RU2476216C1 - Liposomal composition - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to pharmaceutics and concerns a liposomal composition containing eribuline or its pharmaceutically acceptable salt, as well as a method for preparing it.

EFFECT: invention provides high stability of the liposomal composition.

39 cl, 3 dwg, 7 tbl, 10 ex

Description

FIELD OF THE INVENTION

The present invention relates to a novel liposomal composition comprising eribulin or a pharmacologically acceptable salt thereof. The present invention also relates to a method for producing a liposome composition.

BACKGROUND OF THE INVENTION

Liposomes are microscopic closed vesicles having an internal phase surrounded by one or more lipid bilayers and capable of retaining water-soluble material in the internal phase, and lipophilic material in a lipid bilayer. When enclosing an active compound in a liposome and delivering it to the target tissue, important tasks are to capture the active compound in the liposome with high efficiency and ensure stable retention of the active compound by the liposome.

When lipophilic compounds are enclosed in a liposome, a high uptake coefficient can be achieved relatively easily, with the exception of compounds with a very high affinity for membranes, such as amphotericin B (the main active ingredient in the AmBisome liposome drug), serum retention stability is usually low and it is difficult to achieve a sufficient improvement in pharmacokinetics. Regarding methods for enclosing water-soluble compounds in liposomes, various methods exist, such as the lipid film method (vortex method), reverse phase evaporation method, surfactant removal method, freeze-thaw method, and remote loading methods (pH gradient method, method ion gradient). However, only remote loading methods provide a capture rate close to 100%; the capture coefficient in the case of other methods is only about 5-30%.

As remote loading methods, methods using a pH gradient and a gradient of ammonium sulfate ions are known. The pH gradient method, which is a remote loading method using a pH gradient, is a method of encapsulating compounds in liposomes by changing the equilibrium of molecular / ionic dissociation due to the pH of the target compound.

As one example of a compound enclosed in a liposome by a pH gradient method, for example, doxorubicin (DOX, pKa: 8.2). After obtaining a liposome solution with a buffer solution having a pH of 4, the external phase of the liposomes is replaced by a buffer solution with a pH of 7. In the case where DOX is added to this solution of liposomes, since the molecular DOX in the solution with pH 7 is lipophilic, it migrates to the liposome membranes and not in the aqueous phase. In the case when DOX, which migrated into the liposome membranes, is further contacted with the internal phase of the liposomes with pH 4, it becomes ionic and is included in the internal phase of the liposomes. Thus, DOX is transferred from the external phase to the internal phase of liposomes due to a change in the equilibrium of dissociation (see Non-Patent Literature 1, Non-Patent Literature 2 and Patent Literature 1).

Many ways have been reported to improve this type of remote boot method. Non-Patent Literature 3 describes a method for improving the capture coefficient of active compounds by adding ethanol together with the active compound to the external phase of liposomes when the pH gradient method is carried out in liposomes of a special composition called cholesterol-free liposomes.

In Patent Literature 2, in addition to the pH gradient, a method for improving the uptake of active compounds by the presence of copper ions in the liposome internal phase is described.

Instead of the pH gradient in the pH gradient method, the ammonium sulfate method, which is a remote loading method using a gradient of ammonium sulfate ions, is a method for enclosing active compounds in the internal phase of liposomes using an ion gradient such as divalent ammonium sulfate (see Non-Patent Literature 1 and patent literature 3).

In addition to the ion gradient method based on ammonium sulfate, Patent Literature 4 describes a method for enclosing active compounds in liposomes by adding boronic acid together with the active compound to the external phase of the liposomes.

Instead of an ammonium sulfate-based ion gradient, Patent Literature 5 describes a method in which, compared to a situation where ammonium sulfate is used, the release rate of the active compound is improved by encapsulating the active compound in liposomes using the ionic gradient of glucuronic acid anions.

Thus, in terms of capture coefficient, remote loading methods are excellent capture methods. However, in the case where remote loading methods are used, except in special cases, for example with Doxil (DOX liposome preparation), in which the active compound enclosed in the internal phase of the liposomes is crystallized, there is a problem that the active compound tends to leak from liposomes to serum and the stability of retention of the active compound is low.

As described above, using conventional technical methods, the situation is currently such that it is difficult to achieve simultaneously a high coefficient of capture of the active compound in liposomes and stability of retention of the active compound in liposomes.

BACKGROUND OF THE INVENTION

Patent Literature

Patent Literature 1: United States Patent No. 5192549, Specification.

Patent Literature 2: PCT International Publication WO 2006/037230, brochure.

Patent Literature 3: United States Patent No. 5316771, Specification.

Patent Literature 4: United States Patent No. 6051251, Specification.

Patent Literature 5: PCT International Publication WO 2005/046643, brochure.

Non-Patent Literature

Non-Patent Literature 1: Yasuyuki Sazuka, “Liposome Preparation Method”, “New Developments in Liposome Application: Toward the Development of Artificial Cells” (Kazunari Akiyoshi, Shigeru Tsujii, editorial) ”NTS, (2005), pp. 33-37 .

Non-Patent Literature 2: Mayer LD et al., Biochimica et Biophysica Acta, (1986), 857: pp. 123-126.

Non-Patent Literature 3: N. Dos Santos et al., Biochimica et Biophysica Acta, (2004), 1661 (1): pp. 47-60.

Disclosure of invention

The problem solved by the invention

An object of the present invention is to provide a liposomal composition with a high capture coefficient and stability of retention of the active compound.

Ways to solve the problem

As a result of a comprehensive study aimed at solving the above problems, the authors of the present invention have established with respect to the liposomal composition, the active compound of which is eribulin or its pharmacologically acceptable salt, that the capture coefficient and stability of retention of the active compound in the liposomal composition are extremely high, which led to the completion of the present inventions.

In particular, the present invention is as follows.

(one)

A liposome composition containing liposomes and containing the active compound in the internal phase of the liposomes, wherein the active compound is eribulin or a pharmacologically acceptable salt thereof.

(2)

The liposome composition according to claim 1, in solid or liquid form.

(3)

The liposome composition according to 1 or 2, in which the internal phase of the liposomes further comprises an ammonium salt.

(four)

The liposomal composition of claim 3, wherein the concentration of the above ammonium salt is 10 mM or higher.

(5)

The liposome composition according to any one of 1 to 4, in which the internal phase of the liposomes further comprises a salt, acid, base and / or amino acid.

(6)

The liposomal composition according to 5, in which the concentration of the above salt is 1-300 mm.

(7)

The liposomal composition according to 5 or 6, in which the concentration of the above acid is 1-300 mm.

(8)

The liposomal composition according to any one of 5-7, in which the concentration of the above amino acids is 1-300 mm.

(9)

The liposomal composition according to any one of 5-8, in which the concentration of the above base is 1-300 mm.

(10)

The liposomal composition according to any one of 1 to 9, wherein the concentration of the above active compound is 0.01-300 mg / ml.

(eleven)

The liposomal composition according to any one of 1-10, wherein the above active compound is eribulin mesylate.

(12)

The liposome composition according to any one of 1-11, wherein the internal phase of the liposomes further comprises ammonium sulfate, citric acid and the active compound.

(13)

The liposomal composition according to any one of 1-12, in which the external phase of the liposomes contains sugar, electrolyte and / or amino acid.

(fourteen)

The liposome composition according to any one of 1 to 13, wherein the external phase of the liposomes contains sugar or an electrolyte and an amino acid.

(fifteen)

The liposomal composition according to 13 or 14, in which the concentration of the above sugar is 2-20%.

(16)

The liposome composition according to any one of 13-15, wherein the concentration of the above amino acid is 1-300 mM.

(17)

The liposome composition according to any one of 1-16, wherein the external phase of the liposomes contains sucrose or sodium chloride and histidine.

(eighteen)

The liposome composition according to any one of 1-17, wherein the above-mentioned internal phase of the liposomes is substantially free of cyclodextrin.

(19)

The liposome composition according to any one of 1-18, in which the liposomes contain hydrogenated phosphatidylcholine.

(twenty)

The liposome composition according to any one of 1-19, wherein the liposomes contain cholesterol.

(21)

The liposome composition according to any one of 1-20, wherein the liposomes comprise a methoxypolyethylene glycol condensate.

(22)

The liposomal composition according to 21, in which the above methoxypolyethylene glycol condensate is a distearoylphosphatidylethanolamine-polyethylene glycol condensate.

(23)

The liposome composition according to any one of 1-22, wherein the liposomes contain hydrogenated phosphatidylcholine, cholesterol and a distearoylphosphatidylethanolamine-polyethylene glycol condensate.

(24)

The liposomal composition according to 23, which contains from 10 to 80% of the above hydrogenated phosphatidylcholine, from 1 to 60% of the above cholesterol and from 0 to 50% of the above condensate of distearoylphosphatidylethanolamine-polyethylene glycol.

(25)

The liposomal composition according to any one of 1-24, wherein the liposomes contain hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol 2000-phosphatidylethanolamine.

(26)

A method of manufacturing a liposome composition according to any one of 1-25, comprising:

a step in which a liposome dispersion liquid containing liposomes is obtained;

a step in which the aforementioned liposome dispersion liquid is mixed with the aforementioned active compound; and

a step in which the aforementioned active compound is introduced into the internal phase of the liposomes of the aforementioned liposome dispersion liquid.

(27)

The method of claim 26, wherein the aforementioned liposomal dispersion liquid is substantially free of ammonium salt in the external phase of the liposomes.

(28)

The method according to 26 or 27, wherein the pH of the external phase of the liposomes of the above liposome dispersion liquid is 3-10.

(29)

The method according to any one of 26-28, wherein the pH of the external phase of the liposomes of the above liposome dispersion liquid is 7-10.

(thirty)

The method according to 28 or 29, wherein the above pH is the pH of the external phase of the liposomes of the above liposome dispersion liquid in a step in which the above liposome dispersion liquid and the above active compound are mixed.

(31)

The method according to any one of 26-30, wherein the step of producing the above liposomal dispersion liquid comprises the step of: preparing a preliminary liposome solution containing liposomes and containing an ammonium salt in the inner phase of the liposomes and the outer phase of the liposomes; and a step in which the external liposome phase of the aforementioned pre-liposome solution is replaced or diluted.

(32)

The method of claim 31, wherein the step of replacing or diluting the aforementioned external phase of the liposomes is a step of making the pH of the external phase of the liposomes higher than the pH of the internal phase of the liposomes.

(33)

The method of claim 31 or 32, wherein the step of replacing or diluting the aforementioned external phase of the liposomes is a step wherein the difference between the pH of the internal phase of the liposomes and the pH of the external phase of the liposome is 1-5.

(34)

The method according to any one of 26-33, wherein the pH of the above internal phase of the liposomes is 3-9.

(35)

The method according to any one of 26-34, wherein the pH of the above internal phase of the liposomes is 4-9.

(36)

The method according to any one of 26-35, wherein the pH of the above internal phase of the liposomes is 5-8.

(37)

The method according to any one of 26 to 36, wherein the external phase of the liposomes is a solution containing an electrolyte in a step in which the above active compound is administered.

(38)

The method according to any one of 26-37, wherein the above liposome dispersion liquid essentially does not contain cyclodextrin in the inner phase of the liposomes.

(39)

The method according to any one of 26-38, further comprising the step of neutralizing the pH of the external phase of the liposomes.

