WO2007107161A2 - Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis - Google Patents

Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis Download PDF

Info

Publication number
WO2007107161A2
WO2007107161A2 PCT/DK2007/000143 DK2007000143W WO2007107161A2 WO 2007107161 A2 WO2007107161 A2 WO 2007107161A2 DK 2007000143 W DK2007000143 W DK 2007000143W WO 2007107161 A2 WO2007107161 A2 WO 2007107161A2
Authority
WO
WIPO (PCT)
Prior art keywords
lipid
drug delivery
delivery system
based drug
lipid based
Prior art date
Application number
PCT/DK2007/000143
Other languages
French (fr)
Other versions
WO2007107161A3 (en
WO2007107161A8 (en
Inventor
Kent JØRGENSEN
Thomas L. Andresen
Original Assignee
Liplasome Pharma A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Liplasome Pharma A/S filed Critical Liplasome Pharma A/S
Priority to CA002647779A priority Critical patent/CA2647779A1/en
Priority to JP2009500701A priority patent/JP2009530318A/en
Priority to AU2007229160A priority patent/AU2007229160A1/en
Priority to EP07711285A priority patent/EP2004236A2/en
Publication of WO2007107161A2 publication Critical patent/WO2007107161A2/en
Publication of WO2007107161A8 publication Critical patent/WO2007107161A8/en
Publication of WO2007107161A3 publication Critical patent/WO2007107161A3/en

