EP2398463A1 - Procede de chargement en medicaments amphiphiles dans des liposomes par gradient ionique - Google Patents

Procede de chargement en medicaments amphiphiles dans des liposomes par gradient ionique

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Publication number
EP2398463A1
EP2398463A1 EP10714114A EP10714114A EP2398463A1 EP 2398463 A1 EP2398463 A1 EP 2398463A1 EP 10714114 A EP10714114 A EP 10714114A EP 10714114 A EP10714114 A EP 10714114A EP 2398463 A1 EP2398463 A1 EP 2398463A1
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EP
European Patent Office
Prior art keywords
liposomes
drug
edta
tetraacetic acid
egta
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10714114A
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German (de)
English (en)
Inventor
Jerzy Gubernator
Arkadiusz Kozubek
Grzegorz Grynkiewicz
Janusz Obukowicz
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Instytut Farmaceutiyczny
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Instytut Farmaceutiyczny
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Publication of EP2398463A1 publication Critical patent/EP2398463A1/fr
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Classifications

    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1277—Processes for preparing; Proliposomes
    • A61K9/1278—Post-loading, e.g. by ion or pH gradient

Definitions

  • the invention relates to a method for amphiphilic drug loading in liposomes by ion gradient.
  • the method is useful for encapsulation of a wide variety of amphiphilic drug substances of weakly basic nature, especially those selected from anthracyclines, fluoroquinolones, alkaloids of antineoplastic activity, analgesics and anaesthetics.
  • Liposome drug delivery systems are reviewed, among others, in G.V. Betageri, S.A. Jenkins, D.L. Parsons “Liposome Drug Delivery Systems", Technomic Publishing Co., Inc., 1993; D.D. Lasic, “Liposomes: from physics to applications", Elsevier, Amsterdam 1995; D.D. Lasic, F. Martin “Stealth lipoosmes” CRC Press Boca Raton 1995, D.D. Lasic, D. Papahadjopoulos, "Medical applications of liposomes", Elsevier, Amsterdam 1998, Lian T., Ho R. J. Y. "Trends and developments in liposome drug delivery systems", J. Pharm. Sci. 90(6), 667-680, 2001.
  • Liposomes are vesicular structures in which internal aqueous phase is separated by bilayer lipid membrane from external aqueous phase.
  • the size of liposomal vesicles may be from 20 nm for extremely small liposomes to even 20 ⁇ m in case of multilamellar structures.
  • multilamellar liposomes multilamellar vesicles
  • unilamellar liposomes which in turn are divided based on size into small vesicles of below 80 nm (small unilamellar vesicles, SUVs), large vesicles of 80 to 1000 nm (large umilamellar vesicles, LUVs) and giant vesicles reaching diameter of 1-2 ⁇ m (giant unilamellar vesicles, GUVs).
  • Polar hydrophilic groups of amphiphilic lipids forming bilayer are directed towards aqueous phase, whereas lipophilic fragments of both lipid layers form internal hydrophobic layer of a lipid membrane.
  • Polar groups may be derivatives of phosphates, sulfates and nitrogen compounds, but most commonly phospholipids are used, especially of natural origin, as well as synthetic phospholipids and cholesterol derivatives. Multilateral uses of liposomes as drug carriers result from possibility of encapsulation of a wide variety of biologically active substances. While hydrophilic substances are encapsulated in internal aqueous phase, lipophilic ones are incorporated into double phase of lipid membrane, and amphiphilic and charged substances are adsorbed on a lipid membrane.
  • liposomes may reach distant regions of the system, that is not always possible in case of other drug carriers.
  • liposomal preparations show significantly lower side effects in terms of drug toxicity as well as improvement of its therapeutic index. Passive or active targeting of liposomes to certain regions of the system is also possible.
  • the use of liposomal preparations results also in limitation of drug administration frequency.
