WO2010095964A1

WO2010095964A1 - A method for amphiphilic drug loading in liposomes by ion gradient

Info

Publication number: WO2010095964A1
Application number: PCT/PL2010/000014
Authority: WO
Inventors: Jerzy Gubernator; Arkadiusz Kozubek; Grzegorz Grynkiewicz; Janusz Obukowicz
Original assignee: Instytut Farmaceutyczny
Priority date: 2009-02-17
Filing date: 2010-02-16
Publication date: 2010-08-26
Also published as: EP2398463A1; WO2010095964A4; PL387296A1

Abstract

An active loading of amphophilic drugs, especially anthracycline antibiotics, in liposomes using conventional lipid compositions, is achieved by applying salts of polycarboxylic acids with mono- or divalent cations, preferably selected from sodium, potassium, calcium, magnesium or ammonium salt of ethylenediaminotetraacetic acid (EDTA) or ethyleneglycol-O-O'-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA). The liposomal formulations obtained by the method of active loading of drugs to liposomes by EDTA or EGTA salts 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.

Description

A method for amphiphilic drug loading in liposomes by ion gradient

Field of the invention 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.

Background of the invention

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 μm in case of multilamellar structures. Based on size criterion and lamellar structure of liposomes, there are multilamellar liposomes (multilamellar vesicles, MLVs) and 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 μm (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. Additionally, due to their size liposomes may reach distant regions of the system, that is not always possible in case of other drug carriers. As a result of non-occurrence of high concentration of free drug in the circulation directly after its administration and omitting some sensitive organs or tissues, 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. Generally, 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.

Conventional 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. 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. 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. Especially conventional liposomal preparations of taxoids (hardly soluble in water) based on soy lecithin or synthetic phospholipids (Bartoli et al. J. Microencapsulation 7, 1990, 191- 197, Riondel et al. In Vivo 6, 1992, 23-28) show a tendency for aggregation as well instability causing "leakage" of active substance from a liposome and its crystallization.

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).

Efficiency of encapsulation in case of hydrophilic substances is merely several percent.

Other techniques, used mainly in case of hydrophobic drugs, include emulsifying of lipid in biphasic mixture of aqueous and organic phase with simultaneous evaporation of organic solvent (e.g. US 4,522,803, 5,030,453 and 5,169,637), evaporation of organic solvents from water-in-oil emulsion to obtain gel, which upon mixing forms oligolamellar liposomes (US 4,235,871) or multiply freeze-thaw processing (US 5,008,050). 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.

However, the method of passive encapsulation is not efficient in case of substances having weak amphiphilic properties, i.e. low affinity to organic phase in classical test of partition between aqueous and octanol phase. Amphiphilic compounds are difficult to retain inside the liposomes as they can rapidly permeate through and do not bind to lipid bilayers. The attempts to solve the problem of leakage of amphiphilic substances from liposomes consisted, eg., of electrostatic binding of anthracyclines with surface of membrane containing negatively charged phospholipids (WO 9202208, EP 546951 Al) or addition of polyhydric alcohol and tertiary ammonium salt to the liposomes (JP-0625479). However, despite excellent stability during storage, after intravenous administration fast leakage of encapsulated drug was observed.

For molecules having ionizable moieties and weak amphiphilic properties, the method of active loading of liposomes was developed, wherein the molecules are loaded in response to the ion pH gradient by an accumulation of the drug into lipoosmes when their internal pH is lower than the external medium pH. (Chem. Phys. Lipids, 40:333- 345 (1986). Upon ionization of molecules at low or high pH migration of the accumulated drug is not possible, what causes their retention by one side of the membrane.

The method of anthracyclines active loading in liposomes by ion pH gradient created by the use of citric acid was adopted to develop daunomycin hydrochloride formulation, DaunoXome®, while the method of active loading by ion pH gradient achieved by the use of ammonium sulfate was useful in case of preparation of doxorubicin formulation, Doxil®. US 5,316,771 discloses the system wherein the loading of the amphiphilic drug into liposomes is dependent on NH₄ ⁺ and pH gradients, which gradients are created by forming liposomes in ammonium solution, and subsequent ammonium removal from the external aqueous phase, thus forming a pH gradient in which the internal aqueous phase is more acidic then the external phase.

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. However, 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.

