JP3521925B2 - Phospholipid derivatives - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel phospholipid derivative and a liposome using the same, and more particularly to a phosphatidylethanolamine derivative stabilized by having a maleimidocaproyl group and the same. It relates to liposomes.

[0002]

BACKGROUND OF THE INVENTION Liposomes, which are lipid membrane microspheres, can contain many substances, not limited to water solubility and hydrophobicity.
In particular, it is of great interest as a carrier for drug delivery systems (DDS). In recent years, attempts have been made to bind (introduce) functional compounds such as proteins, peptides, sugars and hydrophilic polymers to the liposome surface for the purpose of imparting functionality in addition to the original properties of the liposome.

[0003] For imparting such functionality, a technique for binding a phospholipid on a liposome to such a functional compound is a necessary and important factor, and many methods have been proposed. For example, when trying to bind (introduce) a protein or peptide, N- (3- (2-pyridyldithio) propionyl) is utilized by utilizing the reactivity of the thiol group of the cysteine residue contained therein.
Phosphatidylethanolamine (PDP-PE; N
ature, 288 , 602 (1980), Bioch.
EMISTRY, 20 , 4229 (1981)), a method of introducing an S-S bond into the phospholipid itself and forming a strong S-S bond between this and the thiol group of the protein or peptide, N- (M-maleimidobenzoyl)
Dipalmitoylphosphatidylethanolamine (M
B-PE; Cancer Research, 43 , 5
328 (1983)), N- (4- (p-maleimidophenyl) butyryl) phosphatidylethanolamine (MPB-PE; The Journal of Bi).
logical Chemistry, 257 , 28
6 (1982)), a substituent having a highly reactive double bond such as a maleimide group is introduced into a phospholipid to form a strong thioether bond between this and a thiol group of a protein or peptide. Method etc. Of these, the latter method is being studied more because of its advantages such as less aggregate formation, less activity reduction when the protein or peptide is an antibody, and no limitation on the encapsulated drug.

However, on the other hand, in the case of liposomes containing 4- (p-maleimidophenyl) butyryl group, leakage of the encapsulated substance is caused (Biochemistr).
y, 25 , 5693 (1986)), and the stability of the maleimide group is not high (J. Immunol. Meth.
od, 132 , 25 (1990)) and the like, and the present situation is that a sufficiently satisfactory liposome has not been obtained.

[0005]

Means for Solving the Problems As a result of studies to solve the problems of the conventional phospholipid-maleimide derivative, the present inventors have found that a phosphorus consisting of an ε-maleimidocaproic acid moiety and a phosphatidylethanolamine moiety. It was found that a lipid derivative (hereinafter sometimes abbreviated as "MC-PE") is extremely stable as a component constituting a liposome, and the present invention has been completed.

That is, the gist of the present invention resides in a phospholipid derivative represented by the following general formula (I) and a liposome containing the same.

[0007]

[Chemical 2]

(In the formula, R ¹ and R ² each independently represent a C ₁₁ -C ₁₉ alkyl group or an alkenyl group.) The present invention is described in detail below. The phospholipid derivative of the present invention is represented by the above general formula (I). Examples of the C _{11 to} C ₁₉ alkyl group for R ¹ and R ² include lauroyl group, myristoyl group, palmitoyl group, stearoyl group and the like, and examples of the C _{11 to} C ₁₉ alkenyl group include oleyl group and the like. In the present invention, C _{13 to} C ₁₇
Is preferable, and a palmitoyl group is particularly preferable.

Next, the method for producing the phospholipid derivative of the present invention will be described. The phospholipid derivative of the present invention can be synthesized, for example, by the method (1) or (2) shown below. (1)

[0010]

[Chemical 3]

(Wherein R ¹ and R ² are the same as defined above) (II) N- (ε-maleimidocaproyloxy) succinimide represented by the formula (hereinafter sometimes abbreviated as “EMCS”) Phosphatidylethanolamine represented by the formula (III) is dissolved in an organic solvent such as methanol and chloroform-methanol (1/1 to 10/1) in the presence of a basic compound such as triethylamine and pyridine, and 20 The reaction is carried out at -40 ° C under the condition of an inert gas such as nitrogen gas or argon gas.

(2)

[0013]

[Chemical 4]

(Wherein R ¹ and R ² are as defined above) (IV) ε-maleimidocaproic acid of the formula (I)
II) with phosphatidylethanolamine of the formula (1) in a conventional peptide synthesis reaction (eg protein chemistry 1,
5.3 Peptide bond formation reaction, condensation reaction by carbodiimide method, azide method, isocyanate method, etc. described in Kyoritsu Shuppan Co., Ltd. (1969).

