Peptide-lipid conjugates, liposomes and lipsomal drug delivery

Details Peptide-lipid conjugates, liposomes and lipsomal drug delivery Peptide-lipid conjugates, liposomes and lipsomal drug delivery

1999-06-29

2002-01-15

Nguyen, Dave T. (Department: 1633)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C424S450000, C435S320100, C435S455000, C435S458000

Reexamination Certificate

active

06339069

ABSTRACT:

FIELD OF THE INVENTION
Peptide-lipid conjugates are incorporated into liposomes so as to localize delivery of the liposomes' contents to the vicinity of target cells.
BACKGROUND OF THE INVENTION
Liposomes have been widely used as carriers to deliver a variety of therapeutic and diagnostic agents into cells. Encapsulation of active agents in liposomes protects the agents from premature degradation, and ameliorates side effects resulting from administration of the agents to animals (for a review, see, e.g., A. Bangham, 1992; M. Ostro, 1987; and, M. Ostro and P. Cullis, 1989). However, the efficiency of liposomal drug delivery has heretofore been constrained by the lack of a means of inducing liposomes to preferentially release their contents in the vicinity of, or into, target cells. This invention provides such a means, by incorporating peptide-lipid conjugates into liposomes and then contacting cells with these liposomes.
The lipid portion of the peptide-lipid conjugate is a phosphatidylethanolamine (“PE”). Many of these lipids ordinarily do not organize into bilayers at neutral pH, instead forming hexagonal (H
II
)-phase structures in aqueous environments which tend to destabilize the bilayers of liposomes into which the lipids have been incorporated. These same structures can also enhance the liposomes' fusogenicity (Verkleij, 1984; Cullis & de Kruijff, 1979; Ellens et al., 1989). Conjugation of a peptide to the PE stabilizes the PE in a bilayer conformation and hence, allows the conjugated lipid to be stably incorporated into liposome bilayers. However, once the peptide is cleaved, e.g., in the vicinity of peptidase-secreting cells, the lipid then resumes its nonbilayer-preferring conformation, in which it tends to destabilize the same liposome bilayers. In addition, the peptide linker could bind a blocking group, such as a polyethyleneglycol (PEG), such that it may act to sterically hinder fusion
The peptide portion of the peptide-lipid conjugate is any of those peptides having amino acid sequences that are recognized and cleaved by any of the various peptidases secreted by mammalian cells, e.g., at sites of inflammation and tumor metastases (see, e.g.: Aimes and Quigley, 1995; Fosang et al., 1994; Froelich et al., 1993; Knauper et al., 1996; Liotta et al., 1991; Moehrle et al., 1995; Nagase et al., 1994; Nakajima et al., 1979; Odake et al., 1991; Palmieri et al., 1989; Pei et al., 1994; Prechel et al., 1995; Yamashita et al., 1994). Neither linkage of peptidase-cleavabe peptides nor the incorporation of such peptides into liposomes, let alone for the purpose of promoting controlled liposome destabilization, has previously been described.
Vogel et al. (1993) and Subbaro et al. (1967) both covalently linked peptides to PEs; however, these peptide-lipids are not described therein as being cleavable by cell-secreted peptidases. Rather, the peptide-modified lipids of these documents are pH sensitive, adopting an alpha-helical conformation in low pH endosomal environments. Kirpotin et al. modified distearoyl phosphatidylcholine (“DSPE”) by the attachment thereto of methoxypoly(ethylene glycol) (“mPEG”) to DSPE on the amino group; liposomes containing mPEG-modified DSPE were stable in solution until thiolytic cleavage and removal of the mPEG moiety. Kirpotin does not describe the peptide-based modification of PEs, let alone with peptidase-cleavable peptides.
SUMMARY OF THE INVENTION
This invention provides a means of delivering and localizing the contents of liposomes to the vicinity of cells in a controlled manner, by conjugating certain peptides to phosphatidylethanolamines, and then incorporating these conjugated lipids into liposomes. The resulting liposomes are stable so long as the peptide remains conjugated to the lipid. However, once the peptide portion of the conjugate is cleaved from the lipid, by the action of cell-secreted or associated peptidases, the liposomes tend to destabilize, so as to release their contents in the vicinity of, or into, the secreting cells. Delivery of the liposomes' contents is thus targeted to the peptidase-secreting cells.
Peptide-liquid conjugates of this invention have the formula:
wherein: each of R
1
and R
2
is an acyl chain, X is a single bond or an acyl chain, and Y is a peptidase-cleavable peptide. The acyl chains are preferably oleic acid chains, X is preferably a single bond, and the peptide preferably contains the amino acid sequence Ala-Ala-; more preferably N-Acetyl-Ala-Ala-, or Ala-Ala-Pro-Val- (SEQ ID NO:1); and most preferably, N-methoxysuccinyl-Ala-Ala-Pro-Val (SEQ ID NO:2). Accordingly, the peptide-lipid conjugate preferably has the formula:
Most preferably, the peptide-lipid conjugate has the formula:
The liposomes' lipid component can be entirely composed of the peptide-lipid conjugate, or can comprise one or more additional lipids. 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. Most preferably, the liposome of this invention comprises a peptide-lipid conjugate and the positively charged synthetic lipid 1-N,N-dimethylamino dioleoyl propane (DODAP).
Controlled delivery with the liposomes of this invention can be used to deliver the liposomal drugs and/or bioactive agents in vitro or in vivo, for example, in the treatment of mammals afflicted with various diseases, disorders or conditions, e.g., cancers, amenable to treatment with the bioactive agent associated with the liposome.


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