Liposome Composition

Abstract

A pharmaceutical composition comprising a nucleoside analog encapsulated in a liposome of a liposome delivery system, wherein the nucleoside analog is a purine analog selected from {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid or 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, or a pyrimidine analog selected from 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide.

Description

[Technical field]

[0001] The present invention relates to liposomal compositions, particularly liposomal compositions incorporating nucleoside analogues, and their use in the treatment of cancer. [Background technology]

[0002] Cancer, one of the leading causes of death worldwide, continues to be the focus of considerable research efforts.

[0003] While new potential therapeutic molecules and new targets are being discovered, the key to effectively treating certain disorders, such as cancer, often relies on improving the delivery of active ingredients to target cells.

[0004] Innovative drug delivery systems capable of such delivery are needed and rigorous research is being conducted to provide biocompatible, cost-effective, safe and efficient options that would enable their use in therapy.

[0005] A representative drug delivery system is that of liposomes, which can encapsulate active ingredients inside. Liposomes are nano-sized, can be surface-modified for better targeting, or multiple types of liposomes can be combined to improve results.

[0006] Liposomes are an established tool for drug delivery and were first described in 1963 by Alee Bangham.

[0007] There are already products on the market today in liposomal form that are useful for cancer therapy, such as Onivyde™, Marqibo®, Doxil®, Visudyne® and Depocyt®.

[0008] Liposomes encapsulate both hydrophilic and hydrophobic drugs. Liposomes are primarily composed of phospholipids, which can form monolayers or bilayers in most environments. Hydrophilic drugs are usually trapped inside the liposome (aqueous environment), while hydrophobic molecules remain within the lipid bilayer of the liposome. Phospholipids tend to form particles with an aqueous core when dispersed in an aqueous environment above the transition temperature.

[0009] Liposomes have an added advantage in combination with cytotoxic drugs: the compartmentalization afforded by liposomes can reduce toxicity while at the same time providing more targeted drug delivery, thereby increasing efficacy.

[0010] Patent Document 1 relates to nucleotide or nucleoside therapeutic compositions and their use in the treatment of infectious diseases, viral infections and cancer, and discloses that liposome suspensions may be suitable for the delivery of free nucleosides, acyl nucleosides, or phosphate ester prodrug forms of nucleoside compounds according to inventions known in the literature.

[0011] Furthermore, Patent Document 2 relates to sulfur-containing nucleotide or nucleoside therapeutic compositions and their use in the treatment of infectious diseases, viral infections and cancer, and discloses that liposomal suspensions may be suitable for the delivery of free nucleosides, acyl nucleosides, or phosphate ester prodrug forms of nucleoside compounds according to inventions known in the literature.

[0012] US Patent No. 5,399,633 relates to nucleic acids such as oligonucleotides encapsulated within liposome complexes containing a mixture of cationic lipids, fusogenic lipids and PEG lipids.

[0013] Patent Document 4 relates to nucleoside-lipid conjugates including nucleosides conjugated with phospholipids and nucleoside analogs such as gemcitabine, and further to the use of such nucleoside-lipid conjugates for the treatment of mammalian diseases such as cancer and viral infections. The nucleoside-lipid conjugates can be used in the preparation of liposomes together with phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, cholesterol, [α]-tocopherol, natural or synthetic cardiolipin and its analogs, synthetic cationic cardiolipin analogs and its derivatives, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophosphotidylglycerol, lysophosphatidic acid, lysophosphotidylcholine, lysophosphatidylserine, and PEG-modified lipids.

[0014] Patent document 5 relates to parenteral formulations for certain long-chain saturated and monounsaturated fatty acid derivatives of the nucleoside analogue 2',2'-difluorodeoxycytidine (gemcitabine) in the treatment of cancer, and further to parenteral pharmaceutical compositions of said derivatives in the form of liposomes having an average particle size in the range of 2.5 nm to 30 nm.

[0015] Patent Document 6 relates to a pharmaceutical composition comprising (a) an anionic liposome comprising a phospholipid having a head group selected from the group consisting of sn-glycero-phosphocholine, sn-glycero-phospho-rac-(1-glycerol), sn-glycero-phospho-L-serine, sn-glycero-3-phosphate, and combinations thereof, (b) a bioactive agent which may be a nucleoside analog, and (c) a cation, a buffer, or a combination thereof, wherein the anionic liposome is not a combination of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS) in a ratio of 2:1, having a diameter of 122 nm to 162 nm.