Links

Classifications

    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • 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
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A61K47/544—Phospholipids
    • 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
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • TITLE LIPID BASED DRUG DELIVERY SYSTEMS COMPRISING PHOSPHOLl- PASE A2 DEGRADABLE LIPIDS THAT PERFORM AN INTRAMOLECULAR CY- CLIZATION REACTION UPON HYDROLYSIS.
  • the invention relates to lipid-based pharmaceutical compositions used in the treatment of various disorders, e.g. cancer, infectious, and inflammatory conditions, etc., i.e. disorders and diseases associated with or resulting from increased levels of extracellular PLA 2 activity in the diseased tissue.
  • Mono-ether lyso-phospholipids and alkyl phosphocholines are known to be effective anticancer agents (see e.g. US 3,752,886 and later references).
  • One specific example of a well-studied mono-ether alkyl phosphocholine is i-O-octadecyl-2-O-methyl-sn- glycero-3-phosphocholine (ET 18-OCH 3 ).
  • ether-lipids Several mechanisms of the toxic action of ether-lipids towards cancer cells have been proposed involving lack of alkyl-cleavage enzymes in cancer cells. This leads to an accumulation of the ether-lipids in the cell membranes which induce membrane defects and possibly subsequent lysis. Other potential mechanisms of action include effects on intracellular protein phosphorylation and disruption of the lipid metabolism. Normal cells typically possess alkyl-cleavage enzymes, which enable them to avoid the toxic effect of ether-lipids. However, some normal cells e.g., red blood cells, have like cancer cells no means of avoiding the disruptive effect of the etherlipids. 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 etherlipid to the diseased tissue.
  • prodrugs with enhanced penetration into cells, which are particular useful for treating a condition or disease in a human related to supranormal intracellular enzyme activity.
  • the prodrugs may be C-2 esters of lysophospholipids.
  • Such drugs are designed so as to be cleaved by intracelluar phospholipase A 2 .
  • drug delivery systems for targeted delivery of drug substances which are able to treat or alleviate conditions such as cancer and inflammation. Due to the fact that drugs for the treatment of cancer may be particularly harmful to tissue in general, it is of particular importance to suppress liberation of the drug substance or substances at locations other than the diseased tissue.
  • the present invention is directed to drug delivery systems which are particularly useful in the treatment or alleviation of diseases which are characterised by localised activity of extracelluar PLA 2 activity.
  • lipid-based prodrugs that performs an intramolecular cyclization upon PLA 2 hydrolysis.
  • a specific lipid-analogue compound may be incorporated into the polymer or polysaccharide chains "grafted" carrier liposome and act as a prodrug which is turned into an active drug by hydrolysis via the extracellular phospholipase.
  • Possible examples, but not limited to this, could be certain ester containing lipids that cyclize to form 5 or 6 membered rings after PLA 2 hydrolysis resulting in lipid analogues of lipids which have been found to exhibit anticancer activity as illustrated in Fig 2.
  • Fig. 1 The principle of drug targeting, release and absorption by extracellular phospholipase A2 (PLA 2 ) which is illustrated in Fig. 1 , can be applied to a case also involving lipid- based prodrugs, where a drug is attached to the lipid and liberated after a intramolecular cyclization, as a consequence of hydrolysis by extracellular PLA 2 that is present in elevated concentrations in the diseased target tissue.
  • Possible examples, but not limited to these examples, are shown in Fig. 3 and Fig 4.
  • therapeutically active substances e.g. prostaglandins, peptides, anti-cancer ether lipids or retinol derivatives will be liberated at the desired target site.
  • Drugs of particular interest are drugs with free alcohol groups or bioisosters thereof. Furthermore, some of the hydrolysis products can act as local permeability enhancers facilitating the transport of the generated anti-cancer drug into the cell.
  • Pharmaceutical compositions containing the lipid-based system can be used therapeutically, for example, in the treatment of cancer, infectious and inflammatory conditions. This invention provides such a delivery system in the form of lipid-based carriers, e.g.
  • liposomes or micelles composed of novel lipid-bilayer forming lipids such as glyc- erophospholipids containing an alkyl-linkage or acyl-linkage and/or another organic moiety in the 1 -position and an acyl-linkage or another PLA 2 degradable bond, e.g. a bioisoster of a PLA 2 hydrolysable ester, in a position with the right distance and stereochemistry from a phospolipid headgroup or a bioisoster hereof.
  • novel lipid-bilayer forming lipids such as glyc- erophospholipids containing an alkyl-linkage or acyl-linkage and/or another organic moiety in the 1 -position and an acyl-linkage or another PLA 2 degradable bond, e.g. a bioisoster of a PLA 2 hydrolysable ester, in a position with the right distance and stereochemistry from a phospolipid headgroup or a biois
  • the carrier system may contain lipid-bilayer stabilising components, e.g. lipopolymers, glycolipids and sterols which lead to an increased vascular circulation time and as a consequence an accumulation in the diseased target tissue.
  • lipid-bilayer stabilising components e.g. lipopolymers, glycolipids and sterols which lead to an increased vascular circulation time and as a consequence an accumulation in the diseased target tissue.
  • PLA 2 - catalyzed hydrolysis of the acyl-linkage releases the therapeutically active components, typically lyso-etherlipids and ester-linked derivatives that cyclize upon hydrolysis.
  • extracellular PLA 2 activity is elevated in cancer tissue.
  • extracellular PLA 2 activity is elevated in diseased regions such as inflammatory tissue.
  • the present invention thus provides a lipid-based drug delivery system for administration of an active drug substances selected from either lysolipid derivatives, or from any drug substance that can be attached to a lipid that can release the drug substance upon an intramolecular cyclization reaction as a consequence of PLA 2 hydrolysis.
  • the active drug substance is thus present in the lipid-based system in the form of a prod- rug.
  • the corresponding prodrugs are lipid derivatives having (a) an aliphatic group of a length of at least 2 carbon atoms and an organic radical having at least 2 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 un- affected, whereby the active drug substance is liberated in the form of a lysolipid derivative that performs a intramolecular cyclization, said system comprising lipopolymers and/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 administra- tion of at least one second drug substance, wherein the at least one second drug substance is incorporated in the system, said system including lipid derivatives which has (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, placed in the right distance and with the right stereochemistry relative to each other, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment that performs a intramolecular cyclization, said system comprising lipopolymers and/or gly- colipids so as to present hydrophilic chains on the surface of the system.
  • lipid derivatives which has (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, placed in the right distance and with the right stereochemistry
  • 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 comprises lipopolymers and/or glycolipids, have the properties of circulating in the blood stream sufficiently 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 also provides a class of novel lipid derivatives which are particu- larly useful as constituents of the drug delivery systems described herein.
  • Fig. 1 Schematic illustration of the lipid-based drug-targeting and drug-trigering princi- pie involving accumulation of the liposomal drug carriers in porous diseased tissue and subsequent release of drug 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).
  • the lysolipids will subsequently conduct an intramolecular cyclization.
  • Fig. 2 An example of one particular prodrug where the generated lysolipid, which is generated by PLA 2 hydrolysis, performs an intramolecular cyclization resulting in a cyclic anticancer lipid.
  • Fig. 3 An example of one particular prodrug where PLA 2 hydrolysis results in a lysolipid that cyclize to form a cyclic anticancer lipid. In addition a drug auxiliary is released as a result of the intramolecular cyclization.
  • Fig. 4 An example of one particular prodrug where PLA 2 hydrolysis results in the re- lease of a drug auxiliary as a consequence of an intramolecular cyclization.
  • extracellular PLA 2 is capable of cleaving monoether/monoester lipid derivatives as well as lipids with sub- tituents at the glycerol backbone which produce lipid derivatives 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 in one instance on lipid derivatives having (a) an aliphatic group of a length of at least 2 carbon atoms and an organic radical having at least 2 carbon atoms, and (b) a hydro- philic moiety, said prodrug furthermore being a substrate for extracellular phospholi- pase A2 to the extent that the organic radical can be hydrolytically cleaved off resulting in an intramolecular cyclization, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a cyclized lysolipid derivative, said system comprising lipopolymers and/or glycolipids so as to present hy- drophilic 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 substituted glycerol of the following formula: R D R D
  • R A and R 6 are linked to the glycerol backbone via ester bonds.
  • R D is an ester moiety ((CH 2 ) n C(O)D 1 ) ), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D 1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity.
  • the hydrogens in the CH 2 groups in R D can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl.
  • the CH 2 groups in R D can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group.
  • Al- tematively R D is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA 2 hydrolysis.
  • lysolipid is intended to mean a lipid where the R 6 fatty acid group is absent (e.g. hydrolytically cleaved off), i.e. a glycerol derivative of the formula above where R 6 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 cellular as well as extracellular PLA 2 .
  • lipid derivative and “lysolipid derivative” are intended to cover possible de- rivatives of the above possible compounds within the groups “lipid” and “lysolipid”, respectively.
  • Examples of biologically active lipid derivatives and lysolipid derivatives are given in Houlihan, et al., Med. Res. Rev., 15, 3, 157-223.
  • the extension “derivative” should be understood in the broadest sense.
  • lipid derivatives and lysolipids will in the above described case fulfil certain functional criteria (see above) and/or structural requirements. It is particularly relevant to note that one example of suitable lipid derivatives are those which have (a) an aliphatic group of a length of at least 2, 3, 4, 5, 6, 7, 8, preferably at least 9, carbon atoms and an organic radical having at least 2, 3, 4, 5, 6, 7, 8, preferably at least 9 carbon atoms, and (b) a hydrophilic moiety. It will be evident that the ali- phatic 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.
  • the organic radical and the phospholipid head group, or bioisosters of a phosphor head group should be placed in the right distance and with the right stereochemistry relative to each other, e.g. with the organic radical in the 2 or and a head group in the 3-position in a phospholipid.
  • 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. Ii- pases, which can induce hydrolytic cleavage of the organic radical corresponding to the fatty acid in the 2-position in a lipid.
  • extracellular PLA 2 activity is significantly increased towards organised substrates as compared to monomeric substrates.
  • the organic radical e.g. aliphatic group
  • the organic radical is preferably linked via an ester functionality which can be cleaved by extracellular PLA 2 , preferably so that the group which is cleaved off is a carboxylic acid.
  • 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 .
  • the drug delivery systems (liposomes or micelles) of the present invention relies in another instance on lipid derivatives having (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for ex- tracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off resulting in an intramolecular cyclization, said system comprising lipopoly- mers and/or glycolipids so as to present hydrophiiic chains on the surface of the system.
  • lipid is intended to mean tri-esters of the following formula:
  • R B is linked to the glycerol backbone via ester bonds.
  • D 2 is an organic moiety with biological activity, i.e. a drug substance in its broadest sense, n is an integer from 0-5, preferably 0 - 2.
  • the hydrogens in the CH 2 groups can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl.
  • the CH 2 group alpha to the carbonyl can also be substituted with O, S, NH or bioisosters thereof.
  • (CH 2 ) n -C(O)-D 2 may in some embodiments be equal to R D .
  • lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
  • lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
  • R D is an ester moiety ((CH 2 ) n C(O)D 1 ), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D 1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity.
  • the hydrogen's in the CH 2 groups in R D can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl.
  • the CH 2 groups in R D can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group.
  • R D is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA 2 hydrolysis.
  • D 2 is an organic moiety with biological activity, i.e. a drug substance in its broadest sense, n is an integer from 0-5, preferably 0 - 2.
  • the hydrogen's in the CH 2 groups can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl.
  • the CH 2 groups can furthermore be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group.
  • 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 phosphatide 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 connected 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.
  • R 1 and R 2 are aliphatic groups of the formula Y 1 Y 2 where Y 2 is
  • 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 ⁇ 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 un-branched, that is, they preferably have no double bonds between adjacent carbon atoms, each of n2, n4, n6 and n8 then being equal to zero.
  • Y 1 is preferably (CH 2 ) ⁇ i.
  • R 1 and R 2 are each independently (CH 2 ) ⁇ iCH 3 , and most preferably, (CH 2 )i 7 CH 3 or (CH 2 ) I sCH 3 .
  • the groups can have one or more double bonds, that is, they can be un- saturated, 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 mono-ether lipids where 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, 15 or 16, in particular 14; Y is -OC(O)-, Y then being con- nected to R 2 via the carbonyl carbon atom.
  • hydrophilic moiety which corresponds to R 3
  • R 3 hydrophilic moiety
  • 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 is typically selected from phosphatidic acid (PO 2 -OH), phosphatidylcholine (PO 2 -O- CH 2 CH 2 N(CHs) 3 ), phosphatidylethanolamine (PO 2 -O-CH 2 CH 2 NH 2 ), N-methyl- phosphatidylethanolamine (PO 2 -O-CH 2 CH 2 NHCH 3 ), phosphatidylserine, phosphatidy- linositol, and phosphatidylglycerol (PO 2 -O-CH 2 CHOHCH 2 OH).
  • phosphatidic acid Other possible deriva- tives 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.
  • dicarboxylic acids such as glutaric, sebacic, succinic and tartaric acids
  • a hydrophilic polymer or polysaccharide is typically covalently attached to the phosphatidyl part of the lipid derivative.
  • Another particular lipid derivative comprises 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
  • 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.
  • the polymer is polyethyleneglycol 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).
  • 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, poly- glycolic 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 de- rivatives of arachidonoyl (including the carbonyl from Y), e.g. prostaglandins such as prostaglandin E 1 , as arachidonic acid derivatives are know regulators of hormone action 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-I2 ) fatty acids.
  • 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), monoheterocyclic carboxylic acids (e.g.
  • 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),
  • nicotinic acid and polyheterocyclic carboxylic acids (e.g. penicillins and cephalosporins), diclofenac, indomethacin, ibuprofen, naproxen, 6-methoxy-2 ⁇ naphthylacetic acid.
  • polyheterocyclic carboxylic acids e.g. penicillins and cephalosporins
  • diclofenac indomethacin
  • ibuprofen 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 (corresponding 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.
  • 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 can- cer, e.g. a brain, breast, lung, colon or ovarian cancer, or leukaemia, lymphoma, sarcoma, carcinoma, 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.
  • the present invention provides a lipid-based drug delivery system for administration of an active drug substance selected from lysolipid derivatives and/or drug substances that can be covalently attached to a lipid, wherein the active drug sub- stance is present in the lipid-based system in the form of a prodrug, said prodrug being being a lipid derivative having (a) an organic radical having at least 2 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, leading to an intramolecular cyclization reaction, whereby the active drug sub- stance is liberated in the form of a lysolipid derivative, said system comprising lipopoly- mers and/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/or drugs with a therapeutic effect with a chemical structure that makes it possible to attach it to a lipid that can be set up for an intramolecular cyclization upon PLA 2 hydrolysis, and as it 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 mono-ether 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 (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 example:
  • both the substituent R 2 and/or the substituent D 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 vari- ous 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).
  • the above is also applica- ble for D.
  • lipid-based drug delivery system should encompass macromolecular structures which as the main constituent comprise lipid or lipid derivatives. Suitable exam- pies 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, micelle systems are also believed to offer interesting embodiments within the pre- sent invention.
  • the lipid derivative (e.g. the prodrug) is comprised in liposomes either as the only constituent or - which is more common - in combination with other constitu- ents (other lipids, sterols, etc.).
  • the lipid-based systems described herein are preferably in the form of liposomes, wherein the liposomes are building up of layers comprising the lipid derivative (e.g. a prodrug).
  • “Liposomes” are known as self-assembling structures comprising one or more lipid bi- layers, each of which surrounds an aqueous compartment and comprises two opposing mono-layers of amphipathic lipid molecules.
  • Amphipathic lipids i.e. lipid derivatives
  • lipid derivatives comprise a polar (hydrophilic) head group 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 head groups 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 me- dium.
  • Liposomes can have a single lipid bilayer (unilamellar liposomes, "ULVs”), or multiple lipid bi-layers (multilamellar liposomes, "MLVs”), and can be made by a variety of methods (for a review, see, for example, Deamer and Uster, Liposomes, Marcel Dek- ker, N.Y., 1983, 27-52).
  • UUVs unilamellar liposomes
  • MLVs multilamellar liposomes
  • MLVs multilamellar liposomes
  • Lenk's, Fountain's and Cullis' methods for making MLVs with substantially equal interlamellar solute distribution
  • US 4,235,871 Papahadjopoulos et al.'s reverse-phase evaporation method
  • ULVs can be produced from MLVs by such methods as sonication (see Papahadjopoulos 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 deriva- tives 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 mono-ether lysolipids like ET-18-OCH3, which can only constitute a minor part of the liposomes.
  • liposomes advantageously comprise 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 at least one second drug substance (see below) 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 lipo- somes 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 parti- cle 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.
  • 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.
  • the lipid derivatives will become more or less inert or even "invisible” when "packed” in liposomes in which lipopolymers and/or glycolipids are comprised. This means than any potential disadvantageous effect, e.g. haemolytic 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.
  • liposomes may comprise 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.
  • lipopolymers Two very important groups of compounds to be comprised in liposomes as modifiers are the stabilising compound lipopolymers and glycolipids, such as lipopolymers (e.g. polyethyleneoxide-dipalmitoylphosphatidyl ethanolamine, DPPE-PEG, polyethyleneox- ide-distearoylphosphatidylethanolamine, DSPE-PEG) with PEG molecular weight from 100 to 10000 Daltons.
  • lipopolymers function as stabilisers for the liposome, i.e. lipopolymer increases the circulation time, and - which is highly interesting in the present context, as activators for extracellular PLA 2 .
  • 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 bi-layers lipids whose polar head groups 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 nor- mally 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 polyethyleneglycol 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. 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 Hp- ids, 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 com- prised 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 head group size and charge.
  • 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.
  • PE's phosphatidylethanolamines
  • EPE egg phosphatidylethanolamine
  • DOPE dioleoyl phosphatidylethanolamine
  • Lipids may be modified in various way, e.g. by head group derivatisation with dicarboxylic ac- ids, such as glutaric, sebacic, succinic and tartaric acids, preferably the dicarboxylic acid is glutaric acid (“GA").
  • G glutaric acid
  • suitable head group-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 compounds comprised in the liposome may generally affect the fluidity of lipid bi-layers. Accordingly, sterol interactions with surrounding hydrocarbon groups generally inhibit emigration of these groups from the bilayer.
  • An example of a sterolic compound (sterol) to be comprised 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 gener- ally 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.
  • PEG-ceramides polyethylene glycol derivatives of ceramides
  • 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 envi- ronment (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 comprise 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 over expressed 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 bi-layers (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.
  • 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 Lipid derivative liposomes as drug carrier systems
  • the liposomes including the lipid derivatives of the present invention may also comprise at least one second drug substance.
  • the lipid-based drug delivery system described above is in the form of liposomes wherein the at least one second drug substance is incorporated.
  • second drug substances may comprise pharmaceutically active ingredients which may have an individual or synergistic pharmaceutical effect in combination with the lipid derivative and lysolipid derivatives.
  • the term "second" does not necessarily imply that the pharmaceutical effect of the at least one second drug substance is infe- rior 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: antiviral agents such as acyclovir, zidovudine and the in- terferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene antibiotics, imidazoles and triazoles; antime- tabolic agents such as folic acid, and purine and pyrimidine analogs; antineoplastic agents such as the anthracycline 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; bron- chodilators; local ane
  • 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 at least one second drug substance 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 liposome 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 at least one second drug substance may be any of a wide variety of known and possible pharmaceutically active ingredients, but is preferably a therapeuti- cally 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 at least one 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) antitumour agents such as anthracyline derivatives, cisplatin, paclitaxel, 5-fluoruracil, exisulind, cis-retinoic acid, suldinac sulfide, vincristine, interleukins, oligonucleotides, peptides, proteins and cytokines (ii) antibiotics and antifungals, and (iii) antiinflammatory agents such as ster- oids and non-steroids.
  • 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 per- meation across the target membranes when the carriers locally are broken down in the diseased tissue.
