WO2006086992A2 - Drug delivery systems containing phqspholipase a2 degradable lipid prodrug derivatives and the therapeutic uses thereof as. e.g. wound healing agents and peroxisome proliferator activated receptor ligands - Google Patents

Drug delivery systems containing phqspholipase a2 degradable lipid prodrug derivatives and the therapeutic uses thereof as. e.g. wound healing agents and peroxisome proliferator activated receptor ligands Download PDF

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WO2006086992A2
WO2006086992A2 PCT/DK2006/000097 DK2006000097W WO2006086992A2 WO 2006086992 A2 WO2006086992 A2 WO 2006086992A2 DK 2006000097 W DK2006000097 W DK 2006000097W WO 2006086992 A2 WO2006086992 A2 WO 2006086992A2
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lipid
drug delivery
delivery system
acid
liposomes
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PCT/DK2006/000097
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French (fr)
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WO2006086992A3 (en
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Kent JØRGENSEN
S. Jensen Simon
Thomas L. Andresen
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Liplasome Pharma A/S
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/555—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the lipid based prodrug and drug delivery systems are useful for local as well as sys- temic treatment of inflammatory conditions or damaged tissue, wounds, injuries, and burns that are associated with increased levels of extracellular phospholipase A 2 (PLA 2 ).
  • the liposomal prodrug and drug carries can be used in combination with incorporated drugs and a wound coverage material.
  • Lysophosphatidic acid (LPA) bioactive lipids are known to be potent biological mediators with growth stimulating and wound healing activities (7).
  • LPA has been shown to bind to specific G protein-coupled receptors (Edg-receptors) (7,8) thereby eliciting a biological response in mammalian cells including cell proliferation (9), wound healing (10), cell survival (11 ,12,13), accelerated development of embryos (14), preservation of organs (15), monocyte migration (16), and growth stimulating activity in salvia (17).
  • Lysophosphatidic acid is an endogeneous soluble glycerophospholipid in the body that shows similar biological activity as the cell growth factor.
  • LPA 7-transmembrane Edg-receptors
  • Edg-receptors for example LPA1 , LPA2, LPA3
  • Various new LPA analogs that are peroxisome proliferator activated receptor ligands have been synthesized to improve healing and treat or prevent cancer or diabetes (described in WO200492188-A2).
  • LPA also displays activity as a ligand for inhibiting neutrophil accumulation in tissues for treating or preventing diseases or disorders associated with excessive accumulation of neutrophils in e.g. inflammatory diseases (described in WO2004052375-A1 ).
  • the present invention describes a lipid based prodrug and drug delivery system that can be used for treatment or alleviation of disorders that are associated with or result- ing from increased levels of secretory phospholipase A 2 (PLA 2 ) in the diseased or injured tissue (1 ,2,3,4,5,6).
  • the lipid based delivery systems are useful for administration of lipid prodrugs of lysophosphatidic acid (LPA) bioactive analogs, which are known to be potent biological mediators with e.g. growth stimulating and wound healing activities (7).
  • LPA lysophosphatidic acid
  • the present invention is directed to drug delivery systems, which are particularly useful in the treatment or alleviation of diseases that are characterised by localised activity of extracelluar PLA 2 activity.
  • lipid-based prodrugs as illustrated in Figs. 1 and 2 for systemic or local administration of prodrugs of LPA bioactive analogs that can be activated to LPA active agents at the desired target site.
  • Specific lipid-analogue compounds may be in- corporated into the lipid based carrier, which furthermore can have polymer or polysaccharide chains "grafted" to the surface and act as a prodrug particulate carrier that is turned into active drugs by extracellular phospholipase A2 hydrolysis.
  • Possible examples include certain lysophosphatidic acid analogs that are peroxisome proliferator activated receptor ligands useful as, e.g. wound healing agents and for treatment or pre- vention of cancer or diabetes (WO200492188-A2).
  • the bioactive LPA lipids can be generated through the action of secretory PLA 2 on lipid substrates containing phosphatidic acid lipids (7,19). Degradation of the liposomal prodrug carriers by PLA 2 leads to release of acyl and/or alkyl LPAs and fatty acid de- rivatives as shown in Fig. 3, which are designed to be, e.g. effective growth promoting agents.
  • the liposomal carriers can advantageously be used for targeted transport and delivery of conventional wound healing agents as well as novel biological growth promoting substances like peptides, proteins, and DNA ana- logues that are encapsulated in the interior of the prodrug liposomes.
  • novel principle behind the site-specific activation and delivery of growth promoting agents to diseased tissue such as wounds and bums is illustrated in Figs. 1 and 2.
  • the liposomal prodrug and drug carries can furthermore be used in combination with a wound coverage material (20)
  • the PLA 2 -triggered hydrolysis of the liposomal prodrug carries leads to a site-specific activation and conversion of the phosphatidic acid prodrug lipids (acyl/alkyl proLPA) to potent growth promoting lysophosphatidic acids (acyl/alkyl LPA) as illustrated in Fig. 3.
  • the substituent R may constitute an auxiliary drug substance or an efficiency modifier (e.g. permeability enhancer or second growth factor) and will simultaneously be released under the action of PLA 2 .
  • the prodrug lipids form stable liposomal membrane structures, which function as favorable substrates for PLA 2 catalyzed lipid hydrolysis.
  • a particular advantage of using liposomal prodrug carriers for targeted delivery of growth promoting agents involves a sig- nificantly increased activity of PLA 2 towards organized lipid substrates such as lipid membranes as compared to monomeric lipid substrates. This leads to a fast and efficient degradation of the prodrug liposomes resulting in a site-specific conversion of the phosphatidic acid lipid analogs to active LPA acting compounds, e.g.
  • lipid derivatives are constituents of the carrier liposome and act as prodrugs, which are turned into active drugs (e.g. ether lipids and fatty acid drug derivatives) by hydrolysis via the extracellular PLA 2 that is present at elevated concentrations in the diseased target tissue.
  • active drugs e.g. ether lipids and fatty acid drug derivatives
  • the prodrug lipids can be modified with a long fatty-acid chain that is ester linked in the C-2 or C-3 position and a head group that is linked in the C-3 or C-2 position, respectively, and therefore can be hydrolysed by extracellular PLA 2 at the target site.
  • a specific example is a lipid prodrug of a certain mono-ether lysolipid, which is bioactive LPA analog.
  • This can be a therapeutically active compound having, e.g. a regulatory fatty acid derivative ester bound to the phospholipid in, e.g. the C-3 or C-2 position and a head group that is linked in the C-2 or C-3 position, respectively, and therefore renders the lipid derivative substrate for extracellular PLA 2 .
  • the lipids are modified with, e.g. a ester-linked derivative in the C-3 or C-2 position and therefore can be hydrolysed by extracellular PLA 2 at the target site, these lipid derivatives constituting the carrier liposome will act as prodrugs that become hydrolysed and turned into drugs by extracellular PLA 2 at the target site.
  • the therapeutically active substances e.g., monoether lipids and ester-linked drug derivatives will be liberated at the desired target site.
  • the hydrolysis product can act as local permeability enhancers facilitating the transport of the released drugs into the cell.
  • Pharmaceutical compositions containing the lipid-based system can be used therapeutically, for example, in the treatment of cancer, infectious and inflam- matory conditions.
  • lipid-based carriers e.g. liposomes or micelles, composed of novel unnatural lipid-bilayer forming lipids such as glycerophospholipids containing an alkyl-linkage or acyl-linkage in the 1 -position and an acyl-linkage or another PLA2 degradable bond, e.g. a bioisoster of a PLA2 hydroly- sable ester in the C-2 or C-3 position on the glycerol backbone and which have polymer or polysaccharide chains grafted thereto in the C-2 or C-3 position.
  • the carrier system may contain lipid-bilayer stabilising components, e.g.
  • lipopolymers gly- colipids and sterols, which lead to an increased vascular circulation time and as a con- sequence an accumulation in the diseased target tissue.
  • PLA 2 -catalyzed hydrolysis of the acyl-linkage releases the therapeutically active components, typically Iy- solipids and ester-linked derivatives.
  • the present invention thus provides a lipid-based drug delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a lipid derivative having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysoiipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
  • the present invention also provides a lipid based drug delivery system for administration of an second drug substance, wherein the second drug substance is incorporated in the system, said system including lipid derivatives which has (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydro- lytically cleaved off, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
  • lipid derivatives which has (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extra
  • the present invention takes advantage of the surprising finding that liposomes (and micelles) including lipid derivatives which can be specifically and only partially cleaved by extracellular phospholipases, and which at the same time includes lipopolymers or glycolipids, have the properties of circulating in the blood stream suffi- ciently long so as to reach target tissue where the extracellular PLA 2 activity is elevated without being recognised by the mammalian reticuloendothelial systems and without penetrating cell walls, whereby the lipid derivatives of the liposomes are specifically cleaved by extracellular PLA 2 so as to liberate therapeutically active ingredients at the desired location.
  • the present invention provides a class of novel lipid derivatives which are particularly useful as constituents of the drug delivery systems described herein.
  • Fig. 1 Schematic illustration of the lipid-based (pro)drug-targeting and (pro)drug- trigering principle involving accumulation of the liposomal (pro)drug carriers in porous diseased tissue and subsequent release and activation of (pro)drugs and transport across the target membrane via extracellular PLA 2 activity.
  • V Drugs (lysolipid and fatty acid derivatives), enhancers (lysolipid + fatty acid), PLA 2 activators (lysolipid + fatty acid)
  • Fig. 2 Local administration (e.g. dermal application) of the targeted liposomal delivery of growth stimulating lysophosphatidic acids (LPA), fatty acid drug derivatives and en- capsulated growth promoting hormones, peptides and proteins to diseased tissue (i.e. wounds, burns, and injuries) using phospholipase A 2 (PLA 2 ) as a site-specific trigger.
  • LPA growth stimulating lysophosphatidic acids
  • PDA 2 phospholipase A 2
  • Phospholipase A 2 generation of lysophosphatidic acids (acyl/alkyl LPA) and fatty acid drug derivatives (RCOOH) from phosphatidic acid prodrug lipids (acyl/alkyl proLPA).
  • Fig. 4 Stimulation of cell proliferation by LPA treatment.
  • LPA was added at indicated concentrations to NIH 3T3 fibroblasts (mouse immortal cell line) and HUVEC cells (human umbilical cord endothelial cells-normal endothelial cells) for 24 h, in media con- taining fatty acid free serum albumin (0,1 %), and no serum. Proliferation was determined by incorporation of radioactive thymidine.
  • Fig. 5 Release of LPA from proLPA liposomes by secretory PLA2 stimulates HUVEC cell proliferation.
  • Media from Colo 205 cells was conditioned for 24 h, and mixed 1 :1 with normal growth media for HUVEC cells.
  • Fatty acid free BSA was added to the system also, to allow LPA to work in an growth stimulating manner. After 22 h incubation, cells were pulse-labelled with radioactive thymidin.
  • Lyso- phosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta. Am. J. Physiol. 267:C204-210.
  • Lysophosphatidic acid a novel growth and survival factor for renal proximal tubular cells. Am. J. Physiol. 273:F575-585.
  • Lysophosphatidic acid is a major serum noncytokine survival factor for murine macrophages which acts via the phosphatidylinositol 3-kinase signaling pathway. J. Clin. Invest. 102:716-727.
  • Lysophosphatidic acid prevents apoptosis in fibroblasts via Gi-protein- mediated activation of mitogen-activated protein kinase. Biochem. J. 352:135-143. 19. Snitko, Y. E. T. Yoon, and W. Cho. 1997. High specificity of human secretory class Il phospholi- pase A 2 for phosphatidic acid. Biochem. J. 321:737-741.
  • extracellular PLA 2 is capable of cleaving lipid derivatives so as to produce LPA lysolipid derivatives, e.g. lipid produgs of LPA analogs which as such, or in combination with other active compounds, will exhibit a therapeutic effect.
  • the drug delivery systems (liposomes or micelles) of the present invention relies on lipid derivatives having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
  • lipid and “lysolipid” (in the context of phospholipids) will be well- known terms for the person skilled in the art, it should be emphasised that, within the present description and claims, the term “lipid” is intended to mean Westers of glycerol of the following formula:
  • R A and R B are linked to the glycerol backbone via ester bonds.
  • lysolipid is intended to mean a lipid where the R B fatty acid group is absent (e.g. hydrolytically cleaved off), i.e. a glycerol derivative of the formula above where R B is hydrogen, but where the other substituents are substantially unaffected. Conversion of a lipid to a lysolipid can take place under the action of an enzyme, specifically under the action of celluar as well as extracellular PLA 2 .
  • the terms “lipid derivative” and “lysolipid derivative” are intended to cover possible derivatives of the above possible compounds within the groups “lipid” and “lysolipid”, respectively. Examples of biologically active lipid derivatives and lysolipid derivatives are given in WO200492188-A2 and WO2004052375-A1. Thus, as will be evident, the ex- tension "derivative" should be understood in the broadest sense.
  • lipid derivatives and lysolipids should however fulfil certain functional criteria (see above) and/or structural requirements. It is particularly relevant to note that the suitable lipid derivatives are those which have (a) an aliphatic group of a length of at least 3, preferably at least 9, carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety. It will be evident that the aliphatic group and the organic radical will correspond to the two fatty acid moieties in a normal lipid and that the hydrophilic moiety will correspond to the phosphate part of a (phospho)lipid or a bioisoster thereof.
  • the lipid derivatives which can be utilised within the present invention should be substrates for extracellular PLA 2 , i.e. the lipid derivatives should be able to undergo hydrolytic, enzymatic cleavage of the organic radical corresponding to a fatty acid in the 2- or 3-position in a lipid.
  • Extracellular PLA 2 is known to belong to the enzyme class (EC) 3.1.1.4. Thus by reference to (extracellular) PLA 2 should be understood all extracellular enzymes of this class, e.g.
  • lipases which can induce hydrolytic cleavage of the organic radical corresponding to the fatty acid in the 2- or 3-position in a lipid.
  • One particular advantage of the lipid based drug delivery system is that extracellular PLA 2 activity is significantly increased towards organised substrates as compared to monomeric substrates.
  • the or- ganic radical e.g. aliphatic group
  • an ester functionality e.g. a bioisoster which can be cleaved by extracellular PLA 2 , preferably so that the group which is cleaved off is a carboxylic acid or a bioisoster therof.
  • the aliphatic group (the group corresponding to the fatty acid in the 1 -position in a lipid) of the lipid derivative i.e. the lysolipid derivative after cleavage by extracellular PLA 2
  • substantially unaffected is meant that the integrity of the aliphatic group is preserved and that less than 1 mol%, preferably less than 0.1 mol%, of the aliphatic group (the aliphatic group in the 1 -position) is cleaved under the action of extracellular PLA 2 .
  • lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
  • X and Z independently are selected from OC(O), O, CH 2 , NH, NMe, S, S(O), OS(O), S(O) 2 , OS(O) 2 , OP(O) 2 , OP(O) 2 O, OAs(O) 2 and OAs(O) 2 O; preferably from O, NH, NMe and CH 2 , in particular O and CH 2 ;
  • Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH 2 C(O)O, NC(O)O, Y then being connected to R 2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom, preferably via the carbonyl carbon atom;
  • R 1 is an aliphatic group of the formula Y 1 Y 2 ;
  • R 2 is an organic radical having at least 2 carbon atoms, such as an aliphatic group having a length of at least 2, preferably at least 9, carbon atoms, preferably a group of the formula Y 1 Y 2 ;
  • R 3 is selected from phosphatidic acid (PO 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid, e.g. P(O)O, P(O) 2 CH 2 , S(O)O, S(O)CH 2 , C(O)O, C(O)N, C(S)O, P(S)O 2 , S(O) 2 CH 2 and derivatives thereof (among others phosphatidic acid derivatives to which a hydrophilic polymer or polysaccharide is covalently attached).
  • P(O)O, P(O) 2 CH 2 S(O)O, S(O)CH 2 , C(O)O, C(O)N, C(S)O, P(S)O 2 , S(O) 2 CH 2 and derivatives thereof (among others phosphatidic acid derivatives to which a hydrophilic polymer or polysaccharide is covalently attached).
  • Y is -OC(O)- where Y is con- nected to R 2 via the carboxyl atom.
  • X and Z are O and that Y is -OC(O)- where Y is connected to R 2 via the carboxyl atom.
  • the lipid derivative is a 1-monoether-2-monoester-phospholipid type compound.
  • Another preferred group of lipid derivatives is the one where the group X is S.
  • n1 is equal to zero or is an integer of from 1 to 23; n3 is equal to zero or is an integer of from 1 to 20; n5 is equal to zero or is an integer of from 1 to 17; n7 is equal to zero or is an integer of from 1 to 14; n9 is equal to zero or is an integer of from 1 to 11 ; and each of n2, n4, n6 and 8 is independently equal to zero or 1.
  • the aliphatic groups may be unsaturated and even substituted with halogens (flouro, chloro, bromo, iodo) and C-i.-io-groups (i.e. yielding branched aliphatic groups), the aliphatic groups as R 1 and R 2 are in one embodiment preferably saturated as well as unbranched, that is, they preferably have no double bonds between adjacent carbon atoms, each of n2, n4, n6 and n8 then being equal to zero. Accordingly, Y 1 is preferably (CH 2 ) n i.
  • R 1 and R 2 are each independently (CH 2 ) n1 CH 3 , and most preferably, (CH 2 )i 7 CH 3 or (CH 2 ) 15 CH 3 .
  • the groups can have one or more double bonds, that is, they can be unsatu- rated, and one or more of n2, n4, n6 and n8 can be equal to 1.
