WO2017064289A1 - Colloidal particles for topical administration with therapeutic agent - Google Patents

Colloidal particles for topical administration with therapeutic agent Download PDF

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Publication number
WO2017064289A1
WO2017064289A1 PCT/EP2016/074783 EP2016074783W WO2017064289A1 WO 2017064289 A1 WO2017064289 A1 WO 2017064289A1 EP 2016074783 W EP2016074783 W EP 2016074783W WO 2017064289 A1 WO2017064289 A1 WO 2017064289A1
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composition
factor
protein
amphipathic lipid
blood
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PCT/EP2016/074783
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French (fr)
Inventor
William Henry
Richard WOLF-GARRAWAY
John Mayo
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Cantab Biopharmaceuticals Patents Limited
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Publication of WO2017064289A1 publication Critical patent/WO2017064289A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/10Dispersions; Emulsions
    • A61K9/127Liposomes
    • A61K9/1271Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021Intradermal administration, e.g. through microneedle arrays, needleless injectors

Definitions

  • the present invention relates to topical formulations of colloidal particles for use in medicine in the therapy of a disease or condition, such as for example a blood factor disease.
  • blood factors have been prepared as pharmaceutical compositions for intravenous administration to treat blood factor diseases.
  • the compositions have been based on the active protein, often conjugated to a polymer such as polyethylene glycol (PEG) to improve the half-life in circulation.
  • PEG polyethylene glycol
  • Intravenous administration of PEGylated blood factors as therapeutic agents is therefore well understood and widely accepted.
  • Liposomal formulations of naked (i.e. unconjugated and without modification) blood factors such as Factor VIII and Factor IX substances are known also, see for example WO 95/04524.
  • compositions comprising Factor VIII and liposomes modified by the presence of polyethylene glycol are described in WO 99/55306 in which the blood factor is not encapsulated in the liposome. However, the formulations are prepared for intravenous administration. Additional formulations of other proteins are described in WO 2004/091723 where the proteins include blood clotting factors. The proteins are said to bind to the liposomes in a non-covalent manner through interaction with the polyethylene glycol present on the surface of the liposomes. However, the formulations of blood clotting factors prepared according to the examples of this document are also for intravenous administration.
  • WO 2013/156488 also describes a dosage form of modified therapeutic agents, including blood factors such as Factor VIII (FVIII) and Factor Vila (FVIIa), for subcutaneous administration.
  • blood factors such as Factor VIII (FVIII) and Factor Vila (FVIIa)
  • the blood factor Factor VIII has also been found to be capable of association with PEGylated liposomes, i.e. the blood factor is not encapsulated inside the liposome (Baru ef al Thromb. Haemost., 93, pages 1061-1068, (2005)).
  • the compositions of FVIII were only prepared as formulations for intravenous administration.
  • composition comprising a biologically active polypeptide or protein and a colloidal particle comprising approximately 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for use in the treatment of a disease or condition wherein the colloidal particles are for topical administration and the biologically active polypeptide or protein are for topical, intravenous or subcutaneous administration.
  • the colloidal particles are suitable for topical administration and the biologically active polypeptide or protein is suitable for topical, intravenous or subcutaneous administration.
  • the biologically active polypeptide or protein may be selected from the group consisting of blood factors, hormones, growth factors, cytokines, antibodies and/or fragments thereof.
  • Reference to polypeptide includes the term peptide and defines a series of amino acids linked by peptide bonds.
  • the colloidal particles may be administered separately, simultaneously or subsequently to the biologically active polypeptide or protein.
  • the composition may therefore be a co-formulation of the colloidal particles and the biologically active polypeptide or protein, or the colloidal particles and the biologically active polypeptide or protein may be formulated for separate administration.
  • the colloidal particles of the present invention therefore provide for the protection and prolongation of the activity of biologically active polypeptides or proteins.
  • the invention provides for the use of biologically active polypeptides or proteins without further derivatization and from any convenient source.
  • the biologically active polypeptide or protein has an increased half-life and thereby has a longer duration of action after administration to a subject.
  • Such combinations of the colloidal particles of the invention with biologically active polypeptides or proteins are also less immunogenic to a subject also.
  • the invention also means that a subject may not be required to receive either as high a dose and/or as many injections as in conventional therapies in order to maintain a therapeutically effective circulating level of the biologically active polypeptide or protein.
  • the biologically active polypeptide or protein is a blood factor it may be selected from the group consisting of Factor VII, Factor Vila, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor Vila, Factor V, Factor XIII, von Willebrand's Factor, prothrombin, Protein C and/or fragments thereof.
  • the blood coagulation factor may be suitably Factor VII, Factor VIII or Factor IX.
  • Factor Vila (FVIIa) and Factor VII (FVII) are also used interchangeably unless the context specifies otherwise.
  • FVIII is used as an abbreviation for Factor VIII
  • FIX is used as an abbreviation for Factor IX
  • References to a blood clotting factor include a blood coagulation factor.
  • hormones and/or cytokines may be selected from the group consisting of (but not limited to) calcitonin, erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor, granulocyte macrophage colony stimulating factor (GMCSF), growth hormone, heparin, human growth hormone (HGH), growth hormone releasing hormone (GHRH), interferon alpha, interferon beta, interferon gamma, interleukin-1 receptor, interleukin-2, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-6, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, amylin, C-peptide, somatostatin, vasopressin, follicle stimulating hormone (FSH), insulin-like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-C
  • the biologically active polypeptide or protein may also be an antibody or an antibody fragment for example; a single-domain antibody, V L , V H , Fab, F(ab') 2 , Fab', Fab3, scFv, di-scFv, sdAb, Fc and/or combinations thereof.
  • the biologically active polypeptide or protein may be used in a lyophilised form when preparing the composition.
  • the disease or condition may be characterised by a loss of function of the native biologically active polypeptide or protein in a subject.
  • the biologically active polypeptide or protein may be absent or only present in low amounts.
  • diseases include but are not limited to blood factor diseases, for example a haemophilia (haemophilia A, B or C), von Willebrand Disease, Factor V deficiency, Factor X deficiency, Factor XII deficiency, acromegaly, Addison's Disease, Cushing's Syndrome, De Quervain's Thyroiditis, obesity, diabetes mellitus (Type 1 diabetes or Type 2 diabetes), diabetes insipidous, Goiter, Graves' Disease, Growth Disorders, Growth Hormone Deficiency, Hashimoto's Thyroiditis, Hyperglycaemia, Hyperparathyroidism, Hypoglycaemia, Hypoparathyroidism, Low Testosterone, Menopause, Osteoporosis, Para
  • the colloidal particles may be substantially neutral and the polymer may carry substantially no net charge.
  • the colloidal particles may have a mean particle diameter of between about 0.03 to about 0.4 microns ( ⁇ ), for example having a mean particle diameter of approximately 0.1 microns ( ⁇ ).
  • mean particle diameter in this range may increase the circulation time of the particles in vivo and prevent their adsorption by the reticuloendothelial system (RES).
  • the composition comprises a fragment of a biologically active polypeptide or protein
  • the fragment may suitably be an active fragment thereof in which the fragment retains the biological activity, or substantially the same biological activity as the native polypeptide or protein.
  • composition may comprise both the native polypeptide or protein and a fragment thereof.
  • the biologically active polypeptide or protein may also be co-administered with another therapeutically active compound or molecule, e.g. an anti-inflammatory drug, analgesic or antibiotic, or other pharmaceutically active agent which may promote or enhance the activity of the biologically active polypeptide or protein.
  • another therapeutically active compound or molecule e.g. an anti-inflammatory drug, analgesic or antibiotic, or other pharmaceutically active agent which may promote or enhance the activity of the biologically active polypeptide or protein.
  • the biologically active polypeptide or protein may be from any suitable source and may be a recombinant protein produced by recombinant DNA technology using molecular biological techniques or synthesised chemically or produced transgenically in the milk of a mammal, or the biologically active polypeptide or protein may be isolated from natural sources (e.g. purified from blood plasma).
  • the biologically active polypeptide or protein factor is mammalian, such as a human biologically active polypeptide or protein.
  • colloidal particles of the invention are typically in the form of lipid vesicles or liposomes as are well known in the art. References to colloidal particles in the present specification include liposomes and lipid vesicles unless the context specifies otherwise.
  • the amphipathic lipid may be a phospholipid from natural or synthetic sources.
  • the amphipathic lipid may comprise approximately 0.5 to about 20 mole percent (%) of the particles, for example approximately about 1 to 20%, or about 1 to 6%, or about 3%.
  • amphipathic lipid refers to a substance including a hydrophilic region and a hydrophobic region, such as phospholipids.
  • Amphipathic lipids can be zwitterionic phospholipids, zwitterionic lipids, lipids having a net negative charge, and lipids having a net positive charge.
  • amphipathetic lipids include, but are not limited to, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelin, soybean lecithin (soya lecithin), egg lecithin, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, cardiolipins, acyl trimethylammonium propane, diacyldimethylammonium propane, stearylamine, ethyl phosphatidylcholine and the like. Soya lecithin is a combination predominantly of naturally- occurring phospholipids; phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidyli
  • amphipathic lipids include a phosphatidylethanolamine (PE), a carbamate-linked uncharged lipopolymer or aminopropanediol distearoyl (DS), or mixtures thereof.
  • phosphatidylethanolamine phospholipids include 1 ,2-Dierucoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Dimyristoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Dipalmitoyl- sn-glycero-3-phosphoethanolamine, 1 ,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine.
  • a suitable example of phosphatidyl ethanolamine (PE) may be 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE).
  • the purpose of the biocompatible hydrophilic polymer is to sterically stabilize the SUVs, thus preventing fusion of the vesicles in vitro, and allowing the vesicles to escape adsorption by the RES in vivo.
  • the colloidal particles may further comprise a second amphipathic lipid obtained from either natural or synthetic sources.
  • the second amphipathic lipid may be a phosphatidylcholine (PC).
  • PC phosphatidylcholine
  • phosphatidylcholine phospholipids include 1 ,2-Didecanoyl-sn-glycero-3-phosphocholine, 1 ,2- Dierucoyl-sn-glycero-3-phosphocholine, 1 ,2-Dilinoleoyl-sn-glycero-3-phosphocholine, 1 ,2-Dilauroyl- sn-glycero-3-phosphocholine, 1 ,2-Dimyristoyl-sn-glycero-3-phosphocholine, 1 ,2-Dioleoyl-sn- glycero-3-phosphocholine, 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine, 1 ,2-Diste
  • phosphatidyl choline may be palmitoyl- oleoyl phosphatidyl choline (POPC) or soy phosphatidyl choline.
  • POPC palmitoyl- oleoyl phosphatidyl choline
  • soy phosphatidyl choline Other natural sources of phosphatidylcholine include egg phosphatidylcholine.
  • the phosphatidylcholine may be hydrogenated or non-hydrogenated.
  • the pharmaceutical composition may be composed of colloidal particles which comprise palmitoyl- oleoyl phosphatidyl choline (POPC) and 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine (DSPE) in a molar ratio (POPC:DSPE) of from 85 to 99: 15 to 1.
  • POPC:DSPE a molar ratio of POPC:DSPE
  • the molar ratio of POPC:DSPE may be from 90 to 99: 10 to 1.
  • the molar ratio of POPC:DSPE may be 97:3. A molar ratio of 97:3 of these lipids is equivalent to a 97: 10 weight ratio.
  • composition of the invention may be supplemented with cholesterol.
