CZ237699A3 - Conjugates of peptide with lipid, liposomes, preparations and method of their addressing - Google Patents

Abstract

Lipid-conjugated peptide conjugates are incorporated into the liposomes so that the lipid conjugates are incorporated into the liposomes selectively destabilize liposomes near target cells secreting peptidases, and also deliver liposomes nearby target cells, or directly into cells. Liposomes may be used to treat mammals in case of illness, disorders or symptoms, tzn. tumor growth, microbial infections and inflammations, characterized by the presence of secreting peptidases

Description

PEPTIDE CONJUGATES WITH LIPIDE, LIPOSOMES, PREPARATIONS AND METHODS OF THEIR

DIRECTION

TECHNICAL FIELD

Lipid-peptide conjugates are incorporated into liposomes to allow targeting of the liposomes and delivery of their contents to the proximity of the target cell.

BACKGROUND OF THE INVENTION

Liposomes are commonly used as carriers to deliver a variety of agents for therapeutic or diagnostic purposes to target cells. The encapsulation of these active substances in liposomes protects them from premature degradation while preventing unwanted side effects of these substances on the organism to which they have been administered (eg A. Bangham, 1992, M. Ostro, 1987 and M.Ostro and P. Culis, 1989) . Unfortunately, the efficacy of liposome delivery has hitherto been greatly limited by the lack of ways to force liposomes to release their contents near or directly within the target cells. The present invention describes methods by which, by incorporating lipid-peptide conjugates into liposomes, these liposomes can be contacted with target cells.

The lipidic portion of the lipid-peptide conjugate is phosphatidylethanolamine ("PE"). This lipid normally does not form a lipid bilayer at neutral pH and instead forms hexagonal (Hn) -phase structures in an aqueous environment that tend to destabilize the bilayer liposomes into which the lipids have been incorporated. However, the same structures increase the ability of liposomes to fuse (Verkleij, 1984, Cullis & de Kruijff, 1979, Ellens et al, 1989). Conjugation of peptides to PE stabilizes PE in the membrane structure and, moreover, allows the conjugate to be stably incorporated into liposomes. However, once the peptide is removed, for example, enzymatically cleaved near the peptidase secreting cells, the lipid in the membrane begins to behave unstable, forming hexagonal conformations, resulting in destabilization of the membrane of the whole liposome.

The peptide portion of the conjugate may be any peptide whose amino acid sequence is recognized and cleaved by any of a number of peptidases secreted by mammalian cells, e.g., at sites of inflammation or tumor metastasis (see 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 the attachment of peptides cleavable by peptidases nor the incorporation of these peptides into liposomes to allow their controllable destabilization has been previously described.

Voegel et al, and Subbaro et al, coupled peptides to PE, but these peptides were never described as peptides cleavable by peptidase secreted cells. The lipid-modified peptides described herein were more sensitive to pH changes as they formed α helical structures at low pH within the endosomes. Kirpotin et al, modified distearoyl phosphatidilcholine ("DSPE") by attaching a methoxypoly (ethylene glycol) group ("mPEG") to the amino group of DSPE. Liposomes containing mPEG-modified DSPE were stable in solution until the mPEG moiety was cleaved thiolytically. Kirpotin has never described modifications of PE by peptides cleavable by peptidases.

The present invention describes a method of delivering liposome contents to a particular site, near the target cells, in a controllable manner by conjugating the peptides to phosphatidylethanolamine and incorporating such conjugates into the liposomes. The resulting liposomes are then stable as long as the peptide is attached to the lipid. Thus, once the peptide portion of the conjugate is cleaved from the lipid by the cell secreted peptidases, the liposomes become unstable and their contents are released either in the vicinity or directly inside the cells secreting the appropriate enzyme. Thus, the targeting of such liposomes is directed against cells secreting the appropriate enzyme. The peptide-lipid conjugate of the present invention should have the following general formula:

H2C-R ¹

^HC-R2

H ₂ C-OP (O) ₂ - (CH ₂ ) 2 -NHX-Y wherein each R ¹ and R ² is an alkyl chain, X is a single bond, or an acyl chain and Y is a peptidase cleavable peptide. The acyl chain is mostly an oleic acid chain, X is mostly a single bond, and the peptide usually contains the amino acid sequence Ala-Ala-Pro-Val, preferably N-methoxysuccinyl-Ala-Ala-Pro-Val. Then a preferred variant is a peptide-lipid conjugate having the following general formula:

H2C-OC (O) _(CH2) 7CH = CH _(CH2) 7 CH ₃

HC-OC (O) (CH ₂ ) 7 CH = CH (CH ₂ ) 7 CH ₃

H2C-OP (O) ₂ - _(CH2) 2-NH-Val-Pro-Ala.

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The lipidic portion of the liposomes may either be composed of only the conjugate or may contain one or more additional lipids. These may be any of the following types of lipids, e.g., phospholipids, glycolipids and sterols, which may be used to make liposomes. It is preferred that the liposomes of the present invention comprise a lipid-peptide conjugate and a positively charged synthetic DODAP lipid.

The controlled liposome targeting described by the present invention can be used to deliver substances contained in liposomes in vitro and in vivo, for example in the treatment of mammals afflicted with various diseases, disorders or symptoms such as cancer, which can be treatable with bioactive substances that can be incorporated into liposomes.

SUMMARY OF THE INVENTION

The present invention provides peptide-lipid conjugates according to the following general formula:

HjC-R ¹

^HC-R2

H ₂ C-OP (O) 2- (CH ₂ ) 2 -NHX-Y wherein each R ¹ and R ² is independently a group of the formula OC (O) (CH ₂ ) _n 1 (Target = CH) _n 2 (CI 12) ) _n3 (CH = CH) 4 "(CH2)" 5 (CH = CH) _{'6 (CH2) n7} (CH = CH) _n8 (CH2) n9CH3, and X is a linking group having a single bond and an acyl chain of the formula 00 (0) (01-12) 111 (013 = 011) ,, 2 (0142) 3 ,, (01-1 = 011) 4 ,, ((3112), 15 (CH = CH) _{n6 (CH2) n7} (CH = CH) _{n8 (CH2) n9.}

N1 is zero or fits 1 to 22, n3 is zero or fits 1 to 19, n5 is zero or fits 1 to 16, n7 is zero or fits 0 to 13, n9 is zero or fits in the range of 1-10, and each value of n2, n4, n6 and n8 is independently zero or 1. For R ¹ and R ² is the sum of nl + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 is in the range from 10 to 22.

X is mostly a single bond, if X is anything other than a single bond then the sum of n1 + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 for X corresponds to the interval from 1 to 22. X is mostly saturated, more preferably -C (O) (CH ₂ ) n -.

Suitably, at least one R or R group contains at least one double bond and the lipid-peptide conjugate is therefore partially or fully unsaturated. More preferably, both R ¹ and R ² contain one double bond and the conjugate is then completely unsaturated. Most preferably both R ¹ and R ² have the formula -OC (O) (CH ₂ ) 7 (CH = CH) (CH ₂ ) 7 CH 3, i. That the lipid-peptide conjugate is a dioleoyl phosphatidylethanolamine from ("DOPE") derived conjugate. Thus, each of R ¹ and R ² may also be a saturated or unsaturated acyl chain according to the formulas (not). But exclusively): -OC (O) (CH ₂ ) ₁₄ CH ₃ , -OC (O) (CH ₂ ) i ₆ CH ₃ , -OC (O) (CH ₂ ) i ₈ CH ₃ or

OC (O) (CH ₂ ) ₈ (CH = CH) (CH ₂ ) ₈ CH ₃ .

Y is then an "enzyme-cleavable peptide" wherein the peptide comprises an amino acid sequence recognized by peptidases present on mammalian cells and surrounding tissues, or produced by microorganisms capable of causing disease in a mammal. The enzymatically cleavable peptide may or may not contain one or more amino acids in addition to the amino acid sequence recognized by the enzyme. These amino acids can then be attached to the amino terminus, carboxy terminus and both ends of the peptide sequence recognized by the cleavage enzyme. Methods of adding amino acids to an amino acid sequence recognized by an enzyme, for example, by an automated peptide synthesizer, as well as a method for detecting peptide cleavage by a peptidase, eg, by chromatographic analysis of the cleavage products are well known to any person skilled in the art.

Enzymatically cleavable peptides, typically 2 to 20 amino acids in length, are of sufficient length to penetrate the surface of the lipid-based carrier into which they have been incorporated. These peptides are well known to one of ordinary skill in the art, including but not limited to these sequences: Ala-Ala-, Ala-Ala-Pro-Val-, Ala-Ala-Met-, Ala-Ala-Pro-Phe, Ala-Ala-Pro-Met-, Ala-Ala-Arg-, Ser -Ala-Ala-Arg-, Ser-Ser-Ala-Ala-Arg-, Ser-S-Carboxy SugarAla-Ala-Arg-, Ala-Ala-Asp-, Ser-Ala-Ala-Asp-, Ser-Ser -Ala-Ala-Asp-, Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Ser-Ser-Arg-Pro-Lys-Pro- Leu-Ala-Nva, _{ProCHO-Gly-Nva-His-Ala-Dpa-NH2, Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2,} ProCHO-Gly-Nva, ProLeu- Gly-Leu-, Gly-Pro-Arg-, Leu-Pro-Arg-, Glu-Gly-Arg-, Pro-Leu-Gly-Leu- and Gly-Pro-Gln-Gly-Ile. Currently, peptides containing the AlaAla- amino acid sequence, more preferably N-methoxysuccinyl-Ala-Ala-Pro-Val- are preferred.

Accordingly, the most preferred peptide-lipid conjugate of the present invention will have the general formula:

H ₂ C-OC (O) (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ CH ₃

HC-OC (O) (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ CH ₃

H ₂ C-P (O) ₂ - _(CH2) 2-NH-Y wherein the peptide comprises the amino acid sequence N-methoxysukcinyl-Ala-Ala-Pro-Val-.

