WO2009012784A2 - Procédés de préparation de complexes cytotoxiques d'émulsifiant et d'acide gras - Google Patents

Procédés de préparation de complexes cytotoxiques d'émulsifiant et d'acide gras Download PDF

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WO2009012784A2
WO2009012784A2 PCT/DK2008/050182 DK2008050182W WO2009012784A2 WO 2009012784 A2 WO2009012784 A2 WO 2009012784A2 DK 2008050182 W DK2008050182 W DK 2008050182W WO 2009012784 A2 WO2009012784 A2 WO 2009012784A2
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acid
emulsifier
fatty acid
range
oleic acid
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WO2009012784A3 (fr
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Teit Agger
Christoffer Bro
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Nya Hamlet Pharma Ab
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • 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
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to methods for preparing complexes of emulsifiers and fatty acids, which have a cell killing effect, in particular on tumour cells or other malignant cells.
  • the complexes obtained according to the methods 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.
  • Alpha-lactalbumin is the major protein in human milk whey. Mature monomeric alpha-lactalbumin consists of 123 amino acid residues (14.2 kDa) in many mammalian species. Human, bovine, equine, caprine, and camelide alpha-lactalbumin all consist of 123 amino acid residues, whereas porcine alpha-lactalbumin consists of 122 amino acids. Human, bovine, caprine and porcine alpha-lactalbumin also comprise a 19 amino acid leader sequence. This 14 KDa protein has been extensively characterised and the crystal structure has been resolved.
  • Alpha-lactalbumin may undergo conformational switching and may adopt the so called apo state when exposed to low pH, or in the presence of chelators, that release the strongly bound Ca 2+ ion.
  • the apo state or molten globule state has native secondary structure, but less well defined tertiary structure than the native state. Similar states of alpha-lactalbumin can also form at neutral pH, upon removal of the tightly bound Ca 2+ ion, reduction of disulphide bonds or at elevated temperatures (the apo-state).
  • the apoptotic activity of this folding variant was discovered by serendipity.
  • human milk induced apoptosis in transformed and nontransformed immature cell lines.
  • the apoptotic activity in human milk was isolated and found to be partially unfolded alpha-lactalbumin in an apo-like conformation with native- like secondary structure, but lacking specific tertiary packing of the side chains.
  • LAC (as used herein) denotes a complex between LA and a fatty acid, said complex having cytotoxic activity.
  • bLAC denotes a complex between bovine LA and a fatty acid, said complex having cytotoxic activity
  • hLAC denotes a complex between human LA and a fatty acid, said complex having cytotoxic activity.
  • LAC is reported as having therapeutic applications both in the field of antibiotics and cancer therapy in particular, LAC may induce apoptotic cell death in cancer cells and immature cells, but not (or only to a low extent) in mature, healthy cells. These observations suggested that the protein acquires novel biological properties when forming an active complex with a fatty acid or lipid.
  • reagents such as fatty acids or lipids, such as oleic acid, have been useful in the conversion of LA to LAC.
  • An active LAC complex has previously been produced by first exposing alpha- lactalbumin in the apo state to a DEAE Trisacyl resin that had been pre-conditioned with oleic acid causing the formation of active complex of alpha-lactalbumin and oleic acid (e.g. Svensson, et al. , (2000) Proc Natl Acad Sci USA, 97,4221 -6, WO 03/098223, WO 2005/082406) or by mixing alpha-lactalbumin and oleic acid prior to exposure to an anion exchange resin (Danish patent application PA 2007 00693). Simple mixing of alpha-lactalbumin and oleic acid did not produce active LAC complexes (e.g. Svensson, et al. , (2000) Proc Natl Acad Sci USA, 97,4221 -6).
  • Whether a complex is cytotoxic may be determined as described herein below in the section "Cytotoxic effect”.
  • the invention relates to the cytotoxic complexes prepared by the method as well as to such complexes for use as a medicament.
  • the invention relates to such complexes for use as a medicament 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.