  • the at least one second drug substance will be distributed in the liposomes according 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.
  • 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.
  • 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 at least one second drug substance, wherein the at least one 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 2 carbon atoms and an organic radical having at least 2 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 alco- hoi fragment and a lysolipid fragment, said system comprising lipopolymers and/or gly- colipids 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 typi- cally selected from cancer, e.g. a brain, breast, lung, colon or ovarian cancer, or leukemia, lymphoma, sarcoma, carcinoma and inflammatory conditions.
  • the prophylactic use 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 administered in a non-particulate form as free agents leading to an increased intracellular cell uptake rendering them favourable substrates for over expressed intracellular PLA2 in the dis- eased target cells.
  • composition comprising optionally 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 liposome 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 at least one second drug substance used, the liposome preparation, its concentration, stability and intended bioavailability; the disease, disorder or condition being treated with the liposome composition; the subject, its age, size and general condition; and the composition's in- tended route of administration, e.g., nasal, oral, ophthalmic, subcutaneous, intramam- mary, 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 exam- pie those which enhance the stability of the active ingredients comprised, 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 effec- tive to inhibit, ameliorate, lessen or prevent establishment, growth, metastasis or invasion 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, ordinarily skilled artisans given the teachings of this invention.
  • Antineoplastic effective amounts of the liposome 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 at least one second drug substance in addition to the liposomal drug, these additional agents being comprised 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 bi-layers, are preferably, but not necessarily, anticancer agents.
  • the pharmaceutical composition is preferably administered parenterally by injection, infusion or implantation (intravenous, intramuscular, intraarticular, and subcutaneous or the like) in dosage forms, formulations or e.g. suitable delivery devices or implants containing 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.
  • 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.
  • solid tumours are those growing in an anatomical site other than the bloodstream (in contrast to blood-borne tumours such as leukaemia's). Solid tumours require the formation of small blood vessels and capillaries to nourish the growing tumour tissue.
  • the anti-tumour or anti-neoplastic 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 liposome formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration.
  • Tumours characterised by an acute increase in permeability of the vasculature in the region of tumour growth 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 bio available, transportable solubilised form.
  • a small elevation in tem- perature as often seen in diseased tissue may further increase the stimulation of extracellular PLA 2 .
  • liposome delivery of an drug requires that the liposome have a long blood half-life, and be capable of pene- trating the continuous endothelial cell layer and underlying basement membrane surrounding 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.
  • the limited drug-carrying capacity of conventional small liposome preparations has limited their effectiveness for such purposes.
  • 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 bar- riers.
  • the resulting liposomal formulation can be administered parenterally to a subject 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.
  • extracellular PLA 2 activity of extracellular PLA 2 is abnormally high in areas of the 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 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 cancer where rather harsh drug (second drug substances) is often needed.
  • the invention thus provides to a method for selectively drug targeting to diseased areas, such as areas comprising neoplastic 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 neoplastic 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 leukaemia, 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 the at least one second drug substance in liposome form is selectively cytotoxic to tumour cells.
  • lipid-based carriers composed of the novel and unnatural lipids
  • novel targeted liposome 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 lected from ether-lysolipid and/or fatty acid derivatives of a lipid prodrug being a substrate for extracellular PLA 2 .
  • the flexible liposome 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 cancer and inflammation.
  • the novel prodrug liposome 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 .
  • Liposome 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, 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 un- derlaying structures are normally so narrow, that the skin only allows passage of entities smaller than 400 Dalton.
  • ultraflexible liposomes 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 application of drugs into the blood via the skin.
  • 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 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 polysaturated 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 render the double prodrug lipid a substrate for PLA 2 .
  • conventional drug substances can be incorporated and transported specifically to the diseased site by the novel lipid-based carriers.
  • the new carriers liposomes 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 .
  • novel lipid-based carrier systems are useful in the targeted diagnosis of diseases such as cancer, infection, and inflammation, which are characterized by localized and elevated activity of extracellular PLA 2 .
  • the increased PLA 2 activity combined with the observation that microparticulates accumulate in the diseased tissue via extravasation through leaky capillaries provide the basis for using contrast agent microcarriers for enhanced imaging.
  • the novel diagnostic system takes advantage of the fact that liposomes (and mi- Scheme), which can be specifically degraded by extracellular PLA 2 also can be designed to circulate in the blood stream sufficiently long to reach the target tissue where the PLA 2 activity is elevated. At the diseased site the lipid derivatives of the liposomes are cleaved by PLA 2 so as to liberate the image enhancing agents.
  • Imaging is widely used in medicine. It requires that an appropriate intensity of signal from an area of interest is achieved in order to differentiate diseased tissue from normal surrounding tissue. Imaging involves the relationship between the three spatial dimensions of the region of interest and a fourth dimension, time, which relates to both the pharmacokinetics of the diagnostic agent and the period necessary to ac- quire the image.
  • the physical properties that can be used to create an image signal include, e.g. emission or absorption of radiation, nuclear magnetic moments and relaxation, and transmission or reflection of ultrasound.
  • emission or absorption of radiation e.g. emission or absorption of radiation
  • nuclear magnetic moments and relaxation e.g., nuclear magnetic moments and relaxation, and transmission or reflection of ultrasound.
  • Imaging contrast agents relate to substances, which are able to absorb certain types of signal much stronger than surrounding tissues.
  • the contrast agents are specific for each imaging technique, and as a result of their accumulation in certain diseased sites of interest, those sites may be visualized when an appropriate imaging technique is applied.
  • the tissue concentration that must be achieved for successful imaging varies between diagnostic techniques.
  • liposomes draw special attention because of their easily controlled properties.
  • liposomes have been recognized as promising carriers for drugs and diagnostic agents for the following reasons: (1) Liposomes are completely biocompatible; (2) they can entrap practically any drug or diagnostic agent into either the internal water compartment or into the membrane itself depending on the physico- chemical properties of the compound; (3) liposome-incorporated compounds are pro- tected from the inactivating effect in the body, yet at the same time do not cause undesirable side-reactions; (4) liposomes also provide a unique opportunity to deliver pharmaceuticals or diagostic agents into cells or even inside individual cellular compartments. Pursuing different in vivo delivery purposes, the size, charge and surface properties of liposomes can easily be changed simply by incorporation of different lipids and/or by variation of the preparation methods.
  • PLA 2 is secreted by malignant cells and immu- nohistochemical staining of various cancers, including cancer of pancreas, breast and stomach has shown increased levels of PLA 2 .
  • An increased expression and secretion of PLA 2 is also found in several cancer cell lines stimulated by interleukines such as IL- 6.
  • interleukines such as IL- 6.
  • elevated extracellular PLA 2 activity has also been described in inflammatory and infected tissues.
  • the main mechanism of liposome accumulation in tumours is via extravasation through leaky tumour capillaries into the interstitial space.
  • the tumour accumulation can be significantly increased by using long-circulating polymer coated liposomes.
  • the lipid-based microcarriers may also be used for visualization of inflammation and infection sites. Similar to what is known with respect to cancer tissue, the use of mi- croparticulate imaging agents for the visualization of infection and inflammation sites characterized by elevated PLA 2 activity is based on the ability of microparticulates to accumulate via extravasation through leaky capillaries.
  • lipid derivatives will be cleaved by extracellular PLA 2 in a well-defined manner in specific extracellular locations of diseased mammalian tissue characterized by an elevated activity of PLA 2 . It has been found that extracellular PLA 2 is capable of cleaving mono- ether/monoester lipid derivatives so as to target the diagnostic label in the relevant diseased tissue. Degradation of the lipid derivatives and the liposomes by PLA 2 leads to a site-specific release of the diagnostic contrast agents in the diseased tissue. Targeting of infected tissues with lipid-based carriers composed of the novel and unnatural lipids
  • 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.
  • Leishmania and Trypanosoma are therefore toxic to the parasites if administered in sufficient amount.
  • 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.
  • ether-cleavage enzymes typically possess ether-cleavage enzymes, which enable them to avoid the toxic effect of ether-lipids.
  • red blood cells have like cancer cells no means of avoiding the disruptive effect of the ether-lipids.
  • 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 determin- ing the therapeutic window "TW", which is a numerical value derived from the relationship between the compound's induction of haemolysis and its ability to inhibit the growth of tumour cells.
  • TW values are defined as HI 5 /GI 50 (wherein “Hl 5 " equals the concentration of compound inducing the haemolysis 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 haemolysis 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.
  • 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 be- lieved 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Epidemiology (AREA)
  • Oncology (AREA)
  • Immunology (AREA)
  • Hematology (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Dispersion Chemistry (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to a lipid-based drug delivery system for administration of anti-cancer drugs in a prodrug form, said prodrug furthermore being a substrate for extracellular phospholipase A2 to the extent that an organic radical can be hydrolytically cleaved off, resulting in an intramolecular reaction, said system comprising lipopolymers and/or glycolipids so as to present hydrophilic chains on the surface of the system. In addition, the lipid prodrug and drug carries can be used in combination with incorporated drugs. Pharmaceutical compositions comprising the drug delivery system can be used in diagnosis and targeted treatment of various disorders, e.g. cancer, infectious, and inflammatory conditions, etc., i.e. disorders and diseases associated with or resulting from increased levels of extracellular PLA2 activity in the diseased tissue.