  • the biocompatible polymer may have a molecular weight of between about 500 to about 5000 Daltons, for example approximately 2000 Daltons.
  • the biocompatible hydrophilic polymer used according to the invention may be selected from the group consisting of polyalkylethers, polylactic acids and polyglycolic acids.
  • the biocompatible hydrophilic polymer may be polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the polyethylene glycol as used in the compositions of the invention may have a molecular weight of between about 500 to about 5000 Daltons, for example it may have a molecular weight of approximately 1000, 2000, or 3000 Daltons. In one embodiment the molecule weight of the PEG may be 2000 Daltons.
  • the polyethylene glycol may be branched or unbranched.
  • An example of a suitable derivatized amphipathic lipid may be 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)]. If the PEG has a molecular weight of 2000 Daltons, the derivatized amphipathic lipid may be described as 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).
  • the concentration of the derivatized amphipathic lipid in the final formulation of colloidal particles may be adjusted accordingly to suit the desired properties of the composition.
  • suitable concentrations may be from 5.0mg/ml_ to 15mg/ml_, for example 7.5mg/ml_ to 12.5mg/ml_, or 7.6mg/ml_ to 9.0mg/ml_.
  • the overall content of lipid in the final formulation may be in the range of from 4% to 12%, for example 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 % or 12%.
  • the composition may comprise any suitable excipient, buffer and/or adjuvant and may be formulated as a pharmaceutical composition.
  • excipients, buffers and/or adjuvants include phosphate buffered saline (PBS), potassium phosphate, sodium phosphate and/or sodium citrate.
  • PBS phosphate buffered saline
  • Other biological buffers can include PIPES, MOPS etc.
  • Suitable pH values for the composition include any generally acceptable pH values for administration in vivo, such as for example pH 5.0 to pH 9.0, suitably from pH 6.8 to pH 7.2, or pH 7.0.
  • the present inventors have surprisingly found that formulations of colloidal particles (liposomes) derivatized with a biocompatible polymer can be successfully administered and achieve a therapeutically effective dose when co-administered with a separate dosage of a biologically active polypeptide or protein to a subject suffering from a disease or condition, for example a blood factor disease, such as a haemophilia.
  • a biologically active polypeptide or protein to a subject suffering from a disease or condition, for example a blood factor disease, such as a haemophilia.
  • the biocompatible polymer is polyethylene glycol.
  • colloidal particles used in accordance with the present invention can potentiate the activity of a biologically active polypeptide or protein in a subject when administered prior to the biologically active polypeptide or protein.
  • a variety of known coupling reactions may be used for preparing vesicle forming lipids derivatized with hydrophilic polymers.
  • a polymer such as PEG
  • a lipid such as phosphatidylethanolamine (PE) through a cyanuric chloride group.
  • PE phosphatidylethanolamine
  • a capped PEG may be activated with a carbonyl diimidazole coupling reagent, to form an activated imidazole compound.
  • a carbamate-linked compound may be prepared by reacting the terminal hydroxyl of MPEG (methoxyPEG) with p-nitrophenyl chloroformate to yield a p-nitrophenyl carbonate.
  • This product is then reacted with 1-amino-2,3-propanediol to yield the intermediate carbamate.
  • the hydroxyl groups of the diol are acylated to yield the final product.
  • a similar synthesis, using glycerol in place of 1-amino-2, 3-propanediol, can be used to produce a carbonate-linked product, as described in WO 01/05873.
  • Other reactions are well known and are described, e.g. in US 5,013,556.
  • Colloidal particles can be classified according to various parameters. For example, when the size and number of lamellae (structural parameters) are used as the parameters then three major types of liposomes can be described: Multilamellar vesicles (MLV), small unilamellar vesicles (SUV) and large unilamellar vesicles (LW).
  • MLV Multilamellar vesicles
  • SUV small unilamellar vesicles
  • LW large unilamellar vesicles
  • MLV are the species which form spontaneously on hydration of dried phospholipids above their gel to liquid crystalline phase transition temperature (cm).
  • the size of the MLVs is heterogeneous and their structure resembles an onion skin of alternating, concentric aqueous and lipid layers.
  • SUV are formed from MLV by sonication or other methods such as extrusion, high pressure homogenisation or high shear mixing and are single layered. They are the smallest species with a high surface-to-volume ratio and hence have the lowest capture volume of aqueous space to weight of lipid.
  • the third type of liposome LUV has a large aqueous compartment and a single (unilamellar) or only a few (oligolamellar) lipid layers. Further details are disclosed in D. Lichtenberg and Y. Barenholz, in “Liposomes: Preparation, Characterization, and Preservation, in Methods of Biochemical Analysis", Vol. 33, pp. 337 - 462 (1988).
  • loading means any kind of interaction of the biopolymeric substances to be loaded, for example, an interaction such as encapsulation, adhesion (to the inner or outer wall of the vesicle) or embedding in the wall with or without extrusion of the biopolymeric substances.
  • liposome refers to colloidal particles and is intended to include all spheres or vesicles of any amphipathic compounds which may spontaneously or non-spontaneously vesiculate, for example phospholipids where at least one acyl group replaced by a complex phosphoric acid ester.
  • the liposomes may be present in any physical state from the glassy state to liquid crystal.
  • the most common phospholipids suitable for use in the present invention are the lecithins (also referred to as phosphatidylcholines (PC)), which are mixtures of the diglycerides of stearic, palmitic, and oleic acids linked to the choline ester of phosphoric acid.
  • the lecithins are found in all animals and plants such as eggs, soybeans, and animal tissues (brain, heart, and the like) and can also be produced synthetically.
  • the source of the phospholipid or its method of synthesis are not critical, any naturally occurring or synthetic phosphatide can be used.
  • Examples of specific phosphatides are L-a-(distearoyl) lecithin, L-a-(dipalmitoyl) lecithin, L-a- phosphatide acid, L-a-(dilauroyl)-phosphatidic acid, L-a(dimyristoyl) phosphatidic acid, L- a(dioleoyl)phosphatidic acid, DL-a (di- palmitoyl) phosphatidic acid, L-a(distearoyl) phosphatidic acid, and the various types of L-a-phosphatidylcholines prepared from brain, liver, egg yolk, heart, soybean and the like, or synthetically, and salts thereof.
  • PC phosphatidylcholines
  • zwitterionic amphipathates which form micelles by themselves or when mixed with the PCs such as alkyl analogues of PC.
  • the phospholipids can vary in purity and can also be hydrogenated either fully or partially. Hydrogenation reduces the level of unwanted peroxidation, and modifies and controls the gel to liquid/crystalline phase transition temperature (try) which effects packing and leakage.
  • try liquid/crystalline phase transition temperature
  • the liposomes can be "tailored” to the requirements of any specific reservoir including various biological fluids, maintains their stability without aggregation or chromatographic separation, and remains well dispersed and suspended in the injected fluid.
  • the fluidity in situ changes due to the composition, temperature, salinity, bivalent ions and presence of proteins.
  • the liposome can be used with or without any other solvent or surfactant.
  • suitable lipids may have an acyl chain composition which is characteristic, at least with respect to transition temperature (Tm) of the acyl chain components in egg or soybean PC, i.e., one chain saturated and one unsaturated or both being unsaturated. However, the possibility of using two saturated chains is not excluded.
  • Tm transition temperature
  • the liposomes may contain other lipid components, as long as these do not induce instability and/or aggregation and/or chromatographic separation. This can be determined by routine experimentation.
  • the biocompatible hydrophilic polymer may be physically attached to the surface of the liposome, or inserted into the membrane of the liposome.
  • the polymer may therefore be covalently bound to the liposome.
  • Either or both of the colloidal particle or the biologically active polypeptide or protein may be modified in order to modulate the dynamics of the association between the colloidal particle and the polypeptide or protein. Such modulation may be achieved by customising regions or binding sequences on the colloidal particle or the biologically active polypeptide or protein.
  • a variety of methods for producing the modified liposomes which are unilamellar or multilamellar are known and available (see Lichtenberg and Barenholz, (1988)): 1. A thin film of the phospholipid is hydrated with an aqueous medium followed by mechanical shaking and/or ultrasonic irradiation and/or extrusion through a suitable filter;
  • liposome down-sizing can be used for reducing the size and size heterogeneity of liposome suspensions.
  • the liposome suspension may be sized to achieve a selective size distribution of vesicles in a size range less than about 5 ⁇ , for example ⁇ 0.4 ⁇ .
  • the colloidal particles have an average particle size diameter of from about 0.03 to 0.4 microns ( ⁇ ), suitably around 0.1 microns ( ⁇ ).
  • Liposomes in this range can readily be sterilized by filtration through a suitable filter. Smaller vesicles also show less of a tendency to aggregate on storage, thus reducing potentially serious blockage or plugging problems when the liposome is injected intravenously or subcutaneously. Finally, liposomes which have been sized down to the submicron range show more uniform distribution.
  • Several techniques are available for reducing the sizes and size heterogeneity of liposomes, in a manner suitable for the present invention. Ultrasonic irradiation of a liposome suspension either by standard bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) between 0.02 and 0.08 ⁇ in size.
  • SUVs small unilamellar vesicles
  • Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones.
  • the liposome suspension is recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 ⁇ are observed.
  • the particle size distribution can be monitored by conventional laser-beam particle size determination.
  • Extrusion of liposomes through a small-pore polycarbonate filter or equivalent membrane is also an effective method for reducing liposome sizes down to a relatively well-defined size distribution whose average is in the range between about 0.02 and 5 ⁇ , depending on the pore size of the membrane.
  • the suspension is cycled through one or two stacked membranes several times until the desired liposome size distribution is achieved.
  • the liposome may be extruded through successively smaller pore membranes to achieve a gradual reduction in liposome size.
  • Centrifugation and molecular sieve chromatography are other methods which are available for producing a liposome suspension with particle sizes below a selected threshold less than 1 ⁇ . These two respective methods involve preferential removal of large liposomes, rather than conversion of large particles to smaller ones. Liposome yields are correspondingly reduced.
  • the size-processed liposome suspension may be readily sterilized by passage through a sterilizing membrane having a particle discrimination size of about 0.4 ⁇ , such as a conventional 0.45 ⁇ depth membrane filter.
  • the liposomes are stable in lyophilized form and can be reconstituted shortly before use by taking up in water.
  • Suitable lipids for forming liposomes are described above. Suitable examples include but are not limited to phospholipids such as dimirystoylphosphatidylcholine (DMPC) and/or dimirystoyl - phosphatidylglycerol (DMPG), egg and soybean derived phospholipids as obtained after partial or complete purification, directly or followed by partial or complete hydrogenation.
  • DMPC dimirystoylphosphatidylcholine
  • DMPG dimirystoyl - phosphatidylglycerol
  • egg and soybean derived phospholipids as obtained after partial or complete purification, directly or followed by partial or complete hydrogenation.
  • amphipathic substances such as lipids suitable for forming vesicles in water-immiscible organic solvents
  • dried amphipathic substances or mixtures thereof can be used in any form (powder, granular, etc.) directly,
  • step b) taking up the product of step b) into a solution of the biopolymeric substances in a physiologically compatible solution
  • step a) of Method A amphipathic substances suitable for forming vesicles as mentioned above are mixed in a water-immiscible organic solvent.
  • the water-immiscible organic solvent may be a polar-protic solvent such as fluorinated hydrocarbons, chlorinated hydrocarbons and the like.
  • step b) of the method of the invention the solvent is removed in presence of a solid support.