  • n2 is equal to 1
  • n1 is equal to zero or is an integer of from 1 to 21
  • n3 is also zero or is an integer of from 1 to 20, at least one of n1 or n3 not being equal to zero.
  • the lipid derivatives are those which are monoether lipids where X and Z are O, R 1 and R 2 are independently selected from alkyl groups, (CHz) n CH 3 , where n is 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, preferably 14, 15 or 16, in particular 14; Y is -OC(O)-, Y then being connected to R 2 via the carbonyl carbon atom.
  • hydrophilic moiety (often known as the "head group") which corresponds to R 3 , it is believed that a wide variety of groups corresponding to phosphatide acid (PO 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof can be used.
  • Bioisosters to phosphatidic acid and derivatives thereof indeed implies that such groups - as phosphatidic acid - should allow for enzymatic cleavage by extracellular PLA 2 .
  • R 3 can be selected from phosphatidic acid (PO 2 -OH), phosphatidylcholine (PO 2 -O- CH 2 CH 2 N(CH 3 ) 3 ), phosphatidylethanolamine (PO 2 -O-CH 2 CH 2 NH 2 ), N-methyl- phosphatidylethanolamine (PO 2 -O-CH 2 CH 2 NCH 2 ), , phosphatidylserine, phosphatidy- linositol, and phosphatidylglycerol (PO 2 -O-CH 2 CHOHCH 2 OH).
  • phosphatidic acid examples include those where dicarboxylic acids, such as glutaric, sebacic, succinic and tartaric acids, are coupled to the terminal nitrogen of phosphatidyl- ethanolamines, phosphatidylserine, phosphatidylinositol, etc.
  • dicarboxylic acids such as glutaric, sebacic, succinic and tartaric acids
  • a hydrophilic polymer or polysaccharide is typically covalently at- tached to the phosphatidyl part of the lipid derivative.
  • Another particular lipid derivative include an acyl chain attached to the head group of the lipids,
  • Hydrophilic polymers which suitable can be incorporated in the lipid derivatives of the invention so as to form lipopolymers are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e. are biocompatible).
  • Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polygly- colide), a polylactic-polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polyme- thacrylamide, polydimethylacrylamide, and derivatised celluloses such as hydroxy- methylcellulose or hydroxyethylcellulose.
  • PEG polyethylene glycol
  • polylactic also termed polylactide
  • polyglycolic acid also termed polygly- colide
  • a polylactic-polyglycolic acid copolymer polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polyme- thacrylamide, polydimethylacrylamide, and deriva
  • Preferred polymers are those having a molecular weight of from about 100 daltons up to about 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons.
  • the polymer is polyethyleneglycol hav- ing a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons.
  • the polymer is polyethyleneglycol of 750 daltons (PEG(750)).
  • Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilises polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
  • the glycolipid or lipopolymer is represented by a fraction of the lipid derivative
  • a lipid derivative lipid derivative with a polymer or polysaccharide chain
  • the fraction may be even higher, such as from 1-100 mol%, such as 10-100 mol%, of the total dehydrated lipid-based system.
  • Preferred polymers to be covalently linked to the phosphatidyl part are polyethylene glycol (PEG), polyactide, polyglycolic acid, polyactide-polyglycolic acid copolymer, and polyvinyl alcohol.
  • R 2 should be an organic radical having at least 2 carbon atoms) (such as an aliphatic group having a certain length (at least 2, preferably 9, carbon atoms)), a high degree of variability is possible, e.g. R 2 need not necessarily to be a long chain residue, but may represent more complex structures.
  • R 2 may either be rather inert for the environment in which it can be liberated by extracellular PLA 2 or that R 2 may play an active pharmaceutical role, typically as an auxiliary drug substance or as an efficiency modifier for the lysol- ipid derivative and/or any other (second) drug substances present in the environment.
  • the R 1 and R 2 groups will be long chain residues, e.g. a fatty acid residue (the fatty acid will include a carbonyl from the group Y).
  • auxiliary drug substances as R 2 within this subgroups are polyunsaturated acids, e.g. oleate, linoleic, linonleic, as well as derivatives of arachidonoyl (including the carbonyl from Y), e.g. prostaglandins such as prostaglandin E 1 , as arachidonic acid derivatives are know regulators of hormone ac- tion including the action of prostaglandins, thromboxanes, and leukotrines.
  • efficiency modifiers as R 2 are those which enhance the permeability of the target cell membrane as well as enhances the activity of extracellular PLA 2 or the active drug substance or any second drug substances. Examples hereof are short chain (C 8-12 ) fatty acids.
  • organic radical R 2 e.g. vitamin D derivatives, steroid derivatives, retinoic acid (including all-trans- retinoic acid, all-cis-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid), cholecalciferol and tocopherol analogues, pharmacologically active carboxylic acids such as branched-chain aliphatic carboxylic acids (e.g. valproic acid and those described in WO 99/02485), salicylic acids (e.g. acetylsalicylic acid), steroidal carboxylic acids (e.g.
  • lysergic and isolysergic acids include monoheterocyclic carboxylic acids (e.g. nicotinic acid) and polyheterocyclic carboxylic acids (e.g. penicillins and cephalosporins), diclofenac, indomethacin, ibuprofen, naproxen, 6-methoxy-2-naphthylacetic acid.
  • R 2 groups are referred to by the name of a discrete species, rather than the name of the radical.
  • possible examples may include the carbonyl group or oxy group of the bond via which the organic radical is linked to the lipid skeleton (corre- sponding to "Y" in the formula above). This will of course be appreciated by the person skilled in the art.
  • lipid derivatives may be substituted, e.g. in order to modify the cleavage rate by extracellular PLA 2 or simply in order to modify the properties of the liposomes comprising the lipid derivatives.
  • a particular group of novel compounds is lipid derivatives of the following formula:
  • X and Z independently are selected from OC(O), O, CH 2 , NH, NMe, S, S(O), OS(O), S(O) 2 , OS(O) 2 , OP(O) 2 , OP(O) 2 O, OAs(O) 2 and OAs(O) 2 O; preferably from O, NH, NMe and CH 2 , in particular O and CH 2 ;
  • Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH 2 C(O)O, NC(O)O, Y then being connected to R 2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom, preferably via the carbonyl carbon atom;
  • R 1 is an aliphatic group of the formula Y 1 Y 2 ;
  • R 2 is an organic radical having at least 2 carbon atoms
  • the hydrophilic polymer or polysaccharide is typically and preferably selected from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polyme- thacrylamide, polydimethylacrylamide, and derivatised celluloses, in particular from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, and polyvinyl alcohol.
  • a particular subgroups are those wherein X and Z are O, R 1 and R 2 are independently selected from alkyl groups, (CH 2 ) n CH 3 , where n is 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, preferably 14 or 16; Y is -OC(O)-, Y then being connected to R 2 via the carbonyl carbon atom.
  • a specific group of compounds are polyethyleneoxide-i-O-palmityl-sn-2-palmitoyl- phosphatidyl ethanolamine, DPPE-PEG, and polyethyleneoxide-1-0-stearyl-s/>2- stearoylphosphatidylethanolamine, DSPE-PEG, with PEG molecular weight from 100 to 10000 Daltons, in particular from 300-5000 Daltons.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid derivative as defined above.
  • the lipid derivative in such a composition is dispersed in the form of a liposome (see below).
  • the present invention relates to such lipid derivatives for use as a medicament, preferably present in a pharmaceutical composition, and to the use of a lipid derivative as defined above for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
  • diseases or conditions are typically selected from wounds and inflammatory conditions.
  • the present compositions and uses are especially applicable in the instances where the increase in extracellular PLA 2 activity is at least 25% compared to the normal level of activity in the tissue in question, the tissue being that of a mammal, in particular a human.
  • Lipid derivatives as prodrugs are especially applicable in the instances where the increase in extracellular PLA 2 activity is at least 25% compared to the normal level of activity in the tissue in question, the tissue being that of a mammal, in particular a human.
  • the present invention provides a lipid-based drug delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a lipid derivative having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
  • active drug substance any chemical entity which will provide a prophylactic or therapeutic effect in the body of a mammal, in particular a human.
  • the present invention mainly relates to the therapeutic field.
  • prodrug should be understood in the normal sense, namely as a drug which is masked or protected with the purpose of being converted (typically by cleavage, but also by in vivo chemical conversion) to the intended drug substance.
  • prodrug typically by cleavage, but also by in vivo chemical conversion
  • the active drug substance is selected from lysolipid derivatives, and as it will be understood from the present description with claims, the lysolipid derivatives relevant within the present invention will have a therapeutic effect - at least - in connection with diseases and conditions where a local area of the body of the mammal has a level of extracellular PLA 2 activity which can liberate the lysolipid derivative.
  • the lipid derivative will often constitute the prodrug referred to above and the lysolipid derivative will thereby constitute the active drug substance often a monoether lysolipid derivative. It should however be understood that this does not exclude the possibility of including other drug substances, referred to as second drug substances, in the drug delivery systems of the invention, neither does it exclude that the organic radical which can be hydrolytically cleaved by the action of extracellular PLA 2 can have a certain pharmaceutical effect
  • the pharmaceutical effect of the "active drug substance”, i.e. the lysolipid derivative, need not be the most predominant when a second drug substance is included, actually the effect of the second drug substance might very well be the most predominant as will become apparent in the other main embodiment (see “Lipid derivative liposomes as drug delivery systems", below).
  • the active drug substance (lysolipid derivative) release from the prodrug (lipid derivative) is believed to take place as illustrated in the following examples:
  • the substituent R 2 may constitute an auxiliary drug substance or an efficiency modifier for the active drug substance and will simultaneously be released under the action of extracellular PLA 2 :
  • the substituent R 2 may constitute an auxiliary drug substance or an efficiency modifier for the active drug substance and will simultaneously be released under the action of extracellular PLA 2 :
  • R 2 It has been described above under the definition of R 2 how the group R 2 can have various independent or synergistic effects in association with the active drug substance, e.g. as an auxiliary drug substance or an efficiency modifier, e.g. permeability or cell lysis modifier. It should be borne in mind that the groups corresponding to R 2 (e.g. R 2 - OH or R 2 -COOH) might have a pharmaceutical effect which is predominant in relation the effect of the lysolipid derivative (active drug substance).
  • Lipid derivatives formulated as liposomes and micelles
  • lipid-based drug delivery system should encompass macromolecular struc- tures which as the main constituent include lipid or lipid derivatives. Suitable examples hereof are liposomes and micelles. It is presently believed that liposomes offer the broadest scope of applications and those have been described most detailed in the following. Although liposomes currently are believed to be the preferred lipid-based system, micellular systems are also believed to offer interesting embodiments within the present invention. In one important variant which advantageously can be combined with the embodiments described herein, the lipid derivative (e.g. the prodrug) is included in liposomes either as the only constituent or - which is more common - in combination with other constituents (other lipids, sterols, etc.). Thus, the lipid-based systems described herein are preferably in the form of liposomes, wherein the liposomes are build up of layers comprising the lipid derivative (e.g. a prodrug).
  • the lipid derivative e.g. a prodrug
  • “Liposomes” are known as self-assembling structures comprising one or more lipid bilayers, each of which surrounds an aqueous compartment and comprises two opposing monolayers of amphipathic lipid molecules.
  • Amphipathic lipids i.e. lipid derivatives
  • lipid derivatives comprise a polar (hydrophilic) headgroup region (corresponding to the substituent R 3 in the lipid derivatives) covalently linked to one or two non-polar (hydrophobic) aliphatic groups (corresponding to R 1 and R 2 in the lipid derivatives).
  • Energetically unfavourable contacts between the hydrophobic groups and the aqueous medium are generally believed to induce lipid molecules to rearrange such that the polar headgroups are oriented towards the aqueous medium while the hydrophobic groups reorient towards the interior of the bilayer.
  • An energetically stable structure is formed in which the hydrophobic groups are effectively shielded from coming into contact with the aqueous medium.
  • Liposomes can have a single lipid bilayer (unilamellar liposomes, "ULVs”), or multiple lipid bilayers (multilamellar liposomes, "MLVs”), and can be made by a variety of methods (for a review, see, for example, Deamer and Uster, Liposomes, Marcel Dekker, N.Y., 1983, 27-52).
  • UUVs unilamellar liposomes
  • MLVs multilamellar liposomes
  • MLVs multilamel- lar liposomes
  • Lenk's, Fountain's and Cullis' methods for making MLVs with substantially equal interlamellar solute distribution
  • ULVs can be produced from MLVs by such methods as sonication (see Pa- pahadjopoulos et al., Biochem. Biophys. Acta, 135, 624 (1968)) or extrusion (US 5,008,050 and US 5,059,421).
  • the liposome of this invention can be produced by the methods of any of these disclosures, the contents of which are incorporated herein by reference.
  • Liposome sizes can also be determined by a number of techniques, such as quasi-electric light scattering, and with equipment, e.g., Nicomp ® particle sizers, well within the possession of ordinarily skilled artisans.
  • the lipid derivatives of the present invention can constitute the major part of a lipid-based system even if this system is a liposome system. This fact resides in the structural (but not functional) similarity between the lipid derivatives of the present invention and lipids.
  • the lipid derivatives for the present invention can be the sole constituent of liposomes, i.e. up to 100 mol% of the total dehydrated liposomes can be constituted by the lipid derivatives. This is in contrast to the known lysolipids, which can only constitute a minor part of the liposomes.
  • liposomes advantageously include other constituents which may or may not have a pharmaceutical effect, but which will render the liposome structure more stable (or alternatively more unstable) or will protect the liposomes against clearance and will thereby increase the circulation time thereby improving the overall efficiency of a pharmaceutical including the liposome.
  • the particular lipid derivatives will typically constitute from 5-100 mol%, such as 50-100 mol%, preferably from 75-100 mol%, in particular 90-100 mol%, based on the total dehydrated liposome.
  • the liposomes can be unilamellar or multilamellar. Some preferred liposomes are unilamellar and have diameters of less than about 400 nm, more preferably, from greater than about 40 nm to less than about 400 nm.
  • the liposomes are typically - as known in the art - prepared by a method comprising the steps of: (a) dissolving the lipid derivative in an organic solvent; (b) removing the organic solvent from the lipid derivative solution of step (a); and (c) hydrating the product of step (b) with an aqueous solvent so as to form liposomes.
  • the method may further comprise a step of adding an second drug substance (see be- low) to the organic solvent of step (a) or the aqueous phase of step (c).
  • the method may comprise a step of extruding the liposomes produced in step (c) through a filter to produce liposomes of a certain size, e.g. 100 nm.
  • Lipid-based particulate systems i.e. liposomes as well as micelles; of sizes covering a broad range may be prepared according to the above-mentioned techniques.
  • suitable sizes for pharmaceutical applications will normally be in the range of 20-10,000 nm, in particular in the range of 30-1000 nm. Sizes in the range of 50-200 nm are normally preferred because liposomes in this size range are generally believed to circulate longer in the vascular system of mammals than do larger liposomes which are more quickly recognised by the mammals' reticuloendothelial systems ("RES"), and hence, more quickly cleared from the circulation.
  • RES reticuloendothelial systems
  • Longer vascular circulation can enhance therapeutic efficacy by allowing more liposomes to reach their intended site of actions, e.g., tumours or inflammations.
  • the liposomes should preferably have a mean particle size of about 100 nm.
  • the particle size should generally be in the range of 40-400 nm.
  • the liposomes should preferably have a mean particle size from 100 to 5000 nm, and the liposomes can then be uni- or multilayered.
  • the liposome structure in particular when stabilised as described in the following, will have a much longer vascular circulation time that the lipid derivatives as discrete compounds. Furthermore, the lipid derivatives will become more or less inert or even “invisible” when “packed” in liposomes in which lipopolymers or glycolipids are included. This means than any potential disadvantageous effect, e.g. hemolytic effect, can be suppressed.
  • the liposomes should preferably act as inert constituents until they reach the area of interest, e.g. cancerous, infected or inflammatorily diseased areas or tissue. As will be described in the following, liposomes may include a number of other constituents.
  • a drug delivery system according to the invention may further contain a component which controls the release of any second drug substance, extracellular PLA 2 activity controlling agents or permeability enhancer, e.g. short chain lipids and lipopolymers/glycolipids.
  • extracellular PLA 2 activity controlling agents or permeability enhancer e.g. short chain lipids and lipopolymers/glycolipids.
  • lipopolymers and glycolipids such as lipopolymers (e.g. poly- ethyleneoxide-dipalmitoylphosphatidyl ethanolamine, DPPE-PEG, polyethyleneoxide- distearoylphosphatidylethanolamine, DSPE-PEG) with PEG molecular weight from 100 to 10000 Daltons.
  • lipopolymers e.g. poly- ethyleneoxide-dipalmitoylphosphatidyl ethanolamine, DPPE-PEG, polyethyleneoxide- distearoylphosphatidylethanolamine, DSPE-PEG
  • PEG molecular weight from 100 to 10000 Daltons.
  • Liposome outer surfaces are believed to become coated with serum proteins, such as opsonins, in mammals' circulatory systems. Without intending in any way to be limited by any particular theory, it is believed that liposome clearance can be inhibited by modifying the outer surface of liposomes such that binding of serum proteins thereto is generally inhibited.
  • Effective surface modification that is, alterations to the outer surfaces of liposomes which result in inhibition of opsonisation and RES uptake, is believed to be accomplished by incorporating into liposomal bilayers lipids whose polar headgroups have been derivatised by attachment thereto of a chemical moiety which can inhibit the binding of serum proteins to liposomes such that the pharmacokinetic behaviour of the liposomes in the circulatory systems of mammals is altered and the activity of extracellular PLA 2 is enhanced as described for the lipopolymers above.
