WO2005072776A2

WO2005072776A2 - Liposomal formulations of the antineoplastic agents

Info

Publication number: WO2005072776A2
Application number: PCT/PL2005/000007
Authority: WO
Inventors: Arkadiusz Kozubek; Grzegorz Grynkiewicz; Wieslaw Szelejewski; Piotr Slifirski; Jerzy Gubernator; Maria Stasiuk; Krzysztof Wisniewski
Original assignee: Instytut Farmaceutyczny
Priority date: 2004-01-30
Filing date: 2005-01-28
Publication date: 2005-08-11
Also published as: WO2005072776A3

Abstract

The invention relates to the liposomal formulation of an antineoplastic agent encapsulated in the liposomal vesicles which are the composition of lipid components, wherein the ratio of a therapeutically active agent to the lipid components is between about 1:10 and about 1:30 (w/w). The composition of lipid components contains at least one phospholipid derivative and at least one alkylphenol derivative presented by a general formula (I) , wherein: n is an integer 1 - 17; m is an integer 0-15; X is -C=O, -0- or a bond; Y is a group of formula -NR2R3, a monosaccharide moiety or a bond; Z is H, -0-, -N-, -OR4, -NR4 or a monosaccharide moiety, R5 is H or COOH.

Description

Liposomal formulations of the antineoplastic agents

Field of the invention The invention relates to the liposomal formulations of the antineoplastic agents, the process for their preparation and to the anticancer pharmaceutical compositions containing the liposomal preparations. State of art Usefulness of liposomes in pharmacy and medicine as a carrier of the active substances has been postulated since early ' 60, when the phenomenon of encapsulating certain chemical compounds in lipid vesicles has been observed. It was only in the last few years that the objective evidence of therapeutic efficacy of this administration method of pharmaceutically active substances was obtained and first liposomal drug delivery systems have been introduced to medicine. Advantageous effects of administering active substances in liposomes consist in higher bioavailability, lower systemic and/or organ toxicity, direct action in targeted area, e.g. cancer tissue, longer half-life, thus giving a selective bio- distribution and therapeutic index. The current knowledge on liposomes, particularly on their pharmaceutical application, is derived from publications, e.g. D.D. Lasic, "Liposomes: from physics to applications", Elsevier, Amsterdam 1995; D.D. Lasic, F. Martin "Stealth liposomes" 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 spontaneuosly self-assembling structures, uni- or multilamellar, in which the double layer of amphiphilic lipid provides a sheath for the microdrop of water (unilamellar liposomes) or the lipid membranes are arranged spherically alternatively with water layers (multilamellar liposomes) . The amphiphilic lipids which form the bilayer, comprise a polar hydrophilic group and one or more hydrophobic acyl chain (>Cs) . Polar groups may be the derivatives of phosphates, sulfates and nitrogen compounds, however usually phospholipids are used, particularly of natural origin, such as phosphatidylcholines, which are refined vegetable fat; synthetic phospholipids; commercially available phospholipids formulations, including phospholipids chemically modified with ethylene glycol derivatives; and cholesterol derivatives. Depending on the solubility, the active substance is placed in the water layer or lipid layer of the liposome. A variety methods for preparing the liposomes exist. The classical preparation method of multilamellar liposomes consists in evaporation of the lipid - organic solvent mixture and rehydration of thus formed lipid film with aqueous solution of the active substance (J. Mol. Biol., 13 (1965), 238-252). Other methods include emulsification of lipid in a two-phase mixture of water and organic solvent, with concurrent evaporation of organic solvent (US 4,522,803, 5,030,453 and 5,169,637), evaporation of the solvent from oil-in- water emulsion to form a gel, then stirred to give oligolamellar liposomes (US 4,235,871) and repeating freeze-and-thaw (US 5,008,050). Unilamellar liposomes are produced from multilamellar liposomes by the methods of ultrasounds treatment, extrusion (US 4,975,282), homogenization, as well as by injecting ether or ethanol lipid solutions to water phase (Deamer R., Uster P., "Liposome preparation; Methods and mechanisms", in "Liposomes", M. Ostro, Marcel Dekker, New York, 1987) . In many therapeutic groups, both encapsulation efficiency of an active substance in the lipid vesicles and stability of liposomes { in vi tro and in vi vo) makes a serious technical difficulty. Particularly, classical liposomal formulations of water insoluble taxanes based on soybean lecithin or synthetic phospholipid analogs (Bartoli et al, J. Microencapsulation 7, 1990, 191-197, Riondel et al In Vivo 6, 1992, 23-28) exhibit a tendency for aggregation and instability causing the "leakage" of an active substance from the liposome and its crystallization. Producing of liposomal formulations containing an antineoplastic agent of an effective lipid/drug ratio, very often requires specific procedures, such as using of the negatively charged phospholipids (WO 9202208, EP 546951), addition of polyhydroxy alcohol and quaternary ammonium salts (JP 06254379), stabilization of liposomes by polyethylene glycol sheath (WO 9422429) or encapsulation of an active substance in liposomes in an electrostatic gradient (EP 361894) . The so-called "stealth liposomes" are the improved liposome forms which provide a better stability through steric stabilization of the lipid surface (D.D. Lasic, F. Martin "Stealth liposomes", CRC Press Book Raton, 1995) . Stable liposomal formulations of paclitaxel have been obtained by addition of cardiolipin to the lipid composition (US 5,424,073, 5,648,090, 5,939,567, 6,146,659). US 6,146,659 reveal that the encapsulation efficiency of paclitaxel in the lipid vesicles exceeds 90%, with the active substance to the lipid carrier ratio (w/w) at ca. 7%. In general, the possibility of incorporating paclitaxel into the liposomes, due to its high hydrophobia, is limited to 1-10% (w/w) , generally 2-8% (w/w) in relation do lipid carrier. This ratio may only be slightly improved (up to 12-14% w/w) by modification of paclitaxel particle, e.g. by coupling hydrocarbon chains (US 5,919,815, 5,939,567, 6,118,011) . Therefore, there is still a need for development of the liposomal pharmaceutical formulations, particularly liposomal formulations of hydrophobic antineoplastic agents, of the favorable lipid/drug substance ratio, which facilitates transporting the same amount of the active substance by smaller amounts of the lipid carrier.

