JP2000302685A - Liposome preparation containing anti-tumor agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a liposome preparation being a 1-(2'-cyano-2'-deoxy-β-D- arabino-pentofuranosyl)cytosine-containing liposome preparation, having high accumulation and retention in a tumor tissue and excellent antitumor activity. SOLUTION: This preparation has 30-300 mM phospholipid concentration and contains a sterol and a phosphatidylcholine as lipids constituting a liposome. The preparation has 50-200 mM phospholipid concentration and the sterol being one of the lipids constituting the liposome is preferably cholesterol. Further, the preparation preferably contains a lipid chemically modified with a polyethylene glycol as the lipid constituting the liposome.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a novel 1- (2 '
-Cyano-2'-deoxy-β-D-arabino-pentofuranosyl) cytosine-containing liposome formulation.

[0002]

2. Description of the Related Art 1- (2'-cyano-2'-deoxy-)
β-D-arabino-pentofuranosyl) cytosine (hereinafter referred to as CNDAC) is a compound having excellent antitumor activity (Tanaka et al., Cancer Letter 64, 67-74 (199).
2) / Azuma et al., J. Med. Chem. 36, 4183-4189 (1993)
/ See Japanese Patent Application Laid-Open No. 4-235182).
Since AC is water-soluble, it can be administered intravenously by dissolving it in physiological saline or the like.

[0003] However, in general, water-soluble antitumor drugs are easily degraded in the living body, are rapidly excreted from the living body, and have a nonspecific tissue distribution in the living body. In order to obtain tumor activity and reduce side effects, a formulation is generally desired.

[0004] By improving the drug transferability of an antitumor drug to tumor tissue and the retention of the antitumor drug in tumor tissue, the antitumor drug is used for the purpose of obtaining better antitumor activity and reducing side effects. Pharmaceutical innovation to make drugs into liposomal preparations,
Generally done.

[0005] Asai et al. Have already attempted to encapsulate CNDAC in liposomes, but the amount of CNDAC incorporated into liposomes is small, and CNDAC incorporated in liposomes flows out relatively quickly. For,
CNDAC has concluded that it is an antitumor drug that is not suitable for liposome preparations (Drug Delivery System 13, 341).
-346 (1998). ).

[0006]

SUMMARY OF THE INVENTION The present inventors have developed a CND.
As for the liposome preparation of AC, as a result of intensive studies on the lipids constituting the liposome and the composition ratio thereof, the liposome preparation contains a specific lipid, and particularly when the lipid is contained in the liposome preparation at a specific composition ratio, The present inventors have found that CNDAC, which has been unsuitable for use as a liposome preparation, can be used as a liposome preparation that can be put to practical use. The present invention was found to have high retention in tissues and, as a result, to have low toxicity, and completed the present invention.

[0007]

According to the present invention, there is provided 1- (2'-cyano-2'-deoxy-β-D which solves the above-mentioned problems.
-Arabino-pentofuranosyl) cytosine-containing liposome formulations.

[0008] The present invention provides <1> 1- (2'-cyano), wherein the phospholipid concentration is 30 to 300 mM, and the liposome comprises sterols and phosphatidylcholines as lipids. -2'-deoxy-β-D-arabino-pentofuranosyl) cytosine-containing liposome preparation, <2> liposome preparation according to <1>, wherein the phospholipid concentration is 50 to 200 mM, <3>
A liposome preparation according to <1> or <2>, wherein the sterol, which is one of the lipids constituting the liposome, is cholesterol, <4> a lipid constituting the liposome, and polyethylene. The liposome preparation according to <1> to <3>, which contains a lipid chemically modified with a glycol, <5> one of lipids constituting the liposome, a lipid chemically modified with a polyethylene glycol, N-monomethoxy polyethylene glycol succinylphosphatidylethanolamines, N-monomethoxy polyethylene glycol (2-chloro-1,
3,5-triazine-4,6-diyl) succinylphosphatidylethanolamines, N-monomethoxy polyethylene glycol carbonyl phosphatidyl ethanolamines or N-monomethoxy polyethylene glycol ethylene phosphatidyl ethanolamines. The liposome preparation according to 4>, <6>
The liposome preparation according to <4>, wherein the lipid chemically modified with polyethylene glycol, which is one of lipids constituting the liposome, is N-monomethoxy polyethylene glycol succinylphosphatidylethanolamine. <7> The liposome preparation according to <1> to <6>, further comprising a phosphatidylglycerol as a lipid constituting the liposome, <8> a phosphatidylcholine which is a lipid constituting the liposome. And / or wherein the acyl group in the phosphatidylglycerol is an aliphatic acyl group having 10 to 20 carbon atoms, wherein the liposome preparation according to <1> to <7>, <9> comprising a liposome. Acyl groups in phosphatidylcholines and / or phosphatidylglycerols Is a myristoyl group, a palmitoyl group or a stearoyl group, the liposome preparation according to <1> to <8>, <10> a sterol that is a lipid constituting the liposome, Composition ratio to lipid amount is 10 mol
% To 60 mol%, <1> to
<9> The liposome preparation according to <9>, wherein the composition ratio of sterols, which are lipids constituting the liposome, to the total lipid amount constituting the liposome is 30 mol% to 50 m
liposome preparation according to any one of <1> to <10>, wherein the liposome is composed of N-monomethoxy polyethylene glycol succinylphosphatidylethanolamine, which is a lipid constituting the liposome. The liposome preparation according to <5> or <6>, wherein the composition ratio of the liposome to the total lipid amount is 1 mol% to 10 mol%, <13> a liposome comprising a phosphatidylcholine as a lipid constituting the liposome. The composition ratio is 35 mol% to 8 with respect to the total lipid amount.
The liposome preparation according to any one of <1> to <12>, wherein the composition ratio of the phosphatidylcholine, which is a lipid constituting the liposome, to the total lipid amount constituting the liposome is 40 mol. % To 6
The liposome preparation according to any one of <1> to <13>, wherein the composition ratio of phosphatidylglycerols, which are lipids constituting the liposome, to the total lipid amount constituting the liposome is 0 mol%. <7> to <9>, which is 1 mol% to 10 mol%.
<16> The liposome-constituting lipid is (1) a sterol, (2) a phosphatidylcholine, and (3) a monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine. <1> or <2>
The liposome preparation according to <17>, wherein the composition ratio of (1) sterols to the total lipid amount constituting the liposome is:
10 mol% to 60 mol%, and the composition ratio of (2) phosphatidylcholines is 35 mol% to 85 mol%.
The liposome preparation according to <1> or <2>, wherein the composition ratio of (3) monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine is 1 mol% to 10 mol%. <18> The composition ratio of (1) sterols is from 30 mol% to 50 mol%, and (2) the composition ratio of phosphatidyl cholines is from 40 mol% to 60 mol%, based on the total lipid amount constituting the liposome. ,
(3) The liposome preparation according to <1> or <2>, wherein the composition ratio of monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine is 1 mol% to 10 mol%.
9> The lipids constituting the liposome are (1) sterols, (2) phosphatidylcholines, and (3) phosphatidylglycerols, <1> to
<2> The liposome preparation according to <2>, wherein the composition ratio of (1) sterols is 30 mol% to 50 mol%, and (2) the composition ratio of phosphatidylcholines is based on the total amount of lipids constituting the liposomes. , 40 mol% to 60
liposome preparation according to <1> or <2>, wherein the composition ratio of the phosphatidylglycerols is 1 mol% to 10 mol%.
21> The liposome has a volume average particle diameter of 100 nm to 400 nm, <1> to <20>.
The liposome preparation described in 1. above.

