JP2006306794A - Temperature-sensitive liposome and temperature-sensitive medicine-releasing system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a temperature-sensitive liposome having a practically higher medicine-releasing profile. <P>SOLUTION: This temperature-sensitive liposome is characterized by carrying a polymer having a heat-sensitive response portion and a hydrophobic portion and polyethylene glycol on a liposome membrane. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a temperature-sensitive liposome whose membrane structure collapses at a specific temperature, and a temperature-sensitive drug release system comprising the temperature-sensitive liposome and a drug.

  Liposomes are composed of lipid bilayers composed of phospholipids, etc., and are closed vesicles with a particle size of about several tens to several hundreds of nanometers and function as nanocapsules that can encapsulate various substances. It has been. In recent years, a drug delivery system (DDS) for delivering a drug to a target site, which is composed of a liposome prepared using a phospholipid derived from a living body and a drug such as an anticancer drug or a therapeutic gene, has been developed. Liposomes used for such applications encapsulate a hydrophilic drug in the hydrophilic region inside the closed endoplasmic reticulum and / or carry a hydrophobic drug on the lipid bilayer membrane.

  Development of DDS using liposomes has been widely conducted, improving the retention of liposomes in blood vessels, and modifying liposome surfaces with antibodies to ensure delivery of liposomes to the target organ Etc. are done.

  As a mechanism for delivering, for example, an anticancer drug to a target site using liposomes, the following may be considered. There are many holes of about 200 nm in microvessels around cancer cells. Therefore, by setting the particle size of the liposome containing the anticancer agent to about 200 nm, the liposome can leak from the microvessel near the target cancer cell and reach the target cancer cell. In this manner, the anticancer drug-containing liposome can be accumulated specifically and at a high concentration in the site surrounding the target cancer cell. On the other hand, since such a hole does not exist in the micro blood vessel around the normal cell, the liposome does not leak from the blood vessel around the normal cell.

  However, in order to obtain the efficacy of the drug contained in the liposome, it is necessary that the drug is efficiently released from the liposome after the drug-containing liposome reaches the target site by the above mechanism.

  Non-Patent Documents 1 and 2 report that, for liposomes that can be disintegrated at a specific temperature to release inclusions, an excellent therapeutic effect can be obtained by combining liposomes encapsulating an anticancer agent with thermotherapy for cancer. Has been.

  Patent Document 1 discloses a liposome in which a polymer compound having a heat-responsive part and a hydrophobic part is held on a liposome membrane via the hydrophobic part. Since such liposomes can release inclusions at a specific temperature, for example, by administering liposomes containing pharmaceuticals to humans and warming the affected area, the inclusions can be released at that part. Are listed.

  Patent Document 1 describes that in this liposome, the structure of the lipid bilayer collapses in a temperature range of about 10 to 100 ° C., and components contained in the liposome are released.

  However, in the liposome disclosed in Patent Document 1, since the substance contained in the liposome is released to some extent around 36 ° C., which is close to the human body temperature, even when the liposome is administered to the human body, it reaches the target site. The substance was released before and was insufficient to release the substance specifically and efficiently at the target site.

Moreover, the liposome described in Patent Document 1 has a short residence time in blood, and tends to be discharged outside the body before reaching the target site.
Japanese Patent Laid-Open No. 2003-221755 "Clinical Cancer" Vol. 32, No. 13 (1986) "General Clinical" Vol. 37, No. 1 (1988)

In order to make a liposome retaining a drug or the like into a temperature-sensitive liposome having higher practicality in the pharmaceutical field, it is desired to have the following characteristics.
(1) The drug can be stably maintained at a temperature close to the body temperature of mammals, particularly humans, that is, around 34 to 39 ° C .;
(2) A drug can be rapidly released at a temperature away from the body temperature of mammals, particularly humans, that is, 38 ° C. or higher, preferably 40 to 45 ° C .;
(3) Long residence time in blood in mammals, particularly humans.

  As a result of intensive studies, the present inventors have determined that a liposome composed of a liposome membrane-constituting lipid, a polymer compound having a thermosensitive response portion and a hydrophobic portion, and polyethylene glycol (PEG) has a disrupted liposome membrane structure. The present invention was completed by finding that the temperature is 5 to 9 ° C. higher than the liposome membrane structure collapse temperature of the conventional liposome and has the above-mentioned characteristics (1) to (3).