Description of the invention In the present invention, a liposomal formulations of an antineoplastic agents, characterized by high encapsulation efficiencies of an active substance and good stability have been obtained by means of the modification of classical lipid carrier composition. The background of the present invention is an observation made by the present Inventors that the high encapsulation efficiency of an active substance may be achieved by incorporating semi-synthetic polyhydroxyl derivatives of alkylphenols comprising the saturated carbohydrate chains in the molecule, into the composition of the classical lipid carrier. The present invention provides a liposomal formulation of an antineoplastic agent encapsulated in the liposomal vesicles which are the composition of lipid components, wherein the ratio of a therapeutically active agent to the lipid components is between about 1:10 and about 1:30 (w/w). The preferred embodiment of the invention is the liposomal formulation of an antineoplastic agent wherein the ratio of a therapeutically active agent to the lipid components is about 1:20 (w/w) . The lipid composition includes at least one phospholipid derivative and at least one alkylphenol derivative of a general formula (I), wherein: n is an integer 1 - 17; m is an integer 0-15; X is -C=0, -0- or a bond;

Y is a group of formula -NR₂R, a monosaccharide moiety or a bond;

Z is H, -0-, -N-, -0R₄, -NR₄ or a monosaccharide moiety, R₅ is H or COOH; while : a) if X is C=0, then Y is NR₂R₃, in which R₂ is H or Cι~ C₃ alkyl, and R₃ is Cι-C₆ alkyl substituted with at least one hydroxylic group, and

