JP4723380B2 - Polyethylene glycol derivative and drug carrier comprising this as a membrane constituent - Google Patents

Description

  The present invention relates to a novel polyethylene glycol derivative and a drug carrier containing this as one of membrane constituents.

  In recent years, research on drug delivery systems (DDS) that efficiently distribute drugs to target organs has become active. For example, a method of using closed vesicles such as liposomes, emulsions, lipid microspheres, and nanoparticulates as a drug carrier, a method of including or binding a drug to a polymer carrier such as a polymer synthetic polymer micelle or polysaccharide, In addition, these closed vesicles and polymer carriers can be modified with high-molecular functional molecules such as antibodies and proteins, or with low-molecular functional molecules such as specific sugar chains and peptides to increase target directivity. (Cancer Letters, USA, 1997, Vol. 118, No. 2, p. 153; British Journal of Cancer, UK, 1997, Vol. 76, No. 1) , P.83).

However, when these drug carriers (hereinafter also referred to as “drug carriers”) are put to practical use, there are various problems to be overcome. Among them, avoidance from the foreign body recognition mechanism on the living body side, and control of pharmacokinetics. Difficulties are a problem. In particular, closed vesicles are aggregated by interaction with opsonin proteins and plasma proteins in the blood and captured by reticuloendothelial tissues (RES) such as the liver and spleen. It was a difficult situation for highly selective delivery.
As a means to solve the above problems, the surface of the polymer carrier including these closed vesicles is coated with a hydrophilic polymer such as polyethylene glycol (PEG) to adsorb plasma proteins and opsonin proteins. It is possible to prevent the increase in blood stability and avoid the capture by RES.

However, commercially available PEG lipids used for such purposes are phospholipid derivatives (PEG-PE) consisting of polyethylene glycol and diacylphosphatidylethanolamine, which are negatively charged by the phosphate group moiety. is doing. Therefore, the negative surface charge of the drug carrier can reduce the interaction with the cells (Miller et al., Biochemistry, 1998, 37, p. 12875). .
In addition, in order to improve the adhesion of the drug carrier to the target cells, a cationized lipid such as stearylamine is compounded, but when a negatively charged PEG-PE is compounded, It will reduce the effect.

  In order to solve such problems, PEG lipids having no charge that can be used for the above-mentioned purpose have been reported (see Japanese Patent Publication Nos. 10-506622 and 2003-505401). However, these PEG lipids are expensive to produce due to their complex structures such as the inclusion of asymmetric molecules in the molecular structure. Therefore, a PEG lipid having a simpler structure is eagerly desired as a PEG lipid having no charge that can be used for the above purpose.

  Therefore, the present invention enables drug targeting to a target site reliably, efficiently and safely, and is effective as a DDS preparation, and a novel charge constituting a membrane component of the drug carrier It aims at providing the polyethyleneglycol derivative which does not have this.

In order to solve the above problems, the present invention provides the following (1) to (22).
(1) A polyethylene glycol derivative represented by the following general formula (a).

In the formula (a), A is an aromatic ring, and R ¹ and R ² are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms. X and Y are each independently O, S, COO, OCO, CONH or NHCO, and m is a natural number from 1 to 6 . n represents a natural number from 10 to 300. L is OCONH (carbamate bond) or OCO (ester bond), and B is a hydroxyl group, an alkoxy group or a benzyloxy group.
(2) The polyethylene glycol derivative according to (1), wherein A in the general formula (a) is a benzene ring.
(3) The polyethylene glycol derivative of (1) or (2), wherein, in the general formula (a), R ¹ and R ² are each independently an alkyl group or alkenyl group having 12 to 18 carbon atoms.
(4) The polyethylene glycol derivative according to any one of (1) to (3), wherein in the general formula (a), X and Y are both O.
(5) In the general formula (a), A is a benzene ring, R ¹ and R ² are alkyl groups having 15 carbon atoms, X and Y are O, and m is 1. The polyethylene glycol derivative according to any one of (4).

(6) A polyethylene glycol derivative represented by the following general formula (b).

In the formula (b), A ′ is an aromatic ring, and R ³ and R ⁴ are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms. p represents 0 or a natural number from 1 to 6. q represents a natural number from 10 to 300. L ′ is OCONH (carbamate bond) or OCO (ester bond), and B ′ is a hydroxyl group, an alkoxy group or a benzyloxy group.
(7) The polyethylene glycol derivative according to (6), wherein A ′ in the general formula (b) is a benzene ring.
(8) The polyethylene glycol derivative according to (6) or (7), wherein, in the general formula (b), R ³ and R ⁴ are each independently an alkyl group or alkenyl group having 12 to 18 carbon atoms.
(9) In the above general formula (b), A ′ is a benzene ring, R ³ and R ⁴ are each an alkyl group having 18 carbon atoms, and p is 1, and any one of (6) to (8) Polyethylene glycol derivative.

(10) A drug carrier comprising the polyethylene glycol derivative according to any one of (1) to (9) as one of the constituent components.
(11) The drug carrier according to (10), wherein the drug carrier is at least one selected from the group consisting of liposomes, lipid microspheres, and polymer microspheres.
(12) The drug according to (10) or (11), wherein the drug carrier contains at least one selected from the group consisting of phospholipids and derivatives thereof, lipids and derivatives other than phospholipids, cholesterols and surface modifiers. Carrier.
(13) The drug carrier according to any one of (10) to (12), further comprising at least one selected from the group consisting of a stabilizer and an antioxidant.
(14) The supported drug is a nucleic acid, polynucleotide, gene and analog thereof, anticancer agent, antibiotic, enzyme agent, enzyme inhibitor, antioxidant, lipid uptake inhibitor, hormone agent, anti-inflammatory agent, steroid agent , Vasodilator, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, smooth muscle cell proliferation and / or migration inhibitor, platelet aggregation inhibitor, anticoagulant, chemical mediator release inhibitor, vascular endothelial cell proliferation Or inhibitors, aldose reductase inhibitors, mesangial cell growth inhibitors, lipoxygenase inhibitors, immunosuppressants, immunostimulators, antiviral agents, Maillard reaction inhibitors, amyloidosis inhibitors, NOS inhibitors, AGEs (Advanced glycation endproducts ) Inhibitors, radical scavengers Glycosaminoglycans and derivatives thereof, oligosaccharides and / or polysaccharides and derivatives thereof is at least one selected from the group consisting of proteins and peptides (10) to either drug carrier (13).
(15) The carried drug is at least one in-vivo diagnostic agent selected from the group consisting of an X-ray contrast agent, a radioisotope-labeled nuclear medicine diagnostic agent, and a diagnostic agent for nuclear magnetic resonance diagnosis (10) to (14 ) Any drug carrier.

(16) A pharmaceutical composition comprising the drug carrier according to any one of (10) to (15).
(17) A method for delivering a drug carried on a drug carrier to a target site, comprising administering the drug carrier of any one of (10) to (15) to a host.
(18) A method for transporting a drug carrier to a target site, comprising administering the drug carrier of any one of (10) to (15) to a host.
(19) A method for the prophylaxis and / or treatment of a disease, comprising administering an effective amount of a drug carrier according to any one of (10) to (15) to a host.
(20) A method for diagnosing a disease, comprising administering an effective amount of the drug carrier according to any one of (10) to (13) or (15) to a host.
(21) A composition for delivering a drug carried on a drug carrier to a target site, comprising an effective amount of the drug carrier of any one of (10) to (15).
(22) A composition for transporting a drug carrier to a target site, comprising an effective amount of the drug carrier of any one of (10) to (15).

According to the present invention, a novel non-charged polyethylene glycol derivative that can be used as a membrane constituent of a drug carrier is provided. A drug carrier containing the polyethylene glycol derivative of the present invention as one of the membrane constituents is excellent in blood retention. Moreover, the drug carrier containing the polyethylene glycol derivative of the present invention and a cationized lipid as constituents has good binding properties to target cells as shown in Examples and Test Examples described later.
From these characteristics, the drug carrier containing the polyethylene glycol derivative of the present invention as one of the membrane constituents and the pharmaceutical composition containing the drug carrier have an excellent effect in the treatment and / or diagnosis of diseases. .

FIG. 1 is a graph comparing the amount of binding of the liposomes prepared in Example 2 and Reference Example 1 (modification rate (the ratio of the amount of PEG derivative to the total lipid amount) 1%) to cells in Test Example 1. . FIG. 2 is a graph comparing the amount of binding of the liposomes prepared in Example 4 and Reference Example 2 (modification rate (the ratio of the amount of PEG derivative to the total amount of lipid) 1%) to cells in Test Example 2. .