Description

TITLE: LIPID BASED DRUG DELIVERY SYSTEMS COMPRISING PHOSPHOLl- PASE A2 DEGRADABLE LIPIDS THAT PERFORM AN INTRAMOLECULAR CY- CLIZATION REACTION UPON HYDROLYSIS.
FIELD OF THE INVENTION
The invention relates to lipid-based pharmaceutical compositions used in the treatment of various disorders, e.g. cancer, infectious, and inflammatory conditions, etc., i.e. disorders and diseases associated with or resulting from increased levels of extracellular PLA2 activity in the diseased tissue.
BACKGROUND OF THE INVENTION
Mono-ether lyso-phospholipids and alkyl phosphocholines are known to be effective anticancer agents (see e.g. US 3,752,886 and later references). One specific example of a well-studied mono-ether alkyl phosphocholine is i-O-octadecyl-2-O-methyl-sn- glycero-3-phosphocholine (ET 18-OCH3).
Several mechanisms of the toxic action of ether-lipids towards cancer cells have been proposed involving lack of alkyl-cleavage enzymes in cancer cells. This leads to an accumulation of the ether-lipids in the cell membranes which induce membrane defects and possibly subsequent lysis. Other potential mechanisms of action include effects on intracellular protein phosphorylation and disruption of the lipid metabolism. Normal cells typically possess alkyl-cleavage enzymes, which enable them to avoid the toxic effect of ether-lipids. However, some normal cells e.g., red blood cells, have like cancer cells no means of avoiding the disruptive effect of the etherlipids. 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 etherlipid to the diseased tissue.
US 5,985,854, US 6,077,837, US 6,136,796 and US 6,166,089 describe prodrugs with enhanced penetration into cells, which are particular useful for treating a condition or disease in a human related to supranormal intracellular enzyme activity. The prodrugs may be C-2 esters of lysophospholipids. Such drugs are designed so as to be cleaved by intracelluar phospholipase A2. Even in view of the above, an increasing demand for novel drug delivery systems exist, in particular drug delivery systems for targeted delivery of drug substances which are able to treat or alleviate conditions such as cancer and inflammation. Due to the fact that drugs for the treatment of cancer may be particularly harmful to tissue in general, it is of particular importance to suppress liberation of the drug substance or substances at locations other than the diseased tissue.
BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to drug delivery systems which are particularly useful in the treatment or alleviation of diseases which are characterised by localised activity of extracelluar PLA2 activity.
The new principle for liposomal drug targeting by extracellular PLA2 described in this application and illustrated in Fig. 1 - involves lipid-based prodrugs that performs an intramolecular cyclization upon PLA2 hydrolysis. In this case a specific lipid-analogue compound may be incorporated into the polymer or polysaccharide chains "grafted" carrier liposome and act as a prodrug which is turned into an active drug by hydrolysis via the extracellular phospholipase. Possible examples, but not limited to this, could be certain ester containing lipids that cyclize to form 5 or 6 membered rings after PLA2 hydrolysis resulting in lipid analogues of lipids which have been found to exhibit anticancer activity as illustrated in Fig 2.
The principle of drug targeting, release and absorption by extracellular phospholipase A2 (PLA2) which is illustrated in Fig. 1 , can be applied to a case also involving lipid- based prodrugs, where a drug is attached to the lipid and liberated after a intramolecular cyclization, as a consequence of hydrolysis by extracellular PLA2 that is present in elevated concentrations in the diseased target tissue. Possible examples, but not limited to these examples, are shown in Fig. 3 and Fig 4. In this way, therapeutically active substances, e.g. prostaglandins, peptides, anti-cancer ether lipids or retinol derivatives will be liberated at the desired target site. Drugs of particular interest, but not limited to such drugs, are drugs with free alcohol groups or bioisosters thereof. Furthermore, some of the hydrolysis products can act as local permeability enhancers facilitating the transport of the generated anti-cancer drug into the cell. Pharmaceutical compositions containing the lipid-based system can be used therapeutically, for example, in the treatment of cancer, infectious and inflammatory conditions. This invention provides such a delivery system in the form of lipid-based carriers, e.g. liposomes or micelles, composed of novel lipid-bilayer forming lipids such as glyc- erophospholipids containing an alkyl-linkage or acyl-linkage and/or another organic moiety in the 1 -position and an acyl-linkage or another PLA2 degradable bond, e.g. a bioisoster of a PLA2 hydrolysable ester, in a position with the right distance and stereochemistry from a phospolipid headgroup or a bioisoster hereof. Typically this will be as a phospholipid with a glycerol backbone with or without substituents in the 1 or 3 position, including lipids which have polymer or polysaccharide chains grafted to the lipid headgroup. In addition, the carrier system may contain lipid-bilayer stabilising components, e.g. lipopolymers, glycolipids and sterols which lead to an increased vascular circulation time and as a consequence an accumulation in the diseased target tissue. When the carriers reach the target site of therapeutic action, e.g. cancer cells, PLA2- catalyzed hydrolysis of the acyl-linkage releases the therapeutically active components, typically lyso-etherlipids and ester-linked derivatives that cyclize upon hydrolysis. Contradictory to alkyl-cleavage enzymes which are nearly absent in cancer cells, extracellular PLA2 activity is elevated in cancer tissue. In addition, extracellular PLA2 activity is elevated in diseased regions such as inflammatory tissue.
The present invention thus provides a lipid-based drug delivery system for administration of an active drug substances selected from either lysolipid derivatives, or from any drug substance that can be attached to a lipid that can release the drug substance upon an intramolecular cyclization reaction as a consequence of PLA2 hydrolysis. The active drug substance is thus present in the lipid-based system in the form of a prod- rug. For the lysolipid derivatives the corresponding prodrugs are lipid derivatives having (a) an aliphatic group of a length of at least 2 carbon atoms and an organic radical having at least 2 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 un- affected, whereby the active drug substance is liberated in the form of a lysolipid derivative that performs a intramolecular cyclization, said system comprising lipopolymers and/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 administra- tion of at least one second drug substance, wherein the at least one second drug substance is incorporated in the system, said system including lipid derivatives which has (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, placed in the right distance and with the right stereochemistry relative to each other, where the lipid derivative furthermore is a substrate for extracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment that performs a intramolecular cyclization, said system comprising lipopolymers and/or gly- colipids 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 comprises lipopolymers and/or glycolipids, have the properties of circulating in the blood stream sufficiently 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 also provides a class of novel lipid derivatives which are particu- larly useful as constituents of the drug delivery systems described herein.
DESCRIPTION OF THE DRAWINGS
Fig. 1. Schematic illustration of the lipid-based drug-targeting and drug-trigering princi- pie involving accumulation of the liposomal drug carriers in porous diseased tissue and subsequent release of drug 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 analogs e.g. prodrug (lipid), proenhancer (lipid), proactivator (lipid)
(V) Drugs (lysolipid and fatty acid derivatives), enhancers (lysolipid + fatty acid), PLA2 activators (lysolipid + fatty acid). The lysolipids will subsequently conduct an intramolecular cyclization. Fig. 2. An example of one particular prodrug where the generated lysolipid, which is generated by PLA2 hydrolysis, performs an intramolecular cyclization resulting in a cyclic anticancer lipid.
Fig. 3. An example of one particular prodrug where PLA2 hydrolysis results in a lysolipid that cyclize to form a cyclic anticancer lipid. In addition a drug auxiliary is released as a result of the intramolecular cyclization.
Fig. 4. An example of one particular prodrug where PLA2 hydrolysis results in the re- lease of a drug auxiliary as a consequence of an intramolecular cyclization.
DETAILED DESCRIPTION OF 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 extracellular locations of mammalian diseased tissue. It has been found that extracellular PLA2 is capable of cleaving monoether/monoester lipid derivatives as well as lipids with sub- tituents at the glycerol backbone which produce lipid derivatives 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 in one instance on lipid derivatives having (a) an aliphatic group of a length of at least 2 carbon atoms and an organic radical having at least 2 carbon atoms, and (b) a hydro- philic moiety, said prodrug furthermore being a substrate for extracellular phospholi- pase A2 to the extent that the organic radical can be hydrolytically cleaved off resulting in an intramolecular cyclization, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a cyclized lysolipid derivative, said system comprising lipopolymers and/or glycolipids so as to present hy- drophilic 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 substituted glycerol of the following formula: RD RD
I I
CH-O-RA CH-O-RA I I
CH-O-R6 or CH-O-RC
I I
CH2-O-R0 CH2-O-R6
wherein RA and R6 are fatty acid moieties (Cg-30-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 R6 are linked to the glycerol backbone via ester bonds. RD is an ester moiety ((CH2)nC(O)D1) ), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity. The hydrogens in the CH2 groups in RD can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 groups in RD can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group. Al- tematively RD is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA2 hydrolysis.
The term "lysolipid" is intended to mean a lipid where the R6 fatty acid group is absent (e.g. hydrolytically cleaved off), i.e. a glycerol derivative of the formula above where R6 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 cellular as well as extracellular PLA2.
The terms "lipid derivative" and "lysolipid derivative" are intended to cover possible de- rivatives of the above possible compounds within the groups "lipid" and "lysolipid", respectively. Examples of biologically active lipid derivatives and lysolipid derivatives are given in Houlihan, et al., Med. Res. Rev., 15, 3, 157-223. Thus, as will be evident, the extension "derivative" should be understood in the broadest sense.
Within the present application, lipid derivatives and lysolipids will in the above described case fulfil certain functional criteria (see above) and/or structural requirements. It is particularly relevant to note that one example of suitable lipid derivatives are those which have (a) an aliphatic group of a length of at least 2, 3, 4, 5, 6, 7, 8, preferably at least 9, carbon atoms and an organic radical having at least 2, 3, 4, 5, 6, 7, 8, preferably at least 9 carbon atoms, and (b) a hydrophilic moiety. It will be evident that the ali- phatic 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. This in the broadest sense means that the organic radical and the phospholipid head group, or bioisosters of a phosphor head group, should be placed in the right distance and with the right stereochemistry relative to each other, e.g. with the organic radical in the 2 or and a head group in the 3-position in a phospholipid. 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. Ii- pases, which can induce hydrolytic cleavage of the organic radical corresponding to the fatty acid in the 2-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 organic radical (e.g. aliphatic group) is preferably linked via an ester functionality which can be cleaved by extracellular PLA2, preferably so that the group which is cleaved off is a carboxylic acid.
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 unaffected 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. The drug delivery systems (liposomes or micelles) of the present invention relies in another instance on lipid derivatives having (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, said prodrug furthermore being a substrate for ex- tracellular phospholipase A2 to the extent that the organic radical can be hydrolytically cleaved off resulting in an intramolecular cyclization, said system comprising lipopoly- mers and/or glycolipids so as to present hydrophiiic chains on the surface of the system.
The term "lipid" is intended to mean tri-esters of the following formula:
CH2-(CH2)n-C(O)-D2 CH2-(CHs)n-C(O)-D2 i • i
CH-O-RB or CH-O-R0 I I
CH2-O-R0 CH2-O-R8
wherein RB is a fatty acid moieties (C9-3o-alkyl/alkylene/alkyldiene/alkyltriene/- alkyltetraene-C(=O)-) and R° is a phosphatidic acid (PO2-OH) or a derivative of phos- phatidic acid or a bioisoster of phosphatidic acid. Thus, the RB is linked to the glycerol backbone via ester bonds. D2 is an organic moiety with biological activity, i.e. a drug substance in its broadest sense, n is an integer from 0-5, preferably 0 - 2. The hydrogens in the CH2 groups can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 group alpha to the carbonyl can also be substituted with O, S, NH or bioisosters thereof.
(CH2)n-C(O)-D2 may in some embodiments be equal to RD.
One preferred class of lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
RD RD CH2-X-R GH2-X-R
I I
CH-Y-R2 or CH-Z-R3
I I CH2-Z-R3 CH2-Y-R2
Another preferred class of lipid derivatives for incorporation in the drug delivery systems of the invention can be represented by the following formula:
CH2-(CH2)π-C(O)-D2 CH2-(CH2)n-C(O)-D2
I I
CH-Y-R2 or CH-Z-R3
I I
CH2-Z-R3 CH2-Y-R2
Wherein
RD is an ester moiety ((CH2)nC(O)D1), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity. The hydrogen's in the CH2 groups in RD can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 groups in RD can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group. Alternatively RD is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA2 hydrolysis.
D2 is an organic moiety with biological activity, i.e. a drug substance in its broadest sense, n is an integer from 0-5, preferably 0 - 2. The hydrogen's in the CH2 groups can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 groups can furthermore be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group.
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+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, 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 un-substituted,
R3 is selected from phosphatide 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 connected 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)π7(CH=CH)n8-
(CH2)n9; the sum of n1+2n2+n3+2n4+n5+2n6+n7+ 2nδ+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^io-groups (i.e. yielding branched aliphatic groups), the aliphatic groups as R1 and R2 are in one embodiment preferably saturated as well as un-branched, 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)πi. More preferably (in this embodiment), R1 and R2 are each independently (CH2)πiCH3, and most preferably, (CH2)i7CH3 or (CH2)IsCH3. In alternative embodiments, the groups can have one or more double bonds, that is, they can be un- saturated, 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 mono-ether lipids where 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, 15 or 16, in particular 14; Y is -OC(O)-, Y then being con- nected 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 phosphatidic 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 is typically selected from phosphatidic acid (PO2-OH), phosphatidylcholine (PO2-O- CH2CH2N(CHs)3), phosphatidylethanolamine (PO2-O-CH2CH2NH2), N-methyl- phosphatidylethanolamine (PO2-O-CH2CH2NHCH3), phosphatidylserine, phosphatidy- linositol, and phosphatidylglycerol (PO2-O-CH2CHOHCH2OH). Other possible deriva- tives 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 attached to the phosphatidyl part of the lipid derivative. Another particular lipid derivative comprises 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 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).
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 micelle 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, poly- glycolic 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 understood 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 de- rivatives of arachidonoyl (including the carbonyl from Y), e.g. prostaglandins such as prostaglandin E1, as arachidonic acid derivatives are know regulators of hormone action 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-I2) 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 (corresponding 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 understood 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.
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 can- cer, e.g. a brain, breast, lung, colon or ovarian cancer, or leukaemia, lymphoma, sarcoma, carcinoma, 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 and/or drug substances that can be covalently attached to a lipid, wherein the active drug sub- stance is present in the lipid-based system in the form of a prodrug, said prodrug being being a lipid derivative having (a) an organic radical having at least 2 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, leading to an intramolecular cyclization reaction, whereby the active drug sub- stance is liberated in the form of a lysolipid derivative, said system comprising lipopoly- mers and/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/or drugs with a therapeutic effect with a chemical structure that makes it possible to attach it to a lipid that can be set up for an intramolecular cyclization upon PLA2 hydrolysis, and as it will be understood from the present description with claims.
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 mono-ether 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 example:
Figure imgf000017_0001
Prodrug Drug
Furthermore, both the substituent R2 and/or the substituent D 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:
Figure imgf000017_0002
prodrug drug drug drug
It has been described above under the definition of R2 how the group R2 can have vari- ous 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). The above is also applica- ble for D.
Lipid derivatives formulated as liposomes