Y is -H, -N- or NR₄, where R₄ is H or Cι~C₂ alkyl; or Y is -N-, and Ri and R₂ together with Y form a saturated heterocyclic ring; Z is -OR₄, where R₄ is H; whereas b) if X is -0-, then Y is a monosaccharide moiety, and Z is H or monosaccharide moiety attached to X through an oxygen atom. In one embodiment of the invention, the alkylphenol derivative has a formula (I) in which: n is an integer 13 - 17; m is an integer 0-15; X is -0-;

Y is a bond;

Z is -OR₄, wherein R₄ is H;

R₅ is H or COOH. In another embodiment of the invention, the alkylphenol derivative has a formula (I), wherein: n is an integer 13 - 17; m is 1;

X is -C=0; Y is a group of formula -NR₂R₃ ; in which

R₂ is H or Cχ-C₃ alkyl , and R₃ is Ci-Cβ alkyl substituted with at least one hydroxylic group, and

R₄ is H;

R₅ is H or COOH. In yet another embodiment of the invention, the alkylphenol derivative has a formula (I), wherein: n is an integer 13 - 17; m is 1 ;

X is -0-; Y is a monosaccharide moiety; and

Z is H or monosaccharide moiety attached to X through an oxygen atom;

R is H or COOH. As the preferred monosaccharide moiety in the alkylphenol derivative, the sugars: pentose, hexose and heptose, preferably 2-deoxyheptose in pyranose form of the D- or L- configuration, e.g. D-glucose, D- galactose, D-mannose, i-rhamnose or L-fucose; or amino sugars in pyranose form, particularly L-acosamine or L- daunosamine, can be mentioned. Due to the presence of one or more asymmetric centers in the side chain, alkylphenol derivatives of the present invention can exist in the form of enantiomers, diastereoisomers or the racemic mixtures thereof. All of them are encompassed by the scope of the present invention. Alkylphenol derivatives of formula (I) can be prepared by the chemical modifications of the naturally occuring alkylphenols, e.g., by O-alkylation with esters of alkyl halides in the presence of sodium hydride and then the reaction of thus obtained phenoxy acids with hydroxy-amines or cyclic amines to amides. As the result of derivatization of the natural structures, the derivatives of formula (I) may differ in chemical as well as physicochemical properties, such as solubility and lipophilicity. Preferably, the phospholipid and the alkylphenol derivative of formula (I) are present in the composition of lipid components in a molar ratio of about 9:1. The preferred active substance for use in the liposomal formulation according to the present invention may be any therapeutically effective and safe drug accepted for use in mammals, particularly in humans. Particularly, the use of insoluble antineoplastics, selected from the group of anthracyclines, such as mitoxantrone, daunomycine, doxorubicine, epirubicine or idarubicine; alkylating agents; camptothecin derivatives, such as irinotecan and camptothecin; lignans, such as etoposide; and taxanes, such as paclitaxel and docetaxel, may be considered in the context of the present invention. The above mentioned examples are not to be considered as limiting in any way the scope of the invention. Especially preferred active substances for use in the liposomal formulation according to the present invention are taxanes, such as paclitaxel and docetaxel . The lipid fraction of the formulation according to the invention comprises one or more of the phospholipids selected from the group consisting of saturated phospholipids such as dimiristoylphosphatidilcholine, dipalmitoyl- phosphatidylcholine, distearoylphosphatidilcholine and unsaturated phospholipids such as hydrogenated purified soy bean phosphatidilcholine, hydrogenated purified egg phosphatidilcholine, etc. The liposomal formulation according to the present invention, besides the active substance and lipid components able to form liposomes, may further contain additional stabilizers of the vesicle structure and other pharmaceutically acceptable excipients which improve its stability. The composition of the lipid components according to the invention provides a high encapsulation efficiency of the active substance in lipid vesicles. Particularly favorable results are obtained with paclitaxel, where the encapsulation efficiency preferably exceeds 90%, more preferably exceeds 95%. The liposomal formulation may be prepared by any suitable method known to the skilled in the art. In a preferred embodiment of the invention, the process for the preparation of the liposomal formulation of the high encapsulation efficiency of the antineoplastic agent comprises the steps of: (a) combining the solution of the active substance in the suitable solvent, with the solution of lipid components in the suitable organic solvent; (b) removing the solvents to form the lipid film; (c) dissolving the lipid film formed in step (b) in the organic solvent, preferably alkanol, and the freeze- thawing of thus formed dispersion; (d) hydration of the lyophilisate obtained in step (c) with aqueous system, to form the pre-formulation of multilamellar liposomes; (e) further processing of the pre-formulation of step (d) , to form a suspension of calibrated bi-lamellar liposomes of the size 50-200 nm, preferably 100 nm; (f) lyophilisation of the liposomal dispersion together

