Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1998-09-02
2001-05-01
Riley, Jezia (Department: 1656)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C514S001000, C536S022100, C435S006120, C435S007100, C435S007200
Reexamination Certificate
active
06225293
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to the use of fluorescently labeled carriers and macromolecules for following cellular trafficking of certain macromolecule-carrier combinations. In particular embodiments, this invention can be utilized to determine cellular pathways for oligonucleotide uptake and to ascertain biodistribution of macromolecules and carriers in cells.
BACKGROUND OF THE INVENTION
Antisense oligonucleotides specifically target a messenger RNA. They have found a wide range of in vitro functions including roles in diagnostics and research. They are also a potentially important class of novel therapeutics, with several in clinical trials.
In order to find its target in vitro, an oligonucleotide must enter the cell and localize to its target in the cytoplasm and/or nucleus. Unmodified oligonucleotides have demonstrated poor ability to cross cell membranes. Several approaches to enhance the uptake of oligonucleotides have been tried, including liposomal encapsulation, conjugation with other ligands, such as cholesterol, peptides and poly-lysines, and cationic lipids. Cationic lipids have been shown to increase the cellular uptake of oligonucleotides, but the mechanism by which this occurs is not well understood (Bennett, C. F., et al.,
Mol. Pharmacol.
1992 41, 1023-1033).
Oligonucleotide uptake is thought to proceed initially by interaction with cell surface proteins, followed by internalization through an endocytic mechanism (Loke, S. L., et al.,
Proc. Natl. Acad. Sci. USA
1989, 86, 3474-3478). These events lead to a punctate distribution of the oligonucleotides in intracellullar membrane-bound structures, which are thought to represent endosomes and lysosomes (Bennett, C. F., et al.,
Mol. Pharmacol.
1992, 41, 1023-1033). The sequestration of oligonucleotides into endosomal compartments may prevent their interaction with their target mRNA and hence decrease their activity.
One of the most commonly used enhancers is a mixture of a neutral lipid with a cationic lipid. The cationic lipid is thought to be the more crucial part of this mixture, as cationic lipids increase the activity of antisense oligonucleotides, whereas neutral lipids cannot. The mechanism by which cationic lipids increase the activity of antisense oligonucleotides is poorly understood. It was recently demonstrated that oligonucleotide-lipid complexes are taken into the cell via an endocytic mechanism and do not simply fuse with the plasma membrane (Zelphati, O. and Szoka, F. C., Jr.,
Pharmacol. Res.
1996, 13, 1367-1372). It is also known that cationic lipids enhance cellular accumulation of the oligonucleotide by increasing the amount that escapes from the endosomal pathway and thus has an opportunity to interact with its target mRNA (Bennett, C. F., et al.,
Mol. Pharmacol.
1992, 41, 1023-1033). While the mechanism by which an oligonucleotide is released from the endocytic compartments in order to gain access to its RNA target is not well characterized, models have been proposed (Zelphati, O. and Szoka, F. C., Jr.,
Proc. Natl. Acad. Sci. USA
1996, 93, 11493-11498). Fluorescence studies by Zelphati, O. and Szoka, F. C., Jr. have recently shown that when transfected with a cationic
eutral lipid mixture, oligonucleotides reached the nucleus while the neutral lipid stayed in punctate structures within the cytoplasm. These punctate structures were presumed to be endosome. However, the final destination of the cationic lipid was not examined in this study and whether the cationic lipid traffics together with the oligonucleotide to the nucleus was not determined.
Fully understanding the mechanism by which oligonucleotides are taken up by cells and released from endosomes will aid in the design of delivery vehicles to improve both in vitro and in vivo efficacy. Such improved designs are important in the investigation of the therapeutic utility of antisense oligonucleotides and to improve the dosing regimens for antisense therapeutics.
