JPH0276810A - Lipid membrane structure - Google Patents

Abstract

PURPOSE:To obtain the title structure for medical use having specific directional movement properties on tumor cells, etc., producible in a large amount in good reproducibility, containing a specific compound or a salt thereof as an active ingredient. CONSTITUTION:A lipid membrane structure containing a compound shown by formula I (R1 is H or fatty acid residue; R2 is H or noncyclic hydrocarbon residue; R3 is amino or guanidino; r is 1-6; with the proviso that a case where R1 and R2 are simultaneously H is omitted) or a salt thereof. The aimed substance, for example, is obtained by preparing water dispersion using a phospholipid such as phosphatidylcholine or sphingomyelin as a lipid, glycolipid and a membrane-forming lipid such as dialkyl type synthetic surfactant. The water dispersion is blended with the compound formula by formula I in the ratio of the compound to the total lipid component of about 1/40. The compound shown by formula I, for example, is obtained by reacting a compound shown by formula II with nitric acid in the presence of sulfuric acid and reacting the reaction product with a fatty acid chloride in the presence of a base and then catalytically reducing.

Description

Detailed Description of the Invention The present invention relates to the formula (1) (wherein 8R1 represents hydrogen or a fatty acid residue, R1 represents hydrogen or an acyclic hydrocarbon residue, and R3 represents an amino group, an amidino group, or a guanidino group). , n means an integer from 1 to 6.However, except when RI and R2 are hydrogen at the same time, fat containing a compound represented by (RI and R2 are hydrogen at the same time) or a salt thereof,
related.

<Industrial Application Field> The lipid membrane structure of the present invention has specific tropism toward tumor cells and the like, and is medically useful.

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

For example, a multi-day review [Pharmaceutical Journal, 20°843
(taa4) ; Cell engineering, L: 72 (19
82)] and the report by Osawa et al. [Chemical and Pharmaceutical Rublete 4:/, 35.740
(191m7)].

There is a report by Papahad Jopoulos et al. [Cancer e-Research, ■, 4904 (1986)]. In particular, according to the method of Osawa et al., it has been shown that small monolithic liposomes whose surface is modified with anti-carcinoembryonic antigen antibodies are easily taken up by cancer cells and their antitumor effects are enhanced. .

However, these monoclonal antibodies.

The current situation is that mass production is difficult and commercialization is difficult.

<Problems to be Solved by the Invention> The present inventors have conducted intensive studies on a lipid membrane structure that has efficient specific targeting to cancer cells and can be mass-produced with good reproducibility. The invention has been completed.

<Configuration of the Invention> The present invention relates to a lipid membrane structure containing the compound of formula (1) or a salt thereof.

The fatty acid residue in the compound of formula (1) is a group formed by removing a hydroxyl group from a saturated or unsaturated fatty acid that may have a branch; examples thereof include formyl,
Acetyl, propanoyl.

Butanoyl, pentanoyl, hexanoyl, heptanoyl, octanoyl, decanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl,
Nonadecanoyl, eicosanoyl, henicosanoyl, docosanoyl, tricosanoyl, tetracosanoyl, hexacosanoyl, triacontanoyl, 9-hexadecenoyl, 9-octadecenoyl, 9,12-octadecajenoyl, 9,12,15-octadecatrienoyl, 11-eicosanoyl , 11.14-eicosagenoyl, 11°14.17-nicosatrienoyl,
4,8,12.16-nicosatetraenoyl, 1
3-dodecanoyl, 4,8.12°15.19-docosapentaenoyl, 15-tetracomoyl. 2-dodecylhexadecanoyl, 2-tetradecylhexadecanoyl, 2-dodecyltetradecanoyl, 2-tetradecenylhexadecenoyl, 2-tetradecylhexadecenoyl, 2-tetradecenylhexadecanoyl, etc. having 1 to 30 carbon atoms 5, preferably 14 to 20.

In addition, the acyclic hydrocarbon residue in Formula 2 (1) is a group formed by removing one hydrogen from a saturated or unsaturated acyclic hydrocarbon that may have a branch. Examples include methyl, ethyl, propyl, butyl, pentyl,
Hexyl, heptyl.