with the pharmaceutically acceptable excipients and/or

carriers . The active substance in step (a) may be dissolved in a suitable solvent, such as alkanol, methylene chloride, chloroform and the like. This solution is combined with the solution of two or more lipid components in the same or in a different solvent or a solvent system. The solvents are removed in step (b) by any method and with the employment of any apparatus, preferably with a rotary evaporator under reduced pressure. The dispersion formed in step (c) is frozen by plunging the vial with suspension, possibly with the cryoprotectants, in liquid nitrogen, then thawed at 40°C. This process is repeated 7 - 10 times. The studies with the transmission electron microscope exhibit that multiple freezing and thawing of liposome suspension changes the two, pharmaceutically significant properties, reduces the number of liposome layers and standarizes liposome size. Similar advantageous effects are achieved by sonication, i.e. treatment of the liposome suspension with the ultrasounds. The desired liposomal formulation of the active substance is obtained by hydration of the pre- formulation in step (d) . Hydration consists in shaking, mechanical stirring or sonication in the presence of a water system, such as water, a solution of saline, a solution of a buffering agent or a solution of other pharmaceutically acceptable auxiliaries. The hydrating solution can contain one or more cryoprotectants, such as glucose, maltose, lactose, trehalose, dextran or the combination thereof. The hydration of the liposomal formulation obtained in step (d) results in the formation of multi- and unilamellar liposomes of a mean diameter 80-1500 nm. Bilayer liposomes of the desired diameter 100-200 nm are obtained by subjecting the liposome dispersion to the further physical processes, such as freezing, extrusion through a sieve of a desired size, by a homogenization using high pressure homogenizer or by sonication. A suitable method for preparation of the uniform liposomes consists in multiple extrusion of the dispersion through a 50-200 nm, preferably 100 nm size polycarbonate filters. Multiple extrusions allow for carrying out three processes in one operation: encapsulation of the active substance, calibration of the liposomes and the sterilization of the formulation. Hydration of the lyophilisate of the composition of the invention by shaking the lipid components with the aqueous solution of the active substance provides a high encapsulation efficiency of the active substance in the liposomes, preferably exceeding 90%, more preferably 95%. Moreover, the invention provides the pharmaceutical composition, especially the composition for parenteral administration, containing the liposomal formulation of the antineoplastic agent according to the invention, and the pharmaceutically acceptable carriers and/or excipients. Pharmaceutically acceptable carrier is any substance or mixture of substances which do not exhibit its own pharmacologic effect, diluent or excipient used for administration of biologically active substances.

Preferably, carriers for the intravenous administration of the composition according to the invention include sterile water solutions, such as the solution of saline, the solutions of carbohydrates, e.g. glucose, mannitol, dextrose, lactose as well as the aqueous solutions of buffers, e.g. phosphate buffer solution.

