WO2007085413A2 - Traitement d'un accident ischémique transitoire - Google Patents

Traitement d'un accident ischémique transitoire Download PDF

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Publication number
WO2007085413A2
WO2007085413A2 PCT/EP2007/000540 EP2007000540W WO2007085413A2 WO 2007085413 A2 WO2007085413 A2 WO 2007085413A2 EP 2007000540 W EP2007000540 W EP 2007000540W WO 2007085413 A2 WO2007085413 A2 WO 2007085413A2
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Prior art keywords
liposomes
phosphate
bodies
group
glycerol
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PCT/EP2007/000540
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English (en)
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Anthony Ernest Bolton
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Vasogen Ireland Limited
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
    • A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/683—Diesters of a phosphorus acid with two hydroxy compounds, e.g. phosphatidylinositols
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • This invention relates to medical treatments, compositions for use therein and the preparation and manufacture of compositions for medical treatment. More specifically, it relates to treatment of and prophylaxis against transient ischaemic attacks (TIAs), and compositions useful therein.
  • TIAs transient ischaemic attacks
  • TIAs are transient ischaemic events affecting parts of the brain and they last only a few minutes. They occur when the blood supply to part of the brain is briefly interrupted.
  • the symptoms of a TIA are very similar to those of a stroke, and include weakness or numbness in the face, arm or leg, especially on one side of the body, speech impairment (aphasia), confusion, vision impairment in one or both eyes, and loss of balance and co-ordination.
  • speech impairment aphasia
  • confusion vision impairment in one or both eyes
  • loss of balance and co-ordination unlike following stroke, generally most symptoms disappear within one hour, but may persist for up to 24 hours. Since the symptoms are difficult to distinguish from those of an acute stroke, immediate treatment is usually conducted as an emergency measure as if the incident were indeed an acute stroke, without waiting to see if the symptoms go away.
  • a physician may prescribe anti-platelet or anticoagulant drugs (e.g. aspirin, warfarin, ticlodipine) and/or drugs to reduce blood pressure or lipid levels.
  • anti-platelet or anticoagulant drugs e.g. aspirin, warfarin, ticlodipine
  • drugs e.g. aspirin, warfarin, ticlodipine
  • drugs to reduce blood pressure or lipid levels.
  • Surgery namely surgical removal of the carotid artery lining (carotid endarterectomy) may be undertaken in patients with extensive carotid artery blockage.
  • Inflammation results from TIAs due to transient ischemia and the resultant ischaemia-reperfusion injury.
  • TIAs often occur in patients with underlying atherosclerosis, especially of the carotid arteries, intercranial arteries and the aorta. Inflammation is involved in atherosclerosis through the maturation of foam cells and release of inflammatory cytokines by T-lymphocytes to stimulate macrophages and endothelial cells (O'Rourke et al., Current and Future Concepts in Stroke Prevention, CMAJ, (March 2004) 170(7): 1123-1133).
  • TIAs Long term treatment or prophylaxis is normally directed towards the underlying risk factors such as high blood pressure, cigarette smoking, hypercholesterolemia, systemic inflammation reflected in elevated circulating C-reative protein levels, heart disease and diabetes.
  • therapies directed at the inflammation associated with TIAs would be useful in the treatment of and prophylaxis against TIAs.
  • TIAs result in transient cerebral ischaemia.
  • Pro-inflammatory cytokines such as IL-1 , TNF- ⁇ , and IFN- ⁇ have been shown to be involved in cerebral ischaemia (Pantoni et al., Cytokines and Cell Adhesion Molecules in Cerebral Ischemia Experimental Bases and Therapeutic Perspectives, (1998) Arterior. Thromb. Vase.
  • compositions of phosphate-glycerol carrying bodies such as liposomes, useful in the treatment and prophylaxis of a variety of disorders including cerebrovascular disease (stroke).
  • stroke cerebrovascular disease
  • the present invention provides a process of treating a mammalian patient who is suffering from, has suffered or is at high risk of suffering a transient ischaemic attack, which comprises administering to the patient an effective amount of phosphate-glycerol group carrying bodies.
  • an appropriate dosage of three- dimensional synthetic or semi-synthetic phosphate-glycerol group carrying bodies is administered to a mammal who has suffered or is at high risk of suffering a transient ischaemic attack.
  • Such bodies have shapes and dimensions ranging from those resembling mammalian cells to shapes and dimensions approximating to apoptotic bodies produced by apoptosis of mammalian cells, and having phosphate-glycerol molecules on the surface thereof.
  • FIG. 1 is a bar graph presentation of the results of Example 2 below, murine contact hypersensitivity (CHS, acute T-cell mediated inflammatory model) experiments using liposomes in accordance with a preferred embodiment of the invention, in comparison with other liposomes and controls.
  • CHS murine contact hypersensitivity
  • FIG. 2 is a similar graphical presentation, showing the use of liposomes of various phosphatidylglycerol (PG) contents, in the murine CHS model, Example 3 below.
  • PG phosphatidylglycerol
  • FIG. 3 is a similar graphical presentation of the results of Example 4 below where different concentrations of 75% PG liposomes were used in the murine CHS model.
  • FIG. 4 is a similar graphical presentation of the results of Example 5 below, where different concentrations of 100% PG liposomes were used in the murine CHS model.
  • FIG. 5 is a similar graphical presentation of the results of Example 6 below, using liposomes of different sizes in the CHS model.
  • FIG. 6 is a similar graphical presentation of the results of Example 7 below, using a murine model of delayed type hypersensitivity (DHS, chronic T-cell mediated inflammatory model).