Hereinafter, the polyethylene glycol derivative of the present invention (hereinafter also referred to as “PEG derivative”) and a drug carrier containing the PEG derivative as one of membrane constituent components will be described in detail.
In the present invention, a polyethylene glycol derivative (PEG derivative (A)) represented by the following general formula (1) is provided.

In formula (1), A is an aromatic ring. Here, as the aromatic ring, in addition to the benzene ring, a condensed ring such as indene, naphthalene, tetralin, anthracene, and phenanthrene may be used. However, a benzene ring is preferable.
R ¹ and R ² are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms, and preferably are independently an alkyl group or alkenyl group having 12 to 18 carbon atoms.
X and Y are independently of each other O, S, COO, OCO, CONH or NHCO, and preferably both X and Y are O.
m is a natural number from 1 to 6, and preferably a natural number from 1 to 3.
n represents a natural number from 10 to 300, preferably n is a natural number from 20 to 200, more preferably a natural number from 30 to 150.
L is OCONH ₂ (carbamate bond) or OCO (ester bond), preferably a carbamate bond.
B is a hydroxyl group, an alkoxy group or a benzyloxy group, preferably a methoxy group.

  Therefore, in the PEG derivative (A) represented by the general formula (1), it is preferable that A is a benzene ring, B is a methoxy group, X and Y are both O, and m is 1. Specific examples of such a PEG derivative (A) include monomethoxy polyethylene glycol-dialkoxybenzyl carbamate.

Furthermore, in the PEG derivative (A) represented by the general formula (1), A is a benzene ring, R ¹ and R ² are both alkyl groups having 15 carbon atoms, and X and Y are both O. , M is preferably 1. Specific examples of such a PEG derivative (A) include monomethoxy polyethylene glycol-3,5-dipentadecyloxybenzyl carbanate represented by the following structural formula.

The PEG derivative (A) represented by the general formula (1) is a compound represented by the following general formula (4) (Appl. Biochem. Biotech.) On the amino group of the compound represented by the following general formula (3), for example. 11, 141) can be produced.


In formula (3), A is an aromatic ring, and R ¹ and R ² are each independently an alkyl or alkenyl group having 10 to 25 carbon atoms. X and Y are each independently O, S, COO, OCO, CONH or NHCO, and l represents a natural number from 1 to 6 .
In formula (4), n represents a natural number from 10 to 300. B is a hydroxyl group, an alkoxy group or a benzyloxy group.

The compound of the general formula (3) can be synthesized by a general synthesis method. For example, as described below, an ether compound (X, Y = O) is obtained by esterifying the carboxylic acid moiety of a dihydroxy aromatic ring carboxylic acid derivative, then introducing an alkyl group or an alkenyl group through an ether bond, and further adding an ester moiety. After reduction to a hydroxyl group, it can be synthesized by converting the hydroxyl group to an amino group.

Here, in the formula (5), A is an aromatic ring, and m is 0 or a natural number from 1 to 5. In formula (6), A is an aromatic ring, R ¹ and R ² are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms, and m is 0 or a natural number from 1 to 5. In formula (7), A is an aromatic ring, R ¹ and R ² are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms, and l represents a natural number from 1 to 6 .

The compound of the general formula (4) can be synthesized by a general method. For example, it can be synthesized by the method of Veronese et al. (Applied Biochemistry and Biotechnology 11, 141, 1985).
Polyethylene glycol used as a raw material for synthesizing the compound of the general formula (4) can be purchased commercially. In the formula (4), n is a natural number from 10 to 30, and therefore the number average molecular weight of polyethylene glycol. Is from about 500 to about 13000. Hereinafter, the term “average molecular weight” refers to the number average molecular weight. Moreover, the number average molecular weight refers to the average molecular weight based on the mole fraction of each component molecule.

The PEG derivative (A) of the general formula (1) synthesized by the above procedure can be isolated and collected by ordinary purification means such as chromatography and recrystallization. In addition, the manufacturing method and purification method of PEG derivative (A) of General formula (1) are not limited above.
Since the PEG derivative (A) of the general formula (1) has no charge, it is preferably used as a membrane constituent of a drug carrier.

The present invention also provides a polyethylene glycol derivative represented by the following general formula (2) (hereinafter also referred to as “PEG derivative (B)”).

In the formula (2), A ′ is an aromatic ring. Here, as the aromatic ring, in addition to the benzene ring, a condensed ring such as indene, naphthalene, tetralin, anthracene, and phenanthrene may be used. However, a benzene ring is preferable.
R ³ and R ⁴ are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms, and preferably are independently an alkyl group or alkenyl group having 12 to 18 carbon atoms.
p is 0 or a natural number from 1 to 6, and preferably a natural number from 1 to 3.
q represents a natural number from 10 to 300, preferably n is a natural number from 20 to 200, more preferably a natural number from 30 to 150.
L ′ is OCONH ₂ (carbamate bond) or OCO (ester bond), preferably a carbamate bond.
B ′ is a hydroxyl group, an alkoxy group or a benzyloxy group, preferably a methoxy group.

Therefore, in the PEG derivative (B) represented by the general formula (2), A ′ is a benzene ring, B ′ is a methoxy group, R ³ and R ⁴ are alkyl groups independent of each other, p Is preferably 1. Specific examples of such an amide compound include 4- (N ′-(methoxypolyethyleneglycolcarbonyl) 2-aminoethyl) N, N-dialkylbenzamide.

Furthermore, in the amide compound represented by the general formula (2), it is preferable that A ′ is a benzene ring, R ³ and R ⁴ are both alkyl groups having 18 carbon atoms, and p is 1. Specific examples of such an amide compound include 4- (N ′-(methoxypolyethyleneglycolcarbonyl) 2-aminoethyl) N, N-distearoylbenzamide represented by the following structural formula.

The PEG derivative (B) represented by the general formula (2) is produced, for example, by introducing a compound represented by the following general formula (9) into the amino group of the compound represented by the following general formula (8). can do.


In formula (8), A ′ is an aromatic ring, and R ³ and R ⁴ are each independently an alkyl or alkenyl group having 10 to 25 carbon atoms. p represents 0 or a natural number from 1 to 6.
In formula (9), q represents a natural number from 10 to 300. B ′ is a hydroxyl group, an alkoxy group or a benzyloxy group.

The compound of the general formula (8) can be synthesized by a general synthesis method. For example, amino compounds as follows as an example, aminoalkyl aromatic carboxylic acid derivative to an amino group suitable protecting group (Theodora W.Greene of, Peter G.M.Wuts book "Protective Groups in Organic Synthesis" 3 ^rd edition After protecting with John Wiley & Sons Inc), it can be synthesized by forming an amide bond by the condensation reaction of the carboxylic acid portion of the aminoalkyl aromatic ring carboxylic acid derivative and the alkylamine derivative, and then deprotecting the amino group .

Here, in Formula (10), A ′ is an aromatic ring, and p is 0 or a natural number from 1 to 5. In formula (11), A ′ is an aromatic ring, p is 0 or a natural number from 1 to 5, and P represents a protecting group. In formula (12), R ³ and R ⁴ each independently represent an alkyl group or alkenyl group having 10 to 25 carbon atoms. In formula (13), A ′ is an aromatic ring, R ³ and R ⁴ are each independently an alkyl or alkenyl group having 10 to 25 carbon atoms, and p is 0 or a natural number from 1 to 5.

  In the reaction of a carboxylic acid derivative and an amine derivative, examples of the carboxylic acid activator include thionyl chloride, phosphorus pentachloride, chloroformate (methyl chloroformate, ethyl chloroformate), oxalyl chloride, carbodiimides (for example, N, N '-Dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (WSC)), benzotriazol-1-yl-oxy-tris (dimethylamino) -phosphonium hexafluorophosphate (BOP), etc. Can be given. At this time, carbodiimides and N-hydroxybenzotriazole, 4-dimethylaminopyridine or hydroxysuccinimide may be used in combination. This reaction is usually carried out by halogenated hydrocarbons such as methylene chloride and chloroform, ethers such as tetrahydrofuran (THF), dioxane, dimethyl ether, diethyl ether, isopropyl ether, N, N-dimethylformamide, N, N-dimethylacetamide. Alternatively, it is carried out in the presence of a mixed solvent thereof. The reaction temperature is usually −10 ° C. to 50 ° C.

The compound of the general formula (9) can be synthesized by a general method. For example, it can be synthesized by the method of Veronese et al. (Applied Biochemistry and Biotechnology 11, 141, 1985).
Polyethylene glycol used as a raw material for synthesizing the compound of the general formula (9) can be purchased commercially. In the formula (9), q is a natural number from 10 to 30, and therefore the number average molecular weight of polyethylene glycol. Is from about 500 to about 13000. Hereinafter, the term “average molecular weight” refers to the number average molecular weight. Moreover, the number average molecular weight refers to the average molecular weight based on the mole fraction of each component molecule.