Moreover, the composition may contain other auxiliaries, such as iso-osmotic agents, antioxidants, preservatives and others, which are compatible with the active substance and the other components of the lipid bilayer . In the preferred embodiment of the invention, the pharmaceutically acceptable carrier is glucose, which is used in a ratio 5:1 (w/w) in relation to lipid components . Glucose, in the compositions of the invention, plays a function of both a carrier and cryoprotectant . Glucose is added in the amount sufficient to ensure a proper isoosmolarity and isohydria of the composition, so that the formulation may be reconstituted ex tempore exclusively with water for injections, resulting in a solution of a desired glucose concentration (eg. 5%) . In this case, the use of other diluents or excipients is not necessary. The pharmaceutical composition may be in the form of lyophilisate for reconstitution ex tempore, of "ready to administer" dispersion, or in the form of i v concentrate. Prior to an administration, the lyophilisate may be reconstituted with a suitable volume of reconstitution solution, such as a sterile water for injection, the solution of saline, or with the solution of other pharmaceutically acceptable diluent, such as sugar or sugar alcohol. The lyophilisate is supplied in single dosage form, preferably in a vial, containing the effective amount of the active substance. The single dosage form of the composition is prepared by distribution of the sterile suspension under sterile conditions to the vials, lyophilization and tight closure. Lyophilized compositions provided by the present invention are characterized by a long shelf-life at the temperatures between 4°C and 30°C, which is longer than

6 months, preferably longer than 12 months, more preferably more than 24 months.

Description of the figures Fig. 1: Gaussian analysis of the size distribution of PC/KW-23.3 (9:1 m/m) liposomes with paclitaxel (1:30 w/w) , recorded by Photon Correlation Spectroscopy (PCS) . Small unilamellar liposomes are obtained by the extrusion of the large multilayer liposomes through a

100 nm size filter. Analysis: mean diameter (as a function of volume) : 106.6 nm, peak width: 65 nm, quality: passed, index of refraction: 0.118, temperature: 21.3°C, viscosity: 0.97 cps, print data:

312 kcounts. Fig. 2: Gaussian analysis of the size distribution of PC/KW-28 (9:1 m/m) liposomes with paclitaxel (1:30 w/w) , recorded by Photon Correlation Spectroscopy (PCS) . Small single layer liposomes are obtained by extrusion of the large multilayer liposomes through a 100 nm size filter. Analysis: mean diameter (as a function of volume): 129 nm, peak width: 81.8 nm, quality: passed, index of refraction: 0.142, temperature: 21.6°C, viscosity: 0.96 cps, print data:

345 kcounts. Fig. 3: Gaussian analysis of the size distribution of PC/KW-18 (9:1 m/m) liposomes with paclitaxel (1:30 w/w) , recorded by Photon Correlation Spectroscopy (PCS) . Small single layer liposomes are obtained by extrusion of the large multilayer liposomes through a 100 nm size filter. Analysis: mean diameter (as a function of volume): 133.9 nm, peak width: 70.9 nm, quality: passed, index of refraction: 0.133, temperature: 21.5°C, viscosity: 0.97 cps, print data:

343.9 kcounts. Fig. 4: Gaussian analysis of size distribution of

PC/KW-23.3 (9:1 m/m) liposomes with paclitaxel (1:30 w/w) , recorded by Photon Correlation Spectroscopy (PCS) .

A. Liposomes extruded through a 100 nm size filter. Analysis: mean diameter (as a function of volume): 111.7 nm, peak width: 68.2 nm, quality: passed, index of refraction: 0.113, temperature: 21.2°C, viscosity: 0.97 cps: print data: 446 kcounts.

B. Liposomes A, lyophilized in the presence of cryoprotectant (glucose; glucose/lipid = 5:1 w/w) and reconstituted with water. Analysis: mean diameter (as a function of volume):

111.7 nm, peak width: 72.8 nm, quality: passed, index of refraction: 0.121, temperature: 21.1°C, viscosity:

0.98 cps, print data: 421.7 kcounts. Fig. 5: Gaussian analysis of a size distribution of PC/KW-23.3 (9:1 m/m) liposomes with paclitaxel (1:30 w/w) , recorded by Photon Correlation Spectroscopy (PCS) . Liposomes prepared in the Example 1 were stored as a suspension at 4°C for 1 month. Analysis: mean diameter (as a function of volume) : 107.2 nm, peak width: 68.5 nm, quality: passed, index of refraction:

0.123, temperature: 20.7°C, viscosity: 0.99 cps, print data: 296.9 kcounts.