Taking into account the foregoing facts, our efforts has focused on developing a method of active loading of amphiphilic drag utilizing ion gradient, based on assumption that the substance should be precipitated in reaction with an agent being present in the internal aqueous phase of liposomes. The known agents forming hardly soluble complexes with anthracyclines are transition metals. The disadvantage of the method in which precipitates of complexes are formed in the internal aqueous phase of liposomes, e.g. anthracycline-Fe(III), is significant toxicity of such combination related to generation of free radicals and DNA damage (B. CL. Cheung, T.H.T Sun, J.M. Leerihouts, P. Cullis. Loading of doxorubicin into liposomes by forming Mn²⁺ - drug complexes. Biochimica et Biophysica acta 1414 (205-216) 1998).

The studies on antibacterial ciprofloxacin lead to finding the agents which formed hardly soluble complexes in the internal aqueous phase of liposomes, significantly prolonging their stability (LV. Zhigaltsev, N. Maurer, K. Edwards, G.

Karlsson, P. Cullis. Formulation of drug - arylsulfonate complexes inside liposomes: A novel approach to improve drug retention. J. Controlled Release, 110(378-386) 2006).

Summary of the invention

Now, it has been found that 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. In the first aspect, 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.

Brief description of the figures

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 μmoles/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 μmole/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 μmole/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.

Detailed description of the invention The method for active loading of the amphiphilic drug into liposomal structures according to the present invention comprises:

(a) preparing a suspension of primary liposomes in the presence of the polycarboxylic acid salts with mono-or divalent cation,

(b) optionally, calibrating the obtained primary liposomes to form unilamellar liposomes,

(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),

(e) incubating the suspension of liposomes to achieve the final concentration of the drug in the internal phase of liposome that is greater than that in the external phase of liposome,

(f) optionally, removing free drug from the external phase of liposomes.

In the preferred embodiment of the invention, 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.

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.

The 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. As defined herein, 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.

The glycolipids that may be useful in the method according invention, are glyceroglycolipids and glycosphingolipids, especially cerebrosides.

The term 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.

As the modified lipid derivative may be used polyethyleneglycol (PEG) or polyglicerin attached phospholipid, cholesterol or diacylglycerol. Preferably, the lipid composition used in the method of the invention consists of phospholipids, sphingolipids, glycolipids, cholesterol and pegylated derivatives thereof. In one embodiment of the invention, the composition of lipids is the combination of distearylphosphatidylcholine and cholesterol. hi the other embodiment of the invention, 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. hi the process of the invention, 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. hi the method according to the present invention, 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. hi the method according to the present invention, 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. by dialysis of liposomal suspension, desalting of liposomal suspension with the use of molecular sieve or centrifugation, and rinsing of liposomes with aqueous solution free of said EDTA or EGTA salts. 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. When pH of the external phase is higher than 7.0, molecules of drug freely permeate lipid bilayer and meet molecules of polycarboxylic acid, EDTA or EGTA, in the inner phase of liposomes, thus forming sparingly soluble salts. The drug is retained inside liposomal vesicles due to the precipitation of the salt. Efficiency of drug encapsulation at weight ratios drug : lipid 1:16 to 1:6 is 70-100%. Incubation period at the temperature of 37- 6O⁰C, depending upon a degree of amphiphilicity of the drug, is merely 1-15 minutes. The free drug, not encapsulated in liposomal vesicles, may be then removed by dialysis or molecular filtration. 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.

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 (α- or γ- 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.

Examples

Example 1

Liposomes loaded by ETDA disodium salt gradient

A. Preparation of DSPC/cholesterol liposomes (7:3, mol/mol)

To 100 mL round bottom flask 24.78 mg of distearylphosphatidylcholine (DSPC) and 5.22 mg of cholesterol (Choi) was added in the form of chloroform solutions, and organic solvent was evaporated. To the obtained dry lipid film, 1.5 ml of EDTA disodium salt solution was added (pH = 4.3) of 300 mM concentration, and it was hydrated at the temperature of 64⁰C until multilamellar liposomes were obtained. The suspension of liposomes was subjected to freezing and thawing, alternately in N₂ κ_q. and water at 64°C. 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 pre-equilibrated to 64⁰C 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))

B . Creation of ion gradient with ETDA disodium salt

Liposomes prepared in step A, were introduced onto a column (1 x 20 cm) filled with Sephadex G-50 fine gel, pre-equilibrated with PBS buffer, and then desalted by exchanging external solution (300 mM EDTA disodium) to phosphate buffer (PBS, pH = 8.0).

C. Loading of DSPC/Chol liposomes with idarubicin

To the suspension of liposomes prepared in step B, 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 60⁰C. Idarubicin encapsulation efficiency - 97%.

