WO2000048611A1 - Liposome preparations containing antitumor drug - Google Patents

Abstract

Liposome preparations containing 1-(2'-cyano-2'-deoxy-β-D-arabino-pentofranosyl)cytosine which are excellent in the accumulation in a tumor tissue and the retention therein and thus exert a favorable antitumor activity.

Description

 TECHNICAL FIELD The present invention relates to a novel 11- (2,1-cyano1-2,1-deoxy-1) 3-D-arabinontofuranosyl) cytosine-containing liposome preparation. Kagegi Cup

1— (2,1-cyano 2′-doxy-1-D-arabinopentofuranosyl) cytosine (hereinafter referred to as CNDAC) is a compound having excellent antitumor activity (Tanaka et al., Cancer Letter 64, 67- 74 (1992) / Azuma et al., J. Med. Chem. 36, 4183-4189 (1993), Japanese Patent Application Laid-Open No. 4-235182) .Because CNDAC is water-soluble, it can be used in physiological saline and the like. By dissolving, it can be administered intravenously. However, in general, water-soluble antitumor drugs are more likely to be degraded in vivo, are excreted faster from the body, and have a non-specific tissue distribution in the body, so that they have better antitumor activity. Formulations are generally desired in order to obtain and reduce side effects.

 By the way, by improving the drug transferability of an antitumor drug to a tumor tissue and the retention property in the tumor tissue, a superior antitumor activity is obtained, and the antitumor drug is used for the purpose of reducing power, strength, and side effects. Pharmaceutical innovations for converting drugs into ribosome preparations are generally performed.

Already, Asai et al. Have attempted to entrap CNDAC in ribosomes.However, the amount of CNDAC incorporated into ribosomes is small, and CND AC incorporated into ribosomes is also relatively quickly discharged. CNDAC has concluded that it is an unsuitable antitumor drug for ribosome preparations (see Drug Delivery System 13, 341-346 (1998)). DISCLOSURE OF THE INVENTION The present inventors have conducted intensive studies on the lipids constituting ribosomes and their composition ratios regarding the ribosome preparations of CNDAC, and as a result, they contain specific lipids. When contained in a ribosome preparation, it was discovered that CNDAC, which had been previously unsuitable for use as a ribosome preparation, can be made into a ribosome preparation that can be used practically. It has been found that the drug has high drug transferability and high retentivity in tumor tissues, and as a result, has low toxicity, thereby completing the present invention. The present invention provides a 1- (2, -cyano 2'-doxy-1] 3-D-arabino-l-pentfuranosyl) cytosine-containing ribosome preparation which solves the above-mentioned problems. The present invention

1> a phospholipid concentration of 30 to 30 O mM and containing sterols and phosphatidylcholines as lipids constituting liposomes; 3-D-arabinopentofuranosyl) ribosome preparation containing cytosine,

 <2> the liposome preparation according to <1>, wherein the phospholipid concentration is 50 to 20 O mM, and 3) one of the lipids constituting the ribosome, the sterols being cholesterol. The liposome preparation according to <1> or <2>,

<4> The ribosome preparation according to <1> to <3>, further comprising, as a lipid constituting the ribosome, a lipid chemically modified with polyethylene glycols.

<5> One of the liposome-forming lipids, lipids chemically modified with polyethylene glycols, is composed of N-monomethoxy polyethylene glycol succinylphosphatidylethanolamines, N-monomethoxy polyethylene glycol (2-chloro Mouth 1,3,5-triazine-4,6-diyl) Succinylphosphatidyletano Liposomes as described in <4>, characterized in that they are luamines, N-monomethoxy polyethylene glycol carbonyl phosphatidyl ethanolamines or N-mono methoxy polyethylene glycol ethylene phosphatidyl ethanolamines. Formulation,

 <6> A lipid chemically modified with polyethylene glycol, which is one of the lipids constituting the ribosome, is an N-monomethoxypolyethylene glycol succinylphosphatidyl ethanolamine. 4> The ribosome preparation according to any one of <1> to <6>, further comprising: a phosphatidylglycerol as a lipid constituting the liposome.

<8> The liposome-constituting lipid, phosphatidylcholine and Z or phosphatidylglycerol, wherein the acyl group is an aliphatic acyl group having 10 to 20 carbon atoms. The ribosome preparation according to 7>,

<9> The liposome-forming lipid, phosphatidylcholine and Z or the acyl group in phosphatidylglycerol is a myristoyl group, palmitoyl group, or stearoyl group. <10> The liposome preparation according to <8>, wherein the composition ratio of sterols, which are lipids constituting the ribosome, to the total amount of lipids constituting the ribosome is 1 Omo 1% to 6 Omo 1%. The ribosome preparation according to <1> to <9>,

 <11> The composition ratio of sterols, which are lipids constituting the ribosome, to the total lipid amount constituting the ribosome is 3 Omo 1% to 5 Omo 1%, <1> to < Ribosome preparation according to 10>,

 <12> The composition ratio of N-monomethoxypolyethyleneglycol succinylphosphatidylethanolamines, which are liposome-forming lipids, to the total amount of ribosome-forming lipids should be 1 mo 1% to 1 Omo 1%. The ribosome preparation according to 5> to 6>, characterized in that:

<13> The composition ratio of phosphatidylcholines, which are lipids constituting ribosomes, to the total amount of lipids constituting ribosomes is 35 mo 1% to 85 mo 1%, and 1> to <12. Ribosome preparation according to> <14> The composition ratio of phosphatidylcholines, which are lipids constituting ribosomes, to the total amount of lipids constituting ribosomes is 4 Omo 1% to 60 mo 1%, <1> to <13> Ribosome preparation according to,

 <15> The composition ratio of phosphatidylglycerols, which are lipids constituting ribosomes, to the total amount of lipids constituting liposomes is lmo 1% to 1 Omo 1%. Ribosome preparation according to>

<16> The lipids that make up the ribosome

 (1) sterols,

 (2) phosphatidylcholines, and

 (3) The liposome preparation according to (1) or (2), wherein the liposome preparation is characterized by being monomethoxypolyethylene glycol succinirugestearoylphosphatidyleethanolanolamines.