In the present invention, the term “liposome” is known to those skilled in the art (DDLasic, “Liposomes: from bas
ic to applications '', Elsevier Science Publisher
s, pp. 1-171 (1993)), meaning closed vesicles composed of lipids and an internal aqueous phase, assembled in a membrane.

In the liposome of the present invention, the surface of the liposome and the polyethylene glycol are bonded by a non-covalent bond such as an electrostatic interaction or a hydrophobic interaction, or the liposome is chemically modified with a polyethylene glycol described later. The lipid may be contained as a lipid constituting the liposome.

The liposome preparation of the present invention has a phospholipid concentration of usually 30 to 300 mM, preferably 50 to 300 mM.
To 200 mM.

The "sterols" which are essential lipid components constituting the liposome preparation of the present invention include, for example, cholesterol, cholesterol hemisuccinate, 3β-
[N- (N ', N'-dimethylaminoethane) carbamoyl] cholesterol, ergosterol, lanosterol and the like can be mentioned, preferably cholesterol, and sterols are the total lipid amount constituting the liposome. Is preferably 10 to 60 mol%,
More preferably, 30 to 50 mol% is contained in the liposome.

The "phosphatidylcholines", which are essential lipid components constituting the liposome preparation of the present invention, include, for example, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine. , Dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine or distearoylphosphatidylcholine, and the phosphatidylcholine is preferably 35 to 85 mol%, more preferably 40 to 60 mol%, based on the total lipid amount constituting the liposome. , Contained in liposomes.

The liposome preparation of the present invention is preferably
Contains "phosphatidylglycerols" or "lipids chemically modified with polyethylene glycols."

Examples of the above-mentioned "phosphatidyl glycerols" include dilauroyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol and distearoyl phosphatidyl glycerol, and preferably dimyristoyl phosphatidyl glycerol and dipalmitoyl phosphatidyl. It is glycerol or distearoyl phosphatidyl glycerol, and phosphatidyl glycerol is preferably contained in the liposome at 1 to 10 mol% based on the total lipid amount constituting the liposome.

The above-mentioned "lipid chemically modified with polyethylene glycols" means a lipid in which polyethylene glycols having various molecular weights and a lipid are covalently bonded, and the lipid is preferably phosphatidyl. Ethanolamines, for example, of the general formula

[0017]

Embedded image (In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamine.) N-monomethoxy polyethylene glycol succinylphosphatidylethanolamines represented by the general formula:

[0018]

Embedded image (In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamine.) N-monomethoxy polyethylene glycol (2-chloro-1,3,5)
-Triazine-4,6-diyl) succinylphosphatidylethanolamines, general formula

[0019]

Embedded image (In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamine.) N-monomethoxy polyethylene glycol carbonyl phosphatidylethanolamine represented by the general formula or

[0020]

Embedded image (In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamine.) N-monomethoxy polyethylene glycol ethylene phosphatidylethanolamines, more preferably N-monomethoxy Polyethylene glycol ethylene phosphatidylethanolamines, more preferably those having a molecular weight of about 500 to about 5,000, more preferably those having a molecular weight of about 1,000 to about 3,000. Yes,
Most preferably, the moiety has a molecular weight of about 2000 (D.
D. Lasic, `` Liposomes: from basic to application
s ", Elsevier Science Publishers, pp. 294-296 (199
3)).

In the liposome preparation of the present invention, in addition to the above-mentioned "sterols", "phosphatidylcholines", "phosphatidylglycerols" and "lipids chemically modified with polyethylene glycols", they can be generally used in liposome preparations. Phosphatidylinositols such as dilauroyl phosphatidylinositol, dimyristoyl phosphatidylinositol, dipalmitoyl phosphatidylinositol or distearoyl phosphatidylinositol, which may contain lipids;
Phosphatidylserines such as dilauroylphosphatidylserine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine or distearoylphosphatidylserine; dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine or distearoylphosphatidylethanolamine Glycerophospholipids such as phosphatidylethanolamines: digalactosyl diglycerolides such as digalactosyl dilauroyl glyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoyl glyceride, digalactosyl distearoyl glyceride; galactosyl dilauroyl glyceride, galactosyl Glyceroglycolipids such as galactosyl diglycerides such as toyl glyceride, galactosyl dipalmitoyl glyceride and galactosyl distearoyl glyceride: sphingolipids such as ceramide sialatin and sphingomyelin: sterols such as cerebroside and ganglioside: sterols: tetra Long-chain alkylamines such as decylamine, hexadecylamine and stearylamine: long-chain fatty acid hydrazides such as myristic hydrazide, palmitic hydrazide and stearic hydrazide: N- [1- (2,3-dioleyloxy) Propyl] -N, N, N-trimethylammonium chloride, N-α-trimethylammonioacetyldidodecyl-D-glutamate chloride, and positively charged lipids.

In the above "lipids", phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, phosphatidylserines, phosphatidylethanolamines, sulfoxyribosyl diglycerides, digalactosyl diglycerides, galactosyl diglycerides, sphingomyelin, cerebroside, ganglioside, Etc. each have two saturated or unsaturated aliphatic acyl chains, the chain preferably having 14 to 18 carbon atoms (particularly myristoyl, palmitoyl or stearoyl groups).

The volume average particle diameter of the liposome of the liposome preparation of the present invention is preferably 100 to 400 nm, and the volume average particle diameter can be determined based on a principle such as a dynamic light scattering method (DDLasic, "Liposomes: from basic
to applications '', Elsevier Science Publishers, p.
p.1-171 (1993)).