The phospholipid derivative of the present invention thus obtained can be purified by a conventional separation / purification means such as thin layer chromatography and silica gel chromatography. Furthermore, MC-PE can be made into liposome using a known method. For example, phosphatidylcholine, cholesterol, and MC-PE of the present invention can be used as constituents, and phosphatidic acid such as dipalmitoylphosphatidic acid can be added if necessary.
Specifically, dipalmitoylphosphatidylcholine (DPPC), cholesterol (Chol) and MC
-A liposome composed of PE. The use ratio of each component is such that cholesterol is 0.3 to 1 mol, preferably 0.4 to 0.6 mol, and MC-PE is 0.005 to 0.
The composition ratio is 3 mol, preferably 0.01 to 0.1 mol, and when phosphatidic acid is added, the composition ratio is 0.4 mol or less, preferably 0.15 mol or less.

Next, for example, a lipid mixture obtained by removing the solvent is hydrated, emulsified with a homogenizer or the like, and then freeze-thawed to obtain a multilamellar liposome. In addition, ultrasonic treatment, high-speed homogenization, or pressure filtration with a membrane having a uniform pore size (Biochimica)
et Biophysica Acta, 812 , 55.
(1985)) or the like to adjust to an appropriate particle size. The preferable particle size at this time is 10 to 300 n.
m, more preferably 30 to 200 nm.

The liposome can also encapsulate various drugs. Examples of the drug include anti-cancer agents such as adriamycin, daunomycin, mitomycin, cisplatin, vincristine, epirupicin, methotrexate, 5-fluorouracil, aclacinomycin, and aminoglycosides such as gentamicin and β-such as sulpenicillin.
Lactam antibiotics, ricin A, toxins such as diphtheria toxin, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), ra
Antisense RNA for the s gene and the like can be mentioned.
To encapsulate these drugs in liposomes, in the case of water-soluble drugs, by hydrating the lipid with an aqueous drug solution,
In the case of a fat-soluble drug, once the drug and lipid are mixed in a volatile organic solvent and the solvent is distilled off,
It can be embedded within liposomes by hydrating the lipid mixture.

For the purpose of imparting functionality to the liposome, proteins, peptides, and
It is preferable to bond (introduce) sugar, hydrophilic polymer and the like. In the present invention, it is preferable to bind (introduce) various antibodies, various growth factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF), or proteins such as growth factors, and particularly preferable are antibodies. Is. The antibody is an antibody (IgA, IgG, IgM, etc.) having reactivity with a tissue, cell, bacterial virus or the like to be treated, and is a polyclonal antibody of various animals, a mouse monoclonal antibody,
A human-mouse chimeric antibody, a human monoclonal antibody and the like can be used.

It is desirable to utilize a thiol group for binding these proteins to the liposome. Specifically, N-succinimidyl-3- (2-pyridyldithio) propionate (Biochem. J., 173 ), which is usually used for thiolation of proteins with respect to amino groups of proteins, is used.
723 (1978)), iminothiolane, and mercaptoalkylimidate (Biochemistry, 12 ,
3266 (1973)) and the like,
When the protein is an antibody, a method in which the dithiol group of the internal cysteine residue is reduced to a thiol group can be used. When IgG is used among the antibodies, it is preferable that the thiol group produced in Fab ′ obtained by converting it into F (ab ′) ₂ by an enzymatic treatment with pepsin or the like and further reducing it with dithiothreitol or the like is used for binding to the liposome. (Bi
chemistry, 20 , 4229 (198)
1)). In the case of IgM, the method of Miller et al. (J. Bio
l. Chem. , 257 , 286 (1965)), the thiol group of the Fc portion of IgMs obtained by reducing the J chain under mild conditions is preferably used for binding to liposomes. The binding between the liposome and the protein is achieved by reacting in a suitable neutral buffer solution (pH 6.5 to 7.5) for about 2 to 16 hours.

When the liposome of the present invention is used as a pharmaceutical composition, it can be administered to various diseases by intravascular administration, intravesical administration, intraabdominal administration, local administration and the like. The dose can be appropriately adjusted depending on the desired pharmacological action, type of drug, and the like.

[0021]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the invention is not limited thereto unless it exceeds the gist. Example 1 Dipalmitoylphosphatidylethanolamine 1
27 mg, N- (ε-maleimidocaproyloxy) succinimide (EMCS; Dojin Kagaku Co., Ltd.) 80 mg and triethylamine 44 μl were added to a chloroform / methanol solution (5/1), and the mixture was reacted under a nitrogen stream. Three
After an additional 20 mg of EMCS was added at room temperature for 3
Reacted for hours.

After confirming that the reaction solution had a negative ninhydrin reaction and no amine (dipalmitoylphosphatidylethanolamine) was detected, the reaction solution was dried under reduced pressure and redissolved in a small amount of chloroform. Saturated NaCl
After washing several times with, the separated chloroform layer was dried over sodium sulfate, and separated and purified with a silica gel column. Chloroform / methanol was stepwise eluted to obtain a target product (a compound of the general formula (I) in which both R1 and R2 represent palmitoyl groups: MC-DPPE) with a yield of 70%.

The synthesized MC-DPPE showed a single spot by TLC. The structure is ¹³ C-NMR and ¹ H
-Confirmed by NMR (see Table 1).

[0024]

[Table 1]

[0025]

[Chemical 5]

Example 2 Dipalmitoylphosphatidylethanolamine 1
00 mg, maleimidocaproic acid 44.4 mg (manufactured by Sigma), dicyclohexylcarbodiimide 43.3 m
g and hydroxybenzotriazole 32.2 mg
Was added to 20 ml of chloroform and mixed at 0 ° C. for 1 hour. Then, the mixture was stirred and reacted for 20 hours. This was fractionated using a silica gel column to obtain MC-DPPE. The yield was 38%. Example 3 Dipalmitoylphosphatidylcholine (DPPC),
A chloroform solution composed of cholesterol (Chol) and MC-DPPE (molar ratio 18/10 / 0.5) (total lipid amount: 50 mg) was added to an eggplant-shaped flask, and the solvent was distilled off by a rotary evaporator. To the resulting lipid film was added 1 ml of 10 mM phosphate buffer solution pH 7.4 (PBS) containing 0.15 M NaCl, and 60
Violently permeated under heating at ℃. The particles were further sized using EXTRUDER ^™ (manufactured by NOF Liposome Co., Ltd.) equipped with a 200 nm membrane filter to obtain MC-DPPE-containing liposomes.

After incubating the prepared liposomes at 37 ° C. for each time (after 1, 2, 4, 8 and 24 hours), the amount of maleimide group was measured. For the maleimide group, a known amount of cysteine was added to the liposome, and after reacting at room temperature for 15 minutes, the liposome was separated by ultracentrifugation, and the amount of residual cysteine in the supernatant was quantified with 4,4'dithiopyridine. Was calculated as.

As a result, the amount of maleimide in the liposome of the present invention containing MC-DPPE was stable even after 24 hours. Comparative Example In the same manner as in Example 3 except that MC-DPPE was changed to MB-PE, the amount of maleimide in the liposome prepared in the same manner as above was measured in the same manner, and it was undetectable after 8 hours. Example 4 1 ml of 0.1 M carboxyfluorescein solution was added to the lipid film prepared in the same manner as in Example 3 to prepare liposomes in the same manner. After incubating the liposomes,
The liposomes were separated by ultracentrifugation, and the amount of carboxyfluorescein in the supernatant generated by leak (leakage of inclusions) was measured by fluorescence. As a result, it was shown that the leak amount was 3% or less even at 45 ° C. near the phase transition temperature of DPPC. Example 5 Mouse monoclonal antibody (IgG ₁ ) was added to a 1/40 mol amount of pepsin (C) in 0.1 M acetate buffer pH 3.5.
37), and then add 37
The mixture was reacted overnight at 0 ° C. and cleaved into F (ab ′) ₂ .

Further, a cation exchange resin (mono S,
Chromatographic separation by Pharmacia Co. F (a
b ') ₂ was isolated. That is, 0.1 M acetate buffer p
A linear gradient of 0 to 1.0 M NaCl in H4.0 was used. In addition, 0.1% MCl containing 0.1.
Fab ′ was obtained by adding 12 μl of 10% DTT to 1 mg of antibody in 1 M acetate buffer pH 4.5 and reducing at room temperature for 80 minutes. After the reaction, the mixture was desalted with a gel filtration column (PD-10 Pharmacia Co., Ltd.) equilibrated with PBS and used for liposome binding.

With the same composition as in Example 3 above, the total amount of lipids was 10
5 mg of F was added to the liposome obtained as 0 mg.
Ab ′ was added and reacted at 37 ° C. for 8 hours to prepare an immunoliposome. The obtained liposomes were subjected to gel filtration with a Sepharose CL-6B column (Pharmacia) to remove unreacted antibody, and then subjected to SDS-polyacrylamide gel electrophoresis to confirm antibody binding.

[0031]

The phospholipid derivative of the present invention can easily and stably bind (introduce) a functional compound such as a protein or peptide when forming a liposome using the phospholipid derivative.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-5-196624 (JP, A) JP-A-1-226892 (JP, A) Special Table Shou 62-501800 (JP, A) Patent 3236667 (JP, A) B2) (58) Fields surveyed (Int.Cl. ⁷ , DB name) CA (STN) REGISTRY (STN)

Claims (5)

(57) [Claims] 1. A phospholipid derivative represented by the following general formula (I). [Chemical 1] (In the above formula, R1 and R2 are each independently C
₁₅ represents a palmitoyl group) 2. A liposome comprising the phospholipid derivative according to claim 1. 3. The liposome according to claim 2, wherein a protein is bound to the surface of the liposome. 4. The liposome according to claim 3, wherein the protein is an antibody. 5. The liposome according to any one of claims 2 to 4, wherein a hydrophilic polymer is bound to the surface of the liposome.