JP2786482B2 - Lipid membrane structure - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a compound of the formula (I) (In the formula, R ₁ fatty acid residues of hydrogen or C1-30, R ₂
Represents an acyclic hydrocarbon residue having 1 to 30 carbon atoms, R ₃ represents an amino group, an amidino group or a guanidino group, and n represents an integer of 1 to 6. However, when R ₁ is a fatty acid residue and R ₂ is an acyclic hydrocarbon residue, the sum of the carbon numbers of R ₁ and R ₂ is 10 to 40. )) Or a lipid membrane structure containing the compound or a salt thereof.

<Industrial application fields> The lipid membrane structure of the present invention has specific directivity to tumor cells and the like, and is medically useful.

<Prior Art> As a study on liposomes having specific directivity to tumor cells, a method of modifying the surface of liposomes using a monoclonal antibody has been reported at present.

For example, a review of Tadakuma [Medical Journal, 20 , 643 (19
84); Cell Engineering, 1 , 72 (1982), Osawa et al. [Chemical and Pharmaceutical Bulletin, 35 , 740 (1987)], Papahadjopoulos et al. [Cancer Research 46 , 4904 (84); 1986)]. Among them, according to the method of Osawa et al., It has been shown that small unilamellar liposomes whose liposole surface is modified with anti-carcinoembryonic antigen antibodies are easily taken up by cancer cells, and the antitumor effect is enhanced. .

However, mass production of these monoclonal antibodies is difficult, and commercialization is difficult.

<Problems to be Solved by the Invention> The present inventors have intensively studied a lipid membrane structure having specific directivity to cancer cells efficiently and capable of mass production with good reproducibility. Completed the invention.

<Constitution of the Invention> The present invention relates to a lipid membrane structure containing a compound of the formula (I) or a salt thereof.

The lipid acid residue in the compound of the formula (I) is a group formed by removing a hydroxyl group from a saturated or unsaturated fatty acid which may have a branch, and examples thereof include formyl, acetyl, propanoyl, Butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, decanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl, eicosanoyl, henicosanoyl, thocosanoyl, tricosanoyl, tricosanoyl, tricosanoyl, tricosanoyl, tricosanoyl Noyl, 9-hexadecenoyl, 9-octadecenoyl, 9,12-octadecadienoyl, 9,12,15-octadecatrienoyl, 11-eicosenoyl, 11,14-eicosadienoyl, 11,14,17-eico Satrieno Le, 4, 8, 12, 16
−eicosatetraenoyl, 13-docosenoyl, 4,8,12,1
5,19-docosapentaenoyl, 15-tetracosenoyl, 2
-Dodecyl hexadecanoyl, 2-tetradecyl hexadecanoyl, 2-dodecyl tetradecanoyl, 2-tetradecenyl hexadecenoyl, 2-tetradecyl hexadecenoyl, 2-tetradecenyl hexadecanoyl Etc. 1 to carbon number
Thirty, preferably 14 to 20, can be mentioned. The acyclic hydrocarbon residue in the formula (I) is a group formed by removing one hydrogen from a saturated or unsaturated acyclic hydrocarbon which may be branched, and Examples include methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, henicosyl, docosyl, tricosyl, tetracosyl, hexacosyl. , Triacontyl, 9-
Hexadecenyl, 9-octadecenyl, 9,12-octadecadienyl, 9,12,15-octadecatrienyl, 11-eicosenyl, 11,14-eicosadienyl, 11,14,17-eicosatrienyl, 4,8, 1216-eicosatetraenyl, 13-docosenyl, 4,8,12,15,19-docosapentaenyl, 15-tetracosenyl, 2-dodecyltetradecyl, 2-dodecylhexadecyl, 2-tetradecylhexadecyl, 2- Examples thereof include those having 1 to 30 carbon atoms such as tetradecyl hexadecenyl, 2-tetradecenyl hexadecyl, and 2-dodecyl octadecyl, and preferably those having 14 to 20 carbon atoms.

Furthermore, as for n in the formula (I), 4 or 3 can be mentioned as preferable.

For compounds of formula (I), R ₁ is a fatty acid residue and R ₁
Compounds in which ₂ is an acyclic hydrocarbon residue are preferred, in which case the sum of the carbon numbers of R ₁ and R ₂ is preferably 10 to 40.

The compounds of formula (I) have optical isomers, and the isomers and mixtures thereof are also included in the scope of the present invention.

The lipid membrane structure of the present invention means particles having a membrane structure in which polar groups of polar lipids are arranged toward the aqueous phase side of the interface, and examples thereof include liposomes, micelles, and microemulsions.

