Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
2000-01-19
2001-09-25
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S812000, C436S829000
Reexamination Certificate
active
06294191
ABSTRACT:
FIELD OF THE INVENTION
This invention is directed to methods of delivering drugs to cells, by incorporating the drugs into, or associating the drugs with, liposomes containing an N-acyl phosphatidylethanolamine (“NAPE”), and then contacting the cells with the liposomes.
BACKGROUND OF THE INVENTION
Phosphatidylethanolamines (“PEs”) are naturally occurring phospholipids typically having two acyl chains, as well as a phosphorylethanolamine group, attached to the lipid's glycerol backbone. N-acylated phosphorylethanolamines (“NAPEs”) are PEs to which an additional, third acyl chain has been attached, by way of the amino moiety of the lipids' phosphorylethanolamine group. Some NAPEs are also found in biological membranes, in small amounts.
Most PEs ordinarily do not organize into bilayers at neutral pH, instead forming hexagonal (H
II
)-phase structures in aqueous environments. Hexagonal-phase formation is a property that may be associated with enhanced liposome fusogenicity when these lipids are incorporated into liposomal bilayers under appropriate conditions (Verkleij, 1984; Cullis & de Kruijff, 1979; Ellens et al., 1989). NAPEs, by contrast, spontaneously form bilayers in aqueous dispersions, in the absence of added divalent cations (Newman et al., 1986; Akoka et al., 1988; Lafrance et al., 1990; Domingo et al., 1994).
None of the above documents describes a study of NAPEs with regard to their ability to be either fusogenic or bilayer-destabilizing at the desired delivery site, yet still be able to form liposomes that can stably encapsulate material. None of the previous studies describe the use to which NAPEs can be put to for the controlled delivery of liposomal drugs, and none describe tailoring NAPEs so as to optimize such delivery, especially in vivo. Moreover, none have either synthesized or studied the NAPE N-dodecanoyl dioleoyl phosphatidylethanolamine.
SUMMARY OF THE INVENTION
This invention provides liposomes containing a destabilization effective amount of an N-acyl phosphatidylethanolamine (“NAPE”), the liposomes being useful for fusion to cell membranes in the presence of suitable concentrations of cations and when placed adjacent to the cells. NAPEs are glycerol-based phospholipids having 14-24 carbon-long, saturated or unsaturated acyl chains attached at the first and second positions of the glycerol backbone. The third position is occupied by a phosphorylethanolamine, its amino group having a third acyl chain attached to it which is 4-24 carbon atoms long and saturated or unsaturated. Presently, the preferred NAPE is N-C12 DOPE (N-dodecanoyl dioleoyl phosphatidylethanolamine).
The destabilization effective amount of the NAPE contained in the liposome's lipid component is typically from about 10 mole % of the lipid component to about 90 mole %. The liposome into which the NAPE is incorporated can be a unilamellar, oligolamellar or multilamellar liposome, but is preferably unilamellar. The liposome's lipid component comprises, in addition to the NAPE, at least one other lipid. Such additional lipids include, without limitation, any of the types of lipids, e.g., phospholipids, glycolipids and sterols, which may be used in the preparation of liposomes.
Preferably, the additional lipid comprises one or more phospholipids. Most preferably, these include a phosphatidylcholine (“PC”), such as dioleoyl phosphatidylcholine (“DOPC”) or a phosphatidylethanolamine (“PE”), e.g.: a PE selected from the group consisting of transesterified phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, palmitoyl oleoyl phosphatidylethanolamine and dioleoyl phosphatidylethanolamine; or a PE conjugated to a moiety selected from the group consisting of dicarboxylic acids, polyethylene glycols, polyalkyl ethers and gangliosides.
The liposome can have a targeting moiety attached, and can contain, in either a bilayer or an aqueous compartment, one or more bioactive agents. Attachment of targeting moieties to the liposomes is preferred herein, so as to place the liposomes adjacent to the cells being targeted for delivery of the liposomes' contents, where the liposomes, in the presence of suitable concentrations of cations, can become locally destabilized or fuse to the cells.