  • DHS delayed type hypersensitivity
  • FIG. 7 is a similar graphical presentation of the results of Example 8 below, cardiolipin liposomes in a DHS murine model.
  • FIG. 8 is a similar graphical presentation of the results of Example 9 below, cardiolipin liposomes in a CHS murine model.
  • FIG. 9 shows the difference in the concentration of the anti-inflammatory cytokine IL-4 in the hippocampus of control and treated animals, Example 10 below.
  • FIG. 10 shows the difference in the concentration of the pro-inflammatory cytokine IL-1 ⁇ in a single cell suspension of spleen cells of control and treated animals, Example 11 below.
  • FIG. 11 shows the difference in the concentration of TNF- ⁇ in the U937 monocyte cell line treated with varying concentrations of 75% PG liposomes, Example 12 below.
  • phosphate-glycerol group carrying bodies may be administered as liposomes having phosphatidylglycerol on their surfaces.
  • the phosphate-glycerol group carrying bodies have diameters from about 20 nanometers to about 500 micrometers (0.02-500 microns).
  • phosphate-glycerol group carrying bodies are administered in a unit dosage amount of from about 500 to about 10 14 bodies per unit dosage.
  • Such administration may be by any of a number of routes, including, without limitation, intramuscular administration.
  • Phosphate-glycerol group carrying bodies may be used in the preparation of medicaments for treatment and prophylaxis of TIAs and their after-effects, in mammalian subjects.
  • a preferred use of the present invention is in the treatment of human patients who have already suffered at least one TIA, and have apparently wholly or partially recovered from the effects thereof. Such patients can be selected on the basis of their medical history of TIA occurrence, subjected to treatment according to the invention, with the result of a significant lessening of subsequent occurrence of a massive, acute stroke.
  • liposomes and “lipid vesicles” as used herein refer to sealed membrane sacs, having diameters in the micron or sub-micron range, the walls of which consist of layers, typically bilayers, of suitable, membrane-forming amphiphiles. They normally contain an aqueous medium.
  • pharmaceutically acceptable has a meaning that is similar to the meaning of the term “biocompatible.”
  • pharmaceutically acceptable bodies refers to bodies of the invention comprised of one or more materials that are suitable for administration to a mammal, preferably a human, in vivo, according to the method of administration specified (e.g., intramuscular, intravenous, subcutaneous, topical, oral, and the like).
  • phosphate-glycerol-carrying bodies refers to biocompatible, pharmaceutically-acceptable, three-dimensional bodies having on their surfaces phosphate-glycerol groups or groups that can be converted to phosphate- glycerol groups, as described herein.
  • Such synthetically altered phosphate-glycerol groups are capable of expressing phosphate-glycerol in vivo and, accordingly, such altered groups are phosphate- glycerol convertible groups within the scope of the invention.
  • a specific example of a phosphate-glycerol group is the compound phosphatidylglycerol (PG), further defined herein.
  • Phosphatidylglycerol is also abbreviated herein as "PG”. This term is intended to cover phospholipids carrying a phosphate-glycerol group with a wide range of at least one fatty acid chain provided that the resulting PG entity can participate as a structural component of a liposome. Chemically, PG has a phosphate-glycerol group and a pair of similar, but different fatty acid side chains. Preferably, such PG compounds can be represented by the Formula I:
  • R and R 1 are independently selected from Ci -C 24 hydrocarbon chains, saturated or unsaturated, straight chain or containing a limited amount of branching wherein at least one chain has from 10 to 24 carbon atoms.
  • R and R 1 can be varied to include two or one lipid chain(s), which can be the same or different, provided they fulfill the structural function.
  • the fatty acid side chains may be from about 10 to about 24 carbon atoms in length, saturated, mono-unsaturated or polyunsaturated, straight chain or with a limited amount of branching.
  • Laurate (C12), myristate (C14, palmitate (C16), stearate (C18), arachidate (C20), behenate (C22) and lignocerate (C24) are examples of useful saturated fatty acid side chains for the PG for use in the present invention.
  • Palmitoleate (C15) and oleate (C18) are examples of suitable mono-unsaturated fatty acid side chains.
  • Linoleate (C18), linolenate (C18) and arachidonate (C20) are examples of suitable poly-unsaturated fatty acid side chains for use in PG in the compositions of the present invention.
  • Phospholipids with a single such fatty acid side chain, also useful in the present invention, are known as lysophospholipids.
  • PG also includes dimeric forms of PG, namely cardiolipin, but other dimers of Formula I are also suitable.
  • dimers are not synthetically cross-linked with a synthetic cross-linking agent, such as maleimide but rather are cross-linked by removal of a glycerol unit as described by Lehninger, Biochemistry and depicted in the reaction below:
  • phosphatidylglycerol are commercially available, for example, from Sigma-Aldrich (St. Louis, MO).
  • PG can be produced, for example, by treating the naturally occurring dimeric form of phosphatidylglycerol, cardiolipin, with phospholipase D. It can also be prepared by enzymatic synthesis from phosphatidyl choline using phospholipase D (see, for example, U.S. Patent 5,188,951 Tremblay et al., incorporated herein by reference).
  • Phosphate-glycerol group carrying bodies are three-dimensional bodies, as described above, that have surface phosphate-glycerol groups.
  • PG can form the membrane of a liposome, either as the sole constituent of the membrane or as a major or minor component thereof, with other phospholipids and/or membrane forming materials.
  • three-dimensional bodies refer to biocompatible synthetic or semi-synthetic entities, including but not limited to liposomes, solid beads, hollow beads, filled beads, particles, granules and microspheres of biocompatible materials, natural or synthetic, as commonly used in the pharmaceutical industry.