  • 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 trypan blue staining and by luminiscense using the the ViaLight Plus kit (Lonza cat. No. LT07-221 or LT07-121 ). 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. Filled circles - rhMBL saturated with oleic acid (N287-78D), spun at 3000xg, LD 50 76 pg/cell; open circles - rhMBL saturated with oleic acid (N287-78E), spun at 18,000xg, LD 50 123 pg/cell.
  • Figure 15 shows cell killing activity of two preparations of lysozyme saturated and mixed with oleic acid.
  • 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 LD50 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:
  • R is alkyl or alkenyl.
  • the emulsifier is not any of the fatty acids described in the section "Fatty acids" herein below.
  • 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 anionic polypeptides as described below.
  • Anionic emulsifiers may also be bile salts, preferably sodium cholate.
  • 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 is 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.
  • the 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 polypeptide has 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 skilled person will be able to calculate the theoretical pi for a given polypeptide.
  • Theoretical pi for polypeptides are also available in publicly assessable protein databases, such as the Swissprot database. Examples of theoretical pi of a number of polypeptides together with other protein parameters are given in Table 1 below:
  • 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 serum albumin of SEQ ID NO: 5 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 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
  • 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 or more carbohydrates.
  • a lectin i.e. a polypeptide capable of associating with one or more 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 homologies thereof, the following applies.
  • 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. It is preferred within the present invention that 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.
  • the 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).
  • the FABP may for example be a mammalian 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.
  • polypeptide is a polypeptide other than alpha-lactalbumin.
  • polypeptide is alpha- lactalbumin.
  • 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.
  • the liquid solution comprising the emulsifier provided in step a) of the method according to the invention comprises purified alpha-lactalbumin.
  • said 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.
  • conserveed 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 activity of a given polypeptide.
  • 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,)
  • 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,
  • 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.
  • 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, including fluorophenylalanine, norleucine, azetidine-2-carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophan and the like.
  • 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. This may be achieved by techniques of modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be em- ployed 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.
  • Peptides with N-terminal alkylations and C-terminal esterifications are also encom- passed within 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.
  • Functional homologues of alpha-lactalbumin may be functional homologues as described above in this section. More specific examples of useful functional homologues are given below.
  • alpha-lactalbumin sequences are compared between species where alpha-lactalbumin function is conserved, for example but not limited to mammals including rodents, monkeys and apes. Residues under high selective pressure are more likely to represent essential amino acids that cannot easily be substituted whereas residues that change between species may more preferably be substituted.
  • Such an alignment may be performed using ClustalW from EBML-EBI comparing porcine alpha-lactalbumin and human alpha-lactalbumin (see Figure 1 A of PCT application WO2008/058547).
  • Figure 1 A of PCT application WO2008/058547 shows an alignment of the protein sequences of bovine, human, equine, caprine, bovine, camelide and porcine alpha-lactalbumin wherein identical residues (" * ") and residues with conservative (“:”) and semi- conservative (“.”) substitutions are marked.
  • * residues
  • : residues with conservative
  • . semi- conservative substitutions
  • Functional assays can for example be used in order to determine if alpha-lactalbumin function is conserved.
  • Functional assays for evaluating alpha-lactalbumin function include assay for cytotoxic activity in complex with oleic acid and include, but are not limited to, assays described herein in Example 3 and in Danish patent application PA 2007 0693.
  • functional homo- logues of alpha-lactalbumin comprises a sequence with high sequence identity to SEQ ID NO: 1 or SEQ ID NO:2, wherein none of the conserved residues marked with " * " in figure 1 A of PCT application WO2008/058547 are substituted. It is furthermore preferred within this embodiment that the residues marked with ":" in figure 1 A of PCT application WO2008/058547are either not substituted or only substituted by conservative substitution, more preferably by substitution with an amino acid with a high level of similarity as defined herein below.
  • functional homologues of bovine alpha- lactalbumin have a sequence with high sequence identity to SEQ ID NO: 2, wherein residues E1 , L3, E7, V8, L15, Y18, V21 , S22, V27, Q39, A40, 141 , N44, I59, K62, Q65, I85, M90, N102, S1 12, D1 16, K122 are either not substituted or substituted only by conservative substitution, more preferably substituted only an amino acid with a high level of similarity as defined herein below.
