Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1998-12-10
2001-05-01
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C554S035000, C554S079000, C554S085000, C554S101000
Reexamination Certificate
active
06224903
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a polymer-lipid conjugate for promoting fusion between two target membranes. In one embodiment, the conjugate is incorporated into a liposome composition to promote fusion between the liposome and a target membrane for delivery of an agent carried in the liposomes to the cytoplasmic compartment of a cell.
References
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BACKGROUND OF THE INVENTION
The therapeutic benefit of many compounds is limited by low uptake of the compound by the target cells or by intracellular breakdown of the compound after uptake. Generally, for maximum therapeutic benefit, delivery of the compound to the cytoplasmic compartment of the cell, where translation of mRNA and protein synthesis take place and where there is a direct link to the nucleus, is desired. For many small, uncharged compounds, permeation across the cell membrane may allow relatively efficient uptake by the cell. However, for a variety of larger and/or charged compounds, such as proteins, nucleic acids, and highly water soluble charged organic compounds, passive uptake by permeation across the cell membrane is more limited.
Several methods for improving uptake of such compounds into cells have been proposed. For example, a drug can be administered in modified or prodrug form for transport into cells and then undergo enzymatic conversion to an active form within the cells.
Alternatively, the cellular processes of phagocytosis or endocytosis may be used, where drug-containing particles are engulfed by the cells. However, this approach is limited to certain cell types, for example, phagocytosis is limited to cells of monocyte lineage and to certain other myeloid cells, such as neutrophils, and endocytosis is limited to mesenchymal cells, such as vascular endothelial cells and fibroblasts. Another limitation of this approach is that in the normal course of intracellular processing, particles are exposed to the acidic endosome/lysosome compartments and a host of degradative enzymes, including proteases, lipases and nucleases, resulting in degradation of the therapeutic compound, unless an escape from such processing is engineered into the system.
Still another approach to enhancing drug uptake by cells involves the use of fusogenic particles designed to fuse with the surface membrane of a target cell, releasing the particle contents into the cytoplasmic compartment of the cell. Inactivated and reconstituted virus particles have been proposed for this purpose, particularly in gene therapy where large nucleic acid strands are introduced into cells. Virus-like particles composed of fusion-promoting viral proteins embedded in artificial lipid bilayer membranes are another example. However, safety concerns and the expense associated with growing, isolating, and deactivating viral components limit these approaches.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a polymer-lipid conjugate for use in promoting fusion between target membranes, comprising a first segment composed of a hydrophilic polymer and a second segment composed of a hydrophobic polymer. The second segment is joined to the first segment by a bond effective to release the first segment in response to an existing or an induced physiologic condition. Attached to the second segment is a vesicle-forming lipid member.
In one embodiment, the second segment is a hydrophobic homopolymer. For example, the homopolymer is selected from the group consisting of polypropylene oxide, polyethylene, polypropylene, polycarbonate, polystyrene, polysulfone, polyphenylene oxide and polytetramethylene ether. In a preferred embodiment, the hydrophobic polymer is polypropylene oxide having a molecular weight of between 500-3,000 daltons.
In another embodiment, the hydrophobic polymer is a heteropolymer composed of first and second polymer units. For example, in one embodiment, at least one of thee first and second polymer units is selected from the group consisting of polypropylene oxide, polyethylene, polypropylene, polycarbonate, polystyrene, polysulfone, polyphenylene oxide and polytetramethylene ether.
In yet another embodiment, the hydrophobic polymer is a hydrophobic polypeptide, such as a viral fusion polypeptide. Exemplary fusion peptides include SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11.
In another embodiment the hydrophobic polymer is a lytic peptide consisting of a positively charged domain of amino acids and a hydrophobic domain of amino acids. Alternatively, the hydrophobic polymer is a lytic peptide wherein the domain of positively charges amino acid is removed.
In still another embodiment, one of the target membranes is a liposome lipid bilayer membrane. In this embodiment, the conjugate is incorporated into the liposome lipid membrane with the first and second segments oriented to extend from the surface of the liposome.
In one embodiment, the liposome lipid membrane further includes a targeting ligand which extends from the liposome surface. The targeting ligand also extends from the surface of the other target membrane, and a bifunctional or multifunctional binding moiety binds with the targeting ligand extending from the liposome surface and the targeting ligand extending from the other target membrane to bring the liposome and the target membrane into contact for fusion.
In the conjugate of the invention, the releasable bond, in one embodiment, contains a peptide signal sequence that is sensitive to cleavage by an enzyme. For example, the signal sequence is one sensitive to cleavage by enzymes present in the extracellular fluid of a tumor, and examples include matrix metalloproteinase, PLGLWA (SEQ ID NO. 12), enzymes present in the extracellular fluid present in at a site of inflammation or infection, cathepsins, elastases, collagenases, heparinases and gelatinases. The bond can also be one reversible by changes in oxidation/reduction conditions at a region where the target membranes come into contact, such as a disulfide linkage. The bond can also be susceptible to hydrolysis or can be an ester bond, preferably one that is susceptible to non-enzymatic hydrolysis at a rate slower than that required for the target membranes to come into contact.
In anoth
Martin Francis J.
Zalipsky Samuel
Iota Pi Law Group
Kishore Gollamudi S.
Mohr Judy M.
Sequus Pharmaceuticals, Inc.
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