  • Liposomes may be formed of lipids, including phosphatidylglycerol (PG). Beads may be solid or hollow, or filled with a biocompatible material.
  • Such bodies have shapes that are typically, but not exclusively spheroidal, cylindrical, ellipsoidal, including oblate and prolate spheroidal, serpentine, reniform and the like, and have sizes ranging from 20 nm to 500 ⁇ m, preferably measured along the longest axis.
  • phosphate-glycerol-carrying bodies refer to biocompatible, pharmaceutically-acceptable, three-dimensional bodies having on their surfaces phosphate-glycerol groups or groups that can be converted to phosphate-glycerol groups, as described herein.
  • phosphate-glycerol groups useful in the present invention have the general structure:
  • Such phosphate-glycerol groups include synthetically altered versions of the phosphate-glycerol group shown above, and may include all, part of or a modified version of the original phosphate-glycerol group.
  • the fatty acid side chains of the chosen PG will be suitable for formation of liposomes, and incorporate into the lipid membrane(s) forming such liposomes, as described in more detail below.
  • Phosphate-glycerol groups of the present invention are believed to act as ligands, binding to specific sites on a protein or other molecule ("PG receptor") and, accordingly, PG (or derivatives or dimeric forms thereof) are sometimes referred to herein as a "ligand” or a "binding group”.
  • phosphate-glycerol groups, including PG are capable of interacting with one or more receptors in the brain and that such interactions may provide positive effects on synaptic transmission, and, by extension, symptoms of MS, as described herein.
  • phosphate-glycerol group carrying bodies of the present invention carry phosphate-glycerol molecules on their exterior surfaces to facilitate in vivo interaction.
  • Three-dimensional bodies are preferably formed to be of a size or sizes suitable for administration to a living subject, preferably by injection; hence such bodies will preferably be in the range of 20 nm to 500 ⁇ m, more preferably from 20 to 1000 nm (0.02-1 micron), more preferably 20 to 500 nm (0.02-0.5 micron), and still more preferably 20-200 nm in diameter, where the diameter of the body is determined on its longest axis, in the case of non-spherical bodies. Suitable sizes are generally in accordance with blood cell sizes. While bodies of the invention have shapes that are typically, but not exclusively spheroidal, they can alternatively be cylindrical, ellipsoidal, including oblate and prolate spheroidal, serpentine, reniform in shape, or the like.
  • Suitable forms of bodies for use in the compositions of the present invention include, without limitation, particles, granules, microspheres or beads of biocompatible materials, natural or synthetic, such as polyethylene glycol, polyvinylpyrrolidone, polystyrene, and the like; polysaccharides such as hydroxethyl starch, hydroxyethylcellulose, agarose and the like; as are commonly used in the pharmaceutical industry.
  • such materials will have side- chains or moieties suitable for derivatization, so that a phosphate-glycerol group, such as PG, may be attached thereto, preferably by covalent bonding.
  • Bodies of the invention may be solid or hollow, or filled with biocompatible material. They are modified as required so that they carry phosphate-glycerol molecules, such as PG on their surfaces. Methods for attaching phosphate-glycerol in general, and PG in particular, to a variety of substrates are known in the art.
  • the liposome is a particularly useful form of body for use in the present invention.
  • Liposomes are microscopic vesicles composed of amphiphilic molecules forming a monolayer or bilayer surrounding a central chamber, which may be fluid-filled.
  • Amphiphilic molecules also referred to as "amphiphiles”
  • Amphiphiles are molecules that have a polar water-soluble group attached to a water-insoluble (lipophilic) hydrocarbon chain, such that a matrix of such molecules will typically form defined polar and apolar regions.
  • Amphiphiles include naturally occurring lipids such as PG, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, cholesterol, cardiolipin, ceramides and sphingomyelin, used alone or in admixture with one another. They can also be synthetic compounds such as polyoxyethylene alkyl ethers, polyoxyethylene alkyl esters and saccharosediesters. Thus a preferred embodiment of this invention provides liposomal bodies which expose or can be treated or induced to expose, on their surfaces, one or more phosphatidylglycerol groups to act as binding groups.
  • Such lipids should comprise from 10% - 100% of the liposome, with the balance being an inactive constituent, e.g. phosphatidylcholine PC 1 or one which acts through a different mechanism, e.g. phosphatidylserine PS, or mixtures of such. Inactive co-constituents such as PC are preferred. Those used in the present invention have at least 10% by weight PG content.
  • the amphiphilic molecules will include one or more forms of phospholipids of different headgroups (e.g., phosphatidylglycerol, phosphatidylserine, phosphatidylcholine) and having a variety of fatty acid side chains, as described above, as well as other lipophilic molecules, such as cholesterol, sphingolipids and sterols.
  • headgroups e.g., phosphatidylglycerol, phosphatidylserine, phosphatidylcholine
  • other lipophilic molecules such as cholesterol, sphingolipids and sterols.
  • phosphatidylglycerol will constitute the major portion or the entire portion of the liposome layer(s) or wali(s), oriented so that the phosphate-glycerol group portion thereof is presented exteriorly, as described above, while the fatty acid side chains form the structural wall.
  • the bilayer includes phospholipids
  • the resulting membrane is usually referred to as a "phospholipid bilayer,” regardless of the presence of non-phospholipid components therein.
  • Liposomes of the invention are typically formed from phospholipid bilayers or a plurality of concentric phospholipid bilayers that enclose aqueous phases.
  • the walls of the liposomes may be single layered; however, such liposomes (termed "single unilamellar vesicles") are generally much smaller (diameters less than about 70nm) than those formed of bilayers, as described below.
