Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-04-29
2001-05-08
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C514S04400A
Reexamination Certificate
active
06228392
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a composition and solution, as well as use of such composition and solution for delivering substances into the cytosol of living eucaryotic cells.
REFERENCES
BioProbes
28:13. May 1998, Pub. by Molecular Probes, Inc., Eugene, Oreg.
Chakrabarti et al.,
Journal of Biol. Chem.
264:8214 (1989).
Lee et al.,
Cytometry
14:265 (1993).
McElligott & Dice,
Bioscience Reports
4:451 (1984).
Okada & Rechsteiner,
Cell
29:33 (1982).
Rechsteiner,
Methods in Enzymology
149:42 (1987).
2. Background and Prior Art
In the course of biological research and certain medical procedures it is desirable to introduce a variety of substances into the cytosol of living eucaryotic cells. However, most hydrophilic substances, including many drugs, antisense and antigene oligos, RNAs, DNAs, peptides, proteins, carbohydrates, and combinations thereof, enter eucaryotic cells primarily via endocytosis and are subsequently sequestered or degraded in lysosomes, with little of no intact substance achieving the desired entry into the cytosol of the cells.
A variety of methods have been devised to deliver hydrophilic substances directly across the cytoplasmic membrane, including: complexing such substances with or incorporating such substances within liposomes (Capaccioli et al.,
Biochem. Biophys. Res. Comm.
197:818 (1993)); contacting cells with such substances while generating transient pores in the membrane with Streptolysin O (Spiller & Tidd,
Antisense Res.
&
Dev.
5:13 (1995)) or by scraping adherent cells from a surface (Partridge et al.,
Antisense Nuc. Acid Drug Dev.
6:169 (1996)), or by treating cells with an amphiphilic peptide under acidic conditions (Pichon et al.,
Antisense
&
Nuc. Acid Drug Dev.
7:335 (1997)); and, covalently attaching a special amphiphilic transport peptide or protein (Pooga et al.,
Nature BioTech.
16:857 (1998)) to the substance to be delivered.
In contrast to the foregoing direct-entry approaches, only a few delivery methods have been reported which exploit the natural endocytotic route into cells as the first step toward cytosolic delivery. One such indirect method was devised by Summerton and Weller (
Nucleosides
&
Nucleotides
16:1785 (1997)), and entails covalent linkage of a molecular transport engine to the substance to be delivered, said engine being powered by the pH differential between the late endosome and the cytosol.
Another indirect method, which bears directly on the instant invention, is the three-solution osmotic delivery method devised by Okada & Rechsteiner (
Cell
29:33 (1982)), which entails: 1) loading endosomes with an aqueous hypertonic solution of sucrose and poly(ethylene glycol)-1000 and the substance to be delivered; followed by, 2) exposure of the cells to a hypotonic solution to effect release of the endosomal contents into the cytosol due to osmotic pressure generated within the endosome; and, 3) replacing the hypotonic solution with isotonic solution to return the cells to their normal metabolic state. This osmotic delivery method is potentially very useful because it can routinely be used for delivering a broad range of substances into both adherent and non-adherent cells.
While this osmotic delivery method does achieve delivery of substances into the cytosol of cells, nonetheless, the method generally used heretofore (Okada & Rechsteiner,
Cell
29:33 (1982); McElligott & Dice,
Bioscience Reports
4:451 (1984); Rechsteiner,
Methods in Enzymology
149:42 (1987); Lee et al.,
Cytometry
14:265 (1993);
BioProbes,
28:13 (1998)) is complicated to use because of multiple steps with stringent time constraints. This presents difficulties when one wishes to deliver substances into multiple samples of adherent cells, or into even a single sample of non-adherent cells. Further, delivery efficiencies are less than optimal and vary significantly between cell samples. Finally, contacting the cells with the hypotonic solution often causes losses in cell viability, with such viability losses varying due to minor differences in experimental method and minor differences in time of exposure to the hypotonic solution.
A related two-solution osmotic delivery method was reported by Chakrabarti et al. (
The Journal of Biological Chemistry
264:8214 (1989)). This prior-art two-solution osmotic delivery method suffers from various limitations, including: it affords limited delivery because of a sub-optimal poly(ethylene glycol) component, a sub-optimal treatment time, and use of a less effective and cumbersome volume of solution in the second step. Further, it is of little commercial utility because the excessive volume of solution used in the second step effectively precludes its use with adherent cells and renders it inefficient and cumbersome for extracorporal therapy.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to provide a novel osmotic delivery composition which affords delivery of greater amounts of desired substances into the cytosol of living eucaryotic cells then prior-art osmotic delivery compositions.
Another object of the invention is to provide an osmotic delivery method utilizing the osmotic delivery composition which affords more reproducible delivery of desired substances into the cytosol of living eucaryotic cells than prior-art osmotic delivery methods.
A further object of the invention is to provide an osmotic delivery method which is simpler and easier to use with multiple samples of adherent cells and with non-adherent cells than osmotic delivery methods previously used for this purpose. This object relates to improving the efficiency of research with cultured cells and to reducing the cost and complexity of extracorporal therapies.
The invention includes an osmotic delivery composition comprising a poly(ethylene glycol) component having a designated molecular weight in the range of about 300 to about 900 daltons, and a water-soluble membrane-impermeable carbohydrate component.
In a preferred embodiment, the poly(ethylene glycol) component has a designated molecular weight of 600 and the carbohydrate component is sorbitol.
The osmotic delivery composition may further be dissolved in an aqueous medium.
The aqueous solution of the osmotic delivery composition may further include one or more substances to be delivered into the cytosol of the cells.
Also included in the invention is a method for osmotic delivery of substances into the cytosol of living eucaryotic cells, said method comprising the steps of:
a) contacting the cells with an aqueous solution of the osmotic delivery composition of the invention, which further contains the one or more substances to be delivered into the cytosol of the cells; followed by,
b) adding cell culture medium.
These and other objects and features of the invention will be more fully apparent from the following detailed description of the invention and accompanying figures.
REFERENCES:
patent: 5707648 (1998-01-01), Yiu
patent: 5792471 (1998-08-01), Curatalo
BioProbes 28, p. 13. May 1998, Published by Molecular Probes, Inc., Eugene, Oregon, USA.
Chakrabarti et al., Journal of Biol. Chem. 264:8214 (1989).
Lee et al., Cytometry 14:265 (1993).
McElligott & Dice, Bioscience Reports 4:451 (1984).
Okada & Rechsteiner, Cell 29:33 (1982).
Rechsteiner, Methods in Enzymology 149:42 (1987).
Morcos Paul Anton
Summerton James Edward
Summerton James Patrick
Friedman Lori M.
Gene Tools, LLC
Kishore Gollamudi S.
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