  • beneficial effects of administration of pharmacologically active substances in the liposomal form consists in increasing of bioavailability, decreasing of systemic and/or organ toxicity, targeting to certain regions, e.g. neoplastic tissue, prolongation of half-life, that is, improvement of selectivity of action and therapeutic index.
  • Liposomes are easily detected by the body's immune system, specifically, by the cells of reticuloendothelial system (RES), and consequently they are removed from the circulation too early.
  • RES reticuloendothelial system
  • a second generation of liposomes was developed, "Stealth liposomes", ensuring better stability of the drug in the circulation by sterical stabilization of surface of lipid vesicle with hydrophilic polymers (D.D. Lasic, F. Martin “Stealth liposomes", CRC Press Boca Raton, 1995).
  • Liposomes may be stabilized by hydrophilic polyethylene glycol, that is described among others in publication of the International patent application WO 9422429.
  • Doxil® Liposomal form of doxorubicin coated with poliethylene glycol was introduced into medical practice under trade name Doxil®.
  • Doxil® contains doxorubicin entrapped in liposomal long- circulating carriers composed of three lipid components - N-(carbonyl- methoxypolyethylene glycol 2000)-l,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, hydrogenated soy phosphatidylcholine and cholesterol.
  • Serious challenge in liposomal technology is both yield of entrapment of drug substance in vesicles and stability of liposomes in vitro and in vivo.
  • the classical method of forming multilamellar liposomes is a passive entrapment of water soluble drug substance in the dry lipid film by hydration of lipid component with aqueous solution of the drug (J. MoI. Biol. 13 (1965), 238-252).
  • Unilamellar liposomes are formed from multilamellar liposomes by extrusion or any other appropriate method such as homogenization, sonication or injection of ether or ethanol lipid solutions to aqueous phase (Deamer R., Uster P. "Liposome preparation; Methods and Mechanisms", in “Liposomes”, ed. M. Ostro, Marcel Dekker, New York, 1987).
  • the loading efficiency of hydrophobic drugs is usually high and obtaining of liposomal preparations of such substances most often is not very problematic.
  • a factor which additionally facilitates drug accumulation inside liposomes is their precipitation leading to shift of a balance of balance of loading process, so that practically all of the drug, free in the beginning, is accumulated inside liposomal vesicles. It not only affects a very high efficiency of drug loading into liposomes but, what more important, a rate of drug release in human body.
  • the drug in the form of precipitate undergoes zero order kinetic release, i.e. for initiating its release from liposomes, dissolving as well as deprotonization and then migration through the lipid bilayer is necessary.
  • a method of drug loading utilizing ion pH gradient although effective in the case of daunomycin and doxorubicin, may cause problems with too fast release from liposomes of more hydrophobic drugs (of higher partition coefficient), such as idarabicin or ciprofloxacin.
  • Idarabicin belongs to a group of hydrophobic anthracyclines of high affinity to lipid bilayer. Idarabicin forms complexes with cholesterol and negatively charged phospholipids within lipid bilayer, and consequently relatively fast leakage of the drug from liposomes in vivo is observed.
  • the efficient active loading of amphiphilic drugs in liposomes may be achieved by applying the salts of polycarboxylic organic acids, especially those having chelating properties, said acids forming sparingly soluble salts with drugs in the internal aqueous phase of liposomal vesicles.
  • the invention relates to a method for amphiphilic drugs active loading in liposomes by ion gradient, wherein loading is achieved by applying polycarboxylic acid salts with mono-or divalent cation.
  • the other aspect of the invention is the liposomal formulation comprising the liposomes obtained by the method of drug active loading in liposomes by ion gradient.
  • Fig. 1 illustrates process of idarubicin loading into DSPC/Chol (7:3, mol/mol) liposomes by the method of EDTA ion gradient.
  • Fig. 2 presents the long time stability of DSPC/Chol (7:3, mol/mol) liposomes loaded with idarubicin by the method of EDTA ion gradient.