The PEG derivative (B) of the general formula (2) synthesized by the above procedure can be isolated and collected by ordinary purification means such as chromatography and recrystallization. In addition, the manufacturing method and purification method of PEG derivative (B) of General formula (2) are not limited above.
Since the PEG derivative (B) of the general formula (2) has no charge, it is preferably used as a membrane constituent of a drug carrier.

  The drug carrier of the present invention contains the PEG derivative (A) represented by the above general formula (1) or the PEG derivative (B) represented by the general formula (2) as one of membrane constituent components. In the present invention, the drug carrier refers to a small spherical structure having lipid as a membrane basic constituent material and capable of carrying a drug for diagnosis and / or treatment of a disease.

  The content of the PEG derivative (A) of the formula (1) or the PEG derivative (B) of the formula (2) in the drug carrier of the present invention is usually 0.1 to 20 mol in a ratio to the total lipid amount constituting the drug carrier. %, Preferably 0.1 to 5 mol%, more preferably 0.5 to 5 mol%. The total lipid amount is the amount of all lipids constituting the drug carrier, and the amount of the surface modifier described later is also included in the total lipid amount. The total lipid means a membrane component of the drug carrier excluding the PEG derivative (A) of the formula (1) or the PEG derivative (B) of the formula (2), and is expressed in molar concentration (mM). In other words, the total lipid includes phospholipids, lipids other than phospholipids, or cholesterols that form the basic membrane constituent material described later, and also includes a surface modifier when it contains a surface modifier. Is expressed in molarity. In the present invention, the ratio of the amount of the PEG derivative (A) or (B) to the total amount of lipid is sometimes referred to as “modification rate”.

  The drug carrier of the present invention contains the PEG derivative (A) of the above formula (1) or the PEG derivative (B) of the formula (2) as one of the components, and other components are not limited. As other constituent components, lipids and derivatives thereof as a membrane basic constituent material as described later, other surface modifiers may be included, and other components such as stabilizers and antioxidants may also be included. . On the other hand, the drug carrier of the present invention may contain only the PEG derivative (A) of the above formula (1) or the PEG derivative (B) of the formula (2) as a constituent component. In addition, only the PEG derivative (A) or (B) and the membrane basic constituent material can be contained as constituent components. In that case, it is possible to contain only phospholipids, only lipids other than phospholipids, or only cholesterols as membrane basic constituent materials, or only combinations thereof.

The form of the drug carrier of the present invention is not particularly limited as long as it has a structure capable of supporting a drug, and can take various forms. Specifically, it is desirable to be composed of at least one selected from liposomes, lipid microspheres and polymer microspheres having a potential function capable of encapsulating a drug at a high concentration.
Among these, a preferable embodiment is a closed vesicle having a structure that forms a space separated from the outside by a membrane formed based on the polarity of the hydrophobic group and the hydrophilic group of the lipid molecule. Can be mentioned.

Liposomes are closed vesicles consisting of a lipid bilayer based on phospholipids. A lipid microsphere is a closed vesicle formed by emulsifying one of water and oil in the other. The polymer microsphere is a closed vesicle formed by emulsifying a polymer and then solidifying it by operations such as modification, chemical crosslinking, radiation polymerization, and drying in liquid.
In the present invention, a particularly preferred form of the drug carrier is a liposome.

  The size of the drug carrier of the present invention is not particularly limited, but when it takes a spherical shape or a form close thereto, the outer diameter of the particle is 0.02-250 μm, preferably 0.03-0.4 μm, more preferably Is preferably 0.05 to 0.2 μm. The diameter of the particle outer diameter is an average value of the diameters of all the drug carrier particles measured by the light scattering method.

  The other components constituting the drug carrier together with the PEG derivative (A) of the above formula (1) or the PEG derivative (B) of the formula (2) are not particularly limited as long as the above-mentioned form can be stably formed. However, in view of safety, in vivo stability, and the like, the drug carrier desirably contains at least one selected from lipids, derivatives thereof, and surface modifiers as a membrane component. In particular, it is usually preferable to contain lipid as the basic constituent material of the membrane. Lipids include phospholipids, lipids other than phospholipids, or cholesterols, and may be derivatives thereof.

Phospholipids include phosphatidylcholine (= lecithin), phosphatidylglycerol, phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardioribine, etc. Examples thereof include natural or synthetic phospholipids, or those hydrogenated according to these conventional methods.
Although it does not specifically limit as cholesterol, Cholesterol, cholestanol, etc. can be mentioned.
Examples of lipids other than phospholipids include lipids that do not contain phosphoric acid, and are not particularly limited, and examples thereof include glyceroglycolipids and sphingoglycolipids.

  The surface modifier is used for imparting desired characteristics to the membrane of the drug carrier by changing the structure and physical properties of the lipid that is the membrane component of the drug carrier. Accordingly, the surface modifier can be used as one of the membrane constituents of the drug carrier. Although it does not specifically limit as a surface modifier, The substance etc. which consist of a lipid and the compound couple | bonded with the lipid are mentioned. The “compound binding to lipid” is not particularly limited, and examples thereof include a hydrophilic polymer, a derivative of a water-soluble polysaccharide, or a compound having a basic functional group.

  Examples of the “hydrophilic polymer” include, but are not limited to, polyethylene glycol, Ficoll, polyvinyl alcohol, styrene-maleic anhydride alternating copolymer, divinyl ether-maleic anhydride alternating copolymer, and synthetic polyamino acid. “Derivatives of water-soluble polysaccharides” include, but are not limited to, glucuronic acid, sialic acid, dextran, pullulan, amylose, amylopectin, chitosan, mannan, cyclodextrin, pectin, carrageenan and the like. When the surface modifier contains a hydrophilic polymer or a water-soluble polysaccharide, the lipid to which the hydrophilic polymer or water-soluble polysaccharide binds is not particularly limited, but a compound having a hydrophobic region (hydrophobic compound) is used. be able to. The compound having a hydrophobic region is not particularly limited, and examples thereof include a long-chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl, or a glycerin fatty acid ester. For example, when the hydrophilic polymer is polyethylene glycol and the hydrophobic compound is distearoylphosphatidylethanolamine, the surface modifier is polyethylene glycol-distearoylphosphatidylethanolamine.

  For example, when the drug carrier is a liposome, for example, when a surface modifier containing a hydrophobic compound is used, at least the hydrophobic region of the hydrophobic compound is stabilized in the membrane of the lipid bilayer of the liposome. Is done. As a result, the hydrophilic polymer or water-soluble polysaccharide bound to the hydrophobic compound can be present on the membrane surface of the lipid bilayer (on the outer surface and / or the inner surface of the drug carrier). By doing so, it becomes possible to modify the membrane of the drug carrier, and as a result, there are effects such as improving the blood stability of the drug carrier.

Examples of the “basic functional group” include, but are not limited to, an amino group, an amidino group, and a guanidino group. Examples of the compound having a basic functional group include DOTMA (Patent No. 61161246), DOTAP (Patent No. 5508626), Transfectam (Patent No. 2292246), TMAG (Patent No. 4108391), 3,5-Di Known compounds such as pentadecyloxybenzamidine hydrochloride (WO 97/42166 pamphlet), DOSPA, TfxTM-50, DDAB, DC-CHOL, DMRIE, and the like can be mentioned.
When the surface modifier contains a basic functional group, that is, when the surface modifier is a substance in which a compound having a basic functional group is bound to a lipid, this is called a cationized lipid. The case where the drug carrier is a liposome will be described as an example. The cationized lipid has its lipid moiety stabilized in the lipid bilayer membrane of the liposome, and the basic functional group moiety on the lipid bilayer membrane surface. (On the outer surface and / or on the inner surface of the carrier). By doing so, it becomes possible to modify the membrane of the drug carrier, and as a result, the adhesiveness with the target cells and the like can be improved.

Since the drug carrier of the present invention comprises the PEG derivative (A) or (B) of the present invention having no charge as a constituent component, when a cationized lipid is used as a surface modifier, the modification effect by the cationized lipid is inhibited. Not.
The drug carrier of the present invention can be used while carrying a drug for diagnosis and / or treatment of a disease. Here, “supporting” means that a part or all of the drug is contained in a state in which the drug is enclosed in the closed space of the drug carrier, in the membrane of the drug carrier, for example, in the lipid bilayer in the case of liposomes. It means that the drug is contained or the drug is attached to the outer surface of the drug carrier.
The “loading rate” refers to the ratio between the mixed drug and the supported drug when the drug carrier is mixed with the film constituent material of the drug carrier to form the drug carrier.
“Retention in the blood” means the property that a drug in a state of being supported on the drug carrier is present in the blood in the host to which the drug carrier has been administered.