The present invention is a temperature-sensitive liposome in which a polymer compound having a thermosensitive portion and a hydrophobic portion and polyethylene glycol are supported on a liposome membrane.
The present invention comprises a liposome membrane-constituting lipid, a polymer compound having a heat-sensitive responsive portion and a hydrophobic portion, and polyethylene glycol, and the liposome membrane-constituting lipid: polymer compound: polyethylene glycol molar ratio is 1. : It is also a temperature sensitive liposome obtained by using a liposome membrane-constituting lipid, a polymer compound and polyethylene glycol at a ratio of 0.003 to 0.2: 0.001 to 0.2.
The present invention also provides a temperature sensitive drug release system comprising the above temperature sensitive liposome and a drug.

By using the temperature-sensitive liposome of the present invention, the membrane structure of the liposome collapses in a temperature range higher than the collapse temperature of the conventional liposome, that is, a temperature range away from the human body temperature, and the drug retained by the liposome is released. It is possible to provide a temperature sensitive drug release system that can exhibit a more practical drug release profile. Moreover, since the temperature sensitive liposome of this invention contains polyethyleneglycol, it can also show a longer blood retention.
For example, an anticancer agent is contained as a drug in the temperature-sensitive liposome of the present invention, administered to a subject, and subjected to thermotherapy that heats the affected area to about 40 to 45 ° C., whereby the anticancer drug is specifically and efficiently released at the affected area. Therefore, a more preferable DDS can be obtained.

  The liposome of the present invention is composed of a liposome membrane-constituting lipid, a polymer compound having a heat-responsive part and a hydrophobic part, and polyethylene glycol (PEG). Specifically, a polymer compound and PEG are supported on a liposome membrane. In this specification, the substance is “supported on the liposome membrane” means that the substance may be bonded to the liposome membrane and protrude from the surface of the liposome membrane, or the substance may be present on the lipid bilayer constituting the liposome membrane. A part or the whole of may be embedded.

  The temperature-sensitive liposome of the present invention has a liposome membrane structure that is stably formed at around 34 to 39 ° C., can stably hold a drug that can be held in the liposome, and rapidly has a liposome membrane at around 40 to 45 ° C. It is preferred that the structure can collapse to release the drug. That is, the drug release rate from the liposome after incubation at 37 ° C. for 3 minutes is 5% or less, more preferably 4.5% or less, and the drug release from the liposome after incubation at 45 ° C. for 3 minutes. The rate is preferably 85% or more, more preferably 87% or more. The drug release rate in this specification is a value measured according to the method for measuring the release rate of adriamycin (ADR) described in the following examples.

  In the present invention, the mechanism by which the liposome membrane structure is collapsed at a specific temperature is not clear. However, when the temperature rises, the thermosensitive part of the polymer compound is dehydrated, and the hydrophobicity of the thermosensitive part increases. Thus, it is considered that the whole or a part of the thermosensitive responsive portion initially supported outside the liposome membrane enters the liposome membrane, and the structure of the liposome membrane collapses. In the present invention, a temperature at which the dehydrated polymer compound disrupts the structure of the liposome membrane (hereinafter also referred to as liposome membrane disruption temperature) due to some interaction with the polymer compound dehydrated by amphiphilic PEG. ) Can be made higher than the liposome membrane collapse temperature of conventional liposomes.

  As the polyethylene glycol (PEG) used for the temperature-sensitive liposome of the present invention, those usually used for liposomes can be suitably used. The molecular weight of such PEG is preferably 120 to 12000, more preferably 350 to 12000, and still more preferably 350 to 5000. When PEG having a molecular weight in such a range is used, the liposome disintegration temperature can be set within a preferable range.