  • the solid support may be an inert organic or inorganic material having a bead-like structure.
  • the material of the inorganic support material may be glass and the organic material can be TeflonTM or other similar polymers.
  • the step c) of Method A of the invention is for taking up the product of step b) into a solution of the substances to be encapsulated in a physiologically compatible solution.
  • the physiological compatible solution may be equivalent to a sodium chloride solution up to about 1.5 by weight. It is also possible to use other salts as long as they are physiologically compatible e.g. as a cryoprotectant e.g., sugars and/or amino acids. For example, lactose, sucrose or trehalose may be used as a cryoprotectant.
  • step a) and b) a step of virus inactivation, sterilizing, depyrogenating, filtering the fraction or the like of step a) can be provided.
  • This might be advantageous in order to have a pharmaceutically acceptable solution at an early stage of the preparation.
  • the step d) of the Method A is adding an organic solvent having solubilizing or dispersing properties.
  • the organic solvent may be an organic polar-protic solvent miscible with water.
  • Lower aliphatic alcohols having 1 to 5 carbon atoms in the alkyl chain can also be used, such as tertiary butanol (tert-butanol).
  • virus inactivation sterilizing and/or portioning of the fraction yielded after step d) can be carried out.
  • the step e) of the present invention is drying the fraction obtained in step d) under conditions retaining the function of the substance to be loaded.
  • One method for drying the mixture is lyophilization.
  • the lyophilization may be carried out in presence of a cryoprotectant, for example, lactose or other saccharides or amino acids. Alternatively, evaporation or spray-drying can be used.
  • the dried residue can then be taken up in an aqueous medium prior to use. After taking up of the solid it forms a dispersion of the respective liposomes.
  • the aqueous medium may contain a saline solution and the dispersion formed can optionally be passed through a suitable filter in order to down size the liposomes if necessary.
  • the liposomes may have a size of 0.02 to 5 ⁇ , for example in the range of ⁇ 0.4 ⁇ .
  • compositions of the invention can also be an intermediate product obtainable by isolation of either fraction of step c) or d) of the method A.
  • the formulation of the invention also comprises an aqueous dispersion obtainable after taking up the product of step e) of method A in water in form of a dispersion (liposomes in aqueous medium).
  • compositions of the invention are also obtainable by the following methods which are referred to as Methods B, C, D and E.
  • This method comprises also the steps a), b) and c) of the Method A. However, step d) and e) of Method A are omitted.
  • step d) of method A is replaced by a freeze and thaw cycle which has to be repeated at least two times. This step is well-known in prior art to produce liposomes.
  • Method D excludes the use of any osmotic component.
  • method D the steps of preparation of vesicles, admixing and substantially salt free solution of the substances to be loaded and co-drying of the fractions thus obtained is involved.
  • Method E the steps of preparation of vesicles, admixing and substantially salt free solution of the substances to be loaded and co-drying of the fractions thus obtained is involved.
  • Method E is simpler than methods A - D described above. It requires dissolving the compounds used for liposome preparation (lipids antioxidants, etc.) in a polar-protic water miscible solvent such as tert.-butanol. This solution is then mixed with an aqueous solution or dispersion containing the blood factor. The mixing is performed at the optimum volume ratio required to maintain activity .
  • the invention also includes methods of treatment of a disease or condition in a subject in need of treatment comprising administering subcutaneously a composition or dosage as defined herein to a subject in need thereof.
  • Such methods may include a method of treatment of a disease or condition in a subject wherein the patient has developed antibodies (i.e. inhibitors) to a biologically active polypeptide or protein.
  • this aspect includes treatment of blood clotting diseases or trauma.
  • Blood clotting diseases or disorders may be characterised by a loss of function of a blood clotting factor, or the generation of auto-antibodies.
  • blood clotting diseases include haemophilia, such as haemophilia A and haemophilia B.
  • the present invention therefore extends to a composition as defined above for use in the treatment of a disease or condition, for example a blood clotting disease (e.g. haemophilia) or trauma.
  • a disease or condition for example a blood clotting disease (e.g. haemophilia) or trauma.
  • Uses of the invention in accordance with this aspect also include the use of a colloidal particle and a biologically active polypeptide or protein in the manufacture of a medicament as defined above for use in the treatment of a disease or condition.
  • Factor Vila can be used in the treatment of bleeding episodes in haemophilia A or B, or in treatment of patients who have developed inhibitory antibodies against FVIII or IX, respectively.
  • Factor VIII can be used in the treatment of bleeding episodes in patients with haemophilia A and Factor IX can be used in the treatment of patients with haemophilia B.
  • the term "treatment” includes any regime that can benefit a human or a non-human mammal.
  • the treatment of "non-human mammals” extends to the treatment of domestic mammals, including horses and companion animals (e.g. cats and dogs) and farm/agricultural animals including members of the ovine, caprine, porcine, bovine and equine families.
  • the treatment may be in respect of any existing condition or disorder, or may be prophylactic (preventive treatment).
  • the treatment may be of an inherited or an acquired disease.
  • the treatment may be of an acute or chronic condition. Where the disease or condition to be treated is a blood factor disease, the progress of the therapy can be monitored.
  • Levels of activity in the blood coagulation cascade may be measured by any suitable assay, for example the Whole Blood Clotting Time (WBCT) test or the Activated Partial Thromboplastin Time (APTT).
  • WBCT Whole Blood Clotting Time
  • APTT Activated Partial Thromboplastin Time
  • the Whole Blood Clotting Time (WBCT) test measures the time taken for whole blood to form a clot in an external environment, usually a glass tube or dish.
  • the Activated Partial Thromboplastin Time (APTT) test measures a parameter of part of the blood clotting pathway. It is abnormally elevated in haemophilia and by intravenous heparin therapy. The APTT requires a few millilitres of blood from a vein. The APTT time is a measure of one part of the clotting system known as the "intrinsic pathway". The APTT value is the time in seconds for a specific clotting process to occur in the laboratory test. This result is always compared to a "control" sample of normal blood. If the test sample takes longer than the control sample, it indicates decreased clotting function in the intrinsic pathway.
  • APTT Activated Partial Thromboplastin Time
  • General medical therapy usually aims for a range of APTT of the order of 45 to 70 seconds, but the value may also be expressed as a ratio of test to normal, for example 1.5 times normal.
  • a high APTT in the absence of heparin treatment can be due to haemophilia, which may require further testing.
  • the invention also provides a kit of parts comprising a composition of the invention, and an administration vehicle including injectable solutions for administration, said kit suitably comprising instructions for use thereof.
  • the invention therefore may also suitable provide a dosage form of a composition of the invention.
  • dosage forms may be provided as suitable containers or vials containing the appropriate dose for a patient.
  • compositions or dosage forms of the invention may be administered alone or in conjunction with other compounds, such as therapeutic compounds or molecules, e.g. anti-inflammatory drugs, analgesics or antibiotics, or other pharmaceutically active agents which may promote or enhance the activity of the biologically active polypeptide or protein.
  • other compounds such as therapeutic compounds or molecules, e.g. anti-inflammatory drugs, analgesics or antibiotics, or other pharmaceutically active agents which may promote or enhance the activity of the biologically active polypeptide or protein.
  • Such administration with other compounds may be simultaneous, separate or sequential.
  • the components may be prepared in the form of a kit which may comprise instructions as appropriate.
  • compositions of the invention allow for improved treatment of diseases where a blood factor is administered to treat a patient suffering from of a blood clotting disease or trauma.
  • composition comprising a colloidal particle comprising approximately 1-20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for topical administration and a blood factor for intravenous administration.
  • the blood factor can be suitable formulated in a pharmaceutically acceptable buffer, adjusted to physiological pH suitable for intravenous administration, wherein the blood factor is not encapsulated in said colloidal particle.
  • the dosage of the medicament of the invention is depending on the concentration of the effective biopolymeric substances as well as their efficiency.
  • a dosage up to 2.000 mg/liposomes lipid per kg body weight can be administered to patients.
  • the volume of the formulation for delivery into a patient may be no more than 2ml.
  • the delivery volume may be 5 ⁇ , 10 ⁇ , 25 ⁇ , 50 ⁇ , 10 ⁇ , 250 ⁇ , 500 ⁇ , 750 ⁇ , or 1 ml.
  • the volume of the formulation for delivery may be no more than 1.5ml, 2ml, 2.5ml, 3.0ml or 3.5ml.
  • the formulations of the invention may be for administration at least once per day, at least twice per day, about once per week, about twice per week, about once per two weeks, or about once per month.
  • a dosage regime of once per day will be sufficient, but for others a more frequent dosage regime may be more appropriate or desirable, where the amount delivered in each dosage administered subcutaneously may be reduced relative to a standard intravenous dosage.
  • a formulation of the invention may be administered once per day, twice per day (or more if required).
  • the dosage form of a blood clotting factor may be sufficient to maintain a whole blood clotting time in said subject of less than 15 minutes, or suitably, less than 12 minutes.
  • the dosage form of a blood clotting factor is an at least once per week dosage form, or at least once per month, at least once per two weeks, at least once per half week dosage form.
  • a dosage formulation according to the invention in which the dosage of the blood clotting factor is of from 1 to 1000 lU/kg, or from 5 to 500 lU/kg, or from 100 to 250 lU/kg or from 25 to 50 lU/kg. In other embodiments a dosage of a blood clotting factor of from 5 to 50 lU/kg may be used.
  • the formulations of specific embodiments of the invention wherein the formulation comprises a blood factor may comprise a dosage of from 25 to 50 lU/kg.
  • the dosage may be 25, 30, 35, 40, 45, or 50 lU/kg.
  • the dosage may be from 25IU/Kg to 30IU/Kg, 35IU/Kg to 40IU/Kg, or 40IU/Kg to 50IU/Kg.
  • colloidal particles for use according to the invention may be formulated for administration by any convenient route, such as subcutaneous, intravenous or topical administration.
  • a formulation suitable for topical, subcutaneous or intravenous administration may suitably be prepared as an aqueous or substantially aqueous formulation.
  • the formulation may comprise such additional salts, preservatives and stabilisers and/or excipients or adjuvants as required.
  • the dosage forms of the invention may be provided as anhydrous powders ready for extemporaneous formulation in a suitable aqueous medium.
  • Such dosage forms can be formulated as buffered aqueous formulations.
  • Suitable buffer solutions may include, but are not limited to amino acids (for example histidine), salts of inorganic acids and alkali metals or alkaline earth metals, (for example sodium salts, magnesium salts, potassium salts, lithium salts or calcium salts - exemplified as sodium chloride, sodium phosphate or sodium citrate).
  • Other components such as detergents or emulsifiers (for example, Tween 80 ® or any other form of Tween ® ) may be present and stabilisers (for example benzamidine or a benzamidine derivative). Excipients such as sugars, (for example sucrose) may also be present.
  • Suitable values for pH are physiological pH, e.g. pH 6.8 to 7.4 or pH 7.0. The pH may be adjusted accordingly with a suitable acid or alkali, for example hydrochloric acid.
  • Liquid dosage forms may be prepared ready for use in such administration vehicles, for example as 3.5 ml or 7.0ml vials.
  • composition for intravenous or subcutaneous administration for use in accordance with the invention as follows:
  • Formulations for topical administration may be formulated using a topical gel comprising one or more components selected from the group consisting of: surfactants, preservatives, thickeners, buffers, and water.