  • Liposomes formed in accordance with the present invention are designed to be biocompatible, biodegradable and non-toxic. Liposomes of this type are used in a number of pharmaceutical preparations currently on the market, typically carrying active drug molecules in their aqueous inner core regions. In the present invention, however, the liposomes are not filled with pharmaceutical preparation, i.e they are essentially free of non-liposomal, pharmaceutically active components. The liposomes are active themselves, not acting as drug carrier.
  • Preferred phosphate-glycerol group carrying liposomes of the present invention are constituted to the extent of at least 10% by weight of phosphatidyl glycerol, the balance being phosphatidylcholine (PC) or other such biologically acceptable phospholipids(s), preferably at least 50%, more preferably from 60-100% and most preferably from 70-90%, with the single most preferred embodiment being about 75% by weight of PG.
  • PC phosphatidylcholine
  • phospholipids(s) preferably at least 50%, more preferably from 60-100% and most preferably from 70-90%, with the single most preferred embodiment being about 75% by weight of PG.
  • PG liposomes with inactive liposomes and/or with liposomes of phospholipids acting through a different mechanism can also be used, provided that the total amount of PG remains above the minimum of about 10% and preferably above 60% in the total mixture.
  • Such liposomes are prepared from mixtures of the appropriate amounts of phospholipids as starting materials, by known methods.
  • phosphate- glycerol group carrying bodies comprise less than 50%, preferably less than 40%, still preferably less than 25% and even still preferably less than 10% phosphatidyl choline.
  • the present invention contemplates the use, as phosphate-glycerol group carrying bodies, not only of those liposomes having PG as a membrane constituent, but also liposomes having non-PG membrane substituents that carry on their external surface molecules of phosphate-glycerol, either as monomers or oligomers (as distinguished from phosphatidylglycerol), e.g., chemically attached by chemical modification of the liposome surface of the body, such as the surface of the liposome, making the phosphate-glycerol groups available for subsequent interaction. Because of the inclusion of phosphate-glycerol on the surface of such molecules, they are included within the definition of phosphate-glycerol group carrying bodies.
  • Liposomes may be prepared by a variety of techniques known in the art, such as those detailed in Szoka et al. (Ann. Rev. Biophys. Bioeng. 9:467 (1980)). Depending on the method used for forming the liposomes, as well as any after- formation processing, liposomes may be formed in a variety of sizes and configurations. Methods of preparing liposomes of the appropriate size are known in the art and do not form part of this invention. Reference may be made to various textbooks and literature articles on the subject, for example, the review article by Yechezkel Barenholz and Daan J. A. Chromeline, and literature cited therein, for example New, R. C. (1990), and Nassander, U. K., et al. (1990), and Barenholz, Y and Lichtenberg, D., Liposomes: preparation, characterization, and preservation. Methods Biochem Anal. 1988,.33:337-462.
  • Multilamellar vesicles can be formed by simple lipid-film hydration techniques according to methods known in the art. In this procedure, a mixture of liposome-forming lipids is dissolved in a suitable organic solvent. The mixture is evaporated in a vessel to form a thin film on the inner surface of the vessel, to which an aqueous medium is then added. The lipid film hydrates to form MLVs, typically with sizes between about 100-1000 nm (0.1 to 10 microns) in diameter.
  • a related, reverse evaporation phase (REV) technique can also be used to form unilamellar liposomes in the micron diameter size range.
  • the REV technique involves dissolving the selected lipid components, in an organic solvent, such as diethyl ether, in a glass boiling tube and rapidly injecting an aqueous solution, optionally containing a drug solution to be carried in the interior of the liposome, into the tube, through a small gauge passage, such as a 23-gauge hypodermic needle. The tube is then sealed and sonicated in a bath sonicator. The contents of the tube are alternately evaporated under vacuum and vigorously mixed, to form a final liposomal suspension.
  • organic solvent such as diethyl ether
  • the diameters of the phosphate-glycerol group carrying liposomes of the preferred embodiment of this invention range from about 20 nm to 500 ⁇ m, more preferably from 20 nm to about 1000 nm, more preferably from about 20 nm to about 500 nm, and most preferably from about 20 nm to about 200 nm. Such preferred diameters will correspond to the diameters of mammalian apoptotic bodies, such as may be apprised from the art.
  • One effective sizing method for REVs and MLVs involves extruding an aqueous suspension of the liposomes through a series of polycarbonate membranes having a selected uniform pore size in the range of 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1 , or 0.2 microns.
  • the pore size of the membrane corresponds roughly to the median size of liposomes produced by extrusion through that membrane, particularly where the preparation is extruded two or more times through the same membrane.
  • This method of liposome sizing is used in preparing homogeneous-size REV and MLV compositions.
  • Patents 4,737,323 and 4,927,637 describe methods for producing a suspension of liposomes having uniform sizes in the range of 0.1- 0.4 ⁇ m (100-400 nm) using as a starting material liposomes having diameters in the range of 1 ⁇ m. Homogenization methods are also useful for down-sizing liposomes to sizes of 100 nm or less (Martin, F. J. (1990) In: Specialized Drug Delivery Systems-- Manufacturing and Production Technology, P. TyIe (ed.) Marcel Dekker, New York, pp. 267-316.). Another way to reduce liposomal size is by application of high pressures to the liposomal preparation, as in a French Press.
  • Liposomes can be prepared to have substantially homogeneous sizes of single, bi-layer vesicles in a selected size range between about 0.07 and 0.2 microns (70-200 nm) in diameter, according to methods known in the art.
  • liposomes in this size range are readily able to extravasate through blood vessel epithelial cells into surrounding tissues.
