolymer is polyvinylidene-polyacrylonitrile.
Volatile liquids useful in the heat expansion process will also be well known to those skilled in the art and include: aliphatic hydrocarbons such as ethane, ethylene, propane, propene, butane, isobutane, neopentane, acetylene, hexane, heptane; chlorofluorocarbons such as CC13F, CC12F3, CC1F3, CC1F2-CC12F2, chloroheptafluoro- cyclobutane, and 1,2-dichlorohexafluorocyclobutane; tetraalkyl silanes, such as tetramethyl silane, trimethylethyl silane, trimethylisopropyl silane, and trimethyl n- propyl silane; as well as perfluorocarbons, including the perfluorocarbons described above. In general, it is important that the volatile liquid not be a solvent for the polymer or copolymer being utilized. It is also preferred that the volatile liquid have a boiling point that is below the softening point of the involved polymer or co-polymer. Boiling points of various volatile liquids and softening points of various polymers and copolymers will be readily ascertainable to one skilled in the art, and suitable combinations of polymers or copolymers and volatile liquids will be easily apparent to the skilled artisan. By way of guidance, and as one skilled in the art would recognize, generally as the length of the carbon chain of the volatile liquid increases, the boiling point of that liquid increases also. Also, mildly preheating the vesicles in water in the presence of hydrogen peroxide prior to definitive heating and expansion may pre-soften the vesicle to allow expansion to occur more readily.
For example, to produce vesicles from synthetic polymers, vinylidene and acrylonitrile may be copolymerized in a medium of isobutane liquid using one or more of the foregoing modified or unmodified literature procedures, such that isobutane becomes entrapped within the vesicles. When such vesicles are then heated to a temperature of from about 80°C to about 120°C, the isobutane gas expands, which in turn expands the vesicles. After heat is removed, the expanded polyvinylidene and acrylo-nitrile copolymer vesicles remain substantially fixed in their expanded position. The resulting low density vesicles are extremely stable both dry and suspended in an aqueous media. Isobutane is utilized herein merely as an illustrative liquid, with the understanding that other liquids which undergo liquid/gas transitions at temperatures useful for the synthesis of these vesicles and formation of the very low density vesicles upon heating can be substituted for isobutane. Similarly, monomers other than vinylidene and acrylonitrile may be employed in preparing the vesicles.
In certain preferred embodiments, the vesicles which are formulated from synthetic polymers and which may be employed in the methods of the present invention are commercially available from Expancel, Nobel Industries (Sundsvall, Sweden), including EXPANCEL 551 DE™ microspheres. The EXPANCEL 551 DE™ microspheres are composed of a copolymer of vinylidene and acrylonitrile which have encapsulated therein isobutane liquid. Such microspheres are sold as a dry composition and are approximately 50 microns in size. The EXPANCEL 551 DE™ microspheres have a specific gravity of only 0.02 to 0.05, which is between one-fiftieth and one- twentieth the density of water.
As with the preparation of stabilized compositions, including lipid and/or vesicle compositions, a wide variety of techniques are available for the preparation of stabilized formulations, including stabilized lipid and/or vesicle formulations. For example, lipid and/or vesicle formulations may be prepared from a mixture of lipid compounds, bioactive agent and gas or gaseous precursor. In this case, lipid and/or vesicle compositions may be prepared as described above in which the compositions also comprise bioactive agent. Thus, for example, micelles can be prepared in the presence of a bioactive agent. In connection with lipid and/or vesicle compositions which comprise a gas, the preparation can involve, for example, bubbling a gas directly into a mixture of lipid compounds and one or more additional materials. Alternatively, the lipid and/or vesicle compositions may be preformed from lipid compounds and gas or gaseous precursor. In the latter case, the bioactive agent may be then added to the lipid and/or vesicle composition prior to use. For example, an aqueous mixture of liposomes and gas may be prepared to which the bioactive agent may be added and which is agitated to provide the liposome formulation. The liposome formulation can be readily isolated since the gas and/or bioactive agent filled liposome vesicles generally float to the top of the aqueous solution. Excess bioactive agent can be recovered from the remaining aqueous solution. As those skilled in the art will recognize, various of the compositions containing stabilizing materials which are described herein, including, for example, lipid, polymer, protein and/or vesicle compositions and/or formulations, may be lyophilized for storage, and reconstituted, for example, with an aqueous medium (such as sterile water, phosphate buffered solution, or aqueous saline solution), with the aid of vigorous agitation. To prevent agglutination or fusion of the lipids as a result of lyophilization, it may be useful to include additives which prevent such fusion or agglutination from occurring. Additives which may be useful include sorbitol, mannitol, sodium chloride, glucose, trehalose, polyvinylpyrrolidone and poly(ethylene glycol) (PEG), for example, PEG polymers having a molecular weight of from about 400 to about 10,000, with PEG polymers having molecular weights of about 1000, 3000 (such as PEG3350) and 5000 being preferred. These and other additives are described in the literature, such as in the U.S. Pharmacopeia, USP XXII, NF XVII, The United States Pharmacopeia, The National Formulary, United States Pharmacopeial Convention Inc., 12601 Twinbrook Parkway, Rockville, MD 20852, the disclosures of which are hereby incoφorated herein by reference in their entirety. Lyophilized preparations generally have the advantage of greater shelf life. As discussed above, the compositions of the present invention, including gas and/or gaseous precursor filled vesicles, are useful as contrast agents for diagnostic imaging, including, for example, ultrasound (US) imaging, computed tomography (CT) imaging, including CT angiography (CTA) imaging, magnetic resonance (MR) imaging, magnetic resonance angiography (MRA), nuclear medicine, optical imaging and elastography.
In accordance with the present invention, there are provided methods of imaging one or more regions of a patient. The present invention also provides methods for diagnosing the presence or absence of diseased tissue in a patient. The methods of the present invention involve the administration of a contrast medium in the form, for example, a stabilized compositions, such as a lipid and/or vesicle composition, to a patient. The patient is scanned using diagnostic imaging including, for example ultrasound imaging, to obtain visible images of an internal region of a patient. The methods are especially useful in providing images of the heart region, the gastrointestinal region or the lymphatic system, but can also be employed more broadly to image other internal regions of the patient including, for example, the vasculature. The present methods can also be used in connection with the delivery of a bioactive agent to an internal region of a patient. If desired, the stabilized compositions, such as the lipid and/or vesicle compositions described herein may further comprise a targeting agent to promote targeting of tissues and/or receptors in vivo including, for example, myocardial tissue. The targeting ligands may be associated with the lipid compounds, proteins, polymers and/or vesicles covalently or non-covalently. Thus, in the case of lipid compositions, the targeting ligand may be bound, for example, via a covalent or non-covalent bond, to at least one of the lipids incoφorated in the compositions. In the case of vesicles which are formulated from substances other than lipids, for example, clathrates, aerogels and albumin vesicles, the targeting ligand may be preferably bound covalently or non- covalently to one or more of the materials incoφorated in the vesicle walls. It is generally preferred that the targeting ligand be bound to the lipid and/or vesicles covalently. Preferably, in the case of lipid compositions which comprise cholesterol, the targeting ligand is bound to the cholesterol substantially only non-covalently, and/or that the targeting ligand is bound covalently to a component of the composition, for example, another lipid, such as a phospholipid, other than the cholesterol.
If desired, the targeting ligands may also be bound to other stabilizing materials, for example, biocompatible polymers, which may be present in the compositions. The targeting ligands which are incoφorated in the compositions of the present invention are preferably substances which are capable of targeting receptors and/or tissues in vivo. With respect to the targeting of tissue, as noted above, the targeting ligands are desirably capable of targeting heart tissue, including myocardial cells, and membranous tissues, including endothelial and epithelial cells. In the case of receptors, the targeting ligands are desirably capable of targeting GPIIbllla receptors. It is contemplated that preferred targeting ligands for use in targeting tissues and/or receptors, including the tissues and receptors exemplified above, are selected from the group consisting of proteins, peptides, saccharides, steroids, steroid analogs, bioactive agents and genetic material, including, for example, antibodies, glycoproteins and lectins, with peptides being preferred. An example of a protein which may be preferred for use as a targeting ligand is Protein A, which is protein that is produced by most strains of Staphylococcus aureus. Protein A is commercially available, for example, from Sigma Chemical Co. (St. Louis, MO). Protein A may then be used for binding a variety of IgG antibodies. Generally speaking, peptides which are particularly useful as targeting ligands include natural, modified natural, or synthetic peptides that incoφorate additional modes of resistance to degradation by vascularly circulating esterases, amidases, or peptidases. One very useful method of stabilization of peptide moieties incoφorates the use of cyclization techniques. As an example, the end-to-end cyclization whereby the carboxy terminus is covalently linked to the amine terminus via an amide bond may be useful to inhibit peptide degradation and increase circulating half-life. Additionally, a side chain-to-side chain cyclization is also particularly useful in inducing stability. In addition, an end-to-side chain cyclization may be a useful modification as well. In addition, the substitution of an L-amino acid for a D-amino acid in a strategic region of the peptide may offer resistance to biological degradation. Suitable targeting ligands, and methods for their preparation, will be readily apparent to one skilled in the art, once armed with the disclosure herein. Exemplary targeting agents, methods for their incoφoration into stabilized compositions, and methods for the use of such targeted compositions, are described, for example, in copending U.S. application Serial No.08/660,032, filed June 6, 1996, the disclosures of which are hereby incoφorated herein by reference, in their entirety.