The following examples are given for purposes of the illustration only and not by way of limitation of the scope of the invention. Examples Example 1 To a 30 ml screw cap glass vial, 114 mg of egg phosphatidilcholine in chloroform (10 mg/ml), 6 mg of KW-23.3 ( (2-hexadecyloxy-4-pentadecylbenzoic acid) in chloroform (10 mg/ml) and 4 mg of paclitaxel in methanol solution (5 mg/ml) was added, then organic solvents were removed with a stream of nitrogen, with concurrent heating of the vial to 35°C. The viscous residue was dissolved in 5 ml of tert-butyl alcohol, frozen and freeze-thawed overnight. Dry residue was then hydrated, by vigorous shaking with 2 ml of sterile saline. The obtained multilamellar liposomes containing paclitaxel were subjected to 10 cycles of extrusion with pressure extruder using a 100 nm size polycarbonate filters (Fig. 3) . Upon extrusion a 50μl sample was collected; paclitaxel and lipid components were determined with HPLC (Waters System, diode detector, λ=230 nm; acetonitrile : water 6:4;

KNAUER Lichrosorb-100, RP-18, 4,6 mm, 5μm) . Paclitaxel encapsulation efficiency was evaluated by the comparison of paclitaxel content in extruded liposomes and in the pre-liposomes. Encapsulation efficiency > 95%.

Example 2 To a screw cap vial 113.3 mg of dimyristyloylphosphatidylcholine (DMPC) , 6.72 mg of KW- 28 (2- (3-pentadecylphenoxy) acetic acid) and 4 mg of paclitaxel was weighed and tert-butyl alcohol (5 mL) was added. The mixture was then shaken with microshaker until dissolution of components. The clear solution was then frozen and freeze-thawed over the period of 24 hours. The obtained lyophilisate was hydrated with 2 ml of sterile physiological salt, by vigorous shaking of the vial on the microshaker for 10 minutes. The milky suspension of multilayer liposomes was subjected to 10 cycles of extrusion (Fig. 4) . Encapsulation efficiency of paclitaxel was determined as in Example 1. Encapsulation efficiency > 94%.

Example 3 The liposome paclitaxel formulation was prepared as in Example 1 : KW-18 (2-hydroxy-4-pentadecylbenzoesic acid) - 6 mg egg phosphatidilcholine - 114 mg paclitaxel - 4 mg Analysis of liposomes obtained by calibration is shown in Fig. 5. Encapsulation efficiency > 94%.

Examples 4 - 7 Following the procedure described above but using the different phospholipids and alkylphenol derivatives o formula (I), in the proportion 9:1 (m/m), the liposomal paclitaxel formulations were prepared. The compositions of liposomal formulations and encapsulation efficiencies are presented in the Table 1. Table 1.

EPC = Egg phosphatidilcholine KW-93 = 7-0- [4-0- (2, 3, 4, 5-tetra-O-acetylo-β-D- galaktopiranozylo) -6-0-acetylo-α-D-erytro-heks-2- enopiranozylo] genisteina; KW-87 = 2- (3-pentadecylphenoxy) ethan-1-ol; KW-53 = N- (2-hydroxyethyl) -2- (3-pentadecylphenoxy) acetamide; KW-51 = N- (3S,4S,5S,2R)-2,3,4,5, 6-tetrahydroxyethyl) -N- methyl-2- (3-pentadecylphenoxy) acetamide .

Stability tests A. Liposomal paclitaxel formulations stability tests were performed while adding to 1 mL of calibrated liposome suspension (Example 1) of glucose (300 mg) . Upon dissolution of glucose, the sample was frozen and freeze-thawed over 12 hours. The lyophilisate was reconstituted with 1 mL of distilled water. The liposomes mean diameter was identical with that determined for the liposomal pre-formulation and accounted for 111 nm (+/-4 nm) (Fig. 4A and Fig. 4B) . Within 5 days of storing, in the reconstituted formulation at 25°C no paclitaxel crystals were observed.