  • Fig. 3 illustrates process of idarubicin loading into DSPC/Chol/DSPE-PEG 2000 (6,5:3:0,5, mol/mol) liposomes by the method of EDTA ion gradient.
  • Fig. 4 presents cryo-transmission electron micrograph of idarubicin-containing HSPC/Chol/DSPE-PEG 2000 (6.5:3:0.5, mol/mol) liposomes loaded by the method of EDTA ion gradient.
  • Fig. 5 illustrates process of epirubicin loading into DSPC/Chol/DSPE-PEG 2000 (6,5:3:0,5, mol/mol) liposomes by the method of EDTA ion gradient.
  • Fig. 6 illustrates the stability of liposomes DSPC/Chol (7:3, mol/mol) loaded with idarubicin by the method of EDTA ion gradient in human serum.
  • Fig. 7 A, 7B illustrate size distribution of idarubicin DSPC/Chol/DSPE-PEG 2000 (6,5:3:0,5, mol/mol) liposomes prior and after the process of loading by the method of EDTA ion gradient.
  • Fig. 8 illustrates concentration of free idarubicin and its metabolite idarubicinol in mice plasma after injection of free idarubicin at a dose of 33 ⁇ moles/kg (17,6 mg/kg).
  • Fig. 9 illustrates concentrations of idarubicin in mice plasma after injection of
  • HSPC/Chol/DSPE-PEG 2000 (6.5:3:0.5, mol/mol) IDA/EDTA liposomes, IDA/Citrate liposomes and free IDA at a dose of 33 ⁇ mole/kg (17,6 mg/kg).
  • Fig. 10 illustrates concentrations of idarubicinol in mice plasma after injection of HSPC/Chol/DSPE-PEG 2000 (6.5:3:0.5, mol/mol) IDA/EDTA liposomes, IDA/Citrate liposomes and free IDA at a dose of 33 ⁇ mole/kg (17,6 mg/kg).
  • Fig. 11 illustrates the solubility of idarubicin hydrochloride in 300 mM EDTA disodium salt at increasing pH.
  • Fig. 12 illustrates size distribution of epirubicin liposomes loaded by the method of EDTA ion gradient.
  • Fig. 13 illustrates epirubicin concentrations in mice plasma after injection of HSPC/Chol/DSPE- PEG 2000 (5.5:4:0.5, mol/mol) EPI/EDTA liposomes and free EPI at a dose of 20 mg/kg.
  • Fig. 14 illustrates changes in epirubicin concentrations in mice plasma after administration of free drug at the dose of 20 mg/kg.
  • step (c) replacing the polycarboxylic acid salt in the external phase of liposome with buffer solution of pH 7.5-8.5, thereby creating the ion gradient, (d) adding the amphiphilic drug solution to the suspension of liposomes obtained in step (c),
  • the ion gradient is achieved by applying polycarboxylic acid salts with mono-or divalent cation
  • said polycarboxylic acid salts with mono-or divalent cation is selected from ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA) and glycol-O-O'-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) salts, which are known chelating agents.
  • EDTA ethylenediamine-N,N,N',N'- tetraacetic acid
  • EGTA glycol-O-O'-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
  • the preferred salts applied for creating ion gradient in the process according to the invention are ethylenediamine-N,N,N', N' -tetraacetic acid (EDTA) and glycol-O-0'- bis(2-aminoethylether)-N,N,N', N' -tetraacetic acid (EGTA) salts with sodium, potassium, calcium, magnesium, or ammonium.
  • EDTA ethylenediamine-N,N,N', N' -tetraacetic acid
  • EGTA glycol-O-0'- bis(2-aminoethylether)-N,N,N', N' -tetraacetic acid
  • composition of lipids for preparation of the suspension of initial liposomes used in the present invention may be formed from a variety of vesicle-forming lipids, natural or synthetic, fully saturated or partially hydrogenated, including phospholipids, sphingolipids, glycolipids, sterol lipids and derivatives thereof, alone or in combination.