  In the present invention, specific examples of therapeutic drugs that can be carried on a drug carrier include nucleic acids, polynucleotides, genes and analogs thereof, anticancer agents, antibiotics, enzyme agents, antioxidants, lipid uptake inhibitors, Hormone, anti-inflammatory, steroid, vasodilator, angiotensin converting enzyme inhibitor, angiotensin receptor antagonist, smooth muscle cell proliferation / migration inhibitor, platelet aggregation inhibitor, anticoagulant, chemical mediator release inhibition Agents, vascular endothelial cell growth promoters or inhibitors, aldose reductase inhibitors, mesangial cell growth inhibitors, lipoxygenase inhibitors, immunosuppressants, immunostimulants, antiviral agents, Maillard reaction inhibitors, amyloidosis inhibitors, one Nitric oxide synthesis inhibitors, AGEs (Advanced glycation endproducts ) Inhibitors, radical scavengers, proteins, peptides, glycosaminoglycans and derivatives thereof, oligosaccharides and polysaccharides and derivatives thereof, and the like.

  Examples of the anticancer agent include, but are not limited to, nedaplatin, docetaxel, gemcitabine hydrochloride, cyclophosphamide, ifosfamide, nitrogen mustard-N-oxide, thiotepa, brusphan, carbocon, nimustine hydrochloride, ranimustine, melphalan, and improsul tosylate Fan, dacarbazine, procarbazine hydrochloride, cytarabine, cytarabine oxfate, enositabine, mercaptopurine, thioinosine, fluorouracil, doxyfluridine, tegafur, methotrexate, carmofur, hydroxycarbamide, vincristine sulfate, vinblastine sulfate, vindesine sulfate, etoposide hydrochloride, chromomycin u 3 , Doxorubicin hydrochloride, araclubicin hydrochloride, pirarubicin, epirubicin hydrochloride , Dactinomycin, mitoxantrone hydrochloride, bleomycin hydrochloride, peomycin, sulfate mitomycin C, neocarnostatin, L-asparaginase, acegraton mitopronitol, sodium dextran sulfate, octreotide acetate, cisplatin, carboplatin, tamoxifen citrate, acetic acid Examples include medroxyprogesterone, estramustine phosphate sodium, goserelin acetate, leuprorelin acetate, irinotecan hydrochloride, and paclitaxel. Among these anticancer agents, paclitaxel, docetaxel, irinotecan hydrochloride, gemcitabine hydrochloride, cisplatin, carboplatin, nedaplatin and the like are particularly preferable.

  Antibiotics include, but are not limited to, benzylpenicillin potassium, benzylpenicillin benzathine, phenoxymethylpenicillin potassium, pheneticillin potassium, cloxacillin sodium, flucloxacillin sodium, ampicillin, sultamicillin tosylate, bacampicillin hydrochloride, tarampicillin hydrochloride , Lenampicillin, hetacillin potassium, cyclacillin, amoxicillin, pibmesililin hydrochloride, aspoxicillin, carbenicillin sodium, calindacillin sodium, sulbenicillin sodium, ticarcillin sodium, piperacillin sodium, cephaloridine, cephalothin sodium, cefazolin sodium, cefapirin sodium, cefradine sodium, cefradine sodium , Cephalexin, cephatridin propylene glycol , Cefloxazine, cefaclor, cefadroxyl, cefothiam hydrochloride, cefotiam hexetyl hydrochloride, cefuroxime sodium, cefuroxime axetil, cefamandol sodium, cefdinir, cephemetomepipoxil hydrochloride, cefttipene, cefmetazole sodium, cefoxitin sodium , Cefotetan sodium, cefminox sodium, cefuperazone sodium, cefpyramide sodium, cefthrodine sodium, cefotaxime sodium, cefoperazone sodium, ceftizoxime sodium, cefmenoxime hydrochloride, ceftriaxone sodium, ceftazidime, cefpi Mizole sodium, cefixime, cefterampipoxil, cefzonam sodium, cefpodoxyproxetil, cefodizime, Cefpirom acid, latamoxef sodium, flomoxef sodium, imipenem, silastatin sodium, aztreonam, carmonam sodium, streptomycin sulfate, kanamycin sulfate, fradiomycin sulfate, amikacin sulfate, gentamicin sulfate, paromomycin sulfate, pecanamicin sulfate, Lipostamycin sulfate, dibekacin sulfate, tobramycin, sisomycin sulfate, mylonomycin sulfate, astromycin sulfate, netilmycin sulfate, isepamicin sulfate, arbekacin sulfate, erythromycin, kitasamycin, acetylkitasamycin, oleandomycin phosphate, josamycin, acetylspira Mycin, midecamycin, midecamycin acetate, rokitamycin, roxithromycin, clarithromycin, Tetracycline hydrochloride, oxytetracycline hydrochloride, tetracycline metaphosphate, demethylchlortetracycline hydrochloride, lolitetracycline, doxycycline hydrochloride, minocycline hydrochloride, chloramphenicol, chloramphenicol sodium succinate, chloramphenicol palmitate, thianphenicol, Aminoacetic acid thianphenicol hydrochloride, colistin sulfate, colistin sodium methanesulfonate, polymyxin B sulfate, bacitracin, vancomycin hydrochloride, lincomycin hydrochloride, clindamycin, spectinomycin hydrochloride, fosfomycin sodium, fosfomycin calcium and the like.

Examples of the enzyme agent include, but are not limited to, chymotrypsin, crystalline trypsin, streptokinase / streptodolase, hyaluronidase, urokinase, nasarplase, alteplase, lysozyme chloride, semi-alkaline proteinase, serrapeptase, tisokinase, duteplase, batroxobin, pronase, promeline, etc. Can be mentioned.
Antioxidants include, but are not limited to, tocopherol, ascorbic acid, uric acid and the like.

  Anti-inflammatory agents include, but are not limited to, choline salicylate, sazapyrine, sodium salicylate, aspirin, diflunisal, flufenamic acid, mefenamic acid, fructaphenine, tolfenamic acid, diclofenacnatrim, tolmetine sodium, sulindac, fenbufen, ferbinacethyl, indomethacin, indomethacin Farnesyl, acemetacin, progourmetacin maleate, ampenac sodium, nabumetone, ibuprofen, flurbiprofen, flurbiprofen axetil, ketoprofen, naproxen, protidic acid, pranoprofen, fenoprofen calcium, thiaprofenic acid, oxaprozin , Loxoprofen sodium, aluminoprofen, zaltoprofen, phenylbutazone Clofezone, keto phenylbutazone, piroxicam, tenoxicam, ampiroxicam, tiaramide hydrochloride, hydrochloric tinoridine, benzydamine hydrochloride, epirizole, Erumofazon the like.

  Steroid agents include, but are not limited to, cortisone acetate, hydrocortisone (phosphate ester, acetate), hydrocortisone butyrate, hydrocortisone sodium succinate, prednisolone (acetate, succinate, tertiary butyl acetate, phosphate ester), methylprednisolone (Acetate), methylprednisolone sodium succinate, triamcinolone, triamcinolone acetonide (triamcinolone acetate), dexamethasone (phosphate ester, acetate salt, sodium phosphate salt, sulfate ester), dexamethasone palmitate, betamethasone (phosphate, disodium) Salt), acetic acid parameterzone, fludrocortisone acetate, halopredone acetate, clobetasol propionate, halcinonide, beclomethasone propionate, Kusasan betamethasone, betamethasone acetate, acetic acid cortisone and the like.

  Examples of vasodilators include, but are not limited to, theophylline, diprofylline, proxyphylline, aminophylline, corintheophylline, prostaglandin, prostaglandin derivatives, alprostadil alphadex, alprostadil, limaprost alphadex, papaverine, cyclandelate, cinnarizine , Bencyclane fumarate, cinepazide maleate, dirazep hydrochloride, trapidil, diphenidol hydrochloride, nicotinic acid, inositol hexanicotinate, nicotinate citrate, nicotinic alcohol tartrate, tocopherol nicotinate, hepronicart, isoxpurine hydrochloride, bamethane sulfate, tralizone hydrochloride, mesyl Dihydroergotoxin acid, ifenprodil tartrate, moxysilito hydrochloride, nicergoline, nicaric hydrochloride Pin, nilvadipine, nifedipine, benidipine hydrochloride, diltiazem hydrochloride, nisoldipine, nitrendipine, manidipine hydrochloride, balnidipine hydrochloride, efonidipine hydrochloride, verapamil hydrochloride, trimetazidine hydrochloride, captopril, enalapril maleate, alacepril, delapril hydrochloride, cilazapril hydrochloride, lisinapril hydrochloride, Examples include hydralazine, todralazine hydrochloride, budralazine, cadralazine, indapamide, carbochromene hydrochloride, efloxate, etaphenone hydrochloride, oxyfedrine hydrochloride, nicorandil, amyl nitrite, and isosorbide nitrate.