  • functional homologues of alpha-lactalbumin have a sequence with high sequence identity to SEQ ID NO:1 or SEQ ID NO: 2, wherein residues marked with ".” in figure 1 A of PCT application WO2008/058547 are either not substituted or are only substituted by conservative substitutions, such as with amino acids with lower levels or high level of similarity as defined herein below.
  • functional homologues of bovine alpha-lactalbumin have a sequence with high sequence identity to SEQ ID NO: 2, wherein residues D14, K16, G17, G20, P24, S47, N56, D63, D64, N74, V92, and A109 are either not substituted or only substituted by conservative substitutions, such as with amino acids with lower level or high level of similarity as defined herein below. It is also comprised within the present invention that functional homologies of alpha- lactalbumin may have a sequence with high sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, wherein the unmarkes residues in Figure 1 A of PCT application WO2008/058547 may be substituted with any other amino acid.
  • functional homolgoues of human alpha-lactalbumin may have a sequence with high sequence identity to SEQ ID NO: 1 , wherein residues F9, R10, E1 1 , G19, W25, T29, T30, T33, Q43, D46, T48, N66, P67, H68, S70, I89, K98, V99, L1 18, and L123 are either not substituted or substituted with any other amino acid.
  • Functional homologues of alpha-lactalbumin may also be addition mutants as described herein above, in particular addition mutants may be functional homologues comprising at least one additional methionine.
  • the addition mutant consists of SEQ ID 1 or SEQ ID 2 extended by one additional Methionine at the N- or C- terminus, preferably at the N-terminus.
  • 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.
  • 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.
  • the fatty acid is selected from the group of 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:
  • 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 :1 Ocis,
  • the fatty acid complexed with emulsifier is an unsaturated fatty acid in the cis conformation, preferably selected from the group consisting of C17:1 :1 Ocis, C18:1 :9cis, C18:1 :1 1cis, 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 C18:3:9,12,15, 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 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, C18:3:6,9,12cis, C18:3:9,12,15cis and C18:4
  • 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, preferably all impurities are lipophilic.
  • 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 as 99%.
  • the complexes according to the present invention are prepared by a method comprising the steps of:
  • the method 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 recovered together during a purification. In particular if fatty acid may be recovered together with emulsifier in the aqueous phase after centrifugation for 10 min at 3000 x g, then 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, and preferably the emulsifier is a polypeptide. 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 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:
  • 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.
  • fatty acid at a purity as described herein above in the section "Fatty acid” is mixed with emulsifier in aqueous solution using high shear mixing.
  • 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.
  • 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.
  • 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 example in the range of 10 sec. to 1 hour, such as in the range of 10 sec. to 10 min., such as in the range of 10 sec. to 1 min, for example in the range of 10 sec. to 30 sec, such as in the range of 10 sec. to 20 sec, for example for 10 sec.
  • "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 mixer 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 quick flow through a capillary are described in Jaspe and Hagen, 2006, Biophysical Journal, 91 : 3415-3424.
  • 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. Also within this range, the gap width may preferably be up to about 0.5 millimeter, more preferably up to about
  • 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.
  • 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 may be determined as follows:
  • 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 , however preferably it is higher as described below.
  • 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 2:1 :, for example at least 3:1 , such as at least 4:1 , for example at least 5:1 , such as 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 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 . It is very preferred that 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 obtained by the methods according to the present invention preferably have a cytotoxic effect on tumour cells.
  • the terms "Cytotoxic effect” and “cytotoxic activity” are used interchangeably herein.
  • the complexes obtained according to the methods 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 cells infected by virus, bacteria or mycoplasma. 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 the complexes have a low cytotoxicity or are not cytotoxic to healthy cells in vivo.
  • 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 calculated (see more below). It is preferred that 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%.
  • 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 , respectively.