 <1 7> With respect to the total lipid amount constituting the ribosome,

 (1) The composition ratio of sterols is 10 mol 1% to 60 mol 1%,

 (2) the composition ratio of the phosphatidylcholines is 35 mol 1% to 85 mol 1%,

(3) The composition ratio of monomethoxypolyethylene glycol succiniruyl stearoyl phosphatidyl ethanolamines is 1 mo 1% to 10 mo 1%. The ribosome preparation described,

18> With respect to the total lipid content of the ribosome,

 (1) The composition ratio of sterols is 30 mol 1% to 50 mol 1%,

 (2) the composition ratio of the phosphatidylcholines is 40 mol 1% to 60 mol 1%,

(3) The composition ratio of monomethoxypolyethylene glycol succinate-lugestearoyl phosphatidylethanolamine is 1 mo 1% to 10 mo 1%, and is described in <1> to <2>. Ribosome preparations,

19> The lipids that make up the ribosome

 (1) sterols,

 (2) phosphatidylcholines,

(3) phosphatidylglycerols, described in <1> or <2> Ribosome preparation,

 <20> With respect to the total lipid content of the ribosome,

 (1) The composition ratio of sterols is 30 mol 1% to 50 mol 1%,

 (2) the composition ratio of the phosphatidylcholines is 40 mol 1% to 60 mol 1%,

(3) The liposome preparation according to (1) to (2), wherein the composition ratio of the phosphatidylglycerols is 1% to 1% Omo.

<21> The ribosome preparation according to <1> to <20>, wherein the ribosome has a volume average particle diameter of 100 nm to 400 nm. In the present invention, the term "ribosome" refers to a liposome, which is well known to those skilled in the art (see DDLasic; "shiposomes: from basic to applications", Elsevier Science Publishers pp. 1-171 (1993)). A closed vesicle composed of lipids and an internal aqueous phase. In the ribosome of the present invention, the surface thereof and polyethylene dalicols are bound by non-covalent bonds such as electrostatic interaction or hydrophobic interaction, or chemically modified with polyethylene glycols described below. May be contained as a lipid constituting the ribosome. The phospholipid concentration of the ribosome preparation of the present invention is usually 30 to 30 mM, preferably 50 to 200 mM. “Sterols”, which are essential lipid components constituting the ribosome preparation of the present invention, include, for example, cholesterol, cholesterol monohemisuccinate, 3 / 3- [N- (N ,, N′-dimethinoleamino) Ethane) canoleno moinole] cholesterol, enolegosterone, lanosterol and the like, preferably cholesterol, and sterols are preferably based on the total amount of lipids constituting the ribosome. 10 to 6 Omol%, more preferably 30 to 50 mol%, is contained in the ribosome. The `` phosphatidylcholines '' which are essential lipid components constituting the liposome preparation of the present invention include, for example, dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine. Preferably, dimyristoyl phosphatidylcholine, dipamitoyl ^^ phosphatidinolecholine or distearoylenophosphatidylcholine is preferred.Phosphatidylcholines are used with respect to the total amount of lipids constituting ribosomes. Preferably, the ribosome contains 35 to 85 mo 1%, more preferably 40 to 6 O mo 1%. The ribosome preparation of the present invention preferably contains "phosphatidylglycerols" or "lipids chemically modified with polyethylenedaricols". Examples of the above-mentioned "phosphatidylglycerols" include dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoisolephosphatidinoregglycerol, and disteareinolephosphatidinoregglycerol. Preferable are dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol and distear yl phosphatidyl glycerol. Contains 1 to 10 mol% in the ribosome. The above-mentioned “lipid chemically modified with polyethylene glycols” refers to lipids that are covalently bonded to polyethylene dalicols having various molecular weights and lipids, and the lipid is preferably phosphatidylethanolamine. And, for example, the general formula

0 0

CH ₃ 0—— (CH ₂ CH ₂ 0) -C- -CH ₂ CH _P -C—— NH— PE

n (Wherein, n represents 10 to 100, and PE_NH represents phosphatidylamine.) N-monomethoxypolyethyleneglycolsuccinylphosphatidylethanolamines represented by the following formula:

General formula

CH ₃ 0— (CH ₂ CH ₂ 0) N, .NH—— PE

 n

N, N

CI

(Wherein, n represents 10 to 100, and PE—NH represents phosphatidylamine.) N-monomethoxypolyethylene glycol (2_1,3-, 5-triazine) _ 4,6-diyl) Succilphosphatidylethanolamines, general formula

0

CH ₃ 0— (CH ₂ CH ₂ 0)-■ NH— PE

 n

(In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamin.) N-monomethoxypolyethylene glycol carbonyl phosphatidylethanolamines represented by the following formula:

General formula

CH ₃ 0— (CH CH ₂ 0) CH ₂ CH ₂ NH— PE

 n-1

(In the formula, n represents 10 to 100, and PE-NH represents phosphatidylamine.) Monomonomethoxy polyethylene glycol ethylene phosphatidylethanolamines represented by N, more preferably And N-monomethoxypolyethylene glycol ethylene phosphatidylethanolamines. The molecular weight of the lenglycol moiety is from about 500 to about 500, and most preferably the molecular weight of the polyethylene glycol moiety is from about 100 to about 300, most preferably preferably, the molecular weight of the moiety is from about 2 0 0 0 (DD Lasic, "Liposomes:. from basic to appl icationsj , Elsevier Science Publishers, pp 294- 29o (1993)) 0 to ribosomes formulations of the present invention , The above “sterols”, “phosphatidylcholines”,

In addition to “phosphatidylglycerols” and “lipids chemically modified with polyethylene glycols”, they may contain lipids that can be used in ribosome preparations. Examples include dilauroylphosphatidylinositol and dimyristoyl Phosphatidylinositols such as phosphatidyluinositol, dipalmitoyl phosphatidyltidylinositol or distearoyl phosphatidylinositol; dilauroyl phosphatidylserine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine or distearoylphosphatidylphosphatidyl Apatidylserines: dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolanolamine, dipalmitoylphosphatidylethanol Glycerophospholipids such as phosphatidylluetanolamines such as nolamine or distearoylphosphatidylethanolamine: digalactosyl diradiroyl glyceride, digalactosyl dimyristoyl glyceride, digalactosyl dipalmitoyl glyceride, digalactosyl distearoyl glyceride Glycose glycolipids such as digalactosyl diglycerides such as glyceride; galactosyl diglycerides such as galactosyl dilauroyl glyceride, galactosyl dimyristoyl glyceride, galactosyl dipalmitoyl glyceride, and galactosyl distearyl glyceride: Sphingophospholipids such as ceramidosyriatin and sphingomyelin: Glycosphingolipids such as celeb mouth side and ganglioside: Sterols of: Long-chain alkylamines such as tetradecylamine, hexadecylamine, and stearylamine: long-chain fatty acid hydrazides such as myristic hydrazide, palmitic hydrazide, and stearic hydrazide: N— [1— (2 , 3-dioleyloxy) propinole] _N, N, N-trimethylammonium ammonium chloride, N-a-trimethylammonioacetinoreside decinole Positively charged lipids such as D-glutamate chloride can be exemplified. In the above “lipids”, phosphatidylcholines, phosphatidylglycerols, phosphatidylinositols, phosphatidylserines, phosphatidylethanolamines, sulfoxyribosyldiglycerides, digalactosyldiglycerides, galactosyldiglyceride, sulphamine The mouth side, gandarioside, etc. each have two saturated or unsaturated aliphatic acyl chains, and the chain preferably has 14 to 18 carbon atoms (particularly myristoyl, palmi A tyl or stearoyl group). The ribosome of the ribosome preparation of the present invention preferably has a volume average particle size of 100 to 400 nm, and the volume average particle size can be determined based on a principle such as a dynamic light scattering method (DD Lasic ^). “See iposomes: from basic to applicationsj, Elsevier Science Publishers, pp. 1-171 (1993).” The ribosome preparation of the present invention can be produced according to methods well known to those skilled in the art. In other words, the liposomes can be adjusted by the above-mentioned “lipid” and “aqueous phase” by thin-film method, reverse-phase evaporation method, ethanol injection method, ether injection method, dehydration-rehydration method, etc. It can be manufactured, and the volume average particle diameter can be adjusted by a method such as an ultrasonic irradiation method, an ultrasonic irradiation method after freeze-thawing, an extrusion method, a French press method, and a homogenization method. DD Lasic,