The term "lipid-based drug delivery system" should encompass macromolecular structures which as the main constituent comprise lipid or lipid derivatives. Suitable exam- pies 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, micelle systems are also believed to offer interesting embodiments within the pre- sent invention.
In one important variant which advantageously can be combined with the embodiments described herein, the lipid derivative (e.g. the prodrug) is comprised in liposomes either as the only constituent or - which is more common - in combination with other constitu- ents (other lipids, sterols, etc.). Thus, the lipid-based systems described herein are preferably in the form of liposomes, wherein the liposomes are building up of layers comprising the lipid derivative (e.g. a prodrug).
"Liposomes" are known as self-assembling structures comprising one or more lipid bi- layers, each of which surrounds an aqueous compartment and comprises two opposing mono-layers of amphipathic lipid molecules. Amphipathic lipids (i.e. lipid derivatives) comprise a polar (hydrophilic) head group 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 head groups 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 me- dium.
Liposomes can have a single lipid bilayer (unilamellar liposomes, "ULVs"), or multiple lipid bi-layers (multilamellar liposomes, "MLVs"), and can be made by a variety of methods (for a review, see, for example, Deamer and Uster, Liposomes, Marcel Dek- ker, N.Y., 1983, 27-52). These methods include Bangham's methods for making multilamellar 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 Ii- posomes. ULVs can be produced from MLVs by such methods as sonication (see Papahadjopoulos 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 liposomes 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 deriva- tives 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 mono-ether lysolipids like ET-18-OCH3, which can only constitute a minor part of the liposomes.
Typically, as will be described in detail below, liposomes advantageously comprise 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 at least one second drug substance (see below) 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 lipo- somes 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 intravenous and intramuscular injection, the liposomes should preferably have a mean particle size of about 100 nm. Thus, the parti- cle 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 and/or glycolipids are comprised. This means than any potential disadvantageous effect, e.g. haemolytic 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 comprise 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 comprised in liposomes as modifiers are the stabilising compound lipopolymers and glycolipids, such as lipopolymers (e.g. polyethyleneoxide-dipalmitoylphosphatidyl ethanolamine, DPPE-PEG, polyethyleneox- ide-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 interesting 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 bi-layers lipids whose polar head groups 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 nor- mally 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 polyethyleneglycol 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 Hp- ids, 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 com- prised 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 head group 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 head group derivatisation with dicarboxylic ac- ids, such as glutaric, sebacic, succinic and tartaric acids, preferably the dicarboxylic acid is glutaric acid ("GA"). Accordingly, suitable head group-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 compounds comprised in the liposome may generally affect the fluidity of lipid bi-layers. Accordingly, sterol interactions with surrounding hydrocarbon groups generally inhibit emigration of these groups from the bilayer. An example of a sterolic compound (sterol) to be comprised 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 gener- ally 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 envi- ronment (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 comprise 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 over expressed 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 bi-layers (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. Alternatively, 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 comprise at least one second drug substance. In a particular embodiment, the lipid-based drug delivery system described above is in the form of liposomes wherein the at least one second drug substance is incorporated. It should be understood that second drug substances may comprise pharmaceutically active ingredients which may have an individual or synergistic pharmaceutical effect in combination with the lipid derivative and lysolipid derivatives. The term "second" does not necessarily imply that the pharmaceutical effect of the at least one second drug substance is infe- rior 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: antiviral agents such as acyclovir, zidovudine and the in- terferons; antibacterial agents such as aminoglycosides, cephalosporins and tetracyclines; antifungal agents such as polyene antibiotics, imidazoles and triazoles; antime- tabolic agents such as folic acid, and purine and pyrimidine analogs; antineoplastic agents such as the anthracycline 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; bron- chodilators; 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 at least one second drug substance 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 liposome 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 mi- celle formulation are known in the art (see e.g., US 5,534,499 and US 6,118,011 describing covalent attachment of therapeutic agents to a fatty acid chain of a phospholipid). A micelle formulation of taxol is described in Alkan-Onkyuksel et al., Pharmaceutical Research, 11 :206 (1994).
Accordingly, the at least one second drug substance may be any of a wide variety of known and possible pharmaceutically active ingredients, but is preferably a therapeuti- cally 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 at least one 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) antitumour agents such as anthracyline derivatives, cisplatin, paclitaxel, 5-fluoruracil, exisulind, cis-retinoic acid, suldinac sulfide, vincristine, interleukins, oligonucleotides, peptides, proteins and cytokines (ii) antibiotics and antifungals, and (iii) antiinflammatory agents such as ster- oids and non-steroids. 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 per- meation across the target membranes when the carriers locally are broken down in the diseased tissue.
It is envisaged that the at least one second drug substance will be distributed in the liposomes according 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. Methods for incorporation of 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 at least one second drug substance, wherein the at least one second drug substance is incorporated in the system (e.g. where the second drug substance is encapsulated 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 2 carbon atoms and an organic radical having at least 2 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 alco- hoi fragment and a lysolipid fragment, said system comprising lipopolymers and/or gly- colipids 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 typi- cally selected from cancer, e.g. a brain, breast, lung, colon or ovarian cancer, or 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 administered in a non-particulate form as free agents leading to an increased intracellular cell uptake rendering them favourable substrates for over expressed intracellular PLA2 in the dis- eased target cells. Pharmaceutical preparations and therapeutic uses
Also provided herewith is a pharmaceutical composition comprising optionally 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 liposome 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 at least one second drug substance used, the liposome preparation, its concentration, stability and intended bioavailability; the disease, disorder or condition being treated with the liposome composition; the subject, its age, size and general condition; and the composition's in- tended route of administration, e.g., nasal, oral, ophthalmic, subcutaneous, intramam- mary, 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 exam- pie those which enhance the stability of the active ingredients comprised, 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 liposome lipid derivatives are amounts effec- tive to inhibit, ameliorate, lessen or prevent establishment, growth, metastasis or invasion 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, ordinarily skilled artisans given the teachings of this invention. Antineoplastic effective amounts of the liposome 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 at least one second drug substance in addition to the liposomal drug, these additional agents being comprised 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 bi-layers, are preferably, but not necessarily, anticancer agents.
The pharmaceutical composition is preferably administered parenterally by injection, infusion or implantation (intravenous, intramuscular, intraarticular, and subcutaneous or the like) in dosage forms, formulations or e.g. suitable delivery devices or implants containing 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 text- book 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 treatment of a tumour or neoplasm 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.
As used herein, "solid tumours" are those growing in an anatomical site other than the bloodstream (in contrast to blood-borne tumours such as leukaemia's). Solid tumours require the formation of small blood vessels and capillaries to nourish the growing tumour tissue.
In accordance with the present invention, the anti-tumour or anti-neoplastic 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 liposome formulation can be administered parenterally to a subject in need of such treatment, preferably by intravenous administration. Tumours characterised by an acute increase in permeability of the vasculature in the region of tumour growth 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 bio available, transportable solubilised form. Furthermore, a small elevation in tem- perature as often seen in diseased tissue may further increase the stimulation of 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 pene- trating the continuous endothelial cell layer and underlying basement membrane surrounding 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.
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 bar- riers. The resulting liposomal formulation can be administered parenterally to a subject 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 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 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 cancer where rather harsh drug (second drug substances) is 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 neoplastic 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 leukaemia, lymphoma, sarcoma, carcinoma, which comprises administering a pharmaceutical composition of this invention to the mammal. It is believed that the lipid derivatives and/or the at least one 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 liposome 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 lected from ether-lysolipid and/or fatty acid derivatives of a lipid prodrug being a substrate for extracellular PLA2.
Degradation of the flexible liposome 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 cancer and inflammation. In addition, the novel prodrug liposome 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. cancer, inflammation, psoriasis, and eczemas, or are manifestations of general 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 formula- tions 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 owing to skin impermeability, many therapeutic agents must be applied per os or par- enterally even though the skin is the target organ.
Liposome 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, 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 un- derlaying 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 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. 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 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 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 polysaturated 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 render the double prodrug lipid a substrate for PLA2. Furthermore, conventional drug substances can be incorporated and transported specifically to the diseased site by the novel lipid-based carriers.
Thus the new carriers liposomes 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.
Imaging of diseased tissue with contrast carriers composed of the novel and un- natural lipids
The novel lipid-based carrier systems are useful in the targeted diagnosis of diseases such as cancer, infection, and inflammation, which are characterized by localized and elevated activity of extracellular PLA2. The increased PLA2 activity combined with the observation that microparticulates accumulate in the diseased tissue via extravasation through leaky capillaries provide the basis for using contrast agent microcarriers for enhanced imaging.
Thus, the novel diagnostic system takes advantage of the fact that liposomes (and mi- celles), which can be specifically degraded by extracellular PLA2 also can be designed to circulate in the blood stream sufficiently long to reach the target tissue where the PLA2 activity is elevated. At the diseased site the lipid derivatives of the liposomes are cleaved by PLA2 so as to liberate the image enhancing agents.
Diagnostic imaging is widely used in medicine. It requires that an appropriate intensity of signal from an area of interest is achieved in order to differentiate diseased tissue from normal surrounding tissue. Imaging involves the relationship between the three spatial dimensions of the region of interest and a fourth dimension, time, which relates to both the pharmacokinetics of the diagnostic agent and the period necessary to ac- quire the image. The physical properties that can be used to create an image signal include, e.g. emission or absorption of radiation, nuclear magnetic moments and relaxation, and transmission or reflection of ultrasound. Usually, the imaging of different organs and tissues for early detection and localization of numerous pathologies cannot be successfully achieved without using appropriate contrast agents.
Imaging contrast agents relate to substances, which are able to absorb certain types of signal much stronger than surrounding tissues. The contrast agents are specific for each imaging technique, and as a result of their accumulation in certain diseased sites of interest, those sites may be visualized when an appropriate imaging technique is applied. The tissue concentration that must be achieved for successful imaging varies between diagnostic techniques.
To facilitate the accumulation of contrast agents in the diseased zone, various mi- croparticulates have been suggested as carriers for contrast agents. Among those mi- crocarriers, liposomes draw special attention because of their easily controlled properties. For almost two decades liposomes have been recognized as promising carriers for drugs and diagnostic agents for the following reasons: (1) Liposomes are completely biocompatible; (2) they can entrap practically any drug or diagnostic agent into either the internal water compartment or into the membrane itself depending on the physico- chemical properties of the compound; (3) liposome-incorporated compounds are pro- tected from the inactivating effect in the body, yet at the same time do not cause undesirable side-reactions; (4) liposomes also provide a unique opportunity to deliver pharmaceuticals or diagostic agents into cells or even inside individual cellular compartments. Pursuing different in vivo delivery purposes, the size, charge and surface properties of liposomes can easily be changed simply by incorporation of different lipids and/or by variation of the preparation methods.
One area of important potential application of the new contrast-loaded liposomes is tumour imaging. It is well established that PLA2 is secreted by malignant cells and immu- nohistochemical staining of various cancers, including cancer of pancreas, breast and stomach has shown increased levels of PLA2. An increased expression and secretion of PLA2 is also found in several cancer cell lines stimulated by interleukines such as IL- 6. In addition, elevated extracellular PLA2 activity has also been described in inflammatory and infected tissues.
The main mechanism of liposome accumulation in tumours is via extravasation through leaky tumour capillaries into the interstitial space. The tumour accumulation can be significantly increased by using long-circulating polymer coated liposomes.
The lipid-based microcarriers may also be used for visualization of inflammation and infection sites. Similar to what is known with respect to cancer tissue, the use of mi- croparticulate imaging agents for the visualization of infection and inflammation sites characterized by elevated PLA2 activity is based on the ability of microparticulates to accumulate via extravasation through leaky capillaries.
One of the important features of the novel diagnostic system is that certain lipid derivatives will be cleaved by extracellular PLA2 in a well-defined manner in specific extracellular locations of diseased mammalian tissue characterized by an elevated activity of PLA2. It has been found that extracellular PLA2 is capable of cleaving mono- ether/monoester lipid derivatives so as to target the diagnostic label in the relevant diseased tissue. Degradation of the lipid derivatives and the liposomes by PLA2 leads to a site-specific release of the diagnostic contrast agents in the diseased tissue. Targeting of infected tissues with lipid-based carriers composed of the novel and unnatural lipids
One of the important features of the targeted delivery system is the rapid uptake of Ii- posomes 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. Trap. 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. Ac- cordingly, 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 determin- ing the therapeutic window "TW", which is a numerical value derived from the relationship between the compound's induction of haemolysis and its ability to inhibit the growth of tumour cells. TW values are defined as HI5/GI50 (wherein "Hl5" equals the concentration of compound inducing the haemolysis 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 haemolytic 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% haemolysis. 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 haemolysis 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 sufficient level of tumour growth inhibition without also attaining an unacceptable level of haemolysis. 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 be- lieved 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.