  • 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 exploiting 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 Prior to adding complex and during the time cells are incubated with complex, the cells should otherwise be maintained at standard conditions for in vitro cell culture. It is however preferred that the cells are kept in the absence of serum during incubation with complex.
  • One suitable method for determining to cytotoxicity of complexes to tumour cells is the "cell killing assay" described in Danish patent application PA 2007 00693 in Example 7.
  • 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 cells infected by virus, bacteria or mycoplasma. 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 obtained by the methods 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.
  • 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
  • a range of different types of wart have been identified, which differ in shape and site affected, including:
  • 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.
  • 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
  • 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 diameter and gradually enlarge into broader, more elevated lesions. Actinic keratosis (AK) is also referred to as solar keratosis, sun spots, or precancerous spots.
  • actinic keratoses may develop into invasive squamous cell carcinoma and Actinic keratosis is thus a precursor of skin cancer.
  • 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 obtained by the conversion methods 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical 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 and intra-peritonal injection. 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- lactalbumin or a functional homologue thereof) and a fatty acid (e.g. oleic acid), said complex having cytotoxic activity
  • 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.
  • 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. formulating said complex in a liquid solution having a pH of at least 6.5, thereby obtaining 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 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 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.
  • the resulting mixture is ready for use for example in the cell killing assay as described herein below in Example 3.
  • 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. 50 ⁇ l cell suspension in serum free RPMI 1640 medium with 1% sodium pyruvate and 1 % non-essential amino acids containing 100,000 L1210 cells is added to each well and the plate is incubated for 1 hour at 37 0 C and 5% CO 2 .
  • 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.
  • 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.
  • the results are displayed as % viability compared to the viability of cells treated with 0.9% NaCI.
  • LD 50 and LC 50 values were calculated by evaluating the fit for protein dose in ⁇ l_ per well versus the luminescence using a four parameter logistic fit (given by
  • 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.
  • concentration 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 protein concentration in the protein solution was 12 mg/mL and 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.
  • 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).
  • Example 3 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.
  • 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. Table 1
  • 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 bLA and bLG.
  • 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.
  • Acid treatment An acid treatment was performed on the CMP to destroy potential virus.
  • 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. Ratio of lipid to alpha-lacatalbumin
  • 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 WO2008/058547.
  • One preparation purified from whole milk had a bLA 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
  • 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.
  • 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.
  • bLA prepared as described in Example 10
  • oleic acid Various complexes prepared from bLA (prepared as described in Example 10) saturated with oleic acid were prepared essentially as described in Example 10, method A.
  • an 8.6 mg/mL bLA solution was saturated with oleic acid and mixed under high shear conditions.
  • the water phase was sterile filtered (the sterile filtration did not result in changed cell killing activity), and the preparation incubated at -2O 0 C, 2- 8 0 C and 25 0 C.
  • Cell killing activities and bLA+bLAC concentrations by A 28 o nm were determined after 0, 2 and 4 weeks of incubation at the three incubation conditions. More specifically, the protein content was determined by A 28 o nm nanodrop spectrometry.
  • 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
  • the following fatty acids were purchased from Larodan (Sweden) for the conversion of bLA to complex.
  • Oleic acid 13 20 0.25 20 20.27 3
  • Alfa Linolenic acid -1 1 20 0.25 20 20.27 3
  • Elaidic acid is the fatty acid with the highest melting point among those tested and the double bond is a trans double bond.
  • the molar ratio between EDTA and bLA and fatty acid and bLA should have been 15 and 30 respectively. Other molar ratios were applied as the bLA reference for the SE- HPLC analysis was changed. With the new reference the bLA concentration is approximately 15% lower. The sample conditioning calculations were done on basis of the concentration measured.
  • 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). RESULTS
  • 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.
  • Linoleic acid 18:2(n-6) 1.16
  • 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.
  • the fatty acid used for the conversion was detected in all the corresponding samples analysed by Gas chromatography, except for the sample converted with gamma Linolenic acid.
  • the amount of fatty acid was below the detection limit of the analysis.