Next, a method for producing a lipid membrane structure containing the compound of the formula (I) or a salt thereof of the present invention will be described.

a) Method for producing liposome containing compound of formula (I) or a salt thereof Known methods using phospholipids such as phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, glycolipids and membrane-forming lipids such as dialkyl type synthetic surfactants. Method [Annual Review of Biophysics and Bioengineering 9 , 467 (19
80)] to prepare an aqueous dispersion of liposomes. Such a liposome may contain in its membrane sterols such as cholesterol and cholestanol, dialkyl phosphate, diacylphosphatidic acid, stearylamine, α-tocopherol and the like. An aqueous solution of the compound of formula (I) or a salt thereof is added to the prepared aqueous dispersion of liposomes, and the mixture is allowed to stand for a certain period of time, preferably heated to a temperature equal to or higher than the phase transition temperature of the membrane or equal to or higher than 40 ° C. A liposome containing the compound of the formula (I) or a salt thereof can be produced. Alternatively, the desired liposome can be produced by previously mixing the membrane-forming lipid with the compound of the formula (I) or a salt thereof and treating the mixture according to a known liposome production method.

b) Method for producing micelle containing compound of formula (I) or a salt thereof Polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil derivative, polyoxyethylene hydrogenated castor oil derivative, fatty acid sodium, sodium bile acid, polyoxyethylene A micelle-forming interfacial substance, such as a fatty acid ester or polyoxyethylene alkyl ether, is added to water at a micelle-forming critical concentration or higher to prepare an aqueous dispersion of micelles. An aqueous solution of the compound of the formula (I) or a salt thereof is added to the prepared aqueous dispersion of micelles, left for a certain period of time, preferably heated to 40 ° C. or higher, and then left to cool, whereby the desired compound of the formula (I) Micelles containing the compound or a salt thereof can be produced. Alternatively, the desired micelle can be produced by previously mixing the micelle-forming substance and the compound of the formula (I) or a salt thereof, and then treating the mixture according to a known micelle production method.

c) Method for producing microemulsion containing compound of formula (I) or a salt thereof A fat or oil such as soybean oil is added to the micelle produced according to b) to saturate the inside of the micelle and irreversible oil phase separation does not occur. By increasing the oil phase to such an extent, a microemulsion containing the desired compound of formula (I) or a salt thereof can be produced. The desired microemulsion can also be prepared by adding an aqueous solution of the compound of the formula (I) or a salt thereof to a microemulsion prepared according to a known method, allowing the mixture to stand for a certain period of time, preferably heating to 40 ° C. or higher, and then allowing it to cool. Can be manufactured.

By changing the ratio of the compound of the formula (I) or the salt thereof to the total lipid components in the above-mentioned method for producing a lipid membrane structure, the type of the lipid membrane structure to be produced may be changed in some cases. . For example, when only phosphatidylcholine is used as a lipid, liposomes can be formed if the ratio of the compound of the formula (I) or a salt thereof to the total lipid component is about 2/3 or less, and if it is more than that, micelles can be formed. Or a microemulsion may form.

In order for the lipid membrane structure of the present invention thus produced to have directivity to tumor cells or the like, the ratio of the compound of formula (I) or a salt thereof to the total lipid component is usually about 1 in the production process. It is desirable that the molar ratio be at least / 40.

The compound of the formula (I) can be produced by appropriately combining known methods, and typical production methods will be described below.

(1) When R ₃ is a guanidino group (Wherein, R ₁₁ represents a fatty acid residue having 1 to 30 carbon atoms, R ₂₁ represents an acyclic hydrocarbon residue having 1 to 30 carbon atoms, and n is the same as described above). The compound of formula (IIIa) can be prepared by reacting the compound of formula (I) with nitric acid in a suitable organic solvent in the presence of sulfuric acid. This is reacted with a fatty acid chloride represented by the formula R ₁₁ Cl in a suitable organic solvent in the presence of a base such as sodium hydroxide to produce a compound of the formula (IVa). This is catalytically reduced in a suitable organic solvent in the presence of a catalyst such as palladium on carbon to obtain a compound of the formula (I
The compound of a) can be produced. This is esterified with a compound represented by the formula R ₂₁ OH in a suitable organic solvent in the presence of an acid to produce a compound of the formula (Ib). Incidentally, by esterifying the compound of the formula (IIa) in the same manner as in the reaction, in the formula (I), R ₁ is hydrogen, R ₃ is a guanidino group, and R ₂ is an acyclic hydrocarbon residue. Compounds that are groups can be prepared.