As one skilled in the art would recognize, administration of the stabilized compositions, including lipid and/or vesicle compositions described herein, can be carried out in various fashions, including parenterally, orally, or intraperitoneally. Parenteral administration, which is preferred, includes administration by the following routes: intravenous; intramuscular; interstitially; intra-arterially; subcutaneous; intraocular; intrasynovial; transepithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular; rectal; and nasal inhalation via insufflation. Intravenous administration is preferred among the routes of parenteral administration. The useful dosage to be administered and the particular mode of administration will vary depending upon the age, weight and the particular mammal and region thereof to be scanned, and the particular contrast agent employed. Typically, dosage is initiated at lower levels and increased until the desired contrast enhancement is achieved. Various combinations of the lipid compositions may be used to alter properties as desired, including viscosity, osmolarity or palatability. In carrying out the imaging methods of the present invention, the contrast medium can be used alone, or in combination with diagnostic, therapeutic or other agents. Such other agents include excipients such as flavoring or coloring materials. CT imaging techniques which are employed are conventional and are described, for example, in Computed Body Tomography, Lee, J.K.T., Sagel, S.S., and Stanley, R.J., eds., 1983, Ravens Press, New York, N.Y., especially the first two chapters thereof entitled "Physical Principles and Instrumentation", Ter-Pogossian, M.M., and "Techniques" , Aronberg, D.J., the disclosures of which are incoφorated by reference herein in their entirety.
In the case of diagnostic applications, such as ultrasound and CT, energy, such as ultrasonic energy, may be applied to at least a portion of the patient to image the target tissue. A visible image of an internal region of the patient may be then obtained, such that the presence or absence of diseased tissue can be ascertained. With respect to ultrasound, ultrasonic imaging techniques, including second harmonic imaging, and gated imaging, are well known in the art, and are described, for example, in Uhlendorf, "Physics of Ultrasound Contrast Imaging: Scattering in the Linear Range", IEEE Transactions on Ultrasonics, Ferroelectncs, and Frequency Control, Vol. 14(1), pp. 70-79 (1994) and Sutherland, et al, "Color Doppler Myocardial Imaging: A New Technique for the Assessment of Myocardial Function", Journal of the American Society of Echocardiography, Vol. 7(5), pp. 441-458 (1994), the disclosures of which are hereby incoφorated herein by reference in their entireties. Ultrasound can be used for both diagnostic and therapeutic puφoses. In diagnostic ultrasound, ultrasound waves or a train of pulses of ultrasound may be applied with a transducer. The ultrasound is generally pulsed rather than continuous, although it may be continuous, if desired. Thus, diagnostic ultrasound generally involves the application of a pulse of echoes, after which, during a listening period, the ultrasound transducer receives reflected signals. Harmonics, ultraharmonics or subharmonics may be used. The second harmonic mode may be beneficially employed, in which the 2x frequency may be received, where x is the incidental frequency. This may serve to decrease the signal from the background material and enhance the signal from the transducer using the targeted contrast media of the present invention which may be targeted to the desired site. Other harmonics signals, such as odd harmonics signals, for example, 3x or 5x, may be similarly received using this method. Subharmonic signals, for example, x/2 and x/3, may also be received and processed so as to form an image.
In addition to the pulsed method, continuous wave ultrasound, for example, Power Doppler, may be applied. This may be particularly useful where rigid vesicles, for example, vesicles formulated from polymethyl methacrylate or cyanomethacrylate, are employed. In this case, the relatively higher energy of the Power Doppler may be made to resonate the vesicles and thereby promote their rupture. This can create acoustic emissions which may be in the subharmonic or ultraharmonic range or, in some cases, in the same frequency as the applied ultrasound. It is contemplated that there may be a spectrum of acoustic signatures released in this process and the transducer so employed may receive the acoustic emissions to detect, for example, the presence of a clot. In addition, the process of vesicle rupture may be employed to transfer kinetic energy to the surface, for example of a clot to promote clot lysis. Thus, therapeutic thrombolysis may be achieved during a combination of diagnostic and therapeutic ultrasound. Spectral Doppler may also be employed. In general, the levels of energy from diagnostic ultrasound are insufficient to promote the rupture of vesicles and to facilitate release and cellular uptake of the bioactive agents. As noted above, diagnostic ultrasound may involve the application of one or more pulses of sound. Pauses between pulses permits the reflected sonic signals to be received and analyzed. The limited number of pulses used in diagnostic ultrasound limits the effective energy which is delivered to the tissue that is being studied.
Higher energy ultrasound, for example, ultrasound which is generated by therapeutic ultrasound equipment, is generally capable of causing rupture of the vesicle species. In general, devices for therapeutic ultrasound employ from about 10 to about 100%> duty cycles, depending on the area of tissue to be treated with the ultrasound. Areas of the body which are generally characterized by larger amounts of muscle mass, for example, backs and thighs, as well as highly vascularized tissues, such as heart tissue, may require a larger duty cycle, for example, up to about 100%).
In therapeutic ultrasound, continuous wave ultrasound is used to deliver higher energy levels. For the rupture of vesicles, continuous wave ultrasound is preferred, although the sound energy may be pulsed also. If pulsed sound energy is used, the sound will generally be pulsed in echo train lengths of from about 8 to about 20 or more pulses at a time. Preferably, the echo train lengths are about 20 pulses at a time. In addition, the frequency of the sound used may vary from about 0.025 to about 100 megahertz (MHz). In general, frequency for therapeutic ultrasound preferably ranges between about 0.75 and about 3 MHz, with from about 1 and about 2 MHz being more preferred. In addition, energy levels may vary from about 0.5 Watt (W) per square centimeter (cm2) to about 5.0 W/cm2 , with energy levels of from about 0.5 to about 2.5 W/cm2 being preferred. Energy levels for therapeutic ultrasound involving hyperthermia are generally from about 5 W/cm2 to about 50 W/cm2 . For very small vesicles, for example, vesicles having a diameter of less than about 0.5 μm, higher frequencies of sound are generally preferred. This is because smaller vesicles may be capable of absorbing sonic energy more effectively at higher frequencies of sound. When very high frequencies are used, for example, greater than about 10 MHz, the sonic energy may penetrate fluids and tissues to a limited depth only. Thus, external application of the sonic energy may be suitable for skin and other superficial tissues. However, it is generally necessary for deep structures to focus the ultrasonic energy so that it is preferentially directed within a focal zone. Alternatively, the ultrasonic energy may be applied via interstitial probes, intravascular ultrasound catheters or endoluminal catheters. Such probes or catheters may be used, for example, in the esophagus for the diagnosis and/or treatment of esophageal carcinoma. In addition to the therapeutic uses discussed above, the compositions described herein can be employed in connection with esophageal carcinoma or in the coronary arteries for the treatment of atherosclerosis, as well as the therapeutic uses described, for example, in U.S. Patent No. 5,149,319, U.S. Patent No. 5,209,720 and U.S. Patent No. 5,542,935, the disclosures of which are hereby incoφorated herein by reference, in their entireties. A therapeutic ultrasound device may be used which employs two frequencies of ultrasound. The first frequency may be x, and the second frequency may be 2x. In preferred form, the device would be designed such that the focal zones of the first and second frequencies converge to a single focal zone. The focal zone of the device may then be directed to the compositions, for example, vesicle compositions, within the tissue in the region of interest. This ultrasound device may provide second harmonic therapy with simultaneous application of the x and 2x frequencies of ultrasound energy. It is contemplated that, in the case of ultrasound involving vesicles, this second harmonic therapy may provide improved rupturing of vesicles as compared to ultrasound energy involving a single frequency. Also, it is contemplated that the preferred frequency range may reside within the fundamental harmonic frequencies of the vesicles. Lower energy may also be used with this device. An ultrasound device which may be employed in connection with the aforementioned second harmonic therapy is described, for example, in Kawabata, K. et al., Ultrasonics Sonochemistry, Vol. 3, pp. 1-5 (1996), the disclosures of which are hereby incoφorated herein by reference, in their entirety. Other therapeutic ultrasound devices are described, for example, in U.S. Patent Nos. 4,620,546, 4,658,828 and 4,586,512, the disclosures of which are hereby incoφorated herein by reference, in their entireties.
Depending on the particular perfluoroether compound employed in the compositions of the present invention, as well as the temperature and pressure conditions to which the compositions are exposed, the perfluoroether compound may exist in a steady state equilibrium between liquid and gaseous (vapor) phases. As ultrasound is applied to a vesicle containing a perfluoroether compound containing, for example, 6 carbon atoms, such as perfluoromethyl butyl ether, the ultrasound energy may be reflected as well as absorbed by the vesicle. The absorbed energy will generally increase the temperature within the vesicle, and ultrasound shock waves may also lower the pressure within the vesicle. As a result, the fraction of the perfluoroether within the vesicle which is in the gaseous state may increase. This increase in the concentration of gas may also provide an increase in backscatter upon further application of ultrasound energy.
In the case of vesicle compositions formulated from lipids, the concentration of lipid required to form a desired stabilized vesicle level may 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- dipalmitoylphosphatidylcholine (DPPC) used to form stabilized vesicles according to the methods of the present invention may be from 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 even more preferably from about 1 mg/mL to about 10 mg/mL of saline solution. The concentration of distearoylphosphatidylcholine (DSPC) used in preferred embodiments may be from 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 even more preferably from about 1 mg/mL to about 10 mg/mL of saline solution. The amount of composition which is administered to a patient can vary. Typically, the IV dose may be less than about 10 mL for a 70 Kg patient, with lower doses being preferred.
