JP2601373B2 - Amphiphilic compound and liposome using the same - Google Patents

Description

The present invention relates to an amphiphilic compound containing a succinic acid and an amino acid moiety designed to form stable unilamellar liposomes, and a membrane composition comprising the same. The present invention relates to a negatively charged liposome as a component.

(Prior Art) Liposomes are closed vesicles composed of lipid bilayers. It is said that a natural biomembrane has a lipid bimolecular structure, and this liposome is widely used for studying the physicochemical properties of a biomembrane as a model membrane. In addition, liposomes can confine various substances in an internal aqueous layer or membrane and are fused with cells or taken up by cells, so that they are used as carriers for delivering substances into living bodies.

Research using liposomes covers a wide range of fields, such as biology, medicine, and pharmacy.Oxygen is used as a carrier to carry anticancer drugs, immunology, interaction with cells, use as a drug delivery system, etc. Has been studied.

As described above, liposomes have a very wide range of applications, but their problems have been pointed out as the weakness of the membrane structure.

In other words, this is a phenomenon in which the orientation of the membrane is disturbed by a chemical or physical change of the lipid which is a membrane-forming substance, leakage of inclusions, association and aggregation of liposomes occur, and a precipitate is eventually formed.

In an attempt to overcome this drawback, there have been many reports of forming vesicles with artificial amphipathic compounds mimicking natural phospholipids (for example, Nojima, Sunamoto, Inoue, "Liposome" (Nankodo), Chapter 8). However, none of them was satisfactory as a drug carrier in terms of vesicle stability and toxicity to the human body.

Examples of amphiphilic compounds having an oligopeptide in the hydrophilic part and two long-chain alkyl groups in the hydrophobic part include those described by Ihara et al. (Polym. Commun., 27 , 282 (1986); Polymer J., 18 , 163)
(1986); Chem. Lett., (1984), 1713; Nikka (1987),
543) and examples of Shimizu et al. (Chem. Lett., (1986), 1341; Thin).
Solid Films. 180 (1989), 179, JP-A-2-69498,
No. 2-71836) is known. However, none of them form a single membrane vesicle, or even if formed, they easily change to other structures, and are not suitable as drug carriers. In addition, all of the molecular aggregates formed by these compounds are positively charged, and cannot be a suitable model of a biological membrane containing an anionic lipid such as phosphatidylserine and phosphatidylglycerol.

(Object of the Invention) It is an object of the present invention to contain a succinic acid and an amino acid moiety designed to form a stable single membrane liposome which contains a small amount of leaking drug and hardly causes association, aggregation and precipitation. And a negatively charged liposome containing the same as an amphiphilic compound and a membrane component thereof.

(Constitution of the Invention) The object of the present invention has been achieved by compounds represented by the general formulas (I) to (III) and liposomes containing the compounds as membrane components.

R ¹ and R ² each have 8 to 24 carbon atoms, preferably 12, 14,
It is a 16 or 20 linear or branched alkyl group or acyl group, and may have a substituent or an unsaturated group. Examples of the substituent include an alkylcarbonyl, an alkoxycarbonyl, a halogen atom, and an aryl group. The unsaturated group is a double bond or a triple bond, and may have two or more in the same chain. R ¹ and R ² may be the same or different. Specific examples of R ¹ and R ² include dodecyl, tetradecyl, hexadecyl, myristoyl, palmitoyl and the like.

R ³ⁿ , R ^{3 (n + 1)} , R ^3m , and R ^{3 (m + 1)} each represent a side chain residue of an α-amino acid. This includes the naturally occurring α-
20 amino acids (for example, “PROTEINS” by CREIGHTON (F
REEMAN)) or its analogs. Particularly preferable are a hydrogen atom, -CH ₂ OH, −CH ₂ CO ₂ H, −CH ₂ CH ₂ CO ₂ H, −CH ₂ CH ₂ CH ₂ CH ₂ NH ₂ Glycine or a more hydrophilic amino acid side chain residue. ^{(N + 1) in} R ^{3 (n + 1)} represents a single digit number. For example, when n = 5, R ^{3 (n + 1)} represents R ³⁶ . R
³¹ , R ³² ,..., R ^{3 (n + 1)} may be the same or different. The same applies to m.

n represents an integer of 1 to 5 in the formula (I), and the formula (I
In I) and (III), n represents an integer of 0 to 5, and particularly preferably 0, 1, 2, and 3. The same applies to m.

The asymmetric carbon present in the molecule may be any of a racemic form and an optically active form. Further, the carboxyl group at the molecular terminal may form a salt with an appropriate cation component. In this case, preferred cation components include Na ⁺ , K ⁺
And the like, alkali metal ions, ammonium ions and the like.