See "Shi lposomes: from basic to applications", Elsevier Science Publishers ^ PP. 1-171 (1993). ). Here, the “aqueous phase” means an aqueous solution constituting the inside of the ribosome, and is not particularly limited as long as it is a commonly used one. Examples thereof include an aqueous sodium chloride solution, a phosphate buffer, and an acetate buffer. , A glucose aqueous solution, a trehalose aqueous solution and the like, and a mixed aqueous solution thereof. In general, to maintain the structure of ribosomes administered in vivo stably, The aqueous phase used in the production is desirably close to isotonic with the extraribosomal solution, that is, body fluid, and has a small osmotic pressure applied to the inside and outside of the ribosome. As the above-mentioned “lipid” used for producing the ribosome preparation of the present invention, a commercially available product and a product that can be easily chemically synthesized from a commercially available product by an ordinary method can be used. Generally, lipids having various carbon numbers and Z or unsaturation, such as lecithin and soybean lecithin, which can be obtained in a mixed state, can be used without separating and purifying them into a single component. . In the ribosome preparation of the present invention, if necessary, a-tocophere or the like can be added to the liposome preparation for the purpose of antioxidant action and the like. When the ribosome preparation of the present invention is administered to humans, the liposome is diluted with various aqueous solutions so that the concentration of lipids constituting the liposome calculated from the composition at the time of formulation is 1 to 30 O mM, or And used by concentrating by centrifugation. The aqueous solution used for dilution is not particularly limited as long as it is a commonly used aqueous solution. Examples thereof include a phosphate buffer solution, an aqueous saccharide solution such as dalcose and trehalose, an aqueous salt solution such as sodium chloride, and physiological saline. When the final liquid volume is 1 to 10 Om1, usually by intravenous injection, and when the final liquid volume is 10 to 100 Oml, by intravenous drip. Administer. The storage stability of the ribosome preparation of the present invention, that is, the physical stability of the ribosome itself and the chemical stability of the included drug and the lipid forming the ribosome may vary depending on the lipid used and the like. , Or stored in a cold place such as a refrigerator, or in the same manner as ordinary injectable preparations for use at the time of use, such as literature (DD Lasic, “l lsomesomes: rrom basic to applications”, Elsevier Science Pub 丄 ishe: rs, pp. 529-532 (1993)), and freeze-dried according to the method described in pp. 529-532 (1993). In the case of freeze-dried preparations, add the desired injection The spray formulation can be reconstituted. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, Examples and Test Examples will be shown to describe the liposomal preparation of the present invention in more detail. However, the ribosome preparation of the present invention is not limited to these. Among the lipids used in the following Examples and Test Examples, cholesterol was purchased from Tokyo Kasei, and all other lipids were purchased from Yomoto Yushi Co., Ltd. Further, CNDAC hydrochloride was produced by the method described in JP-A-Hei 4-235182.

(Example 1) CND AC-containing ribosome preparation

 Dilauroinolephosphatidinorecholine, dimyristoy / lephosphatidinorecholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine; dipalmitoylphosphatidylglycerol, distearylylphosphatidyltidylglycerol, sphingomyelin; Cholesterol monophosphate; N-monomethoxypolyethylene glycol succinyl distearoylphosphatidylethanolamine (hereinafter referred to as PEG2000-DSPE) having a molecular weight of polyethylene glycol of about 2000; CNDAC hydrochloride, glucose Using an aqueous solution and an aqueous trehalose solution, a crude dispersion of multilayer liposomes was obtained according to the method of Bangham et al. (See J. Mol. Biol. 8, 660-668 (1964)).

That is, the prescribed amounts of various lipids shown in Table 1 were added to a 25-mL eggplant-shaped flask, and 5 mL of clonal form per 100 / zmol of total lipid was added thereto. Warm up to dissolve lipids. Using a evaporator under reduced pressure of 10 to 55 OmmHg at 40 ° C., the chloroform was distilled off to form a thin layer of lipid on the inner wall of the eggplant-shaped flask. An appropriate amount of an aqueous phase containing a predetermined amount of solute shown in Table 2 is added to the flask in which the lipid thin layer has been formed, and the lipid per 1 mL of the aqueous phase is adjusted as shown in Table 1, and vortex mixer is used. By shaking, a crude ribosome dispersion was obtained. Hereinafter, the obtained liposome is referred to as CNDAC ribosome. In Tables 1 and 3 below, PC and PG mean phosphatidylcholines and phosphatidylglycerols, respectively. In the parentheses of the numbers, the chain length of the acyl is S (stearoyl), P (palmitoyl), M (mi). Ristoyl) and L (Lauroyl). When sphingomyelin was used instead of PC, SM was described in parentheses of the numbers.