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 comprising (I) lipid de- rivatives comprising (a) an organic radical having at least 2 carbon atoms, and (b) a hydrophilic moiety, where the lipid derivative is a substrate for extracellular phospholi- pase A2 to the extent that the organic radical can be hydrolytically cleaved off leading to an intramolecular cyclization reaction, and (II) lipopolymers and/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 lipid derivative furthermore comprises an aliphatic group which remains substantially unaffected by the hydrolytically activity of phospholipase A2, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment that performs an in- tramolecular cyclization reaction.
3. The lipid based drug delivery system according to claim 1, wherein the lipid derivatives is a prodrug whereby the drug substance is incorporated in the system as a prodrug.
4. The lipid based drug delivery system according to claim 1-3, wherein said system furthermore comprises at least one second drug substance.
5. The lipid based drug delivery system according to claim 1, wherein the lipopolymers and/or glycolipids are represented by a fraction of the lipid derivative.
6. The lipid based drug delivery system according to any of the claims 1-5, 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.
7. The lipid based drug delivery system according to any of the claims 1-6, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the at least one second drug substance.
8. The lipid based drug delivery system according to any of the claims 1-6, wherein the organic moiety which is released upon the intramolecular cyclization, is an auxiliary drug substance or an efficiency modifier for the at least one second drug substance.
9. The lipid based drug delivery system according to any of the claims 1-8, wherein the lipid derivative is a lipid derivative of the following formula:
RD RD I I
CH-X-R1 CH-X-R1
I I
CH-Y-R2 or CH-Z-R3
I I CH2-Z-R3 CH2-Y-R2
Wherein,
RD is an ester moiety ((CH2)πC(O)D1), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity. The hydrogens in the CH2 groups in RD can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 groups in RD can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group. Alternatively RD is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA2 hydrolysis;
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 carbonyl carbon atom;
R1 is an aliphatic group of the formula Y1Y2, where Y1 is -(CH2)ni-(CH=CH)n2-(CH2)n3- (CH=CH)π4-(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 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 substituents;
R2 is an organic radical having at least 2 carbon atoms;
R3 is selected from phosphatidic acid (PO2-OH), derivatives of phosphatidic acid and bio-isosters to phosphatic acid and derivatives thereof.
10. The lipid based drug delivery system according to any of the claims 1-8, wherein the lipid derivative is a lipid derivative of the following formula:
CH2-(CH2)n-C(O)-D2 CH2-(CH2)n-C(O)-D2
I I
CH-Y-R2 or CH-Z-R3
I I CH2-Z-R3 CH2-Y-R2
Wherein,
D2 is an organic moiety with biological activity, i.e. a drug substance in its broadest sense, n is an integer from 0-5, preferably 0 - 2. The hydrogens in the CH2 groups can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl;
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;
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 carbonyl carbon atom;
R2 is an organic radical having at least 2 carbon atoms; R3 is selected from phosphatide acid (PO2-OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof.
11. The lipid based drug delivery system according to claim 9 or 10, wherein R2 is an 5 aliphatic group of a length of at least 2 carbon atoms.
12. The lipid based drug delivery system according to claim 11 , wherein R2 is a group of the formula Y1Y2.
10 13. The lipid based drug delivery system according to any of the claims 1-12, wherein at least a fraction of the lipid is of the formula defined in claim 9, wherein R3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactic acid), poly(glycolic acid), poly(lactic acid)-poly(glycolic acid) copolymers, polyvinyl alcohol, polyvinylpyrrolidone, polymethoxazoline, polyethyloxazoline, polyhy-
15 droxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and deriva- tised celluloses, is covalently attached.
14. The lipid based drug delivery system according to any of the claims 1-13, wherein the lipid derivative constitutes 5-100 mol% of the total dehydrated system.
20
15. The lipid based drug delivery system according to any of the claims 1-14, wherein the lipopolymer constitutes 2-50 mol% of the total dehydrated system.
16. The lipid based drug delivery system according to any of the claims 1-15, wherein 25 the system is in the form of liposomes.
17. The lipid based drug delivery system according to any of the claims 1-16, wherein the at least one second drug substance is a therapeutically and/or prophylactically active substance selected from (i) antitumor agents, (ii) antibiotics and antifungals, or (iii)
30 antiinflammatory agents.
18. A pharmaceutical composition comprising the drug delivery system according to any of the claims 1-17 and optionally a pharmaceutically acceptable carrier.
35 19. A lipid based drug delivery system according to any of the claims 1-17 for use as a medicament.
20. Use of the lipid based drug delivery system according to any of the claims 1-17 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.
21. The use according to claim 20, wherein the diseases or conditions are selected from the group consisting of inflammatory conditions and cancer.
22. The use according to claim 21 , wherein the type of cancer is selected from the group consisting of brain cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, leukemia, lymphoma, sarcoma and carcinoma.
23. The use according to any of the claims 21-22, wherein the increase in extracellular phospholipase A2 activity is a least 25% compared to the normal level of activity in the tissue in question.
24. The lipid based drug delivery system according to any of the claims 1-8, wherein the lipid derivative is a lipid derivative of the following formula:
RD RD
I I
CH-X-R1 CH-X-R1
I I
CH-Y-R2 or CH-Z-R3 I I
CH2-Z-R3 CH2-Y-R2
Wherein,
RD is an ester moiety ((CH2)nC(O)D1), which will facilitate an intramolecular cyclization upon PLA2 hydrolysis, where n is an integer from 0 - 5, preferably 0 - 2, and D1 is an organic moiety, possibly MeOH or EtOH, preferably an organic moiety with biological activity. The hydrogens in the CH2 groups in RD can be substituted with intramolecular cyclization facilitating groups, e.g. fluoride alpha to the carbonyl. The CH2 groups in RD can be substituted, e.g. with O, S, NH, or bioisosters thereof, in particular at the alpha position to the carbonyl group. Alternatively RD is an organic moiety that contains a leaving group, e.g. iodide, i.e. an organic moiety that can facilitate an intramolecular cyclization upon PLA2 hydrolysis;
X = Y;
5
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;
10 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, 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
15 n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer 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 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 independ-
20 ently may be substituted with halogens and aliphatic substituents;
R2 is an organic radical having at least 2 carbon atoms;
R3 is selected from phosphatidic acid (PO2-OH), derivatives of phosphatidic acid and 25 bioisosters to phosphatic acid and derivatives thereof.
25. The lipid based drug delivery system according to claim 23, wherein R2 is an aliphatic group of a length of at least 7 carbon atoms.
30 26. The lipid based drug delivery system according to claim 11, wherein R2 is a group of the formula Y1Y2.
27. The lipid based drug delivery system according to claims 1 and 26, wherein the system is in the form of liposomes. 35
28. The lipid based drug delivery system according to any of the claims 1-8, 24-27, wherein the drug substance is a therapeutically and/or prophylactically active substance selected from (i) antitumor agents, (ii) antibiotics and antifungals, or (iii) antiinflammatory agents.
5
29. A pharmaceutical composition comprising the drug delivery system according to any of the claims 1-8 and 24-28 and optionally a pharmaceutically acceptable carrier.
30. A lipid based drug delivery system according to any of the claims 1-8 and 24-28 for 10 use as a medicament.
31. Use of the lipid based drug delivery system according to any of the claims 1-8 and 24-28 for the preparation of a medicament for the treatment of diseases or conditions associated with a localised increase in extracellular phospholipase A2 activity in mam-
15 malian tissue.
32. The use according to claim 31 , wherein the diseases or conditions are selected from the group consisting of inflammatory conditions and cancer.
20 33. The use according to claim 32, wherein the type of cancer is selected from the group consisting of brain cancer, breast cancer, lung cancer, colon cancer, ovarian cancer, leukemia, lymphoma, sarcoma, and carcinoma.
34. The use according to any of the claims 31-32, wherein the increase in extracellular 25 phospholipase A2 activity is a least 25% compared to the normal level of activity in the tissue in question.
PCT/DK2007/000143 2006-03-23 2007-03-23 Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis WO2007107161A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002647779A CA2647779A1 (en) 2006-03-23 2007-03-23 Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis
JP2009500701A JP2009530318A (en) 2006-03-23 2007-03-23 Lipid-based drug delivery system containing phospholipase A2-degradable lipid that undergoes intramolecular cyclization during hydrolysis
AU2007229160A AU2007229160A1 (en) 2006-03-23 2007-03-23 Lipid based drug delivery systems comprising phospholipase A2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis
EP07711285A EP2004236A2 (en) 2006-03-23 2007-03-23 Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200600411 2006-03-23
DKPA200600411 2006-03-23

Publications (3)

Publication Number Publication Date
WO2007107161A2 true WO2007107161A2 (en) 2007-09-27
WO2007107161A8 WO2007107161A8 (en) 2007-11-22
WO2007107161A3 WO2007107161A3 (en) 2008-04-10

Family

ID=38121721

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK2007/000143 WO2007107161A2 (en) 2006-03-23 2007-03-23 Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis

Country Status (5)

Country Link
EP (1) EP2004236A2 (en)
JP (1) JP2009530318A (en)
AU (1) AU2007229160A1 (en)
CA (1) CA2647779A1 (en)
WO (1) WO2007107161A2 (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011032563A1 (en) 2009-09-17 2011-03-24 Bio-Bedst Aps Medical use of spla2 hydrolysable liposomes
WO2011047689A2 (en) 2009-10-23 2011-04-28 Bio-Bedst Aps Liposomes with improved storage stability
WO2011098578A2 (en) 2010-02-12 2011-08-18 Bioneer A/S Liposome system for ocular administration

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2667848A1 (en) * 2011-01-28 2013-12-04 Koninklijke Philips N.V. Carriers for the local release of hydrophilic prodrugs

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039831A1 (en) * 1995-06-07 1996-12-19 The Regents Of The University Of California Prodrugs of pharmaceuticals with improved bioavailability
EP1484332A2 (en) * 2000-02-10 2004-12-08 Liplasome Pharma A/S Lipid-based drug delivery system for administration of a drug substance

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996039831A1 (en) * 1995-06-07 1996-12-19 The Regents Of The University Of California Prodrugs of pharmaceuticals with improved bioavailability
EP1484332A2 (en) * 2000-02-10 2004-12-08 Liplasome Pharma A/S Lipid-based drug delivery system for administration of a drug substance

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANDERSEN T L ET AL: "Triggered activation and release of liposomal prodrugs and drugs in cancer tissue by secretory phospholipase A2" CURRENT DRUG DELIVERY, BENTHAM SCIENCE PUBLISHERS, HILVERSUM, NL, vol. 2, no. 4, October 2005 (2005-10), pages 353-362, XP008088792 ISSN: 1567-2018 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011032563A1 (en) 2009-09-17 2011-03-24 Bio-Bedst Aps Medical use of spla2 hydrolysable liposomes
CN102630175A (en) * 2009-09-17 2012-08-08 拜奧贝德斯特有限责任公司 Medical use of SPLA2 hydrolysable liposomes
WO2011047689A2 (en) 2009-10-23 2011-04-28 Bio-Bedst Aps Liposomes with improved storage stability
US9820941B2 (en) 2009-10-23 2017-11-21 Bio-Bedst Aps SPLA2 hydrolysable liposomes with improved storage stability
WO2011098578A2 (en) 2010-02-12 2011-08-18 Bioneer A/S Liposome system for ocular administration

Also Published As

Publication number Publication date
EP2004236A2 (en) 2008-12-24
JP2009530318A (en) 2009-08-27
WO2007107161A3 (en) 2008-04-10
WO2007107161A8 (en) 2007-11-22
AU2007229160A1 (en) 2007-09-27
CA2647779A1 (en) 2007-09-27

Similar Documents

Publication Publication Date Title
EP1838283B1 (en) Lipid-based drug delivery systems containing unnatural phospholipase a2 degradable lipid derivatives and the therapeutic uses thereof
EP1272225B1 (en) Lipid-based systems for targeting diagnostic agents
KR100889139B1 (en) Irinotecan preparation
Apolinário et al. Lipid nanovesicles for biomedical applications:‘What is in a name’?
US20090232900A1 (en) Nano-micellar preparation of anthracylcline antitumor antibiotics encapsulated by the phosphatide derivatized with polyethylene glycol
EP1254143B1 (en) Lipid-based drug delivery systems
US20040166151A1 (en) Lipid-based drug delivery systems containing phospholipase A2 degradable lipid derivatives and the therapeutic uses thereof
EP1759699B1 (en) Liposome preparation containing slightly water-soluble camptothecin
WO2006086992A2 (en) 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
EP2004236A2 (en) Lipid based drug delivery systems comprising phospholipase a2 degradable lipids that perform an intramolecular cyclization reaction upon hydrolysis
US6180137B1 (en) Etherlipid-containing multiple lipid liposomes
USRE39042E1 (en) Etherlipid-containing multiple lipid liposomes
KR100768265B1 (en) Heparin coated liposomes to prolong circulation time in bloodstream and preparation method thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07711285

Country of ref document: EP

Kind code of ref document: A2

WWE Wipo information: entry into national phase

Ref document number: 2009500701

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2647779

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2007229160

Country of ref document: AU

NENP Non-entry into the national phase in:

Ref country code: DE

ENP Entry into the national phase in:

Ref document number: 2007229160

Country of ref document: AU

Date of ref document: 20070323

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2007711285

Country of ref document: EP