  • the surfactant may be a nonionic surfactant selected from the group consisting of: polyoxyethylene sorbitans, polyhydroxyethylene stearates or polyhydroxyethylene laurylethers, optionally the surfactant is polysorbate 80 (Tween 80).
  • the ratio of phospholipid to surfactant may be from 30:1 , to 15:1 , 10:1 , suitably 15:1 , 8: 1 , or 2:1 .
  • the surfactant concentration may be from 0.25% to 5% by weight, for example 1 % to 3%, 1 to 2%, some exemplary values may be 0.47%, 0.85%, or 3.5%. Examples of such formulations for topical administration are described in WO 2010/140061 and WO 201 1/022707. The invention will now be further described by way of reference to the following examples which are present for the purposes of illustration only and are not be taken as limitations to the invention:
  • the final preparation had a concentration of 100mM phospholipids.
  • a 15% w/v mixture of lipids was made with a 97:3 molar ratio of POPC:DSPE-2kPEG. The following were weighed and mixed:
  • the mixture was maintained at 35°C in a water bath and stirred intermittently until all solids had dissolved/dispersed.
  • the final material was a clear colourless mixture.
  • the mixture was frozen at - 80°C overnight.
  • the operation was maintained in a fume hood to allow containment during the post-use clean-up of dried/condensed solvent.
  • the Christ Alpha 1-2 LD freeze-drier and vacuum pump were warmed up for 20 minutes, and the frozen lipid/solvent mixture was removed from -80°C storage and dried overnight.
  • the dried lipids were recovered from the drier the following morning. They appeared as a dry crystalline cake. A 100mM lipid solution was required for further processing. The quantities of lipid present calculate through as around 82 moles of DSPE-2kPEG and 2.69mmoles of POPC; so around 2.77mmoles of lipids. Thus 27.7ml_ of diluent was required. 27.7ml_ of 50mM sodium citrate buffer was added to the dried lipids, and the resulting mixture was stirred and heated to around 35°C. After around 120 minutes, a white emulsion with no obvious large solids resulted. This was subjected to extrusion as below.
  • a Sartorius 47mm stainless steel pressure filtration housing was assembled and wrapped with a water jacket (wrapped tubing fed via a thermocirculator) maintained at 35°C.
  • the housing was fitted with a polycarbonate track-etched membrane (details below), covered by a glass-fibre prefilter (Whatman GF/D).
  • the emulsion was poured into the housing and extruded under 4 bar nitrogen gas, with the filtrate collected into 50ml_ tubes. The duration of each extrusion was timed and noted.
  • the filtration sequence was: ⁇ . ⁇ , 0.4 ⁇ , 0.2 ⁇ , 0.2 ⁇ , 0.1 ⁇ and 0.1 ⁇ (i.e. single passes through the larger filters and two passes through the smaller 0.2 and 0.1 ⁇ filters), with the filtrate warmed back to 35°C between passes.
  • the liposomes were extruded, with tabulated data is below:
  • the resulting extruded lipids were stored at +5°C. 15ml_ of 'Extruded Liposomes' were removed from the chilled stock and dispensed into a sterile 50ml_ tube within a MicroBiological Safety Cabinet. The size of the extruded liposomes was analysed using an ALV5000 photon correlation spectrometer. The average radius was determined to be 75.40 ⁇ 0.86nm and the average peak width 22.21 ⁇ 3.86nm, giving an average diameter of 150.80nm and polydispersity index of 0.087.
  • PEGylated Liposomes in citrate buffer are produced in accordance with Example 1 above according to the method of Baru ef al. (2005).
  • the liposome formulation has the following composition; 50mM sodium citrate pH 7.0 containing 100mM phospholipids; comprising a 97:3 molar ratio mixture of palmitoyl-oleoylphosphatidylcholine (POPC) and 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).
  • POPC palmitoyl-oleoylphosphatidylcholine
  • DSPE-PEG 2000 1,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000]
  • Exemplary topical formulations may be prepared according to the following:
  • Example 3 Treatment of haemophilia A comprising topical administration of PEGylated liposomes and subsequent administration of Factor VIII
  • the subject suffering from haemophilia A is dosed with PEGylated liposomes which are formulated for topical administration as described in accordance with Example 2 followed by administration of Factor VIII either subcutaneously or intravenously.
  • the dosage of Factor VIII is prepared according to the following calculation of the volume of drug to be administered:
  • Dose volume (ml) (a x b)/c Where: a is the target dose (100 lU/kg)
  • the subject is dosed with the colloidal particles of the invention topically, such as in a gel or patch.
  • the blood factor is dosed intravenously or subcutaneously as desired.
  • test subject Following dosing, the test subject is observed for clinical signs. Unexpected toxicities are screened for by performing CBC and serum chemistry tests at 48hr and 5 days post-dose. Fibrinogen, FDPs and the thrombin time (TT) are evaluated to test for increased thrombosis risk.
  • Pre-drug administration and at 0.5, 1 , 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours post-dose.
  • the activated Partial Thromboplastin Time (aPTT), Activated Clot Time (ACT) and Thromboelastogram (TEG) assays are conducted on the citrated whole blood.
  • Plasma is prepared by centrifugation of the remaining citrated blood and the resulting plasma samples are stored in aliquots of approximately 100 ⁇ at -80°C.
  • Blood samples are divided between 2 vacutubes, (2 X 0.5 ml) and observed carefully with periodic and judicious levelling of the tube until a clot was determined by interruption of flow in the fully horizontal position.
  • the quality of the clot is observed by holding the tube in the fully inverted position.
  • the whole blood clotting time was recorded as the mean of the total time from sample extraction until visual observation of blood clot for both samples and the quality of the clot in the inverted position is noted.
  • TEG is performed with re-calcified citrated whole blood using a Hemostasis Analyzer Model 5000 (Haemoscope Corporation) thromboelastograph according to the manufacturers' recommendations. Briefly, 1 ml of citrated whole blood is placed in a commercially available (Teg®Hemostasis System Kaolin, Haemonetics) vial containing kaolin. Mixing is ensured by gentle inversion of the kaolin-containing vials 5 times. Pins and cups are placed in the TEG analyzer in accordance with the standard procedure recommended by the manufacturer. Each standard TEG cup is placed in the 37° C pre-warmed instrument holder and is filled with 20 ⁇ of calcium chloride (0.2 M). Then, 340 ⁇ of kaolin-activated citrated whole blood is added for a total volume of 360 ⁇ .
  • a Hemostasis Analyzer Model 5000 Hemostasis Analyzer Model 5000 (Haemoscope Corporation) thromboelastograph according to the manufacturers'
  • Plasma FVIII activity assay (Chromoqenic)
  • FVIII plasma activity is determined using the Coatest Assay (Dia Pharma, West Chester, OH). Plasma samples are diluted 1 :20 to 1 :80 with assay diluent and assayed according to the manufacturer's instructions. Standard curves are established using normal hemostasis reference plasma (american diagnostica inc, Stamford, CT) and the purified FVIII protein.
  • the concentration of FVIII antigen in plasma samples is determined by ELISA using the Visulize FVIII antigen kit from Affinity Biologicals (Ancaster, Ontario, Canada) according to the manufacturer's instructions.
  • Bethesda assays are conducted on 1 :4, 1 : 10 and 1 :20 dilutions of test plasma into FVIII deficient human plasma. Equal volumes of the diluted test plasma and normal human reference plasma are incubated at 37°C for 2 hours and the Bethesda titre determined using the aPTT assay and a normal human plasma standard curve as described above.
  • Example 4 Formulation of PEGylated liposomes for intravenous (IV) or subcutaneous (SQ) administration
  • PEGylated Liposomes in citrate buffer were produced in accordance with Example 1 above according to the method of Baru ef al. (2005) comprising a mixture of palmitoyl- oleoylphosphatidylcholine (POPC) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly- (ethyleneglycol)-2000] (DSPE-MPEG 2000).
  • POPC palmitoyl- oleoylphosphatidylcholine
  • DSPE-MPEG 2000 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly- (ethyleneglycol)-2000]
  • Exemplary IV or SQ formulations may be prepared according to the following:
  • Example 5 Gel and spray formulations of PEGylated liposomes for topical administration with altered levels of surfactant
  • PEGylated Liposomes in citrate buffer were produced in accordance with Example 1 above according to the method of Baru ef al. (2005) comprising a mixture of soy phosphatidylcholine (SPC) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-MPEG 2000).
  • SPC soy phosphatidylcholine
  • DSPE-MPEG 2000 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000]

Abstract

The invention provides a composition comprising a biologically active polypeptide or protein and a colloidal particle comprising approximately 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for use in the treatment of a disease or condition wherein the colloidal particle is for topical administration prior to the biologically active polypeptide or protein which is for topical, intravenous or subcutaneous administration.

Description

COLLOIDAL PARTICLES FOR TOPICAL ADMINISTRATION WITH THERAPEUTIC AGENT
The present invention relates to topical formulations of colloidal particles for use in medicine in the therapy of a disease or condition, such as for example a blood factor disease.
Typically, blood factors have been prepared as pharmaceutical compositions for intravenous administration to treat blood factor diseases. The compositions have been based on the active protein, often conjugated to a polymer such as polyethylene glycol (PEG) to improve the half-life in circulation. Intravenous administration of PEGylated blood factors as therapeutic agents is therefore well understood and widely accepted. Liposomal formulations of naked (i.e. unconjugated and without modification) blood factors such as Factor VIII and Factor IX substances are known also, see for example WO 95/04524.
Pharmaceutical compositions comprising Factor VIII and liposomes modified by the presence of polyethylene glycol are described in WO 99/55306 in which the blood factor is not encapsulated in the liposome. However, the formulations are prepared for intravenous administration. Additional formulations of other proteins are described in WO 2004/091723 where the proteins include blood clotting factors. The proteins are said to bind to the liposomes in a non-covalent manner through interaction with the polyethylene glycol present on the surface of the liposomes. However, the formulations of blood clotting factors prepared according to the examples of this document are also for intravenous administration.
Other examples of formulations of blood factors, Factor VIII and Factor Vila, present as a conjugate with PEG are shown in WO 201 1/135307 and WO 201 1/135308 respectively where the actual formulations prepared were for intravenous administration. WO 2013/156488 also describes a dosage form of modified therapeutic agents, including blood factors such as Factor VIII (FVIII) and Factor Vila (FVIIa), for subcutaneous administration.
The blood factor Factor VIII has also been found to be capable of association with PEGylated liposomes, i.e. the blood factor is not encapsulated inside the liposome (Baru ef al Thromb. Haemost., 93, pages 1061-1068, (2005)). However, the compositions of FVIII were only prepared as formulations for intravenous administration.
Further studies by Peng ef al in The AAPS Journal, 14(1 ), pages 25-42 (201 1 ) disclose an alternative approach based on FVIII encapsulated in liposomes which are subsequently PEGylated by passively adding PEG to the liposomes after preparation. In one experiment in Peng et al the liposomal formulation is administered subcutaneously (SC) to investigate immunogenicity but there is no suggestion of a therapeutic purpose to this administration. In Peng ef al there is also a specific reference to the paper of Baru ef al (2005) and a statement that the approach of Baru ef al "exposed FVIII to plasma components such as proteases and IgGs". Liposomes prepared according to the method of Baru ef al (2005) containing recombinant Factor VIII have been administered intravenously to subjects (Spira ef a/ Blood, 108 (12), pages 3668-3673 (2012)).