Next, specific examples of the compounds represented by formulas (I) to (III) are shown, but the present invention is not limited thereto.

The amphiphilic compound of the present invention is synthesized by using glycerol substituted at the 1- and 2-positions (general formula (IV)) as a raw material and sequentially introducing an amino acid portion and a succinic acid portion. For the introduction of the amino acid moiety, an ordinary method of using an amino acid having an amino group or a carboxyl group protected and condensing with an appropriate condensing agent can be used. Examples of the protecting group and the condensing agent include “PRINCIPLE” by M. Bodanszk.
S OF PEPTIDE STNTHESIS "(Springer-Verlag, New Yor
k, 1984) and “THE PRACTICE OF PEPTIDE STNTHESIS”
(Springer-Verlag, New York, 1984). For the introduction of the succinic anhydride moiety, the method using succinic acid is the simplest and most useful.

The compound represented by the general formula (IV) is, for example, J.Am.C
hem. Soc.) 63 , 3244 (1941), and is commercially available.

The synthesis examples of the compound of the present invention are described below. Abbreviations of amino acids and protecting groups are commonly used abbreviations (eg,
The above-mentioned book by Bodanzky) was used as it is. In addition,
Here, the liquid crystal phase transition point is a value obtained by using a DSC manufactured by Seiko Denshi Co., Ltd. to determine the temperature at which the crystal phase transitions to the liquid crystal phase.

Synthesis Example 1. Synthesis of Compound 3 Compound 3 was synthesized by the following synthesis route.

It was converted to tBoc-GlyGly according to a commercially available GlyGly standard method (Izumiya et al., “Basic and Experimental Peptide Synthesis” (Maruzen)).

1.39 g (6 mmol) of tBoc-GlyGly, 2.42 g (5 mmol) of 1,2-o-ditetradecyl-sn-glycerol, and 60 mg of N, N-dimethylaminopyridine were dissolved in 20 ml of DMF and 10 ml of methylene chloride. This solution is water-cooled, while stirring, DCC1.3g
Was added and stirred at room temperature for 24 hours. The precipitated dicyclohexylurea was separated by filtration, and methylene chloride was distilled off from the filtrate under reduced pressure. Ethyl acetate (50 ml) was added to the remaining liquid, and the mixture was washed and separated with a 10% aqueous citric acid solution, water and brine in this order. The dicyclohexylurea precipitated again in the ethyl acetate layer was filtered off, the filtrate was concentrated, and the residue was purified by silica gel dalography (n-hexane / ethyl acetate = 2/1) to give 3.37 g (4.8 g) of compound (3a). mmol). Yield 90% 3.37 g of the protected compound was dissolved in 60 ml of methylene chloride, 30 ml of trifluoroacetic acid was added, and the mixture was stirred at room temperature for 30 minutes. The solvent was distilled off under reduced pressure, and the residue was recrystallized from a mixed solvent of ethyl acetate and acetonitrile (1/1) to give 2.87 g of compound (3b).
(4.03 mmol) was obtained. Yield 84%. Liquid crystal phase transition point 79 ° C.

(3b) 2.85 g (4 mmol) was dissolved in a mixed solvent of 30 ml of methylene chloride and 1.4 ml of triethylamine, and 0.5 g (5 mmol) of succinic anhydride was added while stirring with water cooling. Under ice cooling 1
After stirring for 2 hours at room temperature, the methylene chloride solution was washed with 1N hydrochloric acid, water and brine in this order. After drying over sodium sulfate, methylene chloride was distilled off under reduced pressure, and the residue was recrystallized from ethyl acetate to obtain 2.49 g (3.56 mmol) of compound (3).

89% yield, liquid crystal phase transition point 103 ° C Synthesis Example 2. Synthesis of compound (1) ^{t Boc-Gly530mg (3mmol),} 1,2-o- ditetradecyl -sn- glycerol 1.21g (2.5mmol), N, was dissolved in methylene chloride 15ml of N- dimethylaminopyridine 37 mg. The solution was cooled with water and stirred while adding 600 mg of DCC and stirred at room temperature for 24 hours. The precipitated dicyclohexylurea was separated by filtration, and methylene chloride was distilled off from the filtrate under reduced pressure. Ethyl acetate (50 ml) was added to the remaining solution, and a 10% aqueous citric acid solution, water,
After washing in the order of saline solution, the solution was separated. The dicyclohexylurea precipitated again in the ethyl acetate layer was filtered off, the filtrate was concentrated and the residue was purified by silica gel chromatography (n-
Hexane / ethyl acetate = 5/1) to give 1.55 g (2.4 mmol) of compound (1a) as a colorless oil. 97% yield.