Octyl, nonyl 8-decyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl.

Eicosyl, henicosyl, tocosyl, tricosyl, tetracosyl, hexacosyl, triacontyl, 9-hexadecenyl, 9-octadecenyl, 9.12-ophtadecashenyl, 9,12.15-octadecanoylnyl, 11-eicosenyl, 11.14 -eicosagenyl, 11,14.17-nicosatrienyl, 4
, 8,1216-nicosatetraenyl, 13-tocosenyl, 4°8.12. Is, 19-docosapentaenyl, 15-tetracosenyl, 2-dodecyltetradecyl, 2-dodecylhexadecyl, 2-tetradecylhexadecyl, 2-tetradecylhexadecenyl, 2-tetradecenylhexadecyl, 2-dodecyloctadecyl Examples include those having 1 to 30 carbon atoms, preferably 14 to 20 carbon atoms.

Furthermore, with respect to n in Formula 9 (I), 4 or 3 can be cited as preferred.

Regarding the compound of formula (1), a compound in which R1 is a fatty acid residue and R2 is an acyclic hydrocarbon residue is preferable, and in this case, the sum of the carbon numbers of R+'EL and R2 is 10 to 40.
It is desirable that

The compound of formula (1) has optical isomers, and these isomers and mixtures thereof are also included within the scope of the present invention.

The lipid membrane structure of the present invention refers to particles having a membrane structure in which the polar groups of polar lipids are arranged toward the aqueous phase side of the interface,
Examples include liposomes, micelles, microemulsions, etc.

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

a) Method for producing liposomes containing the compound of formula (-I) or a salt thereof Phosphatidylcholine, sphingomyelin.

Phospholipids such as phosphatidylethanolamine.

Known methods using membrane-forming lipids such as glycolipids and dialkyl type synthetic surfactants [Annual Review of
An aqueous dispersion of ribosomes is prepared according to Biophysics and Bioengineering 9.467 (1980)]. Such liposomes may contain cholesterol, sterols such as cholestanol, dialkyl phosphate, diacylphosphatidic acid, stearylamine, α-tocopherol, etc. in their membranes.

By adding an aqueous solution of the compound of formula (1) or its salt to the prepared aqueous dispersion of liposomes and allowing it to stand for a certain period of time, preferably by heating it to a temperature above the phase transition B temperature of the membrane or above 40°C, and then allowing it to cool. A liposome containing the target compound of formula (1) or a salt thereof can be produced. or,
The desired liposome can also be produced by pre-mixing the membrane-forming lipid with a compound of the formula (RI) or a salt thereof, and treating the mixture according to a known liposome production method.

b) Process for producing micelles containing the compound of formula (1) or a salt thereof Polyoxyethylene sorbitan fatty acid ester, polyoxyethylene castor oil conductor, polyoxyethylene hydrogenated castor oil derivative, sodium fatty acid, sodium bile acid, polyoxy A micelle-forming interfacial substance such as ethylene fatty acid ester or polyoxyethylene alkyl ether is added to water at a concentration of at least one micelle-forming concentration to prepare an aqueous dispersion of micelles. An aqueous solution of the compound of formula (I) or a salt thereof is added to the prepared aqueous dispersion of micelles, and the mixture is left to stand for a certain period of time, preferably heated to 40°C or higher, and then allowed to cool to obtain the desired compound of formula (I). Micelles containing the compound or its salt can be produced. The desired micelles can also be produced by pre-mixing a micelle-forming substance and a compound of the formula (RI) or a salt thereof, and then treating the mixture according to a known method for producing micelles.

C) A method for producing a microemulsicone containing the compound of formula (1) or a salt thereof Add fats and oils such as soybean oil to the micelles produced according to b) above to saturate the inside of the micelles, causing irreversible oil phase separation. By increasing the oil phase to such an extent that the desired equation (
A microemulsicon containing the compound of 1) or a salt thereof can be produced. Alternatively, the desired microemulsion can also be prepared by adding an aqueous solution of a compound of the formula (R) or its salt to a microemulsion prepared according to a known method, leaving it for a certain period of time, preferably heating it to 40°C or higher, and then allowing it to cool. can be manufactured.