(table 1)

 Formulation amount of lipid per 1 mL of aqueous phase of CNDAC ribosome dispersion (μπιο 1) Formulation example PC Cholesterol PG PEG2000-DSPE

1 45.0 (P) 60.0 37.5 (P) 7.5

2 52.5 (P) 60.0 3 0.0.0 (P) 7.5

3 60. 0 (P) 60. 0 2.3 (P) 7.5

4 67.5 (P) 60.0 0.1 (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 (L) 60.0 0 0 7.5

10 52.5 (P) 90.0 0 7.5

1 1 67.5 (P) 75.0 0 7.5

1 2 1 12.5 (P) 30.0 0 7.5

1 3 127.5 (P) 1 5.0 0 7.5

14 85.5 (P) 60.0 0 0 4.5

1 5 88.5 (P) 60.0 0 0 1.5

16 90.0 (P) 60.0 0 0 1 7 82.5 (P) 60.0 0.3 (P) 4.5

1 8 82.5 (P) 60.0 6.0 (P) 1.5

1 9 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 1 10.0 (P) 80.0 0 1 0.0

23 1 37.5 (P) 100. 0 0 1 2.5

24 1 65. 0 (P) 1 20.0 0 0 15.0

25 82.5 (SM) 60.0 0 7.5 Control 1 42.5 (P) 0 0 7.5 Prescription example

(Table 2)

 Formulation amount of solute per mL of aqueous phase of CNDAC ribosome (mg) Formulation example Torenodulose CNDAC hydrochloride

00 0 10. 0

2 00 0 20. 0 3 00 0 00. 0 4 87 3 10.0 5 61 2 20 0 6 34 9 30 0 7 0 43 3 8 0 00 0 9 83 5 30.0 10 0 200. 0

In the following Production Examples 48, 50 and 52, the volume average particle diameter of the ribosome was particularly determined by repeating the operation of freezing the crude dispersion of CNDAC ribosome at 160 ° C and thawing at 50 ° C three times. The volume average particle diameter was adjusted by an extrusion method or an ultrasonic irradiation method, and in other production examples, the volume average particle diameter was adjusted by a normal extrusion method or an ultrasonic irradiation method.

 In the extrusion method, a crude CND AC ribosome was prepared using an Extorda (Liposofast-Basic, AVESTIN) equipped with a polycarbonate membrane filter (Nomura Microscience) with a pore size of 100 to 200 nm. The dispersion was adjusted by passing through the membrane. (Hereinafter, passing the crude ribosome dispersion through the membrane using an extruder equipped with a polycarbonate membrane filter having a predetermined pore size is referred to as a predetermined size. Extrusion).

 In addition, in the ultrasonic irradiation method, an ultrasonic crusher (Model 7600, manufactured by Seiko Instruments Inc.) was used to apply ultrasonic waves to the CNDAC liposome coarse dispersion at an output of 25 W for 1 to 60 minutes. Irradiated.

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

 Each of the obtained CNDAC ribosome dispersions whose volume average particle size was adjusted was a uniform emulsion, and was stable at 25 ° C for at least one day with no above change. .

The adjusted ribosome volume average particle diameter was measured as follows. That is, after adjusting the ribosome volume average particle diameter (hereinafter referred to as Dv), the obtained CND AC ribosome dispersion is diluted with a 150 mM aqueous sodium chloride solution to adjust the lipid concentration to about 0.1 mM, Particle size analyzer (Nicomp Particle Sizer Model 370, Nicomp Use the Particle Sizing Systems), was measured Dv ₍

(Table 3)

 Volume average particle size of CNDAC ribosome Production example Formulation of lipid Formulation of solute D V nm

 * 2 (average soil SD)

1 1 24. 9 technicians 60. 0

2 2 188. 3rd person 87. 9 3 3 1 95. 8 Sat. 70. 8 4 4 1 57. 9 Sat. 67. 4 5 5 1 58. 5 Sat. 61. 3 6 6 1 12. 7 Sat. 49. 2 7 1 7 107.9 Sat 50.7 8 2 6 165.2 Sat 79.6 9 3 6 1 59.2 Sat 58.4 10 4 6 1 71.0 Sat 73, 3 1 1 5 6 146.5 Sat 65 1 1 2 6 6 1 1 5.5 Sat 46, 0 1 3 6 6 1 56.5 Sat 55.0 14 6 6 143.3 Sat 48 0 1 5 6 7 106. 1 person 38 5 16 6 8 1 21. 2 persons 47, 6 1 7 6 9 1 1 8.0 Saturday 34 5 1 8 7 6 169. 8 persons 57 7 1 9 7 6 274.1 ± 1 54 4 20 7 7 1 81.8 persons 80 5 6 + LLL 9 ^ Z 8 ΐ ⁷

 o

 o * o o c "p O y ヮ

 6 9

6 * o f -r

 + o Ό y 6 6 9 V o

 o + n y L G

 6 b b y U ヮ

 -y 6 C v

U V + Dagger L Z V

V U T 1- D 4 o L o b y D L I V

• ヮ

GL + VAL y 6 I 0 y + Ot ⁷ L 9 8 I 6 ε bv + L 9 X 9 LI 8 ε

LL + «6 t ⁷ L9 9 IL ε

9 9 + t ⁷ 9 SI 9 ε b In y + o UL y VL s ε b L

 V b U L y L ε f 6 • o y L t n y ヮ

 6 L c ε L L 6 ε o F D Ty

 + L L b o o 6 6 Ζ

0 (J + b b fa L 6 8 Ζ + L b U L 9 6 L に

0 b V + b L L b 8 9 Ζ

6 o + o 0 ia L 8 8 96

Z 'I 9 + "S S I L 8 Ζ ε '69 + 6ss x 9 8 ε 2

8 * Z L + S 'S 8 X 6 L τ ζ z' L L + I • ε 8 I 8 L I S

9ΐ

o / oodm d X 1981 ^ / 00 OAV 49 24 7 1 21.8 person 50.8

 50 24 7 1 20.0 Sat 50.8

51 24 7 195.0 ± 108.7

52 24 7 269.5 5 ± 139.9

53 25 6 104.5Sat 41.1

54 25 0 97.4 Sat 41.1 Control Control 6 65.

* Indicates the prescription example number in Table 1.

* 2, Indicates the prescription example number in Table 2.

(Test Example 1) Determination of CND AC inclusion rate in ribosome

 The dispersion of the volume average particle size CND AC ribosome obtained in Example 1 was diluted with physiological saline, so that the lipid concentration in the dispersion calculated from the formulation at the time of production was 0.5 mM. 100 μL of this dispersion was used as a sample for measuring the total CND AC concentration contained in the dispersion.

In addition, 100 ml of the supernatant obtained by precipitating the liposome by ultracentrifugation of 1 mL of this dispersion (140,000 g x 20 minutes) does not contain ribosomes in the dispersion. The sample was used to measure the CNDAC concentration (hereinafter, referred to as “CNDAC concentration in the external water phase”).

To 100 μL of the above sample for CNDAC concentration measurement, add 150 μL of distilled water, 250 μL of methanol, and 250 μL of black-mouthed form, shake with a vortex mixer for 10 minutes, and centrifuge (1000 g x 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 the concentration of CNDAC eluted in 5 minutes was measured. (Table 4)

 Measuring machine LC-1 OA (manufactured by Shimadzu Corporation)

 Column 5C 18—AR (Waters)

 Column temperature 40 ° C

 Detection wavelength 271 nm

 Mobile phase acetonitrile Z distilled water / ion pair reagent *

 = 21/979/1 (volume ratio)

 Flow rate lmLZ min

 Injection volume I O L

* P ic B 7 (W aters Co.) inclusion rate of CNDAC to ribosomes, CNDAC weight which is included in Liposomes one arm (hereinafter referred to as A _L.) And the total CNDAC weight ribosomal dispersion (hereinafter, Alpha _τ ), and is defined by equation (1).