1.55 g of this protected compound was dissolved in 10 ml of methylene chloride, 5 ml of trifluoroacetic acid was added, and the mixture was stirred for 30 minutes. After evaporating the solvent under reduced pressure, ethyl acetate and a 4% aqueous sodium carbonate solution were added, and the mixture was extracted and separated. The organic layer was dried over sodium sulfate, and the solvent was distilled off under reduced pressure. The residue was dissolved in 15 ml of methylene chloride, cooled on ice, and 250 mg of succinic anhydride was added. 3 under ice cooling
After stirring for 0 minute at room temperature for 1 hour, the solvent was distilled off under reduced pressure.
The residue was purified by silica gel chromatography (chloroform / methanol = 10/1), and crystallized from ethyl acetate to obtain 1.1 g (1.68 mmol) of compound 1. 70% yield. (2
Step) Liquid crystal phase transition point 71 ° C.

Synthesis Example 3. Synthesis of Compound 6 In Synthesis Example 2, 1,2-o-ditetradecyl-sn-
1,2-o-dimyristoyl-sn instead of glycerol
-The same operation was performed using glycerol to obtain Compound 6. Liquid crystal phase transition point 70 ° C.

Synthesis Example 4. Synthesis of Compound 4 In Synthesis Example 2, 1,2-o-ditetradecyl-sn-
Instead of glycerol, 1,2-o-dipalmitoyl-s
The same operation was performed using n-glycerol to obtain Compound 4
I got Liquid crystal phase transition point 78 ° C.

Synthesis Example 5 Synthesis of Compound 12 3 g of 1,2-o-ditetradecyl-sn-glycerol (6.2
mmol), and 680 mg of succinic anhydride was added to a methylene chloride solution (30 ml) containing 80 mg of N, N-dimethylaminopyridine, and the mixture was stirred at room temperature for 39 hours. The finished solvent was distilled off under reduced pressure, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate / = 2/1 to 1/1) to give a colorless oil (solidified at 4 ° C.).
2.4 g (4.1 mmol) of compound (12a) was obtained. Yield 66%.

(12a) 1.95 g (2.3 mmol), Gly-OBzlp-toluenesulfonate 1.2 g (3.55 mmol), triethylamine 490 µ
l, 1-hydroxybenzotriazole monohydrate 540mg
Was dissolved in a mixed solvent of methylene chloride (15 ml) and DMF (5 ml), and 750 mg of DCC was added while stirring with ice cooling. After stirring was continued for 2 hours at room temperature under ice cooling, the precipitated dicyclohexylurea was separated by filtration, and methylene chloride was distilled off from the filtrate under reduced pressure. Ethyl acetate was added to the remaining liquid, and the mixture was washed and separated with a 10% aqueous citric acid solution, water and brine in this order. The dicyclohexylurea precipitated again in the ethyl acetate layer was filtered off, the filtrate was concentrated, and the residue was purified by silica gel column chromatography (hexane / ethyl acetate = 3/1 to 2/1) to obtain 2.01 g of compound (12b) ( 2.75 mmol). 83% yield.

1.97 g (2.69 mmol) of compound (12b) was treated with methanol (20 m
In l), the mixture was dissolved in a mixed solvent of ethyl acetate (20 ml), 200 mg of 5% palladium carbon was added, and hydrogenation was performed at room temperature for 3 hours under normal pressure. The catalyst was filtered off through celite, and the filtrate was concentrated and crystallized from acetonitrile to give compound (12) 1.54
g (2.4 mmol) was obtained. Yield 89%, liquid crystal phase transition point 66 ° C.

Synthesis Example 6. Synthesis of Compound 15 Using Compound 12 as a raw material, Gl was prepared in the same manner as in Synthesis Example 5.
condensation with y-OBzl and debenzylation,
Compound 15 was obtained by crystallization from a mixed solvent of ethyl acetate and acetonitrile (5: 1). Liquid crystal phase transition point 86 ° C.

Liposomes containing the compounds (I) to (III) of the present invention as membrane components are prepared by a known method.