  • Liposome preparations have been devised which avoid rapid RES uptake and which thus have an increased half-life in the bloodstream.
  • STEALTH ® liposomes (Liposome Technology Inc., Menlo Park, Calif.) include polyethyleneglycol (PEG)-grafted lipids at about 5 mol% of the total dehydrated liposome .
  • PEG polyethyleneglycol
  • the presence of polymers on the exterior liposome surface decreases the uptake of liposomes by the organs of the RES.
  • the liposome membranes can be constructed so as to resist the disruptive effects of the surfactant contained therein.
  • a liposome membrane which contains as constituents lipids derivatised with a hydrophilic (i.e., water-soluble) polymer normally has increased stability.
  • the polymer component of the lipid bilayer protects the liposome from uptake by the RES, and thus the circulation time of the liposomes in the bloodstream is extended.
  • Hydrophilic polymers suitable for use in lipopolymers are those which are readily water- soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible).
  • Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol.
  • PEG polyethylene glycol
  • polylactic also termed polylactide
  • polyglycolic acid also termed polyglycolide
  • a polylactic-polyglycolic acid copolymer and polyvinyl alcohol.
  • Preferred polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons.
  • the polymer is polyethylenegly- col having a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons.
  • the polymer is polyethyleneglycol of 750 daltons (PEG(750)).
  • Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilises polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
  • hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhy- droxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatised celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • Glycolipids are lipids to which a hydrophilic polysaccharide chain is covalently attached. It will be appreciated that glycolipids can be utilised like lipopolymers although the lipopolymers currently presents the most promising results.
  • the content of lipopolymer advantageously will be in the range of 1-50 mol%, such as 2-25%, in particular 2-15 mol%, based on the total dehydrated liposome.
  • the liposomes' bi- or multilayers may also contain other constituents such as other lipids, sterolic compounds, polymer-ceramides as stabilisers and targeting compounds, etc.
  • the liposomes comprising lipid derivatives may (in principle) exclusively consist of the lipid derivatives. However, in order to modify the liposomes, "other lipids" may be included as well.
  • Other lipids are selected for their ability to adapt compatible packing conformations with the lipid derivative components of the bilayer such that the all the lipid constituents are tightly packed, and release of the lipid derivatives from the bilayer is inhibited.
  • Lipid-based factors contributing to compatible packing conformations are well known to ordinarily skilled artisans and include, without limitation, acyl chain length and degree of unsaturation, as well as the headgroup size and charge. Accordingly, suitable other lipids, including various phosphatidylethanolamines (“PE's”) such as egg phosphatidylethanolamine (“EPE”) or dioleoyl phosphatidylethanolamine (“DOPE”), can be selected by ordinarily skilled artisans without undue experimentation. Lipids may be modified in various way, e.g. by headgroup derivatisation with dicarboxylic acids, such as glutaric, sebacic, succinic and tartaric acids, preferably the dicarboxylic acid is glutaric acid (“GA").
  • dicarboxylic acids such as glutaric, sebacic, succinic and tartaric acids
  • suitable headgroup-derivatised lipids include phosphatidylethanolamine-dicarboxylic acids such as dipalmitoyl phosphatidyl- ethanolamine-glutaric acid (“DPPE-GA”), palmitoyloleoyl phosphatidylethanolamine- glutaric acid (“POPE-GA”) and dioleoyl phosphatidylethanolamine-glutaric acid (“DOPE-GA”).
  • DPPE-GA dipalmitoyl phosphatidyl- ethanolamine-glutaric acid
  • POPE-GA palmitoyloleoyl phosphatidylethanolamine- glutaric acid
  • DOPE-GA dioleoyl phosphatidylethanolamine-glutaric acid
  • the derivatised lipid is DOPE-GA.
  • the total content of "other lipids” will typically be in the range of 0-30 mol%, in particular 1-10 mol%, based on the total dehydrated liposome.
  • Sterolic compound included in the liposome may generally affects the fluidity of lipid bi- layers. Accordingly, sterol interactions with surrounding hydrocarbon groups generally inhibit emigration of these groups from the bilayer.
  • An examples of a sterolic compound (sterol) to be included in the liposome is cholesterol, but a variety of other sterolic compounds are possible. It is generally believed that the content of sterolic compound, if present, will be in the range of 0-25 mol%, in particular 0-10 mol%, such as 0-5 mol%, based on the total dehydrated liposome.
  • Polymer-ceramides are stabilisers improving the vascular circulation time.
  • examples are polyethylene glycol derivatives of ceramides (PEG-ceramides), in particular those where the molecular weight of the polyethylene glycol is from 100 to 5000. It is generally believed that the content of polymer-ceramides, will be in the range of 0-30 mol%, in particular 0-10 mol%, based on the total dehydrated liposome. Still other ingredients may constitute 0-2 mol%, in particular 0-1 mol%, based on the total dehydrated liposome.
  • the lipid bilayer of a liposome contains lipids derivatised with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the lipid bilayer into the surrounding environment (see e.g. US 5,882,679 and Fig. 1).
  • PEG polyethylene glycol
  • the derivatised lipid components of liposomes according to the present invention may additionally include a labile lipid-polymer linkage, such as a peptide, ester, or disulfide linkage, which can be cleaved under selective patophysiological conditions, such as in the presence of overexpressed peptidase or esterase enzymes at diseased sites or reducing agents.
  • a labile lipid-polymer linkage such as a peptide, ester, or disulfide linkage
  • a labile lipid-polymer linkage such as a peptide, ester, or disulfide linkage
  • liposomes according to the present invention may contain non-polymer molecules bound to the exterior of the liposome, such as haptens, enzymes, antibodies or antibody fragments, cytokines and hormones (see, e.g., US 5,527,528), and other small proteins, polypeptides, single sugar polysaccharide moieties, or non-protein molecules which confer a particular enzymatic or surface recognition feature to the liposome. See published PCT application WO 94/21235.
  • Surface molecules which preferentially target the liposome to specific organs or cell types are referred to herein as "targeting molecules" and include, for example, antibodies and sugar moieties, e.g.
  • gangliosides or those based on mannose and galactose, which target the liposome to specific cells bearing specific antigens (receptors for sugar moieties).
  • Techniques for coupling surface molecules to liposomes are known in the art (see, e.g., US 4,762,915).
  • the liposome can be dehydrated, stored and then reconstituted such that a substantial portion of its internal contents is retained.
  • Liposomal dehydration generally requires use of a hydrophilic drying protectant such as a disaccharide sugar at both the inside and outside surfaces of the liposome bilayers (see US 4,880,635).
  • This hydrophilic compound is generally believed to prevent the rearrangement of the lipids in the liposome, so that the size and contents are maintained during the drying procedure and through subsequent rehydration.
  • Appropriate qualities for such drying protectants are that they are strong hydrogen bond acceptors, and possess stereochemical features that preserve the intramolecular spacing of the liposome bilayer components. Alterna- tively, the drying protectant can be omitted if the liposome preparation is not frozen prior to dehydration, and sufficient water remains in the preparation subsequent to dehydration.
  • the liposomes including the lipid derivatives of the present invention may also include second drug substances.
  • the lipid- based drug delivery system described above is in the form of liposomes wherein a second drug substance is incorporated.
  • second drug substances may comprise pharmaceutically active ingredients which may have an individ- ual or synergistic pharmaceutical effect in combination with the lipid derivative and Iy- solipid derivatives.
  • the term "second” does not necessarily imply that the pharmaceutical effect of the second drug substance is inferior in relation to that of, e.g., the active drug substance derived from the prodrug, but is merely used to differentiate between the two groups of substances.
  • the present invention also provides a drug delivery system which is in the form of liposomes, and wherein a second drug substance is incorporated.
  • a possible "second drug substance” is any compound or composition of matter that can be administered to mammals, preferably humans. Such agents can have biological activity in mammals.
  • Second drug substances which may be associated with liposomes include, but are not limited to: wound healing agents such as sphingosine 1 -phosphate analogs, antiviral agents such as acyclovir, zidovudine and the interferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene antibiotics, imidazoles and triazoles; antimetabolic agents such as fo- lie acid, and purine and pyrimidine analogs; antineoplastic agents such as the anthra- cycline antibiotics and plant alkaloids; sterols such as cholesterol; carbohydrates, e.g., sugars and starches; amino acids, peptides, proteins such as cell receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins; dyes; radio
  • Liposomal second drug substance formulations enhance the therapeutic index of the second drug substances by reducing the toxicity of the drug. Liposomes can also reduce the rate at which a second drug substance is cleared from the vascular circulation of mammals. Accordingly, liposomal formulation of second drug substance can mean that less of the drug need be administered to achieve the desired effect.
  • Liposomes can be loaded with one or more second drug substances by solubilising the drug in the lipid or aqueous phase used to prepare the liposomes.
  • ionis- able second drug substances can be loaded into liposomes by first forming the liposomes, establishing an electrochemical potential, e.g., by way of a pH gradient, across the outermost liposomal bilayer, and then adding the ionisable second drug substance to the aqueous medium external to the liposome (see, e.g., US 5,077,056 and WO 86/01102).
  • the second drug substance may be any of a wide variety of known and possible pharmaceutically active ingredients, but is preferably a therapeutically and/or prophylactically active substance. Due to the mechanism involved in the degradation of the liposomes of the present invention, it is preferred that the second drug substance is one relating to diseases and/or conditions associated with a localised increase in extracellular PLA 2 activity.
  • Particularly interesting second drug substances are selected from (i) wound healing agents (ii) antitumour agents such as anthracyline derivatives, cisplatin, paclitaxel, 5- fluoruracil, exisulind, cis-retinoic acid, suldinac sulfide, vincristine, interleukins, oligonucleotides, peptides, proteins and cytokines (iii) antibiotics and antifungals, (iv) antiinflammatory agents such as steroids and non-steroids, and (v) peroxisome proliferator activated receptor ligands.
  • the steroids can also have a stabilising effect on the liposomes.
  • active agents like peptides and protein derivatives like interferons, interleukins and oligonucleotides can be incorporated into the PLA2 degradable lipid-based carrier.
  • hydrolysis products i.e. monoether lysolipids and/or ester-linked lysolipid derivatives
  • the hydrolysis products act in turn with the released fatty acid derivatives as absorption enhancers for drug permeation across the target membranes when the carriers locally are broken down in the diseased tissue.
  • the second drug substance will be distributed in the liposomes ac- cording to their hydrophilicity, i.e. hydrophilic second drug substances will tend to be present in the cavity of the liposomes and hydrophobic second drug substances will tend to be present in the hydrophobic bilayer.
  • Method for incorporation of second drug substances are know in the art as has been made clear above.
  • the lipid derivatives may - as prodrugs or discrete constituents - posses a pharmaceutical activity.
  • the present invention furthermore relates to a lipid based drug delivery system for administration of an second drug substance, wherein the second drug substance is incorporated in the system (e.g.
  • said system including lipid derivatives which has (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
  • the organic radical which can be hydrolytically cleaved off may be an auxiliary drug substance or an efficiency modifier for the second drug substance.
  • the lipid derivative is a lipid derivative as defined further above. Typically, the lipid derivative constitutes 5-100 mol%, such as 50-100 mol%, of the total dehydrated (liposome) system.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the lipid-based drug delivery systems described above.
  • the composition will be described in detail below.
  • the present invention also relates to the use of any of the lipid-based drug delivery systems described herein as a medicament, and to the use of any of the lipid-based drug delivery systems described herein for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
  • diseases or conditions are typically selected from cancer, e.g. a brain, breast, lung, colon or ovarian cancer, or a leukemia, lymphoma, sarcoma, carcinoma and inflammatory conditions .
  • the prophylactic use is also included.
  • the present compositions and uses are especially applicable in the instances the increase in extracellular PLA 2 activity is at least 25% compared to the normal level of activity in the tissue in question, the tissue being that of a mammal, in particular a human.
  • novel and unnatural lipid analogs can be administred in a non-particulate form as free agents leading to an increased intracellular cell uptake rendering them favourable substrates for overexpressed intracellular PLA2 in the diseased target cells.
  • compositions comprising a pharmaceutically acceptable carrier and the lipid derivative, e.g. as a liposome, of this invention.
  • lipid derivative e.g. as a liposome
  • “Pharmaceutically acceptable carriers” as used herein are those media generally acceptable for use in connection with the administration of lipids and liposomes, including liposomal drug formulations, to mammals, including humans.
  • Pharmaceutically acceptable carriers are generally formulated according to a number of factors well within the purview of the ordinarily skilled artisan to determine and account for, including without limitation: the particular active drug substance and/or second drug substance used, the liposome preparation, its concentration, stability and intended bioavailability; the disease, disorder or condition being treated with the liposomal composition; the subject, its age, size and general condition; and the composition's intended route of administration, e.g., nasal, oral, ophthalmic, subcutaneous, intramammary, intraperitoneal, intravenous, or intramuscular.
  • Typical pharmaceutically acceptable carriers used in parenteral drug administration include, for example, D5W, an aqueous solution containing 5% weight by volume of dextrose, and physiological saline.
  • Pharmaceutically acceptable carriers can contain additional ingredients, for example those which enhance the stability of the active ingredients included, such as preservatives and anti-oxidants.
  • the liposome or lipid derivative is typically formulated in a dispersion medium, e.g. a pharmaceutically acceptable aqueous medium.
  • an amount of the composition comprising an anticancer effective amount of the lipid derivative is administered, preferably intravenously.
  • anticancer effective amounts are amounts effective to inhibit, ameliorate, lessen or prevent establishment, growth, metastasis or inva- sion of one or more cancers in mammals to which the lipid derivatives have been administered.
  • Anticancer effective amounts are generally chosen in accordance with a number of factors, e.g., the age, size and general condition of the subject, the cancer being treated and the intended route of administration, and determined by a variety of means, for example, dose ranging trials, well known to, and readily practised by, ordi- narily skilled artisans given the teachings of this invention.
  • Antineoplastic effective amounts of the liposomal drugs/prodrugs of this invention are about the same as such amounts of free, nonliposomal, drugs/prodrugs, e.g., from about 0.1 mg of the lipid derivative per kg of body weight of the mammal being treated to about 1000 mg per kg.
  • the liposome administered is a unilamellar liposome having an average diameter of from about 50 nm to about 200 nm.
  • the anti-cancer treatment method can include administration of one or more second drug substances in addition to the liposomal drug, these additional agents being included in the same liposome as the lipid derivative.
  • the second drug substances which can be entrapped in liposomes' internal compartments or sequestered in their lipid bilayers, are preferably, but not necessarily, anticancer agents.
  • the pharmaceutical composition is preferably administered parenteral ⁇ by injection, infusion or implantation (intravenous, intramuscular, intraarticular, subcutaneous or the like) in dosage forms, formulations or e.g. suitable delivery devices or implants contain- ing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • the pharmaceutical compositions according to the invention may comprise the active drug substances in the form of a sterile injection.
  • the suitable active drug substances are dispersed in a parenterally acceptable liquid vehicle which conveniently may comprise suspending, solubilising, stabilising, pH- adjusting agents and/or dispersing agents.
  • acceptable vehicles that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1 ,3-butanediol, Ringer's solution and isotonic sodium chloride solution.
  • the aqueous formulation may also contain one or more preservatives, for example, methyl, ethyl or n-propyl p- hydroxybenzoate.
  • liposome-encapsulated drug via the bloodstream requires that the liposome be able to penetrate the continuous (but "leaky”) endothelial layer and underlying basement membrane surrounding the vessels supplying blood to a tumour.
  • liposomes of smaller sizes have been found to be more effective at extravasation into tumours through the endothelial cell barrier and underlying basement membrane which separates a capillary from tumour cells.
  • the second drug agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers.
  • the resulting liposomal formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration.
  • Inflammatory conditions characterised by an acute increase in permeability of the vasculature in the region of 5 the inflamed regions are particularly suited for treatment by the present methods.
  • the liposomes will eventually degrade due to lipase action at the tumour site, or can be made permeable by, for example, thermal or ultrasonic radiation.
  • the drug is then released in a bioavailable, transportable solubilised form.
  • a small elevation in temperature as often seen in diseased tissue may further increase the stimulation of 10 extracellular PLA 2 .
  • liposome delivery of an drug requires that the liposome have a long blood half-life, and be capable of penetrating the continuous endothelial cell layer and underlying basement membrane sur-
  • the anti-inflammatory agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers.
  • the resulting liposomal formulation can be administered parenterally to a subject
  • Inflamed regions characterised by an acute increase in permeability of the vasculature in the region of inflammation are particularly suited for treatment by the present methods.
  • extracellular PLA 2 activity of extracellular PLA 2 is abnormally high in areas of the 30 mammalian body diseased by cancer, inflammation, etc.
  • the present invention have provided a way of exploiting this fact, and it is believed that the extracellular PLA 2 activity should be at least 25% higher in the diseases area of the body (determined in the extracellular environment) compared with a comparative normal area. It is however envisaged that the level of extracellular PLA 2 activity often is much higher, e.g. at least 35 100%, e.g. at least 200% such as at least 400%.
  • treatment of a mammal in need of a treatment with the purpose of cure or relief can be conducted with only minimal influence on tissue having a "normal" level of extracellular PLA 2 activity. This is extremely relevant in particular with the treatment of inflammatory conditions where rather harsh drug (second drug substances) are often needed.