The stabilized compositions described herein, and especially the vesicle compositions, are useful as contrast media in diagnostic imaging, and may also be suitable for use in all areas where diagnostic imaging is employed. However, the stabilized vesicles are particularly useful for perfusion imaging. In accordance with the present invention, there are provided methods of imaging a patient generally, and/or in specifically diagnosing the presence of diseased tissue in a patient. The imaging process of the present invention may be carried out by administering a contrast agent to a patient, and then scanning the patient using, for example, ultrasound, computed tomography, and/or magnetic resonance imaging, to obtain visible images of an internal region of a patient and/or of any diseased tissue in that region. The term "region of a patient" refers to the whole patient or a particular area or portion of the patient. The contrast agent may be particularly useful in providing images of the gastrointestinal and cardiovascular regions, 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 defined by the heart and the vasculature leading directly to and from the heart. The phrase vasculature, as used herein, denotes the blood vessels (including arteries, veins and the like) in the body or in an organ or part of the body. The patient can be any type of mammal, but most preferably is a human. The present invention also provides methods of diagnosing the presence of diseased tissue in a patient. Diseased tissue includes, for example, endothelial tissue which results from vasculature that supports diseased tissue. As a result, the localization and visualization of endothelial tissue to a region of a patient which under normal circumstances is not associated with endothelial tissue provides an indication of diseased tissue in the region.
As noted above, administration of the compositions described herein may be carried out in various fashions, such as intravascularly, orally, rectally, and the like, using a variety of dosage forms. When the region to be scanned is the cardiovascular region, administration of the contrast medium is preferably carried out intravascularly. When the region to be scanned is the gastrointestinal region, administration of the contrast medium 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 mammal and region thereof to be scanned, and the particular contrast medium to be employed. Typically, dosage may be initiated at lower levels and increased until the desired contrast enhancement is achieved. Various combinations of the lipid and/or vesicle compositions 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). The present invention may be performed with ultrasound or computed tomography according to conventional methods known by skilled artisans. 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 examination. 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 interfaces from tissues and fluids. Depending on the acoustic properties of the tissues and fluids in the body, the ultrasound sound waves are partially or wholly reflected or absorbed. When sound waves are reflected by an interface 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. Computed tomography imaging principles and techniques which are employed are conventional and are described, for example, in Computed Body Tomography, Lee, J.K.T., Sagel, S.S., and Stanley, R.J., eds., Ch. 1, pp. 1-7 (Raven Press, NY 1983). In carrying out the magnetic resonance imaging method of the present invention, the contrast agent can be used alone, or in combination with other diagnostic, therapeutic or other agents. Such other agents include excipients such as flavoring 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, Magnetic Resonance Imaging: Principles and Applications, (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.
In preferred embodiments of the present invention, the lipid and/or vesicle compositions may be administered by syringe, that is, by intravenous (IV) injection. As would be apparent to one of ordinary skill in the art, once armed with the present disclosure, the location on the body of the patient at which the stabilized compositions, such as lipid and/or vesicle compositions, are injected may vary and depends upon a variety of factors, including, for example, the particular lipid and/or vesicle composition employed, the contemplated application, such as diagnostic or therapeutic application, and the particular region of interest. For example, in the case of diagnostic ultrasound of myocardial tissue, the lipid and/or vesicle compositions may be injected intravenously, for example, in the arm of a patient.
The IV administration of the contrast agents described herein including, for example, the vesicle compositions, may involve administration via syringe. This may be achieved, for example, by an appropriate medical technician who handles the syringe or syringes manually. Alternatively, administration by syringe may be achieved mechanically, for example, via a mechanical injector, such as a mechanical injector which operates using pneumatic or hydraulic pressure. Suitable mechanical injectors which may be used in the methods of the present invention include a Syringe Pump Model 351, commercially available from Sage Instruments (a division of Orion Research Inc., Boston, MA), a MedRad™ power injector, commercially available from Medrad, Inc. (Pittsburgh, PA) or a Liebel Flarsheim, commercially available from Liebel Flarsheim Co. (Cincinnati, OH). In connection with stabilized compositions, including lipid and/or vesicle compositions which are administered via injection, it may be desirable, and sometimes preferable, to facilitate the movement through the circulating bloodstream of the injected composition. As would be apparent to one of ordinary skill in the art, once armed with the present disclosure, administration by injection generally involves injection of the compositions into a blood vessel. Also as known to the skilled artisan, the blood flow in many blood vessels, and especially smaller blood vessels, may be limited. Due to this limited blood flow, the injected lipid and/or vesicle compositions may pool or accumulate at or near the site of injection. To promote the transport of the lipid and/or vesicle composition from the injection site into the bloodstream and, thereafter, to the region of interest, a flush may be administered. The flush may act, generally by mechanical action, to "push" or "wash" the injected compositions into the bloodstream. Thus, in embodiments which may involve, for example, diagnostic ultrasound of myocardial tissue with a contrast agent that comprises a lipid and/or vesicle composition, a flush may be administered after injection of the lipid and/or vesicle composition to facilitate its movement through the circulatory system and delivery to the region of interest, for example, the heart region. As noted above, embodiments of the present invention may involve perfluoroether compounds which are gaseous precursors, that is, perfluoroether compounds which are liquids at conditions of ambient pressure and temperature. Such perfluoroether compounds are preferably included in compositions in the form of emulsions and/or suspensions. In these embodiments, it may be desirable to activate the perfluoroether compounds, that is, promote the conversion of the perfluoroether compound from a liquid gaseous precursor to a gas, prior to its administration to a patient. This activation may be advantageously achieved hypobarically, for example, by employing suction pressure to a pressure-sealed syringe. For example, in the case of a suspension of a phospholipid and perfluoromethyl butyl ether, contained in a sealed syringe at ambient temperatures and pressures, the perfluoromethyl butyl ether will generally exist in the liquid state. Pulling back on the syringe plunger, while maintaining the seal in the syringe, may produce a decrease in the pressure within the syringe. This may advantageously cause the perfluoromethyl butyl ether to expand, which increases the propensity of the perfluoromethyl butyl ether to vaporize to a gas within the syringe. If desired, the syringe plunger may be rapidly released to promote rupture of the vesicles into smaller sized vesicles during the shock wave within the syringe. In addition, the syringe may be fitted with a filter to promote the break up of any large vesicles prior to injection.
The echogenicity of vesicles, and especially, gas filled vesicles, and the ability to rupture vesicles at, for example, the peak resonant frequency using ultrasound, permits the controlled delivery of bioactive agents to an internal region of a patient. Specifically, the vesicles may be monitored subsequent to their administration to a patient to determine the rate ajt which the vesicles arrive, for example, to a desired region. Furthermore, the vesicles may be ruptured using ultrasound to release the bioactive agent in the region.
The perfluoroethers of the present invention, as well as compositions containing them, may also be employed in a variety of other ways, in addition to those ways described hereinbefore. Such additional applications will be apparent to one of ordinary skill in the art, once placed in possession of the present disclosure, and include, for example, their use as blood substitutes. See, e.g., Long, Jr., U.S. Patent Nos. 4,865,836, 4,987,154 and 4,927,623, the disclosures of which are hereby incoφorated herein by reference, in their entireties. Such additional uses are intended to be within the scope of the present invention.
The invention is further described in the following examples. Examples 1 to 9 are actual examples, while Example 10 is a prophetic example.
Example 1 This example is directed to the preparation of perfluoroether gas filled vesicles within the scope of the present invention. l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (50 mg; MW 734.05; powder, Lot No. 160pc-183) (Avanti-Polar Lipids, Alabaster, AL) was weighed and hydrated with saline solution (5 mL; 0.9%> NaCl) or phosphate buffered saline (5 mL; 0.8%> sodium chloride, 0.02%> potassium chloride, 0.115%) dibasic sodium phosphate and 0.02%) monobasic potassium phosphate, pH adjusted to 7.4) in a centrifuge tube. To this suspension was added perfluoromethyl butyl ether (120 μL) (Fluoro-Seal, Houston TX). The hydrated suspension was then shaken on a vortex machine (Scientific Industries, Bohemia, NY) for 10 minutes at an instrument setting of 6.5. A total volume of 12 mL was noted.
The resulting perfluoromethyl butyl ether (PFMBE) filled vesicles were sized by optical microscopy. It was observed that the vesicles ranged in size from about 0.5 microns to about 50 to about 60 microns. The average size of the vesicles ranged from about 8 to about 12 microns The PFMBE filled vesicles were then filtered through a 10 or 12 micron
"NUCLEPORE™" membrane using a Swin-Lok Filter Holder, (Nuclepore Filtration Products, Costar Corp., Cambridge, MA) and a 20 cc syringe (Becton Dickinsion & Co., Rutherford, NJ). The filter was placed in the Swin-Lok Filter Holder and the cap tightened down securely. The vesicle solution was shaken and transferred to the 20 cc syringe via an 18 gauge needle. Approximately 12 mL of vesicle solution was placed into the syringe, and the syringe was screwed onto the Swin-Lok Filter Holder. The syringe and the filter holder assembly were inverted so that the larger of the perfluoroether filled vesicles could rise to the top. Pressure was gently applied to the syringe to filter the vesicles.
The survival rate of the PFMBE filled vesicles, which corresponds to the percentage of vesicles which remained after the extrusion process described above, was about 83 to about 92%>. Before extrusion, the volume of foam was about 12 mL and the volume of aqueous solution was about 4 mL. After extrusion, the volume of foam was about 10 to 11 mL and the volume of aqueous solution was about 4 mL.
Optical microscopy was used again to determine the size distribution of the extruded PFMBE filled vesicles. It was observed that after filtering, greater than 90%) of the PFMBE filled vesicles were smaller than 10 microns.