  • phospholipids also referred to as glycerophospholipids, are the derivatives of sn-glycero-3 -phosphoric acid, including e.g. phosphatidylcholine
  • lecithin phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, bisphosphatidylglycerol (cardiolipin), egg phosphatidylcholine, partially hydrogenated egg phosphatidylcholine, phosphatidylglycerol, dipalmitoylphosphatidylcholine or distearylphosphatidylcholine.
  • glycolipids that may be useful in the method according invention, are glyceroglycolipids and glycosphingolipids, especially cerebrosides.
  • sphingolipids as used herein is intended to encompass lipids having two fatty acid chains, one of each is the hydrocarbon chain of sphingosine.
  • Example of sphingolipid useful in the method according to the present invention is sphingomyelin.
  • the modified lipid derivative may be used polyethyleneglycol (PEG) or polyglicerin attached phospholipid, cholesterol or diacylglycerol.
  • PEG polyethyleneglycol
  • the lipid composition used in the method of the invention consists of phospholipids, sphingolipids, glycolipids, cholesterol and pegylated derivatives thereof.
  • the composition of lipids is the combination of distearylphosphatidylcholine and cholesterol.
  • the composition of lipids is the combination of distearylphosphatidylcholine, cholesterol and pegylated distearylphosphatidylethanolamine.
  • the suspension of the initial liposomes may be obtained from the lipid composition by any method known in the art, eg. by hydration of dry lipid film with aqueous solution, the emulsifying of lipid in biphasic mixture of aqueous and organic phase with simultaneous evaporation of organic solvent, or by multiply freeze-thaw process.
  • multilamellar liposomes are preferably obtained by hydration of lipid composition with aqueous solution of polycarboxylic acid salt.
  • Concentration of EDTA or EGTA salt used in the process preferably is 50 niM to 300 mM, more preferably from 150 to 300 mM.
  • Unilamellar liposomes may be further formed from multilamellar liposomes by calibration, ie. extrusion or any other appropriate method such as sonication or homogenization.
  • the multilamellar liposomes are subjected to multiple cycles of freezing and thawing, said process increases the content of water soluble substances inside liposomes, and then to calibration process.
  • Convenient method of calibration is an extrusion process through polycarbonate filters of 50, 80 or 100 nm pore size, leading to obtaining unilamellar liposomes.
  • Process of homogenization of liposomes may be also carried out with the use of high pressure homogenizer, thus avoiding freezing and thawing.
  • ion gradient is created by removal of EDTA or EGTA salt from the external aqueous phase of liposomal vesicles, whereas in the internal phase, primary concentration of the salt is retained. Ion gradient is then kept during the course of loading of the liposomes due to the properties of lipid bilayer which prevents drug from migrating outside the vesicles. Removal of EDTA or EGTA salts may be accomplished by any means known in the art, eg.
  • the polycarboxylic acid salt in the external phase of liposome is thus replaced with buffer solution of pH 7.5-8.5,
  • the buffer may be phosphate, citric or sodium bicarbonate in 0.9% sodium chloride solution.
  • the process of active loading of the drug in the liposomes is initiated by the addition of the drug solution to the external phase of liposomes suspension.
  • the pharmacologically active drugs which could be loaded into liposomes by the active loading method according to the present invention, are amphiphilic compounds with weak acidic or basic moieties, and include, without limitation, anthracyclines, eg. doxorubicin, idarubicin, mitoxanthrone, epirubicin, daunomycin; antibacterial fluoroquinolones, eg. ciprofloxacin, ofloxacin; antineoplastic alkaloids, eg. vincristine, vinblastine, vinorelbine; analgesics and anaesthetics, eg. morphine, codeine, lidocaine, and others.