B. Stability of the liposome suspension was evaluated on the basis of the studies carried out with the transmission electron microscope Jeol 100C (5.000- 100.000 x) . The initiation of crystals formation as a result of paclitaxel leakage from the vesicles was the control point of the observation. The liposome suspensions of Examples 1-3 were stored at 4°C. The period of two days before the occurrence of crystals of paclitaxel was regarded as the maximum shelf-life of liposome suspension. The shelf-life was estimated as at least 3 weeks for formulation from Example 1, and 10 days for formulation from examples 2 and 3. Analysis of liposome size did not show degradation of the liposome suspension within 1 month (Fig. 5) .

C. Stability evaluation of lyophilized liposomal composition of Example 2 was performed by HPLC of paclitaxel and 2- (3-pentadecylphenoxy) acetic acid (KW- 28) content after reconstitution with water (5 mg/2.5 mL) . The stability data for lyophilisate stored at 4°C, 25°C and 30°C are presented in the Table 2. The results confirm the stability of the lyophilisate after a 12 month storing period at 25°C and after 24 months at 4°C.

Table 2. Stability test, Paclitaxel lyophilisate 5 mg/2.5 L

* Content determined by HPLC, after reconstitution of lyophilisate with water to 2 5 mL and diluting with methanol to 25 mL The content was determined with the reference to standard

Claims

Claims 1. A liposomal formulation of an antineoplastic agent encapsulated in the liposomal vesicles which are the composition of lipid components, wherein the ratio of a therapeutically active agent to the lipid components is between about 1:10 and about 1:30 (w/w).

2. A liposomal formulation according to claim 1, wherein the ratio of a therapeutically active agent to lipid components is about 1:20 (w/w) .

3. A liposomal formulation according to claims 1-2, wherein the encapsulation efficiency of the antineoplastic agent is greater than 90%.

4. A liposomal formulation according to claims 1-3, wherein the encapsulation efficiency of the antineoplastic agent is greater than 95%. 5. A liposomal formulation according to claim 1, wherein the lipid components consist of at least one phospholipid derivative and at least one alkylphenol derivative of the general formula (I), wherein: n is an integer 1 - 17; m is an integer 0-15; X is -C=0, -0- or a bond; Y is a group of formula -NR₂R₃, a monosaccharide moiety or a bond; Z is H, -0-, -N-, -OR₄, -NR₄ or a monosaccharide moiety, R₅ is H or COOH; while : a) if X is C=0, then Y is NR₂R₃, in which R₂ is H or Cι-C₃ alkyl, and R₃ is Cι-C₆ alkyl substituted with at least one hydroxylic group, and Y is -H, -N- or NR₄, where R₄ is H or Ci-C₂ alkyl; or Y is -N-, and Ri and R₂ together with Y form a saturated heterocyclic ring; Z is -OR4, where R₄ is H; whereas b) if X is -0-, then Y is a monosaccharide moiety, and Z is H or monosaccharide moiety attached to X through an oxygen atom.

5. A liposomal formulation according to claims 1- 4, wherein the lipid components consist of at least one phospholipid derivative and at least one alkylphenol derivative of the general formula (I), wherein: n is an integer 13 - 17; m is an integer 0-15; X is -0-; Y is a bond; Z is -OR4, wherein R₄ is H; R₅ is H or COOH.

6. A liposomal formulation according to claims 1- 4, wherein the lipid components consist of at least one phospholipid derivative and at least one alkylphenol derivative of the general formula (I), wherein: n is an integer 13 - 17; is 1 ; X is -C=0; Y is a group of formula -NR₂R₃; in which R₂ is H or Cι~C₃ alkyl, and R₃ is Ci-Cε alkyl substituted with at least one hydroxylic group, and R₄ is H; R₅ is H or COOH.