That is, the vortex swing method [ADBangham J. Mol. Bi
ol., 13, 238 (1965), sonication method (C. Huang, Bioc
hem., 8 , 344 (1969)], the prevesicle method [H. Trauble, N.
eurosci. Res. Prog. Bull., 9 , 273 (1971)], ethanol injection method [S. Batzri, Biochem. Biophys. Acta., 298 , 1015].
(1973)], French press extrusion method [Y. Barenhollz .. FE
BS. Lett., 99 , 210 (1979)], cholic acid removal method [Y.
a, J. Biol. Chem., 246 , 5477 (1971)], Triton X-100
Batch method [WJ Gerritsen, Eur. J. Biochem., 85 , 255 (197
8)], Ca ²⁺ fusion method [D. PaPahadjopoulos, Biokhem. Bioph
ys. Acta. 394 , 483 (1975)], ether injection method [D. Deame
r. Biokhem. Biophys. Akta., 443 , 629 (1976)], annealing method [R. Lawczeck, Biochem. Biophys. Acta, 443 , 313].
(1976)], freeze-thaw fusion method [M. Kasahara, J. Biol. Chem.,
252 , 7384 (1977)], W / O / W emulsion method [S. Matsumo
to, J. Colloid Interface Sci., 62 , 149 (1977)], the reverse phase evaporation method [F. Szoka, Proc. Natl. Acad. Sci. USA, 75 , 4194 (19)
78)], high-pressure emulsification method [E. Mayhew, Biochem. Biophys.
775,169 (1984)] and JP-A-60-7932 and JP-A-60-793.
Many methods are known, such as the methods described in 3, 60-7934, 60-12127, and 62-152531, but any of the above-mentioned preparation methods may be used in the present invention, and the present invention is not limited thereto. It is not something to be done.

As the encapsulating member used in the present invention, either or both of a hydrophilic drug and a lipophilic drug can be used simultaneously. Such hydrophilic drugs include, for example, adriamycin, actinomycin, mitomycin, 1-β
-Anticancer agents such as arabinofuracyl cytosine, bleomycin, cisplatin, antiviral agents such as interferon, amino acid sugars (e.g., gentamicin), β-lactam compounds (e.g., sulbenicillin, cefotiam,
Antibiotics such as cefmenoxime), peptide hormones such as TRH, leubrolide and insulin, lysozyme,
Oxygen agents such as asparaginase and glycosidase; immunostimulants such as muramyl dipeptide and muramyl tripeptide; immunoglobulins; and proteins such as various toxins.

Examples of lipophilic drugs include anticancer drugs such as ansamitocin and TMD-66 (Gann 74 (2) 192-195 (198
3)), immunostimulants such as MTP-PE (JP-A-59-163389), and phospholipid derivatives (JP-A-59-163389).

Other than the drug, there is no particular limitation as long as the marker or plasmid, DNA, RNA or the like which is useful when administered in vivo is useful.

Next, an aqueous solution in which an appropriate water-soluble substance is dissolved in water using a medium as the filling liquid is used. In some cases, the drug may be simply dissolved in water. Examples of the water-soluble substance include various buffers (eg, phosphate buffer, citrate buffer), various salts (eg, sodium chloride, phosphate-sodium, disodium phosphate), sugars (eg, glucose), Amino acids (eg, 1-arginine) and the like can be used alone or in combination.

If necessary, a preservative (eg, paraban) or the like may be added to this filling solution.

Unencapsulated drugs and liposomes can be easily separated by dialysis, filtration (eg, gel filtration), centrifugation, and the like. At this time, it is desirable to make the osmotic pressures of the inner aqueous phase and the outer aqueous phase as close as possible.

The compounds of the present invention may be used alone or as a mixture of two or more. It may be used in combination with other liposome membrane-forming lipids. Various phospholipids, sphingolipids, or synthetic lipids can be used for this purpose.

Further, in order to further enhance the membrane structure, various known means can be used in combination with the phospholipid liposome.

Typical examples thereof include a mixture of sterol or cholesterol and coating with a polysaccharide polymer (JP-A-61-6961).
No. 801).

The compound of the present invention does not have a hydrophilic portion having a large hydration radius unlike ordinary bilayer membrane-forming lipids. Nevertheless, the formation of stable liposomes is thought to be due to intermolecular hydrogen bonding at the peptide site.

An example of preparing a liposome containing the compound of the present invention as a membrane component will be described below.

Example 1 After dissolving 30 mg of compound 3 in 10 ml of chloroform, chloroform was distilled off using a rotary evaporator and further dried under vacuum to form a thin film of compound 3.
To this, Tris buffer containing 15 mM sodium chloride (6 mM
M, pH 7.0) was added, and Vortex dispersion was performed. At this time, a slight decrease in pH was observed.
Was adjusted to 7. Next, a bath-type ultrasonic irradiation was performed at 50 ° C.
Performed for 10 minutes, and further heated at 80 ° C. for 10 minutes. Disperse the solution using an Extrader (0.2μ polycarbonate filter, 5
(5 ° C.) and pressure filtration (about 11 kg / cm ² ) was performed six times. N
As a result of measuring the particle size by ICOMP, a particle size distribution of a monodisperse mode having an average of 120 nm was obtained. Furthermore, as a result of observation with a TEM after staining with phosphotungstic acid, it was confirmed that the vesicles were single-layered vesicles.