[0016] Non-Patent Document 1 is a review document that discloses liposomes in cancer therapy, such as paclitaxel, triptolide, resveratrol, doxorubicin, docetaxel, hydroxyquinone, curcumin, sorafenib, shikonin, losartan, and liposomes of gemcitabine, 5-fluorouracil, and cordycepin. Gemcitabine is a pyrimidine analogue, particularly a nucleoside analogue, in which the hydrogen atom on the 2' carbon of deoxycytidine is replaced with a fluorine atom. 5-Fluorouracil is a pyrimidine analogue, particularly a nucleobase analogue. Cordycepin is a purine analogue, particularly an adenosine analogue. [Prior art documents] [Patent documents]

[0017] [Patent Document 1] International Publication No. 2017 / 155923 [Patent Document 2] International Publication No. 2015 / 038596 [Patent Document 3] International Publication No. 2010 / 014895 [Patent Document 4] International Publication No. 2006 / 029081 [Patent Document 5] International Publication No. 2011 / 062503 [Patent Document 6] US Patent Application Publication No. 2003 / 0026831 [Non-patent literature]

[0018] [Non-Patent Document 1] Saraf, Shivani, et al. “Advances in liposomal drug delivery to cancer: An overview.” Journal of drug delivery science and technology 56 (2020): 101549 Summary of the Invention

[0019] It is therefore an object of the present invention to provide a pharmaceutical composition comprising a nucleoside analogue encapsulated in a liposome of a liposome delivery system, the nucleoside analogue being a purine analogue selected from {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid or 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, or a pyrimidine analogue selected from 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide. The above-mentioned nucleoside analogues have not been previously known for use in pharmaceutical compositions, particularly in the treatment of cancer.

[0020] The chemical structures of nucleoside analogues are shown below.

[0021] [Table 1]

[0022] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the nucleoside analogue is covalently or non-covalently attached to the liposome.

[0023] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system have a diameter, as determined by SEM, of at least 750, preferably at least 775, more preferably at least 800 nm, preferably between 800 nm and 1000 nm, or between 800 and 950 nm. Liposomes with the preferred size are more stable than liposomes with smaller sizes, especially those with sizes in the range of 10 to 100 nm.

[0024] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system are sterically stabilized liposomes.

[0025] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system comprise at least a phospholipid, preferably at least a phosphatidylcholine and / or a phosphatidylethanolamine, or at least a phospholipid, more preferably at least a phosphatidylcholine and / or a phosphatidylethanolamine, most preferably in addition to one or more phosphoglycerides and / or one or more sphingolipids. The inclusion of phosphatidylethanolamine and phosphatidylcholine has the advantage of providing amino or trimethylamino groups on the surface of the liposome, which may be useful for further functionalization of the liposome, for example by the formation of amide bonds in the case of proteins or amino acids.

[0026] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposome of the liposome delivery system comprises or essentially consists of a PEGylated phospholipid, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, or a combination of PEGylated and non-PEGylated phospholipids, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and non-PEGylated phosphatidylcholine, as lipid components of the liposome, optionally with a sterol, such as cholesterol. Liposomes of the above composition have been found to have better entrapment efficiency during the manufacturing process.

[0027] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the PEGylated phospholipid is PEGylated phosphoethanolamine and the non-PEGylated phospholipid is phosphatidylcholine. The liposome of the above composition has been found to have better entrapment efficiency during the manufacturing process.

[0028] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system are free of fatty acids, in particular free of unsaturated fatty acids, preferably free of monounsaturated fatty acids such as oleic acid, which have been found to improve the entrapment efficiency during the manufacturing process.

[0029] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the nucleoside is {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid, and the liposome of the liposome delivery system comprises or consists of a pegylated phospholipid, such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and a sterol, such as cholesterol, as the sole phospholipid component of the liposome. In the embodiment, the amount of the pegylated phospholipid in the liposome can be about 85% to about 95% by weight, more preferably about 90% to about 95% by weight, based on the total weight of the pegylated phospholipid and the sterol. In the embodiment, the amount of the sterol in the liposome can be about 5% to about 15% by weight, more preferably about 5% to about 10% by weight, based on the total weight of the pegylated phospholipid and the sterol.

[0030] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system comprise a sterol, such as cholesterol, in an amount of from about 5% to less than 50% by weight, preferably from about 5% to less than 25% by weight, more preferably from about 5% to less than 20% by weight, and most preferably from about 6% to less than 16% by weight, based on the weight of the PEGylated phospholipid or the weight of a combination of PEGylated and non-PEGylated phospholipids.

[0031] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposome of the liposome delivery system comprises or consists of 40 to 45% by weight of a pegylated phospholipid such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 40 to 45% by weight of a non-pegylated phospholipid such as phosphatidylcholine, and a sterol such as cholesterol, based on the total weight of the pegylated phospholipid, the non-pegylated phospholipid, and the sterol, and preferably, the pegylated phospholipid and the non-pegylated phospholipid are contained in a weight ratio of 1:1.

[0032] A further object of the present invention is to provide a pharmaceutical composition according to the above objects for use in the treatment of colon cancer or lung cancer or breast cancer, preferably by oral or parenteral route.