  • anthracyclines eg. doxorubicin, idarubicin, mitoxanthrone, epirubicin, daunomycin
  • antibacterial fluoroquinolones eg. ciprofloxacin, ofloxacin
  • the liposomal formulations obtained by the method of amphiphilic drug active loading into liposomes with the use of EDTA or EGTA salt gradient are characterized by high loading efficiency, feature microcrystalline deposits of anthracyclines inside liposomes, which renders them stable and not susceptible to leakage.
  • the liposomes are unilamellar and their size is close to 100 nanometers after drug loading.
  • the liposomal formulations obtained by the method of the invention may further contain excipients such as antioxidants ( ⁇ - or ⁇ - tocopherole, ascorbic acid), cryoprotectants (e.g. glycerol) or osmolality controlling agents (e.g. saccharose).
  • excipients such as antioxidants ( ⁇ - or ⁇ - tocopherole, ascorbic acid), cryoprotectants (e.g. glycerol) or osmolality controlling agents (e.g. saccharose).
  • the rate of drug liberation from the developed liposomes is similar for this observed for liposomal doxorubicin (Doxil®). That offers significant improvement of therapeutic index of drugs, especially anthracyclines, administered in liposomes comparing to drugs delivered in the free form.
  • the invention is illustrated by the following, not limiting, examples.
  • Thermobarrel Extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada).
  • the extruder was pre-equilibrated to 64 0 C prior to liposome extrusion.
  • the mean diameter of the vesicles was measured (multimodal analysis, volume weighted) on a Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK))
  • idarubicin hydrochloride in water (6 mg/ml) was added to achieve a drug : lipid ratio 1:10, wt./wt.. The suspension was incubated for 1 min at 60 0 C. Idarubicin encapsulation efficiency - 97%.
  • Fig. 1 Long time stability of DSPC/Chol liposomes loaded with idarubicin is shown in Fig. 2.
  • Liposomes prepared in Example 1 were analysed in Transmission Cryoelectron Microscope. Liposomal structures containing the drug are observed as circular and rod-shaped precipitates, as shown in Fig. 3.
  • step B DSPC/Chol 7:3
  • DSPC/Chol/DSPE-PEG 2000 long-circulating liposomes (6.5:3:0.5, mol/mol)
  • DSPC distearylphosphatidylcholine
  • Choi cholesterol
  • DSPE-PEG 2000 pegylated distearylphosphatidylethanolamine
  • aqueous solution of idarubicin hydrochloride was added (6 mg/mL), to achieve a drug : lipid ratio of 1 :6.
  • the suspension was incubated with stirring for 10 min at 60 0 C. Idarubicin encapsulation efficiency - 98%.
  • aqueous solution of epirubicin hydrochloride was added (6 mg/mL), to achieve a drug : lipid ratio 1:6.
  • the suspension was incubated with stirring for 15 minutes at 60 0 C. Encapsulation efficiency - 96%. Detailed course of the process of drug encapsulation is shown in Fig. 5.
  • LUVs Large unilamellar vesicles (LUVs) were prepared by extrusion through Nucleopore polycarbonate filters with pore sizes of 100 nm (10 passes) on a Thermobarrel Extruder (Lipex Biomembranes, Vancouver, British Columbia, Canada). The extruder was equilibrated to 64 0 C prior to liposome extrusion. The mean diameter of the vesicles was measured (multimodal analysis, volume weighted) on a Zetasizer Nano-ZS (Malvern Instruments Ltd., Malvern, UK))
  • Liposomal preparation obtained in Example 3B after removal of not encapsulated drug, was diluted with human serum (1:1, v/v) and incubated for 24 hours at 37 0 C. In the predetermined time intervals, samples of the suspension of liposomes were collected and the released drug was separated on a mini-column filled with molecular sieve Sepharose 4B. hi fractions containing free drug, idarubicin was determined by spectrofluorometry. After 24 hours of incubation with 50% human serum, 8% of the primary content of liposomes was released.