The above PEG preferably constitutes the temperature-sensitive liposome of the present invention by bringing the PEG derivative into contact with the polymer compound and the liposome membrane-constituting lipid. The PEG derivative means the following formula (III):
R ′-(PEG) -XR (III)
(In the formula, (PEG) represents polyethylene glycol having a molecular weight as described above, and R ′ is a hydrogen atom, a methoxy group, or a reactivity that may be bonded to (PEG) via a divalent bond. R is a group that is reactive with a group possessed by a liposome membrane-constituting lipid or a group derived from a lipid, and X is a linker)
It is a compound represented by these.

When R ′ in formula (III) is a reactive group, the reactive group is not particularly limited. For example, an amino group, a carboxyl group that may form an ester with N-hydroxysuccinimide, a biotinyl group, Examples thereof include a maleimide group, a nitrophenyl group, an aldehyde group, an acetal group, a tresyl group, and a thiol group. The divalent bonds, for example _{-COO -, - CH 2 -,} - CH 2 CH 2 -, - CH 2 CH 2 CH 2 -, - COCH 2 CH 2 -, - COCH 2 CH 2 CH 2 -, —NHCO—, —NHCO—CH ₂ CH ₂ — and the like can be mentioned.
Linker as X in formula (III) can be suitably selected according to the type of R, for example _{-CO -, - COCH 2 CH 2} CO -, - , and the like COCH ₂ CH ₂ CH ₂ CO- .

  A PEG derivative in which R in the formula (III) is a group reactive with the group possessed by the liposome membrane-constituting lipid is described in, for example, WO98 / 32466, and these can be used. Such a PEG derivative can be bonded to a group of the liposome membrane-constituting lipid, such as an amino group of phosphatidylethanolamine or phosphatidylserine, so that PEG is bonded to the liposome membrane.

  The PEG derivative is preferably a compound in which R in the formula (III) is a group derived from a lipid. Such compounds can be represented as PEG-lipid conjugates. When such a PEG derivative is brought into contact with the liposome membrane-constituting lipid, the lipid portion constituting the PEG-lipid complex can enter the liposome membrane, so that PEG is supported on the liposome membrane.

  The lipid constituting the PEG-lipid complex may be a natural lipid or a synthetic lipid. Examples of natural lipids include glycerophospholipids such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and diphosphatidylglycerol; glycosphingolipids such as cerebroside; glycerides such as diacylglycerol; steroids such as cholesterol, lanosterol, and ergosterol. It is done. Synthetic lipids include synthetic lipids having reactive functional groups such as amino groups, hydroxyl groups, and carboxyl groups. Among them, it is preferable to select from the group consisting of phosphatidylethanolamine, diacylglycerol and cholesterol. These lipids may be used alone or in combination of two or more. Although it does not specifically limit as constituent fatty acid of said phospholipid, Myristic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, linoleic acid, linolenic acid, lauric acid, etc. are mentioned.

Commercially available products can be used as the PEG-lipid complex as described above, and examples thereof include the following (all are trade names manufactured by Avanti Polar Lipids).
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)];
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [poly (ethylene glycol)];
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)];
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N- [poly (ethylene glycol)];
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene glycol)];
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [biotinyl (polyethylene glycol)];

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [carboxy NHS ester (polyethylene glycol)];
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol)];
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)];
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [poly (ethylene glycol)];
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)];
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [poly (ethylene glycol)].

  The above temperature-sensitive liposome is preferably obtained by using PEG in an amount of 0.1 to 30 mol%, more preferably 1 to 30 mol% with respect to the total number of moles of the liposome membrane-constituting lipid, polymer compound and PEG. 6 mol%.

  The polymer compound used in the temperature-sensitive liposome of the present invention is a polymer compound having a heat-sensitive responsive moiety. In particular, a polymer compound having a thermosensitive portion and a hydrophobic portion is preferable. Such a polymer compound is preferably a block copolymer of a thermosensitive vinyl monomer containing one or more hydratable heteroatoms and a hydrophobic vinyl monomer.

Examples of the hydratable hetero atom include an oxygen atom and a nitrogen atom. The upper limit of the number of hydratable heteroatoms is not particularly limited as long as the polymerization can be performed. Examples of the group containing a hydratable hetero atom include —CH ₂ —CH ₂ —O—, —CO—, —COO—, —CONH—, —NHCOO— and the like.