In the method for producing a lipid membrane structure as described above, by changing the ratio of the compound of formula (1) or its salt to the total lipid components, it may be possible to change the type of lipid membrane structure produced. 6 For example, when only phosphatidylcholine is used as the lipid, liposomes can be produced if the ratio of the compound of formula (I) or its salt to the total lipid component is less than about 273 molar ratio, and if it is more than that, micelles or Microemulsions can be produced.

The lipid membrane structure of the present invention produced in this manner
In order to have directivity to cells, etc., it is usually desirable that the ratio of the compound of formula (1) or its salt to the total lipid components be about 1/40 molar ratio or more in the manufacturing process.

The compound of formula (1) can be produced by appropriately combining known methods, and a typical production method will be explained below.

(1) When R3 is a guanidino group (in the formula, 3 units represent a fatty acid residue, R2I represents an acyclic hydrocarbon residue, and n is the same as above), a compound of formula (Ila) is suitably used. A compound of formula (Ota) can be prepared by reacting with nitric acid in the presence of sulfuric acid in an organic solvent. A compound of formula (IVa) can be produced by reacting this with a fatty acid chloride represented by the formula R+tCI in a suitable organic solvent in the presence of a base such as sodium hydroxide. The compound of formula (Ia) can be produced by catalytic reduction of this in a suitable solvent in the presence of a catalyst such as palladium on carbon. A compound of formula (Ib) can be produced by esterifying this with a compound represented by formula R, OH in an appropriate solvent in the presence of an acid. Incidentally, by esterifying the compound of formula (Ila) in the same manner as in the above reaction, a compound in which R1 is hydrogen, R3 is a guanidino group, and R2 is an acyclic hydrocarbon residue in formula (I) can be obtained. can be manufactured.

(B) (If-) (I
C9) (In the formula, R11, R21 and n are the same as above)
Formula (II) can be prepared by reacting a compound of formula (fib) with benzyloxycarbonyl chloride in a suitable organic solvent.
It is also possible to prepare f-hybrid compounds of Ib). Add this to formula R in an appropriate organic solvent! The compound of formula (IVb) can be produced by reacting the fatty acid chloride represented by formula (IVb) in the presence of an alkali such as sodium hydroxide. A compound of formula (Ic) can be produced by catalytic reduction of this in an appropriate organic solvent in the presence of a catalyst such as palladium on carbon. In a suitable organic solvent and in the presence of an acid, the formula R
A compound of formula (Id) can be produced by esterification with a compound represented by 210H. Furthermore, the formula (
In formula (1), R8 is hydrogen by esterifying the compound of Ilb) in the same manner as in the above reaction.

Compounds can be produced in which R3 is an amino group and R2 is an acyclic hydrocarbon residue.

The drugs that can be retained by the lipid membrane structure of the present invention vary depending on the type of lipid membrane structure. For example, there are no particular restrictions on what the liposome can retain.

Water-soluble drugs and fat-soluble drugs 1 such as methotrexate,
Examples include cisplatin.

Furthermore, in the case of micelles and microemulsicone, it is possible to hold fat-soluble drugs.

In the lipid membrane structure of the present invention, the compound of Formula 8 (1) or its salt is firmly incorporated into the membrane of the lipid membrane structure through hydrophobic interaction, and is free as a monomer. It was confirmed by the gel filtration method and Test Example 1 described below that the amount of the compound or its salt was very small.

<Effects of the Invention> The lipid membrane structure of the present invention has excellent tumor cell tropism and can be mass-produced with good reproducibility.

Next, the present invention will be explained with reference to Examples and Test Examples, but the present invention is not limited thereto.