  • a further advantage is that they can be sterilized by simple filtration methods known in the art.
  • a preferred embodiment of phosphate-glycerol group carrying bodies for use in the present invention is liposomes with PG presented on the external surface thereof, it is understood that the phosphate-glycerol group carrying body is not limited to a liposomal structure, as mentioned above.
  • the phosphate-glycerol group carrying bodies of the invention may be administered to the patient by any suitable route of administration, including oral, nasal, topical, rectal, intravenous, subcutaneous and intramuscularly. At present, intramuscular administration is preferred, especially in conjunction with PG liposomes.
  • the phosphate-glycerol group carrying bodies may be suspended in a pharmaceutically acceptable carrier, such as physiological sterile saline, sterile water, pyrogen-free water, isotonic saline, and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.
  • a pharmaceutically acceptable carrier such as physiological sterile saline, sterile water, pyrogen-free water, isotonic saline, and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.
  • phosphate-glycerol group carrying bodies are constituted into a liquid suspension in a biocompatible liquid such as physiological saline and administered to the patient in any appropriate route which introduces it to the immune system, such as intra-arterially, intravenously, or most preferably intramuscularly or subcutaneously.
  • a preferred manner of administering the phosphate-glycerol group carrying bodies to the patient is a course of injections, administered daily, several times per week, weekly or monthly to the patient, over a period ranging from a week to several months.
  • the frequency and duration of the course of the administration is likely to vary from patient to patient, and according to the condition being treated, its severity, and whether the treatment is intended as prophylactic, therapeutic or curative.
  • One currently preferred dosage schedule is a daily injection for six successive days, followed by a booster injection monthly. It is within routine testing to extrapolate such dosing regimens to other mammalian species.
  • One particular injection schedule in at least some of the indications of the invention, is an injection, via the gluteal muscle, of an appropriate amount of bodies on day 1 , a further injection on day 2, a further injection on day 14, and then "booster" injections at monthly intervals.
  • the quantities of phosphate- glycerol group carrying bodies to be administered will vary depending on the identity and characteristics of the patient. It is important that the effective amount of PG-bodies is non-toxic to the patient.
  • the number of phosphate-glycerol group carrying bodies administered per delivery to a human patient is in the range from about 500 to about 10 14 (about 10 mg by weight at the highest end of the range), preferably from about 5,000 to about 500,000,000, more preferably from about 10,000 to about 10,000,000, and most preferably from about 200,000 to about 2,000,000.
  • dosages from about 2.6 x 10 9 to about 10 14 phosphate-glycerol group carrying bodies.
  • a dose of 5 x 10 8 vesicles, of the order of the dose used in the specific in vivo examples below, is equivalent to 4.06 x 10 13 lipid molecules.
  • Using Avogadro's number for the number of molecules of lipid in a gram molecule (mole), 6.023 x 10 23 one determines that this represents 6.74 x 10 "11 moles which, at a molecular weight of 747 for PG is approximately 5.04 x 10 '8 gm, or 50.4 ng of PG for such dosage.
  • the number of such bodies administered to an injection site for each administration is believed to be a more meaningful quantification than the number or weight of phosphate-glycerol group carrying bodies per unit of patient body weight.
  • effective amounts or numbers of phosphate-glycerol group carrying bodies for small animal use may not directly translate into effective amounts for larger mammals on a weight ratio basis.
  • the person skilled in the art could readily extrapolate from the data and other information contained herein to arrive at appropriate dosing for other mammals.
  • the phosphate-glycerol group carrying bodies may be freeze-dried or lyophilized to a form which may be later re-suspended for administration.
  • This invention therefore also includes a kit of parts comprising lyophilized or freeze-dried phosphate-glycerol group carrying bodies and a pharmaceutically acceptable carrier, such as physiological sterile saline, sterile water, pyrogen-free water, isotonic saline, and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations.
  • a kit may optionally provide injection or administration means for administering the composition to a subject.
  • Example 1 Preparation of Liposomes and compositions for therapeutic use.
  • Lipid Premix A dry mixture (“Lipid Premix”) is prepared, consisting of semi-synthetic POPG (1- palmitoyl-2-oleoly-sn-glycero-3-phosphoglycerol sodium salt), 3 parts by mass, and POPC (i-palmitoyl ⁇ -oleoyl-sn-glycero-S-phosphocholine), 1 part by mass.
  • the Lipid Premix was hydrated with phosphate buffered saline (PBS, pH 7.0, sterilized by filtration through a 0.22 micron sterilizing filter) and unilamellar liposomes of 100 ⁇ 20 nm in average diameter were prepared by known extrusion methods.
  • PBS phosphate buffered saline
  • Vesicle size is verified, in-process, using a Quasi-Elastic Light Scattering (QELS) analysis.
  • QELS Quasi-Elastic Light Scattering
  • the suspension of unilamellar vesicles (liposomes) is immediately removed to a class 1 ,000 clean room, where it is redundantly filtered (0.22 micron) and filled into vials (1 mL per 2 mL amber vial) in a class 100 laminar flow hood.
  • the vials are backfilled with nitrogen and sealed with butyl rubber stopper and aluminium crimp seals.
  • CHS murine contact hypersensitivity
  • LPS lipopolysaccharide
  • PBS phosphate-buffered saline
  • PS 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine], referred to in the examples herein as PS
  • POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1 -glycerol)]], referred to in the examples herein as PG
  • PC i-palmitoyl ⁇ -oleoyl-sn-glycero-S-phosphocholine, referred to in the examples herein as PC
  • Liposomes of 100 ⁇ 20 nm in average diameter were prepared according to standard methods known in the art and had the following compositions:
  • Group C - control no liposomes.
  • a stock suspension of each liposome composition containing 4.8 x 10 14 liposomes per ml was diluted with PBS to give an injection suspension containing 6 x 10 6 particles per ml.
  • the liposomal suspensions were injected into female BALB/c mice (Jackson Laboratories) aged 6-8 weeks and weighing 19-23 g, to determine the effect on ear swelling in the murine contact hypersensitivity (CHS) model.
  • CHS murine contact hypersensitivity
  • Groups A and B received approximately 3 x 10 5 of the above-identified liposomes (i.e., 100% PC and 100% PG, respectively), in a volume of approximately 50 ⁇ l.
  • Group C was a control group, receiving no liposomes.
  • mice of Groups A and B were injected with the respective liposomes preparations. Approximately 300,000 liposomes were injected in 50 ⁇ l volume via intramuscular (IM) injection, for a total administration over the test period of about 1 ,800,000 liposomes. Mice of the control group (Group C) received no liposomes, but were sensitized, challenged and tested in the same way as Groups A and B, as described below.
  • IM intramuscular
  • mice were anaesthetized with 0.2 ml 5 mg/ml sodium pentobarbital via IP injection.
  • the abdominal skin of the mouse was sprayed with 70% EtOH and a scalpel blade was used to remove about a one-inch diameter patch of hair from the abdomen.
  • the shaved area was then painted with 25 ⁇ l of 0.5% 2,4-dinitrofluorobenzene (DNFB) in 4:1 acetone: olive oil using a pipette tip.
  • DNFB 2,4-dinitrofluorobenzene
  • mice were challenged with DNFB by painting 10 ⁇ l of 0.2% DNFB on the dorsal surface of the right ear with a pipette tip and by painting 10 ⁇ l of vehicle on the left ear with a pipette tip.
  • FIG. 1 a bar graph showing the mean values from the three experiments of ear swelling, reported in ⁇ m.
  • FIG. 1 shows that a significant reduction in ear swelling was achieved by injection of liposomes according to the present invention.
  • the reduction achieved with 100% PG liposomes is substantially greater than that from 100% PS liposomes.
  • Liposomes of 100 + 20 nm in average diameter were prepared according to standard methods known in the art and had the following compositions:
  • Group A 100% PG Group B - 75% PG, 25% PC Group C - 50% PG, 50% PC Group D - 25% PG, 75% PC Group E - PBS only Group F - no injection
  • a stock suspension of each liposome containing 4.8 x 10 14 liposomes per ml was diluted to give an injection suspension containing 12 x 10 6 liposomes per ml.
  • the liposomal suspensions were used to inject into mice to determine the effect on ear swelling in the murine CHS model, a biological system useful for assaying Th 1 -mediated inflammatory reactions.
  • female BALB/c mice (Jackson Laboratories) aged 6-8 weeks and weighing 19-23 g were used.
  • mice were assigned to one of 6 groups (Groups A-F, above) with 10 animals in each group. Control groups were also included that received no injections (Group F) or injections of PBS with no liposomes (Group E). Animals in Groups A-D were injected with 50 ⁇ l of the above-identified liposome suspensions, each containing about 6 x 10 5 liposomes.
  • the test involves sensitization (Sens) with a potentially inflammation-causing substance, injection of liposomes (Inj) in test animals or PBS in controls and challenge (Chal) with the potentially inflammation-causing substance following measurement (Meas) to determine whether the injection of liposomes are effective against the development of inflammation by the challenge.
  • Sens sensitization
  • Inj injection of liposomes
  • Chal challenge
  • Meas potentially inflammation-causing substance following measurement
  • mice On days 1-6 the mice were injected with the respective liposomes as indicated above. Liposomes were injected in 50 ⁇ l volume via IM injection, i.e., 600,000 liposomes per injection, for a total administration over the test period of 3,600,000 liposomes. Mice of the control group received no liposomes but were sensitized, challenged and tested in the same way as the other groups of mice, as described below.
  • mice were anaesthetized with 0.2 ml 5 mg/ml sodium phenobarbital via IP injection.
  • the abdominal skin of the mouse was sprayed with 70% EtOH and a blade was used to remove about a one inch diameter of hair from the abdomen.
  • the bare area was painted with 25 ⁇ l of 0.5% 2,4-dinitrofluorobenzene (DNFB) in 4:1 acetone: olive oil using a pipette tip.
  • DNFB 2,4-dinitrofluorobenzene
  • mice were challenged (Chal) with DNFB as follows: 10 ⁇ l of 0.2% DNFB was painted on the dorsal surface of the right ear with a pipette tip and 10 ⁇ l of vehicle was painted on the left ear with a pipette tip.
  • FIG. 2 shows that a significant reduction in ear swelling with both 100 and 75% PG is achieved, showing that both these concentrations protect against the development of inflammation resulting from contact with the allergenic substance, DNFB.
  • the 50% and the 25% PG liposomes also showed reductions as compared with both controls, but the differences did not reach statistical significance in this experiment.
  • Liposomes of 100 ⁇ 20 nm in average diameter were prepared according to standard methods known in the art and were composed of 75% PG, 25% PC. A stock suspension containing 4.8 x 10 14 liposomes per ml was used as before and diluted in PBS to give an injection suspension containing the following concentrations of liposomes:
  • mice were divided into six groups (Groups A-F) including a control group receiving no liposomes but injected with 50 ⁇ l_ of PBS (Group F).
  • Mice were sensitized on the flank, injected with their selected liposomal dose, intramuscularly to the right leg muscle, on the same day as, but after, sensitisation (day 1) and on days 2, 3, 4, and 5.
  • day 1 On day 6 they were both injected and challenged on the ear as described in Example 2. The thickness of the ear was measured as described 24 hours after the challenge.
  • Liposomes of formulation 100% PG and 100 ⁇ 20 nm in average size were prepared according to standard methods.
  • Four groups (Groups A-D) of 10 mice were sensitised, injected and challenged in accordance with the procedure and schedule described in Example 4, with the following numbers of 100% PG liposomes delivered in a 50 ⁇ l suspension.
  • Group A - 6 x 10 7 Group B - 6 x 10 6
  • Liposomes of composition 75% PG, 25% PC and of 50, 100, 200, of 400 nm in average diameter were prepared by standard methods. They were tested in the murine CHS model, as in Examples 4 and 5, using 6 x 10 5 liposomes in 50 ⁇ l suspensions for each injection, and a sensitisation-injection-challenge schedule and procedure as in Example 4. The groups were as follows: Group A - 50 nm liposomes Group B - 100 nm liposomes
  • a stock suspension of 75% PG liposomes of 100 ⁇ 20 nm in average diameter containing 4.8 x 10 14 liposomes per ml was diluted to give an injection suspension containing 6 x 10 5 liposomes per ml.
  • the liposomal suspensions were used to inject into mice, to determine the effect on ear swelling in the murine DHS model.
  • female BALB/c mice (Jackson Laboratories) aged 6-8 weeks and weighing 19-23 g were used.
  • mice were assigned to one of 3 groups with 10 animals in each group.
  • a control group (Group C) received only PBS injections. Animals of Groups A and B were injected with 50 ⁇ l of a suspension containing 6 x 10 5 liposomes.
  • mice On days 13-18 the mice were injected with the 75% PG liposomes as indicated below. Liposomes were injected in 50 ⁇ l volume via IM injection, i.e., 600,000 liposomes per injection, for a total administration over the test period of 3,600,000 liposomes. Sensitization and challenge took place as described in Example 3.
  • Liposomes of composed of 100% cardiolipin (CL) and 100 ⁇ 20 nm in average diameter were prepared, by standard methods. These were used at a dosage of 6 x 10 5 liposomes per 50 ⁇ l per injection in the murine DHS model described in Example 7.
  • Data obtained from animals injected with CL liposomes (Group A; 10 animals) was compared to data obtained from animals receiving only PBS (Group B; 10 animals).
  • the sensitisation, injection and challenge procedures were as described in Example 3.
  • the ear thickness measurement results, taken on day 19, 24 hrs after the 6 th injection, are presented in FIG. 7. The results showed a significant reduction in ear swelling within the CL-injected test (Group A).
  • FIG. 8 shows the mean measurements in each group. Both groups receiving CL liposomes showed a statistically significant suppression of CHS compared to the control group.
  • LPS lipopolysaccharide
  • Liposomes of 100 ⁇ 20 nm in average diameter were prepared as according to standard methods known in the art and were composed of 75% PG and 25% PC.
  • a stock suspension of the liposomes containing about 2.9 x 10 14 liposomes per ml was diluted with PBS to give an injection suspension containing about 1.2 x 10 7 liposomes per ml. This was then used to inject into rats, to determine the effect on LPS-induced cytokine levels.
  • male Wistar rats BioResources Unit, Trinity College, Dublin
  • weighing approximately 300 g were used.
  • the animals were assigned to one of four groups, 8 animals in each group to be treated as follows:
  • each above-identified preparation was injected via IM injection on days 1 , 13, and 14.
  • Groups B and D received a total of 5,400,000 liposomes (1 ,800,000 liposomes per injection).
  • the tissue preparation procedure was carried out on day 0.
  • the rats were killed by decapitation and the hippocampi were dissected on ice, sectioned and frozen in 1 ml of Krebs solution (composition of Krebs in mM: NaCI 136, KCI 2.54, KH 2 PO 4 1.18, MgSO 4 .7H 2 O 1.18, NaHCO 3 16, glucose 10, CaCI 2 1.13) containing 10% DMSO.
  • IL-4 is one of a number of cytokines secreted by the Th2 subclass of lymphocytes and is known for its anti-inflammatory effects.
  • FIG. 9 shows that the IL-4 concentration in the hippocampus was significantly increased in the LPS group that had been pre-treated with the PG liposomes ( * p ⁇ 0.05). Open bars represent control group (Group E) and hashed bars represent the PG treated group (Group F). IL-4 was measured by ELISA and expressed as PG of IL-4 per mg of total protein.
  • This upregulation of the anti-inflammatory cytokine IL-4 in the brain is indicative of the use of the process and composition of preferred embodiments of the present invention in treating TIAs.
  • IL-1 ⁇ is one of a number of cytokines secreted by the Th1 subclass of lymphocytes and is known for its proinflammatory effects.
  • FIG. 10 shows that the IL-1 ⁇ concentration in spleen cells was significantly reduced in the LPS group that had been pre-treated with the PG liposomes ( * p ⁇ 0.05).
  • IL-1 ⁇ was measured by ELISA and expressed as picograms of IL-1 ⁇ per mg of total protein. This indicates a systemic inflammatory effect of the process and compositions of preferred embodiments of the present invention, and is indicative of use of the process and compositions of the preferred embodiments of the present invention in treating TIAs.
  • U937 is a monocytic leukemia cell line that can be differentiated into macrophages by administration of phorbol esters.
  • LPS lipopolysaccharide
  • This model provides an experimental system for the assessment of antiinflammatory therapies.
  • the macrophages can be grown in culture medium in the presence of a suspected anti-inflammatory composition, and the expression of TNF- ⁇ can be measured.
  • Liposomes of 100 ⁇ 20 nm in average diameter were prepared according to standard methods known in the art and had a composition of 75% phosphatidylglycerol (PG), 25% phosphatidylcholine (PC). The stock concentration of liposome was about 40 mM lipid and was diluted to the following final concentrations in the assay: 100 ⁇ M phosphatidylglycerol (PG) 40 ⁇ M PG 10 ⁇ M PG 4.0 ⁇ M PG 1 ⁇ M PG
  • the U937 cells were cultured by growing in RPMI medium (GIBCO BRL) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin and grown at 37°C in an atmosphere containing 5% CO2. 5 x 10 5 cells were seeded into wells of 6-well plates and caused to differentiated into macrophages by treatment with 150 nM phorbol myristate acetate (PMA) for 2-3 days. The cell medium was then replaced with complete medium after the U937 cells had differentiated into macrophages. The cells were then incubated for an additional 24 hrs to minimize pleotropic effects due to PMA treatment. The cells were then incubated with either:
  • PBS Phosphate buffered saline
  • the cells were incubated as described above at 37°C in 5% CO2. After 18 hrs, the supernatants from each treatment were collected and assayed for TNF- ⁇ using a standard Quantikine Enzyme-Linked Immunosorbent Assay (ELISA) kit (R&D systems, Minneapolis, USA).
  • ELISA Quantikine Enzyme-Linked Immunosorbent Assay
  • FIG. 11 shows the amount of secreted TNF- ⁇ in picograms per ml. The results demonstrates that U937-differentiated macrophage cells express very low levels of TNF- ⁇ under normal conditions. However, once exposed to LPS, they secrete
  • TNF- ⁇ a 58% decrease in TNF- ⁇ expression.
  • the reduction in TNF- ⁇ is indicative of the use of the process and compositions of preferred embodiments of the present invention in treating TIAs.
  • the Rice-Vannucci model of hypoxic ischaemic brain injury in the neonatal rat may be used for testing the formulations in the present invention and their potential for use in the treatment and prophylaxis of cerebral ischaemia in humans - see Rice, J. E., R.C.Vannucci and J.B.Brierley, "The influence of immaturity on hypoxia-ischemic brain damage in the rat", Ann. Neurol., 1981.9: p.113-141.
  • There is an evolving inflammatory reaction in the injured brain following hypoxia-ischemia generally mimicking certain aspects, particularly inflammatory, of the pathology seen in a TIA, in the immature rat pup model.
  • Inflammatory cytokines such as IL-1 ⁇ increase in the first 24 hours of recovery (1).
  • the inflammatory reaction is characterized by an increase in activated microglial cells, expression of inflammatory cytokines and chemokines, and even the influx of CD4 lymphocytes after a few days.
  • the following experiments are conducted.
  • Timed pregnant rats (2 per week) are allowed to deliver.
  • rat pups On day one of life, rat pups are numbered sequentially and assigned to one of two groups. Also on day 1 of life, one group is injected subcutaneously, at a fold of skin at the back of the neck, with 75% PG/25% PC liposomes, and the other group similarly injected, on the same schedule, with saline as control.
  • Each injection consists of a volume of 0.01 ml, per gram of body weight of the pup.
  • the concentration of the liposomes in the suspension is 3 x 10 6 liposomes per ml, so that each pup receives 3x10 4 vesicles per gram body weight in each injection.
  • the treatments (injections) are repeated on day 2 of life, day 6 of life and day 8 of life.
  • the rats are subjected to hypoxic-ischaemic insult, imitating certain aspects, particularly inflammatory, of the pathology seen in a TIA.
  • the rats are anesthetized with Halothane/nitrous oxide and the right common carotid artery is permanently ligated with silk.
  • the pups are placed in glass jars resting in a temperature controlled incubator and exposed to a hypoxic gas mixture (8% oxygen, balance nitrogen) for 2.25 hrs. Then the pups are returned to room air. This insult produces atrophy to the right hemisphere of the brain, while leaving the left hemisphere virtually intact.
  • the jars are opened to room air, and the pups are returned to their dams and returned to the animal care facility to recover for 21 days. Then they are sacrificed with a lethal dose of pentobarbital and their brains carefully removed intact and immersed in fixative.
  • the brains are removed from fixative and examined intact, to arrive at a gross assessment.
  • the brains are examined by experienced investigators who are unaware of the identity of the treatment group.
  • the investigators allocate to the brains an injury score (0-4) based on the difference in size of the ipsilateral (right) vs. contralateral (left) hemisphere.
  • the examiners will come to a consensus and agree on each score, using the following criteria:
  • 0 no injury; no difference in size between hemispheres;
  • liposome-treated rats will demonstrate lower categories of injuries in comparison to brains from saline-treated rats, thus reflecting a smaller amount of inflammatory damage to brain tissue. It is contemplated that use of compounds of the present invention would decrease inflammation and thus be useful in the treatment and/or prophylaxis of human patients who have suffered from or who are at risk to suffer from transient ischaemic attacks that lead to inflammatory damage to the brain.

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Abstract

L'invention vise à traiter des accidents ischémiques transitoires (AIT) et leurs conséquences et à réduire leur incidence par l'administration, à un patient présentant un risque d'AIT ou ayant déjà subi un AIT, d'une dose efficace de corps porteurs de groupes phosphate-glycérol tels que des liposomes de phosphatidylglycérol, dont la taille est similaire à celle de cellules sanguines de mammifère.
PCT/EP2007/000540 2006-01-24 2007-01-23 Traitement d'un accident ischémique transitoire WO2007085413A2 (fr)

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