7. A liposomal formulation according to claims 1- 4, wherein the lipid components consist of at least one phospholipid derivative and at least one alkylphenol derivative of the general formula (I), wherein: n is an integer 13 - 17; m is 1; X is -0-; Y is a monosaccharide moiety; and Z is H or monosaccharide moiety attached to X through an oxygen atom; R₅ is H or COOH.

8. A liposomal formulation according to any of the preceding claims, wherein the lipid components consist of the phospholipid derivative and alkylphenol derivative of formula (I) which are present in a molar ratio about 9:1.

9. A liposomal formulation according to any of the preceding claims, wherein the antineoplastic agent is selected from the group comprising the anthracyclines, such as mitoxantrone, daunomycine, doxorubicine, epirubicine or idarubicine; alkylating agents; camptothecin derivatives, such as irinotecan and camptothecin; lignans, such as etoposide; and taxanes, such as paclitaxel and docetaxel.

10. A liposomal formulation according to any of the preceding claims, wherein the antineoplastic agent is a taxane derivative.

11. A liposomal formulation according to any of the preceding claims, wherein the taxane derivative is paclitaxel .

12. A liposomal formulation according to any of the preceding claims, wherein the taxane derivative is docetaxel .

13. A process for the preparation of the liposomal formulation of high encapsulation efficiency of the antineoplastic agent, comprising the steps of: (a) combining the solution of the antineoplastic agent in the suitable solvent with the solution of lipid components in the suitable organic solvent;

(b) removing the solvents to form the lipid film; (c) dissolving the lipid film formed in step (b) in the organic solvent, preferably alkanol, and freeze- thawing of thus formed suspension;

(d) hydration of the lyophilisate obtained in step (c) with aqueous system, to form the pre-formulation of multilamellar liposomes;

(e) further processing of the pre-formulation of step (d) , to form a suspension of calibrated bilayer liposomes of the size 50-200 nm, preferably 100 nm;

(f) lyophilisation of the liposomal suspension together with the pharmaceutically acceptable excipients and/or

carriers, and wherein lipid components are a mixture of at least one phospholipid derivative and at least one alkylphenol derivative presented by the general formula 1, where: n is an integer 1 - 17; m is an integer 0-15;

X is -C=0, -0- or a bond;

Y is a group of formula -NR₂R₃, a monosaccharide moiety

or a bond; Z is H, -0-, -N-, -OR₄, -NR₄ or a monosaccharide moiety,

R₅ is H or COOH; while : a) if X is C=0, then Y is NR₂R₃, in which R₂ is H or C_ι~

C₃ alkyl, and R₃ is Cι-C₆ alkyl substituted with at least one hydroxylic group, and

Y is -H, -N- or NR₄, where R₄ is H or Cι-C₂ alkyl; or Y is -N-, and Ri and R₂ together with Y form a saturated heterocyclic ring;

Z is -OR₄, where R is H; whereas b) if X is -0-, then Y is a monosaccharide moiety, and

Z is H or monosaccharide moiety attached to X through an oxygen atom; in a molar ratio of about 9:1.

14. A process according to claim 13, wherein at the step (e) the liposome formulation is extruded through a 50-200 nm size filter, preferably through a

100 nm size filter.

15. A pharmaceutical composition comprising the liposomal formulation of an antineoplastic agent according to claim 1, together with the pharmaceutically acceptable carriers and/or excipients.

16. A pharmaceutical composition according to claim 15, wherein the antineoplastic agent is present in a therapeutically effective amount.

17. A pharmaceutical composition according to claims 15-16, wherein the excipient is glucose.

18. A pharmaceutical composition according to claims 15-17, wherein glucose and lipid components of the liposomal preparation are present in the ratio 5:1 (w/w) .

19. An anticancer pharmaceutical composition according to claims 15-18 in a single dosage form.

20. A composition according to claims 15-19, in a lyophilisate form.