Example 2
This example includes a description of experiments which were conducted to determine the effect of varying the temperature and the particular gaseous perfluorinated compound on the production of gas-filled vesicles. In these experiments, samples of perfluoromethyl butyl ether (PFMBE) and perfiuoropentane (PFP), with and without a stabilizing compound, were mixed on a Wig-L-Bug® shaking device at about 3000 to 3400 φm or a Capmix® shaking device at about 4500 to 5000 φm after incubation at temperatures of -20°C, 4°C or room temperature. Heating produced by the Capmix® device increased the sample temperature by about 20 to about 25°C. Less than 5°C of heating was produced by the Wig-L-Bug® device. The table also includes a comparison of the results obtained when the agitation procedure was conducted at about -20°C or room temperature. The results of these experiments are tabulated below. TABLE
0 I
TABLE
S I
TABLE
DPPC refers to dipalmitoylphosphatidylcholine DOPC refers to dioleoylphosphatidylcholine nwm refers to number weighted mean o
I
- I l l - Inspection of Table 2 above rςveals that improved yields of gas-containing vesicles are generally obtained when the samples are incubated near ambient temperature, independent of the initial temperature of the shaker. This is apparent from an analysis of the experimental data for samples in which a Capmix® shaking device was employed (Samples 2D to 21 and 2M to 2DD). Specifically, this data indicates shaking samples at room temperature (Samples 2D to 2F, 2M to 20 and 2S to 2DD) provide gas filled vesicles having a reduced size variance (nwm), as well as a reduced diameter, generally. For example, the nwm values for PFMBE filled vesicles prepared at room temperature range from 2.2 (Sample 2E) to 2.95 (Sample 2F), as compared to the nwm values for PFMBE filled vesicles prepared at -20°C, which range from 2.9 (Sample 2H) to 5.3 (Sample 21). Similarly, the nwm values for PFP filled vesicles prepared at room temperature range from 3.88 (Sample 2N) to 4.65 (Sample 2V), as compared to the nwm values for PFP filled vesicles prepared at -20°C, which range from 4.5 (Sample 2Q) to 7.72 (Sample 2R). Thus, the samples prepared at room temperature generally possess a more uniform distribution of vesicles. Also, samples prepared at room temperature generally comprise a greater percentage of vesicles sized below lOμ. Inspection of the data in Table 2 also indicates that, if the shaking process induces a phase-transition in the perfluoroether compound, as may occur with an ESPE Capmix® shaking device, vesicles may be obtained with a more uniform size distribution, independent of the initial incubation sample temperature.
Example 3
This example is directed to an analysis of the acoustic activity of aqueous vesicle compositions. The compositions employed in the analysis included a composition of the type prepared in Example 1 above, referred to herein as Example 3A, and a composition containing vesicles prepared from DPPC, DPPA, DPPE- PEG5000 and perfluoropropane, referred to herein as Example 3B.
The acoustic analysis involved a study of the duration of vesicle activity in an ultrasonic field. Also studied were the effects of increasing pressure, followed by a rapid decrease in pressure. These measurements provided information respecting the stability of the vesicles upon exposure to conditions of associated with ultrasound in a diagnostic setting. The results of these studies are depicted graphically in Figures 1 and 2 which show comparative histograms of time-dependent attenuation of vesicles subjected to varying frequencies of ultrasound (Figure 1) and pressure-dependent attenuation at varying frequencies (Figure 2). Inspection of the Figures 1 and 2 reveals that vesicles containing PFMBE (Example 3A) generally demonstrated improved attenuation effects (db) over time, as well as improved stability to increasing pressure, followed by a rapid decrease in pressure.
Example 4
An anesthetized mongrel dog was imaged using an Acoustic Imaging 5200S ultrasound machine (Phoenix, AZ). Physiological parameters monitored included systemic arterial pressure, pulmonary arterial pressure, heart rate and oxygen saturation. Vesicle compositions as in Examples 1 and 3B were prepared by shaking at room temperature on an ESPE Capmix®. A dose of 0.005 cc/kg of each composition was administered via the cephalic vein. Cardiac imaging was carried out using a 7.5Mhz tightly curved linear array (TCLA) transducer. After injection of the Example 3B composition, a distinct attenuation artifact was observed followed by marked ventricular opacification and visible myocardial opacification. The contrast obtained with the Example 3B composition began to clear after 2-3 minutes of continuous imaging. The Example 1 composition, on the other hand, showed reduced attenuation artfacts. In addition, the ventricular contrast obtained with the Example 1 composition was brilliant and long-lasting, and myocardial opacification was easily visualized. Ventricular contrast with the Example 1 composition remained visible after 3-4 minutes of continuous imaging. No changes in physiological parameters were observed with either contrast agent.
Example 5 This example includes an analysis of the effect of varying temperature on the stability, as measured by the attenuation reflectivity, of PFMBE filled vesicles. Figure 3 shows the attenuation reflectivity from 0 to 5 minutes, at 1 minute intervals, of a composition of PFBME filled vesicles when formed and shaken at an initial sample temperature of about -20°C. This shaking protocol involved warming the compositions to beyond the phase transition temperature of PFMBE (35.4°C), so that at time 0, the temperature was relatively warmer than as in subsequent minutes 1 to 5. This experiment was repeated, except that the vesicles were formed at 4°C, but shaken at ambient temperatures. The stability experiments for these latter vesicles are depicted graphically in Figure 4, which provides substantially similar results as those formed and shaken at about -20°C (see Figure 3). When similar studies were conducted to evaluate vesicle stability as pressure was increased (Figures 5 and 6), substantially improved stability was observed in vesicles shaken at room temperature (see Figure 5) as compared to vesicles shaken at -20°C (see Figure 6). Those shaken at room temperature retained higher attenuation levels over time, consistent with the data tabulated above in Table 2. The studies depicted in Figure 5 also involved exposing the vesicles to a rapid decrease in pressure.
Example 6
This example is directed to the preparation of albumin coated vesicles filled with PFMBE gas. Vesicles were prepared by dissolving 5%> bovine serum albumin (Sigma,
St. Louis, MO) in 8:1:1 saline/propylene glycol/glycerol. This solution was then aliquotted into two 2 mL serum vials. One vial sample was shaken immediately while the headspace gas in the other vial was removed and PFMBE was added. Both samples were sized using an Accusizer 770. The sample with the PFMBE had an increased order of magnitude of vesicles with a smaller size profile.
Example 7
This example is directed to the preparation of PFMBE gas vesicles stabilized with a surfactant material (Pluronic).
Vesicles were prepared by dissolving 1% Pluronic F68 (Spectrum, Gardena, CA) in 8:1 :1 saline/propylene glycol/glycerol. This solution was then aliquotted into two 2 mL serum vials. One vial sample was shaken immediately while the headspace gas for the other vial was removed and PFMBE was added. Both samples were sized using an Accusizer 770. The sample with a headspace of air had no visible foam while the sample containing PFMBE contained a large quantity of visible foam. Neither sample was sufficiently stable to size. Example 8
This example is directed to a comparison of the toxicity of PFMBE and perfiuoropentane (PFP) in mice.
Lipid vesicles were prepared as in Example 1 using either PFMBE or PFP. The samples were cooled to -20°C before the addition of the perfluorinated material. The headspace was evacuated briefly with a vacuum after which the samples were warmed to room temperature with shaking for 60 seconds on an ESPE Capmix®.
Groups of outbred mice were immobilized and the vesicle compositions (50 μL) were injected into the mice. No effects were seen in the mice receiving the PFMBE vesicles at a dosage of approximately 2 mL/kg. The group receiving the PFP filled vesicles experienced 2 immediate mortalities, and the third animal was sluggish and in respiratory distress.
Example 9
This is a comparative example directed to the production of PFMBE lipid coated vesicles using lipid in the liquid crystalline state.
A mixture of 82 mole percent DOPC, 10 mole percent DOPA and 8 mole percent DOPE-PEG 5000 was prepared and lyophilized. The phase transition of liquid crystalline to gel state of these lipids is about 4°C. Accordingly, the shaking/gas instillation step described below occurred while the lipids were in the liquid crystalline state. The lipid mix was hydrated in a solution of 8:1 :1 saline/propylene glycol/glycerol at a lipid concentration of 1 mg/mL in a 2 mL vial. PFMBE (20 μL) was added to the vial, the contents were chilled to 4°C and the headspace of air was evacuated under vacuum. The vial was shaken with a ESPE Capmix® at 4500 φm for 60 seconds. The temperature of the solution after shaking was about 26°C, which is well above the phase transition temperature of the lipid. The concentration of vesicles was 6.73 X 107 vesicles/mL as compared to 1.90 X 109 vesicles/mL when gel state lipids were used. (See Sample 2T in data tabulated above.) The vesicle composition was examined for acoustic activity at 37°C, and was found to be substantially inactive as compared to the vesicle compositions which were prepared with gel state lipids as shown in Figures 4 and 6. Example 10
This example is directed to the preparation of a vesicle composition containing a perfluoroether and a bioactive agent. Polylactic acid (4 g; m.w. 15,000-25,000) and dichloromethane (20 mL) are combined in a 30 mL serum bottle. A mixture of 2 mL of an aqueous solution of α-tocopherol (vitamin E) is added after the polylactic acid is dissolved. At this time, PFMBE (20-50 μL) is also added. The vial is attached to a Heavy Duty #6 Wig-L-Bug and shaken for 2 minutes. After the sample is cooled, it is added to a solution of 0.5%) polyvinyl alcohol m.w. 86,000 to 145,000 (Aldrich). This solution is agitated at 7500 φm using a Silverson mixer (Waterside, England). The sample is stirred for an additional 2 minutes and then transferred to a hotplate at 30°C and mixed for 2 hours with a Talboy stirrer (South Montrose, PA). Particles are then observed using a Nikon Diaphot Microscope (Tokyo, Japan) and sized on the Accusizer 770.