  • the invention thus provides to a method for selectively drug targeting to diseased areas, such as areas comprising inflamed cells, e.g., areas within the mammalian body, preferably a human, having a extracellular phospholipase A2 (extracellular PLA 2 ) activity which is at least 25% higher compared to the normal activity in said areas, by administering to the mammal in need thereof an efficient amount of a drug delivery system defined herein.
  • diseased areas such as areas comprising inflamed cells, e.g., areas within the mammalian body, preferably a human, having a extracellular phospholipase A2 (extracellular PLA 2 ) activity which is at least 25% higher compared to the normal activity in said areas, by administering to the mammal in need thereof an efficient amount of a drug delivery system defined herein.
  • a mammal afflicted with a cancer e.g., a brain, breast, lung, colon or ovarian cancer, or a leukemia, lymphoma, sarcoma, carcinoma
  • a pharmaceutical composition of this invention comprises administering a pharmaceutical composition of this invention to the mammal. It is believed that the lipid derivatives and/or second drug substance in liposome form is selectively cytotoxic to tumour cells.
  • the novel targeted liposomal prodrug and drug delivery systems are useful in the treatment or alleviation of disorders in the skin that are associated with or resulting from increased levels of extracellular PLA 2 .
  • the lipid-based prodrug and drug delivery systems are useful for the administration of an active drug substance selected from ether-lysolipid and/or fatty acid derivatives of a lipid prodrug being a sub- strate for extracellular PLA 2 .
  • the flexible liposomal carrier by PLA 2 leads to the release of lysolipid and/or fatty acid derivatives, which are designed to be effective in the treatment of various types of skin diseases such as inflammation.
  • the novel prodrug lipo- somal carriers can be used for targeted delivery of conventional drugs to diseased regions of the skin that is associated with an elevated level of PLA 2 .
  • Liposomal formulations have been the focus of extensive investigation as the mode of skin delivery for many drugs. There is growing evidence that for topical administration, 10 a new generation of flexible liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
  • the major physico-chemical barrier of the skin is localized in the outermost cornified layer of the skin, the stratum corneum.
  • the pores in the stratum corneum and the underlaying structures are normally so narrow, that the skin only allows passage of entities smaller than 400 Dalton.
  • ultraflexible liposomes 20 are able to pass pores in the skin smaller than 30 nm (Cevc et al. (Biochem Biophys Acta (1998) 1368, p 201-215, US Patent 6.180.353).
  • the novel targeted prodrug system addresses both the important issue of ensuring that predominantly the relevant cells are exposed to the drugs, and the fact that only very flexible liposomes that contains "edge active substances" are able to pass through the stratum corneum and penetrate deep into diseased regions of the skin with elevated levels of PLA 2 . It is important to realize that the prodrug liposomes are not intended for
  • extracellular phospholipase A 2 (PLA 2 ) is increased in a number of inflammatory diseases of the skin most notably psoreasis and eczema (Forster et al. (1985) Br J Dermatol 112:135-47; Forster et al. (1983) Br J Dermatol 35 108:103-5). It has furthermore been found that extracellular PLA 2 is capable of cleaving the prodrug lipid derivatives so as to produce ether-lysolipid derivatives, which alone or in combination with other active compounds, will exhibit an effect.
  • the new prodrug liposomes ensure that the pharmaceutical active ether-lysolipids can be delivered specifically at the target cells in the skin using elevated activity of PLA 2 as a site- specific trigger mechanism.
  • specific fatty acid derivatives which are turned into active drugs by PLA 2 hydrolysis may be linked to the C-3 position of the lipid-based carrier composed of the novel unnatural lipids.
  • Possible examples include polysatu- rated fatty acids and other lipophilic groups such as vitamin D derivatives, steroid derivatives, retinoyl derivatives, and tocopherol analogues that can be ester bound to the C-3 position and therefore renders the double prodrug lipid a substrate for PLA 2 .
  • Fur- thermore conventional drug substances can be incorporated and transported specifically to the diseased site by the novel lipid-based carriers.
  • the new carrier liposomes also offer a solution to both the issue of penetration of intact skin and the issue of specific targeting of cells, tissues or part of tissues in the skin that are characterized by an increased level of PLA 2 .
  • the MPS comprises the macrophages, one of the most important components of the immune system involved in the clearance of foreign particles, including liposomes.
  • the macrophages reside in various organs and tissues, e.g. in the spleen and liver (Kupffer cells) and as free and fixed macrophages in the bone marrow and lymph nodes.
  • the novel lipid-based system composed of prodrug lipids is able to deliver various lipid prodrugs and encapsulated drugs directly to the diseased liver or spleen harbouring parasites due to an accumulation of the liposomes and an increased level of the prodrug cleaving PLA 2 enzyme in the infected tissues.
  • One particular advantage of the lipid based drug delivery system is furthermore that extracellular PLA 2 activity is significantly increased towards organized lipid substrates such as the prodrug liposomes as compared to monomeric lipid substrates.
  • the released ether-lipid analogues that are generated by the action of PLA 2 have been shown to result in perturbation of key regulatory enzymes in parasites such as Leishmania and Trypanosoma (Lux et al. Biochem.
  • liver, spleen and bone marrow are organs and tissues wherein high concentrations of macrophages can be found, it is possible to achieve a targeted delivery of the toxic drugs to parasites inhabiting these organs.
  • parasitic infections which are characterized by elevated levels of PLA 2 , such as the malaria causing parasites, are also targets for treatment with the novel prodrug liposomes.
  • the elevated levels of PLA 2 in malaria infections caused by the parasite Plasmodium Falciparum has been described by e.g. Vadas et al. (Infection and Immunity, 60 (1992) 3928-3931 and Am. J. Trop. Hyg. 49 (1993) 455-459).
  • ether-lipids typically possess ether-cleavage enzymes, which enable them to avoid the toxic effect of ether-lipids.
  • some normal cells such as red blood cells have like cancer cells no means of avoiding the disruptive effect of the ether-lipids. Accordingly, therapeutic use of ether-lipids requires an effective drug delivery system that protects the normal cells from the toxic effects and is able to bring the ether-lipids directly to the diseased tissue.
  • the novel targeted prodrug delivery systems can be used for the treatment of parasitic infections, which is characterized by an increased level of PLA 2 in the infected tissue. This is achieved by administering an efficient amount (up to about 1000 mg per kg) of the liposomes wherein the active drug substance is present in the form of a lipid prodrug being a substrate for extracellular PLA 2 .
  • the prodrug liposomes are candidates for targeted transport of encapsulated conventional anti-parasitic drug(s), where a potentiation of the effectiveness of the conventional drug(s) in combination with the PLA 2 generated lysolipids and/or fatty acid derivatives might be obtainable.
  • Toxicity of the liposomes comprising the lipid derivatives can be assessed by determining the therapeutic window "TW", which is a numerical value derived from the relationship between the compound's induction of hemolysis and its ability to inhibit the growth of tumour cells.
  • TW values are defined as HI 5 /GI 50 (wherein “Hl 5 " equals the concentra- tion of compound inducing the hemolysis of 5% of the red blood cells in a culture, and wherein "Gl 50 " equals the dose of compound inducing fifty percent growth inhibition in a population of cells exposed to the agent).
  • the higher its Hl 5 the more therapeutically beneficial is a compound, because more of it can be given before inducing the same amount of hemolysis as an agent with a lower Hl 5 .
  • lower Gl 50 's indicate better therapeutic agents - a lower Gl 50 value indicates that a lesser concentration of an agent is required for 50% growth inhibition. Accordingly, the higher is its Hl 5 value and the lower is its Gl 50 value, the better are a compound's agent's therapeutic properties.
  • a drug's TW when a drug's TW is less than 1 , it cannot be used effectively as a therapeutic agent. That is, the agent's Hl 5 value is sufficiently low, and its Gl 50 value sufficiently high, that it is generally not possible to administer enough of the agent to achieve a suf- ficient level of tumour growth inhibition without also attaining an unacceptable level of hemolysis.
  • the lipid derivative liposomes take advantage of the lower extracellular PLA 2 activity in the bloodstream compared to the activity in the diseased tissue, it is believed that the TW will be much higher that for normal monoether lysolipids.
  • the TW of the liposomes of the invention will be greater than about 3, more preferably greater than about 5, and still more preferably greater than about 8.
  • Lysophosphatidic acid is a wound healing compound that can be delivered using the novel PLA2 degradable liposomes containing proLPA.
  • this lipid has an ester bound acyl chain in the sn-2 position, which is cleaved by PLA2.
  • LPA itself has growth-promoting effects on several cell types, including endothelial cells, fibroblasts, keratinocytes, cancer cells etc., and has growth promoting effects on cells as showed in Fig. 4.
  • LPA stimulated cell growth of NIH 3T3 cells to a level comparable to the growth stimulation seen by addition of 10 % serum, whereas HU- VEC cells were stimulated even more strongly than addition of 10 % serum, during 24 h.
  • ProLPA liposomes were made by sonication in PBS and added to the cell culture as indicated in Fig. 5 (in the presence or absence of the specific sPLA2 inhibitor) in conditioned media from Colo 205 cells with a PLA2 concentration of approximately 30 ng/ml.
  • HUVEC cells were clearly growth-stimulated by incubation with proLPA-liposomes in the presence of PLA2. This effect was reduced when a specific sPLA2 inhibitor was added, proving that PLA2 is required for generation of LPA from proLPA liposomes. A growth stimulation was seen also in the presence of the PLA2 inhibitor. This could indicate that the hydrolysis of the proLPA liposomes by PLA2 is very fast and efficient, and that some hydrolysis occurs, even in the presence of the inhibitor. Alternatively, other secretory PLA2 lipases with less specificity for the inhibitor could be secreted by the HUVEC cells. Similar data was seen with the NIH 3T3 cells upon addition of proLPA liposomes.

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Abstract

The present invention describes a lipid based prodrug and drug delivery system that can be used for treatment or alleviation of disorders that are associated with or resulting from increased levels of secretory phospholipase A2 (PLA2) in the diseased or injured tissue. In particular, the lipid based delivery systems are useful for administration of lipid prodrugs of lysophosphatidic acid (LPA) bioactive analogs, which are known to be potent biological mediators with e.g. growth stimulating and wound healing activities.

Description

TITLE: DRUG DELIVERY SYSTEMS CONTAINING PHQSPHOLIPASE A2 DEGRAD- ABLE LIPID PRODRUG DERIVATIVES AND THE THERAPEUTIC USES THEREOF AS. E.G. WOUND HEALING AGENTS AND PEROXISOME PROLIFERATOR ACTIVATED RECEPTOR LIGANDS
TECHNICAL FIELD
The lipid based prodrug and drug delivery systems are useful for local as well as sys- temic treatment of inflammatory conditions or damaged tissue, wounds, injuries, and burns that are associated with increased levels of extracellular phospholipase A2 (PLA2). In addition, the liposomal prodrug and drug carries can be used in combination with incorporated drugs and a wound coverage material.
BACKGROUND OF THE INVENTION
Lysophosphatidic acid (LPA) bioactive lipids are known to be potent biological mediators with growth stimulating and wound healing activities (7). LPA has been shown to bind to specific G protein-coupled receptors (Edg-receptors) (7,8) thereby eliciting a biological response in mammalian cells including cell proliferation (9), wound healing (10), cell survival (11 ,12,13), accelerated development of embryos (14), preservation of organs (15), monocyte migration (16), and growth stimulating activity in salvia (17). Lysophosphatidic acid is an endogeneous soluble glycerophospholipid in the body that shows similar biological activity as the cell growth factor. It is known that the signal of LPA is transmitted into the cell through the 7-transmembrane Edg-receptors (for example LPA1 , LPA2, LPA3) in the cell membrane. Various new LPA analogs that are peroxisome proliferator activated receptor ligands have been synthesized to improve healing and treat or prevent cancer or diabetes (described in WO200492188-A2). LPA also displays activity as a ligand for inhibiting neutrophil accumulation in tissues for treating or preventing diseases or disorders associated with excessive accumulation of neutrophils in e.g. inflammatory diseases (described in WO2004052375-A1 ).
BRIEF DESCRIPTION OF THE INVENTION
The present invention describes a lipid based prodrug and drug delivery system that can be used for treatment or alleviation of disorders that are associated with or result- ing from increased levels of secretory phospholipase A2 (PLA2) in the diseased or injured tissue (1 ,2,3,4,5,6). In particular, the lipid based delivery systems are useful for administration of lipid prodrugs of lysophosphatidic acid (LPA) bioactive analogs, which are known to be potent biological mediators with e.g. growth stimulating and wound healing activities (7).
The present invention is directed to drug delivery systems, which are particularly useful in the treatment or alleviation of diseases that are characterised by localised activity of extracelluar PLA2 activity.
The new principle for liposomal drug targeting by extracellular PLA2 described in this application involves lipid-based prodrugs as illustrated in Figs. 1 and 2 for systemic or local administration of prodrugs of LPA bioactive analogs that can be activated to LPA active agents at the desired target site. Specific lipid-analogue compounds may be in- corporated into the lipid based carrier, which furthermore can have polymer or polysaccharide chains "grafted" to the surface and act as a prodrug particulate carrier that is turned into active drugs by extracellular phospholipase A2 hydrolysis. Possible examples include certain lysophosphatidic acid analogs that are peroxisome proliferator activated receptor ligands useful as, e.g. wound healing agents and for treatment or pre- vention of cancer or diabetes (WO200492188-A2).
The bioactive LPA lipids can be generated through the action of secretory PLA2 on lipid substrates containing phosphatidic acid lipids (7,19). Degradation of the liposomal prodrug carriers by PLA2 leads to release of acyl and/or alkyl LPAs and fatty acid de- rivatives as shown in Fig. 3, which are designed to be, e.g. effective growth promoting agents. In addition to the site-specific delivery of the bioactive and growth promoting LPAs and fatty acid drug derivatives, the liposomal carriers can advantageously be used for targeted transport and delivery of conventional wound healing agents as well as novel biological growth promoting substances like peptides, proteins, and DNA ana- logues that are encapsulated in the interior of the prodrug liposomes. The novel principle behind the site-specific activation and delivery of growth promoting agents to diseased tissue such as wounds and bums is illustrated in Figs. 1 and 2. The liposomal prodrug and drug carries can furthermore be used in combination with a wound coverage material (20) The PLA2-triggered hydrolysis of the liposomal prodrug carries leads to a site-specific activation and conversion of the phosphatidic acid prodrug lipids (acyl/alkyl proLPA) to potent growth promoting lysophosphatidic acids (acyl/alkyl LPA) as illustrated in Fig. 3. The substituent R may constitute an auxiliary drug substance or an efficiency modifier (e.g. permeability enhancer or second growth factor) and will simultaneously be released under the action of PLA2. In contrast to the released lysophosphatidic acids, the prodrug lipids form stable liposomal membrane structures, which function as favorable substrates for PLA2 catalyzed lipid hydrolysis. A particular advantage of using liposomal prodrug carriers for targeted delivery of growth promoting agents involves a sig- nificantly increased activity of PLA2 towards organized lipid substrates such as lipid membranes as compared to monomeric lipid substrates. This leads to a fast and efficient degradation of the prodrug liposomes resulting in a site-specific conversion of the phosphatidic acid lipid analogs to active LPA acting compounds, e.g. growth promoting and wound healing agents or peroxisome proliferator activated receptor agonists or an- tagonists in the diseased target tissue. Subsequently, the liposomal cargo of various growth factors such as growth hormones, peptides, and proteins will be released in the desired target tissue as illustrated in Figure 2.
The principle of drug targeting, release and absorption mediated by extracellular phos- pholipase A2 (PLA2) which is illustrated in Fig. 1, can be applied to a case involving unnatural lipid-based prodrugs. In this case lipid derivatives are constituents of the carrier liposome and act as prodrugs, which are turned into active drugs (e.g. ether lipids and fatty acid drug derivatives) by hydrolysis via the extracellular PLA2 that is present at elevated concentrations in the diseased target tissue. The prodrug lipids can be modified with a long fatty-acid chain that is ester linked in the C-2 or C-3 position and a head group that is linked in the C-3 or C-2 position, respectively, and therefore can be hydrolysed by extracellular PLA2 at the target site. A specific example is a lipid prodrug of a certain mono-ether lysolipid, which is bioactive LPA analog. This can be a therapeutically active compound having, e.g. a regulatory fatty acid derivative ester bound to the phospholipid in, e.g. the C-3 or C-2 position and a head group that is linked in the C-2 or C-3 position, respectively, and therefore renders the lipid derivative substrate for extracellular PLA2. If the lipids are modified with, e.g. a ester-linked derivative in the C-3 or C-2 position and therefore can be hydrolysed by extracellular PLA2 at the target site, these lipid derivatives constituting the carrier liposome will act as prodrugs that become hydrolysed and turned into drugs by extracellular PLA2 at the target site. In this way, the therapeutically active substances, e.g., monoether lipids and ester-linked drug derivatives will be liberated at the desired target site. Furthermore, the hydrolysis product can act as local permeability enhancers facilitating the transport of the released drugs into the cell. Pharmaceutical compositions containing the lipid-based system can be used therapeutically, for example, in the treatment of cancer, infectious and inflam- matory conditions.
This invention provides such a delivery system in the form of lipid-based carriers, e.g. liposomes or micelles, composed of novel unnatural lipid-bilayer forming lipids such as glycerophospholipids containing an alkyl-linkage or acyl-linkage in the 1 -position and an acyl-linkage or another PLA2 degradable bond, e.g. a bioisoster of a PLA2 hydroly- sable ester in the C-2 or C-3 position on the glycerol backbone and which have polymer or polysaccharide chains grafted thereto in the C-2 or C-3 position. In addition, the carrier system may contain lipid-bilayer stabilising components, e.g. lipopolymers, gly- colipids and sterols, which lead to an increased vascular circulation time and as a con- sequence an accumulation in the diseased target tissue. When the carriers reach the target site of therapeutic action, e.g. in inflammatory conditions PLA2-catalyzed hydrolysis of the acyl-linkage releases the therapeutically active components, typically Iy- solipids and ester-linked derivatives.