Effect of the invention

The present invention provides a novel liposome composition. The liposome composition of the present invention captures the active compound into the internal phase of liposomes with a high degree of efficiency and has a high retention stability of the active compound.

Brief Description of the Drawings

The figure 1 presents the changes in the concentration of eribulin mesylate in the liposome composition in rat serum (37 ° C) in vitro.

The figure 2 presents the antitumor activity in vivo of eribulin mesylate from liposomes in "nude" mice with a cancer tumor FaDu.

Figure 3 shows the in vivo antitumor activity of eribulin mesylate from liposomes in "nude" mice with an ACHN cancer.

The best way of carrying out the invention

The present invention is specifically described using embodiments of the invention, however, the present invention is not limited to the following embodiments of the invention and can be carried out with various modifications.

The content of references cited in the present invention is hereby incorporated by reference.

Definitions

"Liposomes" means microscopic closed vesicles having an internal phase surrounded by a lipid bilayer. In the present invention, liposomes include small single-membrane liposomes (SUV: small single-layered vesicles), large single-membrane liposomes (LUV: large single-layer vesicles), even larger single-membrane liposomes (GUV: giant single-layer vesicles), multilayer liposomes with several concentric membranes (MLV: multilayer vesicles), liposomes having several membranes, not concentric but irregular in shape (MVV: multivesicular vesicles), and so on.

“Internal phase of liposomes” means an aqueous layer enclosed in a lipid bilayer of liposomes and is used in the same meaning as “internal aqueous phase” and “internal aqueous phase of liposomes”. “External phase of liposomes” means a space not enclosed in a lipid bilayer of liposomes (that is, a space outside the internal phase and lipid bilayer) when the liposomes are dispersed in a liquid.

“Liposome composition” means a composition containing liposomes and further comprising eribulin mesylate in the internal phase of liposomes. In the present invention, the liposome composition includes both solid and liquid forms.

“Liposomal dispersion liquid” means a composition containing liposomes, and is a composition before the active compound is introduced into the internal phase of the liposomes.

“Pre-liposome solution” means a composition containing liposomes, and is a composition before adjusting the external phase of the liposomes to capture eribulin mesylate in the internal phase of the liposomes.

“Liposomal reagent” means a liposomal dispersion liquid when it is in liquid form. When it is in solid form, this means a reagent from which the liposome dispersion liquid can be obtained by dissolving or suspending in an established solvent. The solvent is described below. As described below, a solid liposomal reagent can be obtained, for example, by drying the liposomal dispersion liquid.

As used herein, “mixing solid and liquid” includes dissolving and suspending a solid in a liquid, and mixing, dissolving and suspending is used interchangeably. Similarly, a solvent and a dispersion medium are also used interchangeably.

In addition, the liposomal composition, liposomal dispersion liquid, preliminary liposomal solution and liposomal reagent of the present invention are essentially free of cyclodextrin. “Essentially free of cyclodextrin” means that no cyclodextrin is added. It is sufficient if the cyclodextrin is not contained in an amount in which a significant increase in the solubility (nominal solubility) of the active compound due to cyclodextrin is observed, and even when it is added in an amount in which the solubility of the active compound is not significantly increased, this should not be an exception to the embodiments of the present invention.

Furthermore, as a preferred embodiment of the present invention, “liposomal dispersion liquid essentially free of ammonium salt in the external phase of liposomes” means that the ammonium salt is not added to the external phase of the liposomal dispersion liquid. The addition of an ammonium salt in an amount within the range that achieves the objective of the present invention should not be an exception to the embodiments of the present invention. In the case where the ammonium salt is contained in the external phase of the liposomes of the pre-liposome solution, it is possible to obtain a liposomal dispersion liquid, which is essentially free of ammonium salt, by replacing or diluting the external phase of the liposomes of the pre-liposome solution using a solution that essentially does not contain the ammonium salt.

Active connection

The active compound of the present invention is eribulin or a pharmacologically acceptable salt thereof (hereinafter sometimes referred to as "eribulin and so on"). There are no particular restrictions on the pharmacologically acceptable salt, provided that eribulin and salt are formed, whether it is an inorganic acid salt or an organic acid salt. For example, hydrochloric acid salt, sulfuric acid salt, citrate, hydrobromic acid salt, hydroiodic acid salt, nitric acid salt, bisulfate, phosphoric acid salt, superphosphoric acid salt, isonicotinic acid salt, acetic acid salt, lactic acid salt, salicylic salt acids, tartaric acid salt, pantothenic acid salt, ascorbic acid salt, succinic acid salt, maleic acid salt, fumaric acid salt, gluconic acid salt, saccharic acid salt, formic salt acids, benzoic acid salt, glutamic acid salt, methanesulfonic acid salt, ethanesulfonic acid salt, benzenesulfonic acid salt, p-toluenesulfonic acid salt, pamic acid salt (pamoate) and so on. Among these, hydrochloric acid salt, sulfuric acid salt, acetic acid salt, phosphoric acid salt, citrate and mesyl acid salt are preferred, and mesyl acid salt is most preferred. That is, the preferred active compound of the present invention is eribulin mesylate. Moreover, eribulin and salts of aluminum, calcium, lithium, magnesium, calcium [sic], sodium, zinc and diethanolamine are acceptable as pharmacologically acceptable eribulin salts. Eribulin or a pharmacologically acceptable salt thereof is a compound or salt thereof indicated in PCT brochure international publication WO 99/65894 or United States Patent 6,214,865 (the contents of these patents are incorporated herein by reference) and having pharmacological effects, including antitumor activity and antimitotic act. Eribulin or its pharmacologically acceptable salt has an antitumor effect against melanoma, fibrosarcoma, monocytic leukemia, colon cancer, ovarian cancer, breast cancer, bone cancer, prostate cancer, lung cancer and ras-transformed fibroblasts.

However, as active compounds that can be combined with eribulin, and so on, you can choose among the compounds used in the field of medicine (including diagnostic drugs), cosmetic products, food products and so on. As for the active compounds, it is permissible to combine one or more compounds other than eribulin, and so on.

As active compounds, low molecular weight compounds and so on can be mentioned. Among them, compounds useful as antitumor agents, antibacterial agents, anti-inflammatory drugs, anti-myocardial infarction agents and contrast agents are suitable.

Regarding the molecular weight of the active compound, a range of from 100 to 2000, a range of from 200 to 1500, and even more preferred is a range of from 300 to 1000. Within these ranges, the permeability of the liposome membrane for the active compound is generally satisfactory, and the present invention can be applied accordingly.

Active compounds include water-soluble compounds and lipophilic compounds, and the present invention can be applied provided that they are more or less soluble in water or aqueous solvents.

There are no particular restrictions on antitumor agents in the present invention and mention may be made, for example, of camptothecin derivatives such as irinotecan hydrochloride, nogitecan hydrochloride, exatecan, RFS-2000, lurtotecan, BNP-1350, Bay-383441, PNU-166148, IDEC- 132, BN-80915, DB-38, DB-81, DB-90, DB-91, CKD-620, T-0128, ST-1480, ST-1481, DRF-1042, DE-310; taxane derivatives such as docetaxel hydride, docetaxel, paclitaxel, IND-5109, BMS-184476, BMS-188797, T-3782, TAX-1011, SB-RA-31012, SBT-1514 and DJ-927; ifosfamide, nimustine hydrochloride, carvocone, cyclophosphamide, dacarbazine, thiotepa, busulfan, melphalan, ranimustine, estramustine sodium phosphate, 6-mercaptopurine-riboside, enocytabine, gemcitabine hydrochloride, carboxyfurficamide hydroxyflucrate, citrofluorocarbitfin citrate, citrofluorocarbitfin citrate, citrofin flucid , mercaptopurine, fludarabine phosphate, actinomycin D, aclarubicin hydrochloride, idarubicin hydrochloride, pyrarubicin hydrochloride, epirubicin hydrochloride, daunorubicin hydrochloride, doxorubicin hydrochloride, epirubicin, pyranubicin, in, doxorubicin, pyrarubicin hydrochloride, bleomycin hydrochloride, zinostatin stimalamer, neocarcinostatin, mitomycin C, bleomycin sulfate, peplomycin sulfate, etoposide, vinorelbin tartrate, vincristine sulfate, vindesine sulfate, vinbreenbine, ecbin-bencin tinbeneblastin, blinbestin, biblocinestin, biblocinestin, blebecin testin , TAK-165, KW-2401, KW-2170, KW-2871, KT-5555, KT-8391, TZT-1027, S-3304, CS-682, YM-511, YM-598, TAT-59, TAS -101, TAS-102, TA-106, FK-228, FK-317, E7070, (8E, 12E, 14E) -7 - [(4-cycloheptipiperazin-1-yl) carbonyl] oxy-3,6,16 , 21-tetrahydroxy-6,10,12,16,20-pentamethyl-18,19-epoxytricose-8,12,14-triene-11-olide (E7107), KRN-700, KRN-5500, J-107088, Hmn -214, SM-11355, ZD-0473, and so on. As regards the compounds recorded as salts, among the aforementioned antitumor agents, any salt is acceptable and free basic substances are also acceptable. As for the compounds recorded as free basic substances, any salt thereof is acceptable.

There are no specific restrictions on antibacterial agents and mention may be made, for example, of amphotericin B, cefotiam hexyl, cephalosporin, chloramphenicol, diclofenac and so on. As for the compounds of the aforementioned antibacterial agents, any salt thereof is acceptable.

There are no specific restrictions on anti-inflammatory drugs and, for example, prostaglandins (PGE1, PGE2), dexamethasone, hydrocortisone, piroxicam, indomethacin, prednisolone and so on can be mentioned. As for the compounds of the above anti-inflammatory drugs, any salt thereof is acceptable.

There are no specific restrictions on anti-myocardial infarction agents and, for example, adenosine, atenolol, pilsikainide and so on can be mentioned, and contrast agents can be mentioned, for example, yopamidol, yoxaglic acid, yogexol, yomeprol and so on. As for the compounds of the above anti-myocardial infarction and contrast agents, any salt thereof is acceptable.

Lipids

Preferably, the membrane constituents of the liposomes of the present invention include phospholipids and / or derivatives of phospholipids. As phospholipids and phospholipid derivatives can be mentioned, for example, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, cardiolipin, sphingomyelin, ceramide fosforiletanolamin, fosforilglitserin ceramide, ceramide fosforilglitserinfosfat, 1,2-dimyristoyl-1,2-dezoksifosfatidilholin, plasmalogens, phosphatidic acid and so on. One or more of these phospholipids and phospholipid derivatives can be combined.

There are no particular restrictions on fatty acid residues in phospholipids and phospholipid derivatives, and, for example, saturated or unsaturated fatty acid residues with carbon numbers from 12 to 20 can be mentioned. Specifically, acyl groups from fatty acids such as lauric acid, myristic acid can be mentioned. acid, palmitic acid, stearic acid, oleic acid and linoleic acid. You can also use phospholipids obtained from natural substances, such as egg yolk lecithin and soya lecithin, partially hydrogenated egg yolk lecithin, in which the unsaturated fatty acid residue is partially or fully hydrogenated, (fully) hydrogenated egg yolk lecithin, partially hydrogenated soya lecithin, ( fully) hydrogenated soya lecithin and so on.

There are no particular restrictions on the mixed amount (molar fraction) of phospholipids and / or phospholipid derivatives that are used in the preparation of liposomes, however, it is preferable to use from 10 to 80% with respect to the entire composition of the liposome membranes, and more preferably from 30 to 60% .