The enzymatically cleavable protein may be modified at its amino terminus, for example, to increase its hydrophilicity. The increased hydrophilicity improves the interaction of the peptide on the surface of the lipid carrier into which the parent peptide-lipid conjugate has been incorporated. Polar groups suitable for • Fe Fe * ·· Fefe Fefe Fefe fefefefe fefefefe • • Fefe fefefefe fefefefe fefefe fefefe · Fe fefefe fefefe * fe · · · · • Fefe Fefe Fefe Fefe Fefe linkage of peptide so as to increase their hydrophilicity are well known and include, but are not limited to: acetyl (Ac), 3-cyclohexylalanine (Cha), acetylserine (Ac-Ser), acetylseryl-serine (Ac-Ser-Ser), succinyl (Suc), succinylserine (Suc-Ser), succinylseryl-serine (Suc-Ser-Ser), methoxysuccinyl-Meine (MeO-Suc), methoxysuccinyl-serine (MeO-Suc-Ser), methoxysuccinyl-serylserine (MeO-Suc-Ser-Ser), serylserine (Ser-Ser), polyethylene glycol (PEG), polyacrylamide, polyacrylomorpholine, polyvinylpirrolidine, polyhydroxyl groups, and carboxy sugars such as lactobiose, N-acetyl neuraminic and sialic acid. The carboxy groups of these sugars are attached to the N-terminus of the peptide via an amide bond. Currently, the preferred terminal modification is methoxysuccinyl attachment.

Cells secreting peptidases that recognize the appropriate amino acid sequences are also well known to those skilled in the art. These include, but are not limited to, the following peptidases: intercellular metalloproteases, serine proteases, cysteine proteases, elastase, plasmin, plasminogen activator, stromelysin, human collagenases, cathepsin, lysozyme, granzymes, dipeptidyl peptidases, peptide hormone inactivation enzymes, kinases, bacterial peptidases and viral proteases. Elastase, for example, is involved in the reshaping of tissues in case of malignancy. The breast cancer-derived MCF-7 tissue line produces elastase, with secretion rates inversely proportional to the overall survival of breast cancer patients (Yamashita et al.). In addition, stromelysin-3 (ST3) intercellular mass metalloprotease, has been found in the stromal region of tumor cells (Pei et al.), Specifically cleaves the α 1 proteinase inhibitor between amino acids 350 and 351 (Ala-Met). Stromelysin-1 (MMP-3) is also found in tissue reshaping areas, including inflammatory and stromal sites of tumors (Nagase et al.).

Coding cDNAs for human collagenase-3 or another metalloprotease MMP-13 were isolated from a breast tumor library (Knauper et al.). These enzymes cleave the amino acid sequence Pro-Cha-GlyNva-His and Pro-Leu-Gly-Leu. In addition, 72 kDa gelatinase (MMP-2) affects tumor invasiveness and cleaves the Gly-Pro-Gln amino acid sequence of Gly-Ile- between Gly and Ile amino acids (Aimes and Quigley, Liotta et al.). Human neutrophils also secrete collagenase at the site of inflammation, especially MMP-8 (neutrophil collagenase and MMP-9 (type IV collagenase, 92 kDa gelatinase) (Fosang et al.). Kathepsin G is also secreted by human neutrophils at the site of inflammation. specific for peptides containing the amino acid sequence Suc-Ala-Ala-Pro-Phe or MeOSuc-Ala-Ala-Pro-Met (Nakajima et al.) Other enzymes secreted by the neutrophils at the inflammatory site are cathepsins B and D as well as lysozyme. Granzymes A and B are secreted by cytotoxic lymphocytes in the synovial fluid of patients suffering from rheumatoid arthritis (Froehlich et al.) Granzyme A most effectively cleaves peptides containing the Gly-Arg sequence, or »Α ΦΦ

Φ · Φ Φ

Φ Φ Φ Φ

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Ala-Ala-Arg, while granzyme Β cleaves peptides containing the amino acid sequence Ala-AlaAsp (Odake et al.)

Peptidases hydrolyzing enzymatically cleavable peptides also include a group of peptides hormone inactivating enzymes, e.g., aminopeptidase P and angiotensin converting enzyme, located on the surface of epithelial cells. Aminopeptidase P cleaves the bond between Agr-Pro in bradykinin and is located on the surface of epithelial cells in the lung (Prechel et al).

Lipid-peptide conjugates can be prepared by a variety of methods designed to form an amide bond between the amino group of the phosphatidylethanolamine and the carboxy terminus of the amino acid sequence. These methods include, but are not limited to those set forth in Example 1. Generally, an enzymatically cleavable peptide containing an N-terminal blocking group is prepared as an anhydrite, a phosphatidylethanolamine such as DOPE is then allowed to react with the anhydrite in the presence of appropriate reagents such as is triethylamine.

The present invention also provides liposomes composed of lipid components including lipid-peptide conjugates of the present invention. "Liposomes" are spontaneously forming structures composed of one or more lipid bilayers, each surrounded by an aqueous compartment and composed of two opposing layers of amphipathic lipid molecules. Amphipathic lipids comprise a polar (hydrophilic) region covalently linked to one or two non-polar (hydrophobic) acyl chains. The energy-disadvantageous contact between the hydrophobic acyl chains and the aqueous medium is probably the reason that the lipid molecules themselves position such that the polar regions are oriented towards the aqueous medium, while the acyl chains conceal within the resulting structure. This produces an energy-stable bilayer in which the acyl chains are effectively protected by the polar regions from contact with the aqueous environment.

The liposomes of the present invention may be composed of either a single lipid bilayer (unilamellar liposomes, LIL V) or multiple bilayers (multilamellar liposomes, ML V) and may be prepared by a variety of methods well known in the art. Examples are the following methods. Bangham's method of preparing multilamellar liposomes (MLV), the method of Lenka, Fountain and Cullis for preparing MLVs with uniform interlamellar content distribution (see US Patent Nos. 4,522,803, 4,588,578, 5,030,453, 5,169,637 and 4,975,282) phase (US Patent No. 4,235,871). ULVs can then be prepared from MLV using such a method as sonication or extrusion (US Patent No. 5,008,050, and US Patent No. 5,059,421). The liposomes of the present invention may be prepared by any of the above methods, which are incorporated herein by reference.

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A variety of methods, such as sonication, homogenization, use of French Press and milling, can be used to prepare smaller size liposomes in larger liposomes. Extrusion (see US Patent No. 5,008,050) can be used to reduce the size of liposomes to form liposomes with a desired size such that the liposome mixture is forcefully passed through a filter with a defined and desired aperture size. Tangential flow filtration (see WO89 / 008846) can also be used to prepare a population of liposomes with smaller size differences and a homogeneous defined size distribution of liposomes. The contents of these documents are incorporated herein by reference. A variety of techniques can be used to determine the size of the liposomes, such as quasi-elastic light scattering, or by using a Nicomp® instrument, for example, with which only a trained specialist must work.

The liposomes of the present invention may contain only peptide-lipid conjugates. However, it is preferred that these liposomes contain, in addition to the conjugate, one or more other lipids, including such lipids as phospholipids, glycolipids and sterols, which are commonly used for the preparation of liposomes. Preferably, these lipids are positively charged lipids, even more preferably selected from the group consisting of DOTAP, 1N, N-dimethylaminodioleoyl propane (DODAP), 1-oleoyl-2-hydroxy-3N, N-dimethylaminopropane 1,2-diacyl-3-N, N-dimethylamino-propane and 1,2-di-decanoyl-1 N, N-dimethylamino-propane, DC-Chol, DMRIE and DORI.

Most preferably, the positively charged lipid is DODAP. The positively charged lipids are incorporated into the liposomes, preferably at maximum equimolar concentration relative to the peptide-lipid conjugate, to ensure cross-linking of the carrier. By increasing the positive charge on the lipid-based carrier, the electrostatic interaction between the carrier and the bio membrane is improved and their mutual fusion is facilitated.

These additional lipids may also be certain phospholipids such as phosphatidylcholine (PC), which is generally added to the lipid carrier to increase the stability of the structure, or phosphatidyethanolamine (PE). PE may be one of transesterified phosphatidylethanolamine (tPE), dipalmitoyl phosphatidyl ethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE) and DOPE, and these additional PE may be fuzogenic because their hydrophobic "head" group is relatively dehydrated.

Another possibility is that the PE used is a PE on whose hydrophilic moiety is attached a group of the dicarboxylic acid type, polyethylene glycol, polyalkyl ether and gangliosides. These modified PEs, also known as hydrophobic-modified lipids, can inhibit the binding of serum proteins to a lipid carrier and thereby alter the pharmacokinetic behavior of the carrier in the animal circulatory system (see Blume et al., Gabizon et al., Park et al., Woodle. et al. and Allen et al. are * 99

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999 999 99 · 9 99 99 listed below as references). The amount of lipids with modified hydrophobic groups that are incorporated into the liposome depends on a number of factors well known to one skilled in the art, or determinable without much experimentation. In particular, it is the type of lipid and modification of its hydrophobic group, the type and size of liposomes, and independently of the therapeutic use of the drug. The concentration of lipids with modified hydrophobic groups in the liposome must be sufficient to prolong the half-life of the liposome circulating in the animal, but not too high both did not induce unwanted side effects in animals and typically is at least five mole percent of the lipid present in the liposome. Preferred lipids with altered hydrophobic groups are phosphatidylethanolamine dicarboxylic acids (PEDCA) and PEGylated lipids (see Woodle et al. And Allen et al. For description).

The liposomes of the present invention may also comprise a "targeting structure", i. a moiety that can be attached to a liposome and that will direct the liposome to a specific site in the mammalian body. This routing is based on the recognition of some structure on the cell surface against which the routing is directed by the appropriate routing structure. A typical but non-limiting example of such a structure may be, for example, an antibody, a cell receptor ligand, a lectin, and the like. The targeting structure may be attached to the liposome by any method known in the art, both by covalent and noncovalent binding of this moiety to the liposome.