The present invention thus provides a lipid-based drug delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a lipid derivative having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysoiipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
The present invention also provides a lipid based drug delivery system for administration of an second drug substance, wherein the second drug substance is incorporated in the system, said system including lipid derivatives which has (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydro- lytically cleaved off, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
Thus, the present invention takes advantage of the surprising finding that liposomes (and micelles) including lipid derivatives which can be specifically and only partially cleaved by extracellular phospholipases, and which at the same time includes lipopolymers or glycolipids, have the properties of circulating in the blood stream suffi- ciently long so as to reach target tissue where the extracellular PLA2 activity is elevated without being recognised by the mammalian reticuloendothelial systems and without penetrating cell walls, whereby the lipid derivatives of the liposomes are specifically cleaved by extracellular PLA2 so as to liberate therapeutically active ingredients at the desired location.
The present invention provides a class of novel lipid derivatives which are particularly useful as constituents of the drug delivery systems described herein.
DESCRIPTION OF THE DRAWINGS
Fig. 1. Schematic illustration of the lipid-based (pro)drug-targeting and (pro)drug- trigering principle involving accumulation of the liposomal (pro)drug carriers in porous diseased tissue and subsequent release and activation of (pro)drugs and transport across the target membrane via extracellular PLA2 activity.
(I) Pathological tissue with leaky capillaries
(II) Prodrug and drug carrying liposome
(III) Target cell and cell membrane
(IV) Novel and unnatural proLPA analogs (lipids) of LPA analogs, proenhancers (Np- ids), proactivators (lipids)
(V) Drugs (lysolipid and fatty acid derivatives), enhancers (lysolipid + fatty acid), PLA2 activators (lysolipid + fatty acid)
Fig. 2. Local administration (e.g. dermal application) of the targeted liposomal delivery of growth stimulating lysophosphatidic acids (LPA), fatty acid drug derivatives and en- capsulated growth promoting hormones, peptides and proteins to diseased tissue (i.e. wounds, burns, and injuries) using phospholipase A2 (PLA2) as a site-specific trigger.
Fig. 3. Phospholipase A2 (PLA2) generation of lysophosphatidic acids (acyl/alkyl LPA) and fatty acid drug derivatives (RCOOH) from phosphatidic acid prodrug lipids (acyl/alkyl proLPA).
Fig. 4. Stimulation of cell proliferation by LPA treatment. LPA was added at indicated concentrations to NIH 3T3 fibroblasts (mouse immortal cell line) and HUVEC cells (human umbilical cord endothelial cells-normal endothelial cells) for 24 h, in media con- taining fatty acid free serum albumin (0,1 %), and no serum. Proliferation was determined by incorporation of radioactive thymidine.
Fig. 5. Release of LPA from proLPA liposomes by secretory PLA2 stimulates HUVEC cell proliferation. Addition of proLPA liposomes in the presence of secretory sPLA2 se- creted from Colo 205 cells. Media from Colo 205 cells was conditioned for 24 h, and mixed 1 :1 with normal growth media for HUVEC cells. Fatty acid free BSA was added to the system also, to allow LPA to work in an growth stimulating manner. After 22 h incubation, cells were pulse-labelled with radioactive thymidin.
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16. Zhou, D., W. Luini, S. Bemasconi, L. Diomede, M. Salmona, A. Mantovani, and S. Sozzani. 1995. Phosphatidic acid and lysophosphatidic acid induce haptotactic migration of human monocytes. J. Biol Chem. 270:25549-25556. 17. Sugiura, T., S. Nakane, S. Kishimoto, K. Waku, Y. Yoshioka, and A. Tokumura. 2002. Lysophosphatidic acid, a growth factor-like lipid, in the saliva. J. Lipid Res. 43:2049-2055. 18. Fang, X., S. Yu, R. Lapushin, Y. Lu, T. Furui, L. Z. Penn, D. Stokoe, J. R. Erickson, R. C. Bast, and G. B. Mills. 2000. Lysophosphatidic acid prevents apoptosis in fibroblasts via Gi-protein- mediated activation of mitogen-activated protein kinase. Biochem. J. 352:135-143. 19. Snitko, Y. E. T. Yoon, and W. Cho. 1997. High specificity of human secretory class Il phospholi- pase A2 for phosphatidic acid. Biochem. J. 321:737-741.
20. Herndon, D., J. R. Perez-Polo, R. E. Barrow. Enhanced wound coverage to enhance wound healing. 2000. WO 00/78259 A1.
BEST MODE FOR CARRYING OUT THE INVENTION
One of the important features of the present invention is the realisation that certain lipid derivatives will be cleaved by extracellular PLA2 in a well-defined manner in extracellu- lar locations of mammalian diseased tissue. It has been found that extracellular PLA2 is capable of cleaving lipid derivatives so as to produce LPA lysolipid derivatives, e.g. lipid produgs of LPA analogs which as such, or in combination with other active compounds, will exhibit a therapeutic effect.
Lipid derivatives Thus, the drug delivery systems (liposomes or micelles) of the present invention relies on lipid derivatives having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
Although the terms "lipid" and "lysolipid" (in the context of phospholipids) will be well- known terms for the person skilled in the art, it should be emphasised that, within the present description and claims, the term "lipid" is intended to mean Westers of glycerol of the following formula:
CH2-O-RA CH2-O-RA I I
CH-O-R0 or CH-O-R8
I I
CH2-O-R6 CH2-O-R0
wherein RA and RB are fatty acid moieties (C9-3o-alkyl/alkylene/alkyldiene/alkyltriene/- alkyltetraene-C(=O)-) and Rc is a phosphatidic acid (PO2-OH) or a derivative of phos- phatidic acid or a bioisoster of phosphatidic acid. Thus, the groups RA and RB are linked to the glycerol backbone via ester bonds.
The term "lysolipid" is intended to mean a lipid where the RB fatty acid group is absent (e.g. hydrolytically cleaved off), i.e. a glycerol derivative of the formula above where RB is hydrogen, but where the other substituents are substantially unaffected. Conversion of a lipid to a lysolipid can take place under the action of an enzyme, specifically under the action of celluar as well as extracellular PLA2. The terms "lipid derivative" and "lysolipid derivative" are intended to cover possible derivatives of the above possible compounds within the groups "lipid" and "lysolipid", respectively. Examples of biologically active lipid derivatives and lysolipid derivatives are given in WO200492188-A2 and WO2004052375-A1. Thus, as will be evident, the ex- tension "derivative" should be understood in the broadest sense.
Within the present application, lipid derivatives and lysolipids should however fulfil certain functional criteria (see above) and/or structural requirements. It is particularly relevant to note that the suitable lipid derivatives are those which have (a) an aliphatic group of a length of at least 3, preferably at least 9, carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety. It will be evident that the aliphatic group and the organic radical will correspond to the two fatty acid moieties in a normal lipid and that the hydrophilic moiety will correspond to the phosphate part of a (phospho)lipid or a bioisoster thereof.
Thus, as the general idea behind the present invention is to exploit the increased level of extracellular PLA2 activity in localised areas of the body of a mammal, in particular diseased tissue, the lipid derivatives which can be utilised within the present invention should be substrates for extracellular PLA2, i.e. the lipid derivatives should be able to undergo hydrolytic, enzymatic cleavage of the organic radical corresponding to a fatty acid in the 2- or 3-position in a lipid. Extracellular PLA2 is known to belong to the enzyme class (EC) 3.1.1.4. Thus by reference to (extracellular) PLA2 should be understood all extracellular enzymes of this class, e.g. lipases, which can induce hydrolytic cleavage of the organic radical corresponding to the fatty acid in the 2- or 3-position in a lipid. One particular advantage of the lipid based drug delivery system (as liposomes and micelles) is that extracellular PLA2 activity is significantly increased towards organised substrates as compared to monomeric substrates.
In view of the requirement to hydrolability by extracellular PLA2, it is clear that the or- ganic radical (e.g. aliphatic group) is preferably linked via an ester functionality e.g. a bioisoster which can be cleaved by extracellular PLA2, preferably so that the group which is cleaved off is a carboxylic acid or a bioisoster therof.
Furthermore, it is an important feature of the present invention that the aliphatic group (the group corresponding to the fatty acid in the 1 -position in a lipid) of the lipid derivative, i.e. the lysolipid derivative after cleavage by extracellular PLA2, is substantially un- affected by the action of extracellular PLA2. By "substantially unaffected" is meant that the integrity of the aliphatic group is preserved and that less than 1 mol%, preferably less than 0.1 mol%, of the aliphatic group (the aliphatic group in the 1 -position) is cleaved under the action of extracellular PLA2.
One preferred class of lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
CH2-X-R CH2-X-R I I
CH- Z-R3 or CH-Y-R2
I I
CH2-Y-R2 CH2-Z-R3
wherein
X and Z independently are selected from OC(O), O, CH2, NH, NMe, S, S(O), OS(O), S(O)2, OS(O)2, OP(O)2, OP(O)2O, OAs(O)2 and OAs(O)2O; preferably from O, NH, NMe and CH2, in particular O and CH2;
Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH2C(O)O, NC(O)O, Y then being connected to R2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom, preferably via the carbonyl carbon atom;
R1 is an aliphatic group of the formula Y1Y2;
R2 is an organic radical having at least 2 carbon atoms, such as an aliphatic group having a length of at least 2, preferably at least 9, carbon atoms, preferably a group of the formula Y1Y2;
where Y1 is -(CH2)nr(CH=CH)n2-(CH2)n3-(CH=CH)n4-(CH2)n5-(CH=CH)n6-(CH2)n7- (CH=CH)n8r-(CH2)n9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 2 to 29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 ; and each of n2, n4, n6 and nδ is inde- pendently zero or 1; and Y2 is CH3, OH, SH, S(O)2OH, P(O)2OH, OP(O)2OH, NH2 or CO2H; where each Y1 -Y2 independently may be substituted with halogens and aliphatic substituents, but preferably Y1 -Y2 is unsubstituted,
R3 is selected from phosphatidic acid (PO2-OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid, e.g. P(O)O, P(O)2CH2, S(O)O, S(O)CH2, C(O)O, C(O)N, C(S)O, P(S)O2, S(O)2CH2 and derivatives thereof (among others phosphatidic acid derivatives to which a hydrophilic polymer or polysaccharide is covalently attached).
As mentioned above, preferred embodiments imply that Y is -OC(O)- where Y is con- nected to R2 via the carboxyl atom. The most preferred embodiments imply that X and Z are O and that Y is -OC(O)- where Y is connected to R2 via the carboxyl atom. This means that the lipid derivative is a 1-monoether-2-monoester-phospholipid type compound.
Another preferred group of lipid derivatives is the one where the group X is S.
In one embodiment, R1 and R2 are aliphatic groups of the formula Y1 Y2 where Y2 is CH3, OH, SH, S(O)2OH, P(O)2OH, OP(O)2OH, NH2 or CO2H, but preferably CH3, and where Y1 is -(CH2)n1(CH=CH)n2(CH2)n3(CH=CH)n4(CH2)n5(CH=CH)n6(CH2)n7(CH=CH)n8- (CH2)n9; the sum of n1+2n2+n3+2n4+n5+2n6+n7+ 2n8+n9 is an integer of from 2 to 29; that is, the aliphatic group, Y1Y2, is from 2-29 carbon atoms in length. n1 is equal to zero or is an integer of from 1 to 23; n3 is equal to zero or is an integer of from 1 to 20; n5 is equal to zero or is an integer of from 1 to 17; n7 is equal to zero or is an integer of from 1 to 14; n9 is equal to zero or is an integer of from 1 to 11 ; and each of n2, n4, n6 and 8 is independently equal to zero or 1.
Although the aliphatic groups may be unsaturated and even substituted with halogens (flouro, chloro, bromo, iodo) and C-i.-io-groups (i.e. yielding branched aliphatic groups), the aliphatic groups as R1 and R2 are in one embodiment preferably saturated as well as unbranched, that is, they preferably have no double bonds between adjacent carbon atoms, each of n2, n4, n6 and n8 then being equal to zero. Accordingly, Y1 is preferably (CH2)ni. More preferably (in this embodiment), R1 and R2 are each independently (CH2)n1CH3, and most preferably, (CH2)i7CH3 or (CH2)15CH3. In alternative embodiments, the groups can have one or more double bonds, that is, they can be unsatu- rated, and one or more of n2, n4, n6 and n8 can be equal to 1. For example, when the unsaturated hydrocarbon has one double bond, n2 is equal to 1, n4, n6 and n8 are each equal to zero and Y1 is (CH2)ni CH=CH(CH2)n3. n1 is equal to zero or is an integer of from 1 to 21 , and n3 is also zero or is an integer of from 1 to 20, at least one of n1 or n3 not being equal to zero.
In one particular embodiment, the lipid derivatives are those which are monoether lipids where X and Z are O, R1 and R2 are independently selected from alkyl groups, (CHz)nCH3, where n is 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, preferably 14, 15 or 16, in particular 14; Y is -OC(O)-, Y then being connected to R2 via the carbonyl carbon atom.
With respect to the hydrophilic moiety (often known as the "head group") which corresponds to R3, it is believed that a wide variety of groups corresponding to phosphatide acid (PO2-OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof can be used. As will be evident, the crucial requirement to R3 is that the groups should allow for enzymatic cleavage of the R2 group (e.g. R2-C(=O) or R2- OH) by extracellular PLA2. "Bioisosters to phosphatidic acid and derivatives thereof indeed implies that such groups - as phosphatidic acid - should allow for enzymatic cleavage by extracellular PLA2.
R3 can be selected from phosphatidic acid (PO2-OH), phosphatidylcholine (PO2-O- CH2CH2N(CH3)3), phosphatidylethanolamine (PO2-O-CH2CH2NH2), N-methyl- phosphatidylethanolamine (PO2-O-CH2CH2NCH2), , phosphatidylserine, phosphatidy- linositol, and phosphatidylglycerol (PO2-O-CH2CHOHCH2OH). Other possible derivatives of phosphatidic acid are those where dicarboxylic acids, such as glutaric, sebacic, succinic and tartaric acids, are coupled to the terminal nitrogen of phosphatidyl- ethanolamines, phosphatidylserine, phosphatidylinositol, etc.
In the particular embodiment where a fraction of the lipid derivative also is a lipopoly- mer or glycolipid, a hydrophilic polymer or polysaccharide is typically covalently at- tached to the phosphatidyl part of the lipid derivative. Another particular lipid derivative include an acyl chain attached to the head group of the lipids,
Hydrophilic polymers which suitable can be incorporated in the lipid derivatives of the invention so as to form lipopolymers are those which are readily water-soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e. are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polygly- colide), a polylactic-polyglycolic acid copolymer, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polyme- thacrylamide, polydimethylacrylamide, and derivatised celluloses such as hydroxy- methylcellulose or hydroxyethylcellulose.
Preferred polymers are those having a molecular weight of from about 100 daltons up to about 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethyleneglycol hav- ing a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)). Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilises polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons).
When the glycolipid or lipopolymer is represented by a fraction of the lipid derivative, such a lipid derivative (lipid derivative with a polymer or polysaccharide chain) typically constitutes 1-80 mol%, such as 2-50 mol% or 3-25 mol% of the total dehydrated lipid- based system. For micellular compositions, however, the fraction may be even higher, such as from 1-100 mol%, such as 10-100 mol%, of the total dehydrated lipid-based system.
Preferred polymers to be covalently linked to the phosphatidyl part (e.g. via the terminal nitrogen of phosphatidylethanolamine) are polyethylene glycol (PEG), polyactide, polyglycolic acid, polyactide-polyglycolic acid copolymer, and polyvinyl alcohol.
One highly interesting aspect of the present invention is the possibility of modifying the pharmaceutical effect of the lipid derivative by modifying the group R2. It should be un- derstood that R2 should be an organic radical having at least 2 carbon atoms) (such as an aliphatic group having a certain length (at least 2, preferably 9, carbon atoms)), a high degree of variability is possible, e.g. R2 need not necessarily to be a long chain residue, but may represent more complex structures.
Generally, it is believed that R2 may either be rather inert for the environment in which it can be liberated by extracellular PLA2 or that R2 may play an active pharmaceutical role, typically as an auxiliary drug substance or as an efficiency modifier for the lysol- ipid derivative and/or any other (second) drug substances present in the environment.
In some embodiments, the R1 and R2 groups will be long chain residues, e.g. a fatty acid residue (the fatty acid will include a carbonyl from the group Y). This has been described in detail above. Interesting examples of auxiliary drug substances as R2 within this subgroups are polyunsaturated acids, e.g. oleate, linoleic, linonleic, as well as derivatives of arachidonoyl (including the carbonyl from Y), e.g. prostaglandins such as prostaglandin E1, as arachidonic acid derivatives are know regulators of hormone ac- tion including the action of prostaglandins, thromboxanes, and leukotrines. Examples of efficiency modifiers as R2 are those which enhance the permeability of the target cell membrane as well as enhances the activity of extracellular PLA2 or the active drug substance or any second drug substances. Examples hereof are short chain (C8-12) fatty acids.