  As the hydrophobic vinyl monomer, vinyl monomers having various aliphatic, alicyclic or aromatic hydrocarbon groups having about 3 to 40 carbon atoms, particularly about 4 to 30 carbon atoms can be used. .

  In the block copolymer, the copolymerization ratio of the thermoresponsive vinyl monomer containing one or more heteroatoms and the hydrophobic vinyl monomer varies depending on the type of each monomer, but is usually 300: 1 to 3: About 1, especially about 200: 1 to 10: 1 is preferable. Within this range, the polymer compound can be stably retained on the liposome membrane.

  The amount corresponding to the thermosensitive portion of the number average molecular weight of the polymer compound is not particularly limited, but is usually about several hundred to several hundred thousand. In particular, it is preferably about 1,000 to 30,000, more preferably about 10,000 to 20,000, whereby the collapse temperature of the liposome membrane structure can be set to a narrower temperature range.

  The molecular weight of the entire polymer compound is not particularly limited, but it may be usually several hundred to several hundreds of thousands in terms of number average molecular weight. Within this range, the liposome membrane is stably held. More preferably, it is about 2,000 to 50,000, more preferably about 10,000 to 21,000, and particularly preferably about 11,000 to 21,000, whereby the collapse temperature of the liposome membrane structure is narrower. It can be a range.

The molecular weight distribution (weight average molecular weight / number average molecular weight) of the above polymer compound is usually about 1 to 4, particularly about 1.0 to 1.5. Within this range, the collapse temperature of the liposome membrane structure can be set to a narrower temperature range.
The number average molecular weight and the weight average molecular weight of the polymer compound used in the present invention are values measured by the methods described in the following examples, respectively.

  The temperature-sensitive liposome of the present invention is preferably obtained by using the above polymer compound in an amount of about 0.1 to 20 mol% with respect to the total number of moles of the liposome membrane-constituting lipid, polymer compound and PEG, and more preferably. Is about 0.5 to 5 mol%. Within this range, the liposome can be made sufficiently temperature sensitive and a practically stable liposome can be obtained.

  Preferred specific examples of the polymer compound include the following formula (I):

[Wherein R ¹ is a hydrogen atom, the following formula (II):

(In the formula, X is the same or different and is a hydrogen atom or a halogen atom, and R ¹¹ is a hydrogen atom, an alkyl group having 1 to 20 carbon atoms or an aryl group.)
Or a group derived from a polymerization initiator, and R ² is an alkyl group having 1 to 8 carbon atoms, a halogenated alkyl group having 1 to 8 carbon atoms, an aldehyde group, a phenyl group, a biphenyl group, or a carbon number. An aralkyl group having 7 to 14 carbon atoms, R ³ is a hydrocarbon group having 4 to 30 carbon atoms, R ⁴ is a hydrogen atom, an alkoxy group having 1 to 10 carbon atoms, or a group derived from a polymerization terminator, m Is 5 to 500, n is 1 to 10, and y is 0 to 100. ]
The compound represented by these is mentioned.

The polymer compound is preferably obtained by living cationic polymerization.
As a polymerization initiator for living cationic polymerization, for example, a polymerization initiator described in JP-A-1-108202 or JP-A-1-108203, a polymerization initiator used in the inifer method (US Pat. No. 4,276,394) ( Substituents obtained from chlorine compounds having an aromatic ring at the α-position (JP 2001-055408 A), polymerization initiators reported by Higashimura et al. in Macromolecules, 17, 265, 1984 (combining hydrogen iodide and iodine) And polymerization initiators described in JP-A-62-48704 and JP-A-64-62308.

R ² in the above formula (I) is particularly preferably an alkyl group having about 1 to 4 carbon atoms among the groups listed above.

R ³ in the above formula (I) is a hydrocarbon group having about 4 to 30 carbon atoms, and examples of such a group include an alkyl group, an aryl group, and an aralkyl group. In particular, a linear alkyl group having about 8 to 30 carbon atoms is preferable.

The ratio of m to n in the above formula (I) varies depending on the type of R ¹ , R ² , R ³ , R ⁴ and the length of the oxyethylene chain, but m: n is 300: 1 to 3 : About 1, especially about 200: 1 to 10: 1.