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The liposome preparation of the present invention can be produced according to a method well known to those skilled in the art, and the average volume particle diameter can be adjusted. That is, using the "lipid" and "aqueous phase", a thin film method, a reverse phase evaporation method,
Liposomes can be produced by the ethanol injection method, ether injection method, dehydration-rehydration method, etc., ultrasonic irradiation method, ultrasonic irradiation method after freeze-thawing, extrusion method, French press method, homogenization The volume average particle diameter can be adjusted by a method such as the method (D.
D. Lasic, `` Liposomes: from basic to application
s ", Elsevier Science Publishers, pp. 1-171 (1993)
reference. ). Here, the “aqueous phase” means an aqueous solution constituting the inside of the liposome, and if it is a commonly used one,
Although there is no particular limitation, buffers such as an aqueous sodium chloride solution, a phosphate buffer and an acetate buffer, an aqueous glucose solution, an aqueous trehalose solution, and a mixed aqueous solution thereof are used.

Generally, in order to maintain the structure of liposomes administered in vivo stably, the aqueous phase used for the production of liposomes is close to isotonic with the liposome external solution, that is, the body fluid, and is applied to the inside and outside of the liposome. It is desirable that the osmotic pressure be low.

As the above-mentioned “lipid” used for producing the liposome preparation of the present invention, a commercially available product and a product which can be easily chemically synthesized from a commercially available product by an ordinary method can be used. In general, lipids having various carbon numbers and / or degrees of unsaturation, such as egg yolk lecithin and soybean lecithin, which can be obtained in a mixed state, can be used without separating and purifying them into a single component. it can.

In the liposome preparation of the present invention, if necessary, α-tocopherol and the like can be added to the liposome preparation for the purpose of antioxidant action and the like.

When the liposome preparation of the present invention is administered to humans, it is diluted with various aqueous solutions or centrifuged so that the concentration of the lipid constituting the liposome calculated from the composition at the time of formulation is 1 to 300 mM. It is concentrated and used by the separation method. The aqueous solution used for dilution is not particularly limited as long as it is a commonly used aqueous solution, and examples thereof include a phosphate buffer solution, glucose, an aqueous saccharide solution such as trehalose, an aqueous salt solution such as sodium chloride, and physiological saline. When the final volume is 1 to 100 ml, it is usually administered by intravenous injection, and when the final volume is 100 to 1000 ml, it is administered by intravenous drip.

The storage stability of the liposome preparation of the present invention, that is, the physical stability of the liposome itself, and the chemical stability of the included drug and the lipid forming the liposome,
Since it may change depending on the lipids used, etc., generally stored in a cold place such as a refrigerator, or
As with ordinary injectable preparations that are dissolved before use, the literature (DDLasi
c, "Liposomes: from basic to applications", Else
See vier Science Publishers, pp. 529-532 (1993). It is preferable to freeze-dry by the method described in (1) and store in that state. In the case of a freeze-dried preparation, a desired injection preparation can be reconstituted by adding distilled water or the like at the time of use.

[0030]

The following examples and test examples illustrate the liposome preparation of the present invention in more detail. However, the liposome preparation of the present invention is not limited to these. In addition,
Among the lipids used in the following Examples and Test Examples, cholesterol was purchased from Tokyo Kasei, and all other lipids were purchased from NOF Corporation. Further, CNDAC hydrochloride was produced by the method described in JP-A-4-235182. (Example 1) CNDAC-containing liposome preparation Dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine; dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol; sphingomyelin; cholesterol; polyethylene glycol moiety has a molecular weight of about 2000. Using N-monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine (hereinafter referred to as PEG2000-DSPE); CNDAC hydrochloride, aqueous glucose solution and aqueous trehalose solution, Bangham et al. (J. Mol. Biol.
8, see 660-668 (1964). According to the method of (1), a crude dispersion of multilayer liposome was obtained.

That is, predetermined amounts of various lipids shown in Table 1 were added to a 25 mL eggplant-shaped flask, and a total lipid amount of 1 was added thereto.
After adding 5 mL of chloroform per 00 μmol,
The mixture was heated at 0 ° C. to dissolve the lipid. Chloroform was distilled off at 40 ° C. under reduced pressure of 10 to 550 mmHg using an evaporator to form a lipid thin layer on the inner wall of the eggplant-shaped flask. In the flask with this lipid thin layer,
Add an appropriate amount of an aqueous phase containing a predetermined amount of solute shown in Table 2,
The lipid per 1 mL of the aqueous phase was adjusted as shown in Table 1 and shaken using a vortex mixer to obtain a crude liposome dispersion. Hereinafter, the obtained liposome is referred to as CNDAC liposome.

In Tables 1 and 3 below, PC and PG are:
These mean phosphatidylcholines and phosphatidylglycerols, respectively.
(Stearoyl), P (palmitoyl), M (myristoyl), and L (lauroyl). When sphingomyelin was used instead of PC, SM was described in parentheses of the numbers.

[0033]

[Table 1] Formulation amount of lipid per 1 mL of aqueous phase of CNDAC liposome dispersion (μmol) ----------------------------- ------------------------------------- Formulation example PC cholesterol PG PEG2000-DSPE ----- -------------------------------------------------- ----------- 145.0 (P) 60.0 37.5 (P) 7.5 2 52.5 (P) 60.0 30.0 (P) 7.5 3 60.0 (P) 60.0 2.3 (P) 7.5 4 67.5 (P) 60.0 1.5 (P) 7.5 5 75.0 (P) 60.0 0.8 (P) 7.5 6 82.5 (P) 60.0 0 7.5 7 82.5 (S) 60.0 0 7.5 8 82.5 (M) 60.0 0 7.5 9 82 5.5 (L) 60.0 0 7.5 10 52.5 (P) 90.0 0 7.5 11 67.5 (P) 75.0 0 7.5 12 112.5 (P) 30.0 0 7.5 13 127.5 (P) 15.0 0 7.5 14 85.5 (P) 60.0 0 4.5 15 88.5 (P) 60.0 0 1.5 16 90.0 (P) 60.0 00 17 82.5 (P) 60.0 3.0 (P) 4.5 18 82.5 (P) 60.0 6.0 (P) 1.5 19 82.5 (P) 60.0 7.5 (P) 0 20 82.5 (P) 60.0 7.5 (S) 0 21 55.0 (P) 40.0 0 5.0 22 110. 0 (P) 80.0 0 10.0 23 137.5 (P) 100.0 0 12.5 24 165.0 (P) 120.0 0 15.0 25 82.5 (SM) 60.0 0 7.5 ----------------------------------------------- -------------------- Control 142.5 (P) 0 0 7.5 Prescription example ---------------- ---------------------------- -----------------------

[0034]

Table 2 Formulation amount of solute per 1 mL of aqueous phase of CNDAC liposome (mg) ------------------------------- ---------------------------------- Prescription example Trehalose CNDAC hydrochloride ---------- -------------------------------------------------- ----- 1 100.0 10.0 2 100.0 20.0 3 100.0 100.0 4 87.3 10.0 5 61.2 20.0 6 34.9 30.0 70 43 .3 8 0 100.0 9 183.5 30.0 10 0 200.0 ------------------------------- ---------------------------------- The volume average particle diameter of the liposome
In 8, 50 and 52, the operation of freezing the CNDAC liposome dispersion at −60 ° C. and thawing at 50 ° C. was repeated three times, and then the volume average particle diameter was adjusted by extrusion or ultrasonic irradiation. And in other manufacturing examples,
It was adjusted by the usual extrusion method or ultrasonic irradiation method.