However, it is also envisaged that other groups might be useful as the organic radical R2, e.g. vitamin D derivatives, steroid derivatives, retinoic acid (including all-trans- retinoic acid, all-cis-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid), cholecalciferol and tocopherol analogues, pharmacologically active carboxylic acids such as branched-chain aliphatic carboxylic acids (e.g. valproic acid and those described in WO 99/02485), salicylic acids (e.g. acetylsalicylic acid), steroidal carboxylic acids (e.g. lysergic and isolysergic acids), monoheterocyclic carboxylic acids (e.g. nicotinic acid) and polyheterocyclic carboxylic acids (e.g. penicillins and cephalosporins), diclofenac, indomethacin, ibuprofen, naproxen, 6-methoxy-2-naphthylacetic acid.
It should be understood that the various examples of possible R2 groups are referred to by the name of a discrete species, rather than the name of the radical. Furthermore, it should be understood that the possible examples may include the carbonyl group or oxy group of the bond via which the organic radical is linked to the lipid skeleton (corre- sponding to "Y" in the formula above). This will of course be appreciated by the person skilled in the art.
Even though it has not specifically been indicated in the general formula for the suitable examples of lipid derivatives to be used within the present invention, it should be un- derstood that the glycol moiety of the lipid derivatives may be substituted, e.g. in order to modify the cleavage rate by extracellular PLA2 or simply in order to modify the properties of the liposomes comprising the lipid derivatives.
Some of the above defined lipid derivatives may already be known, but is believed that some subgroups thereof are uniquely novel compounds.
A particular group of novel compounds is lipid derivatives of the following formula:
CH2-X-R CH2-X-R I I
CH- Z-R3 or CH-Y-R2
I I
CH2-Y-R2 CH2-Z-R3
wherein
X and Z independently are selected from OC(O), O, CH2, NH, NMe, S, S(O), OS(O), S(O)2, OS(O)2, OP(O)2, OP(O)2O, OAs(O)2 and OAs(O)2O; preferably from O, NH, NMe and CH2, in particular O and CH2;
Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH2C(O)O, NC(O)O, Y then being connected to R2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom, preferably via the carbonyl carbon atom;
R1 is an aliphatic group of the formula Y1Y2;
R2 is an organic radical having at least 2 carbon atoms;
where Y1 is -(CH2)n1-(CH=CH)n2-(CH2)n3-(CH=CH)n4-(CH2)n5-(CH=CH)n6-(CH2)n7- (CH=CH)n8r-(CH2)n9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2nδ+n9 is an integer of from 2 to 29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 ; and each of n2, n4, n6 and nδ is independently zero or 1 ; and Y2 is CH3, OH, SH, S(O)2OH, P(O)2OH, OP(O)2OH, NH2 or CO2H; where each Y1 -Y2 independently may be substituted with halogen or C1-4-alkyl, but preferably Y1 -Y2 is unsubstituted, R3 is selected from derivatives of phosphatidic acid to which a hydrophilic polymer or polysaccharide is attached. The hydrophilic polymer or polysaccharide is typically and preferably selected from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polyme- thacrylamide, polydimethylacrylamide, and derivatised celluloses, in particular from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, and polyvinyl alcohol.
A particular subgroups are those wherein X and Z are O, R1 and R2 are independently selected from alkyl groups, (CH2)nCH3, where n is 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, preferably 14 or 16; Y is -OC(O)-, Y then being connected to R2 via the carbonyl carbon atom.
A specific group of compounds are polyethyleneoxide-i-O-palmityl-sn-2-palmitoyl- phosphatidyl ethanolamine, DPPE-PEG, and polyethyleneoxide-1-0-stearyl-s/>2- stearoylphosphatidylethanolamine, DSPE-PEG, with PEG molecular weight from 100 to 10000 Daltons, in particular from 300-5000 Daltons.
Furthermore, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a lipid derivative as defined above. Preferably, the lipid derivative in such a composition is dispersed in the form of a liposome (see below).
Also, the present invention relates to such lipid derivatives for use as a medicament, preferably present in a pharmaceutical composition, and to the use of a lipid derivative as defined above for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue. Such diseases or conditions are typically selected from wounds and inflammatory conditions. The present compositions and uses are especially applicable in the instances where the increase in extracellular PLA2 activity is at least 25% compared to the normal level of activity in the tissue in question, the tissue being that of a mammal, in particular a human. Lipid derivatives as prodrugs
As described above, the present invention provides a lipid-based drug delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a lipid derivative having (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
By the term "active drug substance" is meant any chemical entity which will provide a prophylactic or therapeutic effect in the body of a mammal, in particular a human. Thus, the present invention mainly relates to the therapeutic field.
The term "prodrug" should be understood in the normal sense, namely as a drug which is masked or protected with the purpose of being converted (typically by cleavage, but also by in vivo chemical conversion) to the intended drug substance. The person skilled in the art will recognise the scope of the term "prodrug".
The active drug substance is selected from lysolipid derivatives, and as it will be understood from the present description with claims, the lysolipid derivatives relevant within the present invention will have a therapeutic effect - at least - in connection with diseases and conditions where a local area of the body of the mammal has a level of extracellular PLA2 activity which can liberate the lysolipid derivative.
As will be understood from the present description with claims, the lipid derivative will often constitute the prodrug referred to above and the lysolipid derivative will thereby constitute the active drug substance often a monoether lysolipid derivative. It should however be understood that this does not exclude the possibility of including other drug substances, referred to as second drug substances, in the drug delivery systems of the invention, neither does it exclude that the organic radical which can be hydrolytically cleaved by the action of extracellular PLA2 can have a certain pharmaceutical effect
(e.g. as an auxiliary drug substance or an efficiency modifier as described elsewhere herein). Furthermore, the pharmaceutical effect of the "active drug substance", i.e. the lysolipid derivative, need not be the most predominant when a second drug substance is included, actually the effect of the second drug substance might very well be the most predominant as will become apparent in the other main embodiment (see "Lipid derivative liposomes as drug delivery systems", below).
The active drug substance (lysolipid derivative) release from the prodrug (lipid derivative) is believed to take place as illustrated in the following examples:
CH2-X-R1 CH2-X-R1 CH2-X-R1
I PLA2 I I
CH-Z-R3 — -> CH-Z-R3 or CH- Z-R3
I I I
CH2-Y-R2 CH2-OH CH2- CH2-COOH
prodrug drug drug
The substituent R2 may constitute an auxiliary drug substance or an efficiency modifier for the active drug substance and will simultaneously be released under the action of extracellular PLA2:
CH2-X-R1 I PLA2 CH- Z-R3 — > R2-OH or R2-COOH
I
CH2- Y-R2
prodrug drug drug
or CH2-X-R1 CH2-X-R1 CH2-X-R1
I PLA2 I ]
CH-Y-R2 — -> CHOH or CH-CH2-COOH
I I I CH2-Z-R3 CH2-Z-R3 CH2-Z-R3
prodrug drug drug
The substituent R2 may constitute an auxiliary drug substance or an efficiency modifier for the active drug substance and will simultaneously be released under the action of extracellular PLA2:
CH2-X-R I PLA2 CH- Y-R2 — > R2-OH or R2-COOH
I
CH2-Z-R3
prodrug drug drug
It has been described above under the definition of R2 how the group R2 can have various independent or synergistic effects in association with the active drug substance, e.g. as an auxiliary drug substance or an efficiency modifier, e.g. permeability or cell lysis modifier. It should be borne in mind that the groups corresponding to R2 (e.g. R2- OH or R2-COOH) might have a pharmaceutical effect which is predominant in relation the effect of the lysolipid derivative (active drug substance).
Lipid derivatives formulated as liposomes and micelles
The term "lipid-based drug delivery system" should encompass macromolecular struc- tures which as the main constituent include lipid or lipid derivatives. Suitable examples hereof are liposomes and micelles. It is presently believed that liposomes offer the broadest scope of applications and those have been described most detailed in the following. Although liposomes currently are believed to be the preferred lipid-based system, micellular systems are also believed to offer interesting embodiments within the present invention. In one important variant which advantageously can be combined with the embodiments described herein, the lipid derivative (e.g. the prodrug) is included in liposomes either as the only constituent or - which is more common - in combination with other constituents (other lipids, sterols, etc.). Thus, the lipid-based systems described herein are preferably in the form of liposomes, wherein the liposomes are build up of layers comprising the lipid derivative (e.g. a prodrug).
"Liposomes" are known as self-assembling structures comprising one or more lipid bilayers, each of which surrounds an aqueous compartment and comprises two opposing monolayers of amphipathic lipid molecules. Amphipathic lipids (i.e. lipid derivatives) comprise a polar (hydrophilic) headgroup region (corresponding to the substituent R3 in the lipid derivatives) covalently linked to one or two non-polar (hydrophobic) aliphatic groups (corresponding to R1 and R2 in the lipid derivatives). Energetically unfavourable contacts between the hydrophobic groups and the aqueous medium are generally believed to induce lipid molecules to rearrange such that the polar headgroups are oriented towards the aqueous medium while the hydrophobic groups reorient towards the interior of the bilayer. An energetically stable structure is formed in which the hydrophobic groups are effectively shielded from coming into contact with the aqueous medium.
Liposomes can have a single lipid bilayer (unilamellar liposomes, "ULVs"), or multiple lipid bilayers (multilamellar liposomes, "MLVs"), and can be made by a variety of methods (for a review, see, for example, Deamer and Uster, Liposomes, Marcel Dekker, N.Y., 1983, 27-52). These methods include Bangham's methods for making multilamel- lar liposomes (MLVs); Lenk's, Fountain's and Cullis' methods for making MLVs with substantially equal interlamellar solute distribution (see, e.g., US 4,522,803, US 4,588,578, US 5,030,453, US 5,169,637 and US 4,975,282); and Papahadjopoulos et al.'s reverse-phase evaporation method (US 4,235,871 ) for preparing oligolamellar liposomes. ULVs can be produced from MLVs by such methods as sonication (see Pa- pahadjopoulos et al., Biochem. Biophys. Acta, 135, 624 (1968)) or extrusion (US 5,008,050 and US 5,059,421). The liposome of this invention can be produced by the methods of any of these disclosures, the contents of which are incorporated herein by reference.
Various methodologies, such as sonication, homogenisation, French Press application and milling can be used to prepare liposomes of a smaller size from larger liposomes. Extrusion (see US 5,008,050) can be used to size reduce liposomes, that is to produce liposomes having a predetermined mean size by forcing the liposomes, under pressure, through filter pores of a defined, selected size. Tangential flow filtration (see WO 89/08846), can also be used to regularise the size of liposomes, that is, to produce Ii- posomes having a population of liposomes having less size heterogeneity, and a more homogeneous, defined size distribution. The contents of these documents are incorporated herein by reference. Liposome sizes can also be determined by a number of techniques, such as quasi-electric light scattering, and with equipment, e.g., Nicomp® particle sizers, well within the possession of ordinarily skilled artisans.
It is quite interesting to note that the lipid derivatives of the present invention can constitute the major part of a lipid-based system even if this system is a liposome system. This fact resides in the structural (but not functional) similarity between the lipid derivatives of the present invention and lipids. Thus, it is believed that the lipid derivatives for the present invention can be the sole constituent of liposomes, i.e. up to 100 mol% of the total dehydrated liposomes can be constituted by the lipid derivatives. This is in contrast to the known lysolipids, which can only constitute a minor part of the liposomes.
Typically, as will be described in detail below, liposomes advantageously include other constituents which may or may not have a pharmaceutical effect, but which will render the liposome structure more stable (or alternatively more unstable) or will protect the liposomes against clearance and will thereby increase the circulation time thereby improving the overall efficiency of a pharmaceutical including the liposome.
This being said, it is believed that the particular lipid derivatives will typically constitute from 5-100 mol%, such as 50-100 mol%, preferably from 75-100 mol%, in particular 90-100 mol%, based on the total dehydrated liposome.
The liposomes can be unilamellar or multilamellar. Some preferred liposomes are unilamellar and have diameters of less than about 400 nm, more preferably, from greater than about 40 nm to less than about 400 nm.
The liposomes are typically - as known in the art - prepared by a method comprising the steps of: (a) dissolving the lipid derivative in an organic solvent; (b) removing the organic solvent from the lipid derivative solution of step (a); and (c) hydrating the product of step (b) with an aqueous solvent so as to form liposomes.
The method may further comprise a step of adding an second drug substance (see be- low) to the organic solvent of step (a) or the aqueous phase of step (c).
Subsequently, the method may comprise a step of extruding the liposomes produced in step (c) through a filter to produce liposomes of a certain size, e.g. 100 nm.
Lipid-based particulate systems, i.e. liposomes as well as micelles; of sizes covering a broad range may be prepared according to the above-mentioned techniques. Depending on the route of administration, suitable sizes for pharmaceutical applications will normally be in the range of 20-10,000 nm, in particular in the range of 30-1000 nm. Sizes in the range of 50-200 nm are normally preferred because liposomes in this size range are generally believed to circulate longer in the vascular system of mammals than do larger liposomes which are more quickly recognised by the mammals' reticuloendothelial systems ("RES"), and hence, more quickly cleared from the circulation. Longer vascular circulation can enhance therapeutic efficacy by allowing more liposomes to reach their intended site of actions, e.g., tumours or inflammations.
It is believed that for a drug delivery system as defined in the embodiments herein, which is adapted to be administered via intraveneous and intramuscular injection, the liposomes should preferably have a mean particle size of about 100 nm. Thus, the particle size should generally be in the range of 40-400 nm.
Furthermore, for a drug delivery system adapted to be administered via subcutaneous injection, the liposomes should preferably have a mean particle size from 100 to 5000 nm, and the liposomes can then be uni- or multilayered.
One of the advantages by including the lipid derivatives in liposomes is that the liposome structure, in particular when stabilised as described in the following, will have a much longer vascular circulation time that the lipid derivatives as discrete compounds. Furthermore, the lipid derivatives will become more or less inert or even "invisible" when "packed" in liposomes in which lipopolymers or glycolipids are included. This means than any potential disadvantageous effect, e.g. hemolytic effect, can be suppressed. The liposomes should preferably act as inert constituents until they reach the area of interest, e.g. cancerous, infected or inflammatorily diseased areas or tissue. As will be described in the following, liposomes may include a number of other constituents. In particular, a drug delivery system according to the invention may further contain a component which controls the release of any second drug substance, extracellular PLA2 activity controlling agents or permeability enhancer, e.g. short chain lipids and lipopolymers/glycolipids.
Two very important groups of compounds to be included in liposomes as modifiers are the stabilising compound lipopolymers and glycolipids, such as lipopolymers (e.g. poly- ethyleneoxide-dipalmitoylphosphatidyl ethanolamine, DPPE-PEG, polyethyleneoxide- distearoylphosphatidylethanolamine, DSPE-PEG) with PEG molecular weight from 100 to 10000 Daltons. It has been shown that lipopolymers function as stabilisers for the liposome, i.e. lipopolymer increases the circulation time, and - which is highly interest- ing in the present context, as activators for extracellular PLA2. The stabilising effect will be described in the following.
Liposome outer surfaces are believed to become coated with serum proteins, such as opsonins, in mammals' circulatory systems. Without intending in any way to be limited by any particular theory, it is believed that liposome clearance can be inhibited by modifying the outer surface of liposomes such that binding of serum proteins thereto is generally inhibited. Effective surface modification, that is, alterations to the outer surfaces of liposomes which result in inhibition of opsonisation and RES uptake, is believed to be accomplished by incorporating into liposomal bilayers lipids whose polar headgroups have been derivatised by attachment thereto of a chemical moiety which can inhibit the binding of serum proteins to liposomes such that the pharmacokinetic behaviour of the liposomes in the circulatory systems of mammals is altered and the activity of extracellular PLA2 is enhanced as described for the lipopolymers above.
Liposome preparations have been devised which avoid rapid RES uptake and which thus have an increased half-life in the bloodstream. STEALTH® liposomes (Liposome Technology Inc., Menlo Park, Calif.) include polyethyleneglycol (PEG)-grafted lipids at about 5 mol% of the total dehydrated liposome . The presence of polymers on the exterior liposome surface decreases the uptake of liposomes by the organs of the RES. The liposome membranes can be constructed so as to resist the disruptive effects of the surfactant contained therein. For example, a liposome membrane which contains as constituents lipids derivatised with a hydrophilic (i.e., water-soluble) polymer normally has increased stability. The polymer component of the lipid bilayer protects the liposome from uptake by the RES, and thus the circulation time of the liposomes in the bloodstream is extended.
Hydrophilic polymers suitable for use in lipopolymers are those which are readily water- soluble, can be covalently attached to a vesicle-forming lipid, and which are tolerated in vivo without toxic effects (i.e., are biocompatible). Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycolic acid (also termed polyglycolide), a polylactic-polyglycolic acid copolymer, and polyvinyl alcohol. Preferred polymers are those having a molecular weight of from about 100 or 120 daltons up to about 5,000 or 10,000 daltons, and more preferably from about 300 daltons to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethylenegly- col having a molecular weight of from about 100 to about 5,000 daltons, and more preferably having a molecular weight of from about 300 to about 5,000 daltons. In a particularly preferred embodiment, the polymer is polyethyleneglycol of 750 daltons (PEG(750)). Polymers may also be defined by the number of monomers therein; a preferred embodiment of the present invention utilises polymers of at least about three monomers, such PEG polymers consisting of three monomers (approximately 150 daltons). Other hydrophilic polymers which may be suitable for use in the present invention include polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhy- droxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatised celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
Glycolipids are lipids to which a hydrophilic polysaccharide chain is covalently attached. It will be appreciated that glycolipids can be utilised like lipopolymers although the lipopolymers currently presents the most promising results.