[0033] Yet a further object of the present invention is to provide the use of a pharmaceutical composition according to above object for inhibiting the proliferation of a cancer cell line in vitro, preferably at a concentration between 0.01 μM and 10 μM, and / or the cancer cell line is preferably a lung cancer or colon cancer or breast cancer cell line, and / or the nucleoside analogue is {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid or 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one, preferably present at a concentration between 0.01 μM and 1 μM.

[0034] Another object of the present invention is a method for preparing a liposomal delivery system comprising a nucleoside analogue encapsulated in liposomes, wherein the liposomes of the liposomal delivery system are composed of a combination of PEGylated and non-PEGylated phospholipids, preferably 40-45% by weight of PEGylated phospholipids, such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, based on the total weight of the PEGylated phospholipids, non-PEGylated phospholipids and sterols. and a sterol such as cholesterol, more preferably the pegylated phospholipid and the non-pegylated phospholipid are contained in a weight ratio of 1:1 or 4:5 to 5:4, and the nucleoside analog is selected from 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide, The method comprises: a. solubilizing PEGylated phospholipids, non-PEGylated phospholipids and sterols, and nucleoside analogs in a polar solvent, preferably a mixture of polar solvents; b. evaporating the solvent; c. adding an aqueous solution or water, preferably an aqueous buffer solution, to form a first liposome suspension; d. sonicating the first liposome suspension to form a second liposome suspension; e. annealing the second liposome suspension to form a liposome delivery system comprising the nucleoside analog encapsulated in the liposome; The object of the present invention is to provide a method comprising the steps of:

[0035] Another object of the present invention is a method for preparing a liposomal delivery system comprising a nucleoside analogue encapsulated in liposomes, wherein the liposomes of the liposomal delivery system are composed of a combination of PEGylated and non-PEGylated phospholipids, preferably 40-45% by weight of PEGylated phospholipids such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, based on the total weight of the PEGylated phospholipids, non-PEGylated phospholipids and sterols. the composition comprises or consists of a PEGylated phospholipid, 40-45% by weight of a non-PEGylated phospholipid such as phosphatidylcholine, and a sterol such as cholesterol, more preferably the PEGylated phospholipid and the non-PEGylated phospholipid are contained in a weight ratio of 1:1 or 4:5 to 5:4, the nucleoside analogue is selected from {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, The method comprises: a. solubilizing PEGylated phospholipids, non-PEGylated phospholipids and sterols in a polar solvent, preferably a mixture of polar solvents; b. evaporating the solvent; c. adding an aqueous solution of a nucleoside analog, preferably a buffered aqueous solution of a nucleoside analog, to form a first liposome suspension; d. sonicating the first liposome suspension to form a second liposome suspension; e. annealing the second liposome suspension to form a liposome delivery system comprising the nucleoside analog encapsulated in the liposome; The object of the present invention is to provide a method comprising the steps of:

[0036] Another object of the present invention is a method for producing a liposomal delivery system comprising a nucleoside analogue encapsulated in a liposome, comprising the steps of: the liposome of the liposome delivery system comprises or consists of a pegylated phospholipid, such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and a sterol, such as cholesterol, as the only phospholipid components of the liposome; the amount of the pegylated phospholipid in the liposome is preferably about 85% by weight to about 95% by weight, more preferably about 90% by weight to about 95% by weight, based on the total weight of the pegylated phospholipid and the sterol; and / or the amount of the sterol in the liposome is about 5% by weight to about 15% by weight, more preferably about 5% by weight to about 10% by weight, based on the total weight of the pegylated phospholipid and the sterol; the nucleoside analog is {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid; The method comprises: a. solubilizing the PEGylated phospholipids and sterol in a polar solvent, preferably a mixture of polar solvents; b. evaporating the solvent; c. adding an aqueous solution of a nucleoside analog, preferably a buffered aqueous solution of a nucleoside analog, to form a first liposome suspension; d. sonicating the first liposome suspension to form a second liposome suspension; e. annealing the second liposome suspension to form a liposome delivery system comprising the nucleoside analog encapsulated in the liposome; The object of the present invention is to provide a method comprising the steps of:

[0037] Further embodiments of the invention are defined in the dependent claims.

[0038] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the present invention will now be described with reference to the drawings, which are intended to illustrate presently preferred embodiments of the invention and are not intended to be limiting thereof. [Brief description of the drawings]