Control Example 1 Dipalmitoylphosphatidylcholine (hereinafter abbreviated as DPPC) and cholesterol in molar ratio 1:1, total lipid amount 8
After dissolving the mixture in chloroform in μmoles, the chloroform was removed in a nitrogen gas stream to form a lipid film on the glass wall of the test tube. Add to this phosphate buffered saline (9) 17.4. 2 ml of PBS (hereinafter abbreviated as PBS) was added, stirred and shaken using a portex mixer, and further lightly treated with ultrasound to prepare a liposome suspension. This was heated to 45-50℃ and then 0
．． A suspension of liposomes with particle size Q of 9 and 2μ or less was prepared by passing through a polycarbonate membrane filter with a pore size of 2μ.
After 10,000 x g for 1 hour, 2 times), only the supernatant was removed, and 5 ml of PBS was added to obtain a liposome suspension.

Example! DPPC, cholesterol and N-cocoyl-arginine ethyl ester (hereinafter abbreviated as CAEE) in a molar ratio of 1:1:Q, 05. A total lipid amount of 8 μmol was taken into a test tube, dissolved in a mixture of 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 in a molar ratio of l:
1:0.1, and the total lipid amount was 8 μmol in a test tube, and a liposome suspension was obtained in the same manner as in Control Example 1.

Example 3 DPPC, cholesterol and CAEE in a molar ratio of 1:
1:0.15. Transfer a total lipid amount of 8 μmol to a test tube and add a mixture of chloroform and methanol (volume ratio 9).
:1) to obtain a liposome suspension in the same manner as in Control Example 1.

Example 4 DPPC, cholesterol and N-valmitoyl-
Arginine (hereinafter abbreviated as FAA) in a molar ratio of 1:1:0
．． 05. Transfer a mixture of chloroform and methanol (volume ratio 9:1) to a test tube so that the total lipid amount is 8 μmol.
A liposome suspension was obtained in the same manner as in Control Example 1.

Example 5 DPPC, cholesterol and FAA in a molar ratio of 1:l
:Q, 1. Take a total lipid amount of 8 μmol into a test tube and add a mixture of chloroform and methanol (volume ratio 9:
1) to obtain a liposome suspension in the same manner as in Control Example 1.

Example 6 DPPC, cholesterol and FAA in molar ratio i:1
:Q, 15. Transfer a total lipid amount of 8 μmol to a test tube, add a mixture of chloroform and methanol (volume ratio 9:
1) to obtain a liposome suspension in the same manner as in Control Example 1.

Control example 2 Egg yolk lecithin 5.54 μmol, cholesterol 1.85
0.62 μmol of phosphatidic acid was dissolved in a mixture of chloroform and methanol (volume ratio 9:1) in a test tube, and 4.0 μCk of 3-todivalmitoylphosphatidylcholine was added in a nitrogen gas stream. In the test tube, the organic solvent was removed to form a liquid film on the glass wall of the test tube. PBS 5a+1 was added to this, and the mixture was stirred and shaken using a portex mixer, and further subjected to light sonication to prepare a liposome suspension. This was heated to 40-45°C and then passed through a polycarbonate membrane filter with a pore size of 0.2 μl, each particle size being 0.2 μl.
A suspension of less than 100ml of liposomes was obtained.

Example 7 Egg yolk water soup 7 Tidylcholine 4.8 μmol 6 Cholesterol 1.6 μmol, Phosphatidic acid 1.07 μmol, C
Take 53 μmol of AEE O into a test tube.

Dissolve in a mixture of chloroform and methanol (volume ratio 9:1) and add 3 dipalmitoylphosphatidylcholine 2
μCL was added. A liposome suspension was obtained in the same manner as in Control Example 2.

Example 8 A liposome suspension was obtained in the same manner as in Example 7, except that 4.24 μmol of egg yolk phosphatidylcholine, 1.41 μmol of cholesterol, 1.41 μmol of phosphatidic acid, and 0.94 μmol of CAEE were used.