It is generally believed that the content of lipopolymer advantageously will be in the range of 1-50 mol%, such as 2-25%, in particular 2-15 mol%, based on the total dehydrated liposome.
The liposomes' bi- or multilayers may also contain other constituents such as other lipids, sterolic compounds, polymer-ceramides as stabilisers and targeting compounds, etc. The liposomes comprising lipid derivatives may (in principle) exclusively consist of the lipid derivatives. However, in order to modify the liposomes, "other lipids" may be included as well. Other lipids are selected for their ability to adapt compatible packing conformations with the lipid derivative components of the bilayer such that the all the lipid constituents are tightly packed, and release of the lipid derivatives from the bilayer is inhibited. Lipid-based factors contributing to compatible packing conformations are well known to ordinarily skilled artisans and include, without limitation, acyl chain length and degree of unsaturation, as well as the headgroup size and charge. Accordingly, suitable other lipids, including various phosphatidylethanolamines ("PE's") such as egg phosphatidylethanolamine ("EPE") or dioleoyl phosphatidylethanolamine ("DOPE"), can be selected by ordinarily skilled artisans without undue experimentation. Lipids may be modified in various way, e.g. by headgroup derivatisation with dicarboxylic acids, such as glutaric, sebacic, succinic and tartaric acids, preferably the dicarboxylic acid is glutaric acid ("GA"). Accordingly, suitable headgroup-derivatised lipids include phosphatidylethanolamine-dicarboxylic acids such as dipalmitoyl phosphatidyl- ethanolamine-glutaric acid ("DPPE-GA"), palmitoyloleoyl phosphatidylethanolamine- glutaric acid ("POPE-GA") and dioleoyl phosphatidylethanolamine-glutaric acid ("DOPE-GA"). Most preferably, the derivatised lipid is DOPE-GA.
The total content of "other lipids" will typically be in the range of 0-30 mol%, in particular 1-10 mol%, based on the total dehydrated liposome.
Sterolic compound included in the liposome may generally affects the fluidity of lipid bi- layers. Accordingly, sterol interactions with surrounding hydrocarbon groups generally inhibit emigration of these groups from the bilayer. An examples of a sterolic compound (sterol) to be included in the liposome is cholesterol, but a variety of other sterolic compounds are possible. It is generally believed that the content of sterolic compound, if present, will be in the range of 0-25 mol%, in particular 0-10 mol%, such as 0-5 mol%, based on the total dehydrated liposome.
Polymer-ceramides are stabilisers improving the vascular circulation time. Examples are polyethylene glycol derivatives of ceramides (PEG-ceramides), in particular those where the molecular weight of the polyethylene glycol is from 100 to 5000. It is generally believed that the content of polymer-ceramides, will be in the range of 0-30 mol%, in particular 0-10 mol%, based on the total dehydrated liposome. Still other ingredients may constitute 0-2 mol%, in particular 0-1 mol%, based on the total dehydrated liposome.
According to an embodiment of the present invention, the lipid bilayer of a liposome contains lipids derivatised with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the lipid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the lipid bilayer into the surrounding environment (see e.g. US 5,882,679 and Fig. 1).
A variety of coupling methods for preparing a vesicle-forming lipid derivatised with a biocompatible, hydrophilic polymer such as polyethylene glycol are known in the art (see, e.g., US 5,213,804; US 5,013,556).
The derivatised lipid components of liposomes according to the present invention may additionally include a labile lipid-polymer linkage, such as a peptide, ester, or disulfide linkage, which can be cleaved under selective patophysiological conditions, such as in the presence of overexpressed peptidase or esterase enzymes at diseased sites or reducing agents. Use of such linkages to couple polymers to phospholipids allows the attainment of high blood levels of such liposomes for several hours after administration, followed by cleavage of the reversible linkages and removal of the polymer from the exterior liposome bilayer. The polymer-less liposomes are then subject to rapid uptake by the RES system (see, e.g., US 5,356,633).
Additionally, liposomes according to the present invention may contain non-polymer molecules bound to the exterior of the liposome, such as haptens, enzymes, antibodies or antibody fragments, cytokines and hormones (see, e.g., US 5,527,528), and other small proteins, polypeptides, single sugar polysaccharide moieties, or non-protein molecules which confer a particular enzymatic or surface recognition feature to the liposome. See published PCT application WO 94/21235. Surface molecules which preferentially target the liposome to specific organs or cell types are referred to herein as "targeting molecules" and include, for example, antibodies and sugar moieties, e.g. gangliosides or those based on mannose and galactose, which target the liposome to specific cells bearing specific antigens (receptors for sugar moieties). Techniques for coupling surface molecules to liposomes are known in the art (see, e.g., US 4,762,915). The liposome can be dehydrated, stored and then reconstituted such that a substantial portion of its internal contents is retained. Liposomal dehydration generally requires use of a hydrophilic drying protectant such as a disaccharide sugar at both the inside and outside surfaces of the liposome bilayers (see US 4,880,635). This hydrophilic compound is generally believed to prevent the rearrangement of the lipids in the liposome, so that the size and contents are maintained during the drying procedure and through subsequent rehydration. Appropriate qualities for such drying protectants are that they are strong hydrogen bond acceptors, and possess stereochemical features that preserve the intramolecular spacing of the liposome bilayer components. Alterna- tively, the drying protectant can be omitted if the liposome preparation is not frozen prior to dehydration, and sufficient water remains in the preparation subsequent to dehydration.
Lipid derivative liposomes as drug carrier systems As mentioned above, the liposomes including the lipid derivatives of the present invention may also include second drug substances. In a particular embodiment, the lipid- based drug delivery system described above is in the form of liposomes wherein a second drug substance is incorporated. It should be understood that second drug substances may comprise pharmaceutically active ingredients which may have an individ- ual or synergistic pharmaceutical effect in combination with the lipid derivative and Iy- solipid derivatives. The term "second" does not necessarily imply that the pharmaceutical effect of the second drug substance is inferior in relation to that of, e.g., the active drug substance derived from the prodrug, but is merely used to differentiate between the two groups of substances.
This being said, the present invention also provides a drug delivery system which is in the form of liposomes, and wherein a second drug substance is incorporated.
A possible "second drug substance" is any compound or composition of matter that can be administered to mammals, preferably humans. Such agents can have biological activity in mammals. Second drug substances which may be associated with liposomes include, but are not limited to: wound healing agents such as sphingosine 1 -phosphate analogs, antiviral agents such as acyclovir, zidovudine and the interferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene antibiotics, imidazoles and triazoles; antimetabolic agents such as fo- lie acid, and purine and pyrimidine analogs; antineoplastic agents such as the anthra- cycline antibiotics and plant alkaloids; sterols such as cholesterol; carbohydrates, e.g., sugars and starches; amino acids, peptides, proteins such as cell receptor proteins, immunoglobulins, enzymes, hormones, neurotransmitters and glycoproteins; dyes; radiolabels such as radioisotopes and radioisotope-labeled compounds; radiopaque compounds; fluorescent compounds; mydriatic compounds; bronchodilators; local anesthetics; and the like.
Liposomal second drug substance formulations enhance the therapeutic index of the second drug substances by reducing the toxicity of the drug. Liposomes can also reduce the rate at which a second drug substance is cleared from the vascular circulation of mammals. Accordingly, liposomal formulation of second drug substance can mean that less of the drug need be administered to achieve the desired effect.
Liposomes can be loaded with one or more second drug substances by solubilising the drug in the lipid or aqueous phase used to prepare the liposomes. Alternatively, ionis- able second drug substances can be loaded into liposomes by first forming the liposomes, establishing an electrochemical potential, e.g., by way of a pH gradient, across the outermost liposomal bilayer, and then adding the ionisable second drug substance to the aqueous medium external to the liposome (see, e.g., US 5,077,056 and WO 86/01102).
Methods of preparing lipophilic drug derivatives which are suitable for liposome or micelle formulation are known in the art (see e.g., US 5,534,499 and US 6,118,011 de- scribing covalent attachment of therapeutic agents to a fatty acid chain of a phospholipid). A micellar formulation of taxol is described in Alkan-Onkyuksel et al., Pharmaceutical Research, 11 :206 (1994).
Accordingly, the second drug substance may be any of a wide variety of known and possible pharmaceutically active ingredients, but is preferably a therapeutically and/or prophylactically active substance. Due to the mechanism involved in the degradation of the liposomes of the present invention, it is preferred that the second drug substance is one relating to diseases and/or conditions associated with a localised increase in extracellular PLA2 activity. Particularly interesting second drug substances are selected from (i) wound healing agents (ii) antitumour agents such as anthracyline derivatives, cisplatin, paclitaxel, 5- fluoruracil, exisulind, cis-retinoic acid, suldinac sulfide, vincristine, interleukins, oligonucleotides, peptides, proteins and cytokines (iii) antibiotics and antifungals, (iv) antiinflammatory agents such as steroids and non-steroids, and (v) peroxisome proliferator activated receptor ligands. In particular the steroids can also have a stabilising effect on the liposomes.
Also active agents like peptides and protein derivatives like interferons, interleukins and oligonucleotides can be incorporated into the PLA2 degradable lipid-based carrier.
The cytotoxic effects of a broad range of anticancer agents are likely to improve when encapsulated in the carriers of this invention. Furthermore, it is expected that the hydrolysis products, i.e. monoether lysolipids and/or ester-linked lysolipid derivatives, act in turn with the released fatty acid derivatives as absorption enhancers for drug permeation across the target membranes when the carriers locally are broken down in the diseased tissue.
It is envisaged that the second drug substance will be distributed in the liposomes ac- cording to their hydrophilicity, i.e. hydrophilic second drug substances will tend to be present in the cavity of the liposomes and hydrophobic second drug substances will tend to be present in the hydrophobic bilayer. Method for incorporation of second drug substances are know in the art as has been made clear above.
It should be understood from the above, that the lipid derivatives may - as prodrugs or discrete constituents - posses a pharmaceutical activity. However, in a particular embodiment, the present invention furthermore relates to a lipid based drug delivery system for administration of an second drug substance, wherein the second drug substance is incorporated in the system (e.g. where the second drug substance is encap- sulated in the interior of the liposome or in the membrane part of the liposome or the core region of micelle), said system including lipid derivatives which has (a) an aliphatic group of a length of at least 3 carbon atoms and an organic radical having at least 3 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
As mentioned above for the system according to the other embodiment, the organic radical which can be hydrolytically cleaved off, may be an auxiliary drug substance or an efficiency modifier for the second drug substance. It should be understood that the lipid derivative is a lipid derivative as defined further above. Typically, the lipid derivative constitutes 5-100 mol%, such as 50-100 mol%, of the total dehydrated (liposome) system.
As should be understood from the above, the present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and any of the lipid-based drug delivery systems described above. The composition will be described in detail below.
The present invention also relates to the use of any of the lipid-based drug delivery systems described herein as a medicament, and to the use of any of the lipid-based drug delivery systems described herein for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue. Such diseases or conditions are typically selected from cancer, e.g. a brain, breast, lung, colon or ovarian cancer, or a leukemia, lymphoma, sarcoma, carcinoma and inflammatory conditions . Also included is the prophylactic use. The present compositions and uses are especially applicable in the instances the increase in extracellular PLA2 activity is at least 25% compared to the normal level of activity in the tissue in question, the tissue being that of a mammal, in particular a human.
It should be mentioned that the novel and unnatural lipid analogs can be administred in a non-particulate form as free agents leading to an increased intracellular cell uptake rendering them favourable substrates for overexpressed intracellular PLA2 in the diseased target cells.
Pharmaceutical preparations and therapeutic uses
Also provided herewith is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the lipid derivative, e.g. as a liposome, of this invention. "Pharmaceutically acceptable carriers" as used herein are those media generally acceptable for use in connection with the administration of lipids and liposomes, including liposomal drug formulations, to mammals, including humans. Pharmaceutically acceptable carriers are generally formulated according to a number of factors well within the purview of the ordinarily skilled artisan to determine and account for, including without limitation: the particular active drug substance and/or second drug substance used, the liposome preparation, its concentration, stability and intended bioavailability; the disease, disorder or condition being treated with the liposomal composition; the subject, its age, size and general condition; and the composition's intended route of administration, e.g., nasal, oral, ophthalmic, subcutaneous, intramammary, intraperitoneal, intravenous, or intramuscular. Typical pharmaceutically acceptable carriers used in parenteral drug administration include, for example, D5W, an aqueous solution containing 5% weight by volume of dextrose, and physiological saline. Pharmaceutically acceptable carriers can contain additional ingredients, for example those which enhance the stability of the active ingredients included, such as preservatives and anti-oxidants.
The liposome or lipid derivative is typically formulated in a dispersion medium, e.g. a pharmaceutically acceptable aqueous medium.
An amount of the composition comprising an anticancer effective amount of the lipid derivative, typically from about 0.1 to about 1000 mg of the lipid derivative per kg of the mammal's body, is administered, preferably intravenously. For the purposes of this invention, "anticancer effective amounts" of liposomal lipid derivatives are amounts effective to inhibit, ameliorate, lessen or prevent establishment, growth, metastasis or inva- sion of one or more cancers in mammals to which the lipid derivatives have been administered. Anticancer effective amounts are generally chosen in accordance with a number of factors, e.g., the age, size and general condition of the subject, the cancer being treated and the intended route of administration, and determined by a variety of means, for example, dose ranging trials, well known to, and readily practised by, ordi- narily skilled artisans given the teachings of this invention. Antineoplastic effective amounts of the liposomal drugs/prodrugs of this invention are about the same as such amounts of free, nonliposomal, drugs/prodrugs, e.g., from about 0.1 mg of the lipid derivative per kg of body weight of the mammal being treated to about 1000 mg per kg.
Preferably, the liposome administered is a unilamellar liposome having an average diameter of from about 50 nm to about 200 nm. The anti-cancer treatment method can include administration of one or more second drug substances in addition to the liposomal drug, these additional agents being included in the same liposome as the lipid derivative. The second drug substances, which can be entrapped in liposomes' internal compartments or sequestered in their lipid bilayers, are preferably, but not necessarily, anticancer agents.
The pharmaceutical composition is preferably administered parenteral^ by injection, infusion or implantation (intravenous, intramuscular, intraarticular, subcutaneous or the like) in dosage forms, formulations or e.g. suitable delivery devices or implants contain- ing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
The formulation and preparation of such compositions is well-known to those skilled in the art of pharmaceutical formulation. Specific formulations can be found in the textbook entitled "Remington's Pharmaceutical Sciences".
Thus, the pharmaceutical compositions according to the invention may comprise the active drug substances in the form of a sterile injection. To prepare such a composition, the suitable active drug substances are dispersed in a parenterally acceptable liquid vehicle which conveniently may comprise suspending, solubilising, stabilising, pH- adjusting agents and/or dispersing agents. Among acceptable vehicles that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1 ,3-butanediol, Ringer's solution and isotonic sodium chloride solution. The aqueous formulation may also contain one or more preservatives, for example, methyl, ethyl or n-propyl p- hydroxybenzoate.
Where systemic treatment of inflammatory diseases is desired, effective delivery of a liposome-encapsulated drug via the bloodstream requires that the liposome be able to penetrate the continuous (but "leaky") endothelial layer and underlying basement membrane surrounding the vessels supplying blood to a tumour. Liposomes of smaller sizes have been found to be more effective at extravasation into tumours through the endothelial cell barrier and underlying basement membrane which separates a capillary from tumour cells.
In accordance with the present invention, the second drug agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers. The resulting liposomal formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration. Inflammatory conditions characterised by an acute increase in permeability of the vasculature in the region of 5 the inflamed regions are particularly suited for treatment by the present methods. The liposomes will eventually degrade due to lipase action at the tumour site, or can be made permeable by, for example, thermal or ultrasonic radiation. The drug is then released in a bioavailable, transportable solubilised form. Furthermore, a small elevation in temperature as often seen in diseased tissue may further increase the stimulation of 10 extracellular PLA2.
Where site-specific treatment of inflammation is desired, effective liposome delivery of an drug requires that the liposome have a long blood half-life, and be capable of penetrating the continuous endothelial cell layer and underlying basement membrane sur-
15 rounding blood vessels adjacent to the site of inflammation. Liposomes of smaller sizes have been found to be more effective at extravasation through the endothelial cell barrier and into associated inflamed regions. However, the limited drug-carrying capacity of conventional small liposome preparations has limited their effectiveness for such purposes.
20
In accordance with the present invention, the anti-inflammatory agent of choice is entrapped within a liposome according to the present invention; the liposomes are formulated to be of a size known to penetrate the endothelial and basement membrane barriers. The resulting liposomal formulation can be administered parenterally to a subject
25 in need of such treatment, preferably by intravenous administration. Inflamed regions characterised by an acute increase in permeability of the vasculature in the region of inflammation are particularly suited for treatment by the present methods.
It is known that the activity of extracellular PLA2 is abnormally high in areas of the 30 mammalian body diseased by cancer, inflammation, etc. The present invention have provided a way of exploiting this fact, and it is believed that the extracellular PLA2 activity should be at least 25% higher in the diseases area of the body (determined in the extracellular environment) compared with a comparative normal area. It is however envisaged that the level of extracellular PLA2 activity often is much higher, e.g. at least 35 100%, e.g. at least 200% such as at least 400%. This means that treatment of a mammal in need of a treatment with the purpose of cure or relief, can be conducted with only minimal influence on tissue having a "normal" level of extracellular PLA2 activity. This is extremely relevant in particular with the treatment of inflammatory conditions where rather harsh drug (second drug substances) are often needed.