The disclosures of each patent, patent application and publication cited or described in this document are hereby incoφorated by reference, in their entirety.
Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Claims

CLAIMS What is claimed is:
1. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a lipid, protein or polymer, and a gaseous compound of the formula R,-X-R2
(I) wherein:
X is O or S;
Rj is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
2. A contrast agent according to Claim 1 which comprises a lipid.
3. A contrast agent according to Claim 2 wherein said lipid comprises a phospholipid.
4. A contrast agent according to Claim 3 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
5. A contrast agent according to Claim 4 wherein said phosphatidylcholine is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl- phosphatidylcholine.
6. A contrast agent according to Claim 5 wherein said phosphatidylcholine comprises dipalmitoylphosphatidylcholine.
7. A contrast agent according to Claim 4 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol- amine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and 1 -hexadecyl-2-palmitoylglycerophosphoethanolamine.
8. A contrast agent according to Claim 7 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
9. A contrast agent according to Claim 4 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
10. A contrast agent according to Claim 2 wherein said lipid further comprises a polymer.
11. A contrast agent according to Claim 10 wherein said polymer comprises a hydrophilic polymer.
12. A contrast agent according to Claim 11 wherein said hydrophilic polymer comprises polyethylene glycol.
13. A contrast agent according to Claim 1 which comprises a protein.
14. A contrast agent according to Claim 13 wherein said protein comprises albumin.
15. A contrast agent according to Claim 1 which comprises a polymer.
16. A contrast agent according to Claim 15 wherein said polymer comprises synthetic polymers or copolymers which are prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N- substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p-aminobenzyl- styrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacryloyloxytrimethyl-ammonium chloride.
17. A contrast agent according to Claim 15 wherein said polymer comprises synthetic polymers or copolymers selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyanomethacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e- caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate and polystyrene-polyacrylonitrile.
18. A contrast agent according to Claim 17 wherein said polymer comprises a polyvinylidene-polyacrylonitrile copolymer.
19. A contrast agent according to Claim 1 which comprises vesicles.
20. A contrast agent according to Claim 19 wherein said vesicles are selected from the group consisting of micelles and liposomes.
21. A contrast agent according to Claim 19 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
22. A contrast agent according to Claim 21 wherein said vesicles comprise unilamellar vesicles.
23. A contrast agent according to Claim 22 wherein said vesicles comprise a monolayer.
24. A contrast agent according to Claim 23 which comprises a phospholipid.
25. A contrast agent according to Claim 22 wherein said vesicles comprise a bilayer.
26. A contrast agent according to Claim 25 which comprises a phospholipid.
27. A contrast agent according to Claim 21 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
28. A contrast agent according to Claim 27 wherein said vesicles comprise monolayers.
29. A contrast agent according to Claim 28 which comprises a phospholipid.
30. A contrast agent according to Claim 27 wherein said vesicles comprise bilayers.
31. A contrast agent according to Claim 30 which comprises a phospholipid.
32. A contrast agent according to Claim 1 wherein X is O.
33. A contrast agent according to Claim 1 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
34. A contrast agent according to Claim 33 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
35. A contrast agent according to Claim 34 wherein R, and R2 are independently perfluoroalkyl.
36. A contrast agent according to Claim 35 wherein R, and R2 are independently perfluoroalkyl of 1 to about 6 carbons.
37. A contrast agent according to Claim 36 wherein R, and R2 are independently perfluoroalkyl of 1 to about 4 carbons.
38. A contrast agent according to Claim 37 wherein one of R, and R^ is perfluorobutyl and the other of R, and R2 is perfluoromethyl.
39. A contrast agent according to Claim 1 wherein R2 is halo.
40. A contrast agent according to Claim 39 wherein R2 is chloro or bromo.
41. A contrast agent according to Claim 39 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
42. A contrast agent according to Claim 41 wherein Rt is perfluoroalkyl.
43. A contrast agent according to Claim 1 wherein R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
44. A contrast agent according to Claim 43 wherein R, and R2 are linked together to form perfluoroheterocyclyl.
45. A contrast agent according to Claim 1 in which said lipid, polymer or protein is substantially crosslinked or substantially non-crosslinked.
46. A contrast agent according to Claim 45 in which said lipid, polymer or protein is substantially crosslinked.
47. A contrast agent according to Claim 45 in which said lipid, polymer or protein is substantially non-crosslinked.
48. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a stabilizing compound and a gaseous compound of the formula
R| -X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and ^ are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R^ are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
49. A contrast agent according to Claim 48 wherein said stabilizing compound is selected from the group consisting of a lipid, protein and polymer.
50. A contrast agent according to Claim 49 wherein said stabilizing compound comprises a lipid.
51. A contrast agent according to Claim 50 wherein said lipid comprises a phospholipid.
52. A contrast agent according to Claim 51 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
53. A contrast agent according to Claim 52 wherein said phosphatidyl-choline is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl-phosphatidylcholine.
54. A contrast agent according to Claim 53 wherein said phosphatidyl-choline comprises dipalmitoylphosphatidylcholine.
55. A contrast agent according to Claim 52 wherein said phosphatidyl-ethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol-amine, dioleoylphosphatidylethanolamine, N- succinyldioleoylphosphatidylethanolamine and l-hexadecyl-2- palmitoylglycerophosphoethanolamine.
56. A contrast agent according to Claim 55 wherein said phosphatidyl-ethanolamine comprises dipalmitoylphosphatidylethanolamine.
57. A contrast agent according to Claim 52 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
58. A contrast agent according to Claim 50 wherein said lipid further comprises a polymer.
59. A contrast agent according to Claim 58 wherein said polymer comprises a hydrophilic polymer.
60. A contrast agent according to Claim 59 wherein said hydrophilic polymer comprises polyethylene glycol.
61. A contrast agent according to Claim 49 wherein said stabilizing compound comprises a protein.
62. A contrast agent according to Claim 61 wherein said protein comprises albumin.
63. A contrast agent according to Claim 49 wherein said stabilizing compound comprises a polymer.
64. A contrast agent according to Claim 63 wherein said polymer comprises synthetic polymers or copolymers which are prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N- substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p-aminobenzyl- styrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacιyloyloxytrimethyl-ammonium chloride.
65. A contrast agent according to Claim 63 wherein said polymer comprises synthetic polymers or copolymers selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyanomethacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e- caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate and polystyrene-polyacrylonitrile.
66. A contrast agent according to Claim 65 wherein said polymer comprises a polyvinylidene-polyacrylonitrile copolymer.
67. A contrast agent according to Claim 49 which comprises vesicles.
68. A contrast agent according to Claim 67 wherein said vesicles are selected from the group consisting of micelles and liposomes.
69. A contrast agent according to Claim 67 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
70. A contrast agent according to Claim 69 wherein said vesicles comprise unilamellar vesicles.
71. A contrast agent according to Claim 70 wherein said vesicles comprise a monolayer.
72. A contrast agent according to Claim 70 wherein said vesicles comprise a bilayer.
73. A contrast agent according to Claim 68 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
74. A contrast agent according to Claim 73 wherein said vesicles comprise monolayers.
75. A contrast agent according to Claim 73 wherein said vesicles comprise bilayers.
76. A contrast agent according to Claim 48 wherein X is O.
77. A contrast agent according to Claim 48 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
78. A contrast agent according to Claim 77 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
79. A contrast agent according to Claim 78 wherein R, and R2 are independently perfluoroalkyl.
80. A contrast agent according to Claim 79 wherein R, and 2 are independently perfluoroalkyl of 1 to about 6 carbons.
81. A contrast agent according to Claim 80 wherein R, and R2 are independently perfluoroalkyl of 1 to about 4 carbons.
82. A contrast agent according to Claim 81 wherein one of R, and R^ is perfluorobutyl and the other of R, and R2 is perfluoromethyl.
83. A contrast agent according to Claim 48 wherein R2 is halo.
84. A contrast agent according to Claim 83 wherein R2 is chloro or bromo.
85. A contrast agent according to Claim 83 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
86. A contrast agent according to Claim 85 wherein R, is perfluoroalkyl.
87. A contrast agent according to Claim 48 wherein R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
88. A contrast agent according to Claim 87 wherein R, and R2 are linked together to form perfluoroheterocyclyl.
89. A contrast agent according to Claim 48 in which said stabilizing compound is substantially crosslinked or substantially non-crosslinked.
90. A contrast agent according to Claim 89 in which said stabilizing compound is substantially crosslinked.
91. A contrast agent according to Claim 89 in which said stabilizing compound is substantially non-crosslinked.
92. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a stabilizing compound which is substantially non-crosslinked or substantially crosslinked, and a gaseous compound of the formula
(I) wherein: X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
93. A contrast agent according to Claim 92 wherein said stabilizing compound is substantially non-crosslinked.
94. A contrast agent according to Claim 93 wherein said stabilizing compound is selected from the group consisting of lipids, proteins and polymers.
95. A contrast agent according to Claim 94 wherein said stabilizing compound comprises a lipid.
96. A contrast agent according to Claim 95 wherein said lipid comprises a phospholipid.
97. A contrast agent according to Claim 96 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
98. A contrast agent according to Claim 97 wherein said phosphatidyl-choline is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl-phosphatidylcholine.
99. A contrast agent according to Claim 97 wherein said phosphatidyl-choline comprises dipalmitoylphosphatidylcholine.
100. A contrast agent according to Claim 97 wherein said phosphatidyl-ethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol-amine, dioleoylphosphatidylethanolamine, N- succinyldioleoylphosphatidylethanolamine and l-hexadecyl-2- palmitoylglycerophosphoethanolamine.