This may be any of the methods described in the following documents, which are incorporated herein by reference: U.S. Pat. No. 5,399,331 discloses binding of a protein to a liposome via a linker comprising at least one maleimide group and an amine reactive group; Nos. 4,885,172, 5,059,421 and 5,171,578 disclose binding of proteins to a liposome using the streptavidin glycoprotein, Sato and Sunamoto disclose binding of polysaccharides to the surface of a targeted liposome.

The liposomes of the present invention contain one or more "bioactive agents" that contain or represent substances with biological function, including therapeutic or diagnostic activity. The bioactive agent may be, for example, antiviral agents such as acyclovir, zidovudine and interferons, antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines, antifungal agents such as polyene antibiotics, imidazoles and triazoles, antimetabolics such as folate, purine and pyrimine analogs are anthracycline antibiotics and plant alkaloids, sterols such as cholesterol, carbohydrates; sugars and starches, amino acids, peptides, proteins such as cellular receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins, dyes, radiolabeled probes such as radioisotopes and radiolabelled substances, contrast agents, fluorescent probes, bronchodilators, local anesthetics, sequences nucleic acids such as mRNA, cDNA, genomic DNA and plasmids, bioactive lipids such as ether lipids and ceramides, and the like.

A preferred bioactive agent is, for example, a nucleotide sequence, an antimicrohial agent, an anticancer drug, and an anti-inflammatory drug.

Preferably, the liposome consists of lipid components represented by positively charged lipids and a peptide-lipid conjugate according to the general formula:

• flfl *

H ₂ C-OC (O) (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ CH 3

HC-OC (O) (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ CH ₃

H ₂ C-OP (O) ₂ - (CH ₂ ) 2 -NH-Y wherein the peptide will contain the amino acid sequence N-methoxysuccinyl-Ala-Ala-Pro-Val- and the positively charged lipid is DODAP. More preferably, the lipid component is DODAP and the peptide-lipid conjugate is present at a molar ratio of 50:50.

Further disclosed herein is a composition comprising liposomes and a pharmaceutically acceptable carrier, which composition is acceptable for use in combination with administering liposomes to mammals, including humans. The pharmaceutically acceptable carrier is prepared in accordance with a variety of factors entirely dependent upon the judgment of one skilled in the art and will include, for example, the particular liposomal bioactive agent used, its concentration, stability and desired bioavailability, the type of disease, disorder or complication to be treated with the liposomal formulation. , the patient, his age, size and general condition, and the intended route of administration of the medicinal product, eg, nasally, orally, opthalmally, topically, transdermally, vaginally, subcutaneously, intramamma, intraperitoneally, intravenously or intramusculaly (see, eg, Nairn 1985). A typical pharmaceutically acceptable carrier used for parenteral administration of a bioactive agent is, for example, D5W, an aqueous solution containing 5% by weight dextrose and saline. The pharmaceutically acceptable carrier may contain other ingredients, such as stability enhancers of the active ingredients used, for example preservatives and antioxidants.

Further, the present invention describes a method of directing the liposome content to a desired cell, requiring contact of the cell with the liposome of the present invention and the presence of a protease capable of cleaving the lipid-peptide conjugate. The targeting may be used both in vitro, for example, for diagnostic purposes, as well as for ex vivo delivery of a therapeutic agent or nucleic acid to bone marrow cells. In vitro contact of the biomembrane with a lipid-based carrier is the addition of a composition comprising the carrier of the present invention to a culture of protease secreting cells, such as a number of different

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For example, it is possible to add endogenous proteases to a culture medium containing membranes and carriers.

An alternative is in vivo contact, in particular mammalian cells, using a pharmaceutically acceptable carrier and targeting structure. Administration in vivo is by administering the composition of the present invention to a mammal by one of the routes, e.g., intravenously, which is an acceptable method for administering pharmaceutical compositions to mammals in the prior art. The carrier then circulates in the mammalian circulatory system and becomes fuzogenic in the presence of a sufficient amount of a peptidase capable of cleaving the peptide-lipid conjugate in the carrier. As described above, this peptidase is found in mammals, for example, at the site of inflammation, microbial infection, or tumor. In addition, incorporating lipids with a modified hydrophobic group into a lipid carrier will increase the time that the carrier circulates in the circulatory system and thereby proportionally increase the amount of carrier that reaches the desired site in the mammalian body. Tumors have a higher degree of vascularization than surrounding tissue, and the vessels passing through the tumor are considerably more permeable to structures of the type described by the lipid carriers. Therefore, the carrier accumulates in the tumor and an increased rate of accumulation at the desired site improves the therapeutic effect. The carrier can then fuse with both protease-forming cells and neighboring cells in surrounding tissue.

In an in vivo system, the described liposomal carrier is capable of delivering a bioactive agent to a desired site. It may be a therapeutically or diagnostically active amount of a therapeutic or diagnostic agent delivered to a mammalian cell affected by a disease, disorder or symptom that is detected or treated by the agent. That is, the method of delivery can be used to diagnose or treat mammals in the event of disease, disorder or injury.

The method of the present invention may also be used to treat a mammal afflicted with an inflammatory disease by administering to the mammal a liposome containing an effective amount of an anti-inflammatory drug. Treatable inflammatory diseases may be, for example, arthritic disorders, autoimmune disorders, atherosclerotic plaque, acute respiratory stress syndrome, internal inflammatory syndrome, acute nephritis, or gout. Suitable anti-inflammatory drugs may be, for example, non-steroidal anti-inflammatory agents, glucocorticoids, bioactive lipids such as ceramides and ether lipids, and prostaglandins. Peptidases present at the site of inflammation can be, for example, Ala-Ala-recognizing elastase, and cleavage peptides such as Ala-Ala-, Ala-Ala-Ala-, AlaAla-Pro-Val-, Ala-Ala-Pro-Met-, Ala. -Ala-Pro-Ala-, stromelysin-1 recognizing peptides containing the amino acid sequences Arg-Pro-Lys-Pro-Leu-Ala-Nva-, such as Ac-ArgPro-Lys-Pro-Leu-Ala-Nva-, MeOSuc-Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Carboxy Sugar-Arg-ProLys-Pro-Leu-Ala-Nva-Suc-Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Ser -Arg-Pro-Lys-Pro-Leu-AIa-Nva ··················· · · · Ro-leu-al-nva-. Ser11, Ac-Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva-, MeOSuc-Ser-Arg-Pro-Lys-Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Ac-Ser- Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva- and MeOSucSer-Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva- and which cleaves peptides in Ala-Nva- binding and cathepsin G secreted by human neutrophils at the inflammatory site and cleave peptides such as Suc-Ala-Ala-Pro-Phe-, carboxy sugar-Ala-Ala-Pro-Phe-, MeOSuc-Ala-Ala-Pro-Met-, Suc-Ala-Ala-Pro-Phe- -Met- and carboxy sugar-Ala-Ala-Pro-Met-.

Peptide substrates for the enzymes granzyme A and granzyme B secreted by cytotoxic lymphocytes in the synovial fluid of patients with rheumatoid arthritis are, for example, Ac-Ala-Ala-Arg-, MeOSuc-Ala-Ala-Arg-, Ala-Ala-Arg-, Setr-Ala- Ala-Arg-, Ac-Ser-Ala-Ala-Arg-, MeOSuc-SerAla-Ala-Arg-, Ser-Ser-Ala-Ala-Arg-, Ac-Ser-Ser-Ala-Al-Arg-, MeOSuc -Ser-Ser-Ala-Ala-Arg-, Carboxylic Sugar-Ala-Ala-Arg-, etc. Ac-Ala-Ala-Asp-, MeOSuc-Ala-Ala-Asp-, Ala-Ala-Asp-, Setr -Ala-Ala-Asp-, Ac-Ser-Ala-Ala-Asp-, MeOSuc-Ser-Ala-Ala-Asp-, Ser-Ser-Ala-Ala-Asp-, Ac-Ser-Ser-Ala-Ala- -Asp-, MeOSuc-Ser-Ser-Ala-Ala-Asp-, Carboxylic Sugar-Ala-Ala-AspDipeptidylaminopeptidase IV (DAP IV, EC 3.4.14.5), belonging to the dipeptyl peptidase family, is found to be increasingly on vascular cells passing smooth muscle in pigs (Palmieri et al.). Damage to the vessel wall, for example by angioplasty, or during other inflammatory conditions, increases peptidase production. For example, inflammatory edema is associated with disruption of the endothelial layer and uncovering of smooth muscle cells. Appropriate substrates must then be used to direct the liposomes to such sites.

The method described by the present invention can also be used to treat mammals afflicted with cancer by administering liposomes containing an effective amount of an anticancer drug. Treatable types of cancer are brain, breast, intestine, lung, ovarian, prostate and stomach cancer as well as cancers, sarcomas, leukemias, lymphomas and melanomas. Useful anticancer drugs may be, for example, anthracycline antibiotics, bioactive lipids such as ceramides and ether lipids, taxanes and vinca alkaloids. Peptidases present in the vicinity of tumors may be, for example, elastase cleavage peptides having the sequence Ala-Ala-, Ala-Ala-Pro-Val- (Nakajima et al., Castillo et al.), Stromelysin-3 cleavage peptides containing the Ala-Met sequence , stromelysin-1 cleavage peptides with the sequence Ala-Nva-, human collagenase-3 recognition sequences such as MeOSuc-Pro-Cha-Gly-Nva-, Suc-Pro-Cha-Gly-Nva-, Pro-Cha-Gly-Nva -, Pro-Leu-Gly-Leu-, MeOSuc-Pro-Leu-Gly-Leu- and Suc-Pro-Leu-Gly-Leu-, a 72 kDa gelatinase cleavage peptide containing the sequence Gly-Pro-Gln-Gly-Ile- (See Pei et al., Knauper et al., Boyd, Unden et al. and Kossakowska et al.), a urokinase plasminogen activator cleaving Glu-Gly-Arg- and Ac-Lys (Wohl et al., Johnson et al. , Petkov et al., Ascenzi et al.) And cathepsin B recognizing and cleaving the amino acid sequence Arg-Arg- (Knight, Barrett & Kirschke, Kirschke et al.).