(2) When R ₃ is an amino group Wherein R ₁₁ , R ₂₁ and n are the same as described above. The compound of formula (IIb) is reacted with benzyloxycarbonyl chloride in a suitable organic solvent to obtain a compound of formula (III)
The compound of b) can be prepared. This is reacted with a fatty acid chloride represented by the formula R ₁₁ Cl in an appropriate organic solvent in the presence of an alkali such as sodium hydroxide to produce a compound of the formula (IVb). This can be catalytically reduced in a suitable organic solvent in the presence of a catalyst such as palladium on carbon to produce a compound of formula (Ic). This is esterified with a compound of the formula R ₂₁ OH in the presence of an acid in a suitable organic solvent to give a compound of the formula (Id)
Can be produced. Incidentally, by esterifying the compound of the formula (IIb) in the same manner as in the reaction, in the formula (I), R ₁ is hydrogen, R ₃ is an amino group,
Compounds wherein R ₂ is an acyclic hydrocarbon residue can be prepared.

The drug that the lipid membrane structure of the present invention can hold depends on the type of the lipid membrane structure. For example, what can be retained by the liposome is not particularly limited, and examples thereof include water-soluble drugs and fat-soluble drugs such as methotrexate and cisplatin. Further, in the case of micelles and microemulsions, those which can hold fat-soluble drugs can be mentioned.

In the lipid membrane structure of the present invention, the compound of the formula (I) or a salt thereof is firmly incorporated into the membrane of the lipid membrane structure via hydrophobic interaction, and the compound of the formula (I) is liberated as a monomer. It was confirmed by gel filtration and Test Example 1 described later that the compound or salt thereof was very small.

<Effect of the Invention> The lipid membrane structure of the present invention has excellent directivity to tumor cells and can be mass-produced with good reproducibility.

Next, the present invention will be described with reference to examples and test examples, but the present invention is not limited thereto.

Comparative Example 1 Dipalmitoyl phosphatidylcholine (hereinafter abbreviated as DPPC) and cholesterol were placed in a test tube in a molar ratio of 1: 1, 8 μmol of total lipid, dissolved in chloroform, and then chloroform was removed in a stream of nitrogen gas. To form a lipid film on the glass wall of the test tube. 2 ml of a phosphate-buffered saline (pH 7.4, hereinafter abbreviated as PBS) was added thereto, and the mixture was stirred and shaken with a vortex mixer, and further slightly sonicated to prepare a liposome suspension. This is 45
Heated to ℃ 50 ° C., then passed through a polycarbonate membrane filter having a pore size of 0.2 μm,
A suspension of liposomes of 2 μm or less was prepared. Next, this was ultracentrifuged (150,000 × g, 1 hour, twice), and then only the supernatant was removed. Further, 5 ml of PBS was added to obtain a liposome suspension.

1 cocoyl -L- arginine ethyl ester (hereinafter abbreviated as CAEE) at a molar ratio - Example 1 DPPC, cholesterol, and N ^alpha:
1: 0.05, place in a test tube so that the total lipid content is 8 μmol, and mix chloroform and methanol (volume ratio 9: 1)
And a liposome suspension was obtained in the same manner as in Control Example 1.

Example 2 DPPC, cholesterol and CAEE were placed in a test tube in a molar ratio of 1: 1: 0.1 and the total lipid amount was 8 μmol, and a liposome suspension was obtained in the same manner as in Control Example 1.

Example 3 DPPC, cholesterol and CAEE were placed in a test tube in a molar ratio of 1: 1: 0.15 and the total lipid amount to 8 μmol, and dissolved in a mixed solution of chloroform and methanol (volume ratio 9: 1). A liposome suspension was obtained in the same manner as in 1.

Control Example 2 Yolk lecithin 5.54μ mol, cholesterol 1.85μ mol, chloroform and methanol mixture phosphatidic acid 0.62μ moles in a test tube (volume ratio 9: 1) was dissolved in further ³
H-dipalmitoyl phosphatidylcholine 4.0 μCi was added. Next, the organic solvent was removed in a stream of nitrogen gas to form a lipid film on the glass wall of the test tube. To this PBS
Add 5 ml, shake with a vortex mixer and shake.
Further, the suspension was slightly sonicated to prepare a liposome suspension. This was heated to 40 to 45 ° C., and then passed through a polycarbonate membrane filter having a pore size of 0.2 μm to obtain a liposome suspension having a particle size of 0.2 μm or less.

Example 4 Egg yolk phosphatidylcholine 4.8 μmol, cholesterol 1.6 μmol, phosphatidic acid 1.07 μmol, CAEE 0.53 μm
The mole was taken in a test tube, dissolved in a mixed solution of chloroform and methanol (volume ratio: 9: 1), and 2 μCi of ³ H-dipalmitoylphosphatidylcholine was further added. Thereafter, a liposome suspension was obtained in the same manner as in Control Example 2.

Example 5 Egg yolk phosphatidylcholine 4.24 μmol, cholesterol 1.41 μmol, phosphatidic acid 1.41 μmol, CAEE 0.94
A liposome suspension was obtained in the same manner as in Example 4 except that μmol was used.