  • the sample was sent for retest by GC analysis. Stearidonic acid was not detected in the sample recovered from the conversion run with this fatty acid In this case no converted bLA peak was obtained, and the sample corresponded to non- converted bLA was sent to GC analysis.
  • Table 22 Lipid composition by GC analysis i idt m yr sc ac i Fatty acids identified by Gas chromatography l dt p am ii c ac i
  • N277- Li ⁇ oleic acid 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0
  • N289- Palmitoleic acid 0 0 100 0 0 0 0 0 0 0 0 0 0 h ti cosa p enaeno i c 0
  • N289- Heptadecenoic 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, Le 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.
  • Table 24 EC50 by histone binding assay Sample ID Plate ID: Fatty acid used EC50 Binding (%) to ref. for conversion ( ⁇ g/mL)
  • Table 25 EC50 ranking by histone binding assay bLA and Fatty acid histone binding Histione binding Molar ratio Mpid/bLA ranking capacity
  • Table 27 LD50 ranking by cell killing assay bLA and Fatty acid Molar ratio lipid/bLA

Abstract

La présente invention concerne des procédés de préparation de complexes d'émulsifiants et d'acides gras, qui présentent un effet destructeur sur des cellules, en particulier des cellules tumorales ou autres cellules malignes. Les procédés comprennent le mélange d'un émulsifiant avec un acide gras en utilisant un mélange par cisaillement élevé. L'émulsifiant peut être l'alpha-lactalbumine et l'acide gras peut être de l'acide oléique. Les complexes obtenus selon les procédés 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/DK2008/050182 2007-07-20 2008-07-21 Procédés de préparation de complexes cytotoxiques d'émulsifiant et d'acide gras WO2009012784A2 (fr)

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WO2012045437A1 (fr) * 2010-10-04 2012-04-12 Centro De Investigacion Principe Felipe Méthodes de traitement de tumeurs et différenciation de l'adipogenèse
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WO2014202956A1 (fr) * 2013-06-21 2014-12-24 Noakes, David Complexes à base de vitamine d avec de-vdbp et un acide gras insaturé, et leur utilisation en thérapie
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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
WO2009135494A3 (fr) * 2008-05-09 2010-07-15 Nya Hamlet Pharma Ab Composition d'alpha-lactalbumine destinée au traitement de la kératose actinique
CN102762115A (zh) * 2010-02-24 2012-10-31 美赞臣营养品公司 供给营养素的制剂和方法
WO2012045437A1 (fr) * 2010-10-04 2012-04-12 Centro De Investigacion Principe Felipe Méthodes de traitement de tumeurs et différenciation de l'adipogenèse
US20120115955A1 (en) * 2010-10-04 2012-05-10 Centro De Investigacion Principe Felipe Methods for tumor treatment and adipogenesis differentiation
US10711051B2 (en) 2011-03-03 2020-07-14 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
EP2681245A4 (fr) * 2011-03-03 2015-02-18 Zymeworks Inc Conception et constructions d'échafaudage hétéromultimère multivalent
US9499605B2 (en) 2011-03-03 2016-11-22 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
US10155803B2 (en) 2011-03-03 2018-12-18 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
EP2681245A1 (fr) * 2011-03-03 2014-01-08 Zymeworks, Inc. Conception et constructions d'échafaudage hétéromultimère multivalent
US9388231B2 (en) 2012-07-13 2016-07-12 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
US10358479B2 (en) 2012-07-13 2019-07-23 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
US11248037B2 (en) 2012-07-13 2022-02-15 Zymeworks Inc. Multivalent heteromultimer scaffold design and constructs
US11103561B2 (en) 2012-08-09 2021-08-31 Hamlet Pharma Ab Prophylactic and nutraceutical therapy
US11865161B2 (en) 2012-08-09 2024-01-09 Hamlet Pharma Ab Prophylactic and nutraceutical therapy
WO2014202956A1 (fr) * 2013-06-21 2014-12-24 Noakes, David Complexes à base de vitamine d avec de-vdbp et un acide gras insaturé, et leur utilisation en thérapie

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