9 9 9 9 9 9 9 9 9 9 9 999 9 999 999

9 9 9 9 9

999 999 99 99 99 99

Specific peptidases are also found in nerve tissue (vis. O'Leary and OConnor), suggesting that liposomes can be prepared to be used to treat certain neuropathies. Specific aminopeptidases are present on the membrane to the placenta! tissues and later are secreted indicating their primary placental location (Rogi et al.). Some kinases are specifically present in the kidney. For example, renin is found in the glomerulosa zone and / or in the adrenal medula (Berka et al.). Some peptidases have also been found in skeletal muscle (Ward et al.).

Detection of high alanylaminopeptidase activity in basal cell stroma and DAP IV in tumor cells (Moehrle et al.) Shows that alanyl phospholipid or the corresponding dipeptides are useful for triggering liposomal fusion with a cancer cell.

The method of the present invention can also be used to treat a mammal afflicted with a microbial infection by administering an effective amount to an infected mammal by administering liposomes containing an anti-infective agent, which can be a variety of antibiotics. A variety of bacterial-specific peptidases can be used to direct liposome contents to the site of infection (see Spratt et al.). The HIV virus contains proteases with particular specificity (see Hoog et al.), Which can be expressed on infected cells and can be used to target lysogenic liposomes as therapy.

The contents of the above documents, with descriptions of secreted enzymes and their target peptides, are incorporated herein by reference.

Liposome targeting of drugs can, according to the present invention, deliver liposome contents to proximity to target cells. It is also possible to deliver liposome contents directly to cells via fusion of liposomes and cells. By fusion of cells with liposomes is meant both the binding of the liposome to the cell and the mixing of the liposomal and cellular membrane lipids. Lipid binding and mixing can be accomplished in a variety of ways well known to those skilled in the art, including those set forth below.

Briefly, liposomes are labeled with a fluorescent label enclosed within the liposome and then mixed with cell shadows of red blood cells that are prepared as described below. The cell shadows of the red blood cells are incapable of endocytosis and therefore any transfer of fluorescence to the rest of the cell must be due to fusion. Thus, by determining the degree of fluorescence of cell shadows, the degree of fusion of liposomes with shadows is determined. Thus, cleavage of the lipid-peptide conjugate converts a non-fusogenic liposome to fuzogenic. The liposome may also contain one or more additional fusogenic PE-type lipids such as DOPE and synthetic lipids such as DOTAP and DODAP. These lipids facilitate the fusion of the maternal liposome with the adjacent lipid membrane due to a structure disrupting the bilayer arrangement of the lipid in an aqueous environment.

• · · · · · · · ···

The peptide conjugate may also contain "blocking groups" such as carboxy sugars such as lactobiose acid and N-acetyl neuraminic acid, or polymeric components such as small polyethylene glycol derivatives, polyhydroxyl polymers, or a number of other amino acids in the range of 1-10 in a mixture containing amino acids with a hydrophilic side chain such as serine or threonine. These blocking groups are attached to the N-terminus of the peptide and inhibit or prevent the liposome to come close enough to the lipid membrane to merge. Cleavage of the peptide by the protease removes these blocking groups from the peptide, thereby allowing the liposome to fuse with the lipid membrane. Thus, peptide-mediated cleavage of the peptide portion of the conjugate results in the formation of a fusogenic lipid.

The present invention will become clearer in light of the following examples. Of course, it will be apparent to one skilled in the art that the examples are illustrative only and the invention is defined only by the following claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1: Structure of the N-Ac-AA-DOPE conjugate and depiction of the breakdown into DOPE by enzyme digestion.

Giant. 2: N-Ac-AA-DOPE protease digestion determined by TLC. N-Ac-AA-DOPE SUVs were incubated with A) elastase or B) proteinase K (1mg enzyme / 100 nmol lipid / 0.1 ml buffer) overnight at 37 ° C. The lipid was isolated and separated by TLC. Lipid bands were developed using the method below. Lane 1, N-Ac-AA-DOPE without enzyme, lane 2, NAc-AA-DOPE after incubation with enzyme, lane 3, DOPE control.

Giant. 3; Cleavage of N-Ac-AA-DOPE with proteinase K. DOTAP / N-Ac-AA-DOPE (1: 1) SUVs were incubated with or without elastase, proteinase K, or heat inactivated proteinase K (95 ° C, 1 hour) ) at 1 mg protease / 0.1 nmol / 0.1 ml lipid concentration buffer and incubate overnight at 37 ° C. The lipid was isolated and analyzed by HPLC. The N-Ac-AA-DOPE peak was quantified and the cleavage rate was determined as a percentage of the starting lipid.

Giant. 4: Determination of ideal liposome composition. Liposomes were prepared in given molar ratios of DOTAP, N-Ac-AA-DOPE, PE. In all cases of liposome preparation, fluorescent labels of 1 mol% of N-NBD-PE and N-Rho-PE were used. Liposomes were mixed with A) unlabeled PE / PS or PC / PS (80/20 mol%, 1:10 effector to acceptor ratio, 60 pmol total lipid) or B) 2 χ 10 ⁸ RBC cell shadows at 37 ° C for for 1 hour.

• · · · ·

The lipid mixture was calculated as a percentage of N-NBD-PE FDQ relative to the maximum FDQ determined by detergent addition. Binding of liposomes to RBC cell shadows was quantified after washing the cells with buffer, comparing the amount of N-Rho-PE fluorescence bound to the cell pellet relative to the total fluorescence added.

Giant. 5: Liposomal fusion activated by elastase and proteinase K. DOTAP / N-Ac-AA-DOPE / PE (15/15/70 mol%) liposomes containing fluorescent membrane tags were pre-incubated with human leukocyte elastase or proteinase K (1 mg protein / 100 nmol lipid / 0.1 ml buffer) overnight. 10 nmol aliquots were incubated with unlabeled PE / PS acceptor liposome (80/20 mol%, 1:10 effector: acceptor ratio) for 60 min at 37 ° C. The lipid mixture was determined by determination of N-NBD-PE FDQ.

Giant. 6: Conditions necessary for activation of DOTAP / N-Ac-AA-DOPE / PE liposomes by proteinase K for fusion with PS / PE liposomes. DOTAP / N-Ac-AA-DOPE / PE (15/15/70 mol%) liposomes (100 nmol) containing fluorescent membrane labels were preincubated with or without 1 mg proteinase K or heat inactivated proteinase K (1 hr, 95 ° C) C) at 37 ° C in 0.1 µm buffer. 10 nmol aliquots were incubated with unlabeled PE / PS acceptor liposome (80/20 mol%, 1:10 effector: acceptor ratio), after which time the lipid mixing rate was determined. Proteinase K residual activity = effect of proteinase K residue transferred to incubation mixture with PE / PS acceptor liposomes was tested by incubation of unmodified DOTAP / N-Ac-AADOPE / PE (15/15/70 mol%) liposomes with PE / PS liposomes in the presence of freshly added proteinase K equivalent to the expected amount transferred.

Giant. 7: Concentration and time dependence of proteinase K activity. DOTAP / N-AcAA-DOPE / PE (15/15/70 mol%) liposomes (100 nmol) containing fluorescent membrane labels were incubated overnight at 37 ° C either A) overnight with a given amount of proteinase K or B) with 1 mg proteinase K over a given period. 10 nmol aliquots were incubated with unlabeled PE / PS acceptor liposomes (80/20 mol%, 1:10 effector acceptor ratio), and then the composition of the lipid mixture was determined. N-Ac-AA-DOPE cleavage: unlabeled DOTAP / N-Ac-AADOPE (1: 1 molar ratio) liposomes were incubated as described for fusion activation and then the lipid extract was extracted by HPLC.

Giant. 8: Activation of DOTAP / N-Ac-AA-DOPE / PE liposomes by proteinase K for fusion with RBC cell shadows. DOTAP / N-Ac-AA-DOPE / PE (20/10/70 mole%) liposomes (100 nmol) • φφφφ ·· ·· φ · · · φ · φ φ

containing fluorescent membrane labels were incubated overnight at 37 ° C with or without 1 mg proteinase K in 0.1 ml buffer. 10 nmol aliquots of DOTAP / N-Ac-AA-DOPE / PE liposomes as well as DOTAP / PE (20/80 mol%) liposomes were incubated with 1x10 ⁸ RBC cell shadows in buffer containing 0.5 mM PMSF for 30 minutes at 37 ° C and the composition of the lipid mixture was determined. The effect of added proteinase K on the composition of the lipid mixture was monitored by incubating untreated liposomes with RBC cell shadows in the presence of an appropriate amount of proteinase K (prot. K, control).

Giant. 9: DOTAP / N-Ac-AA-DOPE / PE liposomes with RBC cell shadows: continuous kinetics of lipid mixture formation. 10 nmol of DOTAP / N-Ac-AA-DOPE / PE (20/10/70 mol%) liposomes incubated with (a) with or (b) without proteinase K overnight at 37 ° C were added to a cuvette containing 2 ml of buffer with 0.5 mM PMSF with stirring at 37 ° C. Changes in N-NBD-PE fluorescence were measured from the time when 1x10 ⁸ RBC cell shadows were added for 30 sec. The residual proteinase K activity on the lipid mixture was assayed by incubating unmodified liposomes with RBC cell shadows in the presence of a freshly equivalent amount of proteinase K.

Giant. 10: Dextran-loaded DOTAP / N-Ac-AA-DOPE / PE liposomes fuse with RBC cell shadows. DOTAP / N-Ac-AA-DOPE / PE (20/10/70 mol%) liposomes were loaded with 10 kD dextran conjugated with Tx-red. Liposomes were incubated with proteinase K overnight at 37 ° C. 40 nmol aliquots of dextran-loaded liposomes (A, C) or empty liposomes + free dextran (B, D) were incubated with 1x10 ⁸ RBC cell shadows in 1 ml buffer for 30 minutes at 37 ° C, after which the cells were washed and observed by fluorescence microscopy (Α, Β) or by Nomarski differential interference contrast (C, D).