In the extrusion method, a pore size of 1
Using an extruder (Liposofast-Basic, AVESTIN) equipped with a polycarbonate membrane filter (Nomura Microscience) of 00 to 200 nm,
The CNDAC liposome crude dispersion was adjusted by passing through the membrane (hereinafter, the passage of the crude liposome dispersion through the membrane using an extruder equipped with a polycarbonate membrane filter having a predetermined pore size was determined. Extrusion of size.).

In the ultrasonic irradiation method, an ultrasonic crusher (Model 7600, manufactured by Seiko Instruments Inc.) is used at a power of 25 W for 1 to 60 minutes using the CNDA.
Ultrasonic waves were applied to the crude dispersion of C liposome.

In Table 3, the volume average particle diameter of the liposome was adjusted by performing extrusion at 100 nm in Production Examples 14, 42, 51 and 52, and performing extrusion at 200 nm in Production Examples 13, 19 and 41. did. In other production examples, the volume average particle diameter of the liposome was adjusted by an ultrasonic irradiation method.

The obtained C having a controlled volume average particle diameter.
Each of the NDAC liposome dispersions was a homogeneous emulsion, and was stable at 25 ° C. for at least one day without any change in appearance.

The volume average particle diameter of the liposome after adjustment is
It was measured as follows. That is, after adjusting the volume average particle diameter (hereinafter referred to as Dv) of the liposome, the obtained CNDAC liposome dispersion is diluted with a 150 mM aqueous sodium chloride solution to adjust the lipid concentration to about 0.1 mM. (Nicomp Particle Sizer Model 37
0, manufactured by Nicomp Particle Sizing Systems).

[0040]

[Table 3] Volume average particle size of CNDAC liposome --------------------------------------- --------------------------- Production example Formulation of lipid Formulation of solute Dv (nm) ^* 1 ^* 2 (mean ± SD)- -------------------------------------------------- -------------- 1 11 124.9 ± 60.0 21 2 188.3 ± 87.9 3 13 195.8 ± 70.8 4 14 157.9 ± 67.4 5 15 158.5 ± 61.3 6 16 112.7 ± 49.2 7 17 77.9 ± 50.7 8 26 165.2 ± 79.6 9 36 159.2 ± 58.4 10 46 171.0 ± 73.3 11 5 6 146.5 ± 65.1 12 66 115.5 ± 46.0 13 6 6 156.5 ± 55.0 14 6 6 143.3 ± 48.0 15 67 106.1 ± 38.5 16 68 121.2 ± 47.6 176 9 118.0 ± 34.5 18 7 6 169.8 ± 57.7 19 7 6 274.1 ± 154.4 20 7 7 181.8 ± 80.5 21 7 8 183.1 ± 77.2 22 7 9 185.5 ± 77.8 23 8 6 153.9 ± 59.3 24 8 7 152.4 ± 61.2 25 8 8 150.2 ± 48.2 26 8 9 141.5 ± 49.0 279 6 103.1 ± 51.1 289 7 99.9 ± 50.0 299 8 97.7 ± 38.5 309 9 107.2 ± 55.0 31 106 150.1 ± 54.8 32 11 6 136.5 ± 54.3 33 12 6 137.6 ± 76.2 34 13 6 109.0 ± 47.9 35 146 150.8 ± 65.3 36 15 6 157.4 ± 65.6 37 16 6 142.8 ± 77.5 38 17 146.7 ± 49.5 39 186 148.5 ± 62.6 40 196 147.4 ± 72.3 41 196 321.8 ± 140.4 42 196 196.3 ± 48.0 43 20 6 75.6 ± 39.2 44 21 6 105.3 ± 51.8 45 22 6 91.3 ± 25.2 46 23 6 85.4 ± 38.8 47 246 124.2 ± 54.0 48 246 113.7 ± 37.2 49 24 7 121.8 ± 50.8 50 24 7 120.0 ± 50.8 51 24 7 195.0 ± 108.7 52 24 7 269.5 ± 139.9 53 256 104.5 ± 41.1 54 25 10 97.4 ± 41.1 --------------------------------- ---------------------------------- Control Control 665.1 ± 30.7 Production example Formulation example- -------------------------------------------------- --------------- ^* 1, represents the prescription example number in Table 1. ^* 2, represents the prescription example number in Table 2.

(Test Example 1) Determination of CNDAC inclusion rate in liposome The dispersion of the volume average particle size CNDAC liposome obtained in Example 1 was diluted with physiological saline to calculate from the formulation at the time of production. 0.5m lipid concentration in the dispersion
M. 100 μL of this dispersion was used as a sample for measuring the total CNDAC concentration contained in the dispersion. Also, 1 mL of this dispersion was subjected to ultracentrifugation (140000 g ×
20 minutes), 100 μL of the supernatant obtained by precipitating the liposome was used to remove the CNDAC concentration not contained in the liposome in the dispersion (hereinafter referred to as “CND in the external aqueous phase”).
AC concentration ". ) Was used as a sample for measurement.

Sample 10 for CNDAC concentration measurement
To 0 μL, 150 μL of distilled water, 250 μL of methanol and 250 μL of chloroform were added, and the mixture was shaken for 10 minutes using a vortex mixer, and centrifuged (1000 g).
× 5 minutes) to separate the system into two layers. The obtained upper layer was analyzed using reverse phase high performance liquid chromatography under the conditions shown in Table 4, and CNDA eluted in 5 minutes.
The concentration of C was measured.

[0043]

[Table 4] Measuring machine LC-10A (manufactured by Shimadzu Corporation) Column 5C18-AR (manufactured by Waters) Column temperature 40 ° C Detection wavelength 271 nm Mobile phase acetonitrile / distilled water / ion pair reagent ^* = 21/979/1 (volume ratio) ) Flow rate 1 mL / min injection volume 10 [mu] L ^* Pic B7 (Waters Co.) inclusion rate of CNDAC to liposomes, CNDAC weight which is included in the liposome (hereinafter, the total CNDAC weight.) and liposomes dispersion and a _L ( Hereinafter, this is defined as A _T ).

[0044]

[Number 1] inclusion rate _{(%) = (A L /} A T) × 100 (1) where, A _L is, CNDAC weight of the external aqueous phase from A _T (hereinafter referred to. As A _S) deduct It is required by things. Therefore, if the volume of the lipid in the liposome dispersion is ignored, the equations (2) and (3) hold.