As for membrane components, in addition to phospholipids and / or phospholipid derivatives, the liposomes of the present invention may also include sterols, such as cholesterol and cholestenol, as membrane stabilizers, fatty acids containing saturated or unsaturated acyl groups with carbon numbers from 8 to 22, and antioxidants such as α-tocopherol.

There are no particular restrictions on the mixed amount (mole fraction) of these sterols used in the preparation of liposomes, however, it is preferable to use from 1 to 60% with respect to the entire composition of the liposome membranes, more preferably from 10 to 50% and even more preferably from 30 to 50%.

In addition, there are no particular restrictions on the mixed amount (molar fraction) of fatty acids, however, it is preferable to use from 0 to 30% with respect to the entire composition of the liposome membranes, more preferably from 0 to 20% and even more preferably from 0 to 10%. As for the mixed amount (mole fraction) of antioxidants, it is sufficient if an amount capable of providing an antioxidant effect is added, however, it is preferable to use from 0 to 15% with respect to the entire composition of the liposome membranes, more preferably from 0 to 10% and even more preferably from 0 to 5%.

The liposomes of the present invention may also contain functional lipids and modified lipids as membrane components.

As functional lipids, mention may be made of lipid derivatives stored in the blood, heat-sensitive lipid derivatives, pH-sensitive lipid derivatives, and so on. As modified lipids, mention may be made of PEG lipids, sugar lipids, antibody-modified lipids, peptide-modified lipids, and so on.

As derivatives of lipids retained in the blood, mention may be made, for example, of glycophorin, ganglioside GM1, ganglioside GM3, derivatives of glucuronic acid, derivatives of glutamic acid, derivatives of polyglycerolphospholipids, derivatives of polyethylene glycol (methoxypolyethylene glycol condensates, etc., methoxypolyethylene glycol-2000] -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-5000] -1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol- 750] -1,2-distear yl-sn-glycero-3-phosphoethanolamine, N- [carbonyl-methoxypolyethylene glycol-2000] -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, (MPEG 2000-distearoylphosphatidylethanolamine) and N- [carbonyl-methoxypolyethylene glycol-5000 -1,2-distearoyl-sn-glycero-3-phosphoethanolamine, which are condensates of phosphoethanolamine and methoxypolyethylene glycol. In the presence of liposomes containing lipid derivatives with the ability to persist in the blood, it becomes possible to improve the ability of liposomes to remain in the blood, since the capture of liposomes as impurities in the liver and so on becomes difficult.

As thermosensitive derivatives of lipids, mention may be made, for example, of dipalmitoylphosphatidylcholine and so on. In the presence of liposomes containing thermosensitive derivatives of lipids, it becomes possible to cause the destruction of liposomes at certain temperatures and cause changes in the surface properties of liposomes. Moreover, when this is combined with an increase in temperature in the target area of the tumor, and so on, it becomes possible to destroy liposomes in the target area and release the active compound in the target area.

As pH-sensitive lipid derivatives, mention may be made, for example, of dioleoylphosphatidylethanolamine and so on. In the presence of liposomes containing pH-sensitive lipid derivatives, it becomes possible to stimulate the membrane fusion of liposomes and endosomes when liposomes enter the cell due to endocytosis, and to improve the transfer of the active compound into the cell tissue.

As sugar lipids of antibody-modified lipids and peptide-modified lipids, mention may be made of lipids associated with sugars, antibodies or peptides that are compatible with target cells or target tissues. Through the use of modified lipids, liposomes can be actively transported to target cells or target tissues.

There are no particular restrictions on the miscible amount (mole fraction) of the used lipid derivatives with the ability to remain in the blood upon receipt of liposomes, however, it is preferable to use from 0 to 50% relative to the total number of lipids that make up the liposome membrane, more preferably from 0 to 30% even more preferably 0 to 20%.

Liposomes

As mentioned above, liposomes are microscopic closed vesicles having an internal phase surrounded by a lipid bilayer.

Ideally, with regard to liposomes, it is preferable that for eribulin and so on there is a barrier function preventing it from leaking into the external phase of liposomes after eribulin and so on has been enclosed in the internal phase of liposomes. In the case when they are used as a medicine, it is desirable that the liposomes have stability in vivo and that for eribulin and so on there is a barrier function that prevents its leakage into the external phase of liposomes in the blood when liposomes are administered in vivo.

The composition of the membrane components for liposomes having such membrane permeability at a level that allows practical application can be properly selected by specialists in this field depending on the active compound, target tissue, and the like, referring, if necessary, to the embodiments described below (Hiroshi Kikuchi , et. al., “Liposome I-Preparation Method and Assay Method-”, Cell Technology (1983), 2 (9): pp. 1136-1149, and with references cited in that publication).

When used as a medicine, it is desirable that eribulin and so on is released from the liposomes after the liposomes reach the target tissue, cells or intracellular organelles. As for liposomes, the membrane components themselves are generally biodegradable and ultimately decompose in the target tissue and the like. Trapped eribulin is believed to be released this way. Moreover, it is also acceptable if the liposomes themselves are incorporated into the cells.

The liposomal composition can not only be targeted to target tissue, such as solid cancer, but it can also be used to transfer active compounds for hematologic cancer and so on. It can also be used in the blood as a sustained release drug, a controlled release drug, and so on.

The particle size of liposomes can be set depending on the tasks. For example, if it is necessary to deliver liposomes to cancerous tissue or inflamed tissue using the EPR effect (increased permeability and retention) as an injection product or the like, it is preferable that the particle size of the liposomes is from 30 to 400 nm, and more preferably, the particle size ranged from 50 to 200 nm. In the case where it is necessary to deliver liposomes to macrophages, it is preferable that the particle size of the liposomes is from 30 to 1000 nm, and more preferably, the particle size is from 100 to 400 nm. In the case where the liposome composition is to be used as an oral preparation or transdermal preparation, the particle size of the liposomes can be set to several microns. It should be noted that (1) in normal tissue, the walls of the vessels serve as a barrier (because the walls of the vessels consist of tightly fitting vascular endothelial cells) and microparticles, such as supermolecules and liposomes of a given size, cannot propagate into the tissue. However, in the affected tissue, the walls of the vessels are loose (because there are gaps between the cells of the vascular endothelium), which increases the permeability of the vessels, and supermolecules and microparticles can spread into the tissue outside the vessels (increased permeability). In addition, (2) in the normal tissue, the lymphatic system is well developed, however, it is known that the lymphatic system is not developed in the affected tissue and that supermolecules or microparticles after their inclusion are not processed within the general system, but are stored in the affected tissue (increased retention) - this is called the EPR effect (Matsumura, Maeda, Cancer Research, (1986), 46: pp.6387-6392). Therefore, pharmacokinetics can be controlled by controlling particle sizes of liposomes.

In the present invention, the particle size of liposomes means the weighted average particle size determined by the dynamic light scattering method (quasi-elastic light scattering method). This document describes the particle size measured on dynamic light scattering devices (for example, Zetasizer Nano ZS manufactured by Malvern Instruments Ltd. and ELS-8000 made by Otsuka Electronics Co., Ltd.). Instruments measure the Brownian motion of particles, and the particle size is determined on the basis of an established methodological theory of dynamic light scattering.

There are no particular restrictions on the solvent in the internal phase of the liposomes and, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution and phosphate-buffered saline, physiological saline, cell culture medium and so on can be mentioned. As for the solvent, when a buffer solution is used, it is preferable that the concentration of the buffer agent is 5-300 mM, and a concentration of 10-100 mM is more preferred. There are no particular restrictions on the pH of the internal phase of liposomes, however, values from 3 to 11 are preferable, and from 4 to 9 are more preferred.

Liposomal composition

The present invention provides a liposome composition. The liposomal composition contains liposomes, and also contains eribulin and so on in the internal phase of liposomes. As mentioned above, the liposome composition includes both a solid form and a liquid form. In the case where the liposomes are in solid form, they can be converted into liquid form by dissolving or suspending them in an established solvent, as described below. In the case where the liposome composition is in a frozen solid form, it can be converted into a liquid form, leaving it to thaw at room temperature.

The concentration of liposomes and the concentration of the active compound in the liposome composition can be appropriately adjusted according to the purpose of the liposome composition, preparation, and so on. In the case where the liposome composition is a liquid preparation, the concentration of liposomes as the concentration of all lipids constituting the liposomes can be set in the range from 0.2 to 100 mm, and preferably from 1 to 30 mm. The concentration (dose) of the active compound when the liposome composition is used as a medicine is described below. Regarding the amount of cyclodextrin in the liposome composition, it is preferable that it is less than 0.1 mol equivalent with respect to eribulin and so on, and most preferably it is less than the detection limit.

In the liposome composition of the present invention, eribulin and so on may be distributed into the lipid bilayer.

There are no particular restrictions on the solvent (dispersion medium) in the liposomal composition in the case where the liposomal composition is a liquid preparation, and, for example, buffer solutions such as phosphate buffer solution, citrate buffer solution and phosphate-buffered saline, physiological saline and culture medium for cell culture. There are no particular restrictions on the pH of the external phase of the liposomes, however, values from 3 to 11 are preferable, and from 4 to 9 are more preferred.

The following can also be added to the liposome composition: monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose and maltose; trisaccharides such as raffinose and melicytosis; polysaccharides such as cyclodextrin; and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol and maltitol; polyvalent alcohols such as glycerin, diglycerin, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol, ethylene glycol monoalkyl ether, diethylene glycol monoalkyl ether, 1,3-butylene glycol. Combinations of sugar and alcohol may also be used.

For stable long-term storage of liposomes dispersed in a solvent (dispersion medium), from the point of view of physical stability, including coagulation and so on, it is desirable, as far as possible, to remove the electrolyte from the solvent (dispersion medium). Moreover, from the point of view of the chemical stability of lipids, it is desirable to set the pH of the solvent (dispersion medium) from acidic to close to neutral (pH 3.0-8.0) and remove dissolved oxygen by sparging with nitrogen.

There are no particular restrictions on the concentration of sugar or polyvalent alcohol contained in the liposome composition, however, in a situation where, for example, the liposomes are dispersed in a solvent, it is preferable that the sugar concentration is from 2 to 20% (weight / volume), and more preferably - from 5 to 10% (weight / volume). With regard to the concentration of polyvalent alcohol, preferred values are from 1 to 5% (weight / volume), and more preferred from 2 to 2.5% (weight / volume). These solvents can also be used as the external phase of liposomes in a liposomal dispersion liquid, and by replacing or diluting the external phase of the liposomes of the preliminary liposome solution with these solvents, it is possible to replace the solutions of the external phase of liposomes with these solutions.

Preferably, the solid preparations of the liposome composition include, for example, monosaccharides such as glucose, galactose, mannose, fructose, inositol, ribose and xylose; disaccharides such as lactose, sucrose, cellobiose, trehalose and maltose; trisaccharides such as raffinose and melicytosis; polysaccharides such as cyclodextrin and sugar alcohols such as erythritol, xylitol, sorbitol, mannitol and maltitol. Mixtures of glucose, lactose, sucrose, trehalose and sorbitol are more preferred. Mixtures of lactose, sucrose and trehalose are even more preferred. In this case, solid preparations can be stably stored for a long period of time. When freezing, it is preferable that the solid preparations contain polyvalent alcohols (aqueous solutions) such as glycerol, diglycerin, polyglycerol, propylene glycol, polypropylene glycol, ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol monoalkyl ether of ethylene glycol and glycol. As for polyvalent alcohols (aqueous solutions), glycerol, propylene glycol and polyethylene glycol are preferred, and glycerin and propylene glycol are more preferred. In this case, it is possible to stably store solid preparations for long periods of time. Sugars and polyvalent alcohols can be used in combination.