Giant. 11: TLC determination of OMe-suc-ala-ala-pro-val-DOPE cleavage. A) HLE dose titration: 1/0 pg HLE / 100 nmol lipid, 2/5 pg HLE / 100 nmol lipid, 3/10 pg HLE / 100 nmol lipid, 4 / pure DOPE, 20 pg, 5/20 pg HLE / 100 nmol lipid, 6/40 pg HLE / 100 nmol lipid, 7/40 proteinase K / 100 nmol lipid, 8 / pure DOPE, 20 pg, B) Cleavage of OMe-suc-ala-ala-pro-val-DOPE protein derived from human neutrophil granules: 1 / w / o protease, 2/5 pg HLE / 50 nmol lipid, 3 2.5 pg neutrophil granules / 100 nmol lipid, 4 / pure DOPE, 20 pg, 5/5 pg z neutrophil granules / 100 nmol lipid, 6/10 µg protein from neutrophil granules / 100 nmol lipid, 7/20 µg neutrophil granules protein / 100 nmol lipid, 8 / pure DOPE, 20 µg. HLE cleavage kinetics: 1 / w / o protease, 2/1 hours, 5 µg HLE / 50 nmol lipid, 6 / overnight, 5 Mg HLE / 50 nmol lipid, 7 / pure DOPE, 20 µg.

And a

• ·

EXAMPLES OF EMBODIMENTS OF THE INVENTION

EXAMPLE 1.

A) Chemical synthesis of N-Ac-Ala-Ala-DOPE

N-Acetyl-alanyl-alanyl-dioleoyl phosphatidylethanolamine (N-Ac-Ala-Ala-DOPE) was synthesized from a previously prepared N-Ac-Ala-Ala-OH-OH peptide anhydride or with another suitably blocked carboxyl-terminated peptide. The starting material was incubated with N, Ndicyclohexyl carbodiimide (DCC) in the presence of chloroform for several hours at room temperature. The resulting anhydrite is soluble in chloroform while the intermediate reaction (dicyclohexylurea) is not. Thus, anhydrite is readily separable from the undesired intermediate by collecting chloroform and removing the precipitate.

DOPE is added to the anhydride in the presence of triethylamine, which catalyzes the acylation reaction, then the mixture is incubated overnight at room temperature. The reaction mixture is applied to a preparative thin layer chromatography plate so as to be purified by N-Ac-Ala-Ala-DOPE by separation in a chloroform / methanol / water solution (65/25/4). The lipid band is identifiable by spraying the plate with water. The band is then scraped and dissolved in chloroform / methanol (2/1). The lipid is stored under nitrogen at -70 ° C.

B) Chemical synthesis of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamido-Val-Pro-Ala-Ala-SucMeO (OMe-Suc-Ala-Ala-Pro-Val-DOPE):

i) p-Nitrophenyl-Val-Pro-Ala-Ala-SucMeO ester: to a solution of H-Val-Pro-Ala-Ala-SucMeO peptide (540 mg, 1015 mmol), 142 mg (1.38 mmol) was added. nitrophenol, 175 mg (1.38 mmol) of 1,3-dicyclohexylcarbodiimide and a catalytic amount (pair of crystals) of 4-dimethylaminopyridine in 10 ml of dry chloroform. The reaction mixture was left stirring at room temperature overnight. At this point, TLC analysis shows that the reaction is complete. The precipitate (DCU) formed in the reaction mixture was filtered using a G-2 funnel and the filtrate was concentrated under reduced pressure. The resulting product was used in the next step without any purification, R f 0.43 (CHCl 3 MeOH, 9: 1 v / v).

(ii) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamido-Val-Pro-Ala-Ala-SucMeO: to a solution of nitrophenyl-Val-Pro-Ala-Ala-SucMeO ester (600 mg, 1.01 mmol), 604 mg (0.81 mmol) of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine and 82 mg (113 mL, 0.81 mmol) of triethylamine in 20 mL of chloroform: tetrahydrofuran (1: 4 v / v) solvent mixture were added in). The reaction mixture was allowed to stir overnight at room temperature. At this point, TLC • ftftftft • ftft analysis shows that the reaction is over. The reaction mixture was concentrated under reduced pressure and passed through the activated TMD-8 ion exchange resin in THF: H ₂ O (9: 1 v / v). The phosphorus-positive fractions were combined and concentrated to give the desired product. The obtained material was purified on a silica gel column by liquid chromatography (the column was washed with 5% methanol in chloroform and then rinsed with CILCl 2 / MeOH / OH 65: 25: 4 v / v / v) to give 915 mg of the product (95% yield based on to DOPE), which lyophilized to give a white solid: R f 0.76 (CH ₃ Cl: MeOH: NH 4 OH 65: 25: 4 v / v / v) and R _f 0.43 (CH ₃ Cl: MeOH; H ₂ O) 65: 25: 4 v / v / v). The lipopeptide molecule shows a positive reaction for the molybdenum reagent and a negative test for the ninhydrin reagent. The identity of the lipopeptide molecule was verified by TLC in two solvent systems: (i) CH ₃ Cl: MeOH: NH 4 OH 65: 25: 4 v / v / v and (ii) CH ₃ Cl: MeOH: H ₂ O 65: 25: 4 v / v / v). In both systems, the lipopeptide has formed a single point and its purity is greater than 99%. The lipopeptide was characterized by NMR and FAB mass analysis. 1 H-NMR (CDCl ₃ ) are the characteristic signals at: d 0.87 (t, 3H, J = 7.15 Hz), 1.27 (40Η), 1.56 (4H), 2.0 (8H) 2.23 (t, 4H, > 7.15 Hz), 5.17 (1H), 5.32 (4H, J = 3.12 Hz). ^{The 31} PNMR spectrum gave a single signal. FAB (MIL) calcd for C62H109N5O15P 1195.55, was

1196.8 (MH +) and 1234.9 (MK ^+).

EXAMPLE 2. Cleavage of N-Ac-Ala-Ala-DOPE

Elastase cleavage of N-Ac-Ala-Ala-Ala-DOPE to DOPE was monitored by thin layer chromatography (TLC). 100-200 nmol of N-Ac-AA-DOPE SUV was incubated with 1 mg of enzyme overnight at 37 ° C. The lipid was extracted by organic phase separation (19) twice. The obtained lipid was dried under N 2 flow and left under vacuum for 4 hours overnight. The samples were then dissolved in chloroform and plated on TLC plates. TLC was run in CH ₃ Cl: MeOH: NH 4 OH (65: 25: 4), air dried, molybdenum blue sprayed, and fired on a hot plate. Incubation of N-Ac-AA-DOPE liposomes with elastase gives rise to a DOPE-like product, while unincubated N-Ac-AA-DOPE shows no changes (Fig. 2A). Accordingly, the elastase recognizes N-Ac-AA-DOPE and cleaves the dipeptide to form DOPE.

Some proteases have been tested whether an enzyme with similar substrate specificity can be used as a model for N-Ac-AA-DOPE elastase cleavage. Proteinase K is a serine protease that, like elastase, can cleave a peptide bond at the C-terminal position to an aliphatic residue. By incubating N-Ac-AA-DOPE with proteinase K, the conjugate is cleaved to form DOPE (Fig. 2B).

Fcfc fcfc fcfc fcfc fcfcfc fcfc fcfcfc fcfc

The cleavage of N-Ac-AA-DOPE to DOPE is also monitored by 31 P-NMR analysis. N-Ac-AADOPE LUV were prepared and incubated with proteinase K (1.5 mg protein / 100 nmol lipid) overnight at 37 ° C. Samples were mixed with buffer (10% deoxycholate, 100 mM EDTA, 20 mM HEPES) and deuterium oxide (Cambridge Isotope Laboratories, Woburn, MA) (1: 4: 2) and transferred to 5 mm NMR cuvettes. The samples were monitored at room temperature on a Bruker AC300 spectrometer operating at 121.5 MHz, with a 110 ms 90 ° radio frequency pulse for proton cleavage, and 2 second intervals were set to avoid saturation of the signal.

in

The bandwidth was selected at 50 Hz. The 1 Hz boundary line was applied for the entire spectrum. NAc-AA-DOPE liposomes with proteinase K (1.5 mg protein / 100 nmol lipid) show a peak of 0.3 ppm as opposed to N-Ac-AA-DOPE, which corresponds to pure DOPE.

cleavage of N-Ac-AA-DOPE by elastase and proteinase K was quantified by liposomes generated from a mixture of N-Ac-AA-DOPE and DOTAP. DOTAP was used to balance the positive charge and is used as a standard by which different samples can be recalculated and compared. After incubation with elastase or proteinase K, the loss of N-Ac-AA-DOPE was monitored by HPLC. Liposomes containing DOTAP / N-Ac-AA-DOPE (1: 1) or DOTAP / N-Ac-AA-DOPE / PE (15/15/70 mol%) were incubated with the enzyme under the given conditions. The lipid was extracted twice using the Blight-Dyer procedure.

The obtained lipid was dried under nitrogen and transferred to vacuum for 4 hours to overnight. The samples were resuspended in 100% ethanol and loaded onto a Spherisorb silica column (150 x 4.6 mm, 0.3 µm, Keystone scientific). HPLC was carried out using hexane: isopropanol: water: TFA aqueous phase. Hexane and TFA were kept constant at 37% and 0.2%, respectively. N-Ac-AADOPE peak was detectable using a 59-55% isopropanol: 4-8% water gradient. The flow rate was 1.5 ml / min, the column temperature was 45 ° C, and the peak was detected by a UV detector set at 205 nm. Lipid peaks were quantified relative to standard curves obtained by loading 5-200 nmol of DOTAP or N-Ac-AA-DOPE and monitoring the 205 nm signal. The% cleavage was recalculated relative to the normal peak DOTAP, and then the loss of NAc-AA-DOPE relative to the input amount was determined.