This invention's liposomes can thus be used to deliver bioactive agents to cells, by contacting the cells with the liposomes under conditions in which the NAPE destabilizes the liposomes' bilayers, so as to induce local release of the liposomes' bioactive agent content and/or fusion of the liposome with the cells. Such delivery can be in vitro or in the body of a mammal, and can be used, e.g., for ex vivo stem cell transfection or in vivo delivery of anticancer therapeutic agents.
REFERENCES:
Akoka, et al., “a phosphorus magnetic resonance spectroscopy and a differentially scanning calorimetry study of the physical properties of N-acylphosphatidylethanolamines in aqueous dispersions,” (1988) Chem. Phys. Lipids 46, 43-50.
Bangham, et al., “Diffusion of Univalent Ions across the Lamellae of Swollen Phospholipids,” J. Mol. Bio. (1965), 13, 238-252.
Batzi, et al., “Single bilayer liposomes prepared without sonication”, BBA, 298 (1973), 1015-1019.
Cullis, P.R., et al., “Lipid Polymorphism and the Functional Roles of Lipids in Biological Membranes”, (1979) Biochim. Biophys Acta 559, 399-420.
Deamer, et al.,Liposomes, “Liposome Preparation: Methods and Mechanism”, M. Ostro, ed., Marcel Dekker, Inc., New York, (1983), Chapter 1, p. 27-51.
Domingo, et al., “The influence of N-acyl chain length of the phase behavior of natural and synthetic N-acylethanolamine phospholipids”, Chemistry and Physics of Lipids, 75 (1995) 15-25.
Domingo, et al., “Role of headgroup structure in the phase behavior of N-acylethanolamine phospholipids:hydrogen-bonding ability and headgroup size,” 1994, Chemistry and Physics of Lipids, 69, 229-240.
Domingo, et a., Incorporation of N-acylethanolamine phospholipids into egg phosphatidylcholine vesicles: characterization and permeability properties of the binary systems, (1993), Biochim. Biophys. Acta 1148, 308-316.
Ellens, et al., “Membrane Fusion and Inverted Phases,” (1989) Biochemistry 28, 3692-3703.
Fahey, “Surface Properties of 1,2-Dipalmitoyl-3-acyl-sn-glycerols,” (1986) Biochemistry, 25, 4468-4472.
Gruner, S.M. (1985) Proc. Natl. Acad. Sci, USA 82, 3665-3669.
Lafrance, et al., “Study of the structure of N-Acyldipalmitoylphosphatidylethanolamines in Aqueous Dispersion by Infrared and Raman spectroscopies,” (1990) Biochemistry 29, 4592-4599.
Lee, et al., “Hydrophobic Alkyl Headgroups Stongly Promote Membrane Curvature and Violate the Headgroup Volume Correlation Due to “Headgroup” Insertion”, (1996) Biochemistry 35, 3677-3684.
Leventis, et al., “pH-Dependent Stability and Fusion of Liposomes Combining Protonatable Double-Chain Amphiphiles with Phosphatidylethanolamine,” Biochem 1967, 26, 3267-3276.
Litzinger, et al., “Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic application,” (1992) Biochim. Biophys. Acta 1113, 201-227.
Mercadal, et al., “N-Palmitoylphosphatidylethanolamine stabilizers liposomes in the presence of human serum: effect of lipidic composition and system characterization,” (1995) Biochim. Biophys. Acta 1235, 281-288.
Newman, et al., “Phase Behavior of Synthetic N-Acylethanolamine Phospholipids,” (1986) Chem. Phys. Lipids 42, 249-260.
Ortiz, et al., “Cation-induced aggregation and fusion of N-acyl-N-methyl-phosphatidylethanolamine vesicles,” Chem. Phys Lipids, 61(1992), 185-191.
Portis, et al., “Studies on the Mechanism of Membrane Fusion: Evidence for an Intramembrane Ca2+-Phospholipid Complex, Synergism with Mg2+, and Inhibition by Spectrin,” (1979( Biochemistry 18, 780-790.
Verkleij, A. J. “Lipidic intramembranous particles,” (1984), Biochim. Biophys. Acta 799, 43-63.
Ali Shaukat
Janoff Andrew
Meers Paul R.
Pak Charles
Shangguan Tong
Goodman Rosanne
Kishore Gollamudi S.
The Liposome Company Inc.
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