[0039] [Figure 1] 1 shows a flow chart illustrating one embodiment of the claimed method showing encapsulation of 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one and N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide into liposomal delivery. Note that for 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one and N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide, the nucleoside analog is added at the stage where the lipid components of the liposomal delivery system are dissolved in chloroform:methanol. [Diagram 2] 1 shows a flow chart illustrating one embodiment of the claimed method showing encapsulation of {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol into liposomal delivery. Note that for {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, the nucleoside analog is added during the PBS film hydration step, and the nucleoside analog is dissolved in PBS. [Diagram 3] FIG. 1 shows standard curves of various nucleoside analogues in phosphate buffered saline (PBS) for {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid, 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide, 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one. [Figure 4]1 shows differential scanning calorimetry thermographs of phase transition peaks of liposomes of (1): 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one in liposomes of liposome delivery system 3, (2): blank liposomes of liposome delivery system 3, (3): 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol in liposomes of liposome delivery system 3, (4): blank liposomes of liposome delivery system 2, (5): N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide in liposomes of liposome delivery system 3, and (6): {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid in liposomes of liposome delivery system 2. [Diagram 5] 1A and 1B show SEM images of blank liposomes, where 1a and 1b are photographs of liposome blank 1 with diameters of 642, 630 and 668 nm as can be seen in 1a, which is a magnified portion of 1b, and 2a and 2b are photographs of liposome blank 2 with diameters of 642, 630 and 668 nm. [Figure 6] (A): SEM image of liposomes encapsulating 2-(6-amino-9H-purin-9-yl)quinazolin-4-yl dihydrogen phosphate, and (B) an enlarged cross section of the SEM image of (A). In (B), it can be seen that the diameters of the three selected liposomes are determined to be 807, 816 and 838 nm. [Figure 7] (A): SEM images of liposomes encapsulating 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, and (B) shows an enlarged cross section of the SEM image of (A). In (B), it can be seen that the diameters of the three selected liposomes are determined to be 938, 907, and 925 nm. [Figure 8]FIG. 1 shows SEM images of liposomes encapsulating (A): and N-(9-(4-hydroxyquinazolin-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide, and an enlarged cross section of the SEM image of (A) is shown in (B). In (B), it can be seen that the diameters of the three selected liposomes are determined to be 860, 830 and 755 nm. [Figure 9] (A): SEM image of liposomes encapsulating 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one, and (B) shows an enlarged cross section of the SEM image of (A). In (B), it can be seen that the diameters of the three selected liposomes are determined to be 862, 846, and 834 nm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the nucleoside analogue is covalently or non-covalently attached to the liposome.

[0041] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system have a diameter, as determined by SEM, of at least 750, preferably at least 775, more preferably at least 800 nm, preferably between 800 nm and 1000 nm, or between 800 and 950 nm. Liposomes with the preferred size are more stable than liposomes with smaller sizes, especially those with sizes in the range of 10 to 100 nm.

[0042] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system are sterically stabilized liposomes.

[0043] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system comprise at least a phospholipid, preferably at least a phosphatidylcholine and / or a phosphatidylethanolamine, or at least a phospholipid, more preferably at least a phosphatidylcholine and / or a phosphatidylethanolamine, most preferably in addition to one or more phosphoglycerides and / or one or more sphingolipids. The inclusion of phosphatidylethanolamine and phosphatidylcholine has the advantage of providing amino or trimethylamino groups on the surface of the liposome, which may be useful for further functionalization of the liposome, for example by the formation of amide bonds in the case of proteins or amino acids.

[0044] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposome of the liposome delivery system comprises or essentially consists of a PEGylated phospholipid, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, or a combination of PEGylated and non-PEGylated phospholipids, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and non-PEGylated phosphatidylcholine, as lipid components of the liposome, optionally with a sterol, such as cholesterol. Liposomes of the above composition have been found to have better entrapment efficiency during the manufacturing process.

[0045] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the PEGylated phospholipid is PEGylated phosphoethanolamine and the non-PEGylated phospholipid is phosphatidylcholine. The liposome of the above composition has been found to have better entrapment efficiency during the manufacturing process.

[0046] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system are free of fatty acids, in particular free of unsaturated fatty acids, preferably free of monounsaturated fatty acids such as oleic acid, which have been found to improve the entrapment efficiency during the manufacturing process.

[0047] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the nucleoside is {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid, and the liposome of the liposome delivery system comprises or consists of a pegylated phospholipid, such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, and a sterol, such as cholesterol, as the sole phospholipid component of the liposome. In the embodiment, the amount of the pegylated phospholipid in the liposome can be about 85% to about 95% by weight, more preferably about 90% to about 95% by weight, based on the total weight of the pegylated phospholipid and the sterol. In the embodiment, the amount of the sterol in the liposome can be about 5% to about 15% by weight, more preferably about 5% to about 10% by weight, based on the total weight of the pegylated phospholipid and the sterol.

[0048] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposomes of the liposomal delivery system comprise a sterol, such as cholesterol, in an amount of from about 5% to less than 50% by weight, preferably from about 5% to less than 25% by weight, more preferably from about 5% to less than 20% by weight, and most preferably from about 6% to less than 16% by weight, based on the weight of the PEGylated phospholipid or the weight of a combination of PEGylated and non-PEGylated phospholipids.

[0049] In one embodiment of the pharmaceutical composition according to the above object of the present invention, the liposome of the liposome delivery system comprises or consists of 40 to 45% by weight of a pegylated phospholipid such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 40 to 45% by weight of a non-pegylated phospholipid such as phosphatidylcholine, and a sterol such as cholesterol, based on the total weight of the pegylated phospholipid, the non-pegylated phospholipid, and the sterol, and preferably, the pegylated phospholipid and the non-pegylated phospholipid are contained in a weight ratio of 1:1.