According to the present invention there is provided a composition comprising a biologically active polypeptide or protein and a colloidal particle comprising approximately 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for use in the treatment of a disease or condition wherein the colloidal particles are for topical administration and the biologically active polypeptide or protein are for topical, intravenous or subcutaneous administration. The colloidal particles are suitable for topical administration and the biologically active polypeptide or protein is suitable for topical, intravenous or subcutaneous administration.
The biologically active polypeptide or protein may be selected from the group consisting of blood factors, hormones, growth factors, cytokines, antibodies and/or fragments thereof. Reference to polypeptide includes the term peptide and defines a series of amino acids linked by peptide bonds.
The colloidal particles may be administered separately, simultaneously or subsequently to the biologically active polypeptide or protein. The composition may therefore be a co-formulation of the colloidal particles and the biologically active polypeptide or protein, or the colloidal particles and the biologically active polypeptide or protein may be formulated for separate administration.
The colloidal particles of the present invention therefore provide for the protection and prolongation of the activity of biologically active polypeptides or proteins. The invention provides for the use of biologically active polypeptides or proteins without further derivatization and from any convenient source. The biologically active polypeptide or protein has an increased half-life and thereby has a longer duration of action after administration to a subject. Such combinations of the colloidal particles of the invention with biologically active polypeptides or proteins are also less immunogenic to a subject also. The invention also means that a subject may not be required to receive either as high a dose and/or as many injections as in conventional therapies in order to maintain a therapeutically effective circulating level of the biologically active polypeptide or protein.
Where the biologically active polypeptide or protein is a blood factor it may be selected from the group consisting of Factor VII, Factor Vila, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor Vila, Factor V, Factor XIII, von Willebrand's Factor, prothrombin, Protein C and/or fragments thereof. In some embodiments the blood coagulation factor may be suitably Factor VII, Factor VIII or Factor IX. The terms Factor Vila (FVIIa) and Factor VII (FVII) are also used interchangeably unless the context specifies otherwise. FVIII is used as an abbreviation for Factor VIII, FIX is used as an abbreviation for Factor IX, and so on for all the blood factors described herein mutatis mutandis. References to a blood clotting factor include a blood coagulation factor. Other factors, hormones and/or cytokines may be selected from the group consisting of (but not limited to) calcitonin, erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), thrombopoietin (TPO), alpha-1 proteinase inhibitor, granulocyte macrophage colony stimulating factor (GMCSF), growth hormone, heparin, human growth hormone (HGH), growth hormone releasing hormone (GHRH), interferon alpha, interferon beta, interferon gamma, interleukin-1 receptor, interleukin-2, interleukin-1 receptor antagonist, interleukin-3, interleukin-4, interleukin-6, luteinizing hormone releasing hormone (LHRH), insulin, pro-insulin, amylin, C-peptide, somatostatin, vasopressin, follicle stimulating hormone (FSH), insulin-like growth factor (IGF), insulintropin, macrophage colony stimulating factor (M-CSF), nerve growth factor (NGF), tissue growth factors, keratinocyte growth factor (KGF), glial growth factor (GGF), tumor necrosis factor (TNF), endothelial growth factors, parathyroid hormone (PTH), glucagon-like peptide (GLP) and/or fragments thereof.
The biologically active polypeptide or protein may also be an antibody or an antibody fragment for example; a single-domain antibody, VL, VH, Fab, F(ab')2, Fab', Fab3, scFv, di-scFv, sdAb, Fc and/or combinations thereof.
The biologically active polypeptide or protein may be used in a lyophilised form when preparing the composition.
The disease or condition may be characterised by a loss of function of the native biologically active polypeptide or protein in a subject. The biologically active polypeptide or protein may be absent or only present in low amounts. Such diseases include but are not limited to blood factor diseases, for example a haemophilia (haemophilia A, B or C), von Willebrand Disease, Factor V deficiency, Factor X deficiency, Factor XII deficiency, acromegaly, Addison's Disease, Cushing's Syndrome, De Quervain's Thyroiditis, obesity, diabetes mellitus (Type 1 diabetes or Type 2 diabetes), diabetes insipidous, Goiter, Graves' Disease, Growth Disorders, Growth Hormone Deficiency, Hashimoto's Thyroiditis, Hyperglycaemia, Hyperparathyroidism, Hypoglycaemia, Hypoparathyroidism, Low Testosterone, Menopause, Osteoporosis, Parathyroid Diseases, Pituitary Disorders, Polycystic Ovary Syndrome, Prediabetes, Turner Syndrome.
The colloidal particles may be substantially neutral and the polymer may carry substantially no net charge. The colloidal particles may have a mean particle diameter of between about 0.03 to about 0.4 microns (μιη), for example having a mean particle diameter of approximately 0.1 microns (μιη). A mean particle diameter in this range may increase the circulation time of the particles in vivo and prevent their adsorption by the reticuloendothelial system (RES). Where the composition comprises a fragment of a biologically active polypeptide or protein, the fragment may suitably be an active fragment thereof in which the fragment retains the biological activity, or substantially the same biological activity as the native polypeptide or protein.
It is further possible that the composition may comprise both the native polypeptide or protein and a fragment thereof.
The biologically active polypeptide or protein may also be co-administered with another therapeutically active compound or molecule, e.g. an anti-inflammatory drug, analgesic or antibiotic, or other pharmaceutically active agent which may promote or enhance the activity of the biologically active polypeptide or protein.
The biologically active polypeptide or protein may be from any suitable source and may be a recombinant protein produced by recombinant DNA technology using molecular biological techniques or synthesised chemically or produced transgenically in the milk of a mammal, or the biologically active polypeptide or protein may be isolated from natural sources (e.g. purified from blood plasma). Suitably the biologically active polypeptide or protein factor is mammalian, such as a human biologically active polypeptide or protein.
The colloidal particles of the invention are typically in the form of lipid vesicles or liposomes as are well known in the art. References to colloidal particles in the present specification include liposomes and lipid vesicles unless the context specifies otherwise.
In the colloidal particles, the amphipathic lipid may be a phospholipid from natural or synthetic sources. The amphipathic lipid may comprise approximately 0.5 to about 20 mole percent (%) of the particles, for example approximately about 1 to 20%, or about 1 to 6%, or about 3%.
An "amphipathic lipid" refers to a substance including a hydrophilic region and a hydrophobic region, such as phospholipids. Amphipathic lipids can be zwitterionic phospholipids, zwitterionic lipids, lipids having a net negative charge, and lipids having a net positive charge. For example, amphipathetic lipids include, but are not limited to, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, sphingomyelin, soybean lecithin (soya lecithin), egg lecithin, lysophosphatidylcholines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylserines, phosphatidylinositols, phosphatidic acids, cardiolipins, acyl trimethylammonium propane, diacyldimethylammonium propane, stearylamine, ethyl phosphatidylcholine and the like. Soya lecithin is a combination predominantly of naturally- occurring phospholipids; phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI).
Suitable examples of such amphipathic lipids include a phosphatidylethanolamine (PE), a carbamate-linked uncharged lipopolymer or aminopropanediol distearoyl (DS), or mixtures thereof. Examples of phosphatidylethanolamine phospholipids include 1 ,2-Dierucoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-Dilauroyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Dimyristoyl-sn- glycero-3-phosphoethanolamine, 1 ,2-Dioleoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-Dipalmitoyl- sn-glycero-3-phosphoethanolamine, 1 ,2-Distearoyl-sn-glycero-3-phosphoethanolamine, 1- Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine. A suitable example of phosphatidyl ethanolamine (PE) may be 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE). The purpose of the biocompatible hydrophilic polymer is to sterically stabilize the SUVs, thus preventing fusion of the vesicles in vitro, and allowing the vesicles to escape adsorption by the RES in vivo.
The colloidal particles may further comprise a second amphipathic lipid obtained from either natural or synthetic sources. The second amphipathic lipid may be a phosphatidylcholine (PC). Examples of phosphatidylcholine phospholipids include 1 ,2-Didecanoyl-sn-glycero-3-phosphocholine, 1 ,2- Dierucoyl-sn-glycero-3-phosphocholine, 1 ,2-Dilinoleoyl-sn-glycero-3-phosphocholine, 1 ,2-Dilauroyl- sn-glycero-3-phosphocholine, 1 ,2-Dimyristoyl-sn-glycero-3-phosphocholine, 1 ,2-Dioleoyl-sn- glycero-3-phosphocholine, 1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine, 1 ,2-Distearoyl-sn-glycero- 3-phosphocholine, 1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine, 1-Myristoyl-2-stearoyl-sn- glycero-3-phosphocholine, 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine, 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine, 1- Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine, 1-Stearoyl-2-oleoyl-sn-glycero-3- phosphocholine, 1-Stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine. A suitable example of phosphatidyl choline (PC) may be palmitoyl- oleoyl phosphatidyl choline (POPC) or soy phosphatidyl choline. Other natural sources of phosphatidylcholine include egg phosphatidylcholine. The phosphatidylcholine may be hydrogenated or non-hydrogenated.
In one embodiment, the pharmaceutical composition may be composed of colloidal particles which comprise palmitoyl- oleoyl phosphatidyl choline (POPC) and 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine (DSPE) in a molar ratio (POPC:DSPE) of from 85 to 99: 15 to 1. In some cases, the molar ratio of POPC:DSPE may be from 90 to 99: 10 to 1. In one embodiment, the molar ratio of POPC:DSPE may be 97:3. A molar ratio of 97:3 of these lipids is equivalent to a 97: 10 weight ratio.
In an alternative embodiment, the composition of the invention may be supplemented with cholesterol.
The biocompatible polymer may have a molecular weight of between about 500 to about 5000 Daltons, for example approximately 2000 Daltons.
The biocompatible hydrophilic polymer used according to the invention may be selected from the group consisting of polyalkylethers, polylactic acids and polyglycolic acids. The biocompatible hydrophilic polymer may be polyethylene glycol (PEG). The polyethylene glycol as used in the compositions of the invention may have a molecular weight of between about 500 to about 5000 Daltons, for example it may have a molecular weight of approximately 1000, 2000, or 3000 Daltons. In one embodiment the molecule weight of the PEG may be 2000 Daltons. The polyethylene glycol may be branched or unbranched.
An example of a suitable derivatized amphipathic lipid may be 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)]. If the PEG has a molecular weight of 2000 Daltons, the derivatized amphipathic lipid may be described as 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).
The concentration of the derivatized amphipathic lipid in the final formulation of colloidal particles may be adjusted accordingly to suit the desired properties of the composition. However, suitable concentrations may be from 5.0mg/ml_ to 15mg/ml_, for example 7.5mg/ml_ to 12.5mg/ml_, or 7.6mg/ml_ to 9.0mg/ml_. The overall content of lipid in the final formulation may be in the range of from 4% to 12%, for example 4%, 5%, 6%, 7%, 8%, 9%, 10%, 1 1 % or 12%.
The composition may comprise any suitable excipient, buffer and/or adjuvant and may be formulated as a pharmaceutical composition. Examples of such excipients, buffers and/or adjuvants, include phosphate buffered saline (PBS), potassium phosphate, sodium phosphate and/or sodium citrate. Other biological buffers can include PIPES, MOPS etc.
Suitable pH values for the composition include any generally acceptable pH values for administration in vivo, such as for example pH 5.0 to pH 9.0, suitably from pH 6.8 to pH 7.2, or pH 7.0.