Examples of the angiotensin converting enzyme inhibitor include, but are not limited to, alacepril, imidapril hydrochloride, temocapril hydrochloride, delapril hydrochloride, benazepril hydrochloride, captopril, cilazapril, enalapril maleate, and lisinopril. Examples of the angiotensin receptor antagonist include, but are not limited to, losartan.
The smooth muscle cell migration and / or growth inhibitor includes, but is not limited to, heparin sodium, dalteparin sodium (low molecular heparin), heparin calcium, dextran sulfate and the like.
Examples of the platelet aggregation inhibitor include, but are not limited to, ticlopidine hydrochloride, cilostazol, ethyl icosapentate, beraprost sodium, sarpgrelate hydrochloride, batroxobin, dipyridamole and the like.
Examples of the anticoagulant include, but are not limited to, heparin sodium, dalteparin sodium (low molecular heparin), heparin calcium, dextran sulfate, warfarin potassium, argatroban and the like.
Examples of the chemical mediator release inhibitor include, but are not limited to, tranilast, ketofetin fumarate, azelastine hydrochloride, oxatomide, amlexanox, and repirinast.
Although it does not specifically limit as an immunosuppressant, Cyclosporine etc. are mentioned.
Antiviral agents include, but are not limited to, acyclovir, ganciclovir, didanosine, zidovudine, sorivudine, vidarabine and the like.

  The type of diagnostic drug that can be carried on the drug carrier is not particularly limited as long as the formation of the drug carrier is not impaired. Specific examples include in-vivo diagnostic agents such as X-ray contrast agents, ultrasonic diagnostic agents, radioisotope-labeled nuclear medicine diagnostic agents, and diagnostic agents for nuclear magnetic resonance diagnosis. Examples of the X-ray contrast agent include meglumine amidotrizoate, sodium iotalamate, meglumine iotaramate, gastrografin, iodamid meglumine, lipiodol ultrafluid, adipiodon meglumine, oxaglic acid, meglumine iotroxate, iotrolane, iopanoic acid, iopamidol, Examples include iohexol, ioversol, iopodate sodium, iomeprol, isopaque, iodoxamic acid and the like.

Although it does not specifically limit as an ultrasonic diagnostic agent, For example, gas and a liquid are mentioned. Examples of the gas include air, carbon dioxide, oxygen, nitrogen, helium, and argon. Examples of the liquid include water, physiological saline, a buffer solution, and an aqueous suspension containing metal powder.
The drug carrier of the present invention may further contain a pharmaceutically acceptable stabilizer and / or antioxidant depending on the route of administration. Stabilizers include, but are not limited to, sterols such as cholesterol that reduce membrane fluidity, or sugars such as glycerol and sucrose. Antioxidants include, but are not limited to, ascorbic acid, uric acid or tocopherol analogues such as vitamin E. Tocopherol has four isomers, α, β, γ, and δ, and any of them can be used in the present invention.

The drug carrier of the present invention may further contain a pharmaceutically acceptable additive depending on the administration route. Examples of such additives include water, saline, pharmaceutically acceptable organic solvents, collagen, polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water soluble Dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, casein, gelatin, agar, diglycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin (HSA), Mannitol, sorbitol, lactose, PBS, biodegradable polymer, serum-free medium, surfactants acceptable as pharmaceutical additives There is like a buffer at physiological pH acceptable in vivo.
The additive is appropriately selected from the above depending on the dosage form, or a combination thereof is used, but is not limited thereto.

In this invention, what carried said drug | pharmaceutical on a drug carrier and contained arbitrary additives, such as said stabilizer and antioxidant, can be provided as a pharmaceutical composition. For example, the above-exemplified therapeutic drug carried on a drug carrier can be used as a medical composition for treating a disease. Moreover, what carried the medicine for a diagnosis illustrated above on the drug carrier can be used as a pharmaceutical composition for the diagnosis of a disease. In the pharmaceutical composition of the present invention, the drug carried on the drug carrier may be one kind or plural kinds. When the drug carrier contains an additive, the additive may be one type or a plurality of types.
The pharmaceutical composition of the present invention can be stored according to an ordinary method, for example, refrigerated at 0 to 8 ° C or stored at a room temperature of 15 to 25 ° C.

The drug carrier of the present invention can be obtained by a conventional method. As an example of the drug carrier, a method for producing liposomes is shown below, but is not limited thereto.
In a flask, the PEG derivative (A) or (B) of the present invention and other membrane constituents such as phospholipid are mixed with an organic solvent such as chloroform and vacuum-dried after the organic solvent is distilled off. To form a thin film on the inner wall of the flask. Next, a drug is added to the flask and vigorously stirred to obtain a liposome dispersion. The obtained liposome dispersion can be purified by a commonly used method such as gel filtration, dialysis, membrane separation and / or centrifugation to remove the drug not supported on the liposome. Moreover, the obtained liposome dispersion liquid can adjust the outer diameter of a liposome particle by methods used normally, such as a French press, a pressure filter, or an extruder. Further, after forming liposomes by a conventional method using a lipid mixture obtained by mixing liposome-forming lipids not containing the PEG derivative (A) or (B) of the present invention, the PEG derivative (A) of the present invention or (B) may be added.
In addition to the above method, the liposome can also be obtained by mixing the above-described constituent components and discharging them with a high-pressure discharge type emulsifier. The method for preparing liposomes is specifically described in “Liposomes in Life Science” (Terada, Yoshimura et al .; Springer Fairlark Tokyo (1992)), and is described in this specification with reference to this description. Shall.
A pH gradient method can be used to support the drug on a carrier. This method is disclosed in G. Gregoriadis ed., "Liposome Technology Liposome Preparation and Related Techniques" 2 ^nd edition, Vol. It is described in I-III, CRC Press, and is described in this specification with reference to this description.

The drug carrier of the present invention transports a drug to a target site while carrying the drug, and as a result, delivers the carried drug to the target site. The transportability of the drug to the target site means that the drug carrier carrying the drug reaches the target site. Delivery means that the drug carried by the drug carrier is taken into the target site. In this case, the influence of the drug on the target site or the vicinity thereof is included even if the drug is not taken into the target site.
Here, in this specification, the “target site” of the drug carrier is a specific site for delivering the drug to be carried, and means a cell, tissue, organ or organ specified for each site, and the inside thereof.

The drug carrier having the PEG derivative (A) or (B) of the present invention as one of the membrane components can maintain stability in blood and has transportability to a target site. As a result, the carried drug can be delivered to the target site. Target sites such as cells, tissues, organs or organs and their interior are sites to be treated and / or diagnosed, and include, but are not limited to, tumors, sites of inflammation and the like. Therefore, the target site is a cell, tissue, organ or organ of a tumor or an inflammatory site and the inside thereof.
Specifically, since the PEG derivative (A) or (B) of the present invention having no charge is used as a membrane component, the drug carrier is excellent in blood retention. In addition, when a cationic lipid is included as a surface modifier for the purpose of enhancing adhesion to a target cell, the PEG derivative (A) or (B) of the present invention having no charge is used as a membrane component, The effect of the cationic lipid, that is, the effect of improving cell adhesion is not inhibited.

  The drug carrier of the present invention is used to transport and / or deliver the drug to the desired target site. Therefore, the drug carrier of the present invention is used for the prevention and / or treatment of diseases, for the diagnosis of diseases, for transporting drug carriers to target sites, or for delivering drugs encapsulated in drug carriers to target sites. It can be administered parenterally systemically or locally to a host (patient). Examples of the host to be administered include mammals, preferably humans, monkeys, mice, livestock and the like.

  As the parenteral route of administration, for example, intravenous injection (intravenous injection) such as infusion, intramuscular injection, intraperitoneal injection, or subcutaneous injection can be selected, and an appropriate administration method is selected depending on the age and symptoms of the patient. be able to. The drug carrier of the present invention is administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially block the symptoms of the disease. For example, the effective dose of the drug carried on the drug carrier is selected in the range of 0.01 mg to 100 mg per kg of body weight per day. However, the drug carrier of the present invention is not limited to these doses. The administration time may be administered after the disease has occurred, or may be administered prophylactically to relieve symptoms at the time of onset when the onset of the disease is predicted. The administration period can be appropriately selected depending on the age and symptoms of the patient.