[0050] Use in pharmaceutical compositions, particularly in the treatment of cancer.

[0051] It is understood that the above limitations on composition also apply to methods of making liposomal delivery systems containing any of the nucleoside analogs.

[0052] In a preferred embodiment of the method for producing a liposomal delivery system containing any of the nucleoside analogs entrapped in liposomes, the polar solvent comprises chloroform mixed with ethanol or methanol, preferably in a 3:1 ratio.

[0053] In a preferred embodiment of the method for producing a liposomal delivery system comprising any of the nucleoside analogues encapsulated in liposomes, the annealing of the second liposomal suspension to form the liposomal delivery system comprising the nucleoside analogues encapsulated in liposomes is carried out at a temperature of at least 45°C, more preferably at a temperature above 50°C, and most preferably at a temperature between 55 and 75°C, e.g., 65°C.

[0054] In a preferred embodiment of the method for producing a liposomal delivery system comprising any of the nucleoside analogs encapsulated in liposomes, the aqueous buffer solution is a phosphate buffered saline, preferably a phosphate buffered saline having a neutral pH.

[0055] In a preferred embodiment of the method of producing a liposomal delivery system comprising any of the nucleoside analogs encapsulated in liposomes, the concentration of the nucleoside analog in step c. is about 1 mg / mL in the first liposomal suspension.

[0056] Working Example Materials and Methods LIPOID PE 18:0 / 18:0-PEG 2000, hereafter DSPE-PEG ([N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt]) and LIPOID S PC-3, hereafter PC (hydrogenated phosphatidylcholine from soybean) were purchased from Lipoid (Lipoid GmbH, Frigenstrasse 4, D-67065 Ludwigshafen, Germany). Cholesterol, hereafter CHOL, and oleic acid, hereafter OA, were purchased from Fluorochem (Graphite Way, Hadfield, Derbyshire, UK). Phosphate buffered saline at 0.01 M and pH 7.4, as well as methanol and chloroform were purchased from Sigma-Aldrich (3050 Spruce Street, Saint Louis, MO 63103, USA).

[0057] Preparation of liposomes Liposomes were prepared based on the thin film method. Different lipid concentrations were tested and the ideal system was selected in each case based on their encapsulation efficiency. 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol and {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid were first encapsulated in different systems and the one showing the best results was used for the remaining APIs. All the concentrations used can be seen in Table 1.

[0058] [Table 2]

[0059] Briefly, the preparation of liposomes began with dissolving PE 18:0 / 18:0-PEG 2000, phosphatidylcholine (PC) and cholesterol in 10 mL of chloroform:methanol (3:1) solution in a 50 mL round-bottom flask. The solvent was then removed by vacuum evaporation to complete dryness or, in some cases, a stream of nitrogen was used to completely evaporate the solvent. A thin film of lipid was then formed on the walls of the flask, which was hydrated with 10 mL of 0.01 M, pH 7.4 phosphate buffered saline (PBS) and sonicated in a sonication bath for 30 min, after which the lipid was annealed at 65°C.

[0060] Encapsulation of Nucleoside Analogues Nucleoside analogs were encapsulated based on their properties at various steps of the process. 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one and N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide were added at the beginning of the process along with lipids and chloroform:methanol solution. To improve the solubility of the analogs, magnetic stirring was applied before vacuum evaporation (Process 1). Meanwhile, {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol were added after evaporating the organic solvent and dissolving in PBS solution (Process 2). The concentration of the nucleoside analogs was 1 mg / mL in both cases. 1 and 2 show schematics for the preparation of liposomes according to processes 1 and 2, respectively.

[0061] As a comparative example, the same process as above was followed but without the addition of any nucleoside analogues.

[0062] Liposome purification At the end of the above encapsulation process, the solution was centrifuged at 15000 rpm for 20 minutes and the supernatant was saved for indirect encapsulation determination. The precipitate was lyophilized and saved for further analysis and characterization.

[0063] Freeze drying The glass vials with rubber stoppers were placed in a freeze-dryer (Telstar, LyoBeta Mini) and the liposomes were precipitated in PBS. The freeze-drying process was started by freezing at -40°C for 2.5 hours and holding this temperature for another 3 hours. The condensation preparation was then started and continued for 10 minutes. After that, the vacuum in the chamber was started at 0.5 mbar to start the drying. The primary drying lasted for 2 hours 13 minutes at 0°C and 0.5 mbar. After holding these conditions for 11 hours (primary drying hold), the secondary drying was started. The temperature during the secondary drying was 23°C, 0.1 mbar and lasted for 6 hours 17 minutes. Finally, the cycle was finished.