(Equation 1)

Inclusion rate _{(%) = (A L /} A T) X 100 (1) where, A _L is, CNDAC weight of the external aqueous phase from Alpha _tau (hereinafter referred to. As A _s) determined by subtracting plug the Can be Therefore, if the volume of lipid in the ribosome dispersion is ignored, equations (2) and (3) hold.

(Equation 2)

 (2)

(Equation 3)

= C _T X VC _S XV (1— v) (3) In Formula 2 and Formula 3, C _T and C _s, respectively represent the CND AC concentration in total CND AC concentration and the external phase of the liposome dispersion as measured by the method described above, V is, liposome dispersions V represents the ratio of the volume of the aqueous phase (hereinafter referred to as the internal aqueous phase) constituting the ribosome to the volume of the liposome dispersion. As described above, when the lipid concentration is set to 0.5 mM, the lipid concentration is 100 / gZmL or less, and the ratio of the volume of the lipid to the volume of the dispersion is practically negligible. Further, V is proportional to the particle size and the lipid concentration, and the liposome of the present invention having a particle size of 100 nm to 400 nm is 3 L to 15 L per mole of lipid. (See DD Lasic, “Liposomes: irom oasic to applications”, Elsevier Science Publishers, pp. 106-107 (1993)), and a lipid concentration of 0.5 mM. When diluted up to V, V is less than 0.01, so (1 V) in equation (3) can be approximated to 1. That is, equation (3) can be approximated to equation (4) .

(Equation 4)

A, = C _T X V- C, XV

= (C _T -C _S ) XV (4)

 By substituting equations (2) and (4) into equation (1), equation (5) is obtained.

(Equation 5)

Coverage (%) = (C _T XV) / [(C _T -C _S ) XV] XI 00

= (C _T -C _S ) / CX 100 (5) The ratio of the total amount of CNDAC in the ribosome dispersion to the prescribed amount of CNDAC was determined as CNDAC recovery. When calculating the recovery rate, the lipid volume is determined by temporarily setting the lipid ratio to 1.0 (see DD Lasic _N, “Liposomes: from basic to applications, Elsevier Science Publishers, p.554 (1993). ), The volume of the ribosome dispersion is It was determined by adding the aqueous phase volume and the lipid volume.

 The CNDAC concentration in the ribosome in the liposome dispersion was determined by multiplying the CNDAC concentration in the ribosome dispersion, which was calculated from the composition at the time of liposome production, by the CNDAC recovery rate and the inclusion rate.

 Inclusion rate and recovery rate of CNDAC in the obtained ribosome preparation and CN in ribosome

Table 5 shows the DAC concentration. As shown in Table 5, the coverage of CNDAC is high and sufficient for practical use.

 In addition, even when the aqueous phase containing CNDAC in the aqueous phase at the time of prescription is 100 or 200 mg ZmL and a high concentration of CND AC exceeding isotonicity (43.3 mg / mL) is used as the aqueous phase, it is stable. A new ribosome preparation could be produced. In the ribosome preparation of the present invention, when ribosomes 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. In addition, the inclusion rate was increased by adding a freeze-thaw operation to the CNDAC ribosome crude dispersion.

(Table 5)

 CNDAC recovery rate in CNDAC ribosome dispersion, ribosome inclusion rate, and ribosome CNDAC concentration At the time of prescription (per 1 mL of aqueous phase) After size adjustment

 Total amount of lipid in ribosome CNDAC amount Recovery rate Coverage CNDAC concentration

Production example (μπιο ΐ) (mg) (%) (%) (mg / mL)

2 50 20.0 97.6 36.0 7.02

3 50 100.0 94.1 25.7 24.19 9 Z '0 X 0 i ε ζ "0 XT 0 • ο ε 0 9 I ζ ε ヮ b 9 τ

 D O ι 9 0 I 0 • ο ε 0 S I τ ε

〇

o & L U • ヮ υ L ϋ I 0 ο ε 0 9 I ο ε

L v 9 G 6 6 V X 0 '0 0 \ 0 S I 6 ζ

L 0 8 6 0 6 6 • Z 6 ε 'ε 0 9 X 8 Ζ

9 L 6 6 ■ x ε 8 'T 0 I 0' ο ε 0 S I L Ζ ε 8 • L I 'z ε "9 6 0' ο ε ο e I 9

8 0 'z ε 9 • i ε s' I 0 I 0 ■ 0 0 I 0 S X S ζ s ε * L X 8' 8 ε ε ■ ε o T ε ■ ε 0 9 I ζ

V £ • 6 S ■ ε ε 6 "Z 6 0 'ο ε 0 S I ε ζ

8 L 'T T 9 ■ ε Ρ 0 "0 6 0' ο ε 0 9 I ζ ζ ε 9 * z I 'ε ε' L 0 '0 0 I 0 S X I ζ

0 0 ■ 9 I X '6 ε 9' 88 8 ε ■ ε 0 S I 0 ζ z z '0 I 9 * 6 ε I' 98 0 'ο ε 0 S I 6 I

Z 6 'T I 0 ■ X 6' 9 60 * ο ε 0 9 I 8 I

6 8 • L 0 '9 ζ ΐ "0 T 0 ■ ο ε 0 9 X L I ε z' 0 Z I 'I s 6" S 6 0' 0 0 I 0 S τ 9

0 • 6 'ο ζ 6 * 0 I ε * ε 0 9 I S I

6 6 • 9 8 '6 ζ Ζ' 8 L 0 'ο ε 0 S I ι

9 9 • L V 'I ε ε' T 8 0 * 0 G 0 9 I ε I

8 ε '9 Z' ζ ζ L '9 6 0 ■ ο ε 0 9 I ζ ι

9 6 "8 ε 'ε ε 9' 6 8 0 ■ ο ε 0 9 I I I ε X * 68 * ζ ε 8” Z 6 0 ■ ο ε 0 9 I 0 I

0 I • 6 I • ε ε 9 "I 6 0 * ο ε 0 9 I 6

0 z • 8 8 ε 6 'Q 8 0' ο ε 0 9 τ 8

8 9 • 8 Z '0 Ζ Ζ' 6 6 ε 'ε 0 Q I L

L ε 'L 9' 0 0 I 0 'ο ε 0 9 I 9

IL • Q 'ο ε 6 ■ ε 6 0' ο ζ 0 SIS df / XDd 1198 囊 OAV 33 1 50 30. 0 106.8 18.9 6.06