  As a specific administration method, the pharmaceutical composition can be administered by syringe or infusion. Further, the catheter is inserted into the body of the patient or host, for example, into a lumen, for example, into a blood vessel, the tip thereof is guided to the vicinity of the target site, and blood flow to the desired target site, the vicinity thereof, or the target site is passed through the catheter. It is also possible to administer from the site where is expected.

  EXAMPLES Hereinafter, although this invention is demonstrated in more detail based on an Example and a test example, this invention should not be limited to these Examples and a test example.

Example 1
Synthesis of PEG derivative (A) (1) 15.97 g of 3,5-dihydroxybenzoic acid methyl ester (Tokyo Kasei Kogyo), 58.26 g of 1-bromopentadecane (Tokyo Kasei Kogyo), 27.64 g of potassium carbonate (Kokusan Kagaku) Of N, N-dimethylformamide (DMF) (Kanto Chemical) in 250 ml was stirred at 60 ° C. overnight. Pour the reaction mixture into water, filter the solid, wash with water, methanol (domestic chemistry) and ethyl acetate (domestic chemistry), recrystallize from ethyl acetate, methyl 3,5-dipentadecyloxybenzoate 32.6 g of ester was obtained.
(2) Lithium aluminum hydride (Kanto Chemical) 228 mg of tetrahydrofuran (THF) (Kanto Chemical) 3 ml solution, 3.5-dipentadecyloxybenzoic acid methyl ester 1.77 g of tetrahydrofuran 20 ml solution was added dropwise under ice cooling. And stirred at room temperature for 2 hours. The reaction mixture was poured into water, chloroform (Kokusan Kagaku) was added, and Celite (Kanto Chemical) filtration was performed. The separated organic layer was washed with saturated aqueous ammonium chloride and saturated brine, and anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) And concentrated under reduced pressure to obtain 1.66 g of 3,5-dipentadecyloxybenzyl alcohol.
(3) To a solution of 0.56 g of 3,5-dipentadecyloxybenzyl alcohol in 10 ml of methylene chloride (Kanto Chemical), 0.38 g of triphenylphosphine (Tokyo Chemical Industry) and 0.66 g of tetrabromocarbon (Tokyo Chemical Industry) And stirred at room temperature for 20 minutes. Saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with methylene chloride, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (Fuji Silysia), and 0.59 g of 3,5-dipentadecyloxybenzyl bromide was obtained from the fraction eluted with 10% ethyl acetate (domestic chemistry) -hexane (domestic chemistry).
(4) 0.20 g of potassium phthalimide (Tokyo Kasei Kogyo) was added to a solution of 0.62 g of 3,5-dipentadecyloxybenzyl bromide in 10 ml of DMF (Kanto Chemical), and the mixture was stirred at 60 ° C. for 6 hours. The reaction mixture is poured into water, extracted with chloroform (Kokusan Kagaku), washed with water and saturated brine, dried over anhydrous sodium sulfate (Wako Pure Chemical Industries), concentrated under reduced pressure, and 3,5-dipentadecyloxybenzyl. 0.66 g of phthalimide was obtained.
(5) 3,5-dipentadecyloxybenzylphthalimide To 80 ml of ethanol (domestic chemistry) 80 ml solution of 5.10 g was added 80% hydrazine (Tokyo Kasei Kogyo) and stirred at reflux for 5.5 hours. After the reaction solvent was distilled off under reduced pressure, chloroform (Kokusan Kagaku) was added, insolubles were removed by filtration, and the solvent was distilled off under reduced pressure. The residue was subjected to silica gel chromatography (Fuji Silysia), and 2.11 g of 3,5-dipentadecyloxybenzylamine was obtained from a fraction eluted with 5% methanol (domestic chemistry) -chloroform (domestic chemistry).
(6) Monomethoxypolyethylene glycol (MW = 5194, Nippon Oil & Fats) 5.19 g, p-nitrophenyl chloroformate (Kanto Chemical) 403 mg and triethyleneamine (Wako Pure Chemical Industries) 222 mg in methylene chloride (Kanto Chemical) 25 ml solution Was stirred at room temperature for 15 hours. 4. The reaction solvent was distilled off under reduced pressure, ether was added to the residue, the crude product was collected by filtration, recrystallized from ethyl acetate (domestic chemistry), and monomethoxypolyethylene glycol (MW = 5193) -p-nitrophenyl carbonate. 43 g was obtained.
(7) 3,5-dipentadecyloxybenzylamine 112 mg, monomethoxypolyethylene glycol (MW = 5193) -p-nitrophenyl carbonate 1.07 g and triethylamine (Wako Pure Chemical Industries) 22 mg dehydrated chloroform (Kanto Chemical) 10 ml The solution was stirred at room temperature for 12 hours. The reaction solvent is diluted with chloroform (domestic chemistry), washed with saturated sodium bicarbonate solution, water, saturated brine, dried over anhydrous sodium sulfate (Kanto Chemical), concentrated under reduced pressure, and isopropanol (Kanto Chemical) Recrystallization gave 92 mg of monomethoxypolyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate.
This instrumental analysis data supports the structure of the following formula:
¹ H-NMR (CDCl ₃ ) δ (ppm): 6.40 (2H, bs), 6.36 (1H, bs), 4.27 (4H, m), 3.97-3.40 (472H, m), 3.38 (3H, s), 1.78-1.22 (52H, m), 0.88 (6H, t, J = 8.0 Hz)

(Example 2)
Liposomes containing monomethoxypolyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate synthesized in Example 1 as one of membrane constituents were prepared as follows.
Hydrogenated soybean phosphatidylcholine (HSPC, Lipoid) 0.225 mmol, cholesterol (Solvay) 0.189 mmol, 3,5-dipentadecyloxybenzamidine hydrochloride (compound described in WO 97/42166 pamphlet) 0.036 mmol was weighed into a 25 ml volumetric flask, and the fluorescent dye, Rhodamin-DHPE (Lissamine ^™ , rhodamine B 1,2-dihezadecanyl-sn-glycero3-phosphoethanolamine, triethylaluminum, Triethylammonium Add 12 ml of 1 mg / ml) and dissolve, make up with chloroform, and mix lipid solution (total lipid concentration) 8mM) was obtained. Monomethoxy polyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate 4.5 μmol synthesized in Example 1 was dissolved in 6 ml of chloroform, and monomethoxy polyethylene glycol (MW = 5193) -3,5 was dissolved. -A dipentadecyloxybenzyl carbanate solution (0.75 mM) was obtained. 4 ml of the mixed lipid solution was transferred to the eggplant flask, and 1 ml of monomethoxypolyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate solution (monomethoxypolyethylene glycol (MW = 5193) -3, 5-dipentadecyloxybenzylcarbanate modification rate 1 mol%) or 2 ml (monomethoxypolyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate modification rate 2 mol%) or 3 ml (monomethoxypolyethylene glycol) (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate modification rate 3 mol%). The solvent was removed by evaporation from the mixed lipid solution to which monomethoxy polyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate was added. 7.5 ml of physiological saline was added to the obtained thin film, passed through a filter (pore size 0.4 μm 1 × 1, 0.2 μm × 3, 0.1 μm × 10) at 65 ° C. Was prepared. As a result, a liposome containing monomethoxypolyethylene glycol (MW = 5193) -3,5-dipentadecyloxybenzylcarbanate as one of membrane constituents was obtained.

(Reference Example 1)
Preparation of liposome containing polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (PEG-PE) as one of the membrane components Hydrogenated soybean phosphatidylcholine (HSPC, Lipoid) 0.225 mmol, cholesterol (Solvay) 0.189 mmol, 0.05 mmol of 3,5-dipentadecyloxybenzamidine hydrochloride was weighed into a 25 ml volumetric flask, and Rhodamin-DHPE (Lissamine ^™ , rhodamine B 1,2-dihezadecanyl-sn-glycero3) as a fluorescent dye. -Phosphoethanolamine, triethylammonium salt, Molecular Probes) / chloroform solution (0.1 mg / ml) ) Was added and dissolved, and the volume was made up with chloroform to obtain a mixed lipid solution (total lipid concentration: 18 mM). Polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (PEG-PE, Nippon Oil & Fats) 4.5 μmol was dissolved in chloroform 6 ml to obtain a PEG-PE solution (0.75 mM). 4 ml of the mixed lipid solution was transferred to the eggplant flask, and 1 ml of the PEG-PE solution (PEG-PE modification rate 1 mol%) or 2 ml (PEG-PE modification rate 2 mol%) or 3 ml (PEG-PE modification rate 3 mol%). added. The solvent was removed by evaporation from the mixed lipid solution to which PEG-PE was added. 7.5 ml of physiological saline was added to the obtained thin film, passed through a filter (pore size 0.4 μm × 1 time, 0.2 μm × 3 times, 0.1 μm × 10 times) attached to an extruder at 65 ° C., and liposomes Was prepared. As a result, a liposome containing PEG-PE as one of the membrane constituents was obtained. The “PEG-PE modification rate” means the ratio of the PEG-PE amount to the total lipid amount.