Example 9 Distearoyl phosphatidylcholine 4.21 μmol,
2.11 μmol of dicetyl phosphoric acid and 68 μmol of CAEEL were dissolved in a mixture of chloroform and methanol (volume ratio 9:1) in a test tube. Next, the organic solvent was removed in a nitrogen gas stream and the mixture was applied to the glass wall of the test tube. A lipid film was produced. To this, 5 ml of a PBS solution of 1 inulin containing 300 μCt of 3■-inulin was added.
A liposome suspension was prepared in the same manner as in Control Example 2. The obtained liposome suspension was subjected to ultracentrifugation (150,008 g, 1
Remove inulin that was not retained in the liposomes by removing the supernatant and submerging in PBS.
was added to finally obtain 2.5 ml of a liposome suspension retaining inulin in its internal aqueous phase. When quantified by an enzymatic method using the choline group of distearoylphosphatidylcholine as a marker, the resulting suspension had a total lipid content of 2.2 μmol per ml, and the retention rate of inulin by liposomes was 1.8 zero. .

Test Example 1 The zeta potential of the liposome suspensions obtained in Control Example 1 and Examples 1 to 6 was measured using Zetasizer 11 (manufactured by Malvern). A capillary cell with an inner diameter of 0.7 mm was used for the measurement, and the measurement conditions were: measurement temperature: 25°C;
Solvent viscosity: 0.8903 poise.

Solvent refractive index: 1.33. Cell voltage: 90 volts, cell current: 2 milliamps. The results are shown in Table 1.

Table 1 Results of measurement of zeta potential of various liposome suspensions From the above results, it was confirmed that the compound of formula (I) was incorporated into the liposome membrane.

Test Example 2 1) Take a suspension containing MH-'134, a mouse liver cancer cell line (medium is RPIJI-1640, p7), and sediment the cells by centrifugation (LOOORPM, 10 minutes). After discarding the supernatant, add PBS to resuspend the cells, and add 4XIQ' MH- per tube.
Add 4 ml of cell suspension (a) to a test tube to contain 134.
The mixture was dispensed into 8 pieces and kept warm at 37°C in advance. Next, control example 2 was heated to 37℃ in advance.
Six bottles of each formulation were added to the liposome suspension prepared in Example 7.8 so that the total lipid amount was 0.32 μmol. Incubate this at 37°C (no shaking), sample 3 bottles of each formulation for 1 hour and 3.5 hours, and centrifuge (loOQrpm, 10 minutes, twice with PBS).
After separating only the cells, the radioactivity incorporated into these cells was measured by liquid scintillation method. The results are shown in Table 2. Note that the number of cells after the test was determined by correcting protein quantification using the Lowry method, and was calculated based on the amount of liposome uptake.

2) A test was conducted in the same manner as in 1) except that fetal calf serum 5 was added to the PBS used to resuspend the cancer cells in 1). The results are shown in Table 3.

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

Table 2 Table 3 (Standard deviation above the mean) Table 4 (Standard deviation above the mean) As is clear from Tables 2 to 4, liposomes modified with the compound of formula 0 <r> are taken up by tumor cells more than the control liposomes. Therefore, it was confirmed that the lipid membrane structure of the present invention has excellent tropism toward cancer cells.

Test Example 3 The number of MH-134 cells was RPMI-11i49 culture solution 2
Using the liposome suspension prepared in Example 9, the total lipid amount was 0.
．． 16 μmol was added, and the sampling time was increased to O, S,
t.

The test was conducted in the same manner as Test Example 2 except that the test time was 2.3 hours.

As a control, 2
．． A PBS solution of 62 nmoles of inulin was added to the culture medium of Mto134 cells, and the following tests were conducted in the same manner. Table 5 shows the results.
It was shown to. Note that the number of cells after the test was determined by correcting protein quantification using the Lowry method, and was calculated based on the amount of liposome uptake.

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

Claims (1)

[Claims] Formula ▲ Numerical formula, chemical formula, table, etc. ▼ (In the formula, R_1 is hydrogen or a fatty acid residue, R_2 is hydrogen or an acyclic hydrocarbon residue, R_3 is an amino group, an amidino group or a guanidino group, n means an integer from 1 to 6, except when R_1 and R_2 are hydrogen at the same time) or a lipid membrane structure containing a salt thereof.