The present inventors have surprisingly found that formulations of colloidal particles (liposomes) derivatized with a biocompatible polymer can be successfully administered and achieve a therapeutically effective dose when co-administered with a separate dosage of a biologically active polypeptide or protein to a subject suffering from a disease or condition, for example a blood factor disease, such as a haemophilia. Suitably, the biocompatible polymer is polyethylene glycol.
Without wishing to be bound by theory, it is believed that that the colloidal particles used in accordance with the present invention can potentiate the activity of a biologically active polypeptide or protein in a subject when administered prior to the biologically active polypeptide or protein.
A variety of known coupling reactions may be used for preparing vesicle forming lipids derivatized with hydrophilic polymers. For example, a polymer (such as PEG) may be derivatized to a lipid such as phosphatidylethanolamine (PE) through a cyanuric chloride group. Alternatively, a capped PEG may be activated with a carbonyl diimidazole coupling reagent, to form an activated imidazole compound. A carbamate-linked compound may be prepared by reacting the terminal hydroxyl of MPEG (methoxyPEG) with p-nitrophenyl chloroformate to yield a p-nitrophenyl carbonate. This product is then reacted with 1-amino-2,3-propanediol to yield the intermediate carbamate. The hydroxyl groups of the diol are acylated to yield the final product. A similar synthesis, using glycerol in place of 1-amino-2, 3-propanediol, can be used to produce a carbonate-linked product, as described in WO 01/05873. Other reactions are well known and are described, e.g. in US 5,013,556.
Colloidal particles (liposomes) can be classified according to various parameters. For example, when the size and number of lamellae (structural parameters) are used as the parameters then three major types of liposomes can be described: Multilamellar vesicles (MLV), small unilamellar vesicles (SUV) and large unilamellar vesicles (LW).
MLV are the species which form spontaneously on hydration of dried phospholipids above their gel to liquid crystalline phase transition temperature (cm). The size of the MLVs is heterogeneous and their structure resembles an onion skin of alternating, concentric aqueous and lipid layers.
SUV are formed from MLV by sonication or other methods such as extrusion, high pressure homogenisation or high shear mixing and are single layered. They are the smallest species with a high surface-to-volume ratio and hence have the lowest capture volume of aqueous space to weight of lipid.
The third type of liposome LUV has a large aqueous compartment and a single (unilamellar) or only a few (oligolamellar) lipid layers. Further details are disclosed in D. Lichtenberg and Y. Barenholz, in "Liposomes: Preparation, Characterization, and Preservation, in Methods of Biochemical Analysis", Vol. 33, pp. 337 - 462 (1988).
As used herein the term "loading" means any kind of interaction of the biopolymeric substances to be loaded, for example, an interaction such as encapsulation, adhesion (to the inner or outer wall of the vesicle) or embedding in the wall with or without extrusion of the biopolymeric substances.
As used herein and indicated above, the term "liposome" refers to colloidal particles and is intended to include all spheres or vesicles of any amphipathic compounds which may spontaneously or non-spontaneously vesiculate, for example phospholipids where at least one acyl group replaced by a complex phosphoric acid ester. The liposomes may be present in any physical state from the glassy state to liquid crystal. Most triacylglycerides are suitable and the most common phospholipids suitable for use in the present invention are the lecithins (also referred to as phosphatidylcholines (PC)), which are mixtures of the diglycerides of stearic, palmitic, and oleic acids linked to the choline ester of phosphoric acid. The lecithins are found in all animals and plants such as eggs, soybeans, and animal tissues (brain, heart, and the like) and can also be produced synthetically. The source of the phospholipid or its method of synthesis are not critical, any naturally occurring or synthetic phosphatide can be used.
Examples of specific phosphatides are L-a-(distearoyl) lecithin, L-a-(dipalmitoyl) lecithin, L-a- phosphatide acid, L-a-(dilauroyl)-phosphatidic acid, L-a(dimyristoyl) phosphatidic acid, L- a(dioleoyl)phosphatidic acid, DL-a (di- palmitoyl) phosphatidic acid, L-a(distearoyl) phosphatidic acid, and the various types of L-a-phosphatidylcholines prepared from brain, liver, egg yolk, heart, soybean and the like, or synthetically, and salts thereof. Other suitable modifications include the controlled peroxidation of the fatty acyl residue cross-linkers in the phosphatidylcholines (PC) and the zwitterionic amphipathates which form micelles by themselves or when mixed with the PCs such as alkyl analogues of PC.
The phospholipids can vary in purity and can also be hydrogenated either fully or partially. Hydrogenation reduces the level of unwanted peroxidation, and modifies and controls the gel to liquid/crystalline phase transition temperature (try) which effects packing and leakage.
The liposomes can be "tailored" to the requirements of any specific reservoir including various biological fluids, maintains their stability without aggregation or chromatographic separation, and remains well dispersed and suspended in the injected fluid. The fluidity in situ changes due to the composition, temperature, salinity, bivalent ions and presence of proteins. The liposome can be used with or without any other solvent or surfactant.
Generally suitable lipids may have an acyl chain composition which is characteristic, at least with respect to transition temperature (Tm) of the acyl chain components in egg or soybean PC, i.e., one chain saturated and one unsaturated or both being unsaturated. However, the possibility of using two saturated chains is not excluded.
The liposomes may contain other lipid components, as long as these do not induce instability and/or aggregation and/or chromatographic separation. This can be determined by routine experimentation.
The biocompatible hydrophilic polymer may be physically attached to the surface of the liposome, or inserted into the membrane of the liposome. The polymer may therefore be covalently bound to the liposome.
Either or both of the colloidal particle or the biologically active polypeptide or protein may be modified in order to modulate the dynamics of the association between the colloidal particle and the polypeptide or protein. Such modulation may be achieved by customising regions or binding sequences on the colloidal particle or the biologically active polypeptide or protein. A variety of methods for producing the modified liposomes which are unilamellar or multilamellar are known and available (see Lichtenberg and Barenholz, (1988)): 1. A thin film of the phospholipid is hydrated with an aqueous medium followed by mechanical shaking and/or ultrasonic irradiation and/or extrusion through a suitable filter;
2. Dissolution of the phospholipid in a suitable organic solvent, mixing with an aqueous medium followed by removal of the solvent;
3. Use of gas above its critical point (i.e., freons and other gases such as C02 or mixtures of C02 and other gaseous hydrocarbons) or
4. Preparing lipid detergent mixed micelles then lowering the concentration of the detergents to a level below its critical concentration at which liposomes are formed.
In general, such methods produce liposomes with heterogeneous sizes from about 0.02 to 10 m or greater. Since liposomes which are relatively small and well defined in size are preferred for use in the present invention, a second processing step defined as "liposome down-sizing" can be used for reducing the size and size heterogeneity of liposome suspensions.
The liposome suspension may be sized to achieve a selective size distribution of vesicles in a size range less than about 5 μιη, for example < 0.4 μιη. In one embodiment of the invention, the colloidal particles have an average particle size diameter of from about 0.03 to 0.4 microns (μιη), suitably around 0.1 microns (μιη).
Liposomes in this range can readily be sterilized by filtration through a suitable filter. Smaller vesicles also show less of a tendency to aggregate on storage, thus reducing potentially serious blockage or plugging problems when the liposome is injected intravenously or subcutaneously. Finally, liposomes which have been sized down to the submicron range show more uniform distribution. Several techniques are available for reducing the sizes and size heterogeneity of liposomes, in a manner suitable for the present invention. Ultrasonic irradiation of a liposome suspension either by standard bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) between 0.02 and 0.08 μιη in size. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedure the liposome suspension is recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 μιη are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size determination. Extrusion of liposomes through a small-pore polycarbonate filter or equivalent membrane is also an effective method for reducing liposome sizes down to a relatively well-defined size distribution whose average is in the range between about 0.02 and 5 μιη, depending on the pore size of the membrane.
Typically, the suspension is cycled through one or two stacked membranes several times until the desired liposome size distribution is achieved. The liposome may be extruded through successively smaller pore membranes to achieve a gradual reduction in liposome size.
Centrifugation and molecular sieve chromatography are other methods which are available for producing a liposome suspension with particle sizes below a selected threshold less than 1 μιη. These two respective methods involve preferential removal of large liposomes, rather than conversion of large particles to smaller ones. Liposome yields are correspondingly reduced.
The size-processed liposome suspension may be readily sterilized by passage through a sterilizing membrane having a particle discrimination size of about 0.4 μιη, such as a conventional 0.45 μιη depth membrane filter. The liposomes are stable in lyophilized form and can be reconstituted shortly before use by taking up in water.
Suitable lipids for forming liposomes are described above. Suitable examples include but are not limited to phospholipids such as dimirystoylphosphatidylcholine (DMPC) and/or dimirystoyl - phosphatidylglycerol (DMPG), egg and soybean derived phospholipids as obtained after partial or complete purification, directly or followed by partial or complete hydrogenation.
The following four methods are described in WO 95/04524 and are generally suitable for the preparation of the colloidal particles (liposomes) used in accordance with the present invention.
Method A
- a) mixing amphipathic substances, such as lipids suitable for forming vesicles in water-immiscible organic solvents
- b) removing of the solvent in presence of a solid support, alternatively, dried amphipathic substances or mixtures thereof can be used in any form (powder, granular, etc.) directly,
- c) taking up the product of step b) into a solution of the biopolymeric substances in a physiologically compatible solution
- d) adding an organic solvent having solubilizing or dispersing properties, as well as
- e) drying the fraction obtained in step d) under conditions retaining the function of the biopolymeric substances. According to step a) of Method A amphipathic substances suitable for forming vesicles as mentioned above are mixed in a water-immiscible organic solvent. The water-immiscible organic solvent may be a polar-protic solvent such as fluorinated hydrocarbons, chlorinated hydrocarbons and the like. In step b) of the method of the invention the solvent is removed in presence of a solid support. The solid support may be an inert organic or inorganic material having a bead-like structure. The material of the inorganic support material may be glass and the organic material can be Teflon™ or other similar polymers. The step c) of Method A of the invention is for taking up the product of step b) into a solution of the substances to be encapsulated in a physiologically compatible solution.
The physiological compatible solution may be equivalent to a sodium chloride solution up to about 1.5 by weight. It is also possible to use other salts as long as they are physiologically compatible e.g. as a cryoprotectant e.g., sugars and/or amino acids. For example, lactose, sucrose or trehalose may be used as a cryoprotectant.
Optionally, between step a) and b) a step of virus inactivation, sterilizing, depyrogenating, filtering the fraction or the like of step a) can be provided. This might be advantageous in order to have a pharmaceutically acceptable solution at an early stage of the preparation.
The step d) of the Method A is adding an organic solvent having solubilizing or dispersing properties. The organic solvent may be an organic polar-protic solvent miscible with water. Lower aliphatic alcohols having 1 to 5 carbon atoms in the alkyl chain can also be used, such as tertiary butanol (tert-butanol).
Optionally, subsequent to step d), virus inactivation sterilizing and/or portioning of the fraction yielded after step d) can be carried out.
The step e) of the present invention is drying the fraction obtained in step d) under conditions retaining the function of the substance to be loaded. One method for drying the mixture is lyophilization. The lyophilization may be carried out in presence of a cryoprotectant, for example, lactose or other saccharides or amino acids. Alternatively, evaporation or spray-drying can be used.
The dried residue can then be taken up in an aqueous medium prior to use. After taking up of the solid it forms a dispersion of the respective liposomes. The aqueous medium may contain a saline solution and the dispersion formed can optionally be passed through a suitable filter in order to down size the liposomes if necessary. Suitably, the liposomes may have a size of 0.02 to 5 μιη, for example in the range of < 0.4 μιη.