In Zelphati, O. and Szoka, F. C., Jr., cationic lipids were labeled with rhodamine and oligonucleotides were labeled with fluorescein. The mixture was introduced to cells. However, it is known that fluorescein has a quenching effect with respect to the cell culture medium, which results in loss of the fluorescent signal. Quenching also occurs using fluoroscein in acidic cellular environments, including endosomes, lysosomes and lipophilic cell surfaces. With quenching, it becomes more difficult to follow macromolecules, especially oligonucleotides, to such environments.
Thus, there remains a need for improved methods for tracking the distribution of carriers and macromolecules within a cell.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with this invention there are provided methods of tracking the cellular trafficking of a macromolecule and a carrier in a cell, especially in appropriate cell culture medium comprising selecting a macromolecule with a first label and a carrier with a second label. The second label is selected to be distinguishable from the first label and both of the first and second labels are substantially free from quenching effects with respect to said cell culture medium and acidic cellular environments. The macromolecule labeled with said first fluorescent label and the carrier labeled with said second fluorescent label are then coadministered to the cell and the labels are tracked to distinguish each of said labels within the cell.
Also provided are compounds useful for carrying out the methods of the invention. Preferred from among such compounds are those have the general formula:
Wherein R
1
is preferably an aliphatic group having from 12 to about 20 carbon atoms or a steroid molecule while R
2
is preferably an aliphatic group having from 12 to about 20 carbon atoms. R
3
and R
4
are preferably CH
3
or CH
2
CH
3
while R
5
is preferred to be CH
3
, (CH
2
)
n
CH
3
where n=1 or 2, or (CH
2
)
n
OH where n=1 to 5. The value of X is preferably Br, I, or Cl and Y, and Y
2
are, independently, an ether, an ester, a carbamate, a carbonate, an amide, a peptide linkage, or a urea linkage.
In accordance with other preferred embodiments, compounds of the structure:
are provided for use with the methods of the present invention wherein the values for R
1
, R
2
, R
3
, R
4
, R
5
, Y
1
, Y
2
and X are as set forth above and Z is a label, preferably a fluorescent label.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The methods and compounds of the invention provide improved means to measure the cellular trafficking of a macromolecule and a carrier. A macromolecule is a molecule of interest that is desired to be introduced to the cell. A carrier is the delivery vehicle for that macromolecule. It is desirable to understand and determine the cellular trafficking of such a complex. This will allow further improvements in the design of these macromolecules and carriers.
A macromolecule as used in this invention includes polynucleotides, including DNA, RNA and oligonucleotides, proteins or peptides, and antibodies. These macromolecules may have therapeutic potential as in the case of antisense oligonucleotides, ribozymes, bioactive peptides, and monoclonal antibodies. In preferred embodiments of the invention, the macromolecule is an antisense oligonucleotide.
Carriers as that term is used in this invention, include anything capable of enhancing the delivery of a macromolecule into a cell and to its final destination within a cell. This would include cationic lipids (Bennett, C. F., et al.,
Mol. Pharmacol.
1992, 41, 1023-1033), liposomes (Bennett, C. F.,
Delivery Strategies for Antisense Oligonucleotide Therapeutics,
Akhtar, S. (ed.) CRC Press, Boca Raton, Fla., 1995, pp. 223-232), peptides (Bongartz, J. P., et al.,
Nucleic Acids Res.
1994, 22, 4681-4688), polycations (Boussif, O., et al.,
Proc. Natl. Acad. Sci. USA
1995, 92, 7297-7301), dendrimers (Bielinska, A., et al.,
Nucl. Acids Res.
1996, 24, 2176-2182) and conjugation with cholesterol (Krieg, A. M., et al.,
Proc. Natl. Acad. Sci. USA
1993, 90, 1048-1052).
Bennett Clarence Frank
Bhat Balkrishen
Hebert Normand
Manoharan Muthiah
ISIS Pharmaceuticals Inc.
Riley Jezia
Woodcock Washburn Kurtz Mackiewicz & Norris LLP
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