Detailed course of the process of drug encapsulation is shown in Fig. 1. Long time stability of DSPC/Chol liposomes loaded with idarubicin is shown in Fig. 2.

Liposomes prepared in Example 1 (idarubicin : lipid 1:10, wt./wt.) were analysed in Transmission Cryoelectron Microscope. Liposomal structures containing the drug are observed as circular and rod-shaped precipitates, as shown in Fig. 3.

Example 2

Mitoxantrone liposomes loaded by ETDA disodium salt gradient

To the suspension of liposomes prepared in Example 1, step B (DSPC/Chol 7:3,

300 mM EDTA disodium salt, pH = 4.3), aqueous solution of mitoxanthrone hydrochloride (6 mg/mL) was added to achieve a drug : lipid ratio 1:10. The suspension was incubated with stirring for 1 min at 6O⁰C. Mitoxantrone encapsulation efficiency - 98%.

Example 3

Long-circulating liposomes loaded by ETDA disodium salt gradient

A. DSPC/Chol/DSPE-PEG 2000 long-circulating liposomes (6.5:3:0.5, mol/mol) To 100 mL round bottom flask 20.04 mg distearylphosphatidylcholine (DSPC) was added, 4.53 mg of cholesterol (Choi) and 5.4 mg pegylated distearylphosphatidylethanolamine (DSPE-PEG 2000) as chloroform solutions. Organic solvent was evaporated. To the dry lipid film 1.5 ml of 300 mM EDTA diammonium salt solution (pH = 4.0) was added and hydrated at 64⁰C until multilamellar liposomes were obtained. Procedure of Example IA and IB was further reproduced.

B. Loading of DSPC/Chol/DSPE-PEG 2000 (6.5:3:0.5, mol/mol) liposomes

To the suspension of liposomes prepared in step A, 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 60⁰C. Idarubicin encapsulation efficiency - 98%.

Detailed course of the process of drug encapsulation is shown in Fig. 4. C. Loading of DSPC/Chol/DSPE-PEG 2000 liposomes with epirubicin

To the suspension of liposomes prepared in step B, 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 60⁰C. Encapsulation efficiency - 96%. Detailed course of the process of drug encapsulation is shown in Fig. 5.

Example 4

Liposomes loaded by EGTA disodium salt gradient

A. Preparation of liposomes containing EGTA

To the 100 mL round bottom flask, 24.78 mg DSPC and 5.22 mg Choi were added as chloroform solutions, and organic solvent was evaporated. To the dry lipid film, 1.5 ml of 300 mM EGTA disodium salt solution (pH = 4.3) was added. It was hydrated at 64⁰C until multilamellar liposomes were obtained. The suspension of liposomes were subjected to 10 cycles of freezeing and thawing, alternately in N₂ ϋ_q. and water of 64⁰C. 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 64⁰C 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))

B . Creation of ion gradient of EGTA disodium salt

Liposomes prepared in step A were introduced onto a column (1x20 cm) filled with Sepahdex G-50 fine gel, pre-equilibrated with PBS buffer, and then desalted by exchanging of external solution (300 mM EDTA disodium salt) to phosphate buffer (PBS, pH = 8.0).

C. Loading of DSPC/Chol liposomes with idarubicin To the suspension of DSPC/Chol (7:3, mol/mol) liposomes with encapsulated

300 mM EGTA disodium salt solution prepared in steps A and B, aqueous solution of idarubicin hydrochloride was added (6 mg/mL), to achieve a drug : lipid ratio of 1:10. The suspension was incubated with stirring for 10 minutes at 6O⁰C. Idarubicin encapsulation efficiency - 70%.

Example 5 Stability of idarubicin liposomes in human serum

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 37⁰C. 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.

The stability of idarubicin in DSPC/Chol (7:3, mol/mol) liposomes is shown in Fig. 6.