Residing in the realisations behind the present invention, the invention thus provides to a method for selectively drug targeting to diseased areas, such as areas comprising inflamed cells, e.g., areas within the mammalian body, preferably a human, having a extracellular phospholipase A2 (extracellular PLA2) activity which is at least 25% higher compared to the normal activity in said areas, by administering to the mammal in need thereof an efficient amount of a drug delivery system defined herein.
Provided is also a method of treating of a mammal afflicted with a cancer, e.g., a brain, breast, lung, colon or ovarian cancer, or a leukemia, lymphoma, sarcoma, carcinoma, which comprises administering a pharmaceutical composition of this invention to the mammal. It is believed that the lipid derivatives and/or second drug substance in liposome form is selectively cytotoxic to tumour cells.
Targeting of diseased sites in the skin with lipid-based carriers composed of the novel and unnatural lipids The novel targeted liposomal prodrug and drug delivery systems are useful in the treatment or alleviation of disorders in the skin that are associated with or resulting from increased levels of extracellular PLA2. In particular, the lipid-based prodrug and drug delivery systems are useful for the administration of an active drug substance selected from ether-lysolipid and/or fatty acid derivatives of a lipid prodrug being a sub- strate for extracellular PLA2.
Degradation of the flexible liposomal carrier by PLA2 leads to the release of lysolipid and/or fatty acid derivatives, which are designed to be effective in the treatment of various types of skin diseases such as inflammation. In addition, the novel prodrug lipo- somal carriers can be used for targeted delivery of conventional drugs to diseased regions of the skin that is associated with an elevated level of PLA2.
There are a large number of skin diseases that are either skin-specific, e.g. inflammation, psoriasis, and eczemas, or are manifestations of general inflammatory diseases. A number of these diseases confined to the skin or mucous membranes could be cured or alleviated by a local topical application of pharmaceutical formulations provided that the application results in an efficient uptake of the active component. Many formulations are applied topically to the skin for therapeutic or preventive purposes. However, the skin forms one of the body's most effective barriers to foreign substances and ow- 5 ing to skin impermeability, many therapeutic agents must be applied per os or par- enterally even though the skin is the target organ.
Liposomal formulations have been the focus of extensive investigation as the mode of skin delivery for many drugs. There is growing evidence that for topical administration, 10 a new generation of flexible liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, into the skin.
15
The major physico-chemical barrier of the skin is localized in the outermost cornified layer of the skin, the stratum corneum. The pores in the stratum corneum and the underlaying structures are normally so narrow, that the skin only allows passage of entities smaller than 400 Dalton. However, it has been shown that ultraflexible liposomes 20 are able to pass pores in the skin smaller than 30 nm (Cevc et al. (Biochem Biophys Acta (1998) 1368, p 201-215, US Patent 6.180.353).
As important as getting the substance into the skin is the issue of ensuring that predominantly the relevant cells are exposed to the pharmaceutical active components.
25 The novel targeted prodrug system addresses both the important issue of ensuring that predominantly the relevant cells are exposed to the drugs, and the fact that only very flexible liposomes that contains "edge active substances" are able to pass through the stratum corneum and penetrate deep into diseased regions of the skin with elevated levels of PLA2. It is important to realize that the prodrug liposomes are not intended for
30 application of drugs into the blood via the skin.
It has been found that the activity of extracellular phospholipase A2 (PLA2) is increased in a number of inflammatory diseases of the skin most notably psoreasis and eczema (Forster et al. (1985) Br J Dermatol 112:135-47; Forster et al. (1983) Br J Dermatol 35 108:103-5). It has furthermore been found that extracellular PLA2 is capable of cleaving the prodrug lipid derivatives so as to produce ether-lysolipid derivatives, which alone or in combination with other active compounds, will exhibit an effect. Thus the new prodrug liposomes ensure that the pharmaceutical active ether-lysolipids can be delivered specifically at the target cells in the skin using elevated activity of PLA2 as a site- specific trigger mechanism. In addition, specific fatty acid derivatives, which are turned into active drugs by PLA2 hydrolysis may be linked to the C-3 position of the lipid-based carrier composed of the novel unnatural lipids. Possible examples include polysatu- rated fatty acids and other lipophilic groups such as vitamin D derivatives, steroid derivatives, retinoyl derivatives, and tocopherol analogues that can be ester bound to the C-3 position and therefore renders the double prodrug lipid a substrate for PLA2. Fur- thermore, conventional drug substances can be incorporated and transported specifically to the diseased site by the novel lipid-based carriers.
Thus the new carrier liposomes also offer a solution to both the issue of penetration of intact skin and the issue of specific targeting of cells, tissues or part of tissues in the skin that are characterized by an increased level of PLA2.
Targeting of infected tissues with lipid-based carriers composed of the novel and unnatural lipids
One of the important features of the targeted delivery systyem is the rapid uptake of liposomes in vivo by cells of the mononuclear phagocytic system (MPS). The MPS comprises the macrophages, one of the most important components of the immune system involved in the clearance of foreign particles, including liposomes. The macrophages reside in various organs and tissues, e.g. in the spleen and liver (Kupffer cells) and as free and fixed macrophages in the bone marrow and lymph nodes.
The novel lipid-based system composed of prodrug lipids is able to deliver various lipid prodrugs and encapsulated drugs directly to the diseased liver or spleen harbouring parasites due to an accumulation of the liposomes and an increased level of the prodrug cleaving PLA2 enzyme in the infected tissues. One particular advantage of the lipid based drug delivery system is furthermore that extracellular PLA2 activity is significantly increased towards organized lipid substrates such as the prodrug liposomes as compared to monomeric lipid substrates. The released ether-lipid analogues that are generated by the action of PLA2 have been shown to result in perturbation of key regulatory enzymes in parasites such as Leishmania and Trypanosoma (Lux et al. Biochem. Parasitology 111 (2000) 1-14) and are therefore toxic to the parasites if administered in sufficient amount. As the liver, spleen and bone marrow are organs and tissues wherein high concentrations of macrophages can be found, it is possible to achieve a targeted delivery of the toxic drugs to parasites inhabiting these organs. Furthermore, parasitic infections, which are characterized by elevated levels of PLA2, such as the malaria causing parasites, are also targets for treatment with the novel prodrug liposomes. The elevated levels of PLA2 in malaria infections caused by the parasite Plasmodium Falciparum has been described by e.g. Vadas et al. (Infection and Immunity, 60 (1992) 3928-3931 and Am. J. Trop. Hyg. 49 (1993) 455-459).
Normal cells typically possess ether-cleavage enzymes, which enable them to avoid the toxic effect of ether-lipids. However, some normal cells such as red blood cells have like cancer cells no means of avoiding the disruptive effect of the ether-lipids. Accordingly, therapeutic use of ether-lipids requires an effective drug delivery system that protects the normal cells from the toxic effects and is able to bring the ether-lipids directly to the diseased tissue.
Thus, the novel targeted prodrug delivery systems can be used for the treatment of parasitic infections, which is characterized by an increased level of PLA2 in the infected tissue. This is achieved by administering an efficient amount (up to about 1000 mg per kg) of the liposomes wherein the active drug substance is present in the form of a lipid prodrug being a substrate for extracellular PLA2. In addition, the prodrug liposomes are candidates for targeted transport of encapsulated conventional anti-parasitic drug(s), where a potentiation of the effectiveness of the conventional drug(s) in combination with the PLA2 generated lysolipids and/or fatty acid derivatives might be obtainable.
Toxicity
Toxicity of the liposomes comprising the lipid derivatives can be assessed by determining the therapeutic window "TW", which is a numerical value derived from the relationship between the compound's induction of hemolysis and its ability to inhibit the growth of tumour cells. TW values are defined as HI5/GI50 (wherein "Hl5" equals the concentra- tion of compound inducing the hemolysis of 5% of the red blood cells in a culture, and wherein "Gl50" equals the dose of compound inducing fifty percent growth inhibition in a population of cells exposed to the agent). The higher an agent's Hl5 value, the less hemolytic is the agent - higher Hl5 's mean that greater concentrations of compound are required to be present in order for the compound to induce 5% hemolysis. Hence, the higher its Hl5, the more therapeutically beneficial is a compound, because more of it can be given before inducing the same amount of hemolysis as an agent with a lower Hl5. By contrast, lower Gl50 's indicate better therapeutic agents - a lower Gl50 value indicates that a lesser concentration of an agent is required for 50% growth inhibition. Accordingly, the higher is its Hl5 value and the lower is its Gl50 value, the better are a compound's agent's therapeutic properties.
Generally, when a drug's TW is less than 1 , it cannot be used effectively as a therapeutic agent. That is, the agent's Hl5 value is sufficiently low, and its Gl50 value sufficiently high, that it is generally not possible to administer enough of the agent to achieve a suf- ficient level of tumour growth inhibition without also attaining an unacceptable level of hemolysis. As the lipid derivative liposomes take advantage of the lower extracellular PLA2 activity in the bloodstream compared to the activity in the diseased tissue, it is believed that the TW will be much higher that for normal monoether lysolipids. As the variance in activity is in orders of magnitude and as the liposomes will be "trapped" in tissue with a high extracellular PLA2 activity, it is generally believed the TW of the liposomes of the invention will be greater than about 3, more preferably greater than about 5, and still more preferably greater than about 8.
The invention will be illustrated by the following non-limiting example.
EXAMPLE
Lysophosphatidic acid (LPA) is a wound healing compound that can be delivered using the novel PLA2 degradable liposomes containing proLPA. Made as a lipid prodrug of LPA, this lipid has an ester bound acyl chain in the sn-2 position, which is cleaved by PLA2. LPA itself has growth-promoting effects on several cell types, including endothelial cells, fibroblasts, keratinocytes, cancer cells etc., and has growth promoting effects on cells as showed in Fig. 4. LPA stimulated cell growth of NIH 3T3 cells to a level comparable to the growth stimulation seen by addition of 10 % serum, whereas HU- VEC cells were stimulated even more strongly than addition of 10 % serum, during 24 h. ProLPA liposomes were made by sonication in PBS and added to the cell culture as indicated in Fig. 5 (in the presence or absence of the specific sPLA2 inhibitor) in conditioned media from Colo 205 cells with a PLA2 concentration of approximately 30 ng/ml.
HUVEC cells were clearly growth-stimulated by incubation with proLPA-liposomes in the presence of PLA2. This effect was reduced when a specific sPLA2 inhibitor was added, proving that PLA2 is required for generation of LPA from proLPA liposomes. A growth stimulation was seen also in the presence of the PLA2 inhibitor. This could indicate that the hydrolysis of the proLPA liposomes by PLA2 is very fast and efficient, and that some hydrolysis occurs, even in the presence of the inhibitor. Alternatively, other secretory PLA2 lipases with less specificity for the inhibitor could be secreted by the HUVEC cells. Similar data was seen with the NIH 3T3 cells upon addition of proLPA liposomes.

Claims

1. A lipid based drug delivery system for administration of a drug substance, wherein the drug substance is incorporated in the system, said system including lipid deriva- tives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, said system having included therein lipopolymers or glycolipids so as to present hydrophilic chains on the surface of the system.
2. The lipid based drug delivery system according to claim 1 , wherein the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment.
3. A drug delivery system according to claim 1 , wherein the lipopolymers or glycolipids are represented by a fraction of the lipid derivative.
4. A drug delivery system according to any of the claims 1-3, wherein the polymer of the lipopolymer is selected from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatised celluloses.
5. A drug delivery system according to any of the claims 1-4, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the second drug substance.
6. A drug delivery system according to any of the claims 1-5, wherein the lipid deriva- tive is a lipid derivative of the following formula: CH2-X-R CH2-X-R
I I
CH- Z-R3 or CH-Y-R2
CH2-Y-R2 CH2-Z-R3
wherein
X and Z independently are selected from O, CH2, NH, NMe, S, S(O), OS(O), S(O)2, OS(O)2, OP(O)2, OP(O)2O, OAs(O)2 and OAs(O)2O;
Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH2C(O)O, NC(O)O, Y then being connected to R2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom;
R1 is an aliphatic group of the formula Y1Y2;
R2 is an organic radical having at least 2 carbon atoms;
where Y1 is -(CH2)n1-(CH=CH)n2-(CH2)n3-(CH=CH)n4-(CH2)n5-(CH=CH)n6-(CH2)n7- (CH=CH)n8r-(CH2)n9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 2 to 29; n1 is zero or an integer from 1 to 29, n3 is zero or an integer from 1 to
20, n5 is zero or an integer from 1 to 17, n7 is zero or an integer from 1 to 14, and n9 is zero or an integer from 1 to 11; and each of n2, n4, n6 and n8 is independently zero or
1 ; and Y2 is CH3, CO2H, SH, S(O)2OH, P(O)2OH, OP(O)2OH, OH, NH2; where each carbon of Y1 -Y2 independently may be substituted with halogens and aliphatic substitu- ents,
R3 is selected from phosphatidic acid (PO2-OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof.
7. A drug delivery system according to claim 6, wherein R2 is an aliphatic group of a length of at least 7 carbon atoms.
8. A drug delivery system according to claim 7, wherein R2 is a group of the formula Y1Y2.
9. A drug delivery system according to any of the claims 1-8, wherein at least a fraction of the lipid is of the formula defined in claim 7, wherein R3 is a derivative of phos- phatidic acid to which a polymer selected from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, polyvinyl alcohol,
5 polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and derivatised celluloses, is covalently attached.
10. A drug delivery system according to any of the claims 1-9, wherein the lipid deriva- 10 tive constitutes 5-100 mol% of the total dehydrated system.
11. A drug delivery system according to any of the claims 1-10, wherein the lipopoly- mer constitutes 2-50 mol% of the total dehydrated system.
15 12. A drug delivery system according to any of the claims 1-11 , wherein the system is in the form of liposomes.
13. A drug delivery system according to any of the claims 1-12, wherein the second drug substance is a therapeutically and/or prophylactically active substance selected
20 from (i) wound healing agents (ii) antitumor agents, (iii) antibiotics and antifungals, and (iv) antiinflammatory agents.
14. A pharmaceutical composition comprising the drug delivery system according to any of the claims 1-13 and optionally a pharmaceutically acceptable carrier.
25
15. A drug delivery system according to any of the claims 1-13 for use as a medicament.
16. The use of a drug delivery system according to any of the claims 1-13 for the 30 preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
17. The use of a drug delivery system according to any of the claims 1-13 for the 35 preparation of a medicament for the treatment of wounds or injured tissues associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
18. The use according to claim 16, wherein the diseases or conditions are selected from the group consisting of inflammatory conditions.
19. The use according to any of claims 17-18, wherein the increase in extracellular phospholipase A2 activity is a least 25% compared to the normal level of activity in the tissue in question.
20. The lipid based drug delivery system according to any of the claims 1-5, wherein the lipid derivative is a lipid derivative of the following formula:
CH2-X-R1 CH2-X-R1
I I
CH- Z-R3 or CH-Y-R2
CH2-Y-R2 CH2-Z-R3
wherein X = Y;
Y is selected from OC(O), OC(O)O, OC(O)N, OC(S), SC(O), SC(S), CH2C(O)O, NC(O)O, Y then being connected to R2 via either the oxygen, sulphur, nitrogen or car- bonyl carbon atom;
Z is selected from O, CH2, NH, NMe, S, S(O), OS(O), S(O)2, OS(O)2, OP(O)2, OP(O)2O, OAs(O)2 and OAs(O)2O;
R1 is an aliphatic group of the formula Y1Y2;
R2 is an organic radical having at least 2 carbon atoms;
where Y1 is -(CH2)nr(CH=CH)n2-(CH2)n3-(CH=CH)n4-(CH2)n5-(CH=CH)n6-(CH2)n7- (CH=CH)n8r-(CH2)n9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2nδ+n9 is an integer of from 2 to 29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11; and each of n2, n4, n6 and n8 is independently zero or 1; and Y2 is CH3, CO2H, SH, S(O)2OH, P(O)2OH, OP(O)2OH, OH, 5 NH2; where each carbon of Y1 -Y2 independently may be substituted with halogens and aliphatic substituents,
R3 is selected from phosphatidic acid (PO2-OH), derivatives of phosphatide acid and bioisosters to phosphatic acid and derivatives thereof. 10
21. A drug delivery system according to claim 20, wherein R2 is an aliphatic group of a length of at least 7 carbon atoms.
22. A drug delivery system according to claim 7, wherein R2 is a group of the formula 15 Y1Y2.
23. The lipid based drug delivery system according to claims 1 and 22, wherein the system is in the form of liposomes.
20 24. The lipid based drug delivery system according to any of the claims 1-5, 20-23, wherein the drug substance is a therapeutically and/or prophylactically active substance selected from (i) wound healing agents (ii) antitumor agents, (iii) antibiotics and antifungals, and (iv) antiinflammatory agents.
25 25. A pharmaceutical composition comprising the drug delivery system according to any of the claims 1-5, 20-24 and optionally a pharmaceutically acceptable carrier.
26. The drug delivery system according to any of the claims 1-5, 20-24 for use as a medicament.
30
27. The use of a drug delivery system according to any of the claims 1-5, 20-24 for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
35
28. The use according to claim 27, wherein the diseases or conditions are selected from the group consisting of inflammatory conditions.
29. The use of a drug delivery system according to any of the claims 1-5, 20-24 for the preparation of a medicament for the treatment of wounds or injured tissues associated with a localised increase in extracellular phospholipase A2 activity in mammalian tissue.
30. The use according to any of claims 27-28, wherein the increase in extracellular phospholipase A2 activity is a least 25% compared to the normal level of activity in the tissue in question.
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