35 1 50 30. 0 1 00. 8 32. 4 9.80

 36 1 50 30. 0 99.3 31.1 9.26

37 1 50 30. 0 1 01.4 18.3 5.57

38 1 50 30. 0 98.5 33.3 9.84

 39 1 50 30. 0 95.4 27.8 7.95

40 1 50 30. 0 97.8 16.4 4.81

41 1 50 30. 0 84.5 25.2 6.39

42 1 50 30. 0 95. 0 20. 1 5. 73

43 1 50 30.0 89.1 15.7 4.20

 44 100 30. 0 79.5 1 7.5 4. 1 7

45 200 30.0 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 7 l. 3 45. 4 14.02

51 300 43.3 72.6 40.4 1 2.70

52 300 43.3 68.3 59.0 1 7.45

53 1 50 30. 0 83.1 21.5 5.36

54 1 50 200.0 76.1 17.0 0 25.84 Control 1 50 30.0 94.6 3.3 0.94

(Test Example 2) Elution test of CNDAC from ribosome

To investigate the retention of ribosome CNDAC in solution after a certain time Thus, the CNDAC retention and release properties of the ribosome were examined as follows. 4.50 mL of a 15 OmM aqueous solution of sodium chloride was added to 50 // L of the CNDAC liposome dispersion prepared in Example 1 and having a controlled volume average particle diameter, followed by ultracentrifugation (l 400 000). g X 20 min) to precipitate the ribosome. The supernatant was removed by decantation, and the phosphate buffer (20 mM sodium dihydrogen phosphate and 150 mM sodium chloride was dissolved, and the pH was adjusted to 7.4 with a 1 M aqueous sodium hydroxide solution. The prepared ribosome was redispersed in 95 mL. In this way, CNDAC that was not included in the liposome was removed, 100 μL of the purified ribosome dispersion was taken out, and the initial concentration of all CNDACs in the dispersion (including in the ribosome) was determined. It was determined by the method in Test Example 1. The remaining dispersion was shaken at 37 ° C. Two hours later, 100 μL of the suspension was collected and the total CNDAC concentration in the dispersion (including in the ribosome) was measured. The remaining dispersion (after shaking at 37 ° C. for 2 hours) was subjected to ultracentrifugation (140,000 g × 20 minutes) to precipitate ribosomes, thereby obtaining a supernatant. 100 μL of the supernatant was collected, and the concentration of CNDAC released from the ribosome into the dispersion was measured. The concentration of CNDAC retained in the liposome was determined by subtracting the concentration of CNDAC released into the dispersion from the total CNDAC concentration. As a control, the residual rate of CNDAC after shaking PBS in which 60 μg ZmL of CNDAC was dissolved at 37 ° C. for 22 hours was measured. Assuming the initial concentration of all CNDAC thus measured as 100, the total CNDAC concentration after shaking at 37 ° C for 22 hours in phosphate buffer and the concentration of CNDAC retained in the ribosome The relative values of were calculated and are shown in Table 6. As shown in Table 6, the stability of CN DAC was enhanced by being included in the ribosome, as compared with the case where it was present in an aqueous solution. That is, not only when the aqueous phase is isotonic and isotonic to the ribosome extracellular solution but also when the CNDAC in the aqueous phase is as high as 10 Omg / mL, the trehalose solution is used as a solute. Even in the case where osmolarity is caused by the hypertonicity caused by the addition, the ribosome can stably retain CNDAC and increase the stability of CNDAC itself. (Table 6)

 Ribosomal CNDAC C Percentage after shaking CNDAC in PBS at 37 ° C for 22 hours Preparation Example PC C Percentage of drug concentration in aqueous phase after 22 hours

 (mg / ZmL) (including liposome)

18 (S) 30.0 (Isolated with Trueno ヽ -S * 88 (84)

 20 (S) 43.3 (isotonic) 90 (86)

22 (S) 30.0 (Service extension with trenoperos * ² ) 55 (46)

 21 (S) 100.0 (Takahashi) 99 (86)

 12 (P) 30.0

 15 (P) 43.3 76 (70)

1 7 (P) 30.0 (hypertonic with trenoperose * ² ) 67 (38)

 16 (P) 100.0 (Takahashi) 104 (83)

 23 (M) 30.0 (isotonic with trenoperose * 85 (71)

 24 (M) 43.3 (isotonic) 74 (65)

 25 (M) 100.0 (Takahashi) 76 (47)

 27 (L) 30.0 (isotonic with trenoperose * 61 (45)

 28 (L) 43.3 81 (62)

 29 (L) 100.0 (Hotonic) 80 (45) Aqueous solution 45 (I)

* ^! 3. Isotonicized by adding 49 mg ZmL of trehalose.

* ² 1. 8. Hypertonic by adding 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 newly prepared CNDAC ribosome preparation similar to the CNDAC ribosome preparation obtained in Example 1 (drug recovery of the tested preparations) The ratio and volume average particle size are shown in Table 7.) In any of the ribosome preparations, CNDAC not included in the liposome was removed by the method described in Test Example 2 before use. However, the medium used for ribosome redispersion was a 15 OmM sodium chloride aqueous solution. The dosing schedule was intermittent four doses every three days. Antitumor activity was evaluated using tumor growth inhibitory effect and survival rate as scales.

(Table 7)

C NDAC formulation used in Test Example 3 Production example Purified CNDAC concentration Volume average particle size

 (mg / mL) (nm)

1 2 7 47 99 5 Sat 42 2

 13 8 33 301 0 ± 1 82 9

 18 6 00 1 13 9 Sat 68 6

 19 1 1 08 274 1 ± 1 54 4

 23 4 45 82 4 Sat 32 8

43 3 35 75 6 Sat 39 2

Aqueous solution * ² 22.50 This represents the concentration of the purified or manufactured preparation in the stock solution, and was appropriately diluted and administered with a 150 mM aqueous sodium chloride solution in consideration of the drug dose.

