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
• • 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
  • A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric

Definitions

• the invention is in the field of imaging.
• the invention is in the field of ultrasound imaging.
• ultrasound imaging has greatly increased during the past few decades.
• this method of diagnostic imaging is widespread and inexpensive; reasonable quality real-time images of internal organs can be obtained.
• Ultrasound imaging systems are small, mobile and have low cost.
• a general disadvantage of this modality in comparison with nuclear medicine and magnetic resonance imaging (MRI) techniques is its lower specificity.
• MRI magnetic resonance imaging
• medical ultrasound is unable to distinguish between normal and diseased tissues.
• a major improvement was achieved with the recent introduction of the ultrasound contrast materials, which could be used to aid delineation of blood/tissue boundaries and help visualize blood flow and blood supply in various organs.
• the most efficient ultrasound contrast materials are gas microbubbles, which are strong scatterers of ultrasound. Microbubble dispersions can be administered to the patient intravenously or by other routes.
• microbubble-containing tissues of the body are visible on the screen of the imaging system as bright areas.
• selective enhancement of certain diseased tissues for ultrasound imaging is still far from clinical application. This task of selective enhancement might be achieved with targeted ultrasound contrasts .
• Targeting of various contrast agents used for other imaging modalities has been performed for decades. Small molecules, macromolecules, polymers and particles have been successfully delivered to target sites for specific enhancement of the diseased tissue images. Such imaging agents have slowly made their way into clinical practice. Targeted ultrasound contrast agents, which could selectively bind to the specific sites after in vivo administration, were postulated more than a decade ago. Still, commercially successful targeted ultrasound contrast agents have yet to be reported.
• the invention discloses an ultrasound contrast agent comprising a monolayer microbubble-shell and a composition of the general formula:
• A-P-L wherein A is an ultrasound contrast agent microbubble-shell binding moiety; P is a spacer arm; and L is a ligand.
• the present invention discloses compositions, methods of imaging, and methods for attachment of targeting ligands to ultrasound contrast agents, allowing selective targeting of agents to desired sites.
• EDC carbodiimide
• SPDP thiopropionate
• An advantage of the disclosed invention is the availability of the targeting ligand to accommodate conformation of the receptor at the binding site or sites, due to flexibility of the spacer arm.
• the ligand is spatially separated from the particle; thus reducing steric hindrance for binding to the target from the bulky ultrasound contrast agent particle. This spatial separation improves targeting efficacy.
• more than one type of ligand may be used with the invention, allowing a cocktail approach to binding numerous targets .
• the invention allows multipoint cooperative interaction between a microbubble and target, and will help to accommodate ligand-receptor interaction in the most favorable conformation, allowing stronger binding of the microbubble to the target.
• Use of the A-P-L design will compensate for the irregularities of the shape of the target, which will otherwise make target receptor molecules on its surface inaccessible for the ligand to be directly immobilized on the surface of the microbubble.
• Spacer arms for use with the invention include a branched or linear synthetic polymer or a biopolymer like polyethyleneglycol (PEG) , polyvinylpyrrolidone, polyoxyethylene, polyvinylpyridine, polyvinyl alcohol, polyglycerol, dextran, and starch.
• PEG polyethyleneglycol
• Such lipid-PEG-ligand compounds have been used for the attachment of ligands to liposomes (bilayer of lipids) .
• the lipid molecules will be facing the gas phase so the anchor will be deposited at the gas-liquid interface, the polymer spacer arm will be extended in the aqueous medium in order to allow improved interaction of the ligand with the target surface.
• the lipid molecules face the gas phase.
• the anchor will be deposited at the gas-liquid interface, and the flexible polymer spacer arm will be extended in the aqueous medium allowing improved interaction of the ligand with the target surface.
• a flexible spacer arm can provide advantages and the polymers, when used, generally will have over ten monomer residues.