Both elastase and proteinase K cleave N-Ac-AA-DOPE to the same extent (Fig. 3). To verify that N-Ac-AA-DOPE cleavage is due to proteinase K activity, liposomes were incubated with heat inactivated proteinase K. Proteinase K was heat inactivated by incubation for 1 hour at 95 ° C. The enzyme was then unable to cleave the colored substrate N-Ac-AAA-pNA. Incubation of DOTAP / N-AcAA-DOPE liposomes with a heat-inactivated enzyme does not result in cleavage of N-Ac-AA-DOPE (Fig. 3), demonstrating that active proteinase K is necessary. Since proteinase K has been found to be 9 9 9 9 9 99 99 9 9 9 9 9 9 9

9999 9 99 9 9 99 9

9 9 9 9 9

999 999 99 99 99 99 is able to cleave the same substrate as elastase, and is considerably cheaper than human leukocyte elastase, most of the subsequent experiments were carried out with proteinase K.

EXAMPLE 3. Digestion of OMe-Suc-Ala-Ala-Pro-Val-DOPE by Human Leukocyte Elastase (HLE)

A) HLE dose titration

To determine whether the OMe-Suc-Ala-Ala-Pro-Val-DOPE peptide is also a suitable substrate for elastase cleavage, 50 nmoles of OMe-Suc-Ala-Pro-Val-DOPE liposomes (SUV) were incubated with 20µg HLE (Calbiochem, 20 µ / mg protein, 1 µ = amount of enzyme capable of hydrolyzing 1.0 µmol OMe-Suc-Ala-Ala-Pro-Val-DOPE per minute at 25 ° C and pH = 8) overnight at 37 ° C in a 50 µl volume of 10 mM TES / 154 mM NaCl / 0.1 mM EDTA, pH 7.4 containing 1.5 mM Ca and 1.5 mM Mg.

The lipid was extracted using the Blight-Dyer technique (chloroform / methanol / water: 2 / 1.7 / 1), dried under a nitrogen atmosphere and placed under high vacuum for about 3 hours. Samples were resuspended in 5 µl chloroform and plated on TLC plates. 20 µg of pure DOPE was also loaded as a control. The mobile phase was chloroform / methanol / ammonium hydroxide (65/25/2). The plates were air dried, molybdenum blue sprayed and fired at 180 ° C.

B) Digestion of OMe-Suc-Ala-Ala-Pro-Val-DOPE protein from human neutrophil granules

Since elastase is produced by activated neutrophils, cleavage of OMe-Suc-Ala-Ala-Pro-ValDOPE by unpurified lymphocyte granule enzyme has also been tested as it mimics better the in vivo situation. Neutrophils were obtained from whole human blood by standard density centrifugation. Granules were isolated from these neutrophils by centrifugation after cell cavitation, again followed by assay procedures. The protein concentration obtained from the neutrophil granules was determined after repeated freezing and melting of the granules to release proteases.

nmol of OMe-Suc-Ala-Ala-Pro-Val-DOPE liposomes (SUV) was incubated with 0, 2.5, 5, 10, or 20 µg of neutrophil granule proteins overnight at 37 ° C in a 50 µl volume of 10 mM Mg The samples were treated as described above. The results show that 2.5 µg of neutrophil granule protein is sufficient for detectable cleavage of OMe-Suc-Ala-Ala-Pro-Val-DOPE to DOPE, suggesting that crude lymphocyte granule protein can cleave the conjugate to DOPE and thus liposomes The peptide-lipid containing may be activated for fusion under physiological conditions.

C) The kinetics of OMe-Suc-Ala-Ala-Pro-Val-DOPE cleavage by HLE ftftftft ftft ftft ftft Ftftft ftftft ftft ftft ftft ftft ftft

OMe-Suc-Ala-Ala-Pro-Val-DOPE liposomes were incubated with 0 or 5 µg HLE for 1, 2, 4 hours, or overnight and further treated as above. The peptide is cleaved by HLE in less than 1 hour at 37 ° C, indicating that the cleavage of OMe-Suc-Ala-Ala-Pro-Val-DOPE occurs within a physiologically relevant timeframe

The results of these experiments are shown in FIG. 11.

EXAMPLE 4. Preparation of liposomes

NBD / Rh labeled or unlabelled large monolayer liposomes (LUV) were prepared as described previously (Mayer et al.). Briefly, the lipid mixture in chloroform was dried under a stream of nitrogen to form a thin film, which is then left under vacuum overnight to remove residual solvent. The lipid film was hydrated with TES buffered solution (10 mM TES, 0.1 mM EDTA, 154 mM NaCl, pH = 7.4). The mixture is shaken gently for complete hydration. After ten cycles of freezing and sealing in a liquid nitrogen / water bath at room temperature, the sample was passed ten times through a 0.1 µm polycarbonate membrane filter (Poretics Corp., Livermore, CA). Liposomes were stored at 4 ° C. Multilamellar liposomes were prepared by hydrating a dry lipid film.

The phospholipid concentration in each preparation was determined by the phosphate method (Bartlett). Liposomes of about 0.1 µm in size were determined on a Nicomp submicron particle sizer (Nicomp Instruments, Inc, Goleta, CA) using quasi-elastic light scattering.

EXAMPLE 5. Preparation of reclosed and unclosed human erythrocyte cell shadows

Reclosed cell shadows essentially describe the erythrocyte shadows, unless otherwise specified, and were prepared as described above (see Williamson et al., Clague et al., Incorporated herein by reference). Briefly, fresh human blood is washed several times with cold 10 mM TES saline to remove plasma and white blood cells. Then, 2 ml of washed erythrocytes (50% hematocrit) were puckered in an ice hypotonic solution containing 8 ml of ILO and 9.6 ml of 10 mM TES saline and then centrifuged at 850 x g for 5 minutes. The pellet was resuspended in 40 ml ice lysis buffer (10 mM Tris, 0.1%

BSA, 2 mM MgCL and 0.1 mM EGTA) and incubated on ice for at least 2 minutes. After addition of 4.5 ml of 10x concentrated recovery buffer (1.22 MnCl, 30 mM KCl, 0.15 M Na 2 HPO 4 and 2 mM MgCL), the samples were incubated for 40 minutes at 37 ° C. Resealed cell shadows were centrifuged at 1750 x g for 10 minutes and washed as many times as possible until hemoglobin was visible in the supernatant. Cell shadows are stored at 4 ° C until use for 1 week.

ftftft · ftft 99 ftft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft ft

EXAMPLE 6. Generation of N-Ac-AA-DOPE Liposome with Regulated Fuzogenicity The degree of fuzogenicity was determined by preparing a liposome with increased PE taransesterified from egg PC. This Pe is preferable to DOPE due to its higher H _n transition temperature (-37 ° C versus 10 ° C), which allows the preparation of stable liposomes but does not inhibit fusion. DOTAP was chosen as a positively charged lipid. The fusion technique was tested for DOTAP / N-Ac-AA-DOPE / PE liposomes containing the membrane fluorescent probe NNBD-PE and N-Rho-PE and vice versa corresponding amounts of N-Ac-AA-DOPE and PE. These liposomes were tested to mix lipids with either other unlabeled liposomes or to mix and bind to RBC cell shadows. Lipid mixing between NBD / Rh labeled liposomes and unlabeled cell shadows was measured in 10 mM TES saline using the NBD / Rh resonance energy transfer (RET) technique (Struck et al., Incorporated herein by reference). Liposomes were prepared with 1 mol% N-NBD-PE and 1 mol% N-Rho-PE, resulting in quenching of the N-NBD-PE fluorescence signal. Membrane fusion leads to diffusion of the probe and suppression of self-extinguishment, resulting in an increase in N-NBD-PE fluorescence. Mixing of the lipids with each other is initiated by adding 10 nmol of fluorescently labeled liposomes to 90 nmol of unlabeled liposomes in a microcentrifuge tube containing 1 ml of TES / NaCl / EDTA buffer with 1.5 mM Ca ⁺⁺ / 1.5 mM Mg ⁺⁺ . For fusion with cells, unlabeled liposomes replaced 1 x 10 ⁸ RBC cell shadows. All samples were mixed in an Eppendorf Thermomixer (Brinkman Instruments, Inc., Westbury, NY), 700 rpm / min for 30 minutes at 37 ° C. N-NBD-PE fluorescence was measured on a T-format PTI Alphascan spectrafluorometer (Priceton, NJ) with a xenon arc lamp using 450 nm excitation / 530 nm emission and a 5 nm slit.450 nm band pass and 500 nm reduction filters were used to treat excitation and emission beams, to reduce stray light. Maximum fluorescence was determined by the addition of 0.1% C12E8 detergents.