101. A contrast agent according to Claim 100 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
102. A contrast agent according to Claim 97 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
103. A contrast agent according to Claim 95 wherein said lipid further comprises a polymer.
104. A contrast agent according to Claim 103 wherein said polymer comprises a hydrophilic polymer.
105. A contrast agent according to Claim 104 wherein said hydrophilic polymer comprises polyethylene glycol.
106. A contrast agent according to Claim 94 wherein said stabilizing compound comprises a protein.
107. A contrast agent according to Claim 106 wherein said protein comprises albumin.
108. A contrast agent according to Claim 94 wherein said stabilizing compound comprises a polymer.
109. A contrast agent according to Claim 108 wherein said polymer comprises synthetic polymers or copolymers which are prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N- substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p-aminobenzyl- styrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacryloyloxytrimethyl-ammonium chloride.
110. A contrast agent according to Claim 108 wherein said polymer comprises synthetic polymers or copolymers selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyanomethacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e- caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate and polystyrene-polyacrylonitrile.
111. A contrast agent according to Claim 110 wherein said polymer comprises a polyvinylidene-polyacrylonitrile copolymer.
112. A contrast agent according to Claim 94 which comprises vesicles.
113. A contrast agent according to Claim 112 wherein said vesicles are selected from the group consisting of micelles and liposomes.
114. A contrast agent according to Claim 112 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
115. A contrast agent according to Claim 114 wherein said vesicles comprise unilamellar vesicles.
116. A contrast agent according to Claim 115 wherein said vesicles comprise a monolayer.
117. A contrast agent according to Claim 115 wherein said vesicles comprise a bilayer.
118. A contrast agent according to Claim 114 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
119. A contrast agent according to Claim 1 18 wherein said vesicles comprise monolayers.
120. A contrast agent according to Claim 119 wherein said vesicles comprise bilayers.
121. A contrast agent according to Claim 92 wherein X is O.
122. A contrast agent according to Claim 92 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
123. A contrast agent according to Claim 122 wherein R, and Rj are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
124. A contrast agent according to Claim 123 wherein R, and Rj are independently perfluoroalkyl.
125. A contrast agent according to Claim 124 wherein R, and R2 are independently perfluoroalkyl of 1 to about 6 carbons.
126. A contrast agent according to Claim 125 wherein R, and ^ are independently perfluoroalkyl of 1 to about 4 carbons.
127. A contrast agent according to Claim 126 wherein one of R, and R2 is perfluorobutyl and the other of R and R is perfluoromethyl.
128. A contrast agent according to Claim 92 wherein R2 is halo.
129. A contrast agent according to Claim 128 wherein R2 is chloro or bromo.
130. A contrast agent according to Claim 128 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
131. A contrast agent according to Claim 130 wherein R, is perfluoroalkyl.
132. A contrast agent according to Claim 92 wherein R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
133. A contrast agent according to Claim 132 wherein R, and R2 are linked together to form perfluoroheterocyclyl.
134. A contrast agent according to Claim 92 wherein said stabilizing compound comprises a substantially crosslinked compound.
135. A contrast agent according to Claim 134 wherein said crosslinking comprises linkages other than disulfide linkages.
136. A contrast agent according to Claim 135 wherein said stabilizing compound is selected from the group consisting of substantially crosslinked lipids, proteins and polymers.
137. A contrast agent according to Claim 86 wherein said stabilizing compound comprises a substantially crosslinked lipid.
138. A contrast agent according to Claim 137 wherein said stabilizing compound comprises a substantially crosslinked phospholipid.
139. A contrast agent according to Claim 138 wherein said substantially crosslinked phospholipid is derived from a phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
140. A contrast agent according to Claim 139 wherein said phosphatidyl-choline is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl-phosphatidylcholine.
141. A contrast agent according to Claim 40 wherein said phosphatidyl-choline comprises dipalmitoylphosphatidylcholine.
142. A contrast agent according to Claim 139 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol- amine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and 1 -hexadecyl-2-palmitoylglycerophosphoethanolamine.
143. A contrast agent according to Claim 142 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
144. A contrast agent according to Claim 139 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
145. A contrast agent according to Claim 137 wherein said lipid further comprises a polymer.
146. A contrast agent according to Claim 145 wherein said polymer comprises a hydrophilic polymer.
147. A contrast agent according to Claim 146 wherein said hydrophilic polymer comprises polyethylene glycol.
148. A contrast agent according to Claim 135 wherein said stabilizing compound comprises a substantially crosslinked protein.
149. A contrast agent according to Claim 148 wherein said substantially crosslinked protein is derived from albumin.
150. A contrast agent according to Claim 135 wherein said stabilizing compound comprises a substantially crosslinked polymer.
151. A contrast agent according to Claim 150 wherein said substantially crosslinked polymer is derived from a synthetic polymer or copolymer which is prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N-substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p-aminobenzyl- styrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacryloyloxytrimethyl-ammonium chloride.
152. A contrast agent according to Claim 150 wherein said substantially crosslinked polymer is derived from a synthetic polymer or copolymer selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyanomethacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e-caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethylmethacrylate and polystyrene- polyacrylonitrile.
153. A contrast agent according to Claim 152 wherein said substantially crosslinked polymer is derived from a polyvinylidene-polyacrylonitrile copolymer.
154. A contrast agent according to Claim 135 which comprises vesicles.
155. A contrast agent according to Claim 154 wherein said vesicles are selected from the group consisting of micelles and liposomes.
156. A contrast agent according to Claim 154 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
157. A contrast agent according to Claim 156 wherein said vesicles comprise unilamellar vesicles.
158. A contrast agent according to Claim 157 wherein said vesicles comprise a monolayer.
159. A contrast agent according to Claim 157 wherein said vesicles comprise a bilayer.
160. A contrast agent according to Claim 154 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
161. A contrast agent according to Claim 160 wherein said vesicles comprise monolayers.
162. A contrast agent according to Claim 160 wherein said vesicles comprise bilayers.
163. A contrast agent according to Claim 135 wherein X is O.
164. A contrast agent according to Claim 135 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
165. A contrast agent according to Claim 164 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
166. A contrast agent according to Claim 165 wherein R, and R2 are independently perfluoroalkyl.
167. A contrast agent according to Claim 166 wherein R, and R2 are independently perfluoroalkyl of 1 to about 6 carbons.
168. A contrast agent according to Claim 167 wherein R, and R2 are independently perfluoroalkyl of 1 to about 4 carbons.
169. A contrast agent according to Claim 168 wherein one of R, and R2 is perfluorobutyl and the other of R and R is perfluoromethyl.
170. A contrast agent according to Claim 135 wherein R2 is halo.
171. A contrast agent according to Claim 170 wherein R2 is chloro or bromo.
172. A contrast agent according to Claim 170 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
173. A contrast agent according to Claim 172 wherein R, is perfluoroalkyl.
174. A contrast agent according to Claim 135 wherein R, and Rj are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
175. A contrast agent according to Claim 174 wherein R, and R, are linked together to form perfluoroheterocyclyl.
176. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a lipid which is substantially non-crosslinked or substantially crosslinked, and a gaseous compound of the formula
R|-X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
177. A contrast agent according to Claim 176 wherein said lipid is substantially non-crosslinked.
178. A contrast agent according to Claim 177 wherein said lipid comprises a phospholipid.
179. A contrast agent according to Claim 178 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
180. A contrast agent according to Claim 179 wherein said phosphatidylcholine is selected from the group consisting of dioleoylphosphatidyl- choline, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
181. A contrast agent according to Claim 180 wherein said phosphatidyl-choline comprises dipalmitoylphosphatidylcholine.
182. A contrast agent according to Claim 179 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol- amine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and 1 -hexadecyl-2-palmitoylglycerophosphoethanolamine.
183. A contrast agent according to Claim 182 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
184. A contrast agent according to Claim 179 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
185. A contrast agent according to Claim 177 wherein said lipid further comprises a polymer.
186. A contrast agent according to Claim 185 wherein said polymer comprises a hydrophilic polymer.
187. A contrast agent according to Claim 186 wherein said hydrophilic polymer comprises polyethylene glycol.
188. A contrast agent according to Claim 176 wherein said lipid is substantially crosslinked.
189. A contrast agent according to Claim 188 wherein said crosslinking comprises linkages other than disulfide linkages.
190. A contrast agent according to Claim 189 wherein said substantially crosslinked lipid comprises a substantially crosslinked phospholipid.
191. A contrast agent according to Claim 190 wherein said substantially crosslinked phospholipid is derived from a phospholipid selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
192. A contrast agent according to Claim 191 wherein said phosphatidyl-choline is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl-phosphatidylcholine.
193. A contrast agent according to Claim 192 wherein said phosphatidyl-choline comprises dipalmitoylphosphatidylcholine.
194. A contrast agent according to Claim 191 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol- amine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and 1 -hexadecyl-2-palmitoylglycerophosphoethanolamine.
195. A contrast agent according to Claim 194 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
196. A contrast agent according to Claim 191 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
197. A contrast agent according to Claim 189 wherein said lipid further comprises a polymer.
198. A contrast agent according to Claim 197 wherein said polymer comprises a hydrophilic polymer.
199. A contrast agent according to Claim 198 wherein said hydrophilic polymer comprises polyethylene glycol.
200. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a stabilizing compound selected from the group consisting of lipids, proteins and polymers, and a gaseous perfluoroether compound selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers contain no more than one oxygen atom in the heterocyclyl ring system.