• Other hydrophilic polymers are proposed as liposome coatings in Torchilin V.P., Shtilman M. I., Tfubetskoy V.S., hiteman K., Milstein A.
• the ultrasound contrast agent monolayer microbubble-shell- binding groups for use with the invention include lipids, phospholipids, long-chain aliphatic hydrocarbon derivatives, lipid multichain derivatives, comb-shaped lipid-polymer derivatives (usually including hydrophobic residues attached), steroids, fullerenes, polyaminoacids, native or denatured proteins, albumin, phosphatidylethanolamine (e.g., distearoyl phosphatidylethanolamine), cardiolipin, aromatic hydrocarbon derivatives, and partially or completely fluorinated lipid derivatives. It is preferable to have larger-size derivatives which would be difficult to remove from the bubble surface gas-liquid interface.
• Spreading and/or intermolecule binding at the monolayer interface e.g., covalent, ionic or hydrogen bonding or van der aals forces
• the ligand for use with the invention can be a biomolecule.
• Biomolecule refers to all natural and synthetic molecules that play a role in biological systems. Biomolecules include hormones, amino acids, vitamins, peptides, peptidomimetics, proteins, deoxyribonucleic acid (DNA) ribonucleic acid (RNA) , lipids, albumins, polyclonal antibodies, receptor molecules, receptor binding molecules, monoclonal antibodies, carbohydrates and aptamers . Specific examples of biomolecules include insulins, prostaglandins, cytokines, chemokines, growth factors including angiogenesis factors, liposomes and nucleic acid probes. The advantages of using biomolecules include enhanced tissue targeting through specificity and delivery.
• Coupling of the chelating moieties to biomolecules can be accomplished by several known methods (e.g., Krejcarek and Tucker Biochem. Biophys . Res . Comm, 30, 581 (1977); Hnatowich, et al . Science, 220, 613 (1983).
• a reactive moiety present in one of the R groups is coupled with a second reactive group located on the biomolecule.
• a nucleophilic group is reacted with an electrophilic group to form a covalent bond between the biomolecule and the chelate.
• nucleophilic groups include amines, anilines, alcohols, phenols, thiols and hydrazines.
• Electrophilic group examples include halides, disulfides, epoxides, maleimides, acid chlorides, anhydrides, mixed anhydrides, activated esters, imidates, isocyanates and isothiocyanates .
• Biomolecules can be covalently or noncovalently attached to one of the tips of the polymer chain, while the lipid anchor grouping is attached to the other end of this polymer chain.
• a specific example of using the claimed invention is tumor targeting.
• Ligands designed to bind specifically to receptors for angiogenesis factors expressed in tumor microvasculature and coupled to echogenic contrast agents enhance the specificity and sensitivity of ultrasound tumor detection.
• Angiogenesis is a process associated with tumor growth.
• Several peptides have been identified as promoters of angiogenesis including interleukins 8, and 6, acidic FGF, basic FGF, TNF-alpha, TGF-alpha, TGF-beta, and VEGF/VPF. See Rak, J. .; St. Croix B.D. and Kerbel R.S. (1995), Anti-Cancer Drugs 6, p. 3-18. See also Bicknell, R. (1994), Annals of Oncology 5 (Suppl. 4), p. 545-550.
• a specific ligand useful for targeting tumor vasculature is the chemokine IL- 8 or an analog, homolog, derivative or fragment thereof, or a peptide having specificity for a receptor of interluekin 8.
• Particularly useful are the amino acid residues at the N terminal end of IL-8, including the ⁇ LR" sequence glu- leu-arg found immediately before the initial cysteine residue. It is known that the ELR amino acid sequence of IL-8 is important for the binding interaction with its receptor.
• the ELR motif also apparently imparts the angiogenic properties of IL-8. See Strieter, R.M.; Kunkel, S.L.; Palverini, P.J.; Arenberg, D.A.; Waltz, A.; Opdenakker, G. and Van Damme, J. (1995), Journal of Leukocyte Biology 576, p. 752-762.