Method for the production of liposome composition

The present invention provides a method for producing a liposomal composition comprising eribulin or a pharmacologically acceptable salt thereof. A method for producing a liposome composition includes the step of producing a liposome dispersion liquid containing liposomes; a step in which the aforementioned liposomal dispersion liquid is mixed with the aforementioned active compound (eribulin or a pharmacologically acceptable salt thereof); and a step in which the aforementioned active compound is introduced into the internal phase of the liposomes of the aforementioned liposome dispersion liquid.

Preferably, if the step in which the liposome dispersion liquid containing the liposomes is prepared includes a step in which the pre-liposome solution is prepared, and a step in which the external liposome phase of the above pre-liposome solution is replaced or diluted.

A preliminary liposome solution can be obtained, for example, by creating liposomes in a solution containing an ammonium salt. By preparing a preliminary liposome solution in a solution containing an ammonium salt, it is possible to create a liposomal dispersion liquid, which also contains an ammonium salt in the internal phase of the liposomes.

There are no particular restrictions on the solution containing the ammonium salt used in the preparation of the preliminary liposome solution, and any solution containing the ammonium salt can be used.

As the ammonium salt, mention may be made, for example, of ammonium chloride, ammonium borate, ammonium sulfate, ammonium formate, ammonium acetate, ammonium citrate, ammonium tartrate, ammonium succinate and ammonium phosphate. Among them, ammonium sulfate, ammonium acetate, ammonium citrate, ammonium tartrate and ammonium phosphate are preferred; more preferred are ammonium sulfate, ammonium citrate and ammonium tartrate; and ammonium sulfate is most preferred.

You can use these ammonium salts in combination of two or more.

The concentration of the ammonium salt in the solution containing the ammonium salt can be appropriately set depending on the amount of eribulin and so on to be captured, and the higher the better; preferably 10 mm or more; more preferably 20 mm or more; and even more preferably 50 mm or more. Regarding the pH of the solution containing the ammonium salt, preferably a value of from 3 to 9, a value of from 4 to 9 is more preferable from the point of view of the balance of the capture coefficient and stability, and even more preferably a value of from 5 to 8.

To adjust the pH of the solution containing the ammonium salt, you can use the pH regulator. There are no particular restrictions on the concentration of individual pH regulators in a solution containing an ammonium salt, however, a concentration of from 1 to 300 mm is preferred, and more preferred is a concentration of from 5 to 100 mm.

As a pH regulator, mention may be made, for example, of amino acids such as arginine, histidine and glycine; acids such as ascorbic acid, benzoic acid, citric acid, glutamic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid and sulfuric acid; salts of the above acids, such as sodium salt, potassium salt and ammonium salt; as well as alkaline compounds (bases) such as Tris-hydroxymethylaminomethane, ammonia water (ammonia), sodium hydride and potassium hydride. As pH adjusters, sodium hydride, hydrochloric acid, ammonia water, acetic acid, lactic acid, tartaric acid, succinic acid, citric acid and phosphoric acid are preferred; more preferred are sodium hydride, ammonia water, hydrochloric acid, acetic acid, citric acid and phosphoric acid; and even more preferred are sodium hydride, ammonia water, hydrochloric acid, citric acid, and phosphoric acid. As pH adjusters, two or more of the ammonium salts in combination can be used. In addition, buffers, phosphate buffered saline, citrate buffered saline and saline buffered saline can also be used as pH adjusters.

As a preliminary liposomal solution, it is best to use a solution that is obtained by the manufacture of liposomes without the existing inclusion of cyclodextrin. In a preliminary liposomal solution, the internal phase of the liposomes may also contain a salt, acid, base and / or amino acid. In this case, it is preferable that the internal phase of the liposomes contains an active compound, an ammonium salt and an acid. As the ammonium salt, a preferred example may be ammonium sulfate; citric acid may be a preferred example.

Regarding liposome preparation, we can mention the lipid film method (vortex method), reverse phase evaporation method, ultrasonic method, prevesicular method, ethanol injection method, French press method, bile acid removal method, volumetric treatment with Triton X-100 , Ca ²⁺ fusion method, ether injection method, annealing method, freeze-thaw method, and so on.

Various conditions (the number of membrane components, temperature, and so on) when receiving liposomes can be appropriately selected depending on the method of obtaining liposomes, the composition of the target liposomes, particle size and so on (see Op.cit, Kikuchi (1983), and so on )

The size of liposome particles can be further adjusted as necessary. Particle size can be adjusted, for example, by extrusion (extrusion filtration) under high pressure using a membrane filter with a conventional pore diameter. Particle size adjustments can be made at any time during the production of the liposome composition of the present invention. For example, it can be carried out before adjusting the external phase of liposomes in a preliminary liposome solution, after adjusting the external phase of liposomes in a preliminary liposome solution, or after introducing the active compound into the internal phase of liposomes. It is preferable to adjust the particle size before introducing the active compound into the internal phase of the liposomes, and more preferably carry out it before adjusting the external phase of the liposomes in the preliminary liposome solution.

Liposomal dispersed liquid can be obtained by replacing or diluting the external phase of the resulting preliminary liposome solution. The replacement or dilution of the external phase of liposomes can be carried out once, or various types of replacement or dilution methods in combination can be applied several times.

Dialysis, centrifugation and gel filtration may be mentioned as a method for replacing the external phase of liposomes of a preliminary liposome solution. By replacing the external phase of the liposomes, the present invention can be implemented so that the external phase of the liposomes is substantially free of cyclodextrin or an ammonium salt. Moreover, by replacing or diluting the external phase of the liposomes, it is possible to efficiently capture eribulin or its pharmacologically acceptable salt into the internal phase of the liposomes.

Dialysis can be carried out, for example, using a dialysis membrane. As the dialysis membrane, a membrane with a nominally cut-off molecular weight, such as a cellulose tube or Spectra / Por, can be mentioned.

With regard to centrifugal separation, centrifugal acceleration can be adjusted preferably to 100,000 g or higher, and more preferably to 300,000 g or higher. Replacing the external phase of liposomes by centrifugation, it is also possible to concentrate the liposomes simultaneously with the replacement of the external phase of liposomes.

Gel filtration can be carried out, for example, by molecular weight fractionation using, for example, a Sephadex or Sepharose column.

As the solvent (dispersion medium) used when replacing and / or diluting the external phase of the liposomes, mention may be made, for example, of sucrose solution, physiological saline and culture medium for cell culture. Using these solvents, a stable liposome composition can be obtained.

There is no particular limitation on the pH of said solvent, however, a range of values from 2 to 11 can be set; preferably from 3 to 10, more preferably from 6 to 10, and even more preferably from 7 to 10. As described below, a pH gradient can be used to introduce eribulin and so on into the internal phase of liposomes. In this case, the pH of the solvent can be set so that the target pH is achieved in the external phase of the liposomes.

A pH adjuster may be used to adjust the pH of the indicated solvent. There are no particular restrictions on the concentration used, but a concentration of 1 to 300 mM is preferred, and a concentration of 5 to 100 mM is more preferred.

As a pH regulator, mention may be made, for example, of amino acids such as arginine, histidine, glycine; acids such as ascorbic acid, benzoic acid, citric acid, glutamic acid, phosphoric acid, acetic acid, propionic acid, tartaric acid, carbonic acid, lactic acid, boric acid, maleic acid, fumaric acid, malic acid, adipic acid, hydrochloric acid and sulfuric acid; salts of the above acids, such as sodium salt, potassium salt and ammonium salt; as well as alkaline compounds such as Tris-hydroxymethylaminomethane, ammonia water, sodium hydride and potassium hydride. Preferred are sodium hydride, hydrochloric acid, histidine, tartaric acid, succinic acid, citric acid and phosphoric acid; more preferred are sodium hydride, hydrochloric acid, histidine, tartaric acid, citric acid and phosphoric acid; and even more preferred are sodium hydride, hydrochloric acid, histidine, and phosphoric acid.

In order to improve the capture coefficient of eribulin or its pharmacologically acceptable salt in liposomes, the capture coefficient can be increased by adding a solution (saline solution) containing electrolyte to the external phase of the liposomes to increase the ion intensity. There are no particular restrictions on the electrolyte (salt) contained in the external phase of the liposomes, however, sodium chloride and potassium chloride are preferred, and sodium chloride is more preferred. You can also use physiological saline. Moreover, as the external phase of the liposomes of the liposome dispersion liquid or the like, sugar, electrolyte and / or amino acid can be included, and sugar or electrolyte and amino acid can also be included. As a preferred example of sugar, sucrose may be mentioned; as a preferred example of an electrolyte, physiological saline and sodium chloride may be mentioned; and histidine may be mentioned as a preferred example of an amino acid.

It is preferable that the obtained liposomal dispersion liquid essentially does not contain cyclodextrin or an ammonium salt in the external phase of the liposomes and the internal phase of the liposomes, however, in the present invention, eribulin or its pharmacologically acceptable salt can be introduced into the internal phase of the liposomes even if for some reason cyclodextrin or an ammonium salt has been added to the external phase of the liposome dispersion liquid, and even if the external phase of the liposome dispersion liquid contains cyclodextrin or ammonium salt.

With regard to the lipid concentration of liposomes in a liposome dispersion liquid, a preferred concentration is 1-100 mM, and more preferred is 1-50 mM. In such ranges, a larger number of liposome particles can be appropriately formed without compromising the physical properties of the liposomal dispersion liquid.

The liposomal composition can be obtained by mixing the obtained liposomal dispersion liquid and the active compound of eribulin, and so on, and by introducing the active compound into the internal phase of the liposomes of the liposomal dispersion liquid. Preferably, the introduction step includes a step in which the permeability of the liposome membranes is increased in a mixed solution of the liposome dispersion liquid and the active compound. Due to this, capture of eribulin and so on into liposomes can occur in a shorter period of time. However, even if no specific actions have been taken to enhance the permeability of the liposome membranes after mixing the liposome dispersion liquid and eribulin, and so on, it is possible to capture eribulin and so on into the liposomes if the necessary time is spent.

In the step in which eribulin or a pharmacologically acceptable salt thereof is added, a substance dissolved in a solvent or a solid substance such as eribulin and so on can be used. There are no particular restrictions on the solvent and, for example, a substance identical to the external phase of the liposomes of the liposome dispersion liquid can be used.

If necessary, a pH gradient can be used to introduce eribulin and so on into the internal phase of liposomes. In this case, with regard to the pH of the internal phase of the liposomes of the liposome dispersion liquid, preferred values are from 3 to 9, more preferred from 4 to 9, and even more preferred from 5 to 8.

Moreover, it is possible to make the pH of the external phase of liposomes higher than the pH of the internal phase of liposomes to create a pH gradient. A pH gradient of 1 to 5 is preferred, and 2 to 3 is more preferred.

In addition, it is possible to increase the capture coefficient in liposomes by adjusting the pH of the external phase of the liposomes to values closer to the pKa region of eribulin and so on, preferred values are from 7.5 to 12.5, more preferred from 8.5 to 11.5 , and even more preferred are from 9 to 10.5 (pKa eribulin mesylate is 9.6).

