Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Ultrasound contrast agent
Reexamination Certificate
1997-05-27
2001-06-12
Dudash, Diana (Department: 1619)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Ultrasound contrast agent
C424S009510
Reexamination Certificate
active
06245318
ABSTRACT:
FIELD OF THE INVENTION
The invention is in the field of imaging. In particular, the invention is in the field of ultrasound imaging.
BACKGROUND OF THE INVENTION
Use of ultrasound in medical imaging has greatly increased during the past few decades. Currently, 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. In many instances 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. Subsequent to administration, the microbubble-containing tissues of the body are visible on the screen of the imaging system as bright areas. However, even with the use of these contrast materials, 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.
It would be beneficial to design a targetable microbubble ultrasound contrast agent that would selectively bind to the areas of interest in the body and enhance the target contrast in the ultrasound examination.
SUMMARY OF THE INVENTION
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.
DETAILED DESCRIPTION OF THE INVENTION
Previously, attachment of targeting ligands to ultrasound contrast agents was not performed via flexible polymer chain anchors. Direct coupling via chemical crosslinking agents was typically performed in accordance with standard protein-polymer or protein-lipid attachment methods (e.g., via carbodiimide (EDC) or thiopropionate (SPDP)).
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. Also, 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. Also, 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. Such lipid-PEG-ligand compounds have been used for the attachment of ligands to liposomes (bilayer of lipids). In a lipid monolayer-coated gas microbubble of the invention 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. Unlike the case of liposomes, where a lipid bilayer is formed with the lipid residues facing each other, in the monolayer-coated gas bubble of the invention 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., Trubetskoy V. S., Whiteman K., Milstein A. M.,
Biochim.Biophys.Acta,
1994, 1195:181-184.; Maruyama K., Okuizumi S., Ishida O. et al.,
International J. Pharmaceutics,
1994, 111:103-107; and Woodle M. C., Engbers C. M., Zalipsky S.,
Bioconjugate Chemistry,
1994, 5:493-496.
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 Waals forces) could be used to further strengthen the anchoring of the ligand on the microbubble shell.
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). For example, a reactive moiety present in one of the R groups is coupled with a second reactive group located on the biomolecule. Typically, a nucleophilic group is reacted with an electrophilic group to form a covalent bond between the biomolecule and the chelate. Examples of nucleophilic groups include amines, anilines, alcohols, phenols, thiols and hydrazines. Electrophilic group examples include halides, disulfides, epoxides, maleimides, acid chlorides, anhydrides, mixed anhydrides
Klibanov Alexander L.
Lyle Leon R.
Thomas M. Elizabeth
Dudash Diana
Guffey Wendell Ray
Hartley Michael G.
Limpus Lawrence L.
Mallinckrodt Inc.
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