• IL-8 See U.S. patent 5,436,686 Sep. 13, 1994, incorporated herein by reference.
• Monolayer microbubble-shells include any composition suitable for ultrasound imaging and capable of being gas filled, liquid filled, or combinations of gas and liquid, and includes those with a protein shell, natural polymer shell, synthetic polymer shell, surfactant, lipid, phospholipid, sphingolipid, sulfolipid, oligolipid, polymeric lipid, sterol, terpene, fullerene, wax, or hydrocarbon shell or any combination of these.
• Gases, liquids, and combinations thereof suitable for use with the invention include decafluorobutane, octafluoro- cyclobutane, decafluoroisobutane, octafluoropropane, octafluorocyclopropane, dodecafluoropentane, decafluoro- cyclopentane, decafluoroisopentane, perfluoropexane, perfluorocyclohexane, perfluoroisohexane, sulfur hexafluoride, and perfluorooctaines, perfluorononanes; perfluorodecanes, optionally brominated.
• an aqueous dispersion of phospholipid (DSPC) , surfactant (PEG stearate) and biotinamidocaproyl PEG-DSPE are mixed in an organic solvent, then the solvent evaporated and saline added. After that, the mixture is generally blended (e.g. sonication, colloid mill) in order to create an aqueous dispersion of the components, and then blending is continued in the presence of the flow of a gas such as decafluorobutane gas, which is dispersed in the form of microbubbles in the aqueous phase.
• a gas such as decafluorobutane gas
• DSPC, PEGstearate and Bac-PEG-DSPE are deposited on the gas- aqueous interface, with hydrophobic residue facing the gas phase and hydrophilic part of the molecule (including the ligand part) immersed in the aqueous phase.
• hydrophobic residue facing the gas phase
• hydrophilic part of the molecule including the ligand part
• the whole surface of the microbubble formed is covered by these molecules which thus create a protective shell.
• the microbubbles are purified from the aqueous dispersion such as by flotation: due to the fact that gas- filled microbubbles are much less dense than water, they will float to the top of solution.
• the aqueous phase is aspirated and replaced with fresh saline which does not contain the shell material, or contains the shell material without the targeting ligand. Centrifugation speeds up the flotation process. This step is employed to remove biotinylated material from solution, otherwise it may block the avidin binding sites. In that case, no biotin-mediated binding of microbubbles may occur.
• ligand-PEG-PE may be added as an aqueous solution to preformed microbubbles, so that the anchor will incorporate in the preformed microbubble shell.
• composition of the invention would include biotinamidocaproyl-poly (ethyleneglycol) - distearoylphosphatidylethanol amine, Sialyl Lewis-poly
• a second attachment procedure is based on the avidin-biotin bridge method originally developed for protein immobilization on regular PEG-free liposomes. See Trubetskaya O.V., Trubetskoy V. S., Domogatsky S. P., Rudin A.V., Popov N.V., Danilov S.M., Nikolayeva M.N., Klibanov A.L., Torchilin V.P., FEBS Lett. , 1988, 228: 131-134, and Loughrey H. C, Bally M.B., Cullis P.R. Biochim. Biophys. Acta, 1987, 901: 157- 160.
• Biotinylated phosphatidylethanolamine is incorporated into the liposome membrane, then avidin is added and attached to biotin on liposomes. Free binding sites on avidin are used to bind biotinylated antibodies which are added later.
• the third approach takes advantage of bifunctional polyethylene glycol, which possesses reactive groups on both ends of the polymer chain.
• PEG-PE with an active group on the outer tip of PEG is synthesized, purified and incorporated in liposomes.
• Various chemistries can be applied for this purpose- See Allen T., Agrawal A.K., Ahmad I., Hansen C.B., Zalipsky S., J. Liposome Res., 1993, 4:1-25 and Blume G., Cevc G., Crommelin M.D.J.A. et al . , Biochim. Biophys. Acta 1993, 1149:180-184.