201. A contrast agent according to Claim 200 wherein said stabilizing compound is selected from the group consisting of substantially non-crosslinked compounds and substantially crosslinked compounds.
202. A contrast agent according to Claim 201 wherein said stabilizing compound comprises a substantially non-crosslinked compound.
203. A contrast agent according to Claim 201 wherein said stabilizing compound comprises a substantially crosslinked compound.
204. A contrast agent according to Claim 203 wherein said crosslinking comprises linkages other than disulfide linkages.
205. A contrast agent according to Claim 200 which comprises vesicles.
206. A contrast agent according to Claim 200 wherein said diagnostic imaging is selected from the group consisting of ultrasound imaging, magnetic resonance imaging and computed tomography imaging.
207. A contrast agent according to Claim 206 wherein said diagnostic imaging comprises ultrasound imaging.
208. A composition comprising, in an aqueous carrier, a lipid, protein or polymer, and a gaseous compound of the formula
R,-X-R2
(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and Rj are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and Rj are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
209. A composition according to Claim 208 which comprises a lipid.
210. A composition according to Claim 209 wherein said lipid comprises a phospholipid.
211. A composition according to Claim 210 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
212. A composition according to Claim 211 wherein said phosphatidylcholine is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine and distearoyl- phosphatidylcholine.
213. A composition according to Claim 212 wherein said phosphatidylcholine comprises dipalmitoylphosphatidylcholine.
214. A composition according to Claim 211 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanol- amine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and l-hexadecyl-2-palmitoylglycerophosphoethanolamine.
215. A composition according to Claim 214 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
216. A composition according to Claim 211 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
217. A composition according to Claim 209 wherein said lipid further comprises a polymer.
218. A composition according to Claim 217 wherein said polymer comprises a hydrophilic polymer.
219. A composition according to Claim 218 wherein said hydrophilic polymer comprises polyethylene glycol.
220. A composition according to Claim 208 which comprises a protein.
221. A composition according to Claim 220 wherein said protein comprises albumin.
222. A composition according to Claim 208 which comprises a polymer.
223. A composition according to Claim 222 wherein said polymer comprises synthetic polymers or copolymers which are prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N- substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p-aminobenzyl- styrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacryloyloxytrimethyl-ammonium chloride.
224. A composition according to Claim 222 wherein said polymer comprises synthetic polymers or copolymers selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyanomethacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e- caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile-polymethyl- methacrylate and polystyrene-polyacrylonitrile.
225. A composition according to Claim 224 wherein said polymer comprises a polyvinylidene-polyacrylonitrile copolymer.
226. A composition according to Claim 208 which comprises vesicles.
227. A composition according to Claim 226 wherein said vesicles are selected from the group consisting of micelles and liposomes.
228. A composition according to Claim 226 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
229. A composition according to Claim 228 wherein said vesicles comprise unilamellar vesicles.
230. A composition according to Claim 229 wherein said vesicles comprise a monolayer.
231. A composition according to Claim 230 which comprises a phospholipid.
232. A composition according to Claim 229 wherein said vesicles comprise a bilayer.
233. A composition according to Claim 232 which comprises a phospholipid.
234. A composition according to Claim 228 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
235. A composition according to Claim 234 wherein said vesicles comprise monolayers.
236. A composition according to Claim 235 which comprises a phospholipid.
237. A composition according to Claim 234 wherein said vesicles comprise bilayers.
238. A composition according to Claim 237 which comprises a phospholipid.
239. A composition according to Claim 208 wherein X is O.
240. A composition according to Claim 208 wherein R[ and R2 are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl or perfluoroaryl.
241. A composition according to Claim 240 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
242. A composition according to Claim 241 wherein R, and K, are independently perfluoroalkyl.
243. A composition according to Claim 242 wherein R, and R2 are independently perfluoroalkyl of 1 to about 6 carbons.
244. A composition according to Claim 243 wherein R, and R2 are independently perfluoroalkyl of 1 to about 4 carbons.
245. A composition according to Claim 244 wherein one of R, and R2 is perfluorobutyl and the other of R, and R2 is perfluoromethyl.
246. A composition according to Claim 208 wherein R2 is halo.
247. A composition according to Claim 246 wherein R2 is chloro or bromo.
248. A composition according to Claim 246 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
249. A composition according to Claim 248 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
250. A composition according to Claim 208 wherein R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
251. A composition according to Claim 250 wherein R, and R2 are linked together to form perfluoroheterocyclyl.
252. A composition according to Claim 208 in which said lipid, polymer or protein is substantially crosslinked or substantially non-crosslinked.
253. A composition according to Claim 252 in which said lipid, polymer or protein is substantially crosslinked.
254. A composition according to Claim 252 in which said lipid, polymer or protein is substantially non-crosslinked.
255. A composition comprising, in an aqueous carrier, a stabilizing compound selected from the group consisting of lipids, proteins and polymers, and a gaseous perfluoroether compound selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers comprise no more than one oxygen atom in the heterocyclic ring system.
256. A composition according to Claim 255 wherein said gaseous perfluoroether compound has a phase transition temperature of from about 0°C to about 100°C.
257. A composition according to Claim 256 wherein said gaseous perfluoroether compound has a phase transition temperature of from about 20°C to about 60°C.
258. A composition according to Claim 257 wherein said gaseous perfluoroether compound has a phase transition temperature of from about 25°C to about 45°C.
259. A composition according to Claim 258 wherein said gaseous perfluoroether compound has a phase transition temperature of from about 30°C to about 40°C.
260. A composition according to Claim 255 wherein said gaseous perfluoroether compound comprises a perfluoroaliphatic ether compound.
261. A composition according to Claim 260 wherein said perfluoroaliphatic ether compound has the formula
^ 2n+ 1 n" " m 2m+ ! (II) wherein: each of n and m is independently an integer of 1 to about 7; and X is O or S.
262. A composition according to Claim 261 wherein X is O.
263. A composition according to Claim 261 wherein the sum of m and n is no greater than about 7.
264. A composition according to Claim 263 wherein the sum m and n is about 5.
265. A composition according to Claim 264 wherein one of m and n is about 1 and the other of m and n is about 4.
266. A composition according to Claim 255 wherein said perfluoroether compound is a perfluoroalicyclic aliphatic ether compound.
267. A composition according to Claim 266 wherein said perfluoroalicyclic aliphatic ether compound has the following formula
wherein: m is an integer of from 1 to about 6; n is an integer of from about 2 to about 5; and
X is O or S.
268. A composition according to Claim 267 wherein X is O.
269. A composition according to Claim 267 wherein the sum of m and n is no greater than about 7.
270. A composition according to Claim 269 wherein the sum of m and n is about 5.
271. A composition according to Claim 270 wherein n is about 2 or about 3 and m is 1 or about 2.
272. A composition according to Claim 255 wherein said perfluoroether compound comprises a perfluoroheterocyclyl compound.
273. A composition according to Claim 272 wherein said perfluoroheterocyclyl compound has the formula
(IV)
wherein: m is an integer from 0 to about 3; n is an integer from about 2 to about 6;
X is O or S; and
R is perfluoroalkyl of 1 to about 3 carbons.
274. A composition according to Claim 273 wherein X is O.
275. A composition according to Claim 273 wherein m is an integer of 0 or 1, n is an integer of about 4 or about 5 and R is perfluoromethyl.
276. A composition according to Claim 255 further comprising a bioactive agent.
277. A method for preparing a stabilized composition which comprises a stabilizing compound and a gaseous perfluoroether compound, wherein the method comprises:
(a) providing a vessel containing a stabilizing compound and a perfluoroether compound; and (b) agitating said vessel to provide the stabilized composition.
278. A method according to Claim 277 wherein said stabilizing compound is selected from the group consisting of a lipid, protein and polymer.
279. A method according to Claim 278 wherein said stabilizing compound comprises a lipid.
280. A method according to Claim 279 wherein said lipid comprises a phospholipid.
281. A contrast agent according to Claim 280 wherein said phospholipid is selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid.
282. A method according to Claim 281 wherein said phosphatidylcholine is selected from the group consisting of dioleoylphosphatidylcholine, dimyristoyl-phosphatidylcholine, dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
283. A method according to Claim 282 wherein said phosphatidylcholine comprises dipalmitoylphosphatidylcholine.
284. A method according to Claim 281 wherein said phosphatidylethanolamine is selected from the group consisting of dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, N-succinyldioleoylphosphatidylethanolamine and 1 -hexadecyl-2-palmitoylglycerophosphoethanolamine.
285. A method according to Claim 284 wherein said phosphatidylethanolamine comprises dipalmitoylphosphatidylethanolamine.
286. A method according to Claim 281 wherein said phosphatidic acid comprises dipalmitolylphosphatidic acid.
287. A method according to Claim 279 wherein said lipid further comprises a polymer.
288. A method according to Claim 287 wherein said polymer comprises a hydrophilic polymer.
289. A method according to Claim 288 wherein said hydrophilic polymer comprises polyethylene glycol.
290. A method according to Claim 278 wherein said stabilizing compound comprises a protein.
291. A method according to Claim 290 wherein said protein comprises albumin.
292. A method according to Claim 278 wherein said stabilizing compound comprises a polymer.
293. A method according to Claim 292 wherein said polymer comprises synthetic polymers or copolymers which are prepared from monomers selected from the group consisting of acrylic acid, methacrylic acid, ethyleneimine, crotonic acid, acrylamide, ethyl acrylate, methyl methacrylate, 2-hydroxyethyl methacrylate, lactic acid, glycolic acid, e-caprolactone, acrolein, cyanoacrylate, cyanomethacrylate, bisphenol A, epichlorhydrin, hydroxyalkylacrylates, siloxane, dimethylsiloxane, ethylene oxide, ethylene glycol, hydroxyalkylmethacrylates, N- substituted acrylamides, N-substituted methacrylamides, N-vinyl-2-pyrrolidone, 2,4- pentadiene-1-ol, vinyl acetate, acrylonitrile, styrene, p-amino-styrene, p- aminobenzylstyrene, sodium styrene sulfonate, sodium 2-sulfoxyethyl-methacrylate, vinyl pyridine, aminoethyl methacrylates and 2-methacryloyloxytrimethyl-ammonium chloride.