The compositions of the invention can also be an intermediate product obtainable by isolation of either fraction of step c) or d) of the method A. Accordingly, the formulation of the invention also comprises an aqueous dispersion obtainable after taking up the product of step e) of method A in water in form of a dispersion (liposomes in aqueous medium).
Alternatively, the pharmaceutical compositions of the invention are also obtainable by the following methods which are referred to as Methods B, C, D and E.
Method B
This method comprises also the steps a), b) and c) of the Method A. However, step d) and e) of Method A are omitted.
Method C
In Method C step d) of method A is replaced by a freeze and thaw cycle which has to be repeated at least two times. This step is well-known in prior art to produce liposomes. Method D
Method D excludes the use of any osmotic component. In method D the steps of preparation of vesicles, admixing and substantially salt free solution of the substances to be loaded and co-drying of the fractions thus obtained is involved. Method E
Method E is simpler than methods A - D described above. It requires dissolving the compounds used for liposome preparation (lipids antioxidants, etc.) in a polar-protic water miscible solvent such as tert.-butanol. This solution is then mixed with an aqueous solution or dispersion containing the blood factor. The mixing is performed at the optimum volume ratio required to maintain activity .
The mixture is then lyophilized in the presence or absence of cryoprotectant. Rehydration is required before the use of the liposomal formulation. These liposomes are multilamellar, their downsizing can be achieved by one of the methods described in WO 95/04524. The invention also includes methods of treatment of a disease or condition in a subject in need of treatment comprising administering subcutaneously a composition or dosage as defined herein to a subject in need thereof. Such methods may include a method of treatment of a disease or condition in a subject wherein the patient has developed antibodies (i.e. inhibitors) to a biologically active polypeptide or protein. As an example, this aspect includes treatment of blood clotting diseases or trauma. Blood clotting diseases or disorders may be characterised by a loss of function of a blood clotting factor, or the generation of auto-antibodies. Examples of blood clotting diseases include haemophilia, such as haemophilia A and haemophilia B.
The present invention therefore extends to a composition as defined above for use in the treatment of a disease or condition, for example a blood clotting disease (e.g. haemophilia) or trauma. Uses of the invention in accordance with this aspect also include the use of a colloidal particle and a biologically active polypeptide or protein in the manufacture of a medicament as defined above for use in the treatment of a disease or condition.
As an example, Factor Vila can be used in the treatment of bleeding episodes in haemophilia A or B, or in treatment of patients who have developed inhibitory antibodies against FVIII or IX, respectively. Factor VIII can be used in the treatment of bleeding episodes in patients with haemophilia A and Factor IX can be used in the treatment of patients with haemophilia B.
As used herein, the term "treatment" includes any regime that can benefit a human or a non-human mammal. The treatment of "non-human mammals" extends to the treatment of domestic mammals, including horses and companion animals (e.g. cats and dogs) and farm/agricultural animals including members of the ovine, caprine, porcine, bovine and equine families. The treatment may be in respect of any existing condition or disorder, or may be prophylactic (preventive treatment). The treatment may be of an inherited or an acquired disease. The treatment may be of an acute or chronic condition. Where the disease or condition to be treated is a blood factor disease, the progress of the therapy can be monitored. Levels of activity in the blood coagulation cascade may be measured by any suitable assay, for example the Whole Blood Clotting Time (WBCT) test or the Activated Partial Thromboplastin Time (APTT). The Whole Blood Clotting Time (WBCT) test measures the time taken for whole blood to form a clot in an external environment, usually a glass tube or dish.
The Activated Partial Thromboplastin Time (APTT) test measures a parameter of part of the blood clotting pathway. It is abnormally elevated in haemophilia and by intravenous heparin therapy. The APTT requires a few millilitres of blood from a vein. The APTT time is a measure of one part of the clotting system known as the "intrinsic pathway". The APTT value is the time in seconds for a specific clotting process to occur in the laboratory test. This result is always compared to a "control" sample of normal blood. If the test sample takes longer than the control sample, it indicates decreased clotting function in the intrinsic pathway. General medical therapy usually aims for a range of APTT of the order of 45 to 70 seconds, but the value may also be expressed as a ratio of test to normal, for example 1.5 times normal. A high APTT in the absence of heparin treatment can be due to haemophilia, which may require further testing.
The invention also provides a kit of parts comprising a composition of the invention, and an administration vehicle including injectable solutions for administration, said kit suitably comprising instructions for use thereof.
The invention therefore may also suitable provide a dosage form of a composition of the invention. Such dosage forms may be provided as suitable containers or vials containing the appropriate dose for a patient.
The compositions or dosage forms of the invention may be administered alone or in conjunction with other compounds, such as therapeutic compounds or molecules, e.g. anti-inflammatory drugs, analgesics or antibiotics, or other pharmaceutically active agents which may promote or enhance the activity of the biologically active polypeptide or protein. Such administration with other compounds may be simultaneous, separate or sequential. The components may be prepared in the form of a kit which may comprise instructions as appropriate.
The compositions of the invention allow for improved treatment of diseases where a blood factor is administered to treat a patient suffering from of a blood clotting disease or trauma.
In one embodiment of the invention there is provided a composition comprising a colloidal particle comprising approximately 1-20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for topical administration and a blood factor for intravenous administration. The blood factor can be suitable formulated in a pharmaceutically acceptable buffer, adjusted to physiological pH suitable for intravenous administration, wherein the blood factor is not encapsulated in said colloidal particle.
It is understood by the skilled person that the dosage of the medicament of the invention is depending on the concentration of the effective biopolymeric substances as well as their efficiency.
A dosage up to 2.000 mg/liposomes lipid per kg body weight can be administered to patients.
Accordingly, in another aspect of the present invention, the volume of the formulation for delivery into a patient may be no more than 2ml. Suitably, the delivery volume may be 5μΙ, 10μΙ, 25μΙ, 50μΙ, 10ΟμΙ, 250μΙ, 500μΙ, 750μΙ, or 1 ml. In alternative embodiments the volume of the formulation for delivery may be no more than 1.5ml, 2ml, 2.5ml, 3.0ml or 3.5ml. The formulations of the invention may be for administration at least once per day, at least twice per day, about once per week, about twice per week, about once per two weeks, or about once per month. For certain therapeutic substances, a dosage regime of once per day will be sufficient, but for others a more frequent dosage regime may be more appropriate or desirable, where the amount delivered in each dosage administered subcutaneously may be reduced relative to a standard intravenous dosage. So for example a formulation of the invention may be administered once per day, twice per day (or more if required).
Suitably, the dosage form of a blood clotting factor may be sufficient to maintain a whole blood clotting time in said subject of less than 15 minutes, or suitably, less than 12 minutes. In an embodiment, the dosage form of a blood clotting factor is an at least once per week dosage form, or at least once per month, at least once per two weeks, at least once per half week dosage form.
Also provided is a dosage formulation according to the invention, in which the dosage of the blood clotting factor is of from 1 to 1000 lU/kg, or from 5 to 500 lU/kg, or from 100 to 250 lU/kg or from 25 to 50 lU/kg. In other embodiments a dosage of a blood clotting factor of from 5 to 50 lU/kg may be used.
The formulations of specific embodiments of the invention wherein the formulation comprises a blood factor may comprise a dosage of from 25 to 50 lU/kg. In some embodiments the dosage may be 25, 30, 35, 40, 45, or 50 lU/kg. The dosage may be from 25IU/Kg to 30IU/Kg, 35IU/Kg to 40IU/Kg, or 40IU/Kg to 50IU/Kg.
The colloidal particles for use according to the invention may be formulated for administration by any convenient route, such as subcutaneous, intravenous or topical administration.
A formulation suitable for topical, subcutaneous or intravenous administration may suitably be prepared as an aqueous or substantially aqueous formulation. The formulation may comprise such additional salts, preservatives and stabilisers and/or excipients or adjuvants as required. The dosage forms of the invention may be provided as anhydrous powders ready for extemporaneous formulation in a suitable aqueous medium. Suitably such dosage forms can be formulated as buffered aqueous formulations. Suitable buffer solutions may include, but are not limited to amino acids (for example histidine), salts of inorganic acids and alkali metals or alkaline earth metals, (for example sodium salts, magnesium salts, potassium salts, lithium salts or calcium salts - exemplified as sodium chloride, sodium phosphate or sodium citrate). Other components such as detergents or emulsifiers (for example, Tween 80® or any other form of Tween®) may be present and stabilisers (for example benzamidine or a benzamidine derivative). Excipients such as sugars, (for example sucrose) may also be present. Suitable values for pH are physiological pH, e.g. pH 6.8 to 7.4 or pH 7.0. The pH may be adjusted accordingly with a suitable acid or alkali, for example hydrochloric acid. Liquid dosage forms may be prepared ready for use in such administration vehicles, for example as 3.5 ml or 7.0ml vials.
In one particular embodiment, there is provided a composition for intravenous or subcutaneous administration for use in accordance with the invention as follows:
- 50mM sodium citrate
- pH 7.0
- 100mM phospholipids - 97:3 molar ratio of palmitoyl- oleoyl phosphatidyl choline (POPC) and 1 ,2- distearoyl-sn-glycero-3phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000). Formulations for topical administration may be formulated using a topical gel comprising one or more components selected from the group consisting of: surfactants, preservatives, thickeners, buffers, and water. In one embodiment, the surfactant may be a nonionic surfactant selected from the group consisting of: polyoxyethylene sorbitans, polyhydroxyethylene stearates or polyhydroxyethylene laurylethers, optionally the surfactant is polysorbate 80 (Tween 80). The ratio of phospholipid to surfactant may be from 30:1 , to 15:1 , 10:1 , suitably 15:1 , 8: 1 , or 2:1 . The surfactant concentration may be from 0.25% to 5% by weight, for example 1 % to 3%, 1 to 2%, some exemplary values may be 0.47%, 0.85%, or 3.5%. Examples of such formulations for topical administration are described in WO 2010/140061 and WO 201 1/022707. The invention will now be further described by way of reference to the following examples which are present for the purposes of illustration only and are not be taken as limitations to the invention:
Example 1 : Synthesis of liposomes
Mixed lipids were prepared from palmitoyl- oleoyl phosphatidyl choline (POPC) and 1 ,2- distearoyl- sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] derivatized with PEG-2000 (PEG with molecular weight 2000 Daltons) (DSPE-PEG 2000), as follows:
Molecular weight of POPC: 760.08g/mol
Molecular weight of DSPE-2kPEG: 2789.5g/mol
The final preparation had a concentration of 100mM phospholipids. A 15% w/v mixture of lipids was made with a 97:3 molar ratio of POPC:DSPE-2kPEG. The following were weighed and mixed:
2.04g POPC
0.232g DSPE-2kPEG 14.9ml_ tert-butanol (melted in a 35°C water bath), all placed in a 100ml_ Schott bottle.
The mixture was maintained at 35°C in a water bath and stirred intermittently until all solids had dissolved/dispersed. The final material was a clear colourless mixture. The mixture was frozen at - 80°C overnight.
The operation was maintained in a fume hood to allow containment during the post-use clean-up of dried/condensed solvent. The Christ Alpha 1-2 LD freeze-drier and vacuum pump were warmed up for 20 minutes, and the frozen lipid/solvent mixture was removed from -80°C storage and dried overnight.