(Test Example 1)
(1) Preparation of cells and affinity with cells Mouse melanoma B16F1 (Tohoku University Institute of Aging Medicine, TKG0347) was prepared in a medium to a concentration of 2 × 10 ⁵ cells / ml, and 1 ml / well (1 3 wells per sample). On the next day, 20 μl / well of the liposome of Example 2 or Reference Example 1 having a lipid concentration of 10 mM was added to the plate containing cell culture medium. After 24 hours, the medium was removed and washed twice with PBS. RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Sodium Deoxycholate, 1% IGEPAL CA-630, 0.1% Sodium Lauryl Sulfate) was added at 0.3 ml / well to lyse the cells. The cell lysate was transferred to a 96-well plate for fluorescence measurement at 0.2 ml / well. A calibration curve was prepared with a solution obtained by diluting rhodamine-labeled liposomes with RIPA buffer. The fluorescence intensity (Ex. 544 nm, Em. 590 nm) of rhodamine in the cell lysate was measured with a fluorescence intensity measurement microplate reader (Labsystems Fluoroskan II, Dainippon Pharmaceutical), and the lipid concentration of the lysate was determined from the calibration curve. After fluorescence intensity measurement, protein quantification of the cell lysate was performed using BCA Protein Assay Kit (PIERCE). 20 μl of the lysate was transferred to a 96-well plate for absorbance measurement, 200 μl / well of color developing solution was added, color was developed at 37 ° C. for 30 minutes, and the absorbance (540 nm) was measured with an absorbance measurement microplate reader (Labsystems iEMS Reader MF, Dainippon Pharmaceutical). ) Was measured. A calibration curve was prepared with BSA. The ratio of lipid concentration to protein amount was taken to calculate the amount of liposome binding to cells (lipid amount / protein amount).

FIG. 1 is a graph showing the amount of liposomes bound to target cells when the polyethylene glycol-modified liposomes prepared in Example 2 and Reference Example 1 (modification rate 1%) were used. The “modification rate” means the ratio of the amount of PEG derivative to the total amount of lipid.
As is clear from FIG. 1, in the liposome modified with the polyethylene glycol derivative, the binding amount of the liposome modified with the PEG derivative (A) of the present invention having no charge to the B16F1 cells is negatively charged. More than the amount of PEG-PE modified liposomes bound to the cells. In addition, the binding amount of the liposome with respect to the target cell in the case of modification rate 2% and 3% was as follows.
Liposome binding amount to target cells (μmol lipids / mg protein)
Liposomes of Example 2 Liposome modification rate of Reference Example 1
2% 0.012 0.0023
3% 0.0082 0.0010
That is, it was shown that the liposome containing the PEG derivative (A) of the present invention as a membrane component has higher drug delivery efficiency to tumor cells than the PEG-PE modified liposome.
Moreover, when the PEG derivative (A) of this invention mix | blended the cationized lipid, it became clear that the interaction with the cell of a cationized lipid is not reduced compared with the conventional PEG-PE. That is, it was shown that the liposome containing the PEG derivative (A) of the present invention as a membrane component has higher drug delivery efficiency to tumor cells than the PEG-PE modified liposome.
(2) Acute toxicity As a result of an acute toxicity test by oral administration using ICR male mice (5 weeks old), the LD50 of the liposome of Example 2 is 320 mg / kg or more, and the PEG derivative (A) The safety of the drug carrier containing

(Example 3)
Synthesis of PEG derivative (B) (1) 7.56 g of 4-aminomethylbenzoic acid (Aldrich), 9.38 g of benzyl chloroformate (Kokusan Kagaku) 75 ml of 2N sodium hydroxide (Kanto Chemical) and 75 ml of tetrahydrofuran (Aldrich) Was stirred overnight at room temperature. The reaction solution is acidified with 6N hydrochloric acid (Kanto Chemical), extracted with ethyl acetate, and the organic layer is washed successively with dilute acid, water, dilute alkali, water, dried over anhydrous magnesium sulfate, concentrated under reduced pressure, 4 10.13 g of-(N-benzyloxycarbonyl) aminomethylbenzoic acid was obtained.
(2) 4- (N-benzyloxycarbonyl) aminomethylbenzoic acid 0.57 g methylene chloride (Kanto Chemical) 20 ml In a mixed solution of 20 ml of N-dimethylformamide (Kanto Chemical), 1.04 g of dioctadecylamine (Fluka) and BOP reagent (benzotriazol-1-yl-oxy-trisdimethylaminophosphonium) (domestic chemistry) 0.97 g , 0.25 g of triethylamine (Wako Pure Chemical Industries, Ltd.) was added and stirred overnight at room temperature. Water was added to the reaction solution, followed by extraction with ethyl acetate, and the organic layer was washed with dilute acid, water, dilute alkali, and water in that order, dried over anhydrous magnesium sulfate (Wako Pure Chemical Industries), and concentrated under reduced pressure. The residue was subjected to silica gel chromatography (Fuji Silysia), and 1.57 g of 4- (N-benzyloxycarbonyl) aminomethylbenzoic acid N, N-distearoylbenzamide was obtained from the fraction eluted with 20% ethyl acetate-hexane.
(3) Under a hydrogen stream, 4- (N-benzyloxycarbonyl) aminomethylbenzoic acid N, N-distearoylbenzamide 1.56 g, palladium carbon (Kawaken Fine Chemical) 0.16 g ethanol (Kanto Chemical) 20 ml and chloroform (Kanto Chemical) 10 ml of the mixed solution was stirred overnight at room temperature. The palladium carbon of the reaction solution was removed by filtration, and then concentrated under reduced pressure. The residue was subjected to silica gel chromatography (Fuji Silysia), and 0.68 g of aminomethylbenzoic acid N, N-distearoylbenzamide was obtained from the fraction eluted with 5% methanol-chloroform.
(4) Monomethoxypolyethylene glycol (MW = 5194, Nippon Oil & Fats) 5.19 g, p-nitrophenyl chloroformate (Kanto Chemical) 403 mg and triethyleneamine (Wako Pure Chemical Industries) 222 mg in methylene chloride (Kanto Chemical) 25 ml solution Was stirred at room temperature for 15 hours. 4. The reaction solvent was distilled off under reduced pressure, ether was added to the residue, the crude product was collected by filtration, recrystallized from ethyl acetate (domestic chemistry), and monomethoxypolyethylene glycol (MW = 5193) -p-nitrophenyl carbonate. 43 g was obtained.
(5) Aminomethylbenzoic acid N, N-distearoylbenzamide 0.13 g, monomethoxypolyethylene glycol (MW = 5193) -p-nitrophenyl carbonate 1.07 g and triethylamine (Wako Pure Chemical Industries) 22 mg of dehydrated chloroform (Kanto) Chemical) A 10 ml solution was stirred at room temperature for 12 hours. The reaction solvent is diluted with chloroform (domestic chemistry), washed with saturated sodium bicarbonate solution, water, saturated brine, dried over anhydrous sodium sulfate (Kanto Chemical), concentrated under reduced pressure, and isopropanol (Kanto Chemical) Recrystallization gave 4- (N ′-(methoxypolyethyleneglycol (MW = 5193) carbonyl) 2-aminoethyl) N, N-distearoylbenzamide (0.85 g).
This instrumental analysis data supports the structure of the following formula:
¹ H-NMR (CDCl ₃ ) δ (ppm): 7.30 (4H, bs), 3.88-3.44 (m), 3.38 (3H, s), 1.38-1.16 ( 64H, m), 0.88 (6H, t, J = 8.0 Hz)

Example 4
Liposomes containing the compound synthesized in Example 3 (4- (N ′-(methoxypolyethyleneglycol (MW = 5193) carbonyl) 2-aminoethyl) N, N-distearoylbenzamide) as one of the membrane components. Prepared as follows.
Hydrogenated soybean phosphatidylcholine (HSPC, Lipoid) 0.225 mmol, cholesterol (Solvay) 0.189 mmol, 3,5-dipentadecyloxybenzamidine hydrochloride (compound described in WO 97/42166 pamphlet) 0.036 mmol was weighed into a 25 ml volumetric flask, and the fluorescent dye, Rhodamin-DHPE (Lissamine ^™ , rhodamine B 1,2-dihezadecanyl-sn-glycero3-phosphoethanolamine, triethylaluminum, Triethylammonium Add 12 ml of 1 mg / ml) and dissolve, make up with chloroform, and mix lipid solution (total lipid concentration) 8mM) was obtained. The compound synthesized in Example 3 (4.5 μmol) was dissolved in 6 ml of chloroform to obtain a compound solution (0.75 mM).
4 ml of the mixed lipid solution was transferred to the eggplant flask, and 1 ml (amide compound modification rate 1 mol%), 2 ml (amide compound modification rate 2 mol%) or 3 ml (amide compound modification rate 3 mol%) was added thereto. The solvent was removed by evaporation from the mixed lipid solution to which the compound synthesized in Example 3 was added. 7.5 ml of physiological saline was added to the obtained thin film, passed through a filter (pore size 0.4 μm 1 × 1 time, 0.2 μm × 3 times, 0.1 μm × 10 times) attached to an extruder at 65 ° C., and liposomes Was prepared. As a result, a liposome containing the compound synthesized in Example 3 as one of the membrane constituents was obtained.