[0064] Encapsulation Effect The supernatant after each centrifugation was saved and used to evaluate the amount of free drug. By subtracting the amount of free drug in the supernatant from the total amount of drug added, the remainder was considered to be encapsulated. Briefly, samples were taken from each solution to prepare solutions, which were subsequently evaluated by UV / VIS spectroscopy. The remaining amount was calculated based on a standard curve of various nucleosides in solution measured by UV-VIS spectroscopy. The encapsulation effect was then calculated using the following formula:

number

[0065] yield Upon completion of each freeze-drying protocol, the dry powder of each product was weighed. The yield of each was calculated according to the following formula (2):

number

[0066] Scanning electron microscope A scanning electron microscope (SEM), Phenom ProX (Thermo Fisher Scientific), was used to evaluate the size, shape and appearance of the liposomes. PELCO Tabs™ carbon conductive tabs, double coated, 12 mm OD, were used with an aluminum stage (1 / 2'' slot head and 1 / 8'' pin). These tabs are adhesives used for SEM mounting for better electrical conductivity. The mode chosen was described as "All Materials (5 kV)" with image intensity and BSD full detector. The live view is medium quality with a resolution of 1024 and the acquired images are high quality with a resolution of 2048. The software version is 4.5.2. All image processing (photo acquisition, size analysis, etc.) was performed by the instrument software.

[0067] Differential scanning calorimetry In general, thermal analysis measures the change in a material's properties as a function of temperature. In this study, differential scanning calorimetry was used, which measures the heat absorbed or released during heating or cooling. This is done by measuring the difference in heat flow between a sample and an inert reference, correlated to time and temperature, under a controlled atmosphere. DSC is a powerful tool that determines the physical and chemical changes in materials and systems as a result of endothermic or exothermic events. Processes can be both exothermic or endothermic, depending on whether the sampling releases or absorbs energy, respectively. The basic principle of this method is that in a physical transition, such as a phase transition, more or less heat needs to flow to the sample than to the reference material to keep both at the same temperature.

[0068] Regarding the characterization of liposomes, differential scanning calorimetry (DSC) is a useful tool. These colloidal lyotropic systems can be characterized by various temperature changes. The program used in this study starts with heating the sample from 30 °C to 180 °C at a rate of 10 °C / min and holds it there for 1 min. Then it is heated again from 180 °C to 370 °C. The experiment is carried out with a Mettler Toledo DSC3. The inert gas is N2 and the rate is 50 mL / min. The instrument was previously calibrated with the standard method using indium as a standard substance. The software to evaluate the results is the STARe software.

[0069] Cytotoxicity assay Human breast adenocarcinoma (luminal type) MCF7 cell line (ECACC 86012803), human breast adenocarcinoma (triple negative) MDA-MB-231 cell line (ECACC 92020424), human colon carcinoma HCT116 cell line (ECACC 91091005) and human lung carcinoma COLO699N cell line (ECACC 93052608) were cultured in RPMI 1640 medium, catalog number R0883 (Sigma-Aldrich, Darmstadt, Germany) supplemented with 10% fetal bovine serum, catalog number FB-1001 / 500 (BioSera, Nuaille, France) and 1% L-glutamine, catalog number G7513-100ML (Sigma-Aldrich, Darmstadt, Germany). All cell lines were maintained at 37 °C in a humidified 5% CO2 atmosphere. Cell viability was assessed by AlamarBlue assay. Viable cells were plated at 2 × 10 4Cells were seeded at a density of 100 μl / well and incubated at 37° C. and 5% CO2 for 24 h to form a cell monolayer. After 24 h incubation, the supernatant above the monolayer was aspirated and 200 μl of medium and various concentrations of substances were added and incubated for 24, 48 and 72 h time points. After the specified time points, 20 μl of 0.15 mg / ml resazurin sodium salt, catalogue number 199303 (Sigma-Aldrich, Darmstadt, Germany) in PBS was added to each well and incubated for 3 h at 37° C. and 5% CO2. Fluorescence was measured at an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Experiments were performed in triplicate. The mean fluorescence was calculated for each triplicate. Differences in the mean values ​​were determined by comparing treated samples with untreated controls using a one-sample t-test. A p-value <0.05 was considered to indicate a statistically significant difference. Results were calculated using Microsoft Excel 2016. LC50 (median lethal concentration) values ​​were determined using Microsoft Excel 2016 using the AlamarBlue assay measurements.

[0070] result Preparation of liposomes and encapsulation of nucleoside analogues 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol and {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid were tested in various liposomal systems. In Table 3, the results of the encapsulation efficiency of each system, calculated according to formula (1), can be seen.

[0071] [Table 3]

[0072] Liposome systems 2 and 3 showed the most successful results for {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, respectively. These two systems (liposome systems 2 and 3) were tested for the remaining nucleoside analogues, and the encapsulation efficiency was indirectly calculated based on formula (1) and the standard curve (Figure 3).