The dried lipids were recovered from the drier the following morning. They appeared as a dry crystalline cake. A 100mM lipid solution was required for further processing. The quantities of lipid present calculate through as around 82 moles of DSPE-2kPEG and 2.69mmoles of POPC; so around 2.77mmoles of lipids. Thus 27.7ml_ of diluent was required. 27.7ml_ of 50mM sodium citrate buffer was added to the dried lipids, and the resulting mixture was stirred and heated to around 35°C. After around 120 minutes, a white emulsion with no obvious large solids resulted. This was subjected to extrusion as below. A Sartorius 47mm stainless steel pressure filtration housing was assembled and wrapped with a water jacket (wrapped tubing fed via a thermocirculator) maintained at 35°C. The housing was fitted with a polycarbonate track-etched membrane (details below), covered by a glass-fibre prefilter (Whatman GF/D). The emulsion was poured into the housing and extruded under 4 bar nitrogen gas, with the filtrate collected into 50ml_ tubes. The duration of each extrusion was timed and noted.
The filtration sequence was: Ο.δμιη, 0.4μιη, 0.2μιη, 0.2μιη, 0.1 μιη and 0.1 μιη (i.e. single passes through the larger filters and two passes through the smaller 0.2 and 0.1 μιη filters), with the filtrate warmed back to 35°C between passes. The liposomes were extruded, with tabulated data is below:
Table 1
Figure imgf000018_0001
The resulting extruded lipids were stored at +5°C. 15ml_ of 'Extruded Liposomes' were removed from the chilled stock and dispensed into a sterile 50ml_ tube within a MicroBiological Safety Cabinet. The size of the extruded liposomes was analysed using an ALV5000 photon correlation spectrometer. The average radius was determined to be 75.40 ± 0.86nm and the average peak width 22.21 ± 3.86nm, giving an average diameter of 150.80nm and polydispersity index of 0.087.
Example 2: Formulation of PEGylated liposomes for topical administration
PEGylated Liposomes in citrate buffer are produced in accordance with Example 1 above according to the method of Baru ef al. (2005). The liposome formulation has the following composition; 50mM sodium citrate pH 7.0 containing 100mM phospholipids; comprising a 97:3 molar ratio mixture of palmitoyl-oleoylphosphatidylcholine (POPC) and 1 ,2-distearoyl-sn-glycero-3- phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000).
Exemplary topical formulations may be prepared according to the following:
Table 2
Figure imgf000019_0001
Example 3: Treatment of haemophilia A comprising topical administration of PEGylated liposomes and subsequent administration of Factor VIII According to the invention, the subject suffering from haemophilia A is dosed with PEGylated liposomes which are formulated for topical administration as described in accordance with Example 2 followed by administration of Factor VIII either subcutaneously or intravenously. The dosage of Factor VIII is prepared according to the following calculation of the volume of drug to be administered:
Dose volume (ml) = (a x b)/c Where: a is the target dose (100 lU/kg)
b is the weight of the dog (kg)
c is the rFVIII activity (150 lU/ml)
The subject is dosed with the colloidal particles of the invention topically, such as in a gel or patch. The blood factor is dosed intravenously or subcutaneously as desired.
Following dosing, the test subject is observed for clinical signs. Unexpected toxicities are screened for by performing CBC and serum chemistry tests at 48hr and 5 days post-dose. Fibrinogen, FDPs and the thrombin time (TT) are evaluated to test for increased thrombosis risk.
Blood samples (5 ml) are taken from the subject dosed SQ at the following times points after administration:
Pre-drug administration and at 0.5, 1 , 2, 4, 8, 12, 24, 36, 48, 60, 72, 84, 96, 108 and 120 hours post-dose.
Whole blood (non-citrated; 1 ml) is used for the whole blood clotting assay and the activated clotting time assay. The remaining 4ml blood samples are transferred into tubes containing 0.109M tri-sodium citrate anticoagulant (9: 1 v/v) on ice.
The activated Partial Thromboplastin Time (aPTT), Activated Clot Time (ACT) and Thromboelastogram (TEG) assays are conducted on the citrated whole blood.
Plasma is prepared by centrifugation of the remaining citrated blood and the resulting plasma samples are stored in aliquots of approximately 100 μΙ at -80°C.
Assays
(i) Non-citrated whole blood: Whole blood clotting assay
Blood samples are divided between 2 vacutubes, (2 X 0.5 ml) and observed carefully with periodic and judicious levelling of the tube until a clot was determined by interruption of flow in the fully horizontal position. The quality of the clot is observed by holding the tube in the fully inverted position. The whole blood clotting time was recorded as the mean of the total time from sample extraction until visual observation of blood clot for both samples and the quality of the clot in the inverted position is noted.
(ii) Citrated whole blood: Thromboelastoqram (TEG) assay
TEG is performed with re-calcified citrated whole blood using a Hemostasis Analyzer Model 5000 (Haemoscope Corporation) thromboelastograph according to the manufacturers' recommendations. Briefly, 1 ml of citrated whole blood is placed in a commercially available (Teg®Hemostasis System Kaolin, Haemonetics) vial containing kaolin. Mixing is ensured by gentle inversion of the kaolin-containing vials 5 times. Pins and cups are placed in the TEG analyzer in accordance with the standard procedure recommended by the manufacturer. Each standard TEG cup is placed in the 37° C pre-warmed instrument holder and is filled with 20 μΙ of calcium chloride (0.2 M). Then, 340 μΙ of kaolin-activated citrated whole blood is added for a total volume of 360 μΙ.
(iii) Activated Clotting Time (ACT) and activated Partial Thromboplastin Time (aPTT)
The ACT and aPTT tests are carried out using a Haemachron Jr coagulation analyzer (International Technidyne Corps.) according to the manufacturer's instructions. (iv) Plasma: FVIII activity assay (Chromoqenic)
FVIII plasma activity is determined using the Coatest Assay (Dia Pharma, West Chester, OH). Plasma samples are diluted 1 :20 to 1 :80 with assay diluent and assayed according to the manufacturer's instructions. Standard curves are established using normal hemostasis reference plasma (american diagnostica inc, Stamford, CT) and the purified FVIII protein.
(v) Plasma: FVIII ELISA
The concentration of FVIII antigen in plasma samples is determined by ELISA using the Visulize FVIII antigen kit from Affinity Biologicals (Ancaster, Ontario, Canada) according to the manufacturer's instructions.
(vi) Plasma: Immunoqenicity
Bethesda assays are conducted on 1 :4, 1 : 10 and 1 :20 dilutions of test plasma into FVIII deficient human plasma. Equal volumes of the diluted test plasma and normal human reference plasma are incubated at 37°C for 2 hours and the Bethesda titre determined using the aPTT assay and a normal human plasma standard curve as described above.
Example 4: Formulation of PEGylated liposomes for intravenous (IV) or subcutaneous (SQ) administration
PEGylated Liposomes in citrate buffer were produced in accordance with Example 1 above according to the method of Baru ef al. (2005) comprising a mixture of palmitoyl- oleoylphosphatidylcholine (POPC) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly- (ethyleneglycol)-2000] (DSPE-MPEG 2000).
Exemplary IV or SQ formulations may be prepared according to the following:
Table 3
Figure imgf000022_0001
q.s.(1 ) - Quantity sufficient to adjust pH to 6.9 q s. (2) - Quantity sufficient to adjust volume to 1 mL
Example 5: Gel and spray formulations of PEGylated liposomes for topical administration with altered levels of surfactant
PEGylated Liposomes in citrate buffer were produced in accordance with Example 1 above according to the method of Baru ef al. (2005) comprising a mixture of soy phosphatidylcholine (SPC) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-MPEG 2000). The formulations were prepared to test different physical forms as a gel or spray for topical administration.
Table 4
Figure imgf000022_0002
Figure imgf000023_0001
Quantity sufficient to adjust volume to 1mL.

Claims

1. A composition comprising a biologically active polypeptide or protein and a colloidal particle comprising approximately 0.5 to 20 mole percent of an amphipathic lipid derivatized with a biocompatible hydrophilic polymer for use in the treatment of a disease or condition wherein the colloidal particle is for topical administration prior to the biologically active polypeptide or protein which is for topical, intravenous or subcutaneous administration.
2. The composition of claim 1 wherein the colloidal particles are substantially neutral and the polymer carries substantially no net charge.
3. The composition of claim 1 wherein the colloidal particle has a mean particle diameter of between about 0.03 to about 0.4 microns (μιη). 4. The composition of claim 3 wherein the colloidal particle has a mean particle diameter of approximately 0.1 microns (μιη).
5. The composition of any of claims 1 to 4 wherein said amphipathic lipid is a phospholipid from natural or synthetic sources.
6. The composition of claim 5 wherein said amphipathic lipid is a phosphatidylethanolamine (PE).
7. The composition of any of claims 1 to 4 wherein said amphipathic lipid is a carbamate- linked uncharged lipopolymer.
8. The composition of claim 7 wherein said amphipathic lipid is aminopropanediol distearoyl (DS). 9. The composition of claim 1 wherein said colloidal particles further comprise a second amphipathic lipid obtained from either natural or synthetic sources.
10. The composition of claim 9 wherein said second amphipathic lipid is a phosphatidylcholine. 1 1. The composition of claim 10 wherein the colloidal particle comprises palmitoyl- oleoyl phosphatidyl choline (POPC) and 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine (DSPE) in a ratio (POPC:DSPE) of from 85 to 99:15 to 1 .
The composition of claim 1 1 wherein the ratio of POPC:DPSE is from 90 to 99: 10 to 1
13. The composition of claim 12 wherein the ratio of POPC:DPSE is 97:3.
14. The composition of claim 9 wherein cholesterol is supplemented to the composition. 15. The composition of any one of claims 1 to 14 wherein said biocompatible hydrophilic polymer is selected from the group consisting of polyalkylethers, polylactic acids and polyglycolic acids.
16. The composition of claim 15 wherein said biocompatible hydrophilic polymer is polyethylene glycol.
17. The composition of claim 16 wherein the polyethylene glycol has a molecular weight of between about 500 to about 5000 Daltons. 18. The composition of claim 17 wherein the polyethylene glycol has a molecular weight of approximately 2000 Daltons.
19. The composition of any one of claims 16 to 18 wherein the derivatized amphipathic lipid is 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)].
20. The composition of any one of claims 16 to 18 wherein the derivatized amphipathic lipid is 1 ,2-distearoyl-sn-glycero-3-phosphoethanol-amine-N-[poly-(ethyleneglycol)-2000] (DSPE-PEG 2000). 21. The composition of any one of claims 1 to 20 wherein the biologically active polypeptide or protein is selected from the group consisting of blood factors, hormones, growth factors, cytokines, antibodies and/or fragments thereof.
22. The composition of claim 21 wherein the blood factor is selected from the group consisting of Factor Vila, Factor VII, Factor VIII, Factor IX, Factor X, Factor Xa, Factor XI, Factor V, Factor
XIII, von Willebrand's Factor (vWF), prothrombin or Protein C and/or a fragment thereof.
23. The composition of any one of claims 1 to 22 wherein the composition additionally comprises another therapeutically active compound.
24. The composition of any one of claims 1 to 23 wherein the disease is a blood factor disease.
25. A method of treatment of a patient suffering from a disease or condition comprising subcutaneously administrating to said patient a composition as claimed in any one of claims 1 to 24. 26. A kit of parts comprising a composition of any one of claims 1 to 24 and administration vehicle including injectable solutions for intravenous or subcutaneous administration.
27. A dosage form of a composition of any one of claims 1 to 24.
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