• Blume and Cevc propose to use PEG with two terminal carboxy residues, one of which is coupled to the primary aminogroup of DSPE.
• Blume G., Cevc G. Crommelin M.D.J.A. et al . , Biochim. Biophys ..Acta 1993, 1149:180-184.
• the resulting PE- PEG-COOH is incorporated in the liposome, and the terminus is activated by water-soluble carbodiimide . After the activation the protein is added in mild alkaline medium. Up
• maleimide- PEG-PE a reagent proposed by Liposome Technology Inc. (Fig.5C).
• PEG residue was used as a spacer arm between the lipid anchor and the maleimide group. This chemistry was proposed for antibody binding to the liposome surface more than a decade ago; and at that time, a maleimide bifunctional cross-linking reagent with a short spacer arm was used.
• Martin F. J. Papahadjopoulos D., J. Biol.
• a thiol group generated on the antibody molecule or Fab ' -fragment, is used to attach to the maleimide residue on tip of the PEG with the formation of a stable thioether bond.
• NP-PEG-PE p-nitrophenylcarbonyl-PEG-PE
• compositions of the invention can be formulated into diagnostic compositions for enteral or parenteral administration. These compositions contain an effective amount of the ultrasound agent along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated. Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride.
• Formulations for enteral administration may vary widely, as is well-known in the art.
• such formulations are liquids which include an effective amount of the ultrasound agent in aqueous solution or suspension.
• Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like.
• Compositions for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities.
• the diagnostic compositions are administered in doses effective to achieve the desired enhancement of the ultrasound image. Such doses may vary widely, depending upon the particular agent employed, the organs or tissues which are the subject of the imaging procedure, the imaging procedure, the imaging equipment being used, and the like.
• compositions of the invention are used in the conventional manner.
• the compositions may be administered to a patient, typically a warm-blooded animal, either systemically or locally to the organ or tissue to be imaged, and the patient then subjected to the imaging procedure.
• Protocols for imaging and instrument procedures are readily found in texts.
• the following examples illustrate the specific embodiments of the invention described in this document. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.
• Example 1 In this example we have used the interaction between avidin and biotin (Fig. 2) as a model system for microbubble targeting.
• Avidin can be tightly attached to solid surfaces, e.g. to standard polystyrene 35mm tissue culture plates, by simple incubation.
• the biotin molecule possesses a carboxyl group, so it can be covalently attached to microbubble shell material, either prior to microbubble preparation or after the shell around the bubbles has been formed.
• the avidin solution (2 mg/ml in saline) was incubated in a 35mm tissue culture-treated polystyrene Petri dish for 1 hour.
• the avidin solution either filled the dish completely or was deposited in certain areas of the dish using a micropipet, creating target zones coated with avidin. These areas were marked with a waterproof marker in order to improve visual identification of the target.
• bovine serum albumin solution 300 mg/ml was added to block nonspecific binding sites on the dish. Unbound avidin and albumin were removed by a vigorous wash in a stream of tap deionized water.
• the dish was then filled with normal saline, the microbubble dispersion added, the dish was sealed with mylar self-adhesive tape and inverted. Microbubbles were allowed to float to the dish surface and contact the avidin-coated or the albumin-coated surface to allow avidin-biotin binding to occur (Fig. 2) . Unless otherwise noted, after completion of the incubation, the dish was inverted back, unsealed, and unbound microbubbles were removed by a wash with a stream of deionized water. In a control study, 1 mg/ml biotin was added to the Petri dish in order to block avidin on the surface. Microbubble attachment to the plate was characterized by optical microscopy as described above.
• microbubbles can be selectively targeted via a ligand-receptor system in vitro. Firm binding of microbubbles to avidin-coated surface can be achieved.