Example 6 4.21 μmol of distearoylphosphatidylcholine, 2.11 μmol of dicetyl phosphate, and 1.68 μmol of CAEE were dissolved in a mixed solution of chloroform and methanol (volume ratio 9: 1) in a test tube. Next, the organic solvent was removed in a stream of nitrogen gas to form a lipid film on the glass wall of the test tube. Here in ³ H
-Contains 300 μCi of inulin. A liposome suspension was prepared in the same manner as in Control Example 2, except that 5 ml of a PBS solution of 1 mM inulin was added. The obtained liposome suspension was subjected to ultracentrifugation (150,000 × g, 1 hour, twice), and the supernatant was removed to remove inulin that was not retained by the liposome. Thus, 2.5 ml of a liposome suspension containing inulin in its inner aqueous phase was obtained. When the choline group of distearoylphosphatidylcholine was used as a marker to determine the amount of oxygen by the oxygen method, the resulting suspension was 2.2 μmol of total lipid per ml and the retention rate of inulin in liposomes was 1.8%.

Test Example 1 The zeta potential of the liposome suspensions obtained in Control Example 1 and Examples 1 to 3 was measured with Zetasizer II (Malvern). A capillary type cell with an inner diameter of 0.7 mm was used for the measurement. The measurement conditions were as follows: measurement temperature: 25 ° C., solvent viscosity: 0.8903 poise, solvent refractive index: 1.33, cell voltage: 90 volts, cell current: 2 mA. . Table 1 shows the results.
It was shown to.

From the above results, it was confirmed that the compound of the formula (I) was incorporated into the liposome membrane.

Test Example 2 1) A suspension containing the mouse liver cancer cell line MH-134 (medium: RPMI-1640, pH7) was taken and centrifuged (1000 rpm,
After 10 minutes), the supernatant was discarded. Next, PBS was added to resuspend the cells, and 4 × 10 ⁶ cells
The cell suspension was dispensed into 18 test tubes of 4 ml each containing MH-134, and these were kept at 37 ° C. in advance. Next, Comparative Example 2 and Example, which were previously heated to 37 ° C
The liposome suspension prepared in steps 4 and 5 was
Six of each formulation were added to give 0.32 μmol. this
Incubate at 37 ° C (without shaking), sample 3 tubes of each formulation at 1 hour and 3.5 hours, and centrifuge (1000 rpm, 10 rpm).
The cells were sorted only by PBS twice, and the radioactivity incorporated therein was measured by the liquid scintillation method. The results are shown in Table 2. The number of cells after the test was determined by correction by performing protein quantification by the Lowry method, and expressed as the amount of liposome uptake.

2) A test was performed in the same manner as in 1) for a case where 5% of fetal calf serum was contained in PBS used for resuspending the cancer cells in 1). The results are shown in Table 3.

3) The test was performed in the same manner as in 1) except that HL-60, a human leukemia cancer cell line, was used instead of MH-134 in 2).
The results are shown in Table 4.

As is clear from Tables 2 to 4, since the liposome modified with the compound of the formula (I) is taken up more by the tumor cells than the control liposome, the lipid membrane structure of the present invention is excellently directed to cancer cells. It was confirmed that it had the property.

Test Example 3 A cell containing 1.6 × 10 ⁶ MH-134 cells in 2 ml of RPMI-1640 culture solution was used, and 0.16 μmol of the liposome suspension prepared in Example 6 was added as a total lipid amount. The test was performed in the same manner as in Test Example 2 except that the sampling time was changed to 0.5, 1, 2, and 3 hours. As a control, a PBS solution of 2.62 nmol of inulin containing 0.157 μCi of ³ H-inulin was added to a culture solution of MH-134 cells, and the test was carried out in the same manner. Table 5 shows the results. In addition, the number of cells after the test was obtained by correcting by quantifying the protein by the Lowry method, and expressed as the liposome uptake amount.

As is clear from Table 5, it was confirmed that by incorporating the liposome according to the present invention into the cancer cells, inulin in the aqueous phase in the liposome was also incorporated into the tumor cells.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yasuyoshi Kawato 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo Investigator, Daiichi Pharmaceutical Central Research Institute Keiji Goto (56) References JP-A 62-42733 (JP) , A) JP-A-63-2921 (JP, A) (58) Fields investigated (Int. Cl. ⁶ , DB name) A61K 9/10-9/133 A61K 47/16, 47/18

Claims (1)

(57) [Claims] (1) Expression (Wherein R ₁ is hydrogen or a fatty acid residue having 1 to 30 carbon atoms, R ₂ is an acyclic hydrocarbon residue having 1 to 30 carbon atoms, R ₃ is an amino group,
An amidino group or a guadinikino group, and n represents an integer of 1 to 6. However, when R ₁ is a fatty acid residue and R ₂ is an acyclic hydrocarbon residue, the sum of the carbon numbers of R ₁ and R ₂ is 10 to 40. A) a lipid membrane structure containing the compound or a salt thereof.