It is clear that the degree of fuzogenicity also depends on the target. Liposomes made from DOTAP and PE fuse with both target liposomes and RBC cell shadows. Addition of 10 mol% N-Ac-AA-DOPE and a corresponding increase in PE to 70 mol% yields liposomes that are capable of fusing with PE / PS liposomes but no longer with PC / PS liposomes (Fig. 4A). The requirements for fusion of membranes with RBC cell spirits appear to be somewhat stricter, the addition of 5 mol% N-Ac-AA-DOPE inhibits both lipid mixing and binding considerably (Fig. 4B). By defining a different fusion rate for different targets, a fusion sensitivity gradient can be created which can be used to determine optimal conditions for activating N-Ac-AA-DOPÉ liposomes to fusion. Since PE / PS liposomes are the most sensitive target for fusion, we have focused on the preparation of DOTAP / N-Ac-AA-DOPE / PE liposomes, which may be activatable to fusion. The PE content appears to be between 65-70 mol%. In order to • Φ Φ

Φ Φ Φ

ΦΦΦ ΦΦΦ

OT Φ φ AD Φ Φ Φ Φ Φ Φ Φ Φ φ φ φ φ Φ Φ Φ 11 • • • 11 11 11 11 • 11 • 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 in an appropriate amount to form liposomes composed of DOTAP / N-AcAA-DOPE / PE in a mole ratio of 15/15/70.

v t

EXAMPLE 8: Activation of lipid mixing between liposomes by enzymatic cleavage Since elastase and proteinase K are able to cleave N-Ac-AA-DOPE into DOPE (Fig. 2,3), both enzymes were tested for their ability to activate DOTAP / N-Ac-AA -DOPE / PE (15/15/70 mol%) liposomes for fusion. These liposomes were incubated overnight at 37 ° C with elastase or proteinase K, or without the appropriate enzymes, and then the liposomes were incubated with PE / PS liposomes and lipid mixing was observed by suppressing N-NBD-PE fluorescence quenching. Liposomes were incubated with 1 mg protease / 100 nmol lipid / 0.1 ml buffer unless otherwise indicated. This proteinase K concentration has been found to have comparable course and maximum activity as elastase in the synovial fluid of patients with rheumatoid arthritis (21, unpublished data). The mixtures were incubated at 37 ° C in microcentrifuge tubes with constant agitation in an Eppendorf Thermomixer, 700 rpm / min. The incubated liposomes were then tested for N-Ac-AA-DOPE digestion by HPLC. For fusion experiments, liposomes containing fluorescent membrane probes were incubated with the protease and then the liposome concentration was determined by direct fluorescence measurement of N-Rho-PE (550 ex / 590 em) and compared to a known number of control liposomes. Aliquots of these fluorescently labeled liposomes incubated with protease were mixed with unlabeled target liposomes or cells, and lipid mixing was determined as above. Incubation with any enzyme was characterized by a much higher lipid mixing rate than uncleaved liposomes (Fig. 5). This result, along with the shared substrate specificity of proteinase K and elastase, suggests that activation by proteinase K may serve as a suitable replacement for elastase to activate fusion of N-Ac-AA-DOPE containing liposomes.

Dependence of cleavage of N-Ac-AA-DOPE conjugate and activation of fusion of liposomes containing DOTAP / NAc-AA-DOPE / PE (15/15/70 mol%) was tested by heat inactivated proteinase K. DOTAP / N-Ac-AA-DOPE / PE (15/15/70 mol%) liposomes containing fluorescent membrane probes N-NBD-PE and N-Rho-PE were incubated overnight at 37 ° C with both active and inactive proteinase K, and aliquots of the liposomes were incubated with unlabeled PS / PE acceptor liposomes to monitor the progress of lipid mixing. Incubation of DOTAP / N-Ac-AA-DOPE / PE liposomes with active proteinase K resulted in an approximately 30% increase in fluorescence, i.e. an approximately 6-fold increase in liposome mixing over control liposomes (Fig. 6). Incubation with the same amount of heat inactivated enzyme does not activate flflflfl flflflfl flflflfl flflflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fllflflfl fll liposomes for fusion (Fig. 6). Thus, it is apparent that enzymatic activity is required to activate liposome fuzogenicity and hence the cleavage of N-Ac-AA-DOPE is necessary to trigger the fuzogenic potential.

Since the experimental protocol involves the transfer of a small amount of active proteinase K (10 µg protease / 100 nmol lipid, 100 times diluted) together with pre-incubated liposomes for subsequent liposome-liposome incubation, it is possible that this amount of enzyme causes observed lipid mixing by nonspecific protein effect. This possibility was tested by adding the predicted residual amount of proteinase K to the pre-cleaved DOTAP / N-Ac-AA-DOPE / PE + PE / PS incubation mixture. Lipid mixing was the same as for liposomes incubated without proteinase K (Fig. 6). In addition, continuous fusion kinetics exhibits an immediate increase in fluorescence intensity after mixing DOTAP / N-Ac-AA-DOPE / PE liposomes incubated with proteinase K with PS / PE liposomes (unpublished data). Enzyme-triggered increases will not be achieved immediately, suggesting that non-specific enzymatic cleavage of the target liposomes is not the cause of fusion. Further presence of the protein cannot be responsible for the increase in fluorescence, since heat inactivated proteinase K does not produce a similar effect. In contrast, proteinase K does not physically prevent fusion, and exogenous proteinase K added to a mixture of fusogenic DOTAP / PE liposomes with target liposomes cannot inhibit lipid mixing (unpublished data). Thus, only prior incubation with enzymatically active proteinase K triggers fusion of N-Ac-AA-DOPE liposomes.

The reliability of fusion activation by enzymatic cleavage of N-Ac-AA-DOPE was further determined by testing the concentration and time dependence of both events. DOTAP / N-Ac-AA-DOPE / PE liposomes were incubated with 0, 0.1, 0.25, 0.5 and 1 mg proteinase K / 100 nmol lipid overnight, or with 1 mg proteinase K / 100 nmol lipid for 1, 2, 4 hours or overnight. N-Ac-AA-DOPE digestion was then measured by HPLC or lipid mixing was tested by increasing the fluorescence intensity of NNBD-PE. In both cases, both N-Ac-AA-DOPE cleavage and liposome fusion, similar concentration dependencies were found (Fig. 7A). Incubation with 0.5 or 1 mg proteinase K shows maximum cleavage efficiency and fusion activation. Only a low level of both activities was observed using 0 or 0.1 mg enzyme. The kinetics of proteinase K cleavage and fusion activation were also compared. Incubation overnight showed the highest amount of lipid digestion and mixing (Fig. 7B). These conclusions support the assertion that activation of the DOTAP / N-Ac-AA-DOPE / PE liposome fusion is due to enzymatic cleavage of N-Ac-AA-DOPE.

EXAMPLE 9: Activation of DOTAP / N-Ac-AA-DOPE / PE liposome fusion with RBC cell shadows • 9999 9 «99 ♦ <·· • · 9 9 9 ·· 9 · 9 · • 999 9 9 9 · ··· ·

9999 999 9 ··· 999

9 9 9 9 «9 • 99 999 99 9 · ·· ··

Since DOTAP / N-Ac-AA-DOPE / PE liposomes can be activated to fuse with target liposomes by enzymatic cleavage, we have investigated whether an activated fusion of N-Ac-AA-DOPE liposomes will also be observed with cells. Since the fusion with RBC cell shadows (Fig. 4B) shows different parameters than the fusion with liposomes (Fig. 4A), we prepared DOTAP / N-Ac-AA-DOPE / PE liposomes in a molar ratio of 20/10/70. The overall positive charge of these liposomes improves cell binding, relative to the 15/15/70 mixture, but does not allow the liposome to fuse with the cell in the absence of the activation signal (Figure 4B). After incubation of these liposomes with proteinase K overnight at 37 ° C, the liposomes were mixed with RBC cell shadows in the presence of the protease inhibitor PMSF (Fig. 8). The residual proteinase K activity transferred from the first incubation is negligible (Fig. 8). Specific activation of the DOTAP / N-Ac-AA-DOPE / PE fusion (20/10/70 mol%) with RBC cell shadows was also observed under continuous kinetic conditions. Only liposomes pre-incubated with proteinase K were able to fuse with RBC cell ghosts, while uncleaved liposomes did not (Fig. 9). The addition of active proteinase K to uncleaved liposomes also did not induce an increase in fluorescence (Fig. 9, curve c), indicating that the observed increase after incubation of DOTAP / N-Ac-AA-DOPE / PE liposomes with proteinase K is due to specific fusion activation.

To determine whether the lipid mixing observed after proteinase K activation was indeed due to fusion of liposomes with cells and not to artificially mixed lipids in membranes, such as membrane probe exchange or hemifusion between outer membrane layers, were DOTAP / N-Ac-AADOPE / PE (20 (10/70 mol%) liposomes filled with a 10,000 MW fluorescent hydrophilic probe TX-red dextran. Then the liposomes were incubated with proteinase K and incubated with RBC cell ghosts. After thorough washing of the cells to remove unbound liposomes, RBC cell shadows were observed with a fluorescence microscope. The cell pellet was resuspended in 0.1 ml buffer and observed on an Olympus BH-2 fluorescence microscope (Olympus Corp., Lake Success, NY) using a 40x apochromic objective (N.A. 1.00) in oil immersion. TX-red fluorescence was excited by a xenon lamp and the beam passed through a green excitation cube (580 nm dichroic mirror, 545nm excitation filter). Non-fluorescent images were obtained using Nomarski differential interference contrast microscopy techniques.

Clear diffuse fluorescence was observed in part of the cells (Fig. 10), indicating that there was a complete fusion between the liposome and the cell, thereby transferring the hydrophilic probe. Fluorescence rate differences can be due to the number of liposomes fused to individual cells. The observed fluorescence is not due to non-specific ingestion of dextran from ruptured liposomes, since after incubation of RBC cell shadows with unlabeled liposomes and free TX-red

99

9 9

9 9

No fluorescent probe is visible inside the cells with dextran (Fig. 10). Thus, DOTAP / N-AcAA-DOPE / PE liposomes can be activated by enzymatic cleavage of the peptide-lipid conjugate to fuse with target cells and deliver their hydrophobic content inside.

Example 10: Activation of OMe-Suc-Ala-Ala-Pro-Val-DOPE-Containing Liposomes by HLE to Increase Binding and Fusion

100 nmol of DOTAP / AAPV-PE (3: 1, 1: 1, 1: 3) was incubated with +/- HLE (5 µg / 100 nmol lipid) in 100 µl for 2 hours at 37 ° C, pH 7.4 with stirring. Then, 10 nmol of liposomes were pre-incubated with +/- HLE mixed with 1 to 2 χ 10 ⁶ HL60 (human leukemia) cells at pH

7.4 or 4. Samples were incubated for 2 hours with stirring. After incubation, the cells were washed twice, resuspended in 0.5 ml and transferred to a 24-well Falcon plate (primaria). Fluorescence was measured by +/- detergent in cytofluor. Control liposomes (without cells) were not washed and transferred directly to a 24-well plate.

Liposomes show higher binding at pH 4.0. Binding is also stronger after incubation with HLE. In addition, DODAP / AAPV-PE (1: 1) may exhibit a higher fluorescence intensity after incubation with HLE, indicating that lipids are mixed between liposomes and cells.