[0045]

A _T = C _T × V (2)

[0046]

A _L = A _T -A _S = C _T × V C _S × V (1-v) (3) In Equations 2 and 3, C _T and C _S are measured by the above-described methods, respectively. Represents the total CNDAC concentration in the obtained liposome dispersion and the CNDAC concentration in the external phase, V represents the volume of the liposome dispersion, and v represents the aqueous phase (hereinafter referred to as the internal Represents the volume ratio (called the aqueous phase).

As described above, when the lipid concentration is 0.5 mM, the lipid concentration is 100 μg / mL or less,
The ratio of lipid volume to dispersion volume is virtually negligible. Further, v is proportional to the particle diameter and the lipid concentration, and the liposome of the present invention having a particle diameter of 100 nm to 400 nm has an internal aqueous phase volume of about 3 L to 15 L per mole of lipid (DDLasic, “ Liposomes:
from basic to applications, '' Elsevier Science Pub
lishers, pp. 106-107 (1993). V) When the lipid concentration is diluted to 0.5 mM, v becomes 0.01 or less. Therefore, (1-v) in equation (3) can be approximated to 1. That is, equation (3) can be approximated to equation (4).

[0048]

Equation 4] _{A L = C T × V-} C S × V = (C T -C S) × V (4) Equation (2) and (4) By substituting the equation (1), wherein (5) is obtained.

[0049]

Equation 5] inclusion rate _{(%) = (C T ×} V) / [(C T -C S) × V] × 100 = (C T -C S) / C T × 100 (5) Further, CNDAC formulation The ratio of the total amount of CNDAC in the liposome dispersion to the amount was determined as CNDAC recovery. In determining this recovery, the lipid volume was assumed to have a specific gravity of lipid of 1.0 (DDLasic, “Liposomes: from
basic to applications '', Elsevier Science Publish
ers, p.554 (1993). ), The volume of the liposome dispersion was determined by adding the aqueous phase volume and the lipid volume.

CND in liposome in liposome dispersion
The AC concentration is calculated by adding the CNDAC concentration in the liposome dispersion liquid calculated from the composition at the time of liposome production to the CNDA concentration.
It was determined by multiplying the recovery rate of C and the inclusion rate.

Table 5 shows the inclusion rate, recovery rate and CNDAC concentration in the liposome of the obtained liposome preparation.
Shown in As shown in Table 5, the inclusion rate of CNDAC is high and sufficient for practical use.

Further, the CNDAC concentration in the aqueous phase at the time of prescription was set to 100 or 200 mg / mL, and isotonic (43.3 m / mL).
g / mL), a stable liposome preparation could be produced even when an aqueous solution containing a high concentration of CNDAC exceeding (g / mL) was used as the aqueous phase.

In the liposome preparation of the present invention, when liposomes having different total lipid concentrations were produced with the same lipid composition ratio and aqueous phase composition, it was confirmed that the inclusion ratio increased as the total lipid concentration increased. Was. In addition, by performing a freeze-thaw operation of the CNDAC liposome crude dispersion,
Inclusion rate increased.

[0054]

[Table 5] CNDAC recovery rate in CNDAC liposome dispersion, inclusion rate in liposome, and CNDAC concentration in liposome ------------------------ ----------------------------------------- At the time of prescription (per 1 mL of aqueous phase) Size After adjustment ------------------------- ---------------- Total lipid content in liposome CNDAC content Recovery rate Coverage CNDAC concentration Production example (μmol) (mg) (%) (%) (mg / mL) ------------------------- ---------------------------------------- 2 150 20.0 97.6 36. 0 7.02 3 150 100.0 94.1 25.7 24.19 5 150 20.0 93.9 30.4 5.71 6 150 30.0 100.6 24.4 7.37 7 150 43. 3 99.2 20.2 8.68 8 150 30.0 85.9 31.8 8.20 9 150 3 0.0 91.6 33.1 9.10 10 150 30.0 92.8 32.8 9.13 11 150 30.0 89.6 33.3 8.95 12 150 30.0 95.7 22.2 6.38 13 150 30.0 81.3 31.4 7.66 14 150 30.0 78.2 29.8 6.99 15 150 43.3 104.9 20.7 9.40 16 150 100.0 95.9 21.1 20.23 17 150 30.0 105.1 25.0 7.89 18 150 30.0 96.9 41.0 11.92 19 150 30.0 86.1 39.6 10. 22 20 150 43.3 88.6 39.1 15.00 21 150 100.0 74.4 33.1 24.63 22 150 30.0 90.0 43.6 11.78 23 150 30. 0 92.9 33.5 9.34 24 150 43.3 103.3 38.8 17.35 25 150 100.0 101.5 31.6 32.08 26 150 30.0 96.3 27.1 7 .83 27 150 30.0 101.8 31.9 9.75 28 150 43.3 92.2 20.2 8.07 29 150 100.0 117.4 22.5 26.41 30 150 30.0 107 0.0 23.0 7.38 31 150 30.0 106.1 29.6 9.42 32 150 30.0 110.2 31.0 10.25 33 150 30.0 106.8 18.9 6.06 35 150 30.0 100.8 32.4 9.80 36 150 30.0 99.3 31.1 9.26 37 150 30.0 101.4 18.3 5.57 38 1 0 30.0 98.5 33.3 9.84 39 150 30.0 95.4 27.8 7.95 40 150 30.0 97.8 16.4 4.81 41 150 30.0 84.5 25 .2 6.39 42 150 30.0 95.0 20.1 5.73 43 150 30.0 89.1 15.7 4.20 44 100 30.0 79.5 17.5 4.17 45 200 30 0.0 84.6 28.1 7.13 46 250 30.0 71.4 38.3 8.20 47 300 30.0 73.7 38.8 8.58 48 300 30.0 84.5 42.9 10.88 49 300 43.3 79.7 40.0 13.80 50 300 43.3 71.3 45.4 14.02 51 300 43.3 72.6 40.4 12.70 52 300 43.3 6 0.3 59.0 17.45 53 150 30.0 83.1 21.5 5.36 54 150 200.0 76.1 17.0 25.84 -------------- -------------------------------------------------- -Control 150 30.0 94.6 3.3 0.94 Production Example -------------------------------- ---------------------------------- (Test Example 2) Dissolution test of CNDAC from liposome Liposomes in solution By examining the amount of CNDAC retained after a certain period of time, the CNDAC retention and release characteristics of the liposomes were examined as follows. 4.95 mL of a 150 mM sodium chloride aqueous solution was added to 50 μL of the dispersion liquid of the CNDAC liposome prepared in Example 1 and having an adjusted volume average particle diameter, followed by ultracentrifugation (1400
(00 g × 20 minutes) to precipitate liposomes. The supernatant was removed by decantation, and the phosphate buffer (20
mM sodium dihydrogen phosphate and 150 mM sodium chloride.
The liposome was redispersed in 4.95 mL of an aqueous solution adjusted to 7.4). Thus, CNDAC not included in the liposome was removed, 100 μL of the purified liposome dispersion was collected, and the initial concentration of all CNDAC in the dispersion (including the inside of the liposome) was determined by the method of Test Example 1. I asked. The remaining dispersion was shaken at 37 ° C., and 2 hours later, 100 μL of the suspension was taken, and the total CNDAC concentration in the dispersion (including the inside of the liposome) was measured. Further, the remaining dispersion (after shaking at 37 ° C. for 2 hours) was subjected to ultracentrifugation (140000 g × 20 minutes) to precipitate liposomes, thereby obtaining a supernatant. 100 μL of the supernatant was collected, and the concentration of CNDAC released from the liposome into the dispersion was measured. CNDA held in liposomes
The C concentration is the C release from the total CNDAC concentration into the dispersion.
It was determined by subtracting the NDAC concentration. As a control, PBS in which 60 μg / mL CNDAC was dissolved was used.
The residual ratio of CNDAC after shaking at 7 ° C. for 22 hours was measured.