Example 2 The Vortex dispersion obtained in the same manner as in Example 1 was irradiated with probe-type ultrasonic waves (30 W, 5 minutes). In the same manner as in Example 1, it was confirmed that one cycling could be prepared with an average particle size of about 80 nm.

[Example 3] The gel-liquid crystal phase transition point of the compound of the present invention in a phosphate buffer (20 mM, pH 7.0) was measured using a Prialog-type DSC. Table 1 shows the results.

Example 4 A thin film of Compound 1 (30 mg) was prepared in the same manner as in Example 1, and then 3 ml of a phosphate buffer (20 mM, pH 7.0) containing 50 mM carboxyfluorescein (CF) was added. Next, in the same manner as in Example 1, processing was performed in the order of Vortex dispersion, bath-type ultrasonic waves, heating at 80 ° C., and an extruder. In this case, the decrease in pH observed in Example 1 did not occur. Then, the dispersion was added to a phosphate buffer (20 mM) containing 150 mM sodium chloride.
(M, pH 7.0), and gel filtration was performed with FADEX G-50 to separate unencapsulated CF.

The obtained lipid fraction (average particle size: 120 nm) was incubated at 37 ° C., and the leaked CF was quantified by a fluorescence method.
As a comparative example, CFPC-encapsulated liposomes (average particle size 140 nm) were prepared by the same operation using DPPC (dipalmitoylphosphatidylcholine) instead of compound 1,
And the leakage of CF was quantified.

 Table 2 shows the results.

From Table 2, it was found that the liposome containing Compound 1 of the present invention as a membrane component had a higher barrier ability against CF as compared with DPPC which is a natural phospholipid.

[Example 5] Liposomes containing CF were prepared in the same manner as in Example 4, except that Compounds 3, 4, 6, 12, and 15 were used instead of Compound 1.
The leakage at 37 ° C. was examined. Table 3 shows the CF leakage rate after one hour.

Table 3 shows that many of the compounds of the present invention are
It was found that it had the same or better barrier ability than DPPC.

Further, a compound which is a synthetic intermediate of compound (12), (12
Using a), liposome formation was attempted in a similar manner, but liposomes containing CF could not be produced. (There is no fraction corresponding to the liposome in the gel filtration step.) This result suggests that the peptide bond contained in the compound of the present invention contributes to the stabilization of the liposome.

[Example 6] The liposome containing CF prepared using compound 1 and prepared in Example 4 was incubated at 4 ° C. The CF-encapsulated liposome prepared from DPPC precipitated after 20 days when stored at 4 ° C., but the liposome using Compound 1 maintained a stable dispersed form even after 4 months or more. The leakage of CF after 60 days was only 1.1%.

[Example 7] In Example 4, compound 4 was used instead of compound 1 to prepare liposomes containing CF. Compound 4
Add 20% and 50% cholesterol in molar ratio to
Similarly, liposomes containing CF were prepared. These liposome solutions were incubated at 37 ° C., and the leaked CF was quantified by a fluorescence method. Table 4 shows the leakage amount after one hour.

From Table 4, it was found that the addition of cholesterol significantly improved the barrier ability of the liposome formed by the compound of the present invention. The liposome to which 50% cholesterol was added was incubated at 4 ° C., but the stable dispersed form was maintained for 2 months or more, and the leakage of CF after 60 days was 1% or less.

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Claims (2)

(57) [Claims] A compound represented by the following general formulas (I) to (III): In the formula, R ¹ and R ² are a linear or branched alkyl group or acyl group having 8 to 24 carbon atoms, and may have a substituent or an unsaturated group. R ³ⁿ , R ^{3 (n + 1)} , R ^3m , and R ^{3 (m + 1)} each represent a side chain residue of an α-amino acid. In the formula (I), n represents an integer of 1 to 5, and m represents an integer of 0 to 5. In the formulas (II) and (III), n and m represent 0 to 5
Represents an integer. As for the asymmetric carbon present in the molecule,
Any of optically active substances may be used. In addition, the carboxy group at the molecular terminal may form a salt with an appropriate cation component. 2. The compounds of the general formulas (I) to (II)
A liposome containing the compound represented by I) as a membrane component.