  • idarubicin in DSPC/Chol (7:3, mol/mol) liposomes is shown in Fig. 6.
  • Long-circulating liposomes Long-circulating liposomes, HSPC/Chol/DSPE-PEG 2000 (6.5:3:0.5, mol/mol), were prepared by lyophilization from cyclohexane. Appropriate amounts of phospholipids (180 mg in total) were weighed and put to the 25 niL screwed glass tube, and all lipids were dissolved in 4 mL of cyclohexane with addition of 0.1 mL of methanol. The obtained clear solution was quickly freezed in N 2 ⁇ q . and subjected to 1- hour lyophilization in a barrel lyophilizing cabinet by Savant (USA).
  • Fig. 7A Liposomes size distribution for both formulations after drug encapsulation are shown in Fig. 7A (Tested formulation) and Fig. 7B (Comperative formulation). Encapsulation efficiency was 98% for the drug loaded by EDTA ion gradient and 99% for the drug loaded by citric buffer gradient. Free drug was not removed.
  • the rates of drug release from long-circulating HSPC/Chol/DSPE PEG 2000 (6.5:3:0.5 mol/mol) liposomes were compared in animal study, wherein liposomes were loaded with idarubicin either by an active method using ion gradient of EDTA salt or classical method of active encapsulation of anthracyclines based on pH/ion gradient using citric buffer. Additionally, animals were treated with non-liposomal idarubicin, in order to compare drug concentration in plasma, after injection either in free or liposomal form.
  • mice balb-c males were injected with free idarubicin (Free IDA) and HSPC/Chol/PEG 2000 (6.5:3:0.5 mol/mol) liposomes containing idarubicin encapsulated by a method using gradient of citrate ions - Comparative sample (Lip/Citrate) of the same composition of liposomes in which the drug was encapsulated by gradient of EDTA ions - Tested sample (Lip/EDTA) .
  • the drug was given at the dose of 33 ⁇ mol/kg (17.6 mg/kg) body weight into caudal vein.
  • the number of mice per group was established to be 5.
  • blood was collected from eye artery into tubes containing 50 ⁇ l of EDTA solution. The animals were earlier sacrificed. The collected blood was centrifuged for 10 minutes at 2000 x g at RT, and the obtained plasma was stored at -2O 0 C. To 100 ⁇ l of plasma, 100 ⁇ l of acetonitrile was added. Samples were shaken for 2 minutes and then centrifuged (25 000 x g, 5 min, 25 0 C). Supernatant was collected, drug content was determined by HPLC: XTerra RPl 8 column, 250 mm x 4.6 mm, 5 ⁇ m; 00014
  • the size of the obtained liposomes is comparable to the size of liposomes prepared by the method based on pH/ion gradient using citric acid.
  • a significant slowing down of drug release in vivo was achieved compared to control liposomes.
  • Drug concentration in animal blood plasma area under the curve, AUC
  • AUC drug concentration in plasma of control group that was given free idarubicin.
  • the concentration of idarubicinol, ie. main metabolite of idarubicin, within 4-24 hours in a group that was given liposomes loaded by a method using EDTA ion gradient is significantly higher than the concentration in the blood of animals belonging to two other groups.
  • Long-circulating HSPC/CH/DSPE-PEG 2000 (5.5:4:0.5, mol/mol) liposomes were prepared by a method described in Example 6. After extrusion, the external solution was exchanged by size exclusion chromatography to 150 nM saline. To the obtained suspension of liposomes, after lipid content determination, 200 mM phosphate buffer pH 7.5 was added, to achieve 20 niM final phosphate buffer concentration. To the obtained suspension, 0.5 niL of aqueous solution of epirubicin was added to achieve a ratio drug : lipid 1:7 (wt./wt). Process of loading the drug was initiated by heating to 60 0 C. The encapsulation efficiency was nearly 100% after 10 min.