* ² Manufactured by dissolving CND AC in physiological saline and adjusting the pH to 6.5 to 7.5 with an aqueous sodium hydroxide solution. Mouse colon cancer co 1 on 26 was implanted subcutaneously into 5- to 6-week-old female CDF 1 mice to engraft and grow tumor tissue. On the seventh day after transplantation, 6 animals per group were randomly divided into groups and administered the first dose. The intravenous administration volume of each formulation was 2 OmL / kg. Similarly, on days 10, 13, and 16 of transplantation, the same formulation was administered in the same volume, and a total of four doses were administered. The maximum tolerated dose (MTD) for this dosing schedule is given by the following formula: MTD (mgZkg) = [dose volume (mL / kg) X maximum available dose (mgZmL)] (dose volume is 2 OmLZkg). However, the maximum dose that can be administered is as follows: on the 20th day of transplantation, there were no deaths due to drug side effects, and the weight loss rate on the 7th day of transplantation, when weight loss was most severe, was 20 on average. % Was defined as the maximum drug concentration in the preparation that was not more than 10%. On the 7th day of transplantation, that is, 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 above the skin, and the tumor volume was calculated by the following formula: fl Injured volume (mm ³ ) = major axis (mm) Calculated from X minor axis ² (mm ² ) 2. The relative tumor volume was calculated as a relative value of the tumor volume with the tumor volume on the day of the first administration taken as 1. The smaller this value is, the stronger the tumor growth inhibitory effect is. Here, the relative tumor volume on day 20 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 of each treatment group was calculated by the following equation: (a_b) / bX100 (%). Here, a and b mean the median value of the number of days alive in the treated group and the untreated group, respectively.

Table 8 shows the results of evaluating the antitumor activity near the MTD. The ribosome preparations of Production Examples 12, 13, 18, 18, 23, and 43 are 9 mgZ kg to 44 mg, kg, which are higher than aqueous preparations, even though they are all smaller than aqueous preparations. It showed a tumor growth inhibitory effect and a high survival rate (Table 8). In the case of the aqueous solution preparation, neither 30 Omg / kg nor 45 Omg / kg provided a tumor growth inhibitory effect and a survival rate lower than those of the ribosome preparation (Table 8). From the above results, it was shown that the ribosome preparation of CNDAC of the present invention has an antitumor activity exceeding that of the aqueous preparation.

(Table 8)

 Test results for antitumor activity Production example On day 20 of tumor implantation

 (Dose) Relative tumor volume Survival rate (%) Untreated group 38. 46 * 0

 1 2

 (29mg / kg) 0.84> 136

 (2 Omg / kg) 2.09> 1 36

 13

 (2 Omg / kg) 0.39> 1 26

 (13 mg / kg) 2.40> 149

 18

 (2 Omg / kg) 0.27> 149

 (13 mg / kg) 1.64> 136

 (9mg / kg) 5.57 104

 1 9

(2 Omg / kg) 32 1 28 (1 3mg / kg) 3.40> 1 23

twenty three

 (44 mg / kg) 0.78 91

 (29mgZk g) 1.46 1 1 5

 (2 Omg / kg) 4.04 77

43

 (29mg / kg) 1.04 96

 (2 OmgZk g) 1.63> 149 Aqueous solution

 (45 Omg / kg) 5.66 70

 (30 OmgZk g) 22.38 51

* In the untreated group, two out of six mice died before the 20th day after tumor implantation, so the tumor volume was measured and the relative tumor volume was calculated for the four surviving mice. The ribosome preparation of the present invention showed a higher survival rate (%) at a lower dose than administration in an aqueous solution.

(Test Example 4) Antitumor effect of single administration of CNDAC ribosome preparation The antitumor activity of each of the 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.

(Table 9)

C NDAC formulation used in Test Example 4 Production example Purified CNDAC concentration ^ Volume average particle size

(m gZmL) (nm) 53 5.36 04.5 ± 41. 1 54 25.84 97.4 ± 37.3

Aqueous solution * ² LO O. 00

* 'Represents the concentration of the purified or manufactured drug product in the stock solution. The drug was appropriately diluted with a 150 mM aqueous sodium chloride solution in consideration of the drug dose and administered.

* ² CND AC was dissolved in purified water and adjusted to pH 6.5 to 7.5 with NaOH for production.

Mouse colon cancer co1on26 was implanted subcutaneously into 5- to 6-week-old female CDF1 mice to engraft and grow tumor tissue. On the 7th day of transplantation, 6 animals per group were randomly divided into groups and administered. The intravenous administration volume of each formulation was 20 mLZkg. The maximum tolerated dose in a single dose is the highest when no fatalities occur due to the side effects of the drug, and the average weight loss rate is less than 20% on the 7th day of transplantation on the 13th day of transplantation, when weight loss is most severe. Made a large amount. Even in a single dose, 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 in MTD.

(Table 10) Production example Survival rate on day 20 of tumor implantation

(Dose) relative tumor volume (%) No treatment group 10.10 * 0

53

 (10 Omg / kg) 0.1 5〉 212

 (5 Omg / kg) 0.66> 231

54

 (10 Omg / kg) 0.43> 231

Aqueous solution

 (200 Omg / kg) 6.57 54

 (100 Omg / kg) 9.26 * 10

* In the untreated group and in the group treated with the aqueous solution of 100 Omg / kg, two out of six mice died before the 20th day after tumor implantation, and the tumor volume was measured for the surviving 4 mice, and the relative tumor volume was measured. Was calculated.

 Even in a single dose, the ribosome preparations of Production Examples 53 and 54 showed a high tumor growth inhibitory effect, surpassing that of the aqueous solution, despite the smaller dose of 5 Omg / kg or 10 Omg / kg. And a high survival rate. In addition, in the case of the aqueous solution preparation, at 100 Omg / kg or 200 Omg / kg, the tumor growth inhibitory effect and the survival rate were both lower than those of the ribosome preparation. The liposomal 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 changes in tumor accumulation and retention of CNDAC due to ribosome formation, CND AC concentrations in tumors after administration of various CND AC preparations were measured.

(Table 11)

CND AC formulation whose distribution in the body was examined Production example Purified CNDAC concentration Volume average particle size (mg / mL) (nm)

1 2 4.9 6 1 0 2.0 0 65.9

Aqueous solution * ² 20.00

* i and * ² : See Table 7 respectively. In the same manner as in Test Example 3, mouse colon cancer co1on26 was implanted subcutaneously in 6-week-old CDF1 mice, and 10 to 14 days later, the preparations shown in Table 11 were treated with 150 mM It was appropriately diluted with an aqueous sodium chloride solution and administered once intravenously. One hour or five hours after the administration, blood was collected. Thereafter, the mice were exsanguinated, the tumor tissue and the kidney were removed, and the weight was measured. To these biological samples, 0.5 to 3 mL of 15 OmL aqueous sodium chloride solution was added and subjected to homogenization. From the CNDAC concentration in each homogenate measured in the same manner as in Test Example 1 and the dilution ratio calculated from the weight ratio of the biological sample to the added sodium chloride aqueous solution, the CNDAC concentration in each sample (1 g of tumor tissue The CNDAC concentration was used as the hydrochloride salt). Table 12 shows the results. Compared to the ribosome and aqueous solution at a drug dose of 300 mg / kg, the liposomal formulation increased CNDAC concentration in J3 severe ulcer by about 100-fold at 5 hours after administration. did. This indicates that using CND AC as a ribosome preparation imparts excellent targeting properties to tumor tissues. In addition, comparing the respective formulations at doses close to the MTD at the time of intermittent administration evaluated for antitumor activity in Test Example 3, the ribosome-formulated product (2 Omg / kg) 5 hours after administration, Despite the dose of 1Z20, the drug concentration in the tumor was higher than that of the aqueous solution (400 mgZkg). That is, by using a ribosome preparation, the retention of CNDAC in tumors could be significantly increased. (Table 1 2)