[0073] For example, for the {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid system, code 1, the supernatant is saved after centrifugation and a fraction is taken from it to prepare a solution with a target concentration of 10 ppm. Based on the dilution formula C1V1=C2V2, C1=1000 since the initial solution is considered to have a concentration of 1000 ppm (based on the assumption that all added nucleoside analogues are contained in the supernatant). C2: Target concentration, here 10 ppm V2: Volume of the final solution, here 10 mL

[0074] For this, 100 μL is taken from the supernatant and diluted to a final volume of 10 mL. The samples are quantified by UV / VIS spectroscopy and the actual concentration of the sample is calculated. The concentration is converted to mass and the final mass of nucleoside analogue in the supernatant and in the liposomes can be estimated.

[0075] Liposomes with the remaining nucleoside analogues were developed using liposome systems 2 and 3. Standard curves of nucleoside analogues in PBS can be seen in FIG.

[0076] [Table 4]

[0077] As can be seen from the results summarized in the table above, liposomal system 3 was found to exhibit good encapsulation efficiency and was used as the liposomal system for all nucleoside analogues, except for {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid, which retained analogue system 2.

[0078] yield The yields of the various liposomal systems were determined based on the mass of the product after the lyophilization cycle. The initial mass of each product is calculated according to the lipid and molecules added.

[0079] [Table 5]

[0080] DSC Blank liposomes and nucleoside analogue-encapsulated liposomes show similar endothermic peaks at the beginning of the thermographs. All liposomes, encapsulated or blank, appear to have the same glass transition temperature of around 35° C., with the exception of the {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid liposomes in thermograph (7).

[0081] Characteristic endothermic peaks are observed in the range of 40°C to 100°C for the liposomes in all cases, except for the {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid liposomes. These peaks arise from the phosphatidylcholine in these products, which is not used in the case of the {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid liposomes. Therefore, for these liposomes, there is a different broad endothermic peak at the beginning of the thermograph. Furthermore, in the DSC thermographs where the phase transition peaks are seen, it is clear that those peaks show large changes, since almost all the liposomes had the same formulation. It is believed that these changes can be attributed to the different chemical properties of each nucleoside analogue.

[0082] SEM The lyophilized liposomes were photographed and examined for their morphological characteristics and size. The liposomes all appear to have the same appearance of roughly spherical, uniformly sized particles. Their sizes were estimated using the sizing tool of the instrument.

[0083] The SEM pictures in figures 5, 6 and 7 show the liposomes. In the table below the size of the liposomes is reported, determined using a sizing tool on the SEM pictures.

[0084] [Table 6]

[0085] Based on the above measurements, it can be demonstrated that the size of liposomes encapsulating nucleoside analogues is significantly larger, indicating that encapsulation of active agents within liposomes can increase their size.

[0086] In vitro evaluation of cytotoxic activity The table below shows the cytotoxic effects of five liposomes encapsulating nucleoside analogues against four human cancer cell lines. LC50 values ​​were calculated for conditions where at least 50% cell viability was observed at test concentrations ranging from 0.01 μM to 10 μM. 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol, with or without liposomal encapsulation (not shown in the table), had no statistically significant cytotoxic effects at any time point against any of the human cancer cell lines at concentrations ranging from 0.01 μM to 10 μM. The MDA-MB-231 human breast adenocarcinoma cell line was resistant to all nucleoside analogues tested, with or without liposomal encapsulation (not shown in the table). For the remaining cancer cell lines, COLO699N, HCT116, and MCF7, none of the nucleoside analogs had a statistically significant cytotoxic effect when used alone (not shown in the table), i.e., when not encapsulated in liposomes. However, for each cancer cell line, certain nucleoside analogs showed significant inhibition of cell proliferation when encapsulated in liposomes, often with LC50s lower than the lowest tested concentration (0.01 μM). In particular, liposomes of 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one and {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid in systems 3 and 2, respectively, were highly effective at submicromolar concentrations in COLO699N, HCT116, and MCF7, as was liposomes of N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide in system 3.

[0087] [Table 7]

Claims

1. 1. A pharmaceutical composition comprising a nucleoside analog encapsulated in a liposome of a liposome delivery system, wherein the nucleoside analog is a pyrimidine analog selected from 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide, or a purine analog selected from {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid or 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol.

2. The pharmaceutical composition of claim 1 , wherein the nucleoside analog is covalently or non-covalently attached to the liposome.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the liposomes of the liposomal delivery system have a diameter of at least 750, preferably at least 775, more preferably at least 800 nm, as determined by SEM imaging.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the liposomes of the liposomal delivery system are sterically stabilized liposomes.

5. 5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the liposomes of the liposomal delivery system comprise at least a phospholipid, preferably at least a phosphatidylcholine and / or a phosphoethanolamine, or at least a phospholipid, more preferably at least a phosphatidylcholine and / or a phosphoethanolamine, most preferably in addition to one or more phosphoglycerides and / or one or more sphingolipids.