As a preliminary liposomal solution, it is optimal to use the solution obtained by creating liposomes without the existing inclusion of cyclodextrin.

As a method for increasing the permeability of liposome membranes in the resulting mixed solution, mention may be made of the heating method of the mixed solution, the method of adding a fluidizer to the mixed solution, and so on.

In the case where the mixed solution is heated, the active compound, as a rule, can be more effectively introduced into the internal phase of liposomes by heating to higher temperatures. In particular, it is preferable to set the heating temperature taking into account the thermal stability of the active compound and the liposome membrane components used. It is particularly preferred that the heating temperature is set to the phase transition temperature of the lipid bilayer membranes or higher.

“Phase transition temperature" of lipid bilayer membranes of liposomes means the temperature at which heat absorption begins (the temperature at which the endothermic reaction begins) in the differential thermal analysis of elevated temperature conditions. Differential thermal analysis is a method that allows you to analyze the thermal properties of samples by measuring the temperature difference of a sample or reference preparation as a function of time or temperature when the temperature of the sample or reference preparation changes. In the case when the differential thermal analysis is carried out with respect to the components of the liposome membranes, the components of the liposome membranes liquefy with increasing temperature, and an endothermic reaction is observed. As is widely known in the art, the temperature range in which the endothermic reaction is observed varies significantly depending on the components of the liposome membranes. For example, in the case where the liposome membrane components are composed of pure lipid, the temperature range in which the endothermic reaction is observed is extremely narrow, and the endothermic reaction is often observed in the range of ± 1 ° C relative to the temperature of the endothermic peak. On the other hand, in the case where the components of the liposome membranes are composed of several lipids, and in particular in the case where the components of the liposome membranes are composed of lipids derived from natural materials, the temperature range in which the endothermic reaction is observed tends to expand, and an endothermic reaction is observed, for example, in the range of ± 5 ° C relative to the temperature of the endothermic peak (i.e., a wide peak is observed, and so on). In accordance with the present invention, it is believed that the fluidization of the liposome membranes increases and the permeability of the membranes for the active compound increases due to an increase in temperature above the phase transition temperature of the liposome liposome bilayer membranes.

For example, although taking into account the dependence on thermal stability and so on of the active compound and used components of liposome membranes, it is preferable to have a temperature in the range from the phase transition temperature of the lipid bilayer membranes of liposomes to + 20 ° C from the phase transition temperature; more preferably, a temperature ranging from a phase transition temperature to + 10 ° C. of a phase transition temperature; and even more preferably, a temperature in the range from + 5 ° C from the phase transition temperature to + 10 ° C from the phase transition temperature.

The heating temperature, as a rule, is from 20 to 100 ° C; preferably from 40 to 80 ° C; and more preferably 45 to 65 ° C.

In particular, in the case of liposomal membranes, the main ingredients of which are dipalmitoylphosphatidylcholine (phase transition temperature of a simple substance 41 ° C) and cholesterol, although also being dependent on their composition, a heating temperature of 40 to 60 ° C is usually preferred, and more preferred temperature from 45 to 50 ° C. In addition, in the case of liposomal membranes, the main ingredients of which are hydrogenated soybean phosphatidylcholine (HSPC; phase transition temperature as a simple substance 50-60 ° C) and cholesterol, although also being dependent on their composition, the heating temperature is from 50 to 70 ° C is preferred, and a temperature of from 55 to 65 ° C is more preferred. However, these heating temperatures in no way limit the present invention.

At the heating step, there are no particular restrictions on the time during which the temperature is maintained at or above the phase transition temperature, and it can be appropriately set in the range, for example, from a few seconds to 30 minutes. Given the thermal stability of the active compound and lipids, as well as efficient mass production, it is desirable to carry out the treatment in a short amount of time. That is, it is preferable that the period of maintaining the elevated temperature is from 1 to 30 minutes, and more preferably from 2 minutes to 5 minutes. However, these temperature holding times do not in any way limit the present invention.

Moreover, as mentioned above, it is also possible to increase the permeability of liposome membranes by adding a membrane fluidizer to the resulting mixed solution (i.e., adding it to the side of the external phase of the liposomes). As the fluidizer of the membranes, organic solvents, surfactants, enzymes and the like, which dissolve in aqueous solvents, can be mentioned. More specifically, as organic solvents, mention may be made, for example, of monovalent alcohols, such as ethyl alcohol and benzyl alcohol; polyvalent alcohols such as glycerin and propylene glycol; aprotic polar solvents such as dimethyl sulfoxide (DMSO). As surfactants, mention may be made, for example, of anionic surfactants such as sodium fatty acids, monoalkyl sulfate and monoalkyl phosphate; cationic surfactants, such as alkyltrimethylammonium salt; ampholytic surfactants such as alkyldimethylamine oxide; and nonionic surfactants such as polyoxyethylene alkyl ester, alkyl monoglyceryl ether, fatty acid ester and sorbitan. As enzymes, mention may be made, for example, of cholinesterase and cholesterol oxidase. Those skilled in the art can determine the amount of membrane fluidizer depending on the composition of the liposome membrane components, membrane fluidizer, and so on, and also taking into account the degree of capture efficiency of the active compound by adding a fluidizer membrane, liposome stability, and so on.

A method of manufacturing a liposomal composition of the present invention may include the step of adjusting the pH of the external phase of the liposomes of the obtained liposomal composition after the above step of introducing the substance.

There are no particular restrictions on the pH of the external phase, but values from 4 to 10, more preferred from 5 to 9, and even more preferred are neutral from 6 to 8, from the point of view of the chemical stability of the phospholipids constituting the liposomes.

In addition, you can optionally include the step of drying the resulting liposome composition. That is, when using the liposomal composition in the form of a liquid preparation, the liposomal composition in liquid form obtained at the above stage of substance administration can be used without changes as the final liposomal composition, or the external phase of liposomes in the liquid liposome composition obtained at the above stage of substance administration can be adjusted (replace, and so on) to obtain the final liposome composition. When implementing this, the correction of the external phase of liposomes can be carried out similarly to the adjustment of the external phase of liposomes in the preliminary liposome fluid. In the case where the liposomal composition is a liquid preparation, it can be used without further modifications.

In addition, in the case where the liposomal composition is to be converted into a solid preparation, the liquid liposomal composition obtained at the above stage of administration of the substance can be dried to obtain the final solid liposome composition. Freeze-drying and spray drying can be given as examples of methods for drying the liposome composition. In cases where the liposomal composition is a solid preparation, it can be dissolved or suspended in a suitable solvent and used as a liquid preparation. The solvent for use can be appropriately selected depending on the purpose of use and so on of the liposome composition, and in the case of using the liposome composition as, for example, an injection product, the solvent is preferably sterile distilled water. If the liposome composition is used as a medicine, the doctor or patient can introduce the solvent into the vial in which the solid preparation is located, for example, to receive the drug immediately at the time of use. In the case where the liquid liposome composition is a frozen solid preparation, it can be used as a liquid preparation, storing it frozen and again turning it into a liquid state, leaving it to thaw at room temperature or heating it to quickly thaw at the time of use.

Pharmaceutical compositions and so on.

The liposome composition of the present invention can be used as a medicine in the medical field. In particular, the liposome composition of the present invention can be used as an antitumor pharmaceutical composition.

In the case where the liposome composition of the present invention is used as a pharmaceutical composition, the liposome composition can be administered by injection (intravenous, intra-arterial or local injection), orally, through the nose, subcutaneously, into the lungs or by eye drops and, in particular, topical injection into a target group of cells or organ or other similar injection is preferred in addition to intravenous injection, subcutaneous injection, intradermal injection and intra-arterial injection. Tablets, powder, granules, syrup, capsules, liquid and the like can be cited as examples of a preparation of a liposome composition in the case of oral administration. Injection product, drip injection, eye drops, ointments, suppository, suspension, hot compress, lotion, aerosol, patch and the like can be cited as examples of liposome composition preparations in case of non-oral administration, and an injection product and drip agent are particularly preferred. .

The dosage of the pharmaceutical composition essentially differs depending on the type of the target disease, the type of active compound, as well as the age, gender and weight of the patient, the severity of symptoms, among other factors, but, as a rule, the daily dose of eribulin or its pharmacologically acceptable salt for adults is not has particular limitations, although for eribulin mesylate, which is a suitable salt, it is usually 0.1 to 10 mg. In addition, administration can be divided into more than one dose per day. A liposome composition containing, for example, 0.01-300 mg / ml eribulin or a pharmacologically acceptable salt thereof in the internal phase of liposomes, can be administered as the liposome composition of the present invention.

Set

The present invention provides a kit for preparing a liposome composition. The kit can be used to obtain a liposomal composition as a medicine that can be used by a physician in a clinical setting or by a patient.

The kit includes a liposomal reagent. The liposomal reagent can be in either solid or liquid form. If the liposomal reagent is in liquid form, then the above liposomal dispersion liquid can be used as the liposomal reagent. In addition, if the liposomal reagent is in solid form, the liposomal reagent can be dissolved or suspended in an appropriate solvent to obtain a liposomal dispersion liquid, and the above liposomal dispersion liquid can be dried to obtain a liposomal reagent. Drying can be carried out similarly to the aforementioned drying of the liposome composition. When using the kit, if the liposomal reagent is in solid form, the liposomal reagent can be dissolved or suspended in an appropriate solvent to obtain a liposomal dispersion liquid. The solvent is similar to the external phase of the liposomes in the above liposome dispersion liquid.

The kit of the present invention further comprises eribulin or a pharmacologically acceptable salt thereof (eribulin mesylate is a suitable salt). Eribulin or its pharmacologically acceptable salt can be either in solid or in liquid form (in a state dissolved or suspended in a solvent). When using the kit, if eribulin, and so on, is in solid form, it is preferable to dissolve or suspend it in an appropriate solvent to obtain a liquid form. The solvent can be appropriately selected depending on the physical properties, and the like, eribulin, and the like, and make it similar, for example, to the external phase of liposomes in the above dispersion liquid. The kit of the present invention may include an active compound other than eribulin or a pharmacologically acceptable salt thereof.

In the kit, the liposome reagent and the active compound can be packaged separately or they can be in solid form and mixed.

In the case where the liposomal reagent is in solid form, with the exception of cases of dissolution or suspension to obtain the above liposomal dispersion liquid, the kit can be used by performing a step similar to the step of mixing the liposomal dispersion liquid and the active compound, as well as introducing the active compound into the inner phase of the liposome liposome dispersion liquid in a method for producing the above liposome composition. In this way, a liposomal composition can be produced in which the active compound is introduced into the internal phase of the liposomal reagent.

In the case where the liposomal reagent and the active compound are both in solid form and packaged together, the mixture of the liposomal reagent and the active compound is suitably dissolved or suspended in the solvent. The solvent is similar to the external phase of the liposomes in the above liposome dispersion liquid. Thus, it is possible to create a state in which the liposomal dispersion liquid and the active compound are mixed, after which the use becomes possible as a result of other steps when introducing the active compound into the internal phase of the liposomes of the liposomal dispersion liquid in the production method of the above liposome composition.

Options for implementation

The present invention is specifically described using embodiments and comparative examples, but is not limited to the following embodiments.

Embodiment 1

Obtaining an aqueous solution for the internal phase of liposomes

396.4 mg of ammonium sulfate and 189.1 mg of citric acid monohydrate were dissolved in pure water, and then diluted to 15 ml to obtain an aqueous solution of 200 mm ammonium sulfate / 60 mm citric acid. After adjusting 2.5 ml of an aqueous solution of 200 mm ammonium sulfate / 60 mm citric acid with an aqueous solution of ammonia to pH 5.5, the aqueous solution for the internal phase of liposomes was diluted to 5 ml with pure water.