 Tumor concentration (iv / g tissue) after intravenous injection of CNDAC Preparation Example 1 hour after administration 5 hours after administration

 (Dose)

1 2

 (2 Omg / kg) 10.76 5.81

 (30 Omg / kg) (not measured) 95. 75

Aqueous solution

 (30 Omg / kg) (not measured) 0.93

 (40 Omg / kg) 92.59 Not detected *

* Detection limit is below 0.08 / gZg tissue. These results indicated that ribosome formation significantly improved targeting of CNDAC to tumor tissue and retention in tumor tissue. Possibility of industrial use CNDAC, which contains certain lipids and is particularly unsuitable for use as a ribosome formulation, especially when liposomes are composed of a certain composition ratio of these lipids The resulting ribosome preparation could be obtained.

 Further, the ribosome preparation has high drug transferability to tumor tissues and high retention in tumor tissues, and as a result, is a low-toxicity preparation, and thus is useful as an antitumor agent. is there.

Claims

The scope of the claims 1. A phospholipid concentration of 30 to 30 OmM, which contains sterols and phosphatidylcholines as lipids constituting ribosomes. 1-D-arabinopentofuranosyl) A cytosine-containing ribosome preparation. 2. The ribosome preparation according to claim 1, wherein the phospholipid concentration is 50 to 20 O mM. 3. The liposome preparation according to claim 1, wherein the sterol, which is one of lipids constituting the ribosome, is cholesterol. 4. The ribosome preparation according to any one of claims 1 to 3, further comprising, as a lipid constituting the ribosome, a lipid chemically modified with polyethylene glycols. 5. Lipids that are chemically modified with polyethylene glycols, one of the lipids that make up the ribosome, are N-monomethoxypolyethylene glycol succinylphosphatidylethanolamines and N-monomethoxypolyethylene glycol (2-chloroethylene). 1,3,5-triazine-1,4,6-diinole) succininolephosphatidinolethanolamines, N-monomethoxypolyethylene glycol carbonyl phosphatidylethanolamines or N-monomethoxypolyethylene glycol ethylene phosphatidyl / 5. The liposome preparation according to claim 4, which is a leetano-lamine. 6. The lipid chemically modified with polyethylene glycol, which is one of the lipids constituting the ribosome, is N-monomethoxy polyethylene glycol succinylphosphatidyl ethanolamines. Ribosome according to 4 Formulation. 7. The liposome preparation according to any one of claims 1 to 6, further comprising a phosphatidylglycerol as a lipid constituting the ribosome. 8. The acyl group in phosphatidylcholines and / or phosphatidylglycerols, which are lipids constituting ribosomes, is an aliphatic acyl group having 10 to 20 carbon atoms. 8. The liposome preparation according to 7. 9. The liposome-constituting lipid, phosphatidylcholine and Z or the acyl group in phosphatidylglycerol, is a myristoyl group, palmitoyl group or stearoyl group, according to claim 1 to 8, wherein Liposomal preparations. 10. The composition according to claim 1, wherein the composition ratio of sterols, which are lipids constituting ribosomes, to the total amount of lipids constituting ribosomes is 1 Omo 1% to 6 Omo 1%. The ribosome preparation according to the above. 1 1. The composition ratio of sterols, which are lipids constituting ribosomes, to 3 Omo 1% to 5 Omo 1%, based on the total amount of lipids constituting ribosomes. 8. The ribosome preparation according to item 1. 1 2. is a lipid constituting the ribosome N- Monome butoxy polyethylene Da Ricoh Rusakushi - Le phosphatidyl E pentanol § Min acids, the composition ratio to the total amount of lipid constituting the liposome, l mo 1% to 1 0 111 ₀ 1% The liposome preparation according to any one of claims 5 to 6, characterized in that: 1 3. The composition ratio of phosphatidylcholines, which are liposome-forming lipids, to the total amount of ribosome-forming lipids should be between 35 mo1% and 85 mo1%. The ribosome preparation according to any one of claims 1 to 12, characterized in that: 14. The composition ratio of phosphatidylcholines, which are lipids constituting liposomes, to the total amount of lipids constituting ribosomes is 4 Omo 1% to 6 Omo 1%, wherein the composition ratio is 1 to 13%. 8. The ribosome preparation according to item 1. 1 5. The composition ratio of phosphatidylglycerols, which are lipids constituting liposomes, to lmo 1% to 1 Omo 1% with respect to the total amount of lipids constituting liposomes. 10. The ribosome preparation according to 9. 16. The lipids that make up the ribosome  (1) sterols,  (2) phosphatidylcholines, and  (3) The liposome preparation according to any one of claims 1 to 2, characterized in that the liposome preparation is a monomethoxypolyethylene glycol succinyl monodistearoylphosphatidyl ethanolamine. 1 7. Based on the total lipid content of the ribosome,  (1) The composition ratio of sterols is 10 mol 1% to 60 mol 1%,  (2) The composition ratio of phosphatidylcholines is 35 mol% to 85 mol%. /. And (3) The composition ratio of monomethoxypolyethyleneglycolsuccinyl-distearoylphosphatidyl ethanolamines is 1 mo 1% to 10 mo 1%, according to claim 1 or 2, characterized in that: Ribosome preparations. 18. With respect to the total lipid content of the ribosome,  (1) The composition ratio of sterols is 30 mol 1% to 50 mol 1%,  (2) The composition ratio of phosphatidylcholines is from 40 mol% to 60 mol 1. /. And · (3) Monomethoxypolyethylene glycosole The ribosome preparation according to any one of claims 1 to 2, wherein the composition ratio of the tizyletanolamines is 1 mol% to 10 mol%. 1 9. The lipids that make up liposomes  (1) sterols,  (2) phosphatidylcholines,  (3) The liposomal preparation according to any one of claims 1 to 2, which is a phosphatidylglycerol. 20. For the total amount of lipids that make up the ribosome,  (1) The composition ratio of the sterols is 30 mol 1% to 50 mol 1%,  (2) The composition ratio of the phosphatidylcholines is 40 mo 1% to 60 mo 1%, (3) The liposome preparation according to any one of claims 1 to 2, wherein the composition ratio of the phosphatidylglycerols is 1% to 1% Omo. 21. The ribosome preparation according to any one of claims 1 to 20, wherein the liposome has a volume average particle diameter of 100 nm to 400 nm.