The initial concentration of the total CNDAC thus determined was taken as 100, and the temperature was adjusted to 37 ° C. and 2 ° C. in a phosphate buffer.
The relative value of the total CNDAC concentration after shaking for 2 hours and the concentration of CNDAC retained in the liposome were calculated,
It is shown in Table 6. As shown in Table 6, the inclusion of CNDAC in the liposome increased the stability as compared to the case where it was present in an aqueous solution. That is, the aqueous phase is isotonic,
In addition, not only when the liposome is isotonic with respect to the liposome external solution but also when the CNDAC in the aqueous phase is as high as 100 mg / mL or when trehalose is added as a solute to make it hypertonic and osmotic pressure is generated, Liposomes are C
The NDAC can be kept stable, and the stability of the CNDAC itself can be increased.

[0056]

[Table 6] Percentage of CNDAC remaining after shaking liposomal CNDAC in PBS at 37 ° C for 22 hours ----------------------------------------- Manufacturing example PC Drug concentration in aqueous phase 22 Residual rate after time (mg / mL) (including in liposome) ----------------------------------- ------------------------------- 18 (S) 30.0 (isotonic with trehalose * ¹ ) 88 (84) 20 (S) 43.3 (isotonic) 90 (86) 22 (S) 30.0 (hypertonic with trehalose * ² ) 55 (46) 21 (S) 100.0 (hypertonic) 99 (86) 12 (P) 30.0 (with trehalose, etc.) Tonicity * ¹ ) 86 (77) 15 (P) 43.3 (isotonic) 76 (70) 17 (P) 30.0 (hypertonicity with trehalose * ² ) 67 (38) 16 (P) 100.0 (hypertonic) 104 (83) ) 23 (M) 30.0 (isotonic * ¹ trehalose) 85 71) 24 (M) 43.3 (isotonic) 74 (65) 25 (M ) 100.0 ( hypertonic) 76 (47) 27 (L ) 30.0 ( trehalose isotonic ^{* 1) 61 (45) 28} (L) 43.3 (Isotonic) 81 (62) 29 (L) 100.0 (high tension) 80 (45) ------------------------------ ------------------------------------ Aqueous solution 45 (-) --------- -------------------------------------------------- ------- * ¹ Isotonic by adding 3.49 mg / mL trehalose. * ² Hypertonic by adding 18.35 mg / mL trehalose to a solute concentration equal to 100 mg / mL CNDAC hydrochloride. (Test Example 3) Antitumor activity in repeated administration of various CNDAC liposome preparations A liposome preparation of CNDAC newly prepared in the same manner as the liposome preparation of CNDAC obtained in Example 1 (drug recovery of the preparations tested) And the volume average particle size are shown in Table 7).
In any of the liposome preparations, CNDAC not included in the liposome by the method shown in Test Example 2.
Was used after removal. However, the medium used for redispersing the liposomes was a 150 mM aqueous sodium chloride solution. The administration schedule was intermittently administered four times every three days. The antitumor activity was evaluated using the tumor growth inhibitory effect and the survival rate as scales.

[0057]

[Table 7] CNDAC preparation used in Test Example 3 -------------------------------------- ------------------------- Production example CNDAC concentration after purification * ¹ Volume average particle size (mg / mL) (nm) ---- -------------------------------------------------- --------- 12 7.47 99.5 ± 42.2 13 8.33 301.0 ± 182.9 18 6.00 113.9 ± 68.6 19 11.08 274.1 ± 154.4 23 4.45 82.4 ± 32.8 43 3.35 75.6 ± 39.2 aqueous solution * ² 22.50 -------------------- --------------------------------------------- * ¹ Purification or production It represents the concentration of the prepared formulation in the stock solution, and was appropriately diluted with a 150 mM aqueous sodium chloride solution in consideration of the drug dose and administered. * ² CNDAC was dissolved in physiological saline, and adjusted to pH 6.5 to 7.5 with an aqueous sodium hydroxide solution.

Mouse colon cancer colon 26 was implanted subcutaneously into 5- to 6-week-old female CDF1 mice to engraft and grow tumor tissues. On the seventh day after the transplantation, 6 animals per group were randomly divided into groups and administered for the first time. The intravenous administration volume of each preparation was 20 mL / kg. Similarly, the same formulation was administered on the 10th, 13th, and 16th days after transplantation in the same volume, and a total of four doses were administered.

The maximum tolerated dose (MTD) in this administration schedule is represented by the following formula: MTD (mg / kg) = [dose volume (mL / k)
g) x maximum administrable concentration (mg / mL)] / mouse weight (kg) (administration volume is 20 mL / k
g. ). However, the maximum administrable concentration is
On day 0, there was no death due to drug side effects, and the maximum drug concentration in the preparation was 20% or less on average on the 7th day of transplantation, when weight loss was most severe, on average.

On the 7th day of transplantation, ie, on the day of the first administration and on the 20th day, the major axis and minor axis of the tumor tissue were measured from the skin, and the tumor volume was calculated by the following formula: tumor volume (mm ³ ) = major axis (m
m) × minor axis ² (mm ² ) / 2. The relative tumor volume was calculated as a relative value of the tumor volume when the tumor volume on the day of the first administration was set to 1. The smaller this value is, the stronger the tumor growth inhibitory effect is. Here, the relative tumor volume on the 20th day of transplantation was calculated as a measure of the tumor growth inhibitory effect.