% better bond% NBD FD

DODAP / AAPV-PE pH 7.4 pH4.0 pH 7.4 pH4.0 1: 3 - HLE 2.68 53.58 104.22 33.13 1: 3 + HLE 0.74 70.34 -32.43 32.53 1: 1 -HLE 2.37 56.95 59.94 23.86 1: 3 + HLE 2.87 85.91 77.42 44.81 3: 1 -HLE 6.76 67.88 -5.05 64.52 3: 1 + HLE 7.78 80.82 43.43 69.04

Three independent experiments also show increased binding of DOTAP / AAPV-PE (1: 1) to HL60 cells. One shows an improvement from 64% to 86% increase in fluorescence. Given the overall rate of liposomal fusion, incubation of HLE has been shown to improve lipid mixing, as demonstrated by all three experiments.

• ···· ♦ · φ φ • «« φ φ φ φ φ φ φ φ φ φ φ φ φ • φ φ φ φ φ φ φ φ φ φ φ

W / oHLE w / HLE % of binding 20.29 45.22 # bindings 1.22E + 10 2.72E + 10 % lipid mixing 48.6 44.24 # lipid mixing 5.87E + 11 1, 20E + 12

W / oHLE w / HLE % of binding 7.72 33.17 # bindings 4.60E + 09 2.00E + 10 % lipid mixing 57.12 25.89 # lipid mixing 2.65E + 11 5.17E + 11

W / oHLE w / HLE % of binding 38.09 46.89 # bindings 2,29E +10 3.08E + 10 % lipid mixing 63.74 86.25 # lipid mixing 1.46E + 12 2.43E + 12

• 4

4

4 4 4 4

444 4,444 4 · 4,444

4 4 4

444 ·· 99

44

4 4 4

4 4 4

444 444

4

Citation:

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Claims (16)

1. Peptide-lipid conjugates according to the following general formula: CR ¹ CR ² C (O) -CH ₂ -CH ₂ -NH ₂ -XY Where X is a linker formed by either a single bond or R ³ , each R ¹ , R ² and R ³ is independently a group of formula C (O) (CH ₂ ) _n (CH = CH) _n 2 (CH ₂ ) _n 3 ( CH = CH) _{n4 (CH2) n5} (CH = CH) _n 6 _{(CH2) n7} (Target = CH) _n (CH _{2) n9} CH _3, the value of AD falls within the range from 1 to 22, n3 falls in the range of 1 to 19, n5 is within the range of 1 to 16, n7 is within the range of 1 to 13, n9 is within the range of 1 to 10, for each R * and R ² is a sum of independently nl + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 is in the interval 12 to 22 for each R ³ is the sum of nl + 2n2 + n3 + 2n4 + n5 + 2n6 + n7 + 2n8 + n9 is in the interval from 0 to 22, each of n2, n4, n6 and n 8 is independently 0 or 1, Y is a peptide comprising an amino acid sequence that is a substrate of a cell-generated peptidase. The conjugate of claim 1, wherein X is a single bond. The conjugate of claim 1, wherein for each R 1 and R ² , at least one of n 2, n 4, n 6 and n ⁸ is independently an integer of 1. 4. The conjugate according to claim 3, characterized in that each R 'and R ² is OC (O) (CH ₂₎ 7 (CH = CH) (CH _{2) 7} CH ₃ The conjugate of claim 1, wherein the peptide comprises the Ala-Ala amino acid sequence. The conjugate of claim 5, wherein the peptide comprises one of the following amino acid sequences Ala-Ala-, Ala-Ala-Pro-Val-, Ala-Ala-Met-, Ala-Ala-Pro-Phe-, Ala. -Ala-Pro-Met-, Ala-Ala-Arg-, Ser-Ala-Ala-Arg-, Ser-Ser-Ala-Ala-Arg-, Ser-S-Carboxy Sugar-Ala-Ala-Arg-, Ala -Ala-Asp-, Ser-Ala-Ala-Asp-, Ser-Ser-Ala-Ala-Asp-. « 4 44 • · « • 4 44 44 4 4 4 4 4 4 4 4 4 4 4 444 4,444 4 · 4 4 4 4 44 44 44 Ί. The conjugate of claim 6, wherein the peptide comprises the Ala-Ala-Pro-Val amino acid sequence. The conjugate of claim 1, wherein the peptide comprises one of the following amino acid sequences Arg-Pro-Lys-Pro-Leu-Ala-Nva-, Ser-Arg-Pro-Lys-Pro-LeuAla-Nva-, Ser Ser-Arg-Pro-Lys-Pro-Leu-Ala-Nva, _{Pro-Cha-Gly-Nva-His-Ala-Dpa-NH2,} Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH ₂ , Pro-Cha-Gly-Nva-, Pro-Leu-Gly-Leu-, Gly-Pro-Arg-, Leu-Pro-Arg-, Glu-Gly-Arg-, Pro-Leu-Gly-Leu- and Gly-Pro-Gln-Gly-Ile. The conjugate of claim 1, wherein the peptide is modified at its amino terminus with one of the groups acetyl, methoxy, carboxy sugar, polyethylene glycol, and methoxy substituted carboxy sugar modifications. 10. The conjugate of claim 9, wherein the amino terminus modification is a methoxysuccinyl group. The conjugate of claim 10, wherein the peptide comprises a modified amino acid sequence of N-methoxy-succinyl-Ala-Ala-Pro-Val. Conjugate according to claim 1, characterized in that it corresponds to the formula: H ₂ C-OC (O) (CH ₂ ) t CH-CH (CH ₂ ) ₇ CH ₃ HC-OC (O) (CH ₂ ) ₇ CH-CH (CH ₂ ) ₇ CH ₃ H ₂ C-OP (O) 2- _{(CH2) 2-NH-Val-Pro-Ala-Ala} 13. A liposome comprising a bilayer of lipids and comprising a peptide-lipid conjugate according to claim 1. 14. The liposome of claim 13 comprising at least 20 mol% of the conjugate relative to the remainder of the lipid components in the liposomes. The liposome of claim 14, wherein the conjugate comprises from about 20 mol% to about 80 mol% of the lipid component. 16. The liposome of claim 15, wherein the conjugate comprises about 50 mol% lipid component. The liposome of claim 14, wherein the additional lipids are positively charged lipids. φφφφ ΦΦ • • φ φ φ φ φ φ φ · · · φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ φ 20, 21. 22nd 23. 24. 25. 26. 27 Mar: Liposome according to claim 17, characterized in that the positively charged lipids are DOTAP, 1Ν, Ν-dimethylaminodioleoyl propane (DODAP), 1-oleoyl-2-hydroxy-3N, N-dimethylamino propane, 1,2-diacyl-3-N , N-dimethylamino propane and 1,2-didecanoyl-1 N, N-dimethylamino propane, DC-Chol, DMRIE and DORI. The liposome of claim 18, wherein the positively charged lipid is DODAP. The liposome of claim 14, wherein the additional lipid is phosphatidylethanolamine. The liposome of claim 14, wherein the phosphatidylethanolamine is transesterified phosphatidylethanolamine (tPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), and DOPE. The liposome of claim 20, wherein the phosphatidylethanolamine is linked via its amino group to a dicarboxylic acid, polyethylene glycol, polyalkyl ether, and ganglioside type. The liposome of claim 13 comprising DODAP and DOPE conjugates with a peptide comprising the amino acid sequence N-methoxy-succinyl-Ala-Ala-Pro-Val. The liposome of claim 23, wherein the lipid component content is 50% DODAP and 50 mol% lipid-peptide conjugate. A composition comprising the liposomes of claim 13. The composition of claim 25, further comprising a pharmaceutically acceptable carrier. A method of delivering liposome content to mammalian cells comprising contacting the cells with the composition of claim 26, wherein the mammalian cell is a peptidase secreting cell and the peptide-lipid conjugate comprises an amino acid sequence recognized by a secreted peptidase. The method of claim 27, wherein the liposome comprises a bioactive agent such as antiviral agents, antibacterial agents, antifungal agents, antineoplastic agents, anti-inflammatory agents, radio labels, contrast agents, fluorescent labels, pupil disseminators, bronchodilators, local anesthetics, nucleic acid sequences acids and bioactive lipids. 28. 9999 • • 9 99 99 99 9 99 9 9 99 9 9,999,999 9,999,999 9 9 9 9 9 999 99 99 ·· 99 99 ₃₁ ΐΜΜή The method of claim 27, wherein the liposome is a large unilamellar liposome. The method of claim 27, wherein the peptide-lipid conjugate comprises an amino acid sequence recognized by a peptidase belonging to the group of intercellular mass metalloproteases, serine proteases, or cysteine proteases. 31. The method of claim 27, wherein the peptide-lipid conjugate comprises an amino acid sequence recognized by a peptidase such as elastase, plasmin, plasminogen activator, stromelysin, human collagenases, cathepsins, lysozyme, granzymes, dipeptidyl peptidases, enzyme inactivating peptide hormones, kinases , bacterial peptidases and viral proteases. 32. The method of claim 31, wherein the peptidase is an elastase. 33. The method of claim 31, wherein the peptidase is stromelysin. 34. The method of claim 31, wherein the peptidase is cathepsin. 35. The method of claim 31, wherein the peptidase is plasmin or plasminogen activator. 36. The method of claim 27, wherein the composition comprises liposomes composed of lipid components of DODAP and N-Ala-Ala-Pro-Val-DOPE. The method of claim 36, wherein the lipid components are 50 mol% DODAP and 50 mol% N-Ala-Ala-Pro-Val. 38. The method of claim 27, wherein the mammal is suffering from cancer and the liposome comprises a therapeutically effective amount of an anticancer drug. 39. The method of claim 28, wherein the mammal is suffering from an inflammatory disorder and the carrier particle comprises a therapeutically effective amount of an anti-inflammatory drug. 40. The method of claim 28, wherein the mammal is suffering from a genetic disorder and the carrier particle comprises a nucleic acid encoding a protein capable of correcting the disorder.