6. 6. The pharmaceutical composition of any one of claims 1 to 5, wherein the liposomes of the liposomal delivery system comprise or consist essentially of a PEGylated phospholipid, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, or a combination of PEGylated and non-PEGylated phospholipids, such as PEGylated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine and non-PEGylated phosphatidylcholine, respectively, optionally together with a sterol, such as cholesterol, as lipid components of the liposome.

7. 7. The pharmaceutical composition of claim 6, wherein the pegylated phospholipid is a pegylated phosphoethanolamine and the non-pegylated phospholipid is a phosphatidylcholine.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the liposomes of the liposomal delivery system are free of fatty acids, in particular free of unsaturated fatty acids, preferably monounsaturated fatty acids such as oleic acid.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the liposomes of the liposomal delivery system comprise a sterol, such as cholesterol, in an amount of from about 5% to less than 50% by weight, preferably from about 5% to less than 25% by weight, more preferably from about 5% to less than 20% by weight, and most preferably from about 6% to less than 16% by weight, based on the weight of the PEGylated phospholipid or the weight of a combination of PEGylated and non-PEGylated phospholipids.

10. 10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the liposome of the liposomal delivery system comprises or consists of 40 to 45% by weight of a pegylated phospholipid, such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 40 to 45% by weight of a non-pegylated phospholipid, such as phosphatidylcholine, and a sterol, such as cholesterol, based on the total weight of the pegylated phospholipid, the non-pegylated phospholipid and the sterol, preferably in a weight ratio of 1:1 between the pegylated phospholipid and the non-pegylated phospholipid.

11. A pharmaceutical composition according to any one of claims 1 to 10 for use in the treatment of colon cancer or lung cancer or breast cancer, preferably by oral or parenteral route.

12. Use of the pharmaceutical composition according to any one of claims 1 to 11, preferably at a concentration between 0.01 μM and 10 μM, for inhibiting the proliferation of cancer cell lines in vitro.

13. 13. The use according to claim 12, wherein the cancer cell line is a lung cancer or colon cancer or breast cancer cell line and the nucleoside analogue is 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid, preferably wherein the nucleoside analogue is present at a concentration of 0.01 μM to 1 μM.

14. 1. A method for producing a liposomal delivery system comprising a nucleoside analog encapsulated in a liposome, comprising: the liposomes of the liposomal delivery system comprise or consist of a combination of PEGylated and non-PEGylated phospholipids, preferably 40-45% by weight of a PEGylated phospholipid such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 40-45% by weight of a non-PEGylated phospholipid such as phosphatidylcholine, and a sterol such as cholesterol, based on the total weight of the PEGylated phospholipid, non-PEGylated phospholipid and sterol, more preferably the PEGylated phospholipid and non-PEGylated phospholipid are comprised in a 1:1 weight ratio; the nucleoside analogue is selected from 4-amino-1-(4-hydroxyquinazolin-2-yl)-1,2-dihydropyrimidin-2-one or N-[1-(4-hydroxyquinazolin-2-yl)-2-oxo-1,2-dihydropyrimidin-4-yl]acetamide; The method further comprising: a. solubilizing the PEGylated phospholipids, non-PEGylated phospholipids and sterols, and the nucleoside analogues in a polar solvent, preferably a mixture of polar solvents; b. Evaporating the solvent; c. adding an aqueous solution or water, preferably an aqueous buffer solution, to form a first liposome suspension; d. sonicating the first liposome suspension to form a second liposome suspension; e. annealing the second liposome suspension to form the liposome delivery system comprising the nucleoside analog entrapped in liposomes; A method comprising:

15. 1. A method for producing a liposomal delivery system comprising a nucleoside analog encapsulated in a liposome, comprising: the liposomes of the liposomal delivery system comprise or consist of a combination of PEGylated and non-PEGylated phospholipids, preferably 40-45% by weight of a PEGylated phospholipid such as N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 40-45% by weight of a non-PEGylated phospholipid such as phosphatidylcholine, and a sterol such as cholesterol, based on the total weight of the PEGylated phospholipid, non-PEGylated phospholipid and sterol, more preferably the PEGylated phospholipid and non-PEGylated phospholipid are comprised in a 1:1 weight ratio; the nucleoside analogue is selected from {[2-(6-amino-9H-purin-9-yl)quinazolin-4-yl]oxy}phosphonic acid and 2-(6-amino-9H-purin-9-yl)quinazolin-4-ol; The method further comprising: a. solubilizing the PEGylated phospholipids, non-PEGylated phospholipids and sterol in a polar solvent, preferably a mixture of polar solvents; b. Evaporating the solvent; c. adding an aqueous solution of said nucleoside analogue, preferably a buffered aqueous solution of said nucleoside analogue, to form a first liposome suspension; d. sonicating the first liposome suspension to form a second liposome suspension; e. annealing the second liposome suspension to form the liposome delivery system comprising the nucleoside analog entrapped in liposomes; A method comprising:

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