After the administration of the various preparations, the mice were bred and the survival days of each mouse were determined. The survival rate for each treatment group is:
It was calculated by (ab) / b × 100 (%). Here, a and b mean the median value of the number of surviving days in the treatment group and the no treatment group, respectively.

Table 8 shows the evaluation results of the antitumor activity near the MTD.

The liposome preparations of Production Examples 12, 13, 18, 19, 23, and 43 contained 9 mg / kg to 44 mg.
/ Kg, both showed a higher tumor growth inhibitory effect and a higher survival rate than the aqueous solution preparation, despite the lower dosage than the aqueous solution formulation (Table 8). For aqueous formulations, 3
Neither 00 mg / kg nor 450 mg / kg obtained a tumor growth inhibitory effect and a survival rate lower than those of the liposome preparation (Table 8).

From the above results, it was shown that the CNDAC liposome preparation of the present invention has an antitumor activity exceeding that of the aqueous preparation.

[0065]

[Table 8] Test results of antitumor activity ---------------------------------------- ------------------------ Production example (Dose) Relative tumor volume survival rate (%) on the 20th day of tumor implantation ----- -------------------------------------------------- --------- No treatment group 38.46 * 0 12 (29 mg / kg) 0.84> 136 (20 mg / kg) 2.09> 136 13 (20 mg / kg) 0.39> 126 ( (13 mg / kg) 2.40> 149 18 (20 mg / kg) 0.27> 149 (13 mg / kg) 1.64> 136 (9 mg / kg) 5.57 104 19 (20 mg / kg) 1.32 128 ( 13 mg / kg) 3.40> 123 23 (44 mg / kg) 0.78 91 (29 mg / kg) 1.46 115 (20 mg / kg) 4.04 77 43 (29 mg / k) g) 1.04 96 (20 mg / kg) 1.63> 149 aqueous solution (450 mg / kg) 5.66 70 (300 mg / kg) 22.38 51 --------------- ------------------------------------------------- * In the untreated group, two out of six mice died before the 20th day after tumor implantation, and the tumor volume was measured for the four surviving mice to calculate the relative tumor volume.

The liposome preparation of the present invention showed a high survival rate (%) at a low dose as compared with administration of an aqueous solution. (Test Example 4) Antitumor effect of single administration of CNDAC liposome preparation The antitumor activity of single administration of various preparations of CNDAC shown in Example 1 was examined. The antitumor activity was evaluated in the same manner as in Test Example 3, using the tumor growth inhibitory effect and the survival rate as scales.

[0067]

[Table 9] CNDAC preparation used in Test Example 4 -------------------------------------- ---------------------- Production example CNDAC concentration after purification * ¹ Volume average particle size (mg / mL) (nm) ------- -------------------------------------------------- --- 53 5.36 104.5 ± 41.1 54 25.84 97.4 ± 37.3 aqueous solution * ² 100.00 -------------------- ------------------------------------------ * ^{1 For} purified or manufactured preparations It represents the concentration in the stock solution, and was appropriately diluted with a 150 mM aqueous sodium chloride solution in consideration of the drug dose and administered. * ² CNDAC was dissolved in purified water and adjusted to pH 6.5 to 7.5 with NaOH for production.

A mouse colon cancer colon 26 was implanted subcutaneously into 5- to 6-week-old female CDF1 mice to engraft and grow tumor tissues. On the 7th day of transplantation, 6 animals per group were randomly divided into groups and administered. The intravenous administration volume of each preparation was 20 mL / kg.

The maximum tolerated dose in a single dose is 20% or less on average on the 7th day of transplantation on the 13th day of transplantation when there is no death due to the side effect of the drug and the most severe weight loss occurs. Maximum amount.

Even in a single administration, the relative tumor volume on the 20th day of transplantation relative to the 7th day of transplantation was calculated as a measure of the tumor growth inhibitory effect.

Table 10 shows the results of evaluating the antitumor activity of the MTD.

[0072]

[Table 10] ---------------------------------------------- --------------------- Manufacturing example Relative tumor volume (%) in survival rate (dose) on day 20 of tumor implantation -------- -------------------------------------------------- --------- No treatment group 10.10 * 053 (100 mg / kg) 0.15> 212 (50 mg / kg) 0.66> 231 54 (100 mg / kg) 0.43> 231 aqueous solution (2000mg / kg) 6.557 54 (1000mg / kg) 9.26 * 10 ------------------------------- -------------------------------------- * No treatment group and 1000mg / kg aqueous solution In the group, since 2 out of 6 mice died before the 20th day after tumor implantation, the tumor volume of the 4 surviving mice was measured to calculate the relative tumor volume.

Even in a single administration, the liposome preparations of Production Examples 53 and 54 are 50 mg / kg or 100 mg / kg.
In spite of the smaller dosages than the aqueous solution preparation, both showed a higher tumor growth inhibitory effect and a higher survival rate than the aqueous solution formulation. In the case of an aqueous solution preparation, 100
In each case of 0 mg / kg or 2000 mg / kg, only a tumor growth inhibitory effect and a survival rate lower than those of the liposome preparation were obtained. The liposome preparation showed a high survival rate even with only a single administration. (Test Example 5) Measurement of CNDAC concentration in tumor after administration of various preparations.

In order to examine the change in tumor accumulation and retention of CNDAC due to liposome formation, the concentration of CNDAC in the tumor after administration of various preparations of CNDAC was measured.

[0075]

[Table 11] CNDAC preparations examined for biodistribution --------------------------------------- ------------------------- Production example CNDAC concentration after purification * ¹ Volume average particle size (mg / mL) (nm) ---- -------------------------------------------------- ---------- 12 4.96 102.0 ± 65.9 Aqueous solution * ² 20.00----------------------- ------------------------------------------ * ¹ and * ² : Tables respectively See FIG.

In the same manner as in Test Example 3, 6-week-old CDF1
The mouse colon cancer colon 26 was implanted subcutaneously in the mouse, and 10 to 14 days later, the preparation shown in Table 11 was administered 150 m
It was appropriately diluted with an aqueous M sodium chloride solution and administered once intravenously. One hour or five hours after the administration, blood was collected. Thereafter, the mouse was exsanguinated, the tumor tissue and the kidney were removed, and the weight was measured. 0.5 to 3 mL of 1
50 mL of an aqueous sodium chloride solution was added, and the mixture was homogenized. From the CNDAC concentration in each homogenate measured in the same manner as in Test Example 1 and the CNDAC concentration in each sample (the CNDAC concentration in 1 g of tumor tissue was determined from the dilution calculated from the weight ratio of the biological sample to the added aqueous sodium chloride solution). Hydrochloride). Table 12 shows the results.

