LIPID-BASED DRUG DELIVERY SYSTEMS CONTAINING PHOSPHOLIPASE A2 DEGRADABLE LIPID DERIVATIVES FOR MEDICAL USE AGAINST PARASITIC INFECTIONS

Abstract: The present invention is directed to a lipid-based delivery system for administration of an active drug substance selected from lysolipid derivatives which are particularly useful in the treatment or detection of parasitic infections, especially parasitic infections which cause an elevated PLA2 level in the infected mammal. Preferred parasitic infections are infections wherein the life cycle of the parasite involves the liver and/or spleen of the infected organism.

Description: LIPID-BASED DRUG DELIVERY SYSTEMS CONTAINING PHOSPHOLIPASE A2 DEGRADABLE LIPID DERIVATIVES FOR MEDICAL USE AGAINST PARASITIC INFECTIONS FIELD OF THE INVENTION The invention relates to lipid-based pharmaceutical compositions for use in the treatment or detection of parasitic infections. BACKGROUND OF THE INVENTION US 5,827,836 discloses retinoyl substituted glycerophophoethanolamines. It is stated that the compounds and salts thereof exhibit antitumor, anti-pso atic and anti-inflammatory activities. A possible class of compounds has a fatty ether substituent in the 1 -position, a retinoid ester (retinoyl) substituent in the 2-position and a phosphoethanolamine substituent in the 3-position. It is mentioned that some of the compounds can be presented in a liposome formulation. US 4,372,949 discloses a carcinostatic and immunostimulating agent containing a lysophospholipid and a phospholipid. Examples of compounds are 3-phosphorylcholine having a C 5 _ 22 -acyloxy or C 5 . 22 -alkoxy substituent in the 1 -position, and a hydrogen, hydroxy, C^-acyloxy or Cι. 5 -alkoxy substituent in the 2-position. It is mentioned that the agents can be dispersed in the form of micelles or lipid vesicles. US 5,484,911 discloses nucleoside 5'-diphosphate conjugates of ether lipids which exhibit antiviral activity. The compounds may have a fatty ether/thioether substituent in the sn-1- position and a fatty acid ester substituent in the sn-2-position. The compounds are designed so as to penetrate the cell membrane whereafter the nucleoside drug is liberated by cleavage by intracellular phosphatases. It is furthermore suggested that the also liberated ether lipids may be subsequently cleaved by intracelluar phospholipase A 2 . It is suggested that the conjugates can be presented in the form of micelles which more easily can be taken up by macrophages. US 4,622,392 discloses cytotoxic compounds of the nucleotide-lipid conjugate type. ES 2 034 884 discloses 2-aza-phospholιpιder as PLA 2 inhibitors Similarly, de Haas et al (Biochem Biophys Acta, Lipid and Lipids Metabolism, 1167 (1993) No 3, pp 281-288, discloses inhibition of pancreatic PLA 2 by (R)-2-acylamιno phospholipid analogues Hoffman et al , Blood, Vol 63, No 3 (March), 1984, pp 545-552, discloses the cytotoxicity of PAF and related alkyl-phospholipid analogues in human Leukemia cells WO 94/09014 discloses phosphoric acid esters as PLA inhibitors A group of the inhibitors are 1-0-phospho-2-0-(C 2 2 ι-acyl)-(C 12 2 -alkanes) Xia and Hui discloses the chemical synthesis of a series of ether phosphohpids from D- mannitol and their properties as tumor-cytotoxic agents 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 sn-2-esters of lysophospholipids Such drugs are designed so as to be cleaved by intracelluar phosphohpase A 2 Vadas et al (Infection and Immunity, 60 (1992) 3928-3931 and Am J Trop Hyg 49 (1993) 455-459) describe the induction of circulating PLA 2 expression in humans with malaria infections caused by Plasmodium falciparum Lux et al (Biochem Parasitology 111 (2000) 1-14) describe the anti-leishmania action of certain ether-lipid analogues which is believed to be caused by the interference with important biosyntheses of the parasite BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to drug delivery systems which are particularly useful in the treatment of parasitic infections especially parasitic infections wherein the life cycle of the parasite involves the liver and/or spleen of the infected organism The liposomes of the present invention are only degraded to a very low extend by enzymes present in the blood stream, whereas said liposomes are up-concentrated and degraded in the liver and/or spleen by the macrophages present in these organs This degradation of the liposomes releases constituents which are designed to be toxic for various types of parasites In one embodiment of the present invention the lysohpids released from the liposome are toxic to the parasites, in another embodiment anti-parasitic drugs are encapsulated in the liposomes In a further embodiment a combination of liposomes made from lipids which are toxic to parasites in which anti-parasitic drugs are encapsulated Accordingly, a highly efficient and indirectly specific release of substances toxic to parasites is achieved in the case of parasitic infection of tissues characterised by the presence of macrophages, such as the liver and/or the spleen Yet another embodiment of the present invention is the diagnostic use of the presently described drug delivery systems, wherein the drug delivery system carries a label which, by the action described above, is specifically directed towards the tissue or organs being infected by the parasite DESCRIPTION OF THE DRAWINGS Fig 1 Heat capacity curves obtained using differential scanning caloπmetry (a) Multilamellar, MLV (the upper curve) and unilamellar, LUV (the bottom curve) liposomes made of 1 mM 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholιne (1-O-DPPC) (b) MLV (the upper curve) and LUV (the bottom curve) liposomes made of dipalmitoylphosphatidylcho ne (DPPC) Fig 2 Characteristic reaction time profile at 41 °C for phosphohpase A 2 , PLA 2 , (A piscivorus piscivorus) hydrolysis of unilamellar liposomes composed of 1-O-DPPC The PLA 2 hydrolysis reaction is monitored by intrinsic fluorescence (solid line) from the enzyme and 90° static light scattering (dashed lines) from the hpid suspension After adding PLA 2 , at 800 sec to the equilibrated liposome suspension a characteristic lag-time follows before a sudden increase in the catalytic activity takes place Samples for HPLC were taken before adding the enzyme and 20 minutes after the observed lag time Fig 3 HPLC chromatograms illustrating the effect of phosphohpase A 2 hydrolysis of liposomes composed of 1-O-DPPC The chromatograms show the amount of 1-O-DPPC (100%, solid line) before phosphohpase A 2 (A piscivorus piscivorus) was added to the liposome suspension and the amount of 1-O-DPPC (21 %, dashed line) after the lag-burst Fig 4 PLA 2 -controlled release of the fluorescent model drug calcein from liposomes composed of 25 μM 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholιne (1 -O- DPPC) suspended in a 10 mM HEPES-buffer (pH = 7 5), as a function of time 25 nM phosphohpase A 2 (A piscivorus piscivorus) was added at time 900 sec, the temperature 5 was 37°C The percentage of calcein released is determined as % Release =100 (l F(t) - IBV T - IB), where l F(t) is the measured fluorescence at time t after addition of the enzyme, l B IS the background fluorescence, and l τ is the total fluorescence measured after addition of Triton X-100 which leads to complete release of calcein by breaking up the liposomes 10 Fig 5 PLA 2 -controlled release of the fluorescent model drug calcein across the target membrane of non-hydrolysable membranes (see Fig 11 b), as a function of time for liposomes composed of 25 μM 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3- phosphochohne (1 -O-DPPC) suspended in a 10 mM HEPES-buffer (pH = 7 5) 25 nM phosphohpase A 2 was added at time 0 sec and the temperature was 37°C The 15 percentage of calcein released is determined as described in Fig 4 Fig 6 Hemolysis profile of normal red blood cells in the presence of liposomes composed of 100% 1 -O-DPPC (squares), 95% 1 -O-DPPC and 5% negatively charged 1-0- hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamιne-N-[methoxy(polyethylene 20 glycol)-350] (1-O-DPPE-PEG350) (triangles) and 1-0-octadecyl-2-0-methyl-s/ι-glycero-3- phosphochohne (ET-18-OCH 3 ) (diamonds) The concentrations that yield 5% hemolysis (H 5 ) were well above 2 mM for liposomes composed of 100% 1-O-DPPC, and for liposomes composed of 90% 1 -O-DPPC with 5% DPPE-PEG350 Hemolysis assay was performed as described by Perkins et al , 1997, Biochimica et Biophysica Acta 1327, 61- 25 68 Briefly, each sample was serially diluted with phosphate buffered saline (PBS), and 0 5 ml of each dilute suspension was mixed with 0 5 ml washed human red blood cells (RBC) [4% in PBS (v/v)] Sample and standard were placed in a 37°C incubator and agitated for 20 hours Tubes were centnfuged at low speed (2000 x G) for 10 minutes and 200 μl of the supernatant was quantitated by absorbance at 550 nm 100 percent 30 hemolysis was defined as the maximum amount of hemolysis obtained from the detergent Triton X-100 The hemolysis profile in Fig 6 shows a low hemolysis value (below 5% percent) for 2 mM 1-O-DPPC-hposomes and for 1 -O-DPPC with 5% 1 -O-DPPE-PEG350, liposomes Fig 7 Characteristic reaction time profiles at 41 °C for PLA 2 (A piscivorus piscivorus) hydrolysis of unilamellar liposomes incorporated with 0 and 5% 1-O-DPPE-PEG350 hpopolymers The PLA 2 hydrolysis reaction is monitored by intrinsic fluorescence (solid line) from the enzyme and 90° static light scattering (dashed lines) from the suspension After adding PLA 2 to the equilibrated liposome suspension a characteristic lag-time follows before a sudden increase in the catalytic activity takes place Fig 8 PLA 2 -controlled release of the fluorescent model drug calcein across the target membrane of non-hydrolysable D-O-SPC membranes as a function of time for micelles composed of 25 μM 1-O-DPPE-PEG350 (dotted line), DSPE-PEG750 (dashed line) suspended in a 10 mM HEPES-buffer (pH = 7 5) Phosphohpase A 2 (25 nM) was added at time 1200 sec and the temperature was 41 °C The percentage of calcein released is determined as described in Fig 4 PLA 2 catalysed hydrolysis of 1-O-DPPE-PEG350 induced the fastest and highest release Fig 9 HPLC chromatograms illustrating the effect of phosphohpase A 2 hydrolysis of micelles composed DSPE-PEG750 (0 150 mM) The chromatograms show the amount of stearic acid generated before (solid line) phosphohpase A 2 (A piscivorus piscivorus) was added to the micelle suspension and the amount (dashed line) of DSPE-PEG750 after the lag-burst The dotted line show pure stearic acid (0 4 mM) The percentage hydrolysis was calculated on basis of the integrated area of the stearic acid standard (115850 units) and the integrated area of the sample (45630 units) The concentration of the stearic acid in the sample was calculated to (45630/115850 x 0 4 mM) 0 157 mM, which means that 100% of the DSPE-PEG750 was hydrolysed to lyso-SPE-PEG750 and stearic acid Fig 10 Describes the principle of posomal drug targeting, release and absorption by extracellular enzymes (I) Reticuloendothehal system, e g macrophages in liver and spleen infected with parasites (i e Leishmania) (II) Prodrug carrier liposome (III) Target parasitic cell and cell membrane (IV) Prodrug (i e monoether-hpid), proenhancer (hpid), proactivator (hpid) (V) Drugs (i e ether-lysohpid and fatty acid derivatives) enhancers (lysolipid + fatty acid) PLA 2 activators (lysolipid + fatty acid) Fig 11 Schematic illustration of a hposomal drug-targeting principle involving accumulation of the hposomal drug carriers the RES in porous diseased tissue and subsequent release of drug and transport across the target membrane via extracellular PLA 2 activity (I) Prodrug carrier liposome (II) Non-degradable target hposomal membrane (III) Non-hydrolysable ether- pids (IV) Proenhancer (hpid), prodrug (i e monoether-hpid), proactivator (hpid) (V) Enhancers (lysolipid + fatty acid), drugs (i e ether-lysohpid and fatty acid derivatives), PLA 2 activators (lysolipid + fatty acid) Fig 12 (a) PLA 2 -controlled release of the fluorescent model drug calcein across the target membrane as a function of time for different compositions of the carrier liposomes The temperature is 37°C In comparison with bare DPPC carriers, the rate of release of the model drug is dramatically enhanced for the carriers incorporated with 2 5 mol% of the negatively charged DPPE-PEG2000 A further augmentation of the rate of release is obtained if the carrier also contains a short-chain phospholipid, didecanoylphosphatidylchohne (DCPC), which acts as a local activator for the enzyme The percentage of calcein released is determined as % Release =100 (l F(t) -l B ./(lτ - I B ). where l F(t) is the measured fluorescence at time t after addition of the enzyme, l B is the background fluorescence, and l τ is the total fluorescence measured after addition of Triton X-100 which leads to complete release of calcein by breaking up the target liposomes (b) PLA 2 -controlled release of the fluorescent model drug calcein across the target membrane as a function of time for different temperatures As the temperature is raised, the rate of release is enhanced due to increased activity of the enzyme induced by structural changes in the hpid bilayer substrate of the carrier liposome In the present assay a maximum release of about 70% is achieved in all cases The insert shows the time of 50% calcein release, t 50 %, as a function of temperature The concentration of the target and carrier liposomes are 25 μM, and PLA 2 is added in a 25 nM concentration in a HEPES buffer with pH=7 5 Fig 13 Total release after 20 mm of the fluorescent model drug calcein across the target membrane as a function of adding increasing amounts of lyso-palmitoyl phospholipid and palmitic acid, separately, and in a 1 1 mixture The concentration of the target membranes is 25 μM in a HEPES buffer with pH=7 5 at a temperature of 39°C Fig 14 PLA 2 -controlled release of the fluorescent model drug calcein from liposomes composed of 25 μM 90 mol% 1-O-DPPC and 10 mol% of the negatively charged 1-0- DPPE-PEG350 suspended in a 10 mM HEPES-buffer (pH = 7 5), as a function of time 50 nM (straight line), 1 nM (solid line) and 0 02 nM (dotted line) phosphohpase A 2 (A piscivorus piscivorus) was added at time 300 sec, the temperature was 35 5°C The percentage of calcein released is determined as describe in Fig 4 Fig 15 Emission spectrum of 1 mol% bis-py-DPC incorporated in negatively charged liposomes (0 100 mM) before (solid line) and after (dashed line) adding 100 nM PLA2 (Agkistrodon piscivorus piscivorus) to a liposome suspension equilibrated at 41 °C Fig 16 Characteristic reaction time profile at 41 °C for rat phosphohpase catalysed hydrolysis of negatively charged liposomes The catalytic reaction was initiated by adding cell-free peritoneal fluid to 2 5 ml of the thermostated liposome suspension equilibrated for 60 sec prior to addition of peritoneal fluid The hydrolysis reaction is monitored by monomer fluorescence (solid line) and eximer fluorescence (dashed line) from bis-py- DPC After adding undiluted peritoneal fluid, at 60 sec, to the equilibrated liposome suspension a sudden increase in monomer fluorescence, and a simultaneously decrease in eximer fluorescence is observed as the bis-py-DPC substrate is hydrolysed The insert shows the reaction time profile of the first 120 sec DETAILED DESCRIPTION OF THE INVENTION One of the important features of the present invention is the realisation of the rapid uptake of liposomes in vivo by cells of the mononuclear phagocytic system (MPS) The MPS comprises the macrophages, one of the most important components of the immune system especially in clearance of foreign particles, including liposomes The macrophages resides in various organs and tissues, e g in connective tissue (as histiocytes), in the liver (Kupffer cells) and as free and fixed macrophages in the spleen, bone marrow and lymph nodes When liposomes in general are administered intravenously they are typically removed from the circulation by the macrophages of the liver, spleen and bone marrow This removal consists of a first opsonization by blood proteins followed by macrophage uptake of these marked liposomes The immune opsonins are comprised by immunoglobu ns and complement proteins, and although each opsonin has its own interaction and confers different fates, it seems that liver sequestration is complement mediated and the spleen removes the opsonized liposomes in general The enhanced clearance of the liposomes of the present invention obtained by the opsonization can be characterised as passive targeting As the MPS uptake of liposomes results in the accumulation of the liposomes in tissues like the liver, the spleen and the bone marrow it enables a targeting directed against parasitic infections of these tissues The liposomes of the present invention comprises constituents which can act as a label for detecting parasite infected tissue/organs or can act as anti-parasitic drugs when released from the liposomes to the tissues of a mammal suffering from a parasitic infection One embodiment of the present invention is thus a method for the treatment of parasitic infections which is characterised by an increased level of PLA 2 in a mammal, preferably a human, by administering to the mammal an efficient amount of a lipid-based delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a hpid derivative having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said prodrug furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative which is not a substrate for lysophosphohpase A further embodiment is a method for the treatment of parasitic infections which is characterised by an increased level of PLA 2 in a mammal, preferably a human, by administering to the mammal an efficient amount of the hpid based delivery system described above for administration of an second substance, wherein the second substance is a anti-parasitic drug incorporated in said system In one specific embodiment of the present invention the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the parasite Especially a situation wherein the parasitic infection involves the liver and/or the spleen and/or the bone marrow of the mammal is comprised by the present invention The treatment is preferably performed by systemically administration of the hpid based delivery system of the present invention, even more preferably administered parenterally by injection such as intravenous injection Yet another embodiment of the present invention is a method for diagnosing a parasitic infection which is characterised by an increased level of PLA 2 of the infected tissue by administering to the mammal an efficient amount of the hpid based delivery system described above for administration of an second substance, wherein the second substance is a label incorporated in said system By administering such a construct to a patient suspected of having a parasitic infection it is possible to determine if the patient is infected and in the case of a parasitic infection the localized areas of the patients body harbouring the parasite is identified The localized deliverance of the diagnostic agent - the label - enables the use of diagnostic imaging tools such as positron emission tomography (PET), X-ray, gamma-scmtigraphy, magnetic resonance (MR) imaging, computed tomography (CT) imaging and ultrasonography The labels applicable for the medical imaging are selected from the group consisting of diagnostic radionuclides, such as ln, 99 Tc, 67 Ga, 1 I C, paramagnetic ions, such as Gd and Mn, and iron oxide, gas, such as air, argon, nitrogen, Iodine, bromine and barium Target parasitic organisms The liposomes of the present invention are able to deliver various ether-hpid analogues to tissues harbouring parasites due to increased levels of PLA 2 levels of said tissue The ether-hpid analogues have been shown to result in perturbation of key enzymes involved m e g alkyl-phospho pid biosynthesis of parasites such as Leishmania and Trypanosomas (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/tissues wherein high concentrations of macrophages can be found, targeting parasites inhabiting these organs is preferred because of resulting accumulation of liposomes of the present invention, but other tissues characterized by having increased PLA 2 levels during parasitic infection may also be of interest Furthermore, parasitic infections which are characterised by highly elevated levels of circulating PLA 2 , such as malaria-causing parasites, e g caused by Plasmodium falαparum, are also targets for treatment with liposomes of the present invention This characteristic of elevated levels of circulating PLA 2 of malaria infections caused by the parasite Plasmodium falciparum has been described by e g Vadas et al (Infection and Immunity, 60 (1992) 3928-3931 and Am J Trop Hyg 49 (1993) 455-459) Examples of parasite infections resulting in increased PLA 2 level of the parasite harbouring tissue are given below Leishmania Members of the genus Leishmania has the potential to infect various vertebrate species, including humans, dogs, and rodents and the various types of leishmaniasis are confined primarily, but not exclusively, to Central and South America, central Africa, and parts of southern and central Asia The life cycle of members of the genus involve a vertebrate host e g the human and a vector that transmits the parasite between vertebrate hosts In the case of Leishmania the vector is various species of Phlebotomus sand flies The characteristic morphological form taken by the Leishmania paracite in the vector is the promastigote, and in this stage it reproduces asexually in the vector's gut Upon biting the vertebrate host, promastigotes from the vector are injected into the vertebrate host After the entrance into the vertebrate host he promastigotes change into a form called amastigote The amastigote reproduces in the host's cells, and when the vertebrate host cell eventually dies, the amastigotes are released and will potentially infect other cells The symptoms and pathology associated with leishmaniasis result from the amastigotes killing the host's cells Leishmania is the cause of several different conditions depending on the site of infection of the vertebrate host In some diseases the amastigotes do not spread beyond the site of the vector's bite which then results in a "cutaneous leishmaniasis" also known as oriental sore, Jericho boil, Aleppo boil, or Dehli boil These conditions often heal spontaneously In other instances the amastigotes may spread to the visceral organs, i e the liver and the spleen, which results in "visceral leishmaniasis" also known as kala-azar or Dum-Dum fever Furthermore, the amastigotes may spread to the mucous membranes of the mouth and nose, resulting in the condition of "mucocutaneous leishmaniasis" also known as espundia or uta Left untreated, these latter diseases result in high rates of mortality Non-limiting examples of Leishmania species are Leishmania major Friedhn, Leishmania (viannia) gr , Leishmania mexicana, Leishmania tropica, Leishmania donovani (mfantum), Leishmania aethiopica, Leishmania amazonensis, Leishmania ennettii, Leishmania chagasi and Leishmania pifanoi Traditional anti-leishmamal therapy agents are compounds like the pentavalent antimony compounds sodium stibogluconate (Pentosram™) and meglumme antimonate (Glucantime™), other drugs are amphotericin, metronidazole, allopuπnol and pentamidine, furthermore paromomycin and interferon gamma Both for malaria treatment and Leishmania treatment a new drug, Licochocone A, which was originally isolated from the roots of Chinese licorice, has been proposed Trypanosoma Three mam species of trypanosomes causes disease in humans, these are Trypanosoma gambiense and Trypanosoma rhodesiense, which both cause sleeping sickness in Africa, and Trypanosoma cruzi, which causes Chargas' disease in South America Both types of disease are characterised by bouts of parasitaemia and fever The damage to the organs is caused toxins released by the parasites The most common vector for transmitting the trypanosomes causing sleeping sickness is the tsetse fly Glossma sp The species that cause human African trypanosomiasis (sleeping sickness) also infect wild animals and can be transmitted from these animals to humans (zoonotic infections) In humans, Trypanosoma cruzi is found as both an intracellular form, the amastigote, and as a trypomastigote form in the blood The vector for Chagas' disease is a "true bug" (Hemiptera) such a Tnatoma, Rhodnius, or Panstongylus which ingests amastigotes or trypomastigotes when it feeds In the vector the parasite reproduces asexually and metacychc trypomastigotes are found in the vector's hindgut The vector defecates on the host's skin at the same time that it feeds, and the metacychc trypomastigotes enter the host's body, most often by being "rubbed in" to the vector's bite or the mucous membranes of the eye, nose, or mouth In the human host, Chagas' disease affects primarily the nervous system and heart but also sometimes the liver, spleen, bone and intestine Chronic infections result in various neurological disorders, including dementia, megacolon, and megaesophagus, and damage to the heart muscle Left untreated, Chagas' disease is often fatal The mam traditional drugs used for treatment of sleeping sickness are suramin and pentamidine Traditional drugs for Chargas' disease are pπmaquine, puromycin and nitrofurantom derivatives, but none of these drugs have proven really effective in the treatment for this condition Non-hmiting examples of Trypanosoma species are Trypanosoma cruzi (causing Chargas' disease), Trypanosoma brucei, Trypanosoma equiperdum and Trypanosoma evansi Plasmodium - malaria Malaria is one of the major killer diseases of the world causing an estimated one - two million deaths annually Malaria is caused by various species of plasmodia The female anophehne mosquito injects sporozoites, which in the liver can develop into merozoites The merozoites infect red blood cells and in the red blood cells the merozoites grow and eventually cause the cell to rupture and release more merozoites most of which infect other red blood cells and repeat the cycle Some of the merozoites evolve into gametocytes, which infect the female anophehne mosquito The rupture of the blood cells and the release of merozoites cause the fever associated with malaria The frequency of the fever attacks depends on the species of Plasmodium Plasmodium falciparum, Plasmodium vivax and Plasmodium ovale causes red blood cell rupture after 48 hrs and the malaπa patient therefor experiences violent fever attacks every third day Plasmodium malaπae causes red blood cell rupture every 72 hrs Entamoeba histolvtica This protozoan parasite infects the lower bowel and frequently causes amebic dysentery If untreated can lead to death This amoebae is not restricted to the large intestine but can spread to other soft organs, particularly the liver, where it can produce large abscesses over the course of a few years People become infected by ingesting the amebic cyst in contaminated food or water The oriental liver fluke The life cycle of the oriental liver fluke Chlornorchis smensis starts with a miracidium infecting a snail In the snail the miracidium develops into a cercanae The cercanae leaves the snail and penetrate the skin of a fish In the fish the cercanae encrysts itself in the mussel tissue and when the fish raw or undercooked fish is eaten the immature worm is released and it migrates to the liver where it matures causing damage to the liver Lipid derivatives The lipid-based delivery systems (liposomes or micelles) relies on hpid derivatives having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said prodrug furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative which is not a substrate for lysophosphohpase Although the terms " pid" and "lysolipid" (in the context of phosphohpids) 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 "hpid" is intended to mean tπesters of glycerol of the following formula CH 2 -0-R A CH-O-R 8 I CH 2 -0-R c wherein R A and R B are fatty acid moieties (C 9 30 -alkyl/alkylene/ alkyldiene/ alkyltπene/- alkyltetraene-C(=0)-) and R c is a phosphatidic acid (P0 2 -OH) or a derivative of phosphatidic acid Thus, the groups R A and R B are linked to the glycerol backbone via ester bonds The term "lysolipid" is intended to mean a hpid where the R B fatty acid group is absent (e g hydrolytically cleaved off), i e a glycerol derivative of the formula above where R B is hydrogen, but where the other substituents are substantially unaffected Conversion of a lipid to a lysolipid can take place under the action of an enzyme, specifically under the action of celluar as well as extracellular PLA 2 The terms "hpid derivative" and "lysolipid derivative" are intended to cover possible derivatives of the above possible compounds within the groups "hpid" 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, hpid derivatives and lysohpids should however fulfil certain functional criteria (see above) and/or structural requirements It is particularly relevant to note that the suitable lipid derivatives are those which have (a) an aliphatic group of a length of at least 7, preferably at least 9, carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety It will be evident that the aliphatic group and the organic radical will correspond to the two fatty acid moieties in a normal hpid and that the hydrophihc moiety will correspond to the phosphate part of a (phospho)hpιd or a bioisoster thereof Thus, one element of the idea behind the present invention is to exploit the increased level of extracellular PLA 2 activity in localised areas of the body of a mammal, in particular areas of parasitic infection, the lipid derivatives which can be utilised within the present invention should be substrates for extracellular PLA 2 , i e the hpid derivatives should be able to undergo hydrolytic, enzymatic cleavage of the organic radical corresponding to the fatty acid in the 2-posιtιon in a hpid Extracellular PLA 2 is known to belong to the enzyme class (EC) 3 1 1 4 Thus by reference to (extracellular) PLA 2 should be understood all extracellular enzymes of this class, e g lipases, which can induce hydrolytic cleavage of the organic radical corresponding to the fatty acid in the 2-posιtιon in a hpid One particular advantage of the lipid-based delivery system (as liposomes and micelles) is that extracellular PLA 2 activity is significantly increased towards organised substrates as compared to monomeπc substrates In view of the requirement to hydrolysabihty by extracellular PLA 2 , it is clear that the organic radical (e g aliphatic group) 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 carboxy c acid Furthermore, an important feature is that the aliphatic group (the group corresponding to the fatty acid in the 1 -position in a hpid) of the hpid derivative, i e the lysolipid derivative after cleavage by extracellular PLA 2 , is substantially unaffected by the action of extracellular PLA 2 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 PLA 2 Also, the lysolipid derivative resulting from the hydrolytic cleavage of the organic radical should not in itself be a substrate for lysophospholipase Lysophosphohpase is known to belong to the enzyme class (EC) 3 1 1 5 Thus by reference to lysophosphohpase should be understood all enzymes of this class that catalyses the reaction lyso(phospho)hpιd + water yielding phosphoglycerol + fatty acid The term "not a substrate for lysophospholipase" is intended to mean that lysophospholipase has an activity of less than 1% towards the substrate compared with the corresponding esterhpid, i e virtually not enzymatic activity Suitable examples of such lysolipid derivatives are those which will not undergo hydrolytical cleavage under the action of lysophospholipases Thus, the lysolipid derivatives are in particular not lysohpids and lysolipid derivatives which have an ester linkage in the 1 -position of the lysolipid or the position of a lysolipid derivative which corresponding to the 1 -position of a lysolipid One preferred class of lipid derivatives for incorporation in the lipid-based delivery systems can be represented by the following formula CH 2 -X-R 1 CH-Y-R 2 | CH 2 -Z-R 3 wherein X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , preferably from O, NH, NMe and CH 2 , in particular O, Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or carbonyl carbon atom, preferably via the carbonyl carbon atom, R is an aliphatic group of the formula Y 1 Y 2 , R 2 is an organic radical having at least 7 carbon atoms, such as an aliphatic group having a length of at least 7, preferably at least 9, carbon atoms, preferably a group of the formula Y Y 2 , where Y 1 is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n3 -(CH=CH) n4 -(CH 2 ) n5 -(CH=CH) n6 -(CH 2 ) n7 - (CH=CH) n8r -(CH 2 )n9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to 29; n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 , and each of n2, n4, n6 and nδ is independently zero or 1 , and Y 2 is CH 3 or C0 2 H, where each Y -Y 2 independently may be substituted with halogen or C^-alkyl, but preferably Y 1 -Y 2 is unsubstituted, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof As mentioned above, preferred embodiments imply that Y is -OC(O)- where Y is connected to R 2 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 R 2 via the carboxyl atom This means that the hpid derivative is a 1-monoether-2-monoester-phospholιpιd type compound Another preferred group of hpid derivatives is the one where the group X is S In one embodiment, R 1 and R 2 are aliphatic groups of the formula Y 1 Y 2 where Y 2 is CH 3 or C0 2 H, but preferably CH 3 , and where Y 1 is -(CH 2 ) n1 (CH=CH) n2 (CH 2 ) n3 (CH=CH) n4 - (CH 2 ) n5 (CH=CH) n6 (CH 2 ) n7 (CH=CH) n8 (CH 2 ) n9 , the sum of M +2n2+n3+2n4+n5+2n6+n7+ 2n8+n9 is an integer of from 9 to 23, that is, the aliphatic group, Y 1 Y 2 , is from 10-24 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 In one embodiment, one or more of the aliphatic groups R 1 /R 2 or the R 3 groups include a label, e g halogens (bromo, lodo) or barium atoms which are particular suitable for computed tomography (CT) imaging, or are enriched with unstable isotopes, e g 11 C which is particularly useful for PET scanning purposes Although the aliphatic groups may be unsaturated and even substituted with halogens (flouro, chloro, bromo, todo) and C^-groups (i e yielding branched aliphatic groups), the aliphatic groups as R 1 and R 2 are in one embodiment preferably saturated as well as unbranched, that is, they preferably have no double bonds between adjacent carbon atoms, each of n2, n4, n6 and n8 then being equal to zero Accordingly, Y 1 is preferably (CH 2 ) n1 More preferably (in this embodiment), R 1 and R 2 are each independently (CH 2 ) n1 CH 3 , and most preferably, (CH 2 ) 17 CH 3 or (CH 2 )ι 5 CH 3 In alternative embodiments, the groups can have one or more double bonds, that is, they can be unsaturated, 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 Y 1 is (CH 2 ) n1 CH=CH(CH 2 ) 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 hpid derivatives are those which are monoether lipids where X and Z are O, R and R 2 are independently selected from alkyl groups, (CH 2 ) n CH 3 , where n is 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, preferably 14, 15 or 16, in particular 14, Y is -OC(O)-, Y then being connected to R 2 via the carbonyl carbon atom With respect to the hydrophihc moiety (often known as the "head group") which corresponds to R 3 , it is believed that a wide variety of groups corresponding to phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof can be used As will be evident, the crucial requirement to R 3 is that the groups should allow for enzymatic cleavage of the R 2 group (actually R 2 -C(=0) or R 2 -OH) by extracellular PLA 2 "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 (P0 2 -OH), phosphatidylcholme (P0 2 -0- CH 2 CH 2 N(CH 3 ) 3 ), phosphatidylethanolamine (P0 2 -0-CH 2 CH 2 NH 2 ), N-methyl- phosphatidylethanolamme (P0 2 -0-CH 2 CH 2 NCH 2 ), phosphatidylseπne, phosphatidyl- mositol, and phosphatidylglycerol (P0 2 -0-CH 2 CH0HCH 2 0H) Other possible derivatives of phosphatidic acid are those where dicarboxyhc acids, such as glutaric, sebacic, succinic and tartaπc acids, are coupled to the terminal nitrogen of phosphatidyl- ethanolamines, phosphatidylsenne, phosphatidyhnositol, etc One highly interesting aspect is the possibility of modifying the pharmaceutical effect of the lipid derivative by modifying the group R 2 It should be understood that R 2 should be an organic radical having at least 7 carbon atoms) (such as an aliphatic group having a certain length (at least 7, 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 Generally, it is believed that 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 anti-parasitic drug substance such as allopuπnol, amodiaquine, amphotericin, antifolates, artemether+benflumetol combination, artemisinin, derivatives, chloroproguanil, Chloroqume, combination of atovaquone and proguanil HCL (salesname Malarone™), dapsone, Doxycychne, halofantπne, interferon gamma, Licochalcone A, Mefloquine, meglumme antimonate, metronidazole, nitrofurantoin derivatives, paromomycm, pentamidine, pπmaquine, pπmaquine, Proguanil, (Chloroqume plus proguanil), puromycm, pyπmethamine, pyronaπdine, quinine and sodium stibogluconate or as an efficiency modifier for the lysolipid derivative and/or any other (second) substances present in the environment In one embodiment, one or more of the aliphatic groups R 1 /R 2 or the R 3 groups include a label, e g halogens (bromo, lodo) or barium atoms which are particular suitable for computed tomography (CT) imaging, or are enriched with unstable isotopes, e g 11 C which is particularly useful for PET scanning purposes In some embodiments, the group R 2 will be a long chain residue, 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 R 2 within this subgroups are poiyunsaturated acids, e g oleate, hnoleic, Imolenic, as well as derivatives of arachidonoyl (including the carbonyl from Y), e g prostaglandins such as prostaglandm Ei, as arachidonic acid derivatives are know regulators of hormone action including the action of prostaglandins, thromboxanes, and leukotπnes Examples of efficiency modifiers as R 2 are those which enhance the permeability of the target cell membrane as well as enhances the activity of extracellular PLA 2 or the active drug substance or any second drug substances Examples hereof are short chain (C 8 . 12 ) fatty acids However, it is also envisaged that other groups might be useful as the organic radical R 2 , e g vitamin D derivatives, steroid derivatives, retmoic acid (including all-trans-retmoic acid, all-cis-retmoic acid, 9-cιs-retιnoιc acid, 13-cιs-retιnoιc acιd), 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 acetylsahcyhc acid), steroidal carboxylic acids (e g lysergic and isolysergic acids), monoheterocyclic carboxylic acids (e g nicotmic acid) and polyheterocychc carboxylic acids (e g penicillins and cephalospoπns), diclofenac, mdomethacin, ibuprofen, naproxen, 6-methoxy-2-naphthylacetιc acid It should be understood that the various examples of possible R 2 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 hpid 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 hpid derivatives to be used within the present invention, it should be understood that the glycol moiety of the hpid 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 pid derivatives Lipid derivatives as prodrugs As described above, the present invention provides the use if a lipid-based delivery system for administration of an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the lipid-based system in the form of a prodrug, said prodrug being a hpid derivative having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said prodrug furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative which is not a substrate for lysophospholipase for the treatment and/or prevention of parasitic infections in mammals Typical parasitic infections in mammals are those where the liver, spleen and bone marrow is targeted 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 The term "prodrug" should be understood in the normal sense, namely as a drug which is masked or protected with the purpose of being converted (typically by cleavage but also by in vivo chemical conversion) to the intended drug substance The person skilled in the art will recognise the scope of the term "prodrug" The active drug substance is selected from lysolipid derivatives, and as it will be understood from the present description with claims, the lysolipid derivatives will have a therapeutic effect - at least - in connection with parasitic infections where a local area of the body of the mammal has a level of extracellular PLA 2 activity caused by said parasitic infection which can liberate the lysolipid derivative As will be understood from the present description with claims, the hpid derivative will often constitute the prodrug referred to above and the lysolipid derivative will thereby constitute the active drug substance often a monoether lysolipid derivative It should however be understood that this does not exclude the possibility of including other drug substances, referred to as second drug substances, in the lipid-based delivery systems, 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) or act as a label Furthermore, the pharmaceutical effect of the "active drug substance", i e the lysolipid derivative, need not the 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 mam embodiment (see "Lipid derivative liposomes as drug delivery systems", below) The active drug substance (hpolipid derivative) release from the prodrug (hpid derivative) is believed to take place as illustrated in the following example CH 2 -X-R 1 CH 2 -X-R 1 CH 2 -X-R 1 | PLA 2 | | CH-Y-R 2 — > CHOH or CHCOOH CH 2 -Z-R 3 CH 2 -Z-R 3 CH 2 -Z-R 3 prodrug drug drug Furthermore, the substituent R 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 CH 2 -X-R 1 I PLA 2 CH-Y-R 2 — > R 2 -OH or R 2 -COOH I CH 2 -Z-R 3 prodrug drug drug It has been described above under the definition of R 2 how the group R 2 can have various independent or synergistic effects in association with the active drug substance, e g as an auxiliary drug substance or an efficiency modifier, e g permeability or cell lysis modifier It should be borne in mind that the groups corresponding to R 2 (e g R 2 -OH or R 2 -COOH) might have a pharmaceutical effect which is predominant in relation the effect of the lysolipid derivative (active drug substance) Lipid derivatives formulated as liposomes and micelles The term "lipid-based drug delivery system" should encompass macromolecular structures which as the main constituent include hpid or hpid derivatives Suitable examples hereof are liposomes and micelles It is presently believed that liposomes offer the broadest scope of applications and those have been described most detailed in the following Although liposomes currently are believed to be the preferred lipid-based system, micellular systems are also believed to offer interesting embodiments within the present invention When used herein, the term "label" is intended to mean a species which is capable of being administered to the mammalian body and being detected by extracorporal means for imaging living tissue The label may be selected from radiolabels such as radioisotopes and radioisotope-labeled compounds, radiopaque compounds, fluorescent compounds, etc More specific labels are 111 ln, 99m Tc, 67 Ga, 1 C, Gd, Mn, iron oxide, argon, nitrogen, Iodine, bromine and barium Suitable labels for gamma-scintigraphy are diagnostic radionuclides, such as 111 ln, 99m Tc, 67 Ga For practical purposes the radionucleotides are complexed with e g chelators such as diethylene tπamine pentaacetic acid (DTPA), hexamethylpropyleneamine oxime (HMPAO), dnsopropyl iminodiacetic acid (DISIDA), or even proteins such as human serum albumin (HSA) Alternatively DTPA or similar chelating compounds may be derivatized by the incorporation of a hydrophobic group, which can anchor the chelating moiety on the liposome surface during or after liposome preparation Suitable labels for X-ray are verografin, loxaglate, lohexol, lopromide, lomeprol, lopamidol, iσpentol, lodixanol, loversol different nonionic contrast media, etc which may be incorporated in liposomes and used both for planar X-ray imaging of the liver and spleen and for CT imaging Suitable labels for magnetic resonance (MR) imaging are paramagnetic ions, such as Gd and Mn, and iron oxide coupled to various carrier molecules E g gadolinium diethylenetπamine pentaacetic acid (Gd-DTPA) complex has been demonstrated to be effective contrast agents for MR imaging of liver, spleen, and hepatic metastases Suitable labels for computed tomography (CT) imaging are iodine, bromine, barium, etc Other examples of suitable labels are often given by the selected method of imaging or detection, examples of which are described in detail in Handbook of Medical Imaging, Vol 1 , 2 and 3, SPIE Press, Washington USA, 2000, eds Beutel, Konden and van Metter The label can be adapted so as to be detectable and optionally quantifiable by a detection method selected from the group consisting of positron emission tomography (PET), X-ray, gamma-scintigraphy, magnetic resonance (MR) imaging, computed tomography (CT) imaging and ultrasonography Thus, the present invention also relates to an image enhancing systems (liposomes or micelles) for use in the present invention relies on hpid derivative having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said hpid-conjugated contrast agents furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the hpid-conjugated contrast agents is liberated in the form of a lysolipid derivative which is not a substrate for lysophosphohpase In one important variant which advantageously can be combined with the embodiments described herein, the lipid derivative (e g the prodrug) is included in liposomes either as the only constituent or - which is more common - in combination with other constituents (other lipids, sterols, etc ) Thus, the lipid-based systems described herein are preferably in the form of liposomes, wherein the liposomes are build up of layers comprising the lipid derivative (e g a prodrug) "Liposomes" are known as self-assembling structures comprising one or more lipid bilayers, each of which surrounds an aqueous compartment and comprises two opposing monolayers of amphipathic hpid molecules Amphipathic lipids (herein i a hpid derivatives) comprise a polar (hydrophihc) headgroup region (corresponding to the substituent R 3 in the hpid derivatives) covalently linked to one or two non-polar (hydrophobic) aliphatic groups (corresponding to R 1 and R 2 in the hpid derivatives) Energetically unfavourable contacts between the hydrophobic groups and the aqueous medium are generally believed to induce hpid molecules to rearrange such that the polar headgroups are oriented towards the aqueous medium while the hydrophobic groups reorient towards the interior of the bilayer An energetically stable structure is formed in which the hydrophobic groups are effectively shielded from coming into contact with the aqueous medium Liposomes can have a single pid bilayer (unilamellar liposomes, "ULVs"), or multiple hpid bilayers (multilamellar liposomes, "MLVs"), and can be made by a variety of methods (for a review, see, for example, Deamer and Uster, Liposomes, Marcel Dekker, N Y , 1983, 27-52) These methods include Bangham's methods for making multilamellar liposomes (MLVs), Lenk's, Fountain's and Cullis 1 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 ohgolamellar liposomes 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 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 hpid derivatives 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 hpid derivatives and lipids Thus, it is believed that the hpid derivatives can be the sole constituent of liposomes, i e up to 100 mol% of the total dehydrated liposomes can be constituted by the hpid derivatives This is in contrast to the known mono-ether lysohpides, which can only constitute a minor part of the liposomes Typically, as will be described in detail below, liposomes advantageously include other constituents which may or may not have a pharmaceutical effect, but which will render the liposome structure more stable (or alternatively more unstable) or will protect the liposomes against clearance and will thereby increase the circulation time thereby improving the overall efficiency of a pharmaceutical including the liposome The liposomes may also include co-factors such as calcium, which upon release would increase PLA 2 activity, as well as lyso-hpids, fatty acids which are known to increase PLA 2 activity This being said, it is believed that the particular hpid derivatives will typically constitute from 15-100 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 200 nm, more preferably, from greater than about 50 nm to less than about 200 nm The liposomes are typically - as known in the art - prepared by a method comprising the steps of (a) dissolving the pid derivative in an organic solvent, (b) removing the organic solvent from the hpid derivative solution of step (a), and (c) hydratmg the product of step (b) with an aqueous solvent so as to form liposomes The method may further comprise a step of adding an second 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 The liposomes comprising pid derivatives may (in principle) exclusively consist of the pid derivatives However, in order to modify the liposomes, "other lipids" may be included as well Other lipids are selected for their ability to adapt compatible packing conformations with the hpid derivative components of the bilayer such that the all the pid constituents are tightly packed, and release of the hpid derivatives from the bilayer is inhibited Lipid-based factors contributing to compatible packing conformations are well known to ordinarily skilled artisans and include, without limitation, acyl chain length and degree of unsaturation, as well as the headgroup size and charge Accordingly, suitable other lipids, including various phosphatidylethanolamines ("PE's") such as egg phosphatidylethanolamine ("EPE") or dioleoyl phosphatidylethanolamme ("DOPE"), can be selected by ordinarily skilled artisans without undue experimentation Lipids may be modified in various way, e g by headgroup deπvatisation with dicarboxyhc acids, such as glutaric, sebacic, succinic and tartaric acids, preferably the dicarboxyhc acid is glutaric acid ("GA") Accordingly, suitable headgroup-deπvatised lipids include phosphatidyl- ethanolamme-dicarboxyhc acids such as dipalmitoyl phosphatidylethanolamine-glutaπc acid ("DPPE-GA"), palmitoyloleoyl phosphatidylethanolamine-glutaπc acid ("POPE-GA") and dioleoyl phosphatidylethanolamine-glutaπc acid ("DOPE-GA") Most preferably, the deπvatised hpid 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 Sterohc compound included in the liposome may generally affects the fluidity of hpid bilayers Accordingly, sterol interactions with surrounding hydrocarbon groups generally inhibit emigration of these groups from the bilayer An examples of a sterohc compound (sterol) to be included in the liposome is cholesterol, but a variety of other sterohc compounds are possible It is generally believed that the content of sterohc 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 Although the an element of idea on which the present invention resides is that the liposomes or micelles should be taken up by the RES system, it may be advantageous to include a small fraction of lipids or hpid derivatives on which polymeric chains are attached in order to adjust the rate at which the liposomes and micelles are taken up and thereby degraded Thus, is may be advantageous to partially, but not fully, employ the so- called STEALTH ® liposomes (Liposome Technology Inc , Menlo Park, Calif ) which include polyethyleneglycol (PEG)-grafted lipids at about 5 mol% of the total dehydrated liposome, or other hpopolymers carrying hydrophihc polymer chains The presence of such polymers on the exterior liposome surface should slightly delay, but not prevent, the uptake of liposomes by the organs of the RES If present, the hpopolymers typically constitute 0 1-10 mol% of the total dehydrated system Hydrophihc polymers suitable for use in hpopolymers are those which are readily water- soluble, can be covalently attached to a vesicle-forming hpid, and which are tolerated in vivo without toxic effects (i e , are biocompatible) Suitable polymers include polyethylene glycol (PEG), polylactic (also termed polylactide), polyglycohc acid (also termed polyglycohde), a polylactic-polyglycohc acid copolymer, and polyvmyl 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 hydrophihc polymers which may be suitable for use in the present invention include polyvinylpyrrohdone, polymethoxazohne, polyethyloxazohne, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide and deπvatised celluloses such as hydroxymethylcellulose or hydroxyethylcellulose Glycohpids are lipids to which a hydrophihc polysacchaπde chain is covalently attached It will be appreciated that glycohpids can be utilised like hpopolymers although the hpopolymers currently presents the most promising results It is generally believed that the content of popolymer, i present, advantageously will be in the range of 0 1-5 mol%, such as 0 2-4 mol%, in particular 0 5-3 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, the hpid bilayer of a liposome contains lipids denvatised with polyethylene glycol (PEG), such that the PEG chains extend from the inner surface of the hpid bilayer into the interior space encapsulated by the liposome, and extend from the exterior of the pid bilayer into the surrounding environment (see e g US 5,882,679) 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 hydrophihc drying protectant such as a disacchaπde sugar at both the inside and outside surfaces of the liposome bilayers (see US 4,880,635) This hydrophihc 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 or label carrier systems As mentioned above, the liposomes including the hpid derivatives may also include second substances, where such second substances may be drugs or labels In a particular embodiment, the hpid-based delivery system described above is in the form of liposomes wherein a second 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 Furthermore, that when the second substance is a label, said label may comprise contrast substances or other substances detectable by means of MR, CT, Gamma-scintigraphy or ultrasonography The term "second" does not necessarily imply that the pharmaceutical effect of the second drug substance is inferior in relation to that of, e g , the active drug substance derived from the prodrug, but is merely used to differentiate between the two groups of substances This being said, the present invention also provides the use of a delivery system which is in the form of liposomes, and wherein a second substance is incorporated, either as a drug or as a label 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 are well known anti-paracitic compounds, such as allopuπnol, amodiaquine, amphotericin, antifolates, artemether+ benflumetol, artemisinin, derivatives, chloroproguanil, Chloroqume, combination of atovaquone and proguanil HCL (salesname Malarone™), dapsone, Doxycycline, halofantπne, interferon gamma, Licochocone A, Mefloqume, meglumme antimonate, metronidazole, nitrofurantom derivatives, paromomycin, pentamidine, pπmaquine, pπmaquine, Proguanil, (Chloroqume plus proguanil), puromycm, pyπmethamine, pyronaπdine, quinine and sodium stibogluconate Liposomal second drug substance formulations enhance the therapeutic index of the second drug substances by reducing the toxicity of the drug Liposomes can also reduce the rate at which a second drug substance is cleared from the vascular circulation of mammals Accordingly, liposomal formulation of second drug substance can mean that less of the drug need be administered to achieve the desired effect Liposomes can be loaded with one or more second substances by solubihsing the drug or label in the hpid or aqueous phase used to prepare the liposomes Alternatively, lonisable second 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 hposomal bilayer, and then adding the lonisable second substance to the aqueous medium external to the liposome (see, e g , US 5,077,056 and WO 86/01102) Methods of preparing hpophihc drug or label derivatives which are suitable for liposome or micelle formulation are known in the art (see e g , US 5,534,499 and US 6, 118,011 describing covalent attachment of therapeutic agents to a fatty acid chain of a phospholipid) A micellar formulation of taxol is described in Alkan-Onkyuksel et al , Pharmaceutical Research, 11 206 (1994) Accordingly, the second drug substance may be any of a wide variety of known and possible pharmaceutically active ingredients, but is preferably a therapeutically and/or prophylactically active substance Due to the mechanism involved in the degradation of the liposomes, it is preferred that the second drug substance is one relating to diseases and/or conditions associated with a localised increase in extracellular PLA 2 activity It is envisaged that the second substance will be distributed in the liposomes according to their hydrophihcity, i e hydrophihc second substances will tend to be present in the cavity of the liposomes and hydrophobic second substances will tend to be present in the hydrophobic bilayer Method for incorporation of second substances are know in the art as has been made clear above It should be understood from the above, that the pid derivatives may - as prodrugs or discrete constituents - posses a pharmaceutical or diagnostic activity However, in a particular embodiment, the present invention furthermore relates to a hpid based drug or label delivery system for administration of an second substance, wherein the second substance is incorporated in the system (e g where the second 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 hpid derivatives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, where the hpid derivative furthermore is a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said lysolipid fragment not being a substrate for lysophospholipase As above for the system according to the other embodiment, the organic radical which can be hydrolytically cleaved off, may be an auxiliary drug or label substance or an efficiency modifier for the second substance It should be understood that the hpid derivative is a hpid derivative as defined further above Typically, the hpid derivative constitutes 15-100 mol%, such as 50-100 mol%, of the total dehydrated (liposome) system The present invention relates to the use of any of the hpid-based drug delivery systems described herein for the preparation of a medicament for the treatment of parasitic infections of a mammal, wherein the parasitic infection is characterized by increasing the level of PLA 2 in said mammal, particularly in the specific tissue of infection, preferably of the liver or spleen Furthermore, the present invention relates to the use of any of the pid-based delivery systems described herein for the preparation of a diagnostic agent for the detection or quantification of parasitic infections of a mammal, wherein the parasitic infection is characterized by increasing the level of PLA 2 in said mammal, particularly in the specific tissue of infection, preferably of the liver or spleen Pharmaceutical preparations and therapeutic uses "Pharmaceutically acceptable carriers" as used herein are those media generally acceptable for use in connection with the administration of lipids and liposomes, including hposomal drug formulations, to mammals, including humans Pharmaceutically acceptable carriers are generally formulated according to a number of factors well within the purview of the ordinarily skilled artisan to determine and account for, including without limitation the particular active drug substance and/or second drug or label substance used, the liposome preparation, its concentration, stability and intended bioavailabihty, the disease, disorder or condition being treated with the liposomal composition, the subject, its age, size and general condition, and the composition's intended route of administration, e g , nasal, oral, ophthalmic, subcutaneous, intramammary, intrapeπtoneal, intravenous, or intramuscular Typical pharmaceutically acceptable carriers used in parenteral drug administration include, for example, D5W, an aqueous solution containing 5% weight by volume of dextrose, and physiological saline Pharmaceutically acceptable carriers can contain additional ingredients, for example those which enhance the stability of the active ingredients included, such as preservatives and anti-oxidants The liposome or lipid derivative is typically formulated in a dispersion medium e g a pharmaceutically acceptable aqueous medium An amount of the composition comprising an anti-parasitic effective amount of the pid derivative, typically from about 0 1 to about 1000 mg of the hpid derivative per kg of the mammal's body, is administered, preferably intravenously For the purposes of this invention, "anti-parasitic effective amounts" of hposomal hpid derivatives are amounts effective to inhibit, ameliorate, lessen or prevent establishment, proliferation, growth, etc of parasites in mammals to which the pid derivatives have been administered Anti- parasitic effective amounts are generally chosen in accordance with a number of factors, e g , the age, size and general condition of the subject, the parasitic infection 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 Anti-parasitic effective amounts of the hposomal drugs/prodrugs of this invention are about the same as such amounts of free, nonhposomal, 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 pharmaceutical composition is preferably administered parenterally by injection, infusion or implantation (intravenous, intramuscular, mtraarticular, 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 textbook entitled "Remington's Pharmaceutical Sciences" Thus, the pharmaceutical compositions may comprise the active substances in the form of a sterile injection To prepare such a composition, the suitable active substances are dispersed in a parenterally acceptable liquid vehicle which conveniently may comprise suspending, solubihsing, stabilising, pH-adjustmg 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-butanedιol, 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 Toxicity Toxicity of the liposomes comprising the hpid derivatives can be assessed by determining the therapeutic window "TW\ which is a numerical value derived from the relationship between the compound's induction of hemolysis and its ability to inhibit the growth/proliferation of parasites TW values are defined as HI 5 /GI 50 (wherein "Hl 5 " equals the concentration of compound inducing the hemolysis of 5% of the red blood cells in a culture, and wherein "Gl 50 " equals the dose of compound inducing fifty percent growth inhibition in a population of parasitic cells exposed to the agent) The higher an agent's Hl 5 value, the less hemolytic is the agent - higher Hl 5 's mean that greater concentrations of compound are required to be present in order for the compound to induce 5% hemolysis Hence, the higher its Hl 5 , the more therapeutically beneficial is a compound, because more of it can be given before inducing the same amount of hemolysis as an agent with a lower Hl 5 By contrast, 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 Generally, when a drug's TW is less than 1 , it cannot be used effectively as a therapeutic agent That is, the agent's Hl 5 value is sufficiently low, and its Gl 50 value sufficiently high, that it is generally not possible to administer enough of the agent to achieve a sufficient level of parasite growth inhibition without also attaining an unacceptable level of hemolysis As the hpid derivative liposomes take advantage of the lower extracellular PLA 2 activity in the bloodstream compared to the activity in the diseased tissue, it is believed that the TW will be much higher that for normal monoether lysohpids As the variance in activity is in orders of magnitude and as the liposomes will be "trapped" in tissue with a high extracellular PLA 2 activity, it is generally believed the TW of the liposomes of the invention will be greater than about 3, more preferably greater than about 5, and still more preferably greater than about 8 The invention will be illustrated by the following non-hmiting examples EXAMPLES Example 1 Liposome preparation Unilamellar fully hydrated liposomes with a narrow size distribution were made from 1-0- hexadecyl-2-hexadecanoyl-SA7-glycero-3-phosphochohne (1-O-DPPC) and di-hexa- decanoyl-sr.-glycero-3-phosphochohne (DPPC) DPPC were obtained from Avanti Polar lipids and 1-O-DPPC were synthesised in our laboratory Briefly, weighed amounts of DPPC or 1-O-DPPC were dissolved in chloroform The solvent was removed by a gentle stream of N 2 and the hpid films were dried overnight under low pressure to remove trace amounts of solvent Multilamellar vesicles were made by dispersing the dried lipids in a buffer solution containing 150 mM KCL, 10 mM HEPES (pH = 7 5), 1 mM NaN 3 , 30 μM CaCI 2 and 10 μM EDTA The multilamellar vesicles were extruded ten times through two stacked 100 nm pore size polycarbonate filters as described by Mayer et al , Biochim Biophys Acta, 858, 161-168 Heat capacity curves were obtained using a N-DSC II differential scanning calorimeter (Caloπmetry Sciences Corp , Provo) of the power compensating type with a cell volume of 0 34 mL Before scanning, the liposome suspension was equilibrated for 50 mm in the calorimeter at the starting temperature A scan rate of +10 °C/h was used The pid concentration was 1 mM The gel-to-fluid transition of the multilamellar liposomes (MLV) is characterised as a sharp first-order transition, as reflected by the narrow peak in the heat capacity curves shown in Figs 1a and 1 b (upper curves) for 1-O-DPPC and DPPC The sharp peak reflects the transitional behaviour of multilamellar liposomes and is in contrast to the broader gel-to-fluid transition observed for unilamellar liposomes (LUV) (Pedersen et al , 1996, Biophys J 71, 554-560) as shown in Figs 1a and 1 b (lower curves) for the unilamellar extruded 1-O-DPPC and DPPC liposomes Example 2 Phosphohpase A? reaction profile and lag time measurements Purified snake-venom phosphohpase A 2 (PLA 2 from Agikistrodon piscivorus piscivorus) has been isolated according to the procedure of Maraganore et al , J Biol Chem 259, 13839-13843 This PLA 2 enzyme belongs to the class of low-molecular weight 14kD secretory enzymes which display structural similarity to human extracellular phosphohpase A 2 indicating a common molecular mechanisms of the phosphohpase catalysed hydrolysis at the pid-membrane interface (Wery et al , Nature 352, 79-82, Hønger et al Biochemistry 35, 9003-9006, Vermehren et al , Biochimica et Biophysica Acta 1373, 27-36) Unilamellar fully hydrated liposomes with a narrow size distribution were prepared from 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphochohne (1-O- DPPC) and from 1-O-DPPC with 5 mol% 1-0-hexadecyl-2-hexadecanoyl-SA7-glycero-3- phosphoethanolamιne-N-[methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350) Assay conditions for the PLA 2 reaction time profile shown in Fig 2 and the lag-time and percent hydrolysis reported in Table 1 were 0 15 mM unilamellar liposomes, 150 nM PLA 2 , 150 mM KCL, 10 mM HEPES (pH 7 5), 1 mM NaN 3 , 30 μM CaCI 2 , and 10 μM EDTA Table 1 Lag-time and percent hydrolysed 1-O-DPPC at 41 °C as determined by HPLC The hpid concentration was 0 150 mM in a 10 mM HEPES-buffer (pH = 7 5) The catalytic reaction was initiated by adding 8 9 μL of a 42 μM PLA 2 (150 nM) stock solution to 2 5 ml of the thermostated liposome suspension (0 150 mM) equilibrated for 800 sec prior to addition of PLA 2 The characteristic lag-burst behaviour of PLA 2 towards the liposomes is signalled by a sudden increase in the intrinsic fluorescence from PLA 2 at 340 nm after excitation at 285 nm followed by a concomitant decrease in the 90° light scattering from the hpid suspension (Hønger et al , Biochemistry 35, 9003-9006) Samples for HPLC analysis of the amount of non-hydrolysed 1-O-DPPC remaining and consequently the amount of 1-0-hexadecyl-2-hydroxy-sn-glycero-3-phosphochohne (lyso- 1-O-PPC) generated were taken before adding PLA 2 and 1200 sec after the observed lag- time 100 μl aliquots were withdrawn from the lipid suspension and rapidly mixed with 1 ml chloroform/methanol/acetic acid (2 4 1) solution in order to quench the enzymatic reaction The solution was washed with 1 ml of water and 20 μl of the heavy organic phase was used for HPLC The HPLC chromatograms in Fig 3 show the amounts of 1-O- DPPC before and after (t = 3000 sec) the addition of PLA 2 (t = 800 sec) to the liposome suspension HPLC analysis was made using a 5 μm diol column, a mobile phase composed of chloroform/ methanol/ water (730 230 30, v/v) and an evaporative light scattering detector The turnover of the PLA 2 catalysed hpid hydrolysis of 1-O-DPPC to lyso-1-O-DPPC was measured by HPLC (see Table 1) The intrinsic enzyme fluorescence and 90° light scattering were measured as a function of time as shown in Fig 2 5 Example 3 Phosphohpase A? induced release of an mcapsulated water-soluble model drug Multilamellar 1-O-DPPC-hposomes were made in the presence of fluorescent calcein in a self-quenching concentration of 20 mM by hydratmg a film of 1-0-hexadecyl-2- hexadecanoyl-sπ-glycero-3-phosphochohne in a HEPES buffer solution at pH=7 5 for one 10 hour at 10°C above the phase transition temperature Unilamellar liposomes were formed by extruding the multilamellar liposomes ten times through two stacked 100 nm polycarbonate filters The unilamellar liposomes were rapidly cooled to a temperature below the transition temperature, and the calcein-contaming 1-O-DPPC liposomes were separated from free calcein using a chromatographic column packed with Sephadex G- 15 50 Assay conditions for the PLA 2 induced calcein release were 25 μM unilamellar 1-O- DPPC-hposomes, 25 nM PLA 2 , 150 mM KCL, 10 mM HEPES (pH 7 5 or 8 0), 1 mM NaN 3 , 30 μM CaCI 2 , and 10 μM EDTA PLA 2 was added at time 900 sec to 2 5 ml of the 0 thermostated 1-O-DPPC-hposome suspension equilibrated for at least 20 mm at 37°C prior to addition of PLA 2 The percentage of calcein released is determined as % Release = 100 x (l F(t ) - l B )/(lτ - I B ) > where l F(t ) is the measured fluorescence at time t after addition of the enzyme, l B is the background fluorescence, and l τ is the total fluorescence measured after addition of Triton X-100 which leads to complete release of calcein by 5 breaking up the 1-O-DPPC-hposomes PLA 2 induced at total release of 90 percent of the entrapped calcein in the 1-O-DPPC-lιposomes as shown in Fig 4 Example 4 Phosphohpase A_ controlled permeability increase of a target model membrane 0 Multilamellar model membrane target liposomes were made in the presence of fluorescent calcein in a self-quenching concentration of 20 mM by hydratmg a film of 1 ,2- 0-dιoctadecyl-s/7-glycero-3-phosphatιdylchohnes (D-O-SPC) in a HEPES buffer solution at pH=7 5 for one hour at 10°C above the phase transition temperature (T m =55°C) Unilamellar liposomes were made by extruding the multilamellar target liposomes ten times through two stacked 100 nm polycarbonate filters The unilamellar liposomes were rapidly cooled to a temperature below the transition temperature, and the calcein- contain g liposomes were separated from free calcein using a chromatographic column 5 packed with Sephadex G-50 The unilamellar carrier liposomes composed of 1-0- hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphochohne were prepared as described above Calcein release from the target liposomes is determined by measuring the fluorescent intensity at 520 nm after excitation at 492 nm 10 The concentrations of D-O-SPC and 1-0-DPPC-lιposomes were 25 μM Snake venom PLA2 (Agkistrodon piscivorus piscivorus) was added (25 nM) to initiate the hydrolytic reaction leading to the formation of 1-0-hexadecyl-2-hydroxy-sn-glycero-3- phosphochohne (lyso-1-O-DPPC) and fatty acid hydrolysis products As calcein is released from the D-O-SPC liposomes, due to the incorporation of the non-bilayer forming 15 lyso-1-O-PPC and fatty acid hydrolysis products into the target hpid membrane, a linear increase in the fluorescence at 520 nm after excitation at 492 nm is observed when calcein is diluted into the surrounding buffer medium as shown in Fig 5 The percentage of calcein released is determined as described above (see Example 3) 20 Example 5 Hemolysis assay Unilamellar fully hydrated liposomes with a narrow size distribution were prepared from 1- 0-hexadecyl-2-hexadecanoyl-sπ-glycero-3-phosphochohne (1-O-DPPC), and from 1-O- DPPC with 5 moi% 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamιne- 25 N-[methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350) The lipids were hydrated in phosphate buffered saline (PBS) 1-0-octadecyl-2-0-methyl-sn-glycero-3-phosphocholιne (ET-18-OCH 3 ) in PBS was included in the assay as a reference Hemolysis assay was performed as described by Perkins et al , Biochim et Biophys Acta 30 1327, 61-68 Briefly, each sample was serially diluted with PBS, and 0 5 ml of each dilute suspension of 1-O-DPPC liposomes were mixed with 0 5 ml washed human red blood cells (RBC) [4% in PBS (v/v)] For controls, 0 5 ml of the red blood cell suspension was mixed with either 0 5 ml buffer solution (negative hemolysis control) or 0 5 ml water (positive hemolysis control) Samples and standard were placed in a 37°C incubator and agitated for 20 hours Tubes were centπfuged at low speed (2000 x G) for 10 minutes to form RBCs pellets 200 μl of the supernatant was quantified by absorbance at 550 nm using a Perkm-Elmer 320 scanning spectrophotometer 100 percent hemolysis was defined as the maximum amount of hemolysis obtained from the detergent Triton X-100 5 The hemolysis profile in Fig 6 shows a low hemolysis value (below 5 percent) for 2 mM 1- 0-DPPC-lιposomes Figure 6 also shows that low concentrations of ET-18-OCH 3 induces a significant degree of hemolysis Example 6 10 Enhancement of phosphohpase A2 activity by negatively charged polymer grafted 1-0- lipids Unilamellar fully hydrated liposomes with a narrow size distribution were prepared from 1- 0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphocholιne (1-O-DPPC) and 1-O-DPPC with 5 mol% 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamιne-N- 15 [methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350), as described in example 2 Assay conditions for the PLA 2 lag-time measurements were 0 15 mM unilamellar liposomes, 150 nM PLA 2 , 150 mM KCL, 10 mM HEPES (pH 7 5), 1 mM NaN 3 , 30 μM CaCI 2 , and 10 μM EDTA The catalytic reaction was initiated by adding 8 9 μL of a 42 μM PLA 2 stock solution to 2 5 ml of the thermostated liposomes suspension equilibrated for 20 800 seconds at 41 °C prior to addition of PLA 2 The time elapsed before the onset of rapid enzymatic activity is determined by a sudden increase in the intrinsic fluorescence from PLA 2 at 340 nm after excitation at 285 nm The results shown in Fig 7 show a significant decrease in the lag time when 5 mol% of the negatively charged 1-O-DPPE-PEG 350 ιs incorporated into the 1-O-DPPC liposomes 25 Example 7 Preparation of micelles composed of 1-O-DPPE-PEG350, DSPE-PEG750/ DPPE- PEG750 Micelles were made from 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanol- 30 amιne-N-[methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350), di-octadecanoyl-sn- glycero-3-phosphoethanolamιne-N-[methoxy(polyethylene glycol)-750 (DSPE-PEG750) Briefly, weighed amounts of the polymer hpid were dissolved in chloroform The solvent was removed by a gentle stream of N 2 The hpid films were then dried overnight under low pressure to remove trace amounts of solvent Micelles were made by dispersing the dried polymer lipids in a buffer solution containing 150 mM KCL, 10 mM HEPES (pH = 7 5), 1 mM NaN 3 , 30 μM CaCI 2 and 10 μM EDTA Example 8 Permeability increase of a target model membranes controlled by phosphohpase A, hydrolysis of micelles Multilamellar model membrane target liposomes were made in the presence of fluorescent calcein in a self-quenching concentration of 20 mM by hydratmg a film of 1 ,2- O-dιoctadecyl-sn-glycero-3-phosphatιdylcholιnes (D-O-SPC) in a HEPES buffer solution at pH=7 5 for one hour at 10°C above the phase transition temperature (T m =55°C) Unilamellar liposomes were made by extruding the multilamellar liposomes ten times through two stacked 100 nm polycarbonate filters The unilamellar liposomes were rapidly cooled to a temperature below the transition temperature, and the calcein-containmg liposomes were separated from free calcein using a chromatographic column packed with Sephadex G-50 Micelles composed of 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3- phosphoethanolamιne-N-[methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350), di- octadecanoyl-sn-glycero-3-phosphoethanolamιne-N-[methoxy(polyethylene glycol)-750 (DSPE-PEG750) were prepared as described in example 7 Calcein release from the target liposome is determined by measuring the fluorescent intensity at 520 nm after excitation at 492 nm The concentrations of D-O-SPC and polymer hpid micelles were 25 μM Snake venom PLA2 (Agkistrodon piscivorus piscivorus) was added (25 nM) to initiate the hydrolytic reaction leading to instant formation of the lysolipid and fatty acid hydrolysis products As calcein is released from the D-O-SPC liposomes, due to the incorporation of the non- bilayer forming polymer-lyso-1-O-hpιd and fatty acid into the target hpid membrane, a linear increase in the fluorescence at 520 nm after excitation at 492 nm is observed when calcein is diluted into the surrounding buffer medium as shown in Fig 8 The percentage of calcein released is determined as described in example 3 PLA 2 catalysed hydrolysis of 1-O-DPPE-PEG350 induced the fastest release rate Example 9 Hydrolysis of micelles composed of DSPE-PEG750 The hydrolysis of micelles composed DSPE-PEG750 was followed by analysis of the amount of stearic acid generated The catalytic reaction was initiated by adding 8 9 μL of a 42 μM PLA 2 (150 nM) stock solution to 2 5 ml a thermostated hpid suspension of DSPE- PEG750 (0 150 mM) equilibrated at 45°C for 600 seconds prior to addition of PLA 2 The characteristic burst behaviour of PLA 2 towards the micelles is signalled by a sudden increase in the intrinsic fluorescence from PLA 2 at 340 nm after excitation at 285 nm followed by a concomitant decrease in the 90° light scattering from the hpid suspension (Hønger et al , Biochemistry 35, 9003-9006) Samples for HPLC analysis of the amount of stearic acid generated were taken before adding PLA 2 and 100 sec after the observed lag-time The HPLC chromatograms in Fig 9 shows the amount of stearic acid generated 100 sec after the observed lag time (10 sec) at 45°C The amount (0 156 mM) of stearic acid generated by hydrolysis was equal to 100% hydrolysis of the DSPE-PEG750 polymer-hpids HPLC analysis was made using a 5 μm diol column, a mobile phase composed of chloroform/methanol/ water (730 230 30, v/v) and an evaporative light scattering detector (see example 2) Example 10 Model examples Liposomes composed of neutral and/or negatively charged phosphohpids can act as versatile drug or label delivery systems targeting parasitic diseases in tissues with elevated levels of PLA 2 due to the parasitic infection, organs of special interest are the liver, spleen and bone marrow When administered intravenously these liposomes have a strong tendency to accumulate in the liver and spleen due to a combination of physicochemical factors relating to the hpid composition of the liposomes and physiological factors involving the rich blood supply and abundance of macrophages in the liver and spleen In the examples herein are described an experimental model system illustrating a new principle for improved drug or label delivery which takes advantage of an elevated activity of extracelluar phosphohpase A 2 in the infected liver or spleen tissue The phosphohpase A 2 hydrolyses a lipid-based proenhancer in the carrier liposome, producing lyso-phosphohpid and free fatty acid, which are shown in a synergistic way to lead to enhanced liposome destabihsation and drug release at the same time as the permeability of the target membrane is enhanced The proposed system can be made thermosensitive and offers a rational way for developing smart liposome-based delivery systems by incorporating into the carrier specific pid-based proenhancers, prodestabihsers or prodrugs that automatically become activated by phosphohpase A 2 only at the diseased and infected target sites Liposomes are self-assembled pid systems and their stability is therefore to a large extent controlled by non-specific physical interactions Insight into the molecular control of the physical properties of liposomes is therefore important for manipulating and tailoring the hposomal properties in relation to specific drug-delivery purposes As an example, the thermally induced gel-fluid hpid phase transition has been exploited and optimised design systems for enhanced release of drugs due to hyperthermia It would be desirable if an intelligent and versatile drug-delivery system could be designed which has built in a dual virtual trigger mechanism of simultaneous (i) enhanced drug release selectively in the infected target tissue and (n) enhanced transport of the drug or label into the infected cells This principle is illustrated schematically in Fig 10 By the examples herein is described the development of a simple and operative experimental biophysical model system which sustains such a dual mechanism to be triggered at the infected target sites such as liver and spleen The model assumes elevated activity of extracellular phosphohpase A 2 in the infected tissue as is the case in inflamed and cancerous tissue where the level of extracellular PLA 2 can be manifold magnified Upon exposure to extracellular PLA 2 , the phosphohpids of negatively charged liposomes have been shown to suffer enhanced hydrolysis compared to neutral liposomes This leads to destabihsation of the liposome and enhanced release of the encapsulated drug or label The hydrolysis products, lyso-phosphohpids and free fatty acids, act in turn as absorption enhancers for drug or label permeation across the target membrane In this way the phosphohpids of the carrier liposome behave as prodestabihsers at the site of the carrier and as proenhancers at the site of the target membrane Molecular details of this principle are illustrated schematically in Fig 11 The experimental model system consists of a negatively charged liposome carrier and a model target membrane The carrier is a 100 nm unilamellar liposome made of dipalmitoyl phosphatidylcholme lipids (DPPC) with a small amount (2 5 mol%) of negatively charged lipid of the type dipalmitoyl phosphatidylethanolamine (DPPE)-PEG 20 _ . The target membrane is another liposome made of 1 ,2-O-dioctadecyl-sn-glycero-phosphatidylchohne (D-O-SPC) which is a phospholipid where the acyl linkages of the stearoyl chains are ether bonds In contrast to DPPC, D-O-SPC is inert towards PLA 2 -catalysed hydrolysis thereby mimicking the stability of an intact target cell membrane toward degradation by its own enzymes This experimental assay, which permits simultaneous as well as separate investigation of the effect of destabihsers at the carrier liposomes and the effect of enhancers at the target membrane, involves entrapment of a water-soluble fluorescent calcein model drug in a self-quenching concentration, in the interior of the non- hydrolysable target liposome, rather than in the carrier liposome The enhanced level of extracellular PLA 2 at the target membrane can then be simulated by adding extracellular PLA 2 to initiate the hydrolytic reaction in a suspension of the carrier and target liposomes The permeation of calcein across the D-O-SPC target membrane is subsequently monitored by the increase in fluorescence In order to investigate the effect of the presence of small amounts of negatively charged PEG-hpids in the carrier liposome, a similar experiment was performed with conventional bare DPPC liposomes Furthermore, in order to compare and discriminate the permeability enhancing effect of lysophospholipids from that of free fatty acids, experiments without enzymes were carried out where lyso-phosphohpids and free fatty acids were added simultaneously or separately to the target liposomes In Fig 12 a are shown the results for the release of calcein as a function of time after adding PLA 2 to the system The reaction time-course of the particular PLA 2 used has a characteristic lag-burst behaviour with a so-called lag time which conveniently can be used as a measure of the enzymatic activity A dramatic decrease in the lag time and a concomitant enhancement of the rate of release are observed when the carrier liposomes contain the negatively charged, DPPE-PEG 200 o, in accordance with previous findings of enhanced extracellular PLA 2 degradation of negatively charged polymer-coated liposomes These results suggest that the products of the PLA 2 -catalysed hydrolysis of the DPPC lipids of the DPPC- posomal carrier, lyso-phosphohpid and free fatty acid, which are produced in a 1 1 mixture, are incorporated into the target membrane, leading to a large increase in membrane permeability These products, which have very low water solubility, are known, due to their non-cyhndπcal molecular shapes, to induce a curvature stress field in the membrane or small-scale lateral phase separation which induce membrane defects and increased permeability This is substantiated by the data in Fig 13 which show that the addition of lyso-phospho pid or fatty acid separately to the present target system, in the absence of PLA 2 , leads to an increased rate of calcein release across the target membrane However, the crucial finding is that if lyso-phosphohpid and free fatty acid are added simultaneously in a 1 1 mixture, a dramatic enhancement in the rate of release is observed as shown in Fig 13 This strongly suggests that the two enhancers act in a synergistic fashion, thereby highlighting the unique possibility in exploiting PLA 2 - catalysed hydrolysis for combined destabihsation of the carrier liposome and enhancement of drug transport across the target membrane The synergistic effect is further augmented by the fact that extracellular PLA 2 is activated by its own hydrolysis products revealing the degradable phosphohpids of the carrier liposome as a kind of proactivators It should be pointed out that the effect in the present drug-delivery model system of using lipids as proenhancers and prodestabihsers via extracellular PLA 2 activity is dynamic and refers to an intrinsic time scale This time scale is the effective retention time of the carrier liposomes near the target membrane The more rapidly the enzyme becomes active, the faster is the drug release and the larger the drug absorption during the time which the carrier spends near the target Furthermore, the faster the enzyme works the more readily it becomes available for hydrolysis of other drug-carrying liposomes that approach the diseased target site Once it has been established that extracellular PLA 2 activity can be used to control drug release, several rational ways open up for intelligent improvements of the proposed drug-delivery system via use of well-known mechanisms of altering extracellular PLA 2 activity by manipulating the physical properties of the hpid bilayer to which the enzyme is known to be sensitive Hence the strategy is to modify certain physical properties of the carrier liposomes without significantly changing their vascular circulation time We shall illustrate this general principle by demonstrating the effects of both a physico-chemical factor, the hpid composition of the carrier, and an enviromental (thermodynamic) factor, the local temperature at the target site Short-chain phosphohpids, such as didecanoyl phosphatidylchohne (DCPC), activate extracellular PLA 2 The effect on calcein permeation across the target membranes induced by incorporation of a small amount of DCPC into the carrier PEG-hposomes is also shown in Fig 12 a The release is very fast due to an almost instantaneous activation of the enzyme We have furthermore found that extracellular PLA 2 becomes deactivated (data not shown) when a large amount of cholesterol ( * 20 mol %) is incorporated into liposomes In contrast we find that a small amount of cholesterol (=3 mol%) activates extracellular PLA 2 Temperature is known to have a dramatic and highly non-linear effect on extracellular PLA 2 activation in the region of the gel-fluid phase transition of saturated phospholipid bilayers This effect is not caused by changes in the enzyme but by dramatic lateral structural changes in the pid bilayer It is possible to take advantage of this effect in the present drug-delivery system as suggested by the data in Fig 12 b As the temperature approaches the transition temperature at 41 °C, the rate of calcein release is progressively enhanced as quantified by the time of 50% calcein release, t 50% , shown in the insert to Fig 12 b It has previously been suggested that hypertermia could be exploited to enhance drug release, and that local heating at predefined infected areas could be used to locally destabilise drug-carrying liposomes, by exploiting the enhanced leakiness of liposomes at their phase transition In the new model drug-delivery system proposed here, these thermosensitive possibilities are integrated and fully exploited via the thermal sensitivity of extracellular PLA 2 to the physical properties of the carrier liposome In contrast to the case where the thermic effect can only be achieved by a local temperature increase using external heating sources at a predetermined infected site of some minimal size, the PLA 2 -controlled release will be enhanced everywhere where temperature and extracellular PLA 2 concentration are elevated, e g in infected tissue, independent of the size of the diseased region and without requiring a preceding localisation of the diseased tissue DPPC, DCPC, D-O-SPC, and DPPE-PEG 200. were obtained from Avanti Polar Lipids Purified snake venom PLA 2 (Agkistrodon piscivorus piscivorus) was a generous gift from dr R L Biltonen This PLA 2 enzyme belongs to the class of low-molecular weight, 14kD secretory enzymes which display structural similarity to human extracellular phosphohpase A 2 Multilamellar target liposomes in the presence of fluorescent calcein in a self-quenching concentration of 20 mM were made by hydratmg a film of D-O-SPC in a HEPES buffer solution at pH=7 5 for one hour at 10°C above the phase transition temperature T m =55°C Unilamellar liposomes were made by extruding the multilamellar liposomes ten times through two stacked 100nm polycarbonate filters The unilamellar liposomes were rapidly cooled to a temperature below the transition temperature, and the calcein-containmg liposomes were separated from free calcein using a chromatographic column packed with Sephadex G-50 The unilamellar carrier liposomes of DPPC, DCPC and DPPE-PEG 20 oo were prepared in a similar fashion T m =41 °C) Calcein release from the target liposomes is determined by measuring the fluorescent intensity at 520 nm after excitation at 492 nm All measurements are performed at temperatures where the lipids of both the carrier and target liposomes are in the gel state Example 11 Phosphohpase A? concentration dependent release assay Multilamellar 1-O-DPPC-lιposomes with 10 mol% 1-O-DPPE-PEG350 were made in the presence of fluorescent calcein in a self-quenching concentration of 20 mM by hydratmg a film of 90% 1-0-hexadecyl-2-hexadecanoyl-s/7-glycero-3-phosphochohne and 10% 1-0- hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamιne-N-[methoxy(polyethylene glycol)-350] in a HEPES buffer solution at pH=7 5 for one hour at 10 °C above the phase transition temperature Unilamellar liposomes were formed by extruding the multilamellar liposomes ten times through two stacked 100 nm polycarbonate filters The unilamellar liposomes were rapidly cooled to a temperature below the transition temperature, and the calcein-containmg liposomes were separated from free calcein using a chromatographic column packed with Sephadex G-50 Assay conditions for the PLA 2 induced calcein release were 25 μM unilamellar liposomes, 50, 1 and 0 02 nM PLA 2 , 150 mM KCL, 10 mM HEPES (pH 7 5), 1 mM NaN 3 , 30 μM CaCI 2 , and 10 μM EDTA PLA 2 was added to 2 5 ml of the thermostated micelle suspension equilibrated for at least 300 sec at 35 5°C prior to addition of PLA 2 The percentage of calcein released is determined as % Release = 100 x (l F(t) - l B )/(l T - I B ), where l F(t) is the measured fluorescence at time t after addition of the enzyme, l B is the background fluorescence, and l τ is the total fluorescence measured after addition of Triton X-100 which leads to complete release of calcein by breaking up the 1-O-DPPC- hposomes Figure 14 show that the induced release of calcein was slowest when only 0 02 nM PLA 2 was added to the liposome suspension Example 12 Hydrolysis of negatively charged liposomes by phosphohpase A2 in cell-free rat peritoneal fluid Cell-free peritoneal fluid from rat with casein-induced acute inflammation was prepared by injecting 5 ml 1 % sodium casemate into the peritoneal cavity of a SRPD male rat, weighing 250- 260 g The rat was sacrificed by bleeding after 24 hours and the inflammatory fluid was collected from the peritoneum and centπfuged at 1500 G for 20 mm in order to obtain a cell-free peritoneal fluid Negatively charged fully hydrated unilamellar liposomes with a narrow size distribution were prepared from 89 mol% dι-hexadecanoyl-sπ-glycero-3-phosphoglycerol (DPPG), 10 mol% 1-0-hexadecyl-2-hexadecanoyl-sn-glycero-3-phosphoethanolamιne-N- [methoxy(polyethylene glycol)-350] (1-O-DPPE-PEG350) and 1 mol% 1 ,2-bιs-(1- pyrenedecanoyl) -sn-glycero-3-phosphochohne (bis-py-DPC) Bis-py-DPC is a PLA2 substrate with two adjacent pyrene fluorophores that form excited-state dimers (eximers) emitting at 470 nm upon excitation at 342 nm Phosphohpase catalysed hydrolysis separates the two fluorophores, which then emit at 380 nm (monomers) Figure 15 shows the emission spectra obtained after excitation at 342 nm of bis-py-DPC incorporated in negatively charged liposomes (0 100 mM) before and after adding 100 nM PLA2 (Agkistrodon piscivorus piscivorus) The observed changed in the emissions spectrum after phosphohpase mediated hydrolysis is used in a contmuos assay, measuring the eximer emission at 470 nm simultaneously with the monomer emission at 380, upon excitation at 342 nm Figure 16 shows the reaction time profile of rat phosphohpase A 2 catalysed hydrolysis of the negatively charged liposomes The catalytic reaction was initiated by adding cell-free peritoneal fluid to 2 5 ml of a thermostated liposome suspension equilibrated for 60 sec prior to addition of PLA 2 The characteristic lag-burst behaviour of the phosphohpase is signalled by a sudden increase in the monomer fluorescence at 380 nm and a subsequent decrease in the eximer fluorescence as shown at the insert on Fig 16 Assay conditions for the PLA 2 reaction time profile shown in Fig 16 were 0 100 mM unilamellar negatively charged liposomes, 100 μl undiluted cell-free peritoneal fluid, 10 mM HEPES (pH 7 5), 5 mM CaCI 2 , and 150 mM NaCI

Claims: CLAIMS 1 Use of a pid-based drug delivery system comprising an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the hpid-based system in the form of a prodrug, said prodrug being a hpid derivative having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said prodrug furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative which is not a substrate for lysophosphohpase, for the preparation of a medicament for the treatment or prevention of a parasitic infection, said parasitic infection being characterised by increasing the PLA 2 level in said mammal 2 Use according to claim 1 , wherein the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the parasite 3 Use according to any of claims 1 or 2, wherein the parasitic infection involves the liver and/or the spleen of the mammal 4 Use according to any of the preceding claims, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the active drug substance 5 Use according to any of the preceding claims, wherein the prodrug is a hpid derivative of the following formula CH 2 -X-R 1 | CH-Y-R 2 I CH 2 -Z-R 3 wherein X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or 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 7 carbon atoms, where Y is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n3 -(CH=CH) n4 -(CH 2 ) n5 -(CH=CH) n6 -(CH 2 ) n7 - (CH=CH) n8r -(CH 2 ) n9 , and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 to 29, n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 , and each of n2, n4, n6 and nδ is independently zero or 1 , and Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with halogen or C^-alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 6 Use according to claim 5, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 7 Use according to claim 6, wherein R is a group of the formula Y 1vY2 8 Use according to any of the preceding claims, wherein at least a fraction of the prodrug is of the formula defined in claims 5-7, wherein R 3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactιc acid), poly(glycohc acid), poly(lactιc acιd)-poly(glycohc acid) copolymers, polyvmyl alcohol, polyvmylpyrrohdone, polymethoxazohne, polyethyloxazohne, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and deπvatised celluloses, is covalently attached 9 Use according to any of the preceding claims, wherein the prodrug constitutes 15-100 mol% of the total dehydrated hpid-based system 10 Use according to claim 8-9, wherein the hpopolymer if present constitutes 0 1-10 mol% of the total dehydrated system 5 11 Use according to any of the preceding claims, wherein the hpid-based system is in the form of liposomes 12 Use according to any of the preceding claims which is in the form of liposomes wherein a second drug substance is incorporated 10 13 Use of a hpid based drug delivery system comprising a second drug substance, wherein the second drug substance is incorporated in the system, said system including hpid derivatives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, where 15 the lipid derivative furthermore is a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said lysolipid fragment not being a substrate for lysophospholipase, for the preparation of a medicament for the treatment or 20 prevention of a parasitic infection of a mammal said parasitic infection being characterised by increasing the PLA 2 level in said mammal 14 Use according to claim 13, wherein the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the 25 parasite 15 Use according to any of claims 13 or 14, wherein the parasitic infection involves the liver and/or the spleen of the mammal 30 16 Use according to any of the claims 13-15, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the second drug substance 17 Use according to any of the claims 13-16, wherein the hpid derivative is a hpid 35 derivative of the following formula CH 2 -X-R 1 I CH-Y-R 2 I CH 2 -Z-R 3 wherein X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or 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 7 carbon atoms, where Y is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n 3-(CH=CH) n4 -(CH 2 ) n 5-(CH=CH) n6 -(CH 2 ) n7 - (CH=CH) n8r -(CH 2 ) n 9, and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2nδ+n9 is an integer of from 9 to 29, n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 , and each of n2, n4, n6 and nδ is independently zero or 1 , and Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with halogen or Ci 4 -alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 18 Use according to claim 18, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 19 Use according to claim 19, wherein R 2 ιs a group of the formula Y 1 Y 2 20 Use according to any of the claims 13-19, wherein at least a fraction of the prodrug is of the formula defined in claim 17, wherein R 3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactιc acid), poly(glycolιc acid), poly(lactιc acιd)-poly(glycohc acid) copolymers, polyv yl alcohol, polyvinylpyrrohdone, polymethoxazohne, polyethyloxazohne, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and deπvatised celluloses, is covalently 5 attached 21 Use according to any of the claims 13-20, wherein the hpid derivative constitutes 15- 100 moI% of the total dehydrated system 10 22 Use according to any of the claims 20-21 , wherein the hpopolymer, if present, constitutes 0 1-10 mol% of the total dehydrated system 23 Use according to any of the claims 13-22, wherein the system is in the form of liposomes 15 24 Use according to claim 13-23, wherein the second drug substance is a therapeutically and/or prophylactically active substance selected from (i) anti-parasitic agents, (n) antibiotics, and (in) antnnflammatory agents 20 25 Use of a hpid based system, said system including a second substance and hpid derivatives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, where the hpid derivative furthermore is a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group 25 remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said lysolipid fragment not being a substrate for lysophosphohpase and said second substance being a label, for the preparation of a diagnostic agent for the detection or quantification of a parasitic infection of a mammal, said parasitic infection being characterised by increasing the PLA 2 level in said mammal 30 26 Use according to claim 25, wherein the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the parasite 27 Use according to any of claims 25 or 26, wherein the parasitic infection involves the liver and/or the spleen of the mammal 28 Use according to any of the claims 25-27, wherein the hpid derivative is a pid derivative of the following formula CH 2 -X-R 1 CH-Y-R 2 | CH 2 -Z-R 3 wherein X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or 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 7 carbon atoms, where Y 1 is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n3 -(CH=CH) n4 -(CH 2 ) n5 -(CH=CH) n6 -(CH 2 ) n7 - (CH=CH) n8r -(CH 2 ) n9 , and the sum of n1 +2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 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 Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with halogen or d -alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 29 Use according to claim 28, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 30 Use according to claim 29, wherein R 2 is a group of the formula Y 1 Y 2 31 Use according to any of the claims 25-30, wherein the pid derivative constitutes 15- 5 100 mol% of the total dehydrated system 32 The use according to any of claims 25-31 , wherein the label is selected from the group consisting of 1 1 ln, 99m Tc, 67 Ga, C, Gd, Mn, iron oxide, argon, nitrogen, Iodine, bromine and barium 0 33 The use according to any of the preceding claims, wherein the medicament or diagnostic agent is suitable for systemic administration 34 The use according to claim 33, wherein the medicament or diagnostic agent is 5 formulated as a medicament for intravenous administration 35 The use according to any of the preceding claims, wherein the parasitic infection is caused by a parasitic organism selected from the group consisting of Leishmania major Friedhn, Leishmania (viannia) gr , Leishmania mexicana, Leishmania tropica, Leishmania donovani (mfantum), Leishmania aethiopica, Leishmania amazonensis, Leishmania enπettn, Leishmania chagasi Leishmania pifanoi, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malaπae, Trypanosoma cruzi (causing Chargas' disease), Trypanosoma brucei, Trypanosoma equiperdum, Trypanosoma evansi Entamoeba histolytica and Chlornorchis smensis 36 A method for the treatment of parasitic infections of a mammal, preferably a human, said parasitic infection being characterised by increasing the PLA 2 level in said mammal by administering to the mammal an efficient amount of a hpid-based drug delivery system comprising an active drug substance selected from lysolipid derivatives, wherein the active drug substance is present in the hpid-based system in the form of a prodrug, said prodrug being a hpid derivative having (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, said prodrug furthermore being a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, whereby the active drug substance is liberated in the form of a lysolipid derivative which is not a substrate for lysophospholipase 37 A method according to claim 36, wherein the increased level of PLA 2 is localized to a 5 specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the parasite 38 A method according to claim 36, wherein the parasitic infection involves the liver and/or the spleen of the mammal 10 39 A method according to claim 36, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the active drug substance 15 40 A method according to claim 36, wherein the prodrug is a lipid derivative of the following formula CH 2 -X-R 1 0 CH-Y-R 2 I CH 2 -Z-R 3 wherein 5 X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or carbonyl carbon atom, 0 R 1 is an aliphatic group of the formula Y 1 Y 2 , R 2 is an organic radical having at least 7 carbon atoms, where Y 1 is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n3 -(CH=CH) n4 -(CH 2 ) n5 -(CH=CH) n6 -(CH 2 ) n7 - 5 (CH=CH) n8r -(CH 2 ) n9 , and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 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 Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with 5 halogen or d -alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 10 41 A method according to claim 40, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 42 A method according to claim 41 , wherein R 2 ιs a group of the formula Y 1 Y 2 15 43 A method according to claim 36, wherein at least a fraction of the prodrug is of the formula defined in claim 40, wherein R 3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactιc acid), poly(glycohc acid), poly(lactιc acιd)-poly(glycohc acid) copolymers, polyvinyl alcohol, polyvmylpyrrohdone, polymethoxa- zohne, polyethyloxazohne, polyhydroxypropyl methacrylamide, polymethacrylamide, 20 polydimethylacrylamide, and deπvatised celluloses, is covalently attached 44 A method according to claim 36, wherein the prodrug constitutes 15-100 mol% of the total dehydrated lipid-based system 25 45 A method according to claim 36, wherein the hpopolymer, if present, constitutes 0 1- 10 mol% of the total dehydrated system 46 A method according to claim 36, wherein the hpid-based system is in the form of liposomes 30 47 A method according to claim 36 which is in the form of liposomes wherein a second drug substance is incorporated 48 A method according to claim 47, wherein the second drug substance is a therapeutically and/or prophylactically active substance selected from (i) anti-parasitic agents, (n) antibiotics and antifungals, and (in) antnnflammatory agents 5 49 A method according to claim 36, wherein the parasitic infection is caused by a parasitic organism selected from the group consisting of Leishmania, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Entamoeba histolytica and Chlornorchis smensis 10 50 A method according to claim 36 for systemic treatment 51 A method according to claim 50 for intravenous administration 52 A method for the treatment of parasitic infections of a mammal, preferably a human, 15 said parasitic infection being characterised by increasing the PLA 2 level in said mammal by administering to the mammal an efficient amount of a hpid based drug delivery system comprising a second drug substance, wherein the second drug substance is incorporated in the system, said system including hpid derivatives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, 20 and (b) a hydrophihc moiety, where the hpid derivative furthermore is a substrate for extracellular phosphohpase A2 to the extent that the organic radical can be hydrolytically cleaved off, whereas the aliphatic group remains substantially unaffected, so as to result in an organic acid fragment or an organic alcohol fragment and a lysolipid fragment, said lysolipid fragment not being a substrate for lysophosphohpase 25 53 A method according to claim 52, wherein the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the parasite 30 54 A method according to claim 52, wherein the parasitic infection involves the liver and/or the spleen of the mammal 55 A method according to claim 52, wherein the organic radical which can be hydrolytically cleaved off, is an auxiliary drug substance or an efficiency modifier for the 35 second drug substance 56 A method according to claim 52, wherein the hpid derivative is a pid derivative of the following formula CH 2 -X-R 1 I CH-Y-R 2 CH 2 -Z-R 3 wherein X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or 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 7 carbon atoms, where Y 1 is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n 3-(CH=CH) n4 -(CH 2 ) n5 -(CH=CH) n6 -(CH 2 ) n7 - (CH=CH) n8r -(CH 2 ) n9 , and the sum of n1 +2n2+n3+2n4+n5+2n6+n7+2nδ+n9 is an integer of from 9 to 29, n1 is zero or an integer of from 1 to 29, n3 is zero or an integer of from 1 to 20, n5 is zero or an integer of from 1 to 17, n7 is zero or an integer of from 1 to 14, and n9 is zero or an integer of from 1 to 11 , and each of n2, n4, n6 and nδ is independently zero or 1 , and Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with halogen or d 4 -alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 57 A method according to claim 56, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 5δ A method according to claim 56, wherein R 2 is a group of the formula Y 1 Y 2 59 A method according to claim 52, wherein at least a fraction of the prodrug is of the formula defined in claim 55, wherein R 3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactιc acid), poly(glycolιc acid), poly(lactιc 5 acιd)-poly(glycohc acid) copolymers, polyvinyl alcohol, polyvmylpyrrohdone, polymethoxazohne, polyethyloxazo ne, polyhydroxypropyl methacrylamide, polymethacrylamide, polydimethylacrylamide, and deπvatised celluloses, is covalently attached 10 60 A method according to claim 52, wherein the lipid derivative constitutes 15-100 mol% of the total dehydrated system 61 A method according to claim 53, wherein the hpopolymer, if present, constitutes 0 1- 10 mol% of the total dehydrated system 15 62 A method according to claim 52, wherein the system is in the form of liposomes 63 A method according to claim 53, wherein the second drug substance is a therapeutically and/or prophylactically active substance selected from (i) anti-parasitic 0 agents, (n) antibiotics and antifungals, and (in) antiinflammatory agents 64 A method according to claim 52, wherein the parasitic infection is caused by a parasitic organism selected from the group consisting of Leishmania major Friedhn, Leishmania (viannia) gr , Leishmania mexicana, Leishmania tropica, Leishmania donovani 5 (mfantum), Leishmania aethiopica, Leishmania amazonensis, Leishmania enπettii, Leishmania chagasi Leishmania pifanoi, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malaπae, Trypanosoma cruzi (causing Chargas' disease), Trypanosoma brucei, Trypanosoma equiperdum, Trypanosoma evansi Entamoeba histolytica and Chlomorchis smensis 0 65 A method according to claim 52 for systemic treatment 66 A method according to claim 65 for intravenous administration 67 A method for detecting or quantifying parasitic infections of a mammal, preferably a human, said parasitic infection being characterised by increasing the PLA 2 level in said mammal, by administering to the mammal an efficient amount of a hpid based drug delivery system comprising a second substance which is a label, said system including 5 hpid derivatives which has (a) an aliphatic group of a length of at least 7 carbon atoms and an organic radical having at least 7 carbon atoms, and (b) a hydrophihc moiety, where the hpid derivative furthermore is a substrate for extracellular phosphohpase 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 10 organic alcohol fragment and a lysolipid fragment, said lysolipid fragment not being a substrate for lysophosphohpase and said second substance being a label 68 A method according to claim 67, wherein the increased level of PLA 2 is localized to a specific tissue and/or organ of the mammal, said tissue and/or organ being infected by the 15 parasite 69 A method according to claim 67, wherein the parasitic infection involves the liver and/or the spleen of the mammal 20 70 A method according to claim 67, wherein the hpid derivative is a hpid derivative of the following formula CH 2 -X-R 1 I 25 CH-Y-R 2 I CH 2 -Z-R 3 wherein 30 X and Z independently are selected from O, CH 2 , NH, NMe, S, S(O), and S(0) 2 , Y is -OC(O)-, Y then being connected to R 2 via either the oxygen or carbonyl carbon atom, 5 R 1 is an aliphatic group of the formula Y 1 1 vY2 R 2 is an organic radical having at least 7 carbon atoms where Y is -(CH 2 ) n1 -(CH=CH) n2 -(CH 2 ) n 3-(CH=CH) n4 -(CH 2 ) n 5-(CH=CH) n6 -(CH 2 ) n7 - 5 and the sum of n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is an integer of from 9 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 1 1 , and each of n2, n4, n6 and nδ is independently zero or 1 , and Y 2 is CH 3 or C0 2 H, where each Y 1 -Y 2 independently may be substituted with 10 halogen or d 4 -alkyl, R 3 is selected from phosphatidic acid (P0 2 -OH), derivatives of phosphatidic acid and bioisosters to phosphatic acid and derivatives thereof 15 71 A method according to claim 70, wherein R 2 is an aliphatic group of a length of at least 7 carbon atoms 72 A method according to claim 70, wherein R 2 is a group of the formula Y 1 Y 2 20 73 A method according to claim 67, wherein at least a fraction of the prodrug is of the formula defined in claim 70, wherein R 3 is a derivative of phosphatidic acid to which a polymer selected from polyethylene glycol, poly(lactιc acid), poly(glycolιc acid), poly(lactιc acιd)-poly(glycolιc acid) copolymers, polyvinyl alcohol, polyvmylpyrrohdone, polymethoxa- ohne, polyethyloxazolme, polyhydroxypropyl methacrylamide, polymethacrylamide, 25 polydimethylacrylamide, and deπvatised celluloses, is covalently attached 74 A method according to claim 67, wherein the lipid derivative constitutes 15-100 mol% of the total dehydrated system 30 75 A method according to claim 67, wherein the hpopolymer, if present, constitutes 0 1- 10 mol% of the total dehydrated system 76 A method according to claim 67, wherein the system is in the form of liposomes 77 A method according to claim 67, wherein the parasitic infection is caused by a parasitic organism selected from the group consisting of Leishmania major Fπedlin, Leishmania (viannia) gr , Leishmania mexicana, Leishmania tropica, Leishmania donovani (mfantum), Leishmania aethiopica, Leishmania amazonensis, Leishmania enπettn, 5 Leishmania chagasi Leishmania pifanoi, Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malaπae, Trypanosoma cruzi (causing Chargas' disease), Trypanosoma brucei, Trypanosoma equiperdum, Trypanosoma evansi Entamoeba histolytica and Chlornorchis sinensis 10 78 A method according to claim 67 for systemic treatment 79 A method according to claim 76 for intravenous administration 80 A method according to claim 67, wherein the label is selected from the group 15 consisting of 111 ln, 99m Tc, 67 Ga, 11 C, Gd, Mn, iron oxide, argon, nitrogen, Iodine, bromine and barium

Inventors: DAVIDSEN JESPER, FROEKJAER SVEN, JOERGENSEN KENT, MOURITSEN OLE G, VERMEHREN CHARLOTTE, DAVIDSEN, JESPER, FROEKJAER, SVEN, JOERGENSEN, KENT, MOURITSEN, OLE, G., VERMEHREN, CHARLOTTE
Applicants: DAVIDSEN JESPER, FROEKJAER SVEN, JOERGENSEN KENT, LIPLASOME PHARMA AS, MOURITSEN OLE G, VERMEHREN CHARLOTTE, DAVIDSEN, JESPER, FROEKJAER, SVEN, JOERGENSEN, KENT, LIPLASOME PHARMA A/S, MOURITSEN, OLE, G., VERMEHREN, CHARLOTTE
Application year: 2001
Main classification: A61K9
IPC codes: A61B5, A61B8, A61K8, A61K9, A61K31, A61K38, A61K47, A61K49, A61K51, A61P17, A61P29, A61P33, A61P35, A61P37, A61P43, G01R33