Example 6

Pharmacokinetics of idarubicin in liposomal and free form in animal model

Preparation of long-circulating liposomal formulations of idarubicin

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 N₂ κ_q. and subjected to 1- hour lyophilization in a barrel lyophilizing cabinet by Savant (USA). The obtained dry residue was heated to 62⁰C and 7 mL of 300 mM EDTA diammonium salt (pH = 4.3) or 300 mM citrate buffer (pH 4.0) of the same temperature was added, to obtain tested sample and comparative sample, respectively. Further process of multilamellar liposomes formation was continued at the same temperature for 0.5 h, and then liposomes were subjected to 7 cycles of freezing (N₂ Hq.) and thawing (water, 62⁰C). The obtained multilamellar liposomes were calibrated at 64⁰C (4 times extrusion through 400 nm pore size filter and 5 times through 100 nm pore size filter). External solution of liposomal formulations (300 mM EDTA diammonium salt or 300 mM citric buffer pH = 4.0, respectively) were exchanged by size exclusion chromatography to 150 mM saline. To the obtained suspensions of liposomes, after lipid content determination, 200 mM phosphate buffer pH 7.5 was added, to achieve 20 mM final phosphate solution. To the obtained suspensions, aqueous solution of idarubicin was added (5 mg/mL, partly in the form of suspension), to achieve a ratio of drug : lipid 1:7 (wt./wt.). Process of encapsulation of the drug was carried out for 15 minutes at 37⁰C.

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.

Pharmacokinetics study

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 μmol/kg (17.6 mg/kg) body weight into caudal vein. The number of mice per group was established to be 5. In the appropriate time intervals, blood was collected from eye artery into tubes containing 50 μl 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 -2O⁰C. To 100 μl of plasma, 100 μl of acetonitrile was added. Samples were shaken for 2 minutes and then centrifuged (25 000 x g, 5 min, 25⁰C). Supernatant was collected, drug content was determined by HPLC: XTerra RPl 8 column, 250 mm x 4.6 mm, 5 μm; 00014

water/acetonitrile/THF/H₃PO₄/TEA 312:165:20:1:2 v/v, pH 2.2 as mobile phase, at 1 ml/min flow rate; UV detection at λ= 485 nm and fluorescence detection at excitation wavelength λ=485 and emission wavelength λ= 542 nm. 20 μl samples of extracts were injected onto a column. From the obtained calibration curve, idarubicin and its active metabolite - idarubicinol content was read.

The obtained results are presented in Fig. 8 - Fig. 10.

Results

The method of active loading of HSPC/Chol/PEG 2000 (6.5:3:0.5, mol/mol) liposomes by ion gradient of EDTA, ammonium salt, results in effective encapsulation of idarubicin with encapsulation efficiency ca. 98%. Drug accumulation inside liposomes, possible due to the formation of sparingly soluble idarubicin salts with EDTA, increases retention of the drug inside liposomes both in vitro and in vivo. Solubility of idarubicin in 300 mM EDTA sodium salt solution at various pH is shown in Fig. 11. For comparison, solubility of idarubicin in citric buffer pH 4.0 is about 0.57 mg/mL in respect to 0.012 mg/mL in EDTA solution pH 4.0.

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) in case of delivering both liposomal formulations is many times higher than drug concentration (AUC) 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.

Example 7

Pharmacokinetics of epirubicin in liposomal and free form in animal model

Preparation of long-circulating liposomal formulations of epirubicin

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 60⁰C. The encapsulation efficiency was nearly 100% after 10 min.

Size distribution of liposomes after drug encapsulation is shown in Fig. 12. Free drag was not removed.

Pharmacokinetics study

The rate of drug release in animal plasma from long-circulating HSPC/Chol/DSPE PEG 2000 (5.5:4:0.5, mol/mol) liposomes loaded with epirubicin by the method using ion gradient of EDTA, diammonium salt, was compared with non- liposomal epirubicin.

The obtained results are presented in Fig. 13 — Fig. 14.

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. In the appropriate time intervals, blood was collected from eye artery into tubes containing 50 μl of EDTA solution. The animals were earlier sacrificed. The collected blood was centrifuged (25 000 x g, 5 min, 25⁰C), and the obtained plasma was stored at -2O⁰C. To 100 μl of plasma, 100 μl of acetonitrile was added. Samples were shaken for 2 minutes and then centrifuged (25 000 x g, 5 min, 25°C). Supernatant was collected, drug content was determined by HPLC: XTerra RP 18 column, 250 mm x 4.6 mm, 5 μm; water/acetonitrile/THF/H₃PO₄/TEA 312:165:20:1:2 v/v, pH 2.2 as mobile phase, at 1 ml/min flow rate; UV detection at λ= 480 nm and fluorescence detection at excitation wavelength λ=480 and emission wavelength λ= 542 nm. 100 μl samples of extracts were injected on a column.

