Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1994-10-12
2001-05-15
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S455000, C424S009100, C424S009200, C424S009300, C424S009320, C424S009340, C424S009360, C424S009364, C424S009365
Reexamination Certificate
active
06232295
ABSTRACT:
BACKGROUND OF THE INVENTION
In recent years, magnetic resonance imaging (MRI) has emerged as a powerful tool in clinical settings because it is noninvasive and yields an accurate volume rendering of the subject. The image is created by imposing one or more orthogonal magnetic field gradients upon the specimen while exciting nuclear spins with radio frequency pulses as in a typical nuclear magnetic resonance (NMR) experiment. After collection of data with a variety of gradient fields, deconvolution yields a one, two, or three dimensional image of the specimen. Typically, the image is based upon the NMR signal from the protons of water where the signal intensity in a given volume element is a function of the water concentration and relaxation times (T
1
and T
2
). Local variations in these three parameters provide the vivid contrast observed in MR images. For example, the low water content of bone makes it distinctively dark, while the short T
2
of clotted blood affords it a higher signal intensity than that from non-clotted blood.
The same advantages that have made MRI the technique of choice in medical imaging make it an ideal imaging tool for use in biological experiments. Unlike light-microscope imaging techniques based upon the use of dyes or fluorochromes, MRI does not produce toxic photobleaching by-products. Furthermore, unlike light-microscopy, MRI is not limited by light scattering or other optical aberrations to cells within approximately only one hundred microns of the surface. MRI was originally considered a purely noninvasive approach but more recently it has been found that contrast agents can significantly improve the diagnostic utility of the technique. MRI contrast agents dramatically reduce the relaxation times of protons in the surrounding water. The ion Gd
3+
, in its non-toxic chelated forms, is the most commonly used paramagnetic ion because of its large magnetic dipole and large effect on relaxation times. For example, Gd
3+
chelated with diethylenetriaminepentaacetic acid (DTPA) is a vascular contrast agent now widely used in diagnostic radiology. The chemical structure of DTPA is depicted in FIG.
4
.
Traditional MRI offers high spatial resolution and multiple plane imaging in a fast noninvasive procedure. When MRI contrast agents are used diagnostically, they are vascularly perfused, enhancing the contrast of blood vessels and reporting on organ lesions and infiltration. However, the labeling of specific tissues for diagnostic radiology remains a difficult challenge for MRI. Efforts to develop cell and tissue-specific MRI contrast agents by modifying existing immunological techniques has been the focus of much research in diagnostic radiology. For example, antibodies labeled with paramagnetic ions, generally the gadolinium chelate Gd-DTPA, have been generated and tested for their effects on MRI contrast of tumors and other tissues [Lex,
Acta Biochim. Biophys. Hung.
24:265-281 (1989); U.S. Pat. No. 5,059,415]. It was anticipated that due to reductions in the rate of molecular tumbling, Gd-DTPA when bound to antibodies would show significantly higher relaxivity, a measure of MRI contrast enhancement, than that of unbound Gd-DTPA. This increase in relaxivity per Gd ion, it was hoped, would generate sufficient signal for tissue contrast to be observed using antibodies labeled with 10-50 Gd ions per protein molecule.
Unfortunately, the relaxivity of Gd bound to antibodies has been found to be only slightly better than that of unbound Gd-DTPA [Paajanen et al.,
Magn. Reson. Med
13:38-43 (1990)]. Therefore, to generate detectable contrast enhancement in an antibody-labeled tissue, the immunological reagent must be conjugated with hundreds if not thousands of Gd ions per antibody. Currently this is unattainable using standard techniques.
Several researchers have examined the possibility that the number of Gd ions per antibody could be increased by conjugating polylysine to the antibody, then labeling the polylysine extensively with Gd-DTPA [WO93/01837]. So far, these attempts have shown only limited success in part due to the unfavorable ionic and steric effects of conjugating antibodies to large polymers.
Research in the field of targeted MRI contrast agents has thus turned to the use of iron oxide particles as high signal strength T
2
contrast agents [Shen et al.,
Magnet. Res. Med.
29:599-604 (1993); Weissleder et al.,
Magnetic Resonance Quarterly,
8:55-63 (1992)]. However, no iron oxide particles have yet been approved for use in humans.
Liposomes as carriers of contrast media show promise as tissue-specific MRI agents as well [Schwendener, R. A.,
Chimia
46:69-77 (1992)]. Two classes of such contrast agents have been developed: (i) water soluble contrast agents entrapped between phospholipid bilayers, and (ii) liposomes directly incorporating amphipatic molecules covalently attached to MRI contrast agents such as Gd-DTPA. The former class of liposomal contrast agents suffers from leakiness of the water soluble agent in vivo, and the later from long-term retention of the agent in the liver and spleen. Nevertheless, liposomes show promise as liver, spleen and lung contrast agents.
SUMMARY OF THE INVENTION
Based on the foregoing it is apparent that there exists a need for improved tissue-specific delivery of contrast agents. Accordingly, it is an object of the invention to provide tissue-specific agents that are capable of binding multiple contrast agents without losing tissue-specificity.
It is a further object of the invention to combine novel tissue-specific contrast agents with nucleic acids to provide delivery vehicles useful in gene delivery and therapy. Such gene delivery can be monitored by way of the presence or absence of the contrast agent.
These and other objects and features of the invention will become apparent to those skilled in the art from the following detailed description and appended claims. The objects are achieved by cell-specific delivery vehicles and methods wherein such delivery vehicles are capable of delivering at least an imaging contrast agent to the targeted cell or tissue. In some embodiments the delivery vehicle is constructed to deliver additional specific molecules (e.g. nucleic acids).
In one embodiment of the invention, an MRI contrast agent delivery vehicle is provided that is capable of enhancing the observed contrast of specific cells and tissues by MRI by targeting cell-surface receptors on such cells or tissue. The delivery vehicle comprises a) a first polymeric molecule having a net positive or negative charge, b) at least one second polymeric molecule having a net charge opposite that of the first polymeric molecule and complexed with the first polymeric molecule, the second polymeric molecule having attached thereto at least one cell targeting moiety, and c) at least one contrast agent attached to the first or second polymeric molecule (see
FIGS. 1A and 1B
) or to a third polymeric molecule (see FIG.
1
C), wherein the third polymeric molecule, if present, has a net charge opposite that of the first polymeric molecule and is complexed with the first polymeric molecule. Thus, the invention can use signal amplification following polycation/polyanion complex formation to induce signal enhancement for MRI.
In another embodiment, one of the polymeric molecules comprises a nucleic acid which is complexed with one or more polymeric molecules comprising a polyamine, so that the resulting contrast agent delivery vehicle is capable of delivering genetic material as well as a contrast agent in a cell or tissue-specific manner.
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p
Fraser Scott E.
Kayyem Jon Faiz
Meade Thomas J.
Flehr Hohbach Test Albritton & Herbert LLP
Priebe Scott D.
Silva Robin M.
Trecartin Richard F.
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