Getting preliminary liposomal fluid

After dissolving 317.9 mg of hydrogenated soybean phosphatidylcholine (manufactured by Lipoid), 116.0 mg of cholesterol (manufactured by Sigma) and 130.4 mg of 2000-phosphatidylethanolamine polyethylene glycol (manufactured by Genzyme, MPEG 2000-distearoylphosphatidylethanolamine) in 10 ml of chloroform, the solution was precisely distributed in three vial, after which chloroform from one vial was removed under reduced pressure in a rotary evaporator to create a lipid film. 5 ml of an aqueous solution for the internal phase of the liposomes was heated to about 60 ° C, added to the resulting lipid film and all were mixed to obtain a preliminary liposome liquid. After treating the pre-liposomal fluid with ultrasonic waves for 20 minutes, it was granulated with an extruder (manufactured by Lipex Biomembranes) heated to about 65 ° C. to obtain the pre-liposomal fluid. The particle size of the liposomes in the obtained preliminary liposomal fluid was measured using the method of dynamic light scattering, and in all it was 90-100 nm.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the obtained preliminary liposomal liquid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine (pH 7.6), replacing the external phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine. After replacing the external phase of the liposomes, centrifugation was performed for 30 minutes at 400,000 × g. After centrifugation, the precipitate was redispersed and an aqueous solution of 0.9% sodium chloride / 10 mM histidine was used to obtain 5 ml of liposome dispersion liquid.

Obtaining a solution of the active compound

Eribulin mesylate was dissolved in an aqueous solution of 0.9% sodium chloride / 10 mM histidine to give 1 mg / ml eribulin mesylate.

Obtaining a liposome composition

0.5 ml of liposome dispersion liquid and 0.5 ml of eribulin mesylate solution were mixed in a 10 ml glass vial and incubated for 3 minutes in water at 55 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

Capture Ratio Measurement

The capture coefficient was determined as described below.

The liposome composition with the captured active compound was ultracentrifuged for 30 minutes at 400,000 × g. The concentration of the active substance in the filtrate was measured by HPLC, determining the amount of active compound not enclosed in liposomes. The capture coefficient was calculated using the formula below.

Formula 1

The capture coefficient of eribulin mesylate was 90.9%.

Embodiment 2

Obtaining an aqueous solution for the internal phase of liposomes

Similarly to embodiment 1, 264.3 mg of ammonium sulfate and 126.1 mg of citric acid monohydrate were dissolved in pure water and, using a graduated flask, this solution was diluted to 10 ml to obtain an aqueous solution of 200 mm ammonium sulfate / 60 mm citric acid. 1 ml was taken from it and adjusted to pH 5.5 with ammonia water, after which it was diluted with pure water to 2 ml to obtain an aqueous solution for the internal phase of liposomes.

Getting preliminary liposomal fluid

80 mg of each component of the lipid mixture (hydrogenated soybean phosphatidylcholine: cholesterol: polyethylene glycol 2000-phosphatidylethanolamine = 58.6: 19.2: 22.2 (by weight)) was weighed, 2 ml of an aqueous solution for the internal phase of liposomes were heated to about 80 ° C and added to them, and all this was mixed to obtain a preliminary liposomal fluid. This pre-liposomal fluid was granulated by an extruder (manufactured by Lipex Biomembranes) heated to about 80 ° C to obtain a pre-liposome fluid.

Obtaining liposomal dispersion fluid

The obtained preliminary liposomal liquid was diluted to 10 ml with an aqueous solution of 0.9% sodium chloride / 10 mm histidine (pH 7.6) and centrifuged for 30 minutes at 400,000 × g. After centrifugation, the entire filtrate was removed. The precipitate was redispersed in an aqueous solution of 0.9% sodium chloride / 10 mM histidine and, using a graduated flask, 1 ml of liposomal dispersion liquid was obtained.

Obtaining a solution of a drug

Eribulin mesylate (eribulin mesylate) was dissolved in an aqueous solution of 0.9% sodium chloride / 10 mM histidine and a 5 mg / ml eribulin mesylate solution was obtained.

Obtaining a liposome composition

0.96 ml of liposome dispersion liquid and 0.24 ml of eribulin mesylate solution were mixed in a 10 ml glass vial, and the mixture was incubated for 3 minutes in water at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

Stability in rat plasma

0.2 ml of the resulting liposomes captured with eribulin mesylate and 1.8 ml of rat blood plasma were mixed and shaken at 37 ° C. using a liquid phase incubator. Immediately after preparation, sampling was performed after 6 hours, 12 hours, 24 hours, 48 hours and 72 hours after the start of shaking, and the residual amount of eribulin mesylate in liposomes was measured by HPLC.

The measurement results are presented in figure 1. As can be seen in figure 1, eribulin mesylate was stably maintained in the blood plasma even for a long period of time for 120 hours, and a gradual release was possible.

Embodiment 3

Obtaining an aqueous solution for the internal phase of liposomes

264.3 mg of ammonium sulfate and 126.1 mg of citric acid monohydrate were dissolved in pure water to obtain approximately 15 ml. After adjusting the pH to 7.0 with an aqueous sodium hydroxide solution, the solution was diluted with pure water to 20 ml to obtain an aqueous solution for the internal phase of liposomes (100 mM ammonium sulfate / 30 mM citric acid).

Getting preliminary liposomal fluid

378 mg of the lipid mixture (hydrogenated soybean phosphatidylcholine: cholesterol: polyethylene glycol 2000-phosphatidylethanolamine = 58.6: 19.2: 22.2 (by weight)) was weighed, 10 ml of the above aqueous solution for the internal phase of liposomes was heated to about 80 ° C and added to them, and all this was mixed to obtain a preliminary liposomal fluid. This pre-liposome liquid was granulated by an extruder (manufactured by Lipex Biomembranes) equipped with a 50 nm polycarbonate membrane filter and heated to about 80 ° C. to obtain a pre-liposome liquid with a particle size of about 80 nm.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the obtained preliminary liposomal liquid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine (pH 7.6), replacing the external phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine. After replacing the external phase of the liposomes, centrifugation was performed for 30 minutes at 400,000 × g. After centrifugation, the precipitate was redispersed in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.6) and 10 ml of the solution was diluted to 10 ml to obtain a liposomal dispersion liquid.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.6) and a 5 mg / ml eribulin mesylate solution was obtained.

Obtaining a liposome composition

9.6 ml of liposome dispersion liquid and 1.2 ml of eribulin mesylate solution were mixed in a 10 ml glass vial and sodium hydroxide was used to adjust the pH to 9.5. The solution was incubated for 3 minutes in water at a temperature of 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes. After cooling, chloride was used to adjust the pH to 7.5. As in embodiment 1, the capture coefficient was measured and found to be 99%.

Embodiment 4

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 1, an aqueous solution of 100 mM ammonium sulfate / 30 mM citric acid was obtained (pH 5.5).

Getting preliminary liposomal fluid

Hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol 2000-phosphatidylethanolamine were weighed in the amounts shown in table 1. After each was dissolved in 3 ml of chloroform, chloroform was removed under reduced pressure in a rotary evaporator to create a lipid film. 10 ml of the obtained aqueous solution for the internal phase of the liposomes was heated to about 80 ° C, added to the obtained lipid film and mixed to obtain a preliminary liposome liquid. It was granulated using an extruder (manufactured by Lipex Biomembranes) heated to about 80 ° C. to obtain a granular pre-liposomal fluid. The particle size of liposomes in the obtained preliminary liposomal fluid was measured using the dynamic light scattering method and Rp.1 was 77 nm, Rp.2 was 95 nm, Rp.3 was 79 nm and Rp.4 was 128 nm.

Table 1 Rp. Hydrogenated Soybean Phosphatidylcholine Cholesterol Polyethylene glycol 2000-phosphatidylethanolamine one 234 mg 76 mg 15 mg 2 234 mg 76 mg 15 mg 3 222 mg 73 mg 87 mg four 222 mg 73 mg 87 mg

Obtaining a liposome composition

As in Embodiment 1, a liposomal dispersion liquid was obtained. Also, eribulin mesylate was dissolved in an aqueous solution of 0.9% sodium chloride / 10 mM histidine and 5 mg / ml eribulin mesylate was obtained.

4.8 ml of each of the liposome dispersion liquids and 0.6 ml of eribulin mesylate solution were mixed in 10 ml glass vials, which were incubated for 3 minutes in water at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes. 24.6 ml of an aqueous solution of 0.9% sodium chloride / 10 mM histidine was added to each of the liposomal compositions and a 0.22 μm polyvinylidene fluoride (PVDF) filter was used for filtration and sterilization to obtain an injection sample (eribulin mesylate concentration 0.1 mg / ml).

As in Embodiment 1, the capture coefficient was measured and confirmed that it was at least 90% in each of the prescriptions.

Female nude mice (NU / NU, Charles River Laboratories Japan, Inc.) were subcutaneously inoculated with LOX human melanoma cells and after 11 or 12 days, the samples were injected into the tail veins at a rate of 10 ml / kg body weight (1.0 mg / kg body weight for eribulin mesylate). Blood samples were taken and tumor tissue was extracted by puncture of the heart in a timely manner after administration (15 minutes, 30 minutes, 1, 2, 4, 8, 12, 24, 36, and 48 hours) (n = 3). Blood was collected in heparin tubes and within 30 minutes after sampling, the blood was separated by centrifugation at 1500 × g for 10 minutes at 4 ° C to obtain blood plasma. All tumor tissue was removed, washed with PBS and wiped with blotting paper, and then tissue weight was immediately determined and recorded. The tissue was placed in test tubes and cooled in ice water, and then stored at −80 ° C until analysis.

Eribulin mesylate in blood plasma and in tumor tissue was measured using LC / MS / MS.

PK parameters were calculated using non-chamber model analysis software (WinNonlin version 5.0.1). The results of determination of PK parameters in plasma and PK parameters in tumor tissue for eribulin mesylate are presented, respectively, in table 2 and table 3.

table 2 PK parameters in blood plasma Rp.1-4 and eribulin mesylate in mice with cancer tumor LOX Copy AUC _0-1 (ng · h / ml) AUC _0-∞ (ngh / ml) CL (ml / hour / kg) V _ss (ml / kg) t _1/2 (hour) MRT (hour) Coefficient 1 Rp. 1 253049 258274 3.87 43,99 8.7 11,4 707.1 Rp.2 176148 177893 5.62 56.40 6.8 10.0 487.0 Rp.3 228151 233067 4.29 48.93 8.4 11,4 638.1 Rp.4 221494 230541 4.34 55.88 9,4 12.9 631.2 Eribulin mesylate 363.02 365,247 2420 8032 3,7 3.3 1,0 Coefficient 1 = _{plasma liposome} AUC / _plasma AUC _{eribulin mesylate}

Table 3 PK parameters in blood plasma Rp.1-4 and eribulin mesylate in mice with cancer tumor LOX Copy C _[max] (ng / g) T _[max] (hour) AUC _0-1 (ng · h / ml) AUC _0-∞ (ngh / ml) t _1/2 (hour) MRT (hour) TPI (ml / g) Coefficient 2 Rp. 1 692.1 4.0 24960.7 34581.8 22.8 38.8 0.13 5.5 Rp.2 1002.9 8.0 16759.6 22301.1 22.2 34.5 0.13 3,5 Rp.3 3965.7 12.0 41,643.7 46,297.3 16.1 23.3 0.20 7.4 Rp.4 1132.8 12.0 28377.4 45005.6 23.7 44.3 0.20 7.2 Eribulin mesylate 323,425 0.25 4649,521 6294,283 17.8 27.7 17.23 1,0 Coefficient 2 = AUC of _{liposomes in the tumor} / AUC of _{eribulin mesylate in the tumor}

From table 2 and table 3 it can be seen that the AUC in the blood plasma and in the tumor tissue is increased compared to free eribulin mesylate in all four liposome compositions of Rp.1-4, and as a result, the amount of eribulin mesylate migrated to the tumor and conservation is improved.

Embodiment 5

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 1, an aqueous solution of 100 mM ammonium sulfate / 30 mM citric acid was obtained (pH 5.5).

Getting preliminary liposomal fluid

221.8 mg of hydrogenated soybean phosphatidylcholine, 72.5 mg of cholesterol and 86.9 mg of 2000-phosphatidylethanolamine polyethylene glycol were weighed. After dissolving them in 3 ml of chloroform, chloroform was removed under reduced pressure in a rotary evaporator and a lipid film was obtained. 10 ml of the obtained aqueous solution for the internal phase of the liposomes was heated to about 80 ° C, added to the obtained lipid film and mixed to obtain a preliminary liposome liquid. It was granulated using an extruder (manufactured by Lipex Biomembranes) heated to about 80 ° C. to obtain a granular pre-liposome liquid. When the particle sizes of liposomes in the obtained preliminary liposomal fluid were measured using the dynamic light scattering method, they were approximately 90 nm.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the obtained preliminary liposomal liquid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine (pH 7.6), replacing the external phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine. After replacing the external phase of the liposomes, centrifugation was performed for 30 minutes at 400,000 × g. After centrifugation, the precipitate was redispersed and an aqueous solution of 0.9% sodium chloride / 10 mM histidine was used to obtain 10 ml of liposome dispersion liquid.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 0.9% sodium chloride / 10 mM histidine and a 1 mg / ml solution of eribulin mesylate was obtained. In addition, as samples of the free basic substance for administration, the eribulin mesylate solution was diluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine and a 0.22 μm PVDF filter was used for filtration and sterilization to obtain samples for administration (concentration of eribulin mesylate 0 3 mg / ml and 0.4 mg / ml).

Obtaining a liposome composition

1.8 ml of liposome dispersion liquid and 1.2 ml of eribulin mesylate solution were mixed in a 10 ml glass vial, which was incubated for 3 minutes in water at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes. The resulting liposome composition was diluted with an aqueous solution of 0.9% sodium chloride / 10 mm histidine and a 0.22 μm PVDF filter was used for filtration and sterilization to obtain a sample for injection (eribulin mesylate concentration 0.2 mg / ml). As in Embodiment 1, the capture coefficient was measured and confirmed to be at least 90%.

FaDu cells (obtained from the American Type Culture Collection), which are a line of human pharyngeal squamous cell carcinoma, were cultured and grown in MEM culture medium containing 10% bovine fetal serum. Cells were separated from the mattress using a 0.05% trypsin-EDTA solution and collected. After washing with PBS, cells were suspended in PBS to a density of 5 × 10 ⁷ cells / ml and stored on ice. 0.1 ml of liquid cell suspension was subcutaneously injected into the right side of the abdominal region of 6-week-old nude mice (Charles River Laboratories Japan, Inc.). Each mouse was inspected daily and made appropriate notes in those cases when an abnormal condition was detected. To measure the size of the tumor over time, calipers were used and the tumor size was calculated based on the calculation formula: major axis × (minor axis squared) ÷ 2. At the time when the tumor size was from 100 to 200 mm ³ , the mice were divided into groups so that the average values of the tumor size and body weight of the mice were the same among the experimental groups (five mice in the experimental group), and the drug was injected into the tail vein (0 , 2 ml / 20 g, 3 times at 7-day intervals).

The resulting changes in the average tumor volume after injection of the sample are presented in figure 2.

As shown in figure 2, the effect of reducing tumors was not obtained even with a dose of 4 mg / kg body weight, which is the maximum tolerated dose for the free basic substance, since FaDu is a cell line with low sensitivity to eribulin mesylate. Meanwhile, in the case of the liposomal composite, a clear effect of tumor reduction was observed even with a dose of 2 mg / kg body weight, which is lower than the maximum tolerated dose, this indicated the possibility of achieving an extremely high pharmacological effect even for those types of cancer against which eribulin mesylate is not effective .

Embodiment 6

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 1, an aqueous solution of 100 mM ammonium sulfate / 30 mM citric acid was obtained (pH 5.5).

Obtaining a solution of a drug

As in Embodiment 5, administration samples were obtained (eribulin mesylate concentration: 0.2 mg / ml, 0.3 mg / ml and 0.4 mg / ml) of free basic substances.

Obtaining a liposome composition

With the exception of using an aqueous solution of the internal phase of liposomes [sic: an aqueous solution for the internal phase of liposomes], prepared as described above, the liposome composition (concentration of eribulin mesylate 0.3 mg / ml) was obtained as in Embodiment 5. As in Embodiment 1, the capture coefficient was measured and confirmed to be at least 90%.

ACHN cells (obtained from the American Type Culture Collection), which are a human kidney cancer cell line, were cultured and grown in MEM culture medium containing 10% bovine fetal serum. Cells were separated from the mattress using a 0.05% trypsin-EDTA solution and collected. After washing with PBS, cells were suspended in PBS to a density of 5 × 10 ⁷ cells / ml and stored on ice. 0.1 ml of liquid cell suspension was subcutaneously injected into the right side of the abdominal region of 6-week-old nude mice (Charles River Laboratories Japan, Inc.). Each mouse was inspected daily and made appropriate notes in those cases when an abnormal condition was detected. To measure the size of the tumor over time, calipers were used and the tumor size was calculated based on the calculation formula: major axis × (minor axis squared) ÷ 2. At the time when the tumor size was from 150 to 200 mm ³ , the mice were divided into groups so that the average values of the tumor size and body weight of the mice were the same among the experimental groups (five mice in the experimental group), and the drug was injected into the tail vein (0 , 2 ml / 20 g, 3 times at 7-day intervals).

The obtained changes in the average tumor volume after injection of the sample are presented in figure 3.

As shown in FIG. 3, since ACHN is an eribulin mesylate resistant cell line, no significant difference was found between any of the groups administered 2 mg / kg, 3 mg / kg and 4 mg / kg body weight (maximum tolerated dose ) free basic substance and the untreated group, 45 days after the start of sample administration. Meanwhile, in the group with the introduction of the liposome composition at a dose of 3 mg / kg of body weight, the effect of suppressing tumor growth was observed, and a significantly lower value of the tumor volume occurred in comparison with the untreated group and the groups with the introduction of the free main substance after 45 days after the introduction of the sample. As follows from the data obtained, it is possible to inhibit tumor growth by creating a liposomal preparation for the tumor, for which the therapeutic effect has never been obtained with eribulin mesylate.

Embodiment 7

Obtaining an aqueous solution for the internal phase of liposomes

Prepared 12 types of aqueous solutions for the internal phase of liposomes, shown below in table 4.

Getting preliminary liposomal fluid

120 mg of the lipid mixture (hydrogenated soybean phosphatidylcholine: cholesterol: polyethylene glycol 2000-phosphatidylethanolamine = 58.6: 19.2: 22.2 (by weight)) was weighed in test tubes, and 3 ml of each aqueous sample of the liposome internal phase was heated to 80 ° C.

This pre-liposomal fluid was granulated using an extruder heated to about 80 ° C. to obtain a granular pre-liposomal fluid.

Obtaining a liposomal dispersion solution

Using Sephadex G-50 columns, the resulting preliminary liposomal fluid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine, replacing the outer phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine.

After replacing the external phase of the liposomes, centrifugation was carried out for 1 hour at 400,000 × g and the filtrate was completely removed. The precipitate was resuspended in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5) so as to obtain about 2 ml.

The particle size in the obtained liposomal dispersion liquid was measured using the method of dynamic light scattering and for all it was approximately 80 nm.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine and a 5 mg / ml solution of eribulin mesylate was obtained.

Obtaining a liposome composition

The liposomal dispersion liquid and eribulin mesylate solution were mixed in a 10 ml glass vial so that the concentration of eribulin mesylate was 0.2 mg / ml and the total lipid concentration was 16 μmol / ml. The mixture was heated for 5 minutes at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

Capture Ratio Measurement

The capture coefficient was determined as in embodiment 1 and the results are shown in table 4. As can be seen from table 4, regardless of which ammonium salt was used in the internal phase, the capture coefficient of eribulin mesylate was clearly improved. In particular, an improvement in the capture coefficient was noted when using ammonium sulfate, ammonium citrate, ammonium phosphate and ammonium tartrate.

Table 4 No. Composition pH Osmotic pressure Capture Ratio (%) one 50 mM ammonium sulfate 7.5
(adjusted with hydrochloric acid or sodium hydroxide) 300 mosmol (brought with sucrose) 69,4 2 50 mM sodium sulfate 7.2 3 50 mM ammonium acetate 36.8 four 50 mM sodium acetate 10,2 5 50 mM ammonium phosphate 45.8 6 50 mM sodium phosphate 14.6 7 50 mM ammonium citrate 65.8 8 50 mM sodium citrate 8.7 9 50 mM ammonium succinate 14.7 10 50 mM sodium succinate 10.0 eleven 50 mM ammonium tartrate 74.7 12 50 mM sodium tartrate 11.6

Option exercise 8

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 7, an aqueous solution was prepared for the internal phase of the liposomes from a solution of 100 mM ammonium sulfate / 30 mM aqueous citric acid (pH 7.5).

Getting preliminary liposomal fluid

As in embodiment 7, the above aqueous solution for the internal phase of the liposomes was used to obtain a preliminary liposomal fluid.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the resulting preliminary liposomal fluid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine, replacing the outer phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine.

After replacing the external phase of the liposomes, centrifugation was carried out for 1 hour at 400,000 × g, completely removing the filtrate. The precipitate was re-suspended in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5), the external phase of the liposomes was replaced with an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5), and a liposomal dispersion liquid was obtained. The particle size in the obtained liposomal dispersion liquid was measured using the method of dynamic light scattering and it was approximately 80 nm.

The liposomal dispersion liquid was poured into seven vials and a known amount of ammonium sulfate (adjusted to pH 7.5 with an aqueous solution of sodium hydroxide) was added to the external phase of the liposomes so that the concentrations in the vials are shown in Table 5 and a liposomal dispersion liquid was obtained, in which ammonium sulfate in the external phase of liposomes was a known concentration.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine and a 5 mg / ml solution of eribulin mesylate was obtained.

Obtaining a liposome composition

The liposomal dispersion liquid and eribulin mesylate solution were mixed in a 10 ml glass vial so that the concentration of eribulin mesylate was 0.2 mg / ml and the total lipid concentration was 16 mM. The mixture was heated for 5 minutes at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

Capture Ratio Measurement

The capture coefficient was determined as in Embodiment 1 and the results are shown in Table 5. They indicate that even if 0.4 mM ammonium sulfate is present in the external phase of the liposomes, the capture coefficient noticeably decreases, and in the presence of 10 mM ammonium sulfate, capture is almost absent .

Table 5 No. Inland water phase The concentration of ammonium sulfate in the external phase (mm) Capture Ratio (%) one 100 mM ammonium sulfate
30 mM citric acid
pH = 7.5 0 90,4 2 0.016 90.8 3 0.08 91.3 four 0.4 75.9 5 2 36.8 6 10 16.1 7 fifty 8.6

Embodiment 9

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 7, an aqueous solution was prepared for the internal phase of the liposomes from a solution of 100 mM ammonium sulfate / 30 mM aqueous citric acid (pH 7.5).

Getting preliminary liposomal fluid

As in embodiment 7, the above aqueous solution for the internal phase of the liposomes was used to obtain a preliminary liposomal fluid.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the resulting preliminary liposomal fluid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine, replacing the outer phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine.

After replacing the external phase of the liposomes, centrifugation was carried out for 1 hour at 400,000 × g and the filtrate was completely removed. The precipitate was re-suspended in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5), the external phase of the liposomes was replaced with an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5), and a liposomal dispersion liquid was obtained. The particle size in the obtained liposomal dispersion liquid was measured using the method of dynamic light scattering and it was approximately 80 nm.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine and a 5 mg / ml solution of eribulin mesylate was obtained.

Obtaining a liposome composition

The liposomal dispersion liquid and eribulin mesylate solution were mixed in a 10 ml glass vial so that the concentration of eribulin mesylate was 0.2 mg / ml and the total lipid concentration was 16 mM. As shown in table 6, in each case, the pH of the external phase of the liposomes was adjusted using a 1M aqueous solution of sodium hydroxide. The solution was heated for 5 minutes at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes. Then, hydrochloric acid was used to adjust the pH of the external phase to 7.5.

Capture Ratio Measurement

The capture coefficient was determined as in embodiment 1 and the results are shown in table 6. Together with an increase in the pH of the external phase of the liposomes, the capture coefficient of eribulin substantially increased, reaching a value of about 100%.

Table 6 No. Inland water phase pH of the external phase Capture Ratio (%) one 100 mM ammonium sulfate
30 mM citric acid
pH 7.5 7.5 72.9 2 8.0 79.8 3 8.5 86.4 four 9.0 92.8 5 9.5 98.5 6 10.0 100.0 7 10.5 99.3

Embodiment 10

Obtaining an aqueous solution for the internal phase of liposomes

As in embodiment 7, an aqueous solution was prepared for the internal phase of the liposomes from a solution of 100 mM ammonium sulfate / 30 mM aqueous citric acid (pH 7.5).

Getting preliminary liposomal fluid

As in embodiment 7, the above aqueous solution for the internal phase of the liposomes was used to obtain a preliminary liposomal fluid.

Obtaining liposomal dispersion fluid

Using Sephadex G-50 columns, the resulting preliminary liposomal fluid was eluted with an aqueous solution of 0.9% sodium chloride / 10 mM histidine, replacing the outer phase of the liposomes with an aqueous solution of 0.9% sodium chloride / 10 mM histidine.

The liposomal dispersion liquid was poured into four vials, which were centrifuged for 1 hour at 400,000 × g, and the filtrate was completely removed. The precipitate from two vials was resuspended in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5) and the external phase of the liposomes was replaced with an aqueous solution of 96 mg / ml sucrose / 10 mM histidine (pH 7.5). The precipitate in the remaining two vials was resuspended in an aqueous solution of 0.9% sodium chloride / 10 mM histidine (pH 7.5) and the external phase of the liposomes was replaced with an aqueous solution of 0.9% sodium chloride / 10 mM histidine (pH 7.5). The particle size in the obtained liposomal dispersion liquid was measured using the method of dynamic light scattering and for all it was approximately 80 nm.

Obtaining a solution of a drug

Eribulin mesylate was dissolved in an aqueous solution of 96 mg / ml sucrose / 10 mM histidine and a 5 mg / ml solution of eribulin mesylate was obtained. Similarly, eribulin mesylate was dissolved in an aqueous solution of 0.9% sodium chloride / 10 mM histidine and a 5 mg / ml eribulin mesylate solution was obtained.

Obtaining a liposome composition

The liposomal dispersion liquid and eribulin mesylate solution were mixed in a 10 ml glass vial so that the concentration of eribulin mesylate was 0.2 mg / ml and the total lipid concentration was 16 mM. The pH value of the external phase of liposomes in one of two vials with an aqueous solution of 96 mg / ml sucrose / 10 mm histidine (pH 7.5) was adjusted to 9.5 by adding sodium hydroxide. Similarly, the pH of the external phase of liposomes in one of the two vials with an aqueous solution of 0.9% sodium chloride / 10 mm histidine (pH 7.5) was adjusted to 9.5 by adding sodium hydroxide. The solutions were heated for 5 minutes at 60 ° C to obtain a liposome composition with eribulin mesylate introduced into the liposomes.

Capture Ratio Measurement

The capture coefficient was determined as in embodiment 1, and the results are shown in table 7. Compared with the case where the external phase of the liposomes is sucrose, which is not an electrolyte, in the case of sodium chloride, which is an electrolyte, an extremely high capture coefficient is clearly achieved. In addition to the electrolyte effect, the use of a pH gradient to alkalize the external phase of liposomes leads to a 100% capture coefficient.

Table 7 No. Inland water phase The composition of the external phase Capture Ratio (%) one 100 mM ammonium sulfate
30 mM citric acid
pH 7.5 96 mg / ml sucrose / 10 mM histidine pH 7.5 72.9 2 0.9% sodium chloride / 10 mM histidine pH 7.5 95.9 3 96 mg / ml sucrose / 10 mM histidine pH 9.5 98.5 four 0.9% sodium chloride / 10 mM histidine pH 9.5 100.0

This application is based on the Japanese Patent Application (Japanese Patent Application 2009-082521), filed March 30, 2009, and the provisional patent application of the United States of America (61/164653), and their contents are incorporated herein by reference.

Industrial applicability

The present invention is capable of providing a method for producing liposomes with high retention stability of the active compound in combination with a high capture coefficient.

The liposomal composition of the present invention is useful for therapeutic purposes due to the pharmacological effect of eribulin or its pharmacologically acceptable salt.

Claims (39)

1. Liposome composition containing liposomes and containing the active compound in the internal phase of the liposomes, where the active compound is eribulin or a pharmacologically acceptable salt thereof. 2. The liposome composition according to claim 1, in solid or liquid form. 3. The liposome composition according to claim 1, in which the internal phase of the liposomes further comprises an ammonium salt. 4. The liposomal composition according to claim 3, in which the concentration of the above ammonium salt is 10 mm or higher. 5. The liposome composition according to claim 1, in which the internal phase of the liposomes further comprises a salt, acid, base and / or amino acid. 6. The liposomal composition according to claim 5, in which the concentration of the above salt is 1-300 mm. 7. The liposomal composition according to claim 5, in which the concentration of the above acid is 1-300 mm. 8. The liposomal composition according to claim 5, in which the concentration of the above amino acids is 1-300 mm. 9. The liposomal composition according to claim 5, in which the concentration of the above base is 1-300 mm. 10. The liposome composition according to claim 1, in which the concentration of the above active compounds is from 0.01 to 300 mg / ml 11. The liposome composition according to claim 1, in which the above active compound is eribulin mesylate. 12. The liposome composition according to claim 1, in which the internal phase of the liposomes further comprises ammonium sulfate, citric acid and the active compound. 13. The liposome composition according to claim 1, in which the external phase of the liposomes contains sugar, electrolyte and / or amino acid. 14. The liposome composition according to item 13, in which the external phase of the liposomes contains sugar or electrolyte and an amino acid. 15. The liposomal composition according to item 13, in which the concentration of the above sugar is 2-20%. 16. The liposomal composition according to item 13, in which the concentration of the above amino acids is 1-300 mm. 17. The liposome composition of claim 14, wherein the external phase of the liposomes contains sucrose or sodium chloride and histidine. 18. The liposome composition according to any one of claims 1 to 17, in which the above internal phase of the liposomes essentially does not contain cyclodextrin. 19. The liposome composition according to any one of claims 1 to 17, in which the liposomes contain hydrogenated phosphatidylcholine. 20. The liposome composition according to any one of claims 1 to 17, in which the liposomes contain cholesterol. 21. The liposome composition according to any one of claims 1 to 17, in which the liposomes contain a condensate of methoxypolyethylene glycol. 22. The liposomal composition according to item 21, in which the above methoxypolyethylene glycol condensate is a distearoylphosphatidylethanolamine-polyethylene glycol condensate. 23. The liposomal composition according to any one of claims 1 to 17, in which the liposomes contain hydrogenated phosphatidylcholine, cholesterol and a condensate of distearoylphosphatidylethanolamine-polyethylene glycol. 24. The liposomal composition according to item 23, which contains from 10 to 80% of the above hydrogenated phosphatidylcholine, from 1 to 60% of the above cholesterol and from 0 to 50% of the above condensate distearoylphosphatidylethanolamine-polyethylene glycol. 25. The liposome composition according to any one of claims 1 to 17, in which the liposomes contain hydrogenated soybean phosphatidylcholine, cholesterol and polyethylene glycol 2000-phosphatidylethanolamine. 26. A method of manufacturing a liposome composition according to any one of claims 1 to 25, including:
a step in which a liposome dispersion liquid containing liposomes is obtained;
a step in which the aforementioned liposome dispersion liquid is mixed with the aforementioned active compound; and a step in which the aforementioned active compound is introduced into the internal phase of the liposomes of the aforementioned liposome dispersion liquid. 27. The method according to p, in which the above liposome dispersion liquid, essentially does not contain an ammonium salt in the external phase of the liposomes. 28. The method according to p, in which the pH of the external phase of the liposomes of the above liposome dispersion liquid is 3-10. 29. The method according to p, in which the pH of the external phase of the liposomes of the above liposome dispersion liquid is 7-10. 30. The method according to p, in which the above pH is the pH of the external phase of the liposomes of the above liposome dispersion liquid at the stage in which the above liposome dispersion liquid and the above active compound are mixed. 31. The method according to p. 26, in which the stage on which the above liposomal dispersion liquid is obtained, includes: the stage in which receive a preliminary liposome solution containing liposomes and containing an ammonium salt in the internal phase of the liposomes and the external phase of liposomes; and a step in which the external liposome phase of the aforementioned pre-liposome solution is replaced or diluted. 32. The method according to p, in which the stage at which the above external phase of the liposomes is replaced or diluted, is the stage in which the pH of the external phase of the liposomes is higher than the pH of the internal phase of the liposomes. 33. The method according to p, in which the stage at which the above external phase of liposomes is replaced or diluted, is the stage in which the difference between the pH of the internal phase of liposomes and the pH of the external phase of liposomes is 1-5. 34. The method according to p, in which the pH of the above internal phase of liposomes is 3-9. 35. The method according to p, in which the pH of the above internal phase of liposomes is 4-9. 36. The method according to p, in which the pH of the above internal phase of liposomes is 5-8. 37. The method according to p, in which the external phase of the liposomes is a solution containing an electrolyte at the stage at which the above active compound is administered. 38. The method according to p, in which the above liposomal dispersion liquid, essentially does not contain cyclodextrin. 39. The method according to p, further comprising a step in which the pH of the external phase of the liposomes is neutral.

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