Profile of epirubicin elimination administered at the dose of 20 mg/kg in free form (Free EPI) and liposomes with a drag encapsulated by a method using ion gradient of EDTA, diamonnium salt (EDTA/EPI) is shown in Fig. 13, and changes of epirubicin concentration in mice plasma after administration in free form at the dose of 20 mg/kg - in Fig. 14.

Results

The method of active loading of HSPC/Chol/PEG 2000 (5.5:4:0.5, mol/mol) liposomes using ion gradient of EDTA, ammonium salt, results in effective encapsulation of epirubicin with encapsulation efficiency ca. 100%. The size of the obtained liposomes after the process of drug encapsulation is not changed. The gradual decrease of plasma drug concentration was observed in the studied animals.

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) in case of delivering liposomal formulation is many times higher than drug concentration (AUC) in plasma of control group that was given free epirubicin. The presence of epirubicin metabolite - epirubicinol within 4-24 hours after administration of liposomes loaded by a method using EDTA ions gradient was not detected by HPLC method.

Claims

14

Claims

1. A method for active loading of the amphiphilic drug into liposomal structures, comprising: (a) preparing a suspension of primary liposomes in the presence of the polycarboxylic acid salt with mono-or divalent cation,

(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),

(f) optionally, removing free drug from the external phase of liposomes.

2. The method according to claim 1, characterized in that the ion gradient is achieved by applying salts of polycarboxylic acids selected of ethylenediamine-N,N,N',N'- tetraacetic acid (EDTA) and glycol-O-O'-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) with mono-or divalent cation.

3. The method according to any of preceding claims characterized in that ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) and glycol-O-O'-bis(2- aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) salts are sodium, potassium, calcium, magnesium, or ammonium salts. 4. The method according to any of preceding claims, characterized in that the salt of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) or glycol-O-O'-bis(2- aminoemylether)-N,N,N',N'-tetraacetic acid (EGTA) is used as aqueous solution of concentration of 50 mM to 300 mM. 5. The method according to claim 4, characterized in that the salt of ethylenediamine- N,N,N',N'-tetraacetic acid (EDTA) or glycol-O-O'-bis(2-aminoethylether)-N,N,N',N'- tetraacetic acid (EGTA) is used as aqueous solution of concentration of 150 to 300 mM. L2010/000014

6. The method according to any of preceding claims, characterized in that external phase of the liposome is exchanged to the buffer solution that is phosphate, citric or sodium bicarbonate in 0.9% sodium chloride solution.

8. The method according to any of preceding claims, characterized in that the concentration of ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA) or glycol-O-0'- bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) inside the liposomes is greater than in the external medium.

9. The method according to any of preceding claims, characterized in that the drug is retained within the liposomes due to the precipitation of its salt with ethylenediamine- N,N,N',N'-tetraacetic acid (EDTA) or glycol-O-O'-bis(2-aminoethylether)-N,N,N\N'- tetraacetic acid (EGTA) inside the liposome.

10. The method according to any preceding claim, characterized in that the initial liposomes are formed from conventional lipids, including phospholipids, sphingolipids, glycolipids, sterol lipids and derivatives thereof, alone or in combination. 11. The method according to claim 9, characterized in that the lipids are the combination of distearylphosphatidylcholine and cholesterol.

12. The method according to claim 9, characterized in that the lipids are the combination of distearylphosphatidylcholine, cholesterol and pegylated distearylphosphatidylethanolamine. 13. The method according to any preceding claim, characterized in that ratio of the drug encapsulated in the liposomes to the total lipid composition is from 1:16 to 1:6 (wt./wt). 14. The method according to any of preceding claims, characterized in that the amphiphilic drug is of weakly basic nature, and is selected from anthracyclines, fluoroquinolones, antineoplastic alkaloids, analgesics and anaesthetics. 15. The method according to any of preceding claims, characterized in that amphiphilic drug is idarubicin or its pharmaceutically acceptable salt.

16. The method according to any of preceding claims, characterized in that amphiphilic drug is mithoxantrone or its pharmaceutically acceptable salt.

17. The method according to any of preceding claims, characterized in that amphiphilic drug is epirubicin or its pharmaceutically acceptable salt.

18. The method according to any of preceding claims, characterized in that the efficiency of drug encapsulation is more than 90%.

19. The method according to claim 18, characterized in that the efficiency of drug encapsulation is more than 98%.

20. The liposomal formulation comprising the liposomes obtained by the method according to Claim 1.

21. The liposomal formulation according to Claim 20, further comprising excipients such as antioxidants, cryoprotectants, osmolality controlling agents, and others.