At a drug dose of 300 mg / kg, the liposome preparation and the aqueous preparation were compared at 5 hours after administration.
The AC concentration increased about 100 times. From this, CND
It has been shown that the use of AC as a liposome formulation imparts excellent targeting properties to tumor tissue.

Further, when the respective formulations were compared at doses close to the MTD at the time of intermittent administration evaluated for antitumor activity in Test Example 3, the liposome preparation (2
0 mg / kg), the drug concentration in the tumor was reduced to an aqueous solution (400 mg / k) despite the 1/20 dose.
g) higher. That is, by using a liposome preparation, the retentivity of CNDAC in the tumor could be significantly increased.

[0079]

[Table 12] Concentration in tumor after intravenous injection of CNDAC (μg / g tissue) --------------------------------- Production example 1 hour after administration 5 hours after administration (Dose) --- -------------------------------------------------- ------------ 12 (20 mg / kg) 10.76 5.81 (300 mg / kg) (not measured) 95.75 aqueous solution (300 mg / kg) (not measured) 0.93 ( 400mg / kg) 92.59 Not detected * --------------------------------------- -------------------------- * The detection limit is 0.08 μg / g tissue or less.

From these results, it was found that liposome formation
It was shown that the targeting of CNDAC to tumor tissue and the retention in tumor tissue were significantly improved.

[0081]

EFFECT OF THE INVENTION The liposome preparation of the present invention has high drug transferability to tumor tissues and high retention in tumor tissues, and as a result, is a low-toxicity preparation. Useful as an agent.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Kenji Hanaoka 1-2-58 Hiromachi, Shinagawa-ku, Tokyo Inside Sankyo Co., Ltd. (72) Inventor Shinichi Kurata 1-2-58 Hiromachi, Shinagawa-ku, Tokyo No. Within Sankyo Co., Ltd. Dormitory (C) -57-11

Claims (21)

[Claims] A liposome-constituting lipid comprising a sterol and a phosphatidylcholine having a phospholipid concentration of 30 to 300 mM and 1- (2'-cyano-2'-deoxy). -Β-D-arabino-pentofuranosyl) cytosine-containing liposome preparation. 2. The liposome preparation according to claim 1, wherein the phospholipid concentration is 50 to 200 mM. (3) one of lipids constituting a liposome,
3. The liposome preparation according to claim 1, wherein the sterol is cholesterol. 4. The liposome preparation according to claim 1, further comprising, as a lipid constituting the liposome, a lipid chemically modified with polyethylene glycols. 5. One of the lipids constituting the liposome,
The lipid chemically modified with polyethylene glycols is
-Monomethoxy polyethylene glycol succinyl phosphatidylethanolamines, N-monomethoxy polyethylene glycol (2-chloro-1,3,5-triazine-4,6-diyl) succinyl phosphatidyl ethanolamines, N-monomethoxy polyethylene glycol carbonyl phosphatidyl The liposome preparation according to claim 4, which is ethanolamines or N-monomethoxy polyethylene glycol ethylene phosphatidylethanolamines. 6. One of the lipids constituting the liposome,
The lipid chemically modified with polyethylene glycols is
The liposome preparation according to claim 4, wherein the preparation is a monomethoxy polyethylene glycol succinylphosphatidylethanolamine. 7. The liposome preparation according to claim 1, further comprising a phosphatidylglycerol as a lipid constituting the liposome. 8. The method according to claim 1, wherein the acyl group in the phosphatidylcholines and / or phosphatidylglycerols, which are lipids constituting the liposome, is an aliphatic acyl group having 10 to 20 carbon atoms. 8. The liposome preparation according to 7 above. 9. The method according to claim 1, wherein the acyl group in phosphatidylcholines and / or phosphatidylglycerols, which are lipids constituting the liposome, is a myristoyl group, palmitoyl group or stearoyl group.
9. The liposome preparation according to any one of items 1 to 8. 10. The liposome according to claim 1, wherein the composition ratio of the sterols, which are lipids constituting the liposome, to the total lipid amount constituting the liposome is 10 mol% to 60 mol%. Formulation. 11. The liposome according to claim 1, wherein the composition ratio of sterols, which are lipids constituting the liposome, to the total lipid amount constituting the liposome is 30 mol% to 50 mol%. Formulation. 12. The composition ratio of N-monomethoxy polyethylene glycol succinyl phosphatidylethanolamines, which are lipids constituting liposomes, to the total amount of lipids constituting liposomes is 1 mol% to 10 mol%.
7. The liposome preparation according to claim 5, wherein 13. The liposome according to claim 1, wherein the composition ratio of the phosphatidylcholine, which is a lipid constituting the liposome, to the total lipid amount constituting the liposome is 35 mol% to 85 mol%. Formulation. 14. The liposome according to claim 1, wherein the composition ratio of the phosphatidylcholine, which is a lipid constituting the liposome, to the total lipid amount constituting the liposome is 40 mol% to 60 mol%. Formulation. 15. The composition according to claim 7, wherein the composition ratio of phosphatidylglycerols, which are lipids constituting the liposome, to the total lipid amount constituting the liposome is 1 mol% to 10 mol%. Liposomal preparation. 16. The lipid constituting the liposome comprises (1) sterols, (2) phosphatidylcholines, and
(3) The liposome preparation according to any one of claims 1 to 2, which is a monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine. 17. The composition ratio of (1) sterols is 10 mol% to 60 mol% and (2) phosphatidyl choline is 35 mol% to 85 mol% with respect to the total lipid amount constituting the liposome. Yes,
(3) The liposome preparation according to any one of claims 1 to 2, wherein the composition ratio of monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine is 1 mol% to 10 mol%. 18. The composition wherein (1) the composition ratio of sterols is 30 mol% to 50 mol% and (2) the composition ratio of phosphatidyl cholines is 40 mol% to 60 mol% with respect to the total lipid amount constituting the liposome. Yes,
(3) The liposome preparation according to any one of claims 1 to 2, wherein the composition ratio of monomethoxy polyethylene glycol succinyl-distearoyl phosphatidylethanolamine is 1 mol% to 10 mol%. 19. The lipid constituting the liposome is (1) a sterol, (2) a phosphatidylcholine, or (3) a phosphatidylglycerol.
The liposome preparation according to claim 1. 20. The composition wherein (1) the composition ratio of sterols is 30 mol% to 50 mol% and (2) the composition ratio of phosphatidylcholines is 40 mol% to 60 mol% with respect to the total lipid amount constituting the liposome. Yes,
(3) The composition ratio of phosphatidylglycerols is 1 m
The liposome preparation according to any one of claims 1 to 2, wherein the liposome preparation is contained in an amount of from 10 mol% to 10 mol%. 21. The liposome having a volume average particle size of 100
2. The method of claim 1, wherein
21. The liposome preparation according to any one of items 20 to 20.