  As described above, y is about 0 to 100, and oxyethylene does not exist when y = 0. y is particularly preferably about 0 to 10. The longer the oxyethylene chain, the higher the collapse temperature of the liposome membrane structure.

  The halogen atom as X in the above formula (II) is selected from a fluorine atom, a chlorine atom, a bromine atom and an iodine atom.

  Although the polymer compound of the above formula (I) varies depending on the kind of vinyl monomer, it can be obtained by polymerizing the monomer by a known method such as cationic polymerization. As the polymerization method, living cationic polymerization is particularly preferable. Living cationic polymerization methods are described, for example, in JP-A-1-108202, JP-A-1-108203, JP-A-2001-055408, JP-A-2000-198825, and JP-A-8-269118. Yes.

  According to living cationic polymerization, the growth ends do not disappear until the monomer disappears or a polymerization terminator is added, so that the chain length of the thermosensitive portion and the hydrophobic portion and thus the molecular weight of the polymer compound can be easily set to a desired value. can do. In addition, a polymer material having a narrow molecular weight distribution can be obtained as compared with other polymerization methods.

  In the temperature-sensitive liposome of the present invention, as the liposome membrane-constituting lipid, an amphiphilic lipid usually used as a membrane lipid of the liposome can be used. Examples of such lipids include phospholipids such as phosphatidic acid, phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin, sphingomyelin, soybean phosphatidylcholine, and egg yolk phosphatidylcholine. Examples of fatty acids constituting these phospholipids include myristic acid, palmitic acid, stearic acid, arachidonic acid, oleic acid, linoleic acid, and linolenic acid. These can be used alone or in combination of two or more. In particular, phosphatidylcholine and phosphatidylethanolamine are preferable.

  In addition, when a gene is contained as an active substance, a known cationic synthetic lipid can be used in addition to the above phospholipid. Examples of such cationic synthetic lipids include N- (α-trimethylammonioacetyl) -didodecylglutamate, N- [1- (2,3-dioleyloxy) propyl] -N, N, N- And quaternary ammonium salts such as trimethylammonium chloride and 1,2-bis (oleoyloxy) -3- (trimethylammonio) propane. These lipids can be used alone or in combination of two or more.

  The liposome membrane-constituting lipid may contain sterols such as cholesterol, lanosterol and ergosterol.

  The particle size of the temperature-sensitive liposome of the present invention may be about 0.05 to 10 μm, and can be various particle sizes depending on the purpose of use. For example, when the anti-cancer drug is held in a temperature-sensitive liposome and used as a temperature-sensitive drug release system for delivery to cancer cells, the particle size is usually about 0.05 to 0.2 μm, particularly about 0.05 to 0.1 μm. preferable.

  The temperature-sensitive liposome of the present invention may be either a monolayer liposome composed of a single lipid bilayer membrane or a multilamellar liposome composed of a plurality of lipid bilayer membranes, as long as it falls within the above particle size range. .

In the temperature-sensitive liposome of the present invention, the liposome membrane-constituting lipid, the polymer compound, and the PEG derivative as described above are prepared in a known liposome production method, and the liposome membrane-constituting lipid: polymer compound: PEG molar ratio is as follows. , 1: 0.003 to 0.2: 0.001 to 0.2. The molar ratio of liposome membrane-constituting lipid: polymer compound: PEG is more preferably 1: 0.003 to 0.1: 0.01 to 0.06, and even more preferably 1: 0.01 to 0.05. : 0.01 to 0.03.
Known methods for producing liposomes include an extruder method, an ultrasonic method, a French press method, and the like. Details of these methods are described in “Liposome” (Shinochi Nojima, Junzo Sunamoto, Junzo Inoue, Nanedo) and “Liposome in Life Science” (Hiroshi Terada, Tetsuro Yoshimura, Springer Fairlark Tokyo). ing.

  For example, a method for producing the liposome of the present invention by the extruder method will be described. A solution in which a predetermined amount of liposome membrane-constituting lipid, polymer compound, and PEG derivative are dissolved in an appropriate organic solvent such as chloroform is prepared, and each solution is placed in a container and mixed. Next, the solvent is removed using an evaporator, and a thin film composed of liposome membrