WO2009012785A2 - Complexes d'un agent émulsionnant et d'un acide gras - Google Patents

Complexes d'un agent émulsionnant et d'un acide gras Download PDF

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WO2009012785A2
WO2009012785A2 PCT/DK2008/050183 DK2008050183W WO2009012785A2 WO 2009012785 A2 WO2009012785 A2 WO 2009012785A2 DK 2008050183 W DK2008050183 W DK 2008050183W WO 2009012785 A2 WO2009012785 A2 WO 2009012785A2
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acid
emulsifier
fatty acid
complex according
range
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PCT/DK2008/050183
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WO2009012785A3 (fr
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Teit Agger
Christoffer Bro
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Nya Hamlet Pharma Ab
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Publication of WO2009012785A3 publication Critical patent/WO2009012785A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to complexes of emulsifiers and fatty acids, which have a cell killing effect, in particular on tumour cells.
  • the complexes may be employed in treatment of a variety of disease characterised by the presence of undesired cells, such as various malignant or pre-malignant disease and infections, in particular viral infections.
  • HAMLET human a-lactalbumin made lethal to tumour cells
  • the apoptotic activity of multimeric LA was discovered by serendipity.
  • human milk induced apoptosis in transformed and non-transformed immature cell lines.
  • the apoptotic activity in human milk was isolated from a fraction of human milk casein obtained by precipitation at low pH, and was purified by ion exchange chromatography, eluting as a single peak after 1 M NaCI.
  • the cell killing form of alpha-lactalbumin was named SRTe5d-a-LA (a-LA slowly rotated in trifluoroethanol for 5 days) and did not contain any fatty acids (Xu et al., Biosci.Biotechnol. Biochem. (2005)69,1082-1089 and Xu et al., Biosci.Biotechnol. Biochem. (2005)69, 1 189-1 192).
  • the present invention now discloses that complexes of emulsifier (such as polypeptide) and fatty acid in general may be cytotoxic, in particular such complexes of emulsifier and fatty acid prepared by the methods disclosed herein.
  • emulsifier such as polypeptide
  • fatty acid in general may be cytotoxic, in particular such complexes of emulsifier and fatty acid prepared by the methods disclosed herein.
  • the present invention discloses that different polypeptides may be converted to cytotoxic complexes with fatty acids.
  • complexes comprising a much higher molar ratio of fatty acid to emulsifier (such as alpha-lactalbumin) than the previously disclosed ratios of 1 :1 or 2:1 may have significant cytotoxic activity to tumour cells.
  • emulsifier such as alpha-lactalbumin
  • complexes comprising an emulsifier and at least one fatty acid of the general formula R-COOH, wherein R is alkyl or alkenyl, wherein a. said emulsifier is not a fatty acid of the general formula R-COOH, wherein R is alkyl or alkenyl, and b. said emulsifier is associated with said fatty acid within said complex c. said complex has a cytotoxic effect on tumour cells with the proviso that when the emulsifier is alpha-lactabumin and the fatty acid is not C17:1 :1 OCis then the molar ratio of fatty acid to emulsifier within the complex is greater than 2:1 .
  • cytotoxic effect on tumour cells may be determined as described herein below in the section "Cytotoxic effect on tumour cells”.
  • the invention also provides methods for treatment of a clinical disorder selected from the group consisting of viral infections, disorders associated with aberrant cell proliferation, actinic keratosis and disorders associated with angiogenesis comprising administering the complexes of the invention to an individual in need thereof.
  • Figure 1 shows the viability of L1210 cells as a dose response to bLAC at a concentration of 1 .4x10 6 cells per ml. The viability was determined by tryphan blue P1643PC00
  • the LC 50 for bLAC was 0.02-0.05 mg/ml.
  • Figure 2 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of HSA and/or oleic acid.
  • Figure 3 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of HSA and oleic acid.
  • Figure 4 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of HSA and/or oleic acid.
  • Figure 5 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of bl_A and oleic acid.
  • Figure 6 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with bl_A with oleic acid in a 1 :15 molar ratio, wherein oleic acid was a 99% pure preparation obtained from Sigma- Aldrich, Denmark or a 65-88% pure preparation obtained from Merck.
  • Figure 7 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of bovine bl_G and oleic acid.
  • Figure 8 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of bl_G and/or oleic acid.
  • Figure 9 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of MBL and oleic acid.
  • Figure 10 shows % viability relative to control sample with 0.9% NaCI of L1210 cells at a concentration of 1 .4x10 6 cells per ml when incubated with the indicated preparations of MBL and oleic acid.
  • Figure 1 1 shows cell killing activity of three different preparations of complexes prepared from 12.1 mg/mL bl_A saturated and mixed under high shear conditions with oleic acid. Water phase and fatty acid phase was separated by a final centrifugation at 150 x g, 3,000 x g or 18,000 x g. Filled circles - bLA saturated with oleic acid (N287- 50D); open circles - bLA saturated with oleic acid (N287-55B); triangles - bLA saturated with oleic acid (N287-69C); X - bLAC (N276-77A)
  • Figure 12 shows cell killing activity of three different preparations of complexes prepared from fatty acid free HSA saturated and mixed under high shear conditions with oleic acid. Water phase and fatty acid phase was separated by a final centrifugation at 150 x g, 3,000 x g or 18,000 x g.
  • Figure 13 shows cell killing activity of two preparations of complexes prepared from bLG saturated and mixed under high shear conditions with oleic acid.
  • Water phase and fatty acid phase was separated by a final centrifugation at first 3,000 x g followed by 18,000 x g.
  • Figure 14 shows cell killing by two preparations of complexes of prepared from rhMBL saturated and mixed under high shear conditions with oleic acid.
  • Water phase and fatty acid phase was separated by a final centrifugation at first 3,000 x g followed by 18,000 x g.
  • N287-78D open circles - rhMBL saturated with oleic acid
  • Figure 15 shows cell killing activity of two preparations of lysozyme saturated and mixed with oleic acid.
  • Water phase and fatty acid phase was separated by a final centrifugation at first 3,000 x g (giving N287-82E) followed by 18,000 x g (giving N287- 82F).
  • Figure 16 shows cell killing activit of two preparations with oleic acid in ethanol mixed into a 3 mg/mL bLA solution in a 1 :15 molar ratio (protein vs. oleic acid).
  • One of the preparations was subjected to high shear mixing (on whirleymixer) the other was not.
  • Filled circles - bLA and oleic acid (whirley mixed -high shear), LD 50 43 pg/cell; open circles - bLA and oleic acid (tube turned 3 times), LD 50 407 pg/cell.
  • Figure 17 shows cell killing activity of preparations of complexes prepared from oleic acid in ethanol mixed under high shear conditions with a 3 mg/mL bLA solution in different molar ratios.
  • Figure 18 shows LD 50 of preparation prepared from bLA saturated with oleic acid incubated at -2O 0 C, 2-8 0 C and 25 0 C for the indicated number of weeks and analysed for cell killing activity assuming a constant bLA+bLAC concentration of 8.6 mg/mL.
  • Figure 19 shows bLA content in a preparation prepared from bLA in 0.9% NaCI solution saturated with oleic acid and mixed under high shear conditionds incubated at -2O 0 C, 2-8 0 C and 25 0 C for the indicated number of weeks and analysed for protein (bLA) content by A 28 onm-
  • Figure 20 shows LD 50 of complexes prepared from bLA in 0.9% NaCI solution with or without 10 mM Tris (pH 8.5) and 8 mM EDTA saturated with oleic acid and mixed under high shear conditions.
  • Water phases (N318-65A+B) were incubated at -2O 0 C and 25 0 C for the indicated number of weeks and analysed for cell killing activity assuming a constant bLA+bLAC concentration of 8.6 mg/mL in N318-65B and 7.6 mg/mL in N318-65A.
  • Figure 21 shows the Cell killing activity of preparations resulting from mixing under high shear conditions oleic acid dissolved in ethanol into a 3 mg/mL bl_A solution in a 1 :15 protein to oleic acid molar ratio with different chelators and buffers. A constant bLA+bLAC concentration of 3 mg/mL was assumed.
  • Figure 22 Chromatograms of the conversion runs with bl_A start material N277-64A (described in Example 22).
  • Figure 23 Chromatograms of the conversion runs with bl_A start material N289-56A (described in Example 22).
  • an emulsifier is meant to cover substances capable of forming and stabilising an emulsion of two liquids which are immiscible.
  • an emulsifier according to the present invention is capable of stabilising an emulsion of oil and water, preferably an oil in water emulsion.
  • the emulsifier comprises at least one hydrophobic and at least one hydrophilic domain, frequently the emulsifier will comprise one hydrophobic and one hydrophilic domain. More preferably, overall the emulsifier is more hydrophilic than hydrophobic, however, the emulsifier should contain at least one hydrophobic domain. Thus, it is preferred that the solubility of the emulsifier is higher in water than in oil.
  • the emulsifier according to the present invention is not a fatty acid of the formula:
  • the emulsifier is not any of the fatty acids described in the section "Fatty acids" herein below. In one embodiment, the emulsifier according to the present invention is not a fatty acid.
  • the emulsifier may be selected from the group consisting non-ionic and ionic emulsifiers, wherein ionic emulsifiers may be anionic, cationic or zwitterionic.
  • Nonionic emulsifiers may for example be selected from the group consisting of polymeric emulsifiers, such as alkyl poly(ethylene oxide), copolymers of poly(ethylene oxide) and polypropylene oxide), copolymers of ethylene and vinyl acetate, styrene and butyl acrylates; polyoxyalkylenated alkyl esters, for example with alkyl radical comprising from 10 to 22 carbon atoms; polyalkylene glycols, preferably polyethylene glycols; polyethylene glycol ethers of fatty alcohols; polypropylene glycols; diethylene glycols; polyoxyalkylenated alkyl esters of sorbitan, for example where the alkyl radical comprises from 10 to 22 carbon atoms, such as polyoxyethylene sorbitan monooleate, -laurate (or Tween-20), or -stearate; cellulose derivatives, such as hydroxypropylcellulose, ethylcellulose, methylcellulose or cellulose
  • the emulsifier is ionic.
  • Very preferred emulsifiers to be used with the present invention are polypeptides, which are described in more detail herein below in the section "Polypeptides". Polypeptides are ionic emulsifiers and dependent on the particular amino acid composition of the polypeptide they may be anionic, cationic or zwitterionic. In one preferred embodiment the polypeptide is anionic at neutral pH, and thus is an anionic emulsifier.
  • ionic emulsifiers may for example be the following.
  • Anionic emulsifiers may for example be emulsifiers comprising one or more groups selected from the group consisting of sulfate, sulfonate or carboxylate groups, preferably carboxylate groups (with the exception of the fatty acids described below).
  • the anionic emulsifier may be an anionic polypeptide as describe below.
  • Anionic emulsifiers may also be bile salts, preferably sodium cholate. P1643PC00
  • Cationic emulsifiers may for example be emulsifiers comprising one or more quaternary ammonium cations or cationic amino groups.
  • the cationic emulsifiers may be cationic polypeptides as described below.
  • Zwitterionic emulsifiers may for example be selected from the group consisting of dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine and coco ampho glycinate.
  • the emulsifier may be one or more phospholipids, for example phospholipids selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylglycerol and sphingomyelin, preferably the phospholipid is phosphatidylcholine. Hydrogenated phospholipids may also be employed..
  • the emulsifier may also be a composition comprising phospholipids, such as for example lecithin.
  • Lecithin may for example be egg yolk lecithin or soybean lecithin.
  • lecithin may also comprises glycolipids and/or triglycerides.
  • the emulsifier has a certain minimal size, preferably the emulsifier is at least 0.2 kDa, preferably at least 0.5 kDa, more preferably at least 0.75 kDa, for example at least 1 kDa, such as at least 5 kDa.
  • the polypeptide is at least 5 kDa, more preferably at least 10 kDa, for example at least 15 kDa, such as in the range of 5 to 500 kDa, for example in the range of 10 to 500 kDa, such as in the range of 15 to 500 kDa, for example in the range of 10 to 400 kDa, such as in the range of 10 to 300 kDa, for example in the range of 10 to 200 kDa, for example in the range of 15 to 400 kDa, such as in the range of 15 to 300 kDa, for example in the range of 15 to 200 kDa, such as in the range of 15 to 100 kDa.
  • the emulsifier is not cytotoxic to normal, healthy mammalian cells when not in complex with fatty acid. If tested in vitro, then the emulsifier is preferably not cytotoxic to primary, non-malignant mammalian cells in in vitro culture, when not in complex with fatty acid.
  • the assays described herein below for determining cytotoxicity of the complexes of the invention may readily be adapted for determining whether a given emulsifier are cytotoxic to primary, non-malignant cells.
  • the emulsifier is not cytotoxic in the cell killing assay of Example 3 in the absence of fatty acid at the concentrations used.
  • the emulsifier is Tween 80 it is preferably used at concentrations of less than 0.14% (w/v) and in embodiments wherein the emulsifier is sodium cholate it is preferably used at concentrations lower than 6.7 mM. More preferably, the emulsifier is neither Tween 80 nor sodium cholate.
  • the emulsifier is provided in a purified form.
  • the emulsifier is from a natural source, it is preferred that the emulsifier is purified from said natural source.
  • the emulsifier is from a natural source for example from a cellular extract, a tissue extract, a body fluid (such as saliva, serum, blood or the like) or milk, then said emulsifier has been isolated from said natural source prior to being contacted with the fatty acid.
  • the emulsifier is a polypeptide.
  • a polypeptide is made up of amino acids linked by amide bonds.
  • Polypeptides according to the present invention may comprise any amino acid, however it is preferred that polypeptides only comprises naturally occurring amino acid forming parts of proteins in nature.
  • polypeptides within the present invention may also comprise post-translational modifications.
  • the polypeptides may be covalently linked to other polypeptides, typically by S-S bridges, to phosphate, methyl, saccharides, such as mono-saccharides, oligosaccharide or polysaccharide, which optionally may be branched, lipids and the like. It is preferred that the polypeptide only comprises such post-translational modifications which occur in nature.
  • polypeptides may be derived from any suitable species, such as virus, phage, bacteria, archaebacteria, fungi, yeast, plants or animals.
  • the polypeptide is derived from an animal, such as insects, protozoans or vertebrates, preferably from a vertebrae, even more preferably from a mammal.
  • the polypeptide may be an individual polypeptide chain or the polypeptide may be associated with other polypeptides in oligomers.
  • the oligomers may be oligomers of identical polypeptides or of different polypeptides.
  • polypeptides may be purified from natural sources, such as milk, serum, tissue extracts, eggs, plant extracts, cell extracts, or the polypeptides may be produced by recombinant methods, for example as described herein below.
  • the present invention relates to a complex comprising a polypeptide (optionally in the form of an oligomer) other than alpha-lactalbumin and at least one fatty acid of the general formula R-COOH, wherein R is alkyl or alkenyl, wherein said polypeptide is associated with said fatty acid within said complex and wherein said complex has a cytotoxic effect on tumour cells.
  • Polypeptides other than alpha-lactalbumin may be any polypeptide as described in this section.
  • the polypeptide have a theoretical pi, which is not too high. It is therefore preferred that the theroretical pi is at the most 9, preferably at the most 8, more preferably at the most 7, such as in the range of 3 to 9, for example in the range of 3 to 8, such as in the range of 3 to 7, for example in the range of 3 to 6, such as in the range of 4 to 9, such as in the range of 4 to 8, for example in the range of 4 to 7, such as in the range of 4 to 6, such as in the range of 4.5 to 9, for example in the range of 4.5 to 8, such as in the range of 4,5 to 7, for example in the range of 4.5 to 6.
  • the polypeptide preferably is not lysozyme.
  • the overall content of negatively charged amino acids is higher than the overall content of positively charged amino acids (Arg and Lys).
  • the aliphatic index of the polypeptide according to the present invention is preferably in the range of 50 to 250, such as in the range of 65 to 1 10.
  • polypeptides are given in order to exemplify the invention.
  • other polypeptides may also be useful with the present invention.
  • the polypeptide may for example be a polypeptide belonging to the albumin family, preferably with the proviso that the polypeptide is not alpha-lactalbumin.
  • polypeptide may be an albumin obtainable from serum, such as serum albumin, such as human serum albumin.
  • polypeptide may be human P1643PC00
  • Functional homologues may be any of the functional homologues described herein below.
  • the polypeptide of the albumin family may be purified from natural sources, for example from serum or it may be produced by recombinant methods.
  • the polypeptide may for example also be a member of the globulin family, such as globulins from serum or globulin from milk, for example lactoglobulin, such as bovine, caprine, equine, porcine or camelide lactoglobulin, such as, bovine, caprine, equine, porcine or camelide beta-lactoglobulin.
  • lactoglobulin such as bovine, caprine, equine, porcine or camelide lactoglobulin, such as, bovine, caprine, equine, porcine or camelide beta-lactoglobulin.
  • the polypeptide may be bovine beta-lactoglobulin (bl_G) of SEQ ID NO: 4 or a functional homologue thereof sharing at least 75% sequence identity therewith.
  • the functional homologue may be any of the functional homologues described herein below.
  • the polypeptide of the globulin family may be purified from natural sources, for example from serum or milk, or it may be produced by recombinant methods.
  • bovine beta-lactoglobulin is purified from bovine milk.
  • the polypeptide may also be a polypeptide comprising a collagen domain.
  • a collagen domain according to the present invention is a domain including the motif -G-X-X- several times, in general at least 5 times, preferably at least 10 times, wherein G is glycine and X is any naturally occurring amino acid.
  • Polypeptides comprising collagen domains are frequently capable of oligomerisation, thus the polypeptide may be present as an individual polypeptide or as an oligomer of polypeptides.
  • the polypeptide may also be a lectin, i.e. a polypeptide capable of associating with one oorr mmoorree carbohydrates.
  • a lectin i.e. a polypeptide capable of associating with one oorr mmoorree carbohydrates.
  • the lectin comprises a carbohydrate binding domain.
  • the polypeptide may also be a lectin comprising a collagen domain, preferably a polypeptide comprising a carbohydrate binding domain and a collagen domain.
  • Polypeptides including a collagen domain may for example be ficolins or mannose binding lectin, such as mammalian mannose binding lectin.
  • the polypeptide may be human mannonse binding lectin (hMBL) of SEQ ID NO: 3 or a functional homologue thereof sharing at least 75% sequence identity therewith.
  • Functional homologues may be any of the functional homologues described herein below. Examples of useful functional homologues of MBL are for example described in Danish patent application PA 2006 01555 in the sections "MBL and MBL variants" and "Functional homologues" on pages 7-1 1 .
  • functional homologues of MBL retain at least some MBL function, preferably the functional homologues of MBL are capable of activating C4 in the presence of MASP-2.
  • a useful functional assay for evaluating MBL function includes the assays, which are described in WO03/033522 in the section "Functionality" on pages 27-28.
  • the polypeptide is MBL or functional homologues thereof
  • 3 individual MBL polypeptides in general associate to form a so-called monomer.
  • These monomers, each consisting of 3 MBL polypeptides may then associate to form oligomers, such as dimers, trimers, tetramers, pentamers, hexamers or even higher oligomers.
  • oligomers such as dimers, trimers, tetramers, pentamers, hexamers or even higher oligomers.
  • the majority of MBL is present as oligomers comprising at least two monomers, preferably at least 3 monomers, wherein the monomers each consists of 3 MBL polypeptides.
  • the polypeptide comprising a collagen domain may be purified from natural sources, for example from serum or tissue extracts, or it may be produced by recombinant methods.
  • human mannose binding protein is produced by recombinant methods involving heterologous expression of MBL in in vitro cultured mammalian cells, such as HEK cells and purification from the culture medium by conventional protein purification techniques.
  • Useful methods for purifying MBL include for example the methods described in Examples 1 , 2, 4 and 6 of WO03/033522 and in Examples 1 , 2 and 2 of WO00/70043.
  • the polypeptide may for example be a polypeptide belonging to the fatty acid binding protein (FABP) superfamily.
  • the fatty acid-binding protein (FABP) superfamily is constituted by 14-15 kDa soluble proteins which bind with a high affinity either long- chain fatty acids (LCFAs), bile acids (BAs) or retinoids.
  • FABPs are members of the superfamily of lipid-binding proteins (LBP). The primary role of all the FABP family members is regulation of fatty acid uptake and intracellular transport.
  • the structure of all FABPs is similar - the basic motif characterizing these proteins is beta-barrel, and a single ligand (e.g. a fatty acid, cholesterol, or retinoid) is bound in its internal water- filled cavity.
  • FABPs have a tissue-specific distribution pattern.
  • polypeptide may be a FABP with tissue-specific distribution for example: L-FABP (liver), I-FABP (intestinal), H-FABP (muscle and heart), A-FABP (adipocyte), E-FABP (epidermal), N-FABP (ileal), B-FABP (brain), M-FABP (myelin) or T-FABP (testis).
  • L-FABP liver
  • I-FABP intestinal
  • H-FABP muscle and heart
  • A-FABP adipocyte
  • E-FABP epidermal
  • N-FABP ileal
  • B-FABP brain
  • M-FABP myelin
  • T-FABP T-FABP
  • the FABP may for example be a mammalian FABP such as human, bovine, caprine, equine, porcine or camelide FABP such as human, bovine, caprine, equine, porcine or camelide FABP.
  • the polypeptide may be human adipocyte FABP of SEQ ID NO: 6 or a functional homologue thereof sharing at least 75% sequence identity therewith.
  • Functional homologues may be any of the functional homologues described herein below.
  • the polypeptide of the FABP family may be purified from natural sources, for example from serum or it may be produced by recombinant methods.
  • the polypeptide is not alpha- lactalbumin.
  • the present invention also surprisingly discloses that the complexes of alpha-lactalbumin and fatty acids, such as oleic acid, with a ratio of fatty acid to alpha-lactalbumin higher than 2:1 do posses cytotoxic activity
  • the emulsifier may be alpha-lactalbumin.
  • the fatty acid when the emulsifier is alpha-lactalbumin, the fatty acid may preferably be heptadecenoic acid.
  • the ratio of emulsifier to fatty acid may be lower or higher than 2:1 , although very preferred ratios, are the ratios given herein below in the section ("Ratio").
  • Alpha-lactalbumin may be mammalian lactalbumin, such as human, bovine, caprine, equine, porcine or camelide alpha-lactalbumin.
  • alpha-lactalbumin may be bovine alpha-lactalbumin of SEQ ID NO: 2 or human alpha-lactalbumin of SEQ ID NO: 1 or a functional homologue sharing a sequence identity with any of the aforementioned sequences of at least 75%.
  • Functional homologues may be any of the functional homologues described herein below.
  • alpha-lactalbumins as well as functional homologues thereof are for example described Danish patent application PA 2007 00693 in the sections "Alpha-lactalbumin” and "Functional homologues of alpha-lactalbumin” p. 9-21 .
  • functional homologues of alpha-lactalbumin retain at least some alpha-lactalbumin function, preferably the functional homologues of alpha-lactalbumin are capable of forming a biologically active complex with a fatty acid, preferably oleic acid, said complex comprising cytotoxic activity or cell killing activity.
  • a useful functional assay for evaluating alpha-lactalbumin function includes the assay, which is described in Example 7 of PA 2007 00693.
  • Alpha-lactalbumin may be purified from natural sources, for example from milk, or it may be produced by recombinant methods.
  • bovine alpha-lactalbumin is purified from milk.
  • human alpha- lactalbumin is produced by by recombinant methods involving heterologous expression of alpha-lactalbumin in host cells, for example yeast cells and purification from the culture medium by conventional protein purification techniques.
  • Useful methods for purifying alpha-lactalbumin include for example the methods described in Examples 1 , 2, 4 and 6 of WO03/033522 and in Examples 1 , 2 and 2 of WO00/70043.
  • purified alpha-lactalbumin is used for the methods of the invention and that alpha-lactalbumin is purified prior to contacting with fatty acid. It is thus preferred that the liquid solution comprising the emulsifier provided in step a) of the method according to the invention comprises purified alpha-lactalbumin.
  • liquid solution is preferably essentially devoid of at least one, more preferably at least two, for example of all milk constituents other than water and alpha-lactalbumin, where "essentially devoid of" within the present context means that said components are present at levels below detection level.
  • said liquid solution is preferably essentially devoid of at least one, preferably at least two, for example of all milk polypeptides other than alpha-lactalbumin.
  • Said milk polypeptide may for example be selected from the group consisting of immunoglobulins and casein.
  • a functional homologue of a polypeptide of a given sequence within the present invention is a polypeptide sharing at least some sequence identity with the given sequence and which shares at least one function, preferably, has cytotoxic activity when in complex with oleic acid.
  • a complex of a functional homologue and oleic acid has an LD 50 of at the most 50 mg/ml, preferably at the most 40 mg/ml, even more preferably at the most 30 mg/ml, yet more preferably at the most 20 mg/ml, even more preferably at the most 10 mg/ml, yet more preferably at the most 5 mg/ml, even more preferably at the most 1 mg/ml, for example at the most 0.75 mg/ml, such as at the most 0.5 mg/ml, when determined as described in Example 3 herein below.
  • polypeptide sequences from at least 2, preferably at least 3, more preferably at least four different species where the function of the polypeptide is conserved are compared, for example but not limited to mammals including rodents, monkeys and apes.
  • conserved residues are more likely to represent essential amino acids that cannot easily be substituted than residues that change between species.
  • such an alignment may be performed using ClustalW from EBML-EBI. It is evident from the above that a reasonable number of modifications or alterations of a polypeptide sequence does not interfere with the P1643PC00
  • functional homologues of a given polypeptide comprise all residues, which are conserved between at least 4, such as at least 3, for example at least 2 different species.
  • Functional homologues may thus comprise one or more amino acid substitutions at residues, which are not conserved between at least 4, such as at least 3, for example at least 2 different species.
  • amino acid substitutions may be regarded as “conservative” where an amino acid is replaced with a different amino acid with broadly similar properties.
  • Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.
  • a person skilled in the art will know how to make and assess 'conservative' amino acid substitutions, by which one amino acid is substituted for another with one or more shared chemical and/or physical characteristics. Conservative amino acid substitutions are less likely to affect the functionality of the protein.
  • Amino acids may be grouped according to shared characteristics.
  • a conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within a predetermined group exhibit similar or substantially similar characteristics, preferably the groups are the groups listed below in "Lower levels of similarity", even more preferably the groups are the groups listed below in "High level of similarity".
  • Polarity i) Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser, Thr, Tyr, and Cys,) P1643PC00
  • Hydrophilic or hydrophobic iii) Hydrophobic amino acids (Ala, Cys, GIy, lie, Leu, Met, Phe, Pro, Trp, Tyr, VaI)
  • v) Neutral amino acids Ala, Asn, Cys, GIn, GIy, lie, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, VaI
  • Acidic amino acids ((asp, GIu)
  • More preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine- glutamine. P1643PC00
  • the same functional homologue or fragment thereof may comprise more than one conservative amino acid substitution from more than one group of conservative amino acids as defined herein above.
  • Polypeptides of the invention may comprise Standard and non-standard amino acids or mixtures of both. It is preferred that the polypeptides only comprise Standard amino acids. There are twenty standard naturally occurring amino acids and two special amino acids, selenocysteine and pyrrolysine, as well as a vast number of "nonstandard amino acids" which are not incorporated into protein in vivo. Examples of nonstandard amino acids include the sulfur-containing taurine and the neurotransmitters GABA and dopamine. Other examples are lanthionine, 2-Aminoisobutyric acid, and dehydroalanine. Further non standard amino acids are ornithine and citrulline.
  • Non-standard amino acids are usually formed through modifications to standard amino acids.
  • taurine can be formed by the decarboxylation of cysteine, while dopamine is synthesized from tyrosine and hydroxyproline is made by a posttranslational modification of proline (common in collagen).
  • Examples of non- naturally occurring amino acids are those listed e.g. in 37 C. F. R. section 1.822(b)(4), all of which are incorporated herein by reference.
  • a functional equivalent according to the invention may comprise any amino acid including non-standard amino acids. In preferred embodiments a functional equivalent comprises only standard amino acids.
  • the standard and/or non-standard amino acids may be linked by peptide bonds or by non-peptide bonds, preferably however by peptide bonds.
  • the term peptide also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art. Such post-translational modifications can be introduced prior to partitioning, if desired.
  • Amino acids as specified herein will preferentially be in the L-stereoisomeric form.
  • Amino acid analogs can be employed instead of the 20 naturally-occurring amino acids. Several such analogs are known, P1643PC00
  • a functional homologue within the scope of the present invention is a polypeptide that exhibits at least some sequence identity with a polypeptide of a given sequence, preferably functional homologues have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, for example at least 91 % sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity with a given polypeptide sequence.
  • Sequence identity can be calculated using a number of well-known algorithms and applying a number of different gap penalties.
  • the sequence identity is calculated relative to the full-length sequence of the reference polypeptide.
  • Any sequence alignment tool such as but not limited to FASTA, BLAST, or LALIGN may be used for searching homologues and calculating sequence identity.
  • any commonly known substitution matrix such as but not limited to PAM, BLOSSUM or PSSM matrices may be applied with the search algorithm.
  • a PSSM position specific scoring matrix
  • sequence alignments may be performed using a range of penalties for gap opening and extension.
  • the BLAST algorithm may be used with a gap opening penalty in the range 5-12, and a gap extension penalty in the range 1 -2.
  • Functional homologues may in one embodiment further comprise chemical modifications such as ubiquitination, labeling (e.g., with radionucleotides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids such as ornithine, which do not normally occur in human proteins.
  • chemical modifications such as ubiquitination, labeling (e.g., with radionucleotides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids such as ornithine, which do not normally occur in human proteins.
  • sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention.
  • esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • Functional equivalents also comprise glycosylated and covalent or aggregative conjugates formed with the same molecules, including dimers or unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.
  • Functional homologues may also be deletion or addition mutants.
  • the addition may be addition of at least one amino acid, an addition of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids.
  • a functional homologue may be a deletion mutant which have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85 % sequence identity, for example at least 90 % sequence identity, for example at least 91 % sequence identity, such as at least 92 % sequence identity, for example at least 93 % sequence identity, such as at least 94 % sequence identity, for example at least 95 % sequence identity, such as at least 96 % sequence identity, for example at least 97% sequence identity, such as at least 98 % sequence identity, for example 99% sequence identity.
  • Deletion mutants suitably comprise at least 20 or 40 consecutive amino acid and more preferably at least 80 or 100 consecutive amino acids in length.
  • functional homologues of a given polypeptide comprises at the most 500, more preferably at the most 400, even more preferably at the most 300, yet more preferably at the most 200, such as at the most 175, for example at the most 160, such as at the most 150 amino acids in addition to the sequence of the given polypeptide.
  • Polypeptides may be purified from natural sources, which should be selected according to the occurrence of the polypeptide.
  • natural sources includes cell extracts, tissue extracts, plant extracts, body fluids, such as saliva or serum, milk or eggs.
  • Polypeptides may also be recombinantly produced as described in more details herein below and then optionally be purified from host cells expressing the heterologous protein, from host organisms, such as transgenic plants or animals expressing the heterologous polypeptide or from tissue culture medium from host cells expressing the heterologous polypeptides.
  • Purification of proteins in general involves one or more steps of removal of or separation from contaminating nucleic acids, phages and/or viruses, other proteins and/or other biological macromolecules.
  • the procedure may comprise one or more protein isolation steps. Any suitable protein isolation step may be used with the present invention.
  • the skilled person will in general readily be able to identify useful protein isolation steps for a given polypeptide using routine experimentation.
  • the protein isolation steps useful with the present invention may be commonly used methods for protein purification including for example chromatographic methods such as for example gas chromatography, liquid chromatography, ion exchange chromatography and/or affinity chromatography; filtration methods such as for example gel filtration and ultrafiltration; precipitation, such as ammonium sulphate precipitation and/or gradient separation such as sucrose gradient separation.
  • the purification may comprise one or more of the aforementioned methods in any combination.
  • polypeptides of the invention may also be recombinantly prepared, in particular functional homologues are preferably produced recombinantly.
  • Useful recombinant production methods includes conventional methods known in the art, such as by expression of heterologuos polypeptide or functional homologues thereof in suitable host cells such as E. coli, S. cerevisiae or S. pombe or insect or mammalian cells suitable for production of recombinant proteins (see below).
  • suitable host cells such as E. coli, S. cerevisiae or S. pombe or insect or mammalian cells suitable for production of recombinant proteins (see below).
  • suitable host cells such as E. coli, S. cerevisiae or S. pombe or insect or mammalian cells suitable for production of recombinant proteins (see below).
  • suitable host cells such as E. coli, S. cerevisiae or S. pombe or insect or mammalian cells suitable for production of recombin
  • the polypeptides are produced in a transgene plant or animal.
  • a transgenic plant or animal in this context is meant a plant or animal which has been genetically modified to contain and express a nucleic acid encoding the given polypeptide or functional homolgue hereof.
  • the polypeptide is produced by host cells comprising a first nucleic acid sequence encoding the given polypeptide or a functional homologue thereof operably associated with a second nucleic acid capable of directing expression in said host cells.
  • the second nucleic acid sequence may thus comprise or even consist of a promoter that will direct the expression of protein of interest in said cells.
  • a skilled person will be readily capable of identifying useful second nucleic acid sequence for use in a given host cell.
  • the process of producing recombinant polypeptide or a functional homologue thereof in general comprises the steps of:
  • a gene expression construct comprising a first nucleic acid sequence encoding a given polypeptide or a functional homologue thereof operably linked to a second nucleic acid sequence capable of directing expression of said protein of interest in the host cell
  • the recombinant polypeptide thus produced may be isolated by any conventional method for example by any of the protein purification methods described herein above.
  • the skilled person will be able to identify a suitable protein isolation steps for purifying any protein of interest.
  • the recombinantly produced LA or the functional homologue thereof is excreted by the host cells.
  • the polypeptide is recombinantly produced in vitro in host cells and is isolated from cell lysate, cell extract or from tissue culture supernatant.
  • polypeptide is produced by host cells that are modified in such a way that they express the polypeptide of interest.
  • said host cells are transformed to produce and excrete the polypeptide.
  • the gene expression construct may comprise a viral based vector, such as a DNA viral based vector, a RNA viral based vector, or a chimeric viral based vector.
  • a viral based vector such as a DNA viral based vector, a RNA viral based vector, or a chimeric viral based vector.
  • DNA viruses are cytomegalo virus, Herpex Simplex, Epstein-Barr virus, Simian virus 40, Bovine papillomavirus, Adeno-associated virus, Adenovirus, Vaccinia virus, and Baculo virus.
  • the gene expression construct may for example only comprise a plasmid based vector.
  • the invention provides an expression construct encoding a given polypeptide or functional homologues thereof, featured by comprising one or more intron sequences for example from the native gene. Additionally, it may contain a promoter region derived from a viral gene or a eukaryotic gene, including mammalian and insect genes.
  • the promoter region is preferably selected to be different from the native promoter, and preferably in order to optimize the yield, the promoter region is selected to function most optimally with the vector and host cells in question.
  • the promoter region is selected from a group comprising Rous sarcoma virus long terminal repeat promoter, and cytomegalovirus immediate- early promoter, and elongation factor-1 alpha promoter.
  • the promoter region is derived from a gene of a microorganism, such as other viruses, yeasts and bacteria.
  • the promoter region may comprise enhancer elements, such as the QBI SP163 element of the 5' end untranslated region of the mouse vascular endothelian growth factor gene
  • One process for producing recombinant LA according to the invention is characterised in that the host cell culture is may be eukaryotic, for example a mammalian cell culture or a yeast cell culture.
  • Useful mammalian cells may for example be human embryonal kidney cells (HEK cells), such as the cell lines deposited at the American Type Culture Collection with the numbers CRL-1573 and CRL-10852, chick embryo fibroblast, hamster ovary cells, baby hamster kidney cells, human cervical carcinoma cells, human melanoma cells, human kidney cells, human umbilical vascular endothelium cells, human brain endothelium cells, human oral cavity tumor cells, monkey kidney cells, mouse fibroblast, mouse kidney cells, mouse connective tissue cells, mouse oligodendritic cells, mouse macrophage, mouse fibroblast, mouse neuroblastoma cells, mouse pre-B cell, mouse B lymphoma cells, mouse plasmacytoma cells, mouse teratocarcinoma cells, rat astrocytoma cells, rat mammary epithelium cells, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells.
  • the host cells may also either be prokaryotic cells or yeast cells.
  • Prokaryotic cells may for example be E. coli.
  • Yeast cells may for example be Saccharomyces, Pichia or Hansenula. P1643PC00
  • the present invention regards complexes of emulsifier and fatty acid, wherein the emulsifier may be any of the emulsifiers described above, and the fatty acid may be any of the fatty acids described in this section.
  • the fatty acid according to the present invention has the general formula:
  • R is alkyl or alkenyl, such as C 4 to C 80 alkyl or alkenyl, preferably C 10 to C 30 alkyl or alkenyl, more preferably alkenyl.
  • said alkyl or alkenyl are unbranched.
  • Fatty acids are carboxylic acids, which preferably have a long unbranched aliphatic chain.
  • the aliphatic chain of a fatty acid can be either saturated or unsaturated.
  • Saturated fatty acids are saturated with hydrogen and thus have no double bonds.
  • Unsaturated fatty acids can be either mono-unsaturated (or MUFAs), having one double bond or poly-unsaturated (PUFAs), having 2 or more double bonds.
  • the fatty acids of the present invention may be a saturated fatty acid or an unsaturated fatty acid, however preferably the fatty acid is an unsaturated fatty acid.
  • C4 to C30 for example from C6 to C28, such as from C8 to C26, for example from C10 to C24, such as from C12 to C22, for example from C14 to C20, such as from C16 to C20, for example from the group of C16, C17, C18 and C20, such as from the group of C16, C18 and C20.
  • the fatty acid is selected from the group of C16, C17, C18 and C20.
  • the fatty acid is preferably a C17 fatty acid, more preferably a C17 fatty acid comprising at least one double bond, which preferably is a cis double bond, more preferably all double bonds are cis double bonds, even more preferably said C17 fatty acid comprises a cis double bond in the 10 position.
  • Fatty acids are often described using the number of C-atoms of the chain and the number, location and conformation of double bonds.
  • Steric acid for example, has a chain of 18 C-atoms and no double bonds and can be described as C18:0
  • oleic acid has a chain of 18 C-atoms and one double bond and can be described as C18:1
  • linoleic acid has a chain of 18 C-atoms and two double bonds and can be described as C18:2 and so forth.
  • the double bond is located on the xth carbon-carbon bond, counting from the carboxyl terminus.
  • the Latin prefixes cis (on this side) and trans (across) describe the conformation of the double bonds by describing the orientation of the hydrogen atoms with respect to said double bond. Double bonds in the cis conformation are preferred. The position of the double bond is frequently indicated as the last number, following the integer indicating the number of double bonds.
  • oleic acid having an 18 carbon chain with one double bond between carbon 9 and 10 may be described as C18:1 :9cis and ⁇ -linolenic acid having an 18 carbon chain with three double bonds between carbon 9 and 10, 12 and 13 and 15 and 16, respectively, may be described as C18:3:9,12,15.
  • Cis or trans may be indicated after the position of the double bond. If there is more than one double bond and they all are of the same conformation, then the term cis or trans may be indicated after indication of the position of all double bonds and thus relates to the conformation of all double bonds.
  • Linoleic acid having an 18 carbon chain with 2 double bonds, which are both cis double bonds between carbons 9 and 10 and 12 and 13, respectively may be described as C18:2:9,12cis
  • the fatty acid has in the range of 0 to 6 double bonds, for example in the range of 1 to 5 double bonds, such as the number of double bonds is selected from the group of 1 , 2, 3 or 4 double bonds. In more preferred embodiments of the invention the fatty acid has 1 or 3 double bonds. In a most preferred embodiment of the invention the fatty acid has one double bond.
  • unsaturated fatty acids examples include for example:
  • Heptadecenoic Acid C17:1 :1 Ocis
  • a mono-saturated fatty acid is complexed with emulsifier. More preferred are mono-saturated fatty acids selected from the group of: C17:1 :10cis or trans, C16:1 :6cis or trans, C16:1 :9cis or trans, C16:1 :1 1 cis or trans, C18:1 :6cis or trans, C18:1 :9cis or trans, C18:1 :1 1cis or trans, C18:1 :13cis or trans, C20:1 :9 cis or trans, C20:1 :1 1 cis and trans, C20:1 :13cis or trans.
  • the mono-saturated fatty acid complexed with emulsifier is in the cis conformation such a fatty acid selected from the group of: C17:1 :10cis, C16:1 :6cis, C16:1 :9cis, C16:1 :1 1 cis, C18:1 :6cis, C18:1 :9cis, C18:1 :1 1 cis, C18:1 :13cis, C20:1 :9cis, C20:1 :1 1 cis, C20:1 :13cis.
  • the fatty acid complexed with emulsifier is an unsaturated fatty acid in the cis conformation, preferably selected from the group consisting of C17:1 :10cis, C18:1 :9cis, C18:1 :1 1 cis, C18:1 :6cis, C16:1 :9cis, C18:3:6,9,12cis, C18:3:9,12,15cis, C18:2:9,12cis.
  • the fatty acid complexed with emulsifier is selected from the group consisting of C16 to C20 fatty acids comprising in the range of 1 to 5 cis double bonds.
  • the fatty acid may for example be selected from the group consisting of Vaccenic Acid C18:1 :1 1 cis, Linoleic Acid C18:2:9,12cis, Alpha Linolenic Acid 018:3:9,12,15, Palmitoleic Acid C16:1 :9cis, Heptadecenoic Acid C17:1 :1 Ocis, Gamma Linolenic Acid C18:3:6,9,12cis, Stearidonic acid C18:4:6,9,12,15cis, Eicosenoic Acid C20:1 :11 cis and Eicosapentaenoic Acid C20:5:5,8,1 1 ,14,17cis, such as from the group consisting of Vaccenic Acid C18:1 :1
  • the fatty acid is one or more selected from the group consisting of oleic acid, linoleic acid and vaccenic acid.
  • the fatty acid is Heptadecenoic Acid, in particular when the emulsifier is alpha-lactalbumin, the fatty acid may preferably be heptadecenoic acid.
  • the fatty acid complexed with alpha- lactalbumin is an unsaturated C16 or C18 fatty acid, preferably a C18 fatty acid, wherein all double bonds are cis double bonds.
  • the fatty acid may preferably comprise 1 , for example 2, such as 3, for example 4 double bonds, wherein all double bonds are cis double bonds.
  • the fatty acid may for example be selected from the group consisting of C18:1 :9cis, C18:1 :1 1 cis, C18:1 :6cis, C16:1 :9cis, C18:3:6,9,12cis, C18:3:9,12,15cis, C18:2:9,12cis and C18:4:6, 9, 12, 15cis, preferably selected from the group consisting of C18:1 :9cis, C18:1 :1 1 cis, C18:1 :6cis, C18:3:6,9,12cis, C18:3:9,12,15cis, C18:2:9,12cis and C18:4:6, 9, 12, 15cis, for example selected from the group consisting of C18:1 :9cis, C18:1 :1 1 cis, P1643PC00
  • the fatty acid complexed with alpha-lactalbumin is an unsaturated C17 fatty acid, preferably C17:1 :1 Ocis.
  • fatty acids are according to the invention C17:1 :1 Ocis, C18:1 :9cis and C18:1 :1 1 cis.
  • C18:1 :9cis is highly preferred for the complex of the invention.
  • a polyunsaturated fatty acid is complexed with emulsifier.
  • a polyunsaturated acid selected from the group of C18:2:9,12cis, C18:3:9,12,15cis, C18:3:6,9,12cis, and C20:4:5,8,1 1 15cis.
  • the fatty acid is an artificial fatty acid.
  • the fatty acid may be purified from a natural source, be prepared by organic synthesis or may be commercially purchased. Fatty acids are for example available from Sigma- Aldrich, Denmark or Merck, Darmstadt, Germany.
  • the fatty acid may be provided as a pure fatty acid or in a mixture with impurities and/or other fatty acids.
  • the fatty acid may have a purity of in the range of 50 to 100%, such as in the range of 60 to 100%, for example in the range of 65-99%, such as in the range of 65-88%, for example approx. 99%, such 99%.
  • the complexes according to the present invention may be produced by various methods.
  • the process of preparing cytotoxic complexes of emulsifier and fatty acid is also referred to as “conversion” or “conversion method” herein.
  • an emulsifier is in complex with fatty acid, when said emulsifier is associated with one or more fatty acids.
  • One method for determining whether an emulsifier is associated with a fatty acid is to determine whether emulsifier and fatty acid may be recoved together during a purification. In particular if fatty acid may be recovered together with emulsifier in the aqueous phase P1643PC00
  • the emulsifier is said to be in complex with the fatty acid recovered in the aqueous phase.
  • the emulsifier may be any of the emulsifiers described herein above. Many emulsifiers are commercially available. If the emulsifier is a polypeptide it may be prepared and provided as described herein above in the section "Methods of preparing polypeptides".
  • the emulsifier and the fatty acid may be mixed in any suitable ratio, however preferably the fatty acid is added in molar excess over emulsifier, such as polypeptide.
  • Molar excess means that there are more moles of a fatty acid than there is of emulsifier.
  • the molar excess of a fatty acid over emulsifier added during preparation may be that for one mole emulsifier in the range of 1 .5 moles and 50 moles of a fatty acid are added, such as for one mole emulsifier in the range of 2 moles and 45 moles of a fatty acid are added, for example in the range of 3 moles and 40 moles of a fatty acid are added.
  • the concentration of fatty acid, such as oleic acid is in the range of 0.01 mM to 200 mM, such as in the range of 0.05 mM to 100 mM, for example in the range of 0.1 mM to 50 mM, such as in the range of 0.2 mM to 25 mM, for example in the range of 0.5 mM to 10 mM, such as in the range of 1 mM to 5mM, such as around 2 mM.
  • the complexes of the invention may be prepared by several methods. Preferred methods include methods comprising the steps of:
  • Conversion may be performed by any suitable method, such as the methods described herein below.
  • Preferably said methods involves a step of ion exchange chromatography and/or high shear mixing.
  • the methods may involve the steps of
  • Contacting the ion exchange medium may for example be performed by providing a chromatography column packed with the ion exchange medium and subjecting said emulsifier and/or fatty acid to ion exchange chromatography using said column.
  • the emulsifier is a calcium binding polypeptide then it is preferred that the calcium is removed from said polypeptide prior to or simultaneously with contacting the fatty acid with the emulsifier. Release of calcium may be obtained by any suitable method known to the skilled person. P1643PC00
  • release of calcium may be achieved by contacting the emulsifier, such as the polypeptide with a calcium chelating agent.
  • the calcium chelating agent may be selected from the group of calcium chelators comprising, but not limited to 1 ,2- Bis(2-aminophenoxy)ethane- ⁇ /, ⁇ /, ⁇ /', ⁇ /-tetraacetic acid (BAPTA) or Ethylene glycol- bis(aminoethylether)- ⁇ /, ⁇ /, ⁇ /', ⁇ /-tetraacetic (EGTA) or Ethylene diamine tetraacetic acid (EDTA).
  • the calcium chelator is Ethylene diamine tetraacetic acid (EDTA).
  • the calcium chelating agent is prerably added in molar excess over polypeptide or a functional homologue thereof.
  • the ion exchange medium is preconditioned with a fatty acid, for example any of the fatty acids described herein above in the section "Fatty Acid”.
  • Preconditioning may be performed by any conventional method, for example as described in Svensson, et al. , (2000) Proc Natl Acad Sci USA, 97,4221 -6, WO 03/098223 or WO 2005/082406.
  • pre-conditioning is performed by adding one or more fatty acids to the ion exchange medium, for example the fatty acids may be added in an amount corresponding to in the range of 1 to 30, such as in the range of 2 to 20, for example in the range of 4 to 12, such as in the range of 6 to 10, for example in the range of 7 to 9, such as approximately 8 mg/cm 2 ion exchange resin.
  • the fatty acids may be added in an amount corresponding to in the range of 1 to 30, such as in the range of 2 to 20, for example in the range of 4 to 12, such as in the range of 6 to 10, for example in the range of 7 to 9, such as approximately 8 mg/cm 2 ion exchange resin.
  • the range of 1 o 30, such as in the range of 2 to 20, such as in the range of 3 to 10
  • for example in the range 5 to 8 such as in the range of 6 to 7, for example approximately 6.4 ml fatty acid (for example oleic acid or vaccenic acid or heptadecenoic acid) may be added per
  • the column may be washed, using any suitable wash solution, preferably at least one column volume (CV) wash solution, for example in the range of 2 to 5 CV.
  • the wash solution may comprise suitable buffer and optionally salt, such as in the range pf 0.001 to 0.2M salt, such as around 0.1 M salt (for example NaCI) for example the wash buffer may be the equilibration buffer described in Example 2 of Danish patent application PA 2007 00693.
  • the method may also comprise an isocratic step, for example a step of washing with elution buffer.
  • elution buffer Preferably, at least one column volume (CV) is used for washing, such as in the range of 2 to 5 CV.
  • the elution buffer may be any suitable elution buffer, which in general comprises a buffer and salt, preferably at least 0.1 M , such as at least 0.5M for example around 1 M salt (for example NaCI). , such as solvent B described herein below in Example 2 of Danish patent application PA 2007 00693.
  • an additional wash with a washing solution may be performed as described above.
  • the preconditioning is performed essentially as described in Example 2 of Danish patent application PA 2007 00693.
  • Ion exchange chromatography separates molecules on the basis of differences in their net surface charge. Emulsifiers vary considerably in their charge properties (see herein above) and will exhibit different degrees of interaction with charged chromatography media according to differences in their overall charge, charge density and surface charge distribution.
  • Ion exchange chromatography takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific protein.
  • separation reversible interactions between charged molecules and oppositely charged ion exchange chromatography media are controlled in order to favour binding or elution of specific molecules and achieve separation.
  • a protein has no net charge at a pH equivalent to its isoelectric point (pi) and will not interact with a charged medium.
  • Preferred pi for polypeptides of the invention is described above.
  • An ion exchange medium which may be used with the invention comprises a matrix of spherical particles substituted with ionic groups that are negatively (anionic) or P1643PC00
  • the matrix is usually porous to give a high internal surface area.
  • the medium is in general packed into a column to form a packed bed. The bed is then equilibrated with buffer which fills the pores of the matrix and the space in between the particles.
  • the pH and ionic strength of the equilibration buffer are selected to ensure that, when sample is loaded, proteins of interest bind to the medium.
  • the ion exchange medium to be used in general consists of a matrix and a ligand.
  • matrix as used herein in relation to ion exchange chromatography relates to the material of the resin without ion exchange ligand.
  • the matrix is the base material to which different ion exchange ligands may be bound.
  • the matrix of a Q Sepharose Fast Flow is Sepharose Fast Flow.
  • the term "ligand" as used herein in relation to ion exchange chromatography relates to an ion exchange group coupled to a given matrix.
  • the ligand is main responsible for the ion exchange properties of a given resin.
  • the ligand of a Q Sepharose Fast Flow is the quaternary ammonium ion Q (see below).
  • the ion exchange medium to be used with the invention may have a high porosity of a matrix which offers a large surface area covered by charged groups and so ensures a high binding capacity.
  • the ion exchange medium may comprise a matrix which is carbohydrate based.
  • such a matrix comprises carbohydrate.
  • the matrix may be a polymer of residues, wherein at least some residues are monosaccharide residues, for example at least 25%, such as at least 50%, for example at least 75%, such as at least 95%, for example essentially all, preferably all residues are monosaccharide residues.
  • the monosaccharide residues may for example be aldose or ketose residues or derivatives thereof, such as derivatives obtained by oxidation, deoxygenation, dehydration, introduction of other substituents, alkylation or acylation of hydroxygroups.
  • the monosaccharide residues are aldose or ketose residues, such as galactose or glucose residues or galactose derived residues, such as galactopyranose or anhydrogalactopyranose.
  • anion exchangers functional group examples include:
  • the ion exchange medium is an anion exchange resin
  • the ion exchange medium may be a strong anion exchanger, such as a Quaternary ammonium (Q) based resin.
  • the ion exchange medium has been preconditioned with a fatty acid as described herein below.
  • the ion exchange medium has not been pre-conditioned with a fatty acid.
  • emulsifier and fatty acids are mixed prior to contacting the ion exchange medium.
  • an actual flow rate when performing ion exchange chromatography of in the range of 5 to 1000 cm/h, preferably in the range 5 to 500 cm/h, such as in the range of 5 to 250 cm/h, for example in the range of 10 to 100 cm/h, such as in the range of 10 to 60 cm/h, for example in the range og 15 to 40 cm/h for preparation of active complexes.
  • step one of the step gradient may be in the range of 30 to 60% of buffer, for example 35 to 55% of buffer, such as 40 to 50% of buffer, for example around 45% of buffer.
  • step two of the step gradient may be in the range of 61 to P1643PC00
  • step three of the step gradient may be at least 81 % of buffer, for example at least 85% of buffer, such as at least 90% of buffer, for example at least 95% of buffer, such as at least 99% of buffer, for example around 100% of buffer.
  • Said afore-mentioned buffer may be any buffer suitable for eluting polypeptides from an ion exchange medium.
  • said buffer may comprise:
  • the column may for example be loaded with more than 1 ,5 mg polyepeptide/cm 2 ion exchange medium, such as at least 5 mg/cm 2 , for example more than 10 mg/cm 2 , such as at least 15 mg/cm 2 , for example more than 20 mg/cm 2 , such as with more than 22 mg/cm 2 , for example at least 25 mg/cm 2 , for example in the range of 23 to 27 mg, for example with more than 20 mg polypeptide/cm 2 ion exchange medium, such as at least 30 mg/cm 2 , for example more than 40 mg/cm 2 , such as at least 50 mg/cm 2 , for example more than 60 mg/cm 2 , such as at least 70 mg/cm 2 , for example more than 80 mg/cm 2 , such as at least 90 mg polypeptide/cm 2 ion exchange medium.
  • more than 1 ,5 mg polyepeptide/cm 2 ion exchange medium such as at least 5 mg/c
  • the complexes according to the invention may however also be prepared by methods not involving ion exchange chromatography.
  • the complexes according to the present invention may be prepared by a method comprising the steps of: P1643PC00
  • the emulsifier and the fatty acid may be mixed in any suitable ratio, however preferably the fatty acid is added in molar excess over emulsifier, such as polypeptide.
  • Molar excess means that there are more moles of a fatty acid than there is of emulsifier.
  • the molar excess of a fatty acid over emulsifier added during preparation may be that for one mole emulsifier in the range of 1 .5 moles and 50 moles of a fatty acid are added, such as for one mole emulsifier in the range of 2 moles and 45 moles of a fatty acid are added, for example in the range of 3 moles and 40 moles of a fatty acid are added.
  • the concentration of fatty acid, such as oleic acid is in the range of 0.01 mM to 200 mM, such as in the range of 0.05 mM to 100 mM, for example in the range of 0.1 mM to 50 mM, such as in the range of 0.2 mM to 25 mM, for example in the range of 0.5 mM to 10 mM, such as in the range of 1 mM to 5mM, such as around 2 mM.
  • the method furthermore comprises a step of adding an alcohol.
  • said alcohol is added to the fatty acid, more preferably said alcohol is added to said fatty acid.
  • the method may for example comprise the steps of
  • the alcohol may be any alcohol, preferably, the alcohol has the general formulae: P1643PC00
  • R-OH wherein R is alkyl or alkenyl, even more preferably C 2 - 2 o alkyl or alkenyl; yet more preferably alkyl, even more preferably C 2 - 2 o alkyl, for example C 2 . 10 alkyl, such as C 2 - 5 alkyl, for example ethanol.
  • the alcohol is added in excess of the fatty acid.
  • ratio on a volume basis between alcohol and fatty acid is at least 2:1 , preferably at least 5:1 , for example at least 10:1 , such as in the range of 2 -100:1 , for example in the range of 5-100:1 , such as in the range of 5 -50:1 , for example in the range of 5-20:1 , such as in the range of 10 -100:1 , for example in the range of 10-50:1 , such as in the range of 10-20:1 , for example in the range of 10- 15:1 .
  • the fatty acid is added in molar excess over emulsifier, such as polypeptide.
  • the molar excess of a fatty acid over emulsifier added during preparation may be that for one mole emulsifier in the range of 1 .5 moles to 50 moles of a fatty acid are added, such as for one mole emulsifier in the range of 2 moles to 45 moles of a fatty acid are added, for example in the range of 3 moles to 40 moles of a fatty acid are added, such as for one mole emulsifier in the range of 5 moles to 30 moles of a fatty acid are added, for example in the range of 10 moles to 20 moles of a fatty acid are added.
  • the alcohol/fatty acid mixture is added to emulsifier in aqueous solution so that for each 20 ⁇ l of pure fatty acid in the range of 1 to 100 ml, preferably in the range of 5 to 50 ml, more preferably in the range of 10 to 30, even more preferably in the range of 15 to 25 ml, such as around 20 ml, for example 20 ml emulsifier in aqueous solution is added.
  • concentration of the emulsifier in the aqueous solution is adjusted in order to obtain the above-mentioned molar ratio between fatty acid and emulsifier.
  • the concentration of the emulsifier in the aqueous solution is in general in the range of 0.1 to 50 mg/ml, more preferably in the range of 0.1 to 25 mg/ml, yet more preferably in the range of 0.1 to 20 mg/ml, for example in the range of 0.1 to 15 mg/ml, such as in the range of 1 to 50 mg/ml, for example in the range of 1 to 25 mg/ml, such as in the range of 1 to 20 mg/ml, for example in the range of 1 to 15 mg/ml, such as in the range of 5 to 50 mg/ml, for example in the range of 5 to 25 mg/ml, such as in the range of 5 to 20 mg/ml, for example in the range of 5 to 15 mg/ml, such as in the range of 10 to 50 mg/ml, for example in the range of 10 to 25 mg/ml, such as in the range of 10 to 20 mg/ml, for example in the range of 10 to 15 mg/ml.
  • Mixing the emulsifier and the fatty acid using high shear mixing may be performed using any suitable high shear mixing method known to the skilled person.
  • Examples includes high speed mixing in a stirred tank, preferably with baffles, by vigorous stirring with an impeller capable of producing turbulent flow, such as one or more rushton turbines, mixing by use of a motionless mixer, containing a number of mixing elements successively dividing and combining the fluid stream, mixing in a tank by jet mixing with high flow, mixing using a propeller-like mixer, vortex mixing or subjecting the liquid to a quick flow through a narrow opening or a capillary.
  • high shear may be determined using a variety of different parameters.
  • "high shear mixing” is mixing with a shear at least equivalent to the shear obtained by vortex mixing using an IKA MS2 Minishaker (IKA Works Inc., Wilmington, NC 28405, USA) vortex mixer with at least 1000 rpm, preferably at least 1500 rpm, even more preferably at least 2000 rpm, more preferably at least 2200 rpm, yet more preferably at least 2500 rpm, such as in the range of 1000 to 2500 rpm, for example in the range of 1000 to 2500 rpm, such as in the range of
  • 1000 to 2500 rpm for example in the range of 2000 to 2500 rpm, such as 2500 rpm in the range of 1000 to 10,000 rpm, for example in the range of 1000 to 5000 rpm for at least 5 sec, preferably at least 10 sec, such as in the range of 5 sec. to 1 hour, for example in the range of 5 sec. to 10 min, such as in the range of 5 sec. to 1 min, for example in the range of 5 sec. to 30 sec, such as in the range of 5 sec. to 20 sec, for P1643PC00
  • "high shear mixing” is vortex mixing using a vortex mixer at at least 1000 rpm, preferably at least 1500 rpm, even more preferably at least 2000 rpm, more preferably at least 2200 rpm, yet more preferably at least 2500 rpm, such as in the range of 1000 to 10,000 rpm, for example in the range of 1000 to 5000 rpm, such as in the range of 1000 to 3000 rpm, for example in the range of 2000 to 10,000 rpm, such as in the range of 2000 to 5000 rpm, for example in the range of 2000 to 3000 rpm, for example in the range of 2000 to 3000, such as in the range of 2200 to 2500, such as in the range of 2500 to 10,000 rpm, for example in the range of 2500 to 5000 rpm, such as in the range of 2500 to 3000 rpm, for example in the range of 2400 to 2600 rpm, such as 2500 rpm in the range of 1000 to 10,000 rpm, for example in the range
  • Vortex mixing is sometimes also referred to as whirley mixing and vortex mixers may also be referred to as whirley mixers. Any suitable vortex mixer may be used with the methods of the invention for example an IKA MS2 Minishaker (IKA Works Inc., Wilmington, NC 28405, USA).
  • the "high shear mixing" may be performed by subjecting the liquid to a quick flow through a capillary.
  • said quick flow has a laminar flow rate of at least 1 m/s, preferably at least 5 m/s, such as at least 10 m/s, for example in the range of 1 to 50 m/s, such as in the range of 5 to 25 m/s, for example in the range pf 5 to 15 m/s, such as around 10 m/s.
  • the inner diameter of the capillary is preferably at the most 500 ⁇ m, preferably at the most 250 ⁇ m, more preferably at the most 180 ⁇ m, such as in the range of 50 to 250 ⁇ m, for example in the range of 100 to 200 ⁇ m, such as in the range of 150 to 180 ⁇ m. Examples of methods for subjecting a liquid to a P1643PC00
  • high shear mixing may be performed using an impeller mixer in which the mixture is centrifuged against the walls of the mixer chamber.
  • the speed of the mixer will depend upon the size and capacity of the mixer.
  • the mixing may for example be carried out at a speed of at least 5 ms ⁇ 1 , such as between 30 and 80 ms ⁇ 1 .
  • the high shear mixing may be performed using a propeller-like mixer.
  • the mixing efficiency may be approximated by the turnover rate, where the turnover rate is the stir rate (rev/sec.) times the turnover volume (ml/rev)) divided by the aqueous volume measured in turnovers/sec. It is preferred that the mixing efficiency be greater than about 0.10 turnovers/sec, and preferably greater than 0.5 turnovers/sec and most preferably greater than 1 turnover/sec.
  • the high shear mixing may be achieved using a pump comprising a stator and a rotor.
  • the stator and the rotor define a gap width of about 0.01 to about 1 millimeter. Within this range, the gap width may preferably be at least about 0.05 millimeter, more preferably at least about 0.10 millimeter.
  • the gap width may preferably be up to about 0.5 millimeter, more preferably up to about 0.25 millimeter.
  • the rotor has a circumferential linear velocity of about 1 to about 100 meters per second. Within this range, the velocity may preferably be at least about 5 meters per second, more preferably at least about 10 meters per second. Also within this range, the velocity may preferably be up to about 60 meters per second, more preferably up to about 40 meters per second. Apparatus suitable in this particular embodiment is described, for example, in European Patent No. 135,697 B1 to Schreiber or commercially available as, for example, the Siefer Trigonal SM 180 centrifugal pump from Wilhelm Siefer GmbH & Co., Velbert, Germany. P1643PC00
  • the "high shear mixing" is mixing at a shear rate of at least 10,000 sec '1 , preferably at least 20,000 sec '1 , such as at least 50,000 sec "1 , for example at least 100,000 sec '1 , such as in the range of 10 4 to 10 8 sec "1 , for example in the range of 10 4 to 10 7 sec '1 , such as in the range of 10 5 to 10 8 sec "1 , for example in the range of 10 5 to 10 7 sec "1 .
  • shear rate during a given mixing procedure may be difficult to determine and also the shear rate may not be completely homogenous throughout a sample.
  • shear rates indicate an approximate average shear rate during mixing.
  • Q is the laminar flow rate and R is the inner radius of the capillary. Details regarding methods for determining the shear rate of laminar flow through a capillary may for example be found in Jaspe and Hagen, 2006, Biophysical Journal, 91 :3415- 3424.
  • the conversion may optionally contain a step of centrifugation.
  • the cytotoxic complexes are contained within the aqueous phase and thus following centrifugation the aqueous phase is then preferably recovered.
  • the method comprises a step of centrifugation in order to remove excess fatty acid.
  • the centrifugation is preferably performed at no more than 20,000 x g, such as no more than 18,000 x g, for example at in the range of 100 x g to 20,000 x g, for example in the range of 150 x g to 18,000 x g for in the range of 2 to 20 min., preferably for in the range of 5 to 10 min. It is preferred that only one round of centrifugation is performed.
  • the ratio between the emulsifier (i.e. any of the emulsifiers described herein above in the sections “Emulsifier” and “Polypeptide”) and the fatty acid (i.e. any of the fatty acids described herein above in the section “Fatty acids”) in the complex may be any useful ratio.
  • the ratio between alpha-lactalbumin and oleic acid in complex may be any useful ratio.
  • the molar ratio of fatty acid to emulsifier in the complex is at least 1 :1 .
  • the ratios indicated in this section relates to the ratio in the complex and not to the ratios of the mixture before complex formation.
  • the molar ratio of fatty acid to emulsifier is at least 2:1 , preferably at least 3:1 , even more preferably at least 4:1 , yet more preferably at least 5:1 , such as at least 6:1 , for example at least 7:1 , such as in the range of 2:1 to 30:1 , for example in the range of 3:1 to 30:1 , such as in the range of 4:1 to 30:1 , for example in the range of 5:1 to 30:1 , such as in the range of 2:1 to 25:1 , for example in the range of 2:1 to 20:1 , such as in the range of 2:1 to 15:1 , for example in the range of 2:1 to 13:1 , such as in the range of 3:1 to 25:1 , for example in the range of 3:1 to 20:1 , such as in the range of 3:1 to 15:1 , for example in the range of 3:1 to 13:1 , such as in the range of 3:1 to 15:1 , for example in the range of 3:1 to 13:1
  • the molar ratio of fatty acid to emulsifier is at least 1 : 2:, more preferably at least 2:1 , for example at least 3:1 , such as at least 4:1 , for example at least 5:1 , such as at P1643PC00
  • At least 6:1 for example at least 10:1 , such as at least 1 1 :1 , for example at least 12:1 , such as at least 13:1 , for example at least 14:1 , such as at least 15:1 , for example at least 16:1 , such as at least 17:1 , for example at least 18:1 , such as at least 19:1 , for example at least 20:1 .
  • the molar ratio of fatty acid to emulsifier is approximately 1 : 2:, more preferably at least 2:1 , for example approximately 3:1 , such as approximately 4:1 , for example approximately 5:1 , such as approximately 6:1 , for example approximately 10:1 , such as approximately 1 1 :1 , for example approximately 12:1 , such as approximately 13:1 , for example approximately 14:1 , such as approximately 15:1 , for example approximately 16:1 , such as approximately 17:1 , for example approximately 18:1 , such as approximately 19:1 , for example approximately 20:1 .
  • the ratio of fatty acid to emulsifier is at least 2:1 , however it is even more preferred that the ratio of fatty acid to emulsifier is at least 4.5:1 , preferably at least 5:1 .
  • At least X:1 as used herein is meant to cover any ratio wherein X is the indicated number or a higher number. Thus, by way of example at least 2:1 , covers for example also 5:1 .
  • the ratio of fatty acid to emulsifier before mixing is higher than the ratios described above in the section "Ratio".
  • the ratio before mixing is preferably at least 1 .5, such as at least 1 .6 times, for example at least 1 .7 times, such as at least 1 .8 times, for example at least 1 .9 times, such as at least 2 times higher than the desired ratio in the complex.
  • the ratio of fatty acid to emulsifier before mixing is at least 8:1 , preferably at least 10:1 , for example at least 15:1 .
  • the complex according to the invention may be any combination of any of the emulsifiers mentioned herein above in the sections “Emulsifier” and “Polypeptide” and any of the fatty acids described herein above in the section “Fatty acid”.
  • the complexes of the present invention preferably have a cytotoxic effect on tumour cells.
  • the terms "Cytotoxic effect” and “cytotoxic activity” are used interchangeably herein.
  • the cytotoxic effect may be determined by a number of different in vivo or in vitro assays.
  • In vitro assays in general comprise contacting tumour cells cultivated in vitro with the complex and determining the rate of living cells. On that basis an LC 50 may be P1643PC00
  • the complexes of the invention has at least some cytotoxic effect using such an in vitro assay.
  • In vivo assays in general comprises contacting an animal suffering from cancer, for example a mouse suffering from a cancer, which may for example have been induced or which is a xenograft, with the complex and determining tumour cell mass.
  • the complexes of the invention are capable of reducing tumour cell mass significantly in vivo, such as a reduction to less than 80%, such as less than 50%.
  • a) cultivating tumour cells in vitro b) adding in different containers various predetermined concentrations of complex to a predetermined number of said cells c) incubating said cells with said complex for a predetermined amount of time d) determining the rate of surviving cells e) determining the concentration of complex capable of killing 50% of the cells, thereby determining the LC 50 .
  • the concentration of complex is herein given as the concentration of emulsifier unless otherwise stated.
  • the LC 50 is the concentration given as the concentration of emulsifier capable of killing 50% of cells.
  • the LC 50 of a solution comprising alpha-lactalbumin complexed with oleic acid may be given as the alpha-lactalbumin concentration in mg/mL in the solution required to kill 50% of the cells in the assay.
  • Cytotoxic activity may also be measured as LD 50 .
  • In vitro assays for cytotoxic effect determined as LD 50 in general comprises:
  • the amount of complex is also referred to as dose and is herein given as the amount of emulsifier per cell , i.e. weight of emulsifier per cell (in general measured in pg/cell).
  • the LD 50 is the dose given as the dose of emulsifier capable of killing 50% of cells.
  • the LD 50 of a solution comprising alpha-lactalbumin complexed with oleic acid may be given as the dose of alpha-lactalbumin in pg/cell in the solution required to kill 50% of the cells in the assay.
  • the concentration or dose of complex capable of killing 50 % of a given cell population is calculated and is called LC 50 and LD 50 , repsectively.
  • the rate of surviving cells may be determined using any suitable assay known to the skilled person, for example using tryphan blue staining (tryphan blue only stains dead cells, but not living cells) as described in Example 3 herein below or by determining cellular ATP levels.
  • Cellular ATP levels is a measure of cell viability. ATP level may be determined by a number of different assays, for example assays taking advantage of that luciferase may generate a luminescent signal from luceferin in the presence of ATP, for example as described in Example 3 herein below.
  • tumour cells may be any tumour cells, such as human or murine tumour cells derived from any tumour or otherwise malignant cells.
  • the cells may be directly obtained from a patient or they may be an established cell lined.
  • the cell line is L1210.
  • the predetermined number of cells may be selected individually, however in a preferred embodiment it is preferred that the concentration of cells is in the range of 0.1 to 10 x10 6 , preferably in the range of 1 to 2 x 10 6 , for example about 1 .4 x 10 6 , such as 1 .4 x 10 6 .
  • the complex may be incubated with the cells for any suitable amount of time, in general for in the range of 30 min. to 5 hours, such as in the range of 30 min to 2 hours, such as in the range of 45 min to 75 min. for example for about 1 hour.
  • the cells are kept in the absence of serum during incubation with complex.
  • the complex according to the invention has an LC 50 of at the most 50 mg/ml, preferably at the most 40 mg/ml, even more preferably at the most 30 mg/ml, yet more preferably at the most 20 mg/ml, even more preferably at the most 10 mg/ml, yet more preferably at the most 5 mg/ml, even more preferably at the most 1 mg/ml, for example at the most 0.75 mg/ml, such as at the most 0.5 mg/ml with respect to L1210 cells at a concentration of 1 .4 x 10 6 cells/mL in in vitro culture.
  • the complex according to the invention has an LC 50 of at the most 50 mg/ml, preferably at the most 40 mg/ml, even more preferably at the most 30 mg/ml, yet more preferably at the most 20 mg/ml, even more preferably at the most 10 mg/ml, yet more preferably at the most 5 mg/ml, even more preferably at the most 1 mg/ml, for example at the most 0.75 mg/ml, such as at the most 0.5 mg/ml, when determined as described in Example 3 herein below.
  • the complex according to the invention has an LD 50 of at the most 400 pg/cell, preferably at the most 300 pg/cell, more preferably at the most 200 pg/cell, even more preferably at the most 150 pg/cell, yet more preferably at the most 100 pg/cell, even more preferably at the most 90 pg/cell, yet more preferably at the most 80 pg/cell, even more preferably at the most 70 pg/cell, yet more preferably at the most 60 pg/cell, for example at the most 55 pg/cell, in particular when determined as described in Example 3 herein below.
  • the complexes of the invention may also be cytotoxic to other cells, in particular cells with aberrant proliferation, for example enhanced proliferation.
  • the complexes may also be cytotoxic to infected cells, such as P1643PC00
  • Cytotocity to such cells may be determined as described in Example 3 herein below except that the cells to be tested are used in place of L1210 cells.
  • the complexes have a low cytotoxicity or are not cytotoxic to healthy cells, such as non-malignant, non-infected cells, preferably in vivo.
  • the complexes according to the invention may be used for the manufacture of a medicament for a clinical disorder wherein selective cytotoxicity is desirable.
  • the clinical disorder may be selected from the group consisting of respiratory tract infections, cancer and warts or for the inhibition of angiogenesis.
  • the cancer is bladder cancer.
  • the warts are caused by papiloma infection.
  • the complexes may be used in the treatment of Infections of the respiratory tract, e.g., meningitis, otitis and sinusitis, which are caused by bacteria which enter via the nasopharynx.
  • Viral infections of the respiratory tract may be caused by such as adenovirus, influcenza viruses, respiratory cyncytial virus (RSV), parainfluenza, Phinoviruses and coronaviruses.
  • adenovirus influcenza viruses
  • RSV respiratory cyncytial virus
  • Phinoviruses Phinoviruses and coronaviruses.
  • composition according to the invention is for the treatment of infections of the respiratory tract.
  • the medicament according to the invention may be inhaled in the form of a mist into the upper respiratory airways.
  • tumors of both the benign or malignant type may further be treated using the complexes according to the invention.
  • a wart is generally a small, rough tumour, typically on hands and feet, that resembles a cauliflower. Warts are common, and are caused by a viral infection, specifically by the human papillomavirus (HPV). They typically disappear after a few months but can last for years and can recur.
  • HPV human papillomavirus
  • Flat wart (verruca plana): a small, smooth flattened wart, tan or flesh coloured, which can occur in large numbers; most common on the face, neck, hands, wrists and knees.
  • Filiform or digitate wart a thread- or finger-like wart, most common on the face, especially near the eyelids and lips.
  • Plantar wart (verruca, verruca pedis): a hard sometimes painful lump, often with multiple black specks in the centre; usually only found on pressure points on the soles of the feet.
  • Mosaic wart a group of tightly clustered plantar-type warts, commonly on the hands or soles of the feet.
  • Genital wart (venereal wart, condyloma acuminatum, verruca acuminata): wart affecting the genital areas.
  • the complexes according to the invention is for the treatment of warts, which is preferably treated by topical application of a medicament according to the invention.
  • Papilloma refers to a benign epithelial tumor, which may or may not be caused by Human papillomavirus. Alternative causes are such as Choroid plexus papilloma (CPP).
  • CPP Choroid plexus papilloma
  • the LAC composition according to the invention is for the treatment of papillomas, which is preferably treated by topical application of a medicament comprising the complexes according to the invention.
  • Cancerous diseases are scientifically designated neoplasia or neoplasms and may be benign or malignant. Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. The following general categories are applied:
  • Carcinoma malignant tumors derived from epithelial cells. This group includes the most common cancers, comprising the common forms of breast, prostate, lung and colon cancer.
  • Lymphoma and Leukemia malignant tumors derived from blood and bone marrow cells.
  • Sarcoma malignant tumors derived from connective tissue, or mesenchymal cells
  • Mesothelioma tumors derived from the mesothelial cells lining the peritoneum and the pleura.
  • Glioma tumors derived from glia, the most common type of brain cell
  • Germinoma tumors derived from germ cells, normally found in the testicle and ovary.
  • Choriocarcinoma malignant tumors derived from the placenta.
  • the complexes according to the invention is for the treatment of cancer.
  • Medicaments, as defined herein below, for treatment of cancer are according to the invention preferably applied directly to the tumour.
  • Mucosal tumors The conditions found at mucosal surfaces can be quite unique in terms of properties such as pH. and the like. Mucosal surfaces are found inter alia in the nasal passages, in the mouth, throat, oesophagus, lung, stomach, colon, vagina and bladder.
  • Particular mucosal surfaces that may be treated with in accordance with the invention include throat, lung, colon and bladder surfaces which tumours.
  • Bladder cancer refers to any of several types of malignant growths of the urinary bladder.
  • the most common type of bladder cancer begins in cells lining the inside of the bladder and is called urothelial cell or transitional cell carcinoma (UCC or TCC).
  • UCC urothelial cell or transitional cell carcinoma
  • the complexes according to the invention is for the treatment of bladder cancer.
  • a glioma is a type of primary central nervous system (CNS) tumor that arises from glial cells.
  • CNS central nervous system
  • the most common site of involvement of a glioma is the brain, but they can also affect the spinal cord, or any other part of the CNS, such as the optic nerves.
  • the complexes according to the invention is for the treatment of glioma/glioblastome.
  • Tumour angiogenesis is the proliferation of a network of blood vessels that penetrates in to cancerous growths, supplying nutrients and oxygen and removing waste products. The process of angiogenesis is initiated when tumor cells release molecules signalling to the normal host tissue, activating genes and proteins to encourage growth of new blood vessels. A series of natural inhibitors of angiogenesis have been identified, and are believed to prevent and/or inhibit the growth and spread of cancer cells.
  • the complexes of the invention may also be used for inhibiting angiogenesis for example in treatment and/or inhibition of cancer.
  • Actinic keratosis is a UV light-induced lesion of the skin that may progress to invasive squamous cell carcinoma. Actinic keratosis is a scaly bump that forms on the skin surface ranging from barely perceptible rough spots of skin to elevated, hyperkeratotic plaques several centimeters in diameter. Most often, they appear as multiple discrete, flat or elevated, keratotic lesions. Lesions typically have an erythematous base covered by scale (hyperkeratosis). They are usually 3-10 mm in P1643PC00
  • Actinic keratosis is also referred to as solar keratosis, sun spots, or precancerous spots. .With time, actinic keratoses may develop into invasive squamous cell carcinoma, and Actinic keratosis is thus a precursor of skin cancer. The most aggressive form of keratosis, actinic cheilitis, appears on the lips and can evolve into squamous cell carcinoma.
  • the complexes of the present invention may also be used in the treatment of actinic keratosis.
  • the complexes are preferably formulated for topical application directly to the diseased site.
  • the present invention provides pharmaceutical compositions comprising the complexes of emulsifier and fatty acid described herein.
  • the present invention relates to a pharmaceutical composition.
  • the pharmaceutical composition may be formulated in a number of different manners, depending on the purpose for the particular pharmaceutical composition.
  • composition may be formulated in a manner so it is useful for a particular administration form.
  • Preferred administration forms are described herein below.
  • the pharmaceutical composition is formulated so it is a liquid.
  • the composition may be a solution or a suspension comprising the complexes.
  • Said liquid may be suitable for parenteral administration, for example for injection or infusion.
  • the liquid may be any useful liquid, however it is frequently preferred that the liquid is an aqueous liquid.
  • the liquid is sterile. Sterility may be conferred by any conventional method, for example filtration, irradiation or heating.
  • the liquid has been subjected to a virus reduction step, in particular if the liquid is formulated for parenteral administration.
  • Virus reduction may for example be performed by nanofiltration or virus filtering over a suitable filter, such as a Planova filter consisting of several layers.
  • the Planova filter may be any suitable size for example 75N, 35N, 2ON or 15N or filters of different size may be used, for example Planova 2ON.
  • Virus reduction may also comprise a step of prefiltering with another filter, for example using a filter with a pore size of the the range of 0.01 to 1 ⁇ m, such as in the range of 0.05 to 0.5 ⁇ m, for example around 0.1 ⁇ m.
  • Virus reductions may also include an acidic treatment step.
  • compositions for bolus injections may be packages in dosage units of for example at the most 10 ml, preferably at the most 8 ml, more preferably at the most 6 ml, such as at the most 5 ml, for example at the most 4 ml, such as at the most 3 ml, for example around 2.2 ml.
  • the pharmaceutical composition may be packaged in any suitable container.
  • a single dosage of the pharmaceutical composition may be packaged in injection syringes or in a container useful for infusion.
  • the pharmaceutical composition is a dry composition.
  • the dry composition may be used as such, but for most purposes the composition is a dry composition for storage only. Prior to use the dry composition may be dissolved or suspended in a suitable liquid composition, for example sterile water.
  • the pharmaceutical composition may be applied topically to the site of the site, for example in the form of a lotion, a creme, an ointment, a spray, such as an aerosol spray or a nasal spray, rectal or vaginal suppositories, drops, such as eye drops or nasal drops, a patch, an occlusive dressing or the like.
  • compositions containing the complexes of the invention may be prepared by any conventional technique, e.g. as described in Remington: The Science and Practice of Pharmacy 1995, edited by E. W. Martin, Mack Publishing Company, 19th edition, Easton, Pa. P1643PC00
  • the pharmaceutically acceptable additives may be any conventionally used pharmaceutically acceptable additive, which should be selected according to the specific formulation, intended administration route etc.
  • the pharmaceutically acceptable additives may be any of the additives mentioned in Nema et al, 1997.
  • the pharmaceutically acceptable additive may be any accepted additive from FDA ' s "inactive ingredients list", which for example is available on the internet address http://www.fda.gov/cder/drug/iig/default.htm.
  • the pharmaceutical composition comprises an isotonic agent.
  • an isotonic agent is added.
  • composition may comprise at least one pharmaceutically acceptable additive which is an isotonic agent.
  • the pharmaceutical composition may be isotonic, hypotonic or hypertonic. However it is often preferred that a pharmaceutical composition for infusion or injection is essentially isotonic, when it is administrated. Hence, for storage the pharmaceutical composition may preferably be isotonic or hypertonic. If the pharmaceutical composition is hypertonic for storage, it may be diluted to become an isotonic solution prior to administration.
  • the isotonic agent may be an ionic isotonic agent such as a salt or a non-ionic isotonic agent such as a carbohydrate.
  • ionic isotonic agents include but are not limited to NaCI, CaCI 2 , KCI and MgCI 2
  • non-ionic isotonic agents include but are not limited to mannitol and glycerol.
  • At least one pharmaceutically acceptable additive is a buffer.
  • a buffer for some purposes, for example, when the P1643PC00
  • composition for infusion or injection, it is often desirable that the composition comprises a buffer, which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
  • a buffer which is capable of buffering a solution to a pH in the range of 4 to 10, such as 5 to 9, for example 6 to 8.
  • the pharmaceutical composition may comprise no buffer at all or only micromolar amounts of buffer.
  • the buffer may for example be selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • the buffer is TRIS.
  • TRIS buffer is known under various other names for example tromethamine including tromethamine USP, THAM, Trizma, Trisamine, Tris amino and trometamol.
  • the designation TRIS covers all the aforementioned designations.
  • the buffer may furthermore for example be selected from USP compatible buffers for parenteral use, in particular, when the pharmaceutical formulation is for parenteral use.
  • the buffer may be selected from the group consisting of monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic, dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric, polybasic acids such as citric and phosphoric and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.
  • monobasic acids such as acetic, benzoic, gluconic, glyceric and lactic
  • dibasic acids such as aconitic, adipic, ascorbic, carbonic, glutamic, malic, succinic and tartaric
  • polybasic acids such as citric and phosphoric and bases such as ammonia, diethanolamine, glycine, triethanol
  • the pharmaceutical compositions may comprise at least one pharmaceutically acceptable additive which is a stabiliser.
  • the stabiliser may for example be a detergent, a polymer, a polyhydric alcohol, a metal ion, a reducing agent, a chelating agent, a sugar or a protein, however any other suitable stabiliser may also be used with the present invention.
  • the stabiliser may be selected from the group consisting of poloxamers, Tween-20, Tween-40, Tween-60, Tween-80, Brij, metal ions, amino acids, polyethylene glycol, Triton, EDTA, ascorbic acid, Triton X-100, NP40 or CHAPS.
  • P1643PC00 poloxamers, Tween-20, Tween-40, Tween-60, Tween-80, Brij, metal ions, amino acids, polyethylene glycol, Triton, EDTA, ascorbic acid, Triton X-100, NP40 or CHAPS.
  • the pharmaceutical composition according to the invention may also comprise one or more cryoprotectant agents.
  • the composition comprises freeze-dried protein or the composition should be stored frozen it may be desirable to add a cryoprotecting agent to the pharmaceutical composition.
  • the cryoprotectant agent may be any useful cryoprotectant agent, for example the cryoprotectant agent may be selected from the group consisting of dextran, glycerin, polyethylenglycol, sucrose, trehalose and mannitol.
  • the pharmaceutically acceptable additives may comprise one or more selected from the group consisting of isotonic salt, hypertonic salt, buffer and stabilisers. Furthermore, the pharmaceutically acceptable additives may comprise one or more selected from the group consisting of isotonic agents, buffer, stabilisers and cryoprotectant agents.
  • the pharmaceutically acceptable additives comprise glucosemonohydrate, glycine, NaCI and polyethyleneglycol 3350.
  • the pharmaceutical composition may be prepared so it is suitable for one or more particular administration methods. Furthermore, the method of treatment described herein may involve different administration methods.
  • any administration method wherein the complexes may be administered to an individual in a manner so that active complex may reach the site of disease may be employed with the present invention.
  • compositions of the invention may be administered parenterally, that is by intravenous, intramuscular, subcutaneous intranasal, intrarectal, intravaginal or intraperitoneal administration.
  • the pharmaceutical composition should be a sterile liquid, which preferably also has been subjected to a virus reduction step.
  • Injection may be injection to any preferred site, for example injection may be selected from the group consisting of intravenous, subcutaneous, intra-arterial, intra-muscular P1643PC00
  • Infusion is generally intra-venous infusion. Injection may also be directly to site of the disease. This may in particular be applicable when treating a cancer which is a solid tumour.
  • the route of administration may be topical administration to for example a mucosal membrane or to the skin.
  • the mucosal membrane to which the pharmaceutical preparation of the invention is administered may be any mucosal membrane of the mammal to which the biologically active substance is to be given, e.g. in the nose, vagina, eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum.
  • Topical administration to the skin may for example be in the form of a lotion, cream, ointment, drops, transdermal patch or the like.
  • compositions according to the present invention may be administered once or more than once, for example they may be administered in the range of 2 to 5 times, such as 5 to 10 times, for example 10 to 20 times, such as 20 to 50 times, for example 50 to 100 times, such as more than 100 times.
  • the dosage of complex to be administered depends on the individual to be treated as well as on the clinical condition and the mode of administration. In general, in the range of 0.5 ⁇ g to 50 mg, such as in the range of 1 ⁇ g to 20 mg, for example in the range 1 ⁇ g to 2 mg, such as in the range of 1 ⁇ g to 100 ⁇ g may be administered per administration to a given individual, such as a human being. The amount indicated is in respect to emulsifier.
  • the individual to be treated may be any individual in need thereof, preferably a human being.
  • the treatment may be prophylactic, ameliorating or curative, preferably ameliorating or curative, more preferably at least ameliorating.
  • the invention relates to a method of preparing a stable, pharmaceutical composition
  • a method of preparing a stable, pharmaceutical composition comprising a complex between an emulsifier (e.g. alpha- P1643PC00
  • said method comprising the steps of a. providing an emulsifier in an aqueous solution, wherein said emulsifier is not a fatty acid; and b. providing a fatty acid; and c. contacting the emulsifier with the fatty acid; and d. forming a complex between the emulsifier and the fatty acid; e. thereby obtaining a solution comprising a complex between said emulsifier and said fatty acid, wherein said solution has a starting cytotoxic activity with a starting LD 50 in respect of dose of emulsifier; and f.
  • a pharmaceutical composition comprising a complex between an emulsifier and a fatty acid, said composition retaining cytotoxic activity after storage, wherein the 2 weeks storage LD 50 in respect of dose of emulsifier after storage for 2 weeks at 25 0 C is at the most 3 times higher than the starting LD 50 .
  • the method may in addition comprise addition of a chelator, which for example may be added to the aqueous solution step a), to the solution in step e) or to the liquid solution in step f).
  • the chelator may be any compound capable of binding to divalent cations through more than one coordination site.
  • the chelator may for example be selected from the group consisting of EDTA, EGTA, BAPTA, porphyrins (such as heme, porphine or porphine substitued with one or more entities) and citrate, preferably from the group consisting of EDTA and citrate.
  • the starting LD 50 and the storage LD 50 are both determined in respect of dose of emulsifier (e.g. alpha-lactalbumin).
  • emulsifier e.g. alpha-lactalbumin
  • the initial cytotoxic complex may have disintegrated and accordingly some emulsifier may be present as part of a complex with fatty acid, whereas some emulsifier may not be in complex with fatty acid.
  • Emulsifier not in complex with fatty acid preferably does not have cytotoxic acitivty (see herein above in the section "Emulsifier") and accordingly, the LD 50 in respect of dose of emulsifier will be higher, if some of the emulsifier is present in an inactive not complexed form. This is certainly the case, when the emulsifier is alpha-lactalbumin.
  • the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 2 weeks storage at 25 e C is preferably at the most 3 times, for example at the most 2.7 times, more preferably at the most 2.5 times, for example at the most 2.2 times, yet more preferably at the most 2 times, yet more preferably at the most 1 .9 times, even more preferably at the most 1 .8 times, yet more preferably at the most 1 .7 times, even more preferably at the most 1 .6 times, even more preferably at the most 1 .5, yet more preferably at the most 1.4 times, for example at the most 1.3 times, such as at the most 1 .2 times higher than the starting LD 50 .
  • emulsifier e.g. alpha-lactalbumin or a functional homologue thereof
  • the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 3 weeks storage at 25 e C is preferably at the most 3 times, for example at the most 2.7 times, more preferably at the most 2.5 times, for example at the most 2.2 times, yet more preferably at the most 2 times, yet more preferably at the most 1 .9 times, even more preferably at the most 1 .8 times, yet more preferably at the most 1 .7 times, even more preferably at the most 1 .6 times, even more preferably at the most 1 .5, yet more preferably at the most 1.4 times, for example at the most 1.3 times, such as at the most 1 .2 times higher than the starting LD 50 .
  • emulsifier e.g. alpha-lactalbumin or a functional homologue thereof
  • the LD 50 in respect of dose of emulsifier (e.g. alpha- lactalbumin or a functional homologue thereof) after 4 weeks storage at 25 e C is preferably at the most 4 times, more preferably at the most 3.5 times, yet more preferably at the most 3 times, yet more preferably at the most 2.8 times, even more preferably at the most 2.6 times, yet more preferably at the most 2.4 times, even more preferably at the most 2.3 times, even more preferably at the most 2.2, for example at the most 2 times higher than the starting LD 50 .
  • emulsifier e.g. alpha- lactalbumin or a functional homologue thereof
  • the emulsifier is alpha-lactalbumin or a functional homologue thereof - that the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 2 weeks storage at 25 e C preferably is at the most 400 pg/cell, preferably at the most 300 pg/cell, more preferably at the most 200 pg/cell, even more preferably at the P1643PC00
  • most 150 pg/cell yet more preferably at the most 100 pg/cell, even more preferably at the most 90 pg/cell, yet more preferably at the most 80 pg/cell, even more preferably at the most 70 pg/cell, such as at the most 60 pg/cell, for example at the most 55 pg/cell, in particular when determined as described in Example 3 herein below.
  • the emulsifier is alpha-lactalbumin or a functional homologue thereof - that the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 2 weeks storage at 25 e C preferably is at the most 100 pg/cell, preferably at the most 80 pg/cell when determined as described in Example 3 herein below.
  • the emulsifier is alpha-lactalbumin or a functional homologue thereof - that the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 3 weeks storage at 25 e C preferably is at the most 400 pg/cell, preferably at the most 300 pg/cell, more preferably at the most 200 pg/cell, even more preferably at the most 150 pg/cell, yet more preferably at the most 100 pg/cell, even more preferably at the most 90 pg/cell, yet more preferably at the most 80 pg/cell, even more preferably at the most 70 pg/cell, such as at the most 60 pg/cell, for example at the most 55 pg/cell, in particular when determined as described in Example 3 herein below.
  • the emulsifier is alpha-lactalbumin or a functional homologue thereof - that the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 4 weeks storage at 25 e C preferably is at the most 400 pg/cell, preferably at the most 300 pg/cell, more preferably at the most 200 pg/cell, even more preferably at the most 150 pg/cell, yet more preferably at the most 100 pg/cell, particularly when determined as described in Example 3 herein below.
  • emulsifier e.g. alpha-lactalbumin or a functional homologue thereof
  • the emulsifier is alpha-lactalbumin or a functional homologue thereof - that the LD 50 in respect of dose of emulsifier (e.g. alpha-lactalbumin or a functional homologue thereof) after 2 weeks storage at 25 e C preferably is at the most 100 pg/cell, when determined as described in Example 3 herein below.
  • the complexes of the stable pharmaceutical compositions retain the molar ratio between fatty acid and emulsifier.
  • the starting ratio of fatty acid to emulsifier e.g. alpha-lactalbumin or a functional homologue thereof
  • the ratio after 2 weeks storage at 25 e C preferably is at least 0.4xY:1 , preferably at least 0.5xY:1 , even more preferably at least 0.6xY:1 , yet more preferably at least 0.7xY:1 , even more preferably at least 0.8xY:1 .
  • One preferred method of preparing stable pharmaceutical compositions according to the invention involves a step of formulating said complex in a liquid solution having a pH of at least 6.5.
  • the pH of the final pharmaceutical composition should be at least 6.5.
  • the pH may be adjusted to at least 6.5 at any step and it can thus be done before or after mixing emulsifier and fatty acid. If the pH is adjusted at an early step it is important that it is kept at the high pH of at least 6.5 or it must be adjusted again at the end of the procedure.
  • the pH is at least 6.5, more preferably at least 7, such as at least 7.1 , for example at least 7.2, such as at least 7.3, for example at least 7.4, such as at least 7.5, for example at least 7.6, such as at least 7.7, for example at least 7.8, such as at least 7.9, for example at least 8, preferably at least 8.1 , for example at least 8.2.
  • the pH is not too high and thus preferably the pH is in the range of 6.5 to 1 1 , such as in the range of 6.5 to 10.5, for example in the range of 6.5 to 10, such as in the range of 6.5 to 9.5, for example in the range of 6.5 to 9, such as in the range of 7 to 1 1 , such as in the range of 7 to 10.5, for example in the range of 7 to 10, such as in the range of 7 to 9.5, for example in the range of 7 to 9, such as in the range of 7.5 to 1 1 , such as in the range of 7.5 to 10.5, for example in the range of 7.5 to 10, such as in the range of 7.5 to 9.5, for example in the range of 7.5 to 9, such as in the range of 8 to 1 1 , such as in the range of 8 to 10.5, for example in the range of 8 to 10, such as in the range of 8 to 9.5, for example in the range of 8 to 9, such as in the range of 7.1 to 7.9, for example in the range of 8.1 to 8.9, such
  • the pH is any of the aforementioned with the proviso that the pH is not 8, 9 or 10.
  • the pH is adjusted with the aid of a buffer, which for example may be added to the liquid solution in step f) of above-mentioned method, to the solution in step e) of the method above or to the aqueous solution comprising the emulsifier in step a) of the method above.
  • a buffer for example may be added to the liquid solution in step f) of above-mentioned method, to the solution in step e) of the method above or to the aqueous solution comprising the emulsifier in step a) of the method above.
  • the buffer may be any buffer suitable of buffering to the desired pH such as a buffer selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • a buffer selected from the group consisting of TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycinate, histidine, glycine, succinate and triethanolamine buffer.
  • the buffer is TRIS.
  • TRIS buffer is known under various other names for example tromethamine including tromethamine USP, THAM, Trizma, Trisamine, Tris amino and trometamol.
  • the designation TRIS covers all the aforementioned designations.
  • Larodan AB (Malm ⁇ , Sweden, cat.no. 10-1801 ) is mixed with 250 ⁇ l ethanol.
  • the mixture is added to 20 ml protein solution, wherein the protein concentration in the solution is adjusted to obtain a molar ratio of protein to oleic acid of 1 :15.
  • the protein concentration may also be adjusted to obtain another molar ratio.
  • the molar ratio above is given in respect of the individual polypeptides and not in respect of the number of oligomers.
  • the protein/oleic acid/ethanol mixture is subjected to high shear mixing by vigorous vortex mixing at 2500 rpm for 10 s.
  • 250 ⁇ l 99% pure oleic acid (obtainable from Sigma-Aldrich, St. Louis, USA; cat. No. 01008) or Larodan AB (Malm ⁇ , Sweden. cat.no. 10-1801 ) is mixed with 2500 ⁇ l protein solution (concentration typically in the range of 10-15 mg/mL). The mixture is subjected to high shear mixing by vigorous vortex mixing at 2500 rpm for 20 sec.
  • the mixture is incubated for 10 min. at room temperature and is then centrifuged in the range of 150 x g to 18,000 x g.
  • the aqueous phase is retrieved and ready to use for example in the cell killing assay as described herein below in Example 3.
  • L1210 cells are in general kept in RPMI 1640 medium with 1 % sodium pyruvate and 1 % non-essential amino acids supplemented with 5% fetal calf serum at 37 e C and 5% CO 2 .
  • cells from day 2 after last sub-culturing having a viability of at least 90% as determined by tryphan blue exclusion were gently spun down (10 minutes at 200 x g) and washed in PBS buffer followed by another spin (10 minutes at 200 x g). The cells were then re-suspended in growth medium without serum at a cell concentration of 2-10 6 /ml_, which was verified by a cell count.
  • a 2-fold dilution series (from 2x and up to to 2128x) of a sample prepared as described for example in Example 1 or Example 2 is made in 0.9% NaCI. 20 ⁇ l of each dilution is added to a well of a 96 well plate. In general a control is also made comprising only 0.9% NaCI.
  • the cell viability is assayed using the ViaLight Plus kit from Lonza (formerly Cambrex Bioscience), cat. No. LT07-221 or LT07-121 according to manufacturers instructions.
  • This kit contains lyophilized AMR (ATP Monitoring Reagent) PLUS, cell lysis reagent and assay buffer.
  • AMR PLUs reagent 100 ⁇ l AMR PLUs reagent is added to each well and the plate is incubated for 5 minutes, after which lumiscense is determined by reading the plate in a luminometer (e.g. BMG Lumistar Optima) with an integrated reading time of 1 second.
  • a luminometer e.g. BMG Lumistar Optima
  • Another method is use of the Cytotoxicity Bioassay kit from Lonza and a BMG Lumistar Optima luminometer as described in the Vialight Plus manual.
  • Another method for determining cell viability is by tryphan blue staining.
  • the results obtained with the ViaLight Plus kit correlates with the results of Tryphan blue staining (see figure 1 ) it is generally not required to determine cell viability using both test.
  • Tryphan blue staining may be performed as follows:
  • the viable cells will be slightly opalescent, round and pale with a darker outline.
  • the nonviable cells will be dark blue.
  • X 0 corresponds to the LD 50 expressed as ⁇ L/well.
  • sample concentration LD 50 can be expressed as pg/cell and the LC 50 can be calculated. In cases where the dilution series did not cover the cell killing curve fully, Y 0 was forced to zero in the fit.
  • Constant in the assay' and in LC 50 is defined as the concentration of the protein (e.g. bl_A) which the L1210 cells encounter during the first one hour of incubation (before addition of medium with serum).
  • bovine alpha-lactalbumin and oleic acid bLAC
  • human alpha-lactalbumin and oleic acid hLAC
  • Figure 1 shows the results for bLAC and also demonstrates the comparability of the tryphan blue method and the ViaLight method.
  • the LC 50 of bLAC was in the range of 0.02 to 0.5 mg/ml, whereas the LC50 for hLAC was in the range of 0.05 to 0.1 1 mg/ml.
  • HAS human serum albumin
  • a control comprising 4 mg/ml HSA in the absence of oleic acid.
  • a control sample comprising 0.9% NaCI saturated with oleic acid.
  • the LC 50 for HSA/oleic acid complexes prepared as described in Example 2 was 0.15 mg/ml for samples centrifuged at 150 x g for 5 min and 0.38 mg/ml for samples centrifuged at 3000 x g for 10 min.
  • Figure 4 shows that centrifugation at 3000 x g for 10 min yielded more active complex than centrifugation at first 3000 x g for 10 min followed by 6 min at 18,000 x g.
  • Bovine alpha-lactalbumin (bl_A) was purified from bovine milk as described in Example 1 of Danish Patent application PA 2006 01512 and in Example 1 of PCT application WO2008/058547.
  • bovine alpha-lactalbumin (bl_A) and oleic acid were prepared as described in Example 2.
  • the centrifugation was performed at 150 x g for 5 min, at 3000 x g for 10 min or at 18,000 x g for 10 min.
  • the samples thus obtained were used in a cell killing assay as described in Example 3.
  • the LC 50 was 0.06 mg/ml for samples centrifuged at 150 x g, 0.24 mg/ml for samples centrifuged at 3000 x g and 0.06 mg/ml for samples centrifuged at 18,000 x g.
  • Bovine beta-Lactoglobulin (bl_G) was obtained from Sigma (L3908). In general beta- Lactoglobulin was dissolved in 0.9% NaCI at a concentration of 10 mg/ml.
  • Example 7 the LC 50 for bLG/oleic acid complexes prepared as described in Example 2 was 0.06 mg/ml for samples centrifuged at 3000 x g for 10 min and 0.12 mg/ml for samples centrifuged at 18,000 x g for 10 min.
  • Example 3 and figure 8 displays the results. As is apparent the controls only had very limited impact on viability, whereas the complex of bl_G and oleic acid killed almost all cells.
  • rhMBL Recombinant human mannose binding lectin
  • rhMBL is generally used at a concentration of 10 mg/ml in 10 mM Tris and 140 mM NaCI (pH 7.2).
  • the LC 50 for rhMBL/oleic acid complexes prepared as described in Example 2 was 0.10 mg/ml for samples centrifuged at 3000 x g for 10 min and 0.17 mg/ml for samples centrifuged at 18,000 x g for 10 min.
  • Example 1 except that the molar ratio between rhMBL polypeptides (i.e. not oligomers) and oleic acid was 1 :24.
  • the sample was used in the cell killing assay as described in the Example 3 and figure 10 displays the results.
  • the LC 50 was 0.50 mg/ml. P1643PC00
  • CMP Concentrated Milk Plasma
  • CMP Concentrated Milk Plasma
  • CMP was obtained from ArIa Foods.
  • CMP was prepared by filtration/ultrafiltration from skimmed milk at ArIa foods.
  • CMP preparation is done to remove casein from other milk proteins, primarily bl_A and bl_G.
  • Prior storage, CMP was sterile filtrated and stored frozen.
  • a total of 900 ml_ CMP was used as starting material prior the next step: Acid treatment.
  • the acid treated CMP (presently pH 6.5) was added to an AIEC resin (Q Sepharose XL) equilibrated at pH 8.5.
  • bl_A was expected to be recovered in the flow through fraction due to the competition with bLG on the resin.
  • bLA recovered from the hydrophobic exchange runs was pooled and sterile filtered through 0.2 ⁇ m (Minikleenpak 20 filter from Pall). The pool of bLA was divided in two portions stored at +4 e C prior the conversion step.
  • EDTA and oleic acid were added to one part of bLA pool.
  • the molar ratio between EDTA and bLA and oleic acid and bLA was respectively 15 and 30.
  • the conditioned bLA sample was applied to the anion exchange column of Q Sepharose XL.
  • the unconverted bLA was removed during the first step gradient of 40% B-buffer, and bLAC was eluted in the second step of the salt gradient consisting of 70% B-buffer (0.7 M NaCI).
  • the column was regenerated with a sequence of acetic acid, NaOH, ethanol 20% and ethanol 70%. Between acid and base CIP's, the column was reequilibrated with Tris (100 mM) pH 8.5.
  • bLAC recovered from the conversion runs was pooled and sterile filtered through 0.2 ⁇ m (Minikleenpak 20 filter from Pall). The pool of bLAC was stored at +4 e C prior the next step (Nanofiltration).
  • the bLAC complexes thus obtained did have cell killing activity towards L1210 cells in an assay performed as described int Example 3.
  • the LC 50 was 0.06 mg/mL.
  • test sample typically 250-300 ⁇ l_
  • 10.0 ⁇ l_ diluted nonadecanoic acid (19:0, 12.5 mg/mL in chloroform) was added as internal standard.
  • the solution was then evaporated to dryness with nitrogen, and subsequently the fatty acids were converted into their methyl esters by addition of acidified methanol and heating for 1 hour at 85 1 C.
  • the precipitate was rediluted in 200 ⁇ l_ hexane and the fatty acid methyl esters analysed by GC, using a Varian 3400 System equipped with a split/splitless injector and flame ionization detector (FID).
  • FID flame ionization detector
  • Injection volume was 3.0 ⁇ l_ at 24O 0 C, split ratio is 1 :20, and applied column was a FFAP column (Zebron, 30 m x 0.32 mm x 0.25 ⁇ m).
  • Oven program 140 0 C for 1 min, increasing to 24O 0 C (by 8 0 C per min), and finally 240 0 C for 10 min.
  • FID temperature is 25O 0 C.
  • Lipid content (mass ratio) was calculated in relation to the concentration of the internal standard.
  • the bLA used in this and the following examples was purified from skimmed milk or from whole milk and essentially as described in Example 1 of PCT application P1643PC00
  • WO2008/058547 One preparation purified from whole milk had a bl_A concentration of 12.1 mg/mL, whereas purified from skimmed milk had a concentration of 8.6 mg/mL bl_A. Both of these were not calcium depleted, but were formulated in a 0.9% NaCI solution without calcium or buffer.
  • Method A In the first method a surplus of at least 99% pure oleic acid (obtainable from Sigma-Aldrich, St. Louise, USA cat.no. 01008 or Larodan AB, Malm ⁇ , Sweden, cat.no. 10-1801 ) was added to the protein solution ⁇ e.g. 100 ⁇ l_ oleic acid to 1000 ⁇ l_ protein solution) in a plastic tube. Then the two phases were mixed on a whirleymixer (MS2 Minishaker, IKA, Staufen, Germany) for approximately 20 seconds on setting 2200 min ' 1 to allow the protein solution to absorb oleic acid.
  • MS2 Minishaker MS2 Minishaker, IKA, Staufen, Germany
  • Method B At least 99% pure oleic acid (obtainable from Sigma-Aldrich, St. Louise, USA cat.no. 01008 or Larodan AB, Malm ⁇ , Sweden, cat.no. 10-1801 ) was first dissolved in 96% ethanol (20 ⁇ L oleic acid per 250 ⁇ L 96% ethanol) and then added to the protein solution to the intended protein oleic acid molar ratio. Usually, 14 ⁇ L oleic acid-ethanol mix was added per 1000 ⁇ L protein solution - more was never added. The resulting solution was then mixed on a whirleymixer (MS2 Minishaker, IKA, Staufen, Germany) for approximately 20 seconds on setting 2200 min '1 . In some instances the resulting water phase was sterile-filtrated on a sterile Pall Acrodisc® Syringe filter with 0.8/0.2 ⁇ m Supor membrane.
  • 96% ethanol 20 ⁇ L oleic acid per 250 ⁇ L 96% ethanol
  • the cell killing activity of the complexes thus prepared is shown in Table 2.
  • bl_A at a concentration of 12.1 mg/mL in 0.9% NaCI prepared as described in Example 10 was in three separate assay runs saturated with oleic acid and mixed under high shear conditions.
  • the ratio of bl_A to oleic acid before mixing was 1 :518 (N287-69C) or 1 :368 for the rest of the preparations.
  • After mixing the mixtures were centrifuged at either 150 x g, 3000 x g or 18000 x g for 10 minutes to separate and collect the water phase (see also Example 10, method A for more details).
  • the results of the subsequent test of cell killing activity performed as described in Example 3 are shown in figure 1 1 .
  • N287-69C 42 0.06 N312-31 A 83 0.12 N312-31 B fa 19 0.03 N312-53A 14 0.02 N312-53B fa 23 0.03 N318-06B 37 0.05 N318-65A C 76 0.1 1 N318-65B 74 0.1 1 3 LC 50 at a L1210 cell concentration of 1 .4-10 6 7mL.
  • Oleic acid of lower purity than 99% was used (obtainable from Merck), ⁇ ith 8 mM EDTA and 10 mM Tris-buffer (pH 8.5).
  • 100 % oleic acid - which is not the case) was determined to be 1.05 mM resulting in a 1 :5 molar ratio of protein vs. oleic acid.
  • Oleic acid with lower purity than 99% was used (obtainable from Merck).
  • HSA was obtained as described above in Example 5.
  • the content of oleic acid in the complexes of preparation N287-50C prepared from HSA saturated with oleic acid was measured to 57.9 mM giving a molar ratio of protein to oleic acid of 1 :276.
  • Example 14 bLG mixed with oleic acid bLG was obtained as described in Example 7. bLG at a concentration of 10 mg/mL in 0.9% NaCI was saturated with oleic acid, mixed and centrifuged at first 3000 x g and then 18000 x g to separate and collect the water phase (see details in method A of Example 10). The results of the subsequent test of cell killing activity are shown in figure 13. bLG alone has been tested in the highest concentration shown in the figure, and no effect on cell viability was observed.
  • Lysozyme was obtained from Sigma (cat.no. L7651 ) and was originated from chicken egg white.
  • Lysozyme at a concentration of 10 mg/mL in 0.9% NaCI was saturated with oleic acid, mixed and centrifuged at first 3000 x g and then 18000 x g to separate and collect the water phase (according to method A of Example 10).
  • the results of the subsequent test of cell killing activity are shown in figure 15. Lysozyme alone has been tested in the highest concentration shown in the figure, and no effect on cell viability was observed.
  • Stability in the context of the present example refers to the complexes retaining their biological activity, preferably their cell killing activity during storage.
  • Example 10 from a bl_A solution with or without 10 mM Tris and 8 mM EDTA was saturated with oleic acid and subjected to high shear mixing.
  • the isolated water phases (N318-65A and N318-65B) were sterile filtrated and aliquots incubated at -2O 0 C and 25 0 C.
  • the cell killing activities of the two preparations were determined before and after different periods of incubation at the two temperatures (see figure 20). From the accelerated stability samples at 25 0 C it was concluded that the preparation with 10 mM Tris and 8 mM EDTA still retained its cell killing activity after at least 4 weeks.
  • N318-65A (containing EDTA and Tris-buffer) contained 1 .5 mM oleic acid resulting in a protein to oleic acid molar ratio of 1 :3 (assuming a bLA+bLAC concentration of 7.6 mg/mL).
  • N318-65B contained only 35 ⁇ M oleic acid.
  • Oligomerisation determined by SEC analysis and oleic acid content determined by GC were also measured on the samples (see table 6 and 7).
  • the three preparations with Tris buffer, which retained their cell killing activity at 25 0 C showed a considerable amount of oligomerisation after 5-6 weeks at 25 0 C.
  • the pH in bLA mixed with oleic acid under high shear conditions was checked by making a fresh preparation of bLA in 0.9% NaCI mixed with oleic acid in a 1 :15 molar ratio and measuring pH 6.4. After 9 weeks at -2O 0 C preparation N326-12D, containing bLA mixed with oleic acid after addition of Tris buffer (pH 8.5) and EDTA, was measured to have a pH of 8.2, which may be assumed to be very close to the pH just after mixing.
  • Table 16 Batches of bLA used for conversion bLA batch N277-64A 7x1 1 .7 mL aliquot stored at -20 e C prepared from bLA batch
  • N278-50C produced at Natlmmune.
  • bLA concentration 4.01 mg/mL (SEC-HPLC) with bLA reference
  • the following fatty acids were purchased from Larodan (Sweden) for the conversion of bLA to complex.
  • Alfa Linolenic acid -1 1 20 0.25 20 20.27 3.5
  • Elaidic acid is the fatty acid with the highest melting point among those tested and the double bond is a trans double bond.
  • the yields of the conversion runs were determined by size exclusion HPLC (SE-HPLC) run according to standard methods.
  • the potency of the converted bLA was determined by cell killing abilities. Cell killing was run according to Example 8. The potency of bLAC determined by cell killing assay is given in pg bLAC per cell (LD50). Before testing in cell killing assay, the bLAC solutions were desalted against milli-Q H2O using NAP-10 desalting column (GE Healthcare).
  • the presence of complex bLA and fatty acid was determined by histone binding assay.
  • the histone binding assays was run according to Example 7. Only complex between bLA and fatty acid have histone biding abilities.
  • the amount of lipid and the lipid composition of the bLAC samples were determined by Net-Food lab (Finland) according to traditional methods: After esterification by the Boron trifluoride-methanol method, the fatty acids methyl esters (FAME) in the bLAC samples were analyzed by Gas Chromatography. Lipid content was calculated in relation to the concentration of the internal standard. The method is based on the European Pharmacopoeia (5.6) protocol 2.4.22 (Composition of Fatty Acids by Gas Chromatography, Method C). P1643PC00
  • the first eluted peak corresponding to non converted bl_A was lower. This can be due to the higher molar ratio between bl_A and EDTA and bl_A and oleic acid, and also the lower amount of bl_A loaded on the column. With these considerations, oleic acid and gondoic acid showed the same elution profile, while with all the other fatty acids the first peak is increased. When stearidonic acid was used for conversion, no peak corresponding to a complex between fatty acid and bl_A was obtained. Here all protein eluted in the 1 st step gradient.
  • Table 21 Yield ranking compared to oleic acid (18:1 (n-9))
  • Lipid composition by GC analysis The lipid composition and lipid content of the bl_A in complex with the different fatty acids obtained was determined by GC analysis.
  • N289- Heptadecenoio 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
  • N289- Gondoic acid 0 0 0 0 0 0 0 0 0 0 0 0 92 0
  • the molar ratio between bLA and lipid was found highest when oleic acid was used to conversion. Similar molar ratios to oleic acid were obtained for fatty acids giving the highest yield of conversion, i.e. gondoic acid, vaccenic and linoleic acid. Then the molar ratio lipid/bLA decrease for the two fatty acids were the yields of the conversion was the lowest, i.e. Palmitoleic acid and Eicosapentaenoic acid.
  • the lipid/bLA ratio in the sample prepared using gamma-Linolenic acid could not be measured.
  • the histone binding abilities of the bLAC samples converted with the different fatty acids were compared by histone binding assay. For each plate, a bLAC reference converted with oleic was run. From the binding curve EC50 was determined for each sample based on the SE-HPLC concentration. The EC50 were normalized to the EC50 of the bLAC reference applied in the plate. The results are shown in Table 24.
  • Sample ID Plate ID Fatty acid used EC50 Binding (%) to ref. P1643PC00
  • Table 25 EC50 ranking by histone binding assay

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Abstract

La présente invention concerne des complexes d'émulsifiants et d'acides gras, qui présentent un effet destructeur des cellules, en particulier des cellules tumorales. Les complexes comprennent un émulsifiant, qui est de préférence un polypeptide et un acide gras. Les complexes préférés présentent un rapport d'acide gras sur émulsifiant supérieur à 2 : 1. Les complexes peuvent être utilisés dans le traitement d'une variété de maladies caractérisées par la présence de cellules non souhaitées, telles que diverses maladies et infections malignes ou prémalignes, en particulier des infections virales.
PCT/DK2008/050183 2007-07-20 2008-07-21 Complexes d'un agent émulsionnant et d'un acide gras WO2009012785A2 (fr)

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WO2009135494A2 (fr) * 2008-05-09 2009-11-12 Nya Hamlet Pharma Ab Composition d'alpha-lactalbumine destinée au traitement de la kératose actinique
CN103372202A (zh) * 2012-07-23 2013-10-30 任发政 一种含有乳蛋白和脂肪酸的组合物及其制备方法与应用
US9055745B2 (en) 2011-04-13 2015-06-16 Natureza, Inc. Compositions for internal and external insecticides, ovicides, repellents and wound healing
CN114788872A (zh) * 2022-05-06 2022-07-26 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及制备和用途
CN115607677A (zh) * 2022-05-06 2023-01-17 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及其制备和应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009135494A2 (fr) * 2008-05-09 2009-11-12 Nya Hamlet Pharma Ab Composition d'alpha-lactalbumine destinée au traitement de la kératose actinique
WO2009135494A3 (fr) * 2008-05-09 2010-07-15 Nya Hamlet Pharma Ab Composition d'alpha-lactalbumine destinée au traitement de la kératose actinique
US9055745B2 (en) 2011-04-13 2015-06-16 Natureza, Inc. Compositions for internal and external insecticides, ovicides, repellents and wound healing
US9538748B2 (en) 2011-04-13 2017-01-10 Natureza, Inc. Compositions for internal and external use as an insecticide, ovicide, repellent and for wound healing
CN103372202A (zh) * 2012-07-23 2013-10-30 任发政 一种含有乳蛋白和脂肪酸的组合物及其制备方法与应用
CN114788872A (zh) * 2022-05-06 2022-07-26 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及制备和用途
CN115607677A (zh) * 2022-05-06 2023-01-17 太阳雨林(北京)生物医药有限公司 预防、阻止或治疗微生物感染的复合物及其制备和应用

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