• microbubbles in suspension are strong ultrasound scatterers may not necessarily imply that microbubbles immobilized on the surface will produce a strong ultrasound contrast effect.
• the C-scan ultrasound measurement and imaging system was used to insonify microbubbles deposited on a Petri dish.
• the dish was mounted in a stationary fixture which was submerged in a degassed, room-temperature water bath (Fig. 3).
• the mounting fixture permitted the dish to be tilted at an angle of 10° relative to the axis of insonification, in order to minimize the ultrasonic specular reflection from the dish surface.
• the center of the dish was placed in the focal zone of the transducer in order to maximize sensitivity and spatial resolution of the image.
• the peak-to-peak voltage of the backscattered signal was acquired as the transducer was translated in a two dimensional grid measuring 3.5 by 3.5 cm, in steps of 0.05 cm. A two dimensional peak-to-peak image was then generated to show the contrast between regions of high and low reflectivity.
• FIG. 9B A sharp acoustic image of microbubbles deposited on the target surface was obtained (Fig. 9B) , which corresponded to the pattern of avidin coat on the dish. Presence of microbubbles on avidin-coated areas was verified by optical microscopy (Fig. 9A) .
• the sensitivity of our custom-built ultrasound laboratory apparatus may be higher than that of a standard medical imaging system.
• acoustic imaging studies of Petri dishes coated with targeted microbubbles were performed with a medical ultrasound imaging system and a tissue-mimicing ultrasound phantom.
• the phantom served as a human tissue-equivalent background to permit the ultrasound imaging device receive gain and transmit intensity to be set as they would for imaging of human tissue .
• a plastic tape skirt was placed around the top of a standard ultrasound tissue-mimicking phantom (RMI, 0.5 dB/cm/NnHz) to create a deionized water-filled basin on top of the phantom.
• the reservoir provided a water path stand-off for coupling the imaging probe to the sample
• a plastic tape skirt was placed around the top of a standard ultrasound tissue-mimicking phantom (RMI, 0.5 dB/cm/MHz) to create a deionized water-filled basin on top of the phantom.
• the reservoir provided a water path stand-off for coupling the imaging probe ⁇ to the sample.
• a Petri dish with immobilized microbubbles or a control dish without bubbles was placed inside this water reservoir on top of the phantom.
• the ultrasound medical imaging system probe was coated with coupling gel and placed in a protective rubber sleeve designed for this purpose. The tip of the probe was then immersed in the reservoir and positioned so that the distance between the tip and the dish would not exceed 5 cm (Fig. 4) . The probe was positioned at an angle ( ⁇ 45° to the plane of the dish) to avoid specular reflection from the polystyrene surface.
• Ultrasound images were recorded on video tape. Transmitted intensity and receive gain settings of the medical imaging system were adjusted prior to the experiment, so that a clear view of the tissue phantom could be obtained, i.e. the sensitivity of the imager was in the range generally used for the imaging of patients.
• Video microscopy of microbubbles on the Petri dish was performed prior to ultrasonic imaging. The fraction of the surface area occupied by microbubbles was calculated using image analysis software .
• mouse antibody e.g. a non-specific mouse IgG or an anti-ICAM-1 monoclonal antibody: 200 ul IgG in saline (1 mg IgG)+ 100 ul 1M HEPES pH 7.9
• a 20-kg dog was anesthetized with thiopental, placed on a respirator and continuously anesthetized with isoflurane/air mixture.
• An IV catheter was used for intravenous injections of the microbubble dispersion.
• Ultrasound imaging was performed using a HP Sonos 500 ultrasound medical system equipped with a 5 MHz probe. Images of the dog heart before and after injection of contrast were recorded on a video tape. The images were digitized for storage and printing using a Nova Microsonics digitizer station.

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• 1997
  • 1997-05-27 US US08/863,647 patent/US6245318B1/en not_active Expired - Lifetime
• 1998
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