(Reference Example 2)
Preparation of liposome containing polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (PEG-PE) as one of the membrane components Hydrogenated soybean phosphatidylcholine (HSPC, Lipoid) 0.225 mmol, cholesterol (Solvay) 0.189 mmol, 0.05 mmol of 3,5-dipentadecyloxybenzamidine hydrochloride was weighed into a 25 ml volumetric flask, and Rhodamin-DHPE (Lissamine ^™ , rhodamine B 1,2-dihezadecanyl-sn-glycero3) as a fluorescent dye. -Phosphoethanolamine, triethylammonium salt, Molecular Probes) / chloroform solution (0.1 mg / ml) ) Was added and dissolved, and the volume was made up with chloroform to obtain a mixed lipid solution (total lipid concentration: 18 mM). Polyethylene glycol (molecular weight 5000) -phosphatidylethanolamine (PEG-PE, Nippon Oil & Fats) 4.5 μmol was dissolved in chloroform 6 ml to obtain a PEG-PE solution (0.75 mM). 4 ml of the mixed lipid solution was transferred to the eggplant flask, and 1 ml of the PEG-PE solution (PEG-PE modification rate 1 mol%) or 2 ml (PEG-PE modification rate 2 mol%) or 3 ml (PEG-PE modification rate 3 mol%). added. The solvent was removed by evaporation from the mixed lipid solution to which PEG-PE was added. 7.5 ml of physiological saline was added to the obtained thin film, passed through a filter (pore size 0.4 μm × 1 time, 0.2 μm × 3 times, 0.1 μm × 10 times) attached to an extruder at 65 ° C., and liposomes Was prepared. As a result, a liposome containing PEG-PE as one of the membrane constituents was obtained. The “PEG-PE modification rate” means the ratio of the PEG-PE amount to the total lipid amount.

(Test Example 2)
(1) Preparation of cells and affinity with cells Mouse melanoma B16F1 (Tohoku University Institute of Aging Medicine, TKG0347) was prepared in a medium to a concentration of 2 × 10 ⁵ cells / ml, and 1 ml / well (1 3 wells per sample). On the next day, 20 μl / well of the liposome of Example 4 or Reference Example 2 having a lipid concentration of 10 mM was added to the plate containing cell culture medium. After 24 hours, the medium was removed and washed twice with PBS. RIPA buffer (50 mM Tris, 150 mM NaCl, 0.5% Sodium Deoxycholate, 1% IGEPAL CA-630, 0.1% Sodium Lauryl Sulfate) was added at 0.3 ml / well to lyse the cells. The cell lysate was transferred to a 96-well plate for fluorescence measurement at 0.2 ml / well. A calibration curve was prepared with a solution obtained by diluting rhodamine-labeled liposomes with RIPA buffer. The fluorescence intensity (Ex. 544 nm, Em. 590 nm) of rhodamine in the cell lysate was measured with a fluorescence intensity measurement microplate reader (Labsystems Fluoroskan II, Dainippon Pharmaceutical), and the lipid concentration of the lysate was determined from the calibration curve. After fluorescence intensity measurement, protein quantification of the cell lysate was performed using BCA Protein Assay Kit (PIERCE). 20 μl of the lysate was transferred to a 96-well plate for absorbance measurement, 200 μl / well of color developing solution was added, color was developed at 37 ° C. for 30 minutes, and the absorbance (540 nm) was measured with an absorbance measurement microplate reader (Labsystems iEMS Reader MF, Dainippon Pharmaceutical). ) Was measured. A calibration curve was prepared with BSA. The ratio of lipid concentration to protein amount was taken to calculate the amount of liposome binding to cells (lipid amount / protein amount).

FIG. 2 is a graph showing the amount of liposomes bound to the target cells when the polyethylene glycol-modified liposomes prepared in Example 4 and Reference Example 2 (modification rate 1%) were used. The “modification rate” means the ratio of the amount of PEG derivative to the total amount of lipid.
As is clear from FIG. 2, in the liposome modified with the polyethylene glycol derivative, the binding amount of the liposome modified with the PEG derivative (B) of the present invention having no charge to the B16F1 cell is negatively charged. More than the amount of PEG-PE modified liposomes bound to the cells. In addition, the binding amount of the liposome with respect to the target cell in the case of modification rate 2% and 3% was as follows.
Liposome binding amount to target cells (μmol lipids / mg protein)
Liposomes of Example 4 Liposome modification rate of Reference Example 2
2% 0.142 0.004
3% 0.135 0.000
That is, it was shown that the liposome containing the PEG derivative (B) of the present invention as a membrane component has higher drug delivery efficiency to tumor cells than the PEG-PE modified liposome.
Moreover, when the PEG derivative (B) of this invention was mix | blended with the cationized lipid, it became clear that the interaction with the cell of a cationized lipid is not reduced compared with the conventional PEG-PE. That is, it was shown that the liposome containing the PEG derivative (B) of the present invention as a membrane component has higher drug delivery efficiency to tumor cells than the PEG-PE modified liposome.
(2) Acute toxicity As a result of an acute toxicity test by oral administration using ICR male mice (5 weeks old), the LD50 of the liposome of Example 4 was 320 mg / kg or more, and the PEG derivative (B) The safety of the drug carrier containing

Claims (13)

A polyethylene glycol derivative represented by the following general formula (1).
(In the formula (1), A is an aromatic ring, R ¹ and R ² are each independently an alkyl group or alkenyl group having 10 to 25 carbon atoms. X and Y are each independently O, S, COO, M is a natural number from 1 to 6, n is a natural number from 10 to 300, L is OCONH (carbamate bond) or OCO (ester bond), B is a hydroxyl group, OCO, CONH or NHCO An alkoxy group or a benzyloxy group.) The polyethylene glycol derivative according to claim 1, wherein A in the general formula (1) is a benzene ring. In Formula (1), a polyethylene glycol derivative according to claim 1 or paragraph 2 wherein R ¹ and R ² is an alkyl or alkenyl group having 12 to 18 carbon atoms each independently. The polyethylene glycol derivative according to any one of claims 1 to 3, wherein in the general formula (1), X and Y are both O. In the general formula (1), A is a benzene ring, R ¹ and R ² are alkyl groups having 15 carbon atoms, X and Y are O, and m is 1. 5. The polyethylene glycol derivative according to any one of items 4. A polyethylene glycol derivative represented by the following general formula (2).
(In the formula (2), A ′ is an aromatic ring, R ³ and R ⁴ are each independently an alkyl group or an alkenyl group having 10 to 25 carbon atoms. P represents 0 or a natural number from 1 to 6) Q represents a natural number from 10 to 300. L ′ is OCONH (carbamate bond) or OCO (ester bond), and B ′ is a hydroxyl group, an alkoxy group or a benzyloxy group. The polyethylene glycol derivative according to claim 6, wherein A 'is a benzene ring in the general formula (2). The polyethylene glycol derivative according to claim 6 or 7, wherein in the general formula (2), R ³ and R ⁴ are each independently an alkyl group or an alkenyl group having 12 to 18 carbon atoms. The general formula (2), wherein A ′ is a benzene ring, R ³ and R ⁴ are alkyl groups having 18 carbon atoms, and p is 1. 9. Polyethylene glycol derivative. A drug carrier comprising the polyethylene glycol derivative according to any one of claims 1 to 9 as one of membrane constituents. The drug carrier according to claim 10, wherein the drug carrier is at least one selected from the group consisting of liposomes, lipid microspheres, and polymer microspheres. 12. The drug carrier according to claim 10 or 11, wherein the drug carrier contains at least one selected from the group consisting of phospholipids and derivatives thereof, lipids and derivatives other than phospholipids, cholesterols and surface modifiers. Drug carrier. The drug carrier according to any one of claims 10 to 12, further comprising at least one selected from the group consisting of a stabilizer and an antioxidant.