  • mice balb-c males were injected with free epirubicin (Free EPI) and HSPC/Chol/PEG 2000 (5.5:4:0.5, mol/mol) liposomes loaded by a method using ion gradient of EDTA - Tested sample (Lip/EDTA).
  • the drug was given at the dose of 20 mg/kg body weight into caudal vein.
  • the number of mice per group was established to be 5.
  • blood was collected from eye artery into tubes containing 50 ⁇ l of EDTA solution. The animals were earlier sacrificed. The collected blood was centrifuged (25 000 x g, 5 min, 25 0 C), and the obtained plasma was stored at -2O 0 C.
  • a significant slowing down of drug release in vivo was achieved compared to free drug.
  • Drug concentration in animal blood plasma area under the curve, AUC
  • AUC drug concentration in plasma of control group that was given free epirubicin.
  • AUC drug concentration in plasma of control group that was given free epirubicin.

Abstract

L'invention concerne un chargement actif en médicaments amphiphiles, notamment des antibiotiques d'anthracyclines, dans des liposomes à l'aide de compositions lipidiques, que l'on réalise par application de sels d'acides polycarboxyliques avec des cations monovalents ou divalents, choisis, de préférence, dans le sel de sodium, de potassium, de calcium, de magnésium ou d'ammonium d'acide éthylène-diamino tétracétique (EDTA) ou d'acide éthylène glycol-O-O'-bis(2-aminoéthyl)-N,N,N',N'-tétraacétique (EGTA). Les formulations liposomales obtenues par le procédé de chargement actif en médicaments vers des liposomes par un gradient de sels EDTA ou EGTA se caractérisent par une efficacité de chargement élevée, des dépôts microcristallins caractéristiques d'anthracyclines à l'intérieur de liposomes, ce qui les rend stables et non susceptibles à des fuites. Les liposomes sont unilamellaires et leur taille est approximativement de 100 nanomètres après chargement en médicaments.
EP10714114A 2009-02-17 2010-02-16 Procede de chargement en medicaments amphiphiles dans des liposomes par gradient ionique Withdrawn EP2398463A1 (fr)

Applications Claiming Priority (2)

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PL387296A PL387296A1 (pl) 2009-02-17 2009-02-17 Sposób aktywnego zamykania amfifilowych substancji czynnych w strukturach liposomowych
PCT/PL2010/000014 WO2010095964A1 (fr) 2009-02-17 2010-02-16 Procede de chargement en medicaments amphiphiles dans des liposomes par gradient ionique

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JP5161995B2 (ja) * 2011-01-04 2013-03-13 日本特殊陶業株式会社 プラズマジェット点火プラグの点火装置
JP2012225204A (ja) * 2011-04-18 2012-11-15 Ngk Spark Plug Co Ltd 点火装置及び点火システム
WO2015166986A1 (fr) * 2014-04-30 2015-11-05 富士フイルム株式会社 Composition liposomale et procédé de production associé
EP3518978A1 (fr) 2016-09-27 2019-08-07 Camurus AB Mélanges et préparations comportant un sel d'edta de type alkylammonium

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CA1338702C (fr) * 1987-03-05 1996-11-12 Lawrence D. Mayer Formulations d'agents liposomiques-antineoplasiques a faible teneur en medicaments-lipides
US5714163A (en) * 1994-06-27 1998-02-03 Nexstar Pharmaceuticals, Inc. Vinca alkaloid vesicles with enhanced efficacy and tumor targeting properties
EP0949906A4 (fr) * 1996-10-22 2004-11-24 Hermes Biosciences Inc Liposomes charges de composes et leurs procedes de preparation
AU2002325120A1 (en) * 2001-09-10 2003-03-24 Celator Technologies Inc. Unilamellar vesicles stabilized with short chain hydrophilic polymers

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WO2010095964A4 (fr) 2010-10-21
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