294. A method according to Claim 292 wherein said polymer comprises synthetic polymers or copolymers selected from the group consisting of polyacrylic acid, polyethyleneimine, polymethacrylic acid, polymethylmethacrylate, polycyano-methacrylate, polysiloxane, polydimethylsiloxane, polylactic acid, poly(e- caprolactone), epoxy resin, poly(ethylene oxide), poly(ethylene glycol), polyamide, polyvinylidene-polyacrylonitrile, polyvinylidene-polyacrylonitrile- polymethylmethacrylate and polystyrene-polyacrylonitrile.
295. A method according to Claim 294 wherein said polymer comprises a polyvinylidene-polyacrylonitrile copolymer.
296. A method according to Claim 277 wherein said stabilized composition comprises vesicles.
297. A method according to Claim 296 wherein said vesicles are selected from the group consisting of micelles and liposomes.
298. A method according to Claim 296 wherein said vesicles are selected from the group consisting of unilamellar, oligolamellar and multilamellar vesicles.
299. A method according to Claim 298 wherein said vesicles comprise unilamellar vesicles.
300. A method according to Claim 299 wherein said vesicles comprise a monolayer.
301. A method according to Claim 299 wherein said vesicles comprise a bilayer.
302. A method according to Claim 298 wherein said vesicles are selected from the group consisting of oligolamellar and multilamellar vesicles.
303. A method according to Claim 302 wherein said vesicles comprise monolayers.
304. A method according to Claim 302 wherein said vesicles comprise bilayers.
305. A method according to Claim 277 wherein said perfluoroether compound has the formula
R,-X-R2 (I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and Rj are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
306. A method according to Claim 305 wherein X is O.
307. A method according to Claim 305 wherein R, and ^ are independently perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl.
308. A method according to Claim 307 wherein R, and R2 are independently perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
309. A method according to Claim 308 wherein R, and R2 are independently perfluoroalkyl.
310. A method according to Claim 309 wherein R, and R2 are independently perfluoroalkyl of 1 to about 6 carbons.
311. A method according to Claim 310 wherein R, and R2 are independently perfluoroalkyl of 1 to about 4 carbons.
312. A method according to Claim 311 wherein one of R, and R2 is perfluorobutyl and the other of R, and R2 is perfluoromethyl.
313. A method according to Claim 305 wherein R2 is halo.
314. A method according to Claim 313 wherein R2 is chloro or bromo.
315. A method according to Claim 313 wherein R, is perfluoroalkyl, perfluoroalkenyl or perfluorocycloalkyl.
316. A method according to Claim 315 wherein R, is perfluoroalkyl.
317. A method according to Claim 305 wherein R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl.
318. A method according to Claim 317 wherein R, and R2 are linked together to form perfluoroheterocyclyl.
319. A method according to Claim 277 wherein said stabilizing compound is substantially crosslinked or substantially non-crosslinked.
320. A method according to Claim 319 wherein said stabilizing compound is substantially crosslinked.
321. A method according to Claim 319 wherein said stabilizing compound is substantially non-crosslinked.
322. A method according to Claim 277 wherein said stabilizing compound and said perfluoroether compound are contained in said vessel at a reduced pressure.
323. A method according to Claim 322 wherein said stabilizing compound and said perfluoroether compound are agitated in said vessel at a reduced pressure.
324. A method for providing an image of an internal region of a patient comprising (i) administering to the patient a contrast agent according to Claim 1 , and (ii) scanning the patient using diagnostic imaging to obtain a visible image of the region.
325. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast agent according to Claim 1 , and (ii) scanning the patient using diagnostic imaging to obtain a visible image of any diseased tissue in the patient.
326. A method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a composition according to Claim 276.
327. A method for providing an image of an internal region of a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a lipid, protein or polymer, and a compound of the formula
Rj-X-R2
(I) wherein:
X is O or S; R[ is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of the region.
328. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a lipid, protein or polymer, and a compound of the formula
R|-X-R2
(I) wherein:
X is O or S;
R! is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R^ are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R{ and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of any diseased tissue in the patient.
329. A method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a formulation which comprises, in combination with a bioactive agent, a composition of a lipid, protein or polymer, and a compound of the formula R]-X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
330. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a gaseous compound of the formula R,-X-R2
(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and Rj are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
331. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a gaseous compound of the formula
R,-X-R2 (I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
332. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a gaseous compound of the formula
R,-X-R2
(I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
333. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a gaseous compound of the formula
R,-X-R2
(I) wherein: X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R[ and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
334. A contrast agent for diagnostic imaging comprising, in an aqueous carrier, a gaseous perfluoroether compound selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers contain no more than one oxygen atom in the heterocyclyl ring system.
335. A composition comprising, in an aqueous carrier, a gaseous compound of the formula
(I) wherein: X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or Rt and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
336. A composition comprising, in an aqueous carrier, a gaseous perfluoroether compound selected from the group consisting of perfluoroaliphatic ethers, perfluoroalicyclic ethers, perfluoroheterocyclyl ethers, perfluoroaryl ethers, perfluoroheteroaryl ethers and combinations thereof, with the proviso that the perfluoroheterocyclyl ethers comprise no more than one oxygen atom in the heterocyclic ring system.
337. A method for preparing a composition which comprises a gaseous perfluoroether compound, wherein the method comprises:
(a) providing a vessel containing a perfluoroether compound; and (b) agitating said vessel to provide the composition.
338. A method for providing an image of an internal region of a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a compound of the formula
R|-X-R2 (I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and 2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of the region.
339. A method for diagnosing the presence of diseased tissue in a patient comprising (i) administering to the patient a contrast agent comprising, in an aqueous carrier, a compound of the formula R,-X-R2
(I) wherein:
X is O or S; R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and
R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl; with the proviso that when R, and Rj are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom; and (ii) scanning the patient using ultrasound imaging to obtain a visible image of any diseased tissue in the patient.
340. A method for the therapeutic delivery in vivo of a bioactive agent comprising administering to a patient a therapeutically effective amount of a formulation which comprises, in combination with a bioactive agent, a composition of a compound of the formula
R,-X-R2 (I) wherein:
X is O or S;
R, is perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; and R2 is halo, perfluoroalkyl, perfluoroalkenyl, perfluoroalkynyl, perfluorocycloalkyl, perfluoroheterocyclyl, perfluoroaryl or perfluoroheteroaryl; or
R, and R2 are linked together to form perfluoroheterocyclyl or perfluoroheteroaryl ; with the proviso that when R, and R2 are linked together, the perfluoroheterocyclyl or perfluoroheteroaryl group contains no more than one oxygen atom.
EP97946447A 1996-11-25 1997-11-03 Perfluorinated-ether compositions as diagnostic contrast agents Withdrawn EP1001816A4 (en)

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US80099397A 1997-02-20 1997-02-20
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350571A (en) * 1988-04-29 1994-09-27 Hemagen/Pfc Emulsions of highly fluorinated organic compounds
WO1995031965A1 (en) * 1994-05-20 1995-11-30 Minnesota Mining And Manufacturing Company Physiologically acceptable emulsions containing perfluorocarbon ether hydrides and methods of use
WO1996031196A1 (en) * 1995-04-05 1996-10-10 Imarx Pharmaceutical Corp. Novel compositions of lipids and stabilizing materials
WO1996040281A2 (en) * 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Gas emulsions stabilized with fluorinated ethers having low ostwald coefficients
WO1998004292A2 (en) * 1996-07-29 1998-02-05 Acusphere, Inc. Polymer-lipid microencapsulated gases for use as imaging agents
WO1998042384A1 (en) * 1997-03-21 1998-10-01 Imarx Pharmaceutical Corp. Methods for delivering bioactive agents

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4004430A1 (en) * 1990-02-09 1991-08-14 Schering Ag CONSTRUCTED POLYALDEHYDE CONSTITUENTS
ATE264671T1 (en) * 1993-02-22 2004-05-15 American Bioscience Inc METHOD FOR THE IN VIVO ADMINISTRATION OF BIOLOGICAL SUBSTANCES AND COMPOSITIONS USABLE THEREFOR

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5350571A (en) * 1988-04-29 1994-09-27 Hemagen/Pfc Emulsions of highly fluorinated organic compounds
WO1995031965A1 (en) * 1994-05-20 1995-11-30 Minnesota Mining And Manufacturing Company Physiologically acceptable emulsions containing perfluorocarbon ether hydrides and methods of use
WO1996031196A1 (en) * 1995-04-05 1996-10-10 Imarx Pharmaceutical Corp. Novel compositions of lipids and stabilizing materials
WO1996040281A2 (en) * 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Gas emulsions stabilized with fluorinated ethers having low ostwald coefficients
WO1998004292A2 (en) * 1996-07-29 1998-02-05 Acusphere, Inc. Polymer-lipid microencapsulated gases for use as imaging agents
WO1998042384A1 (en) * 1997-03-21 1998-10-01 Imarx Pharmaceutical Corp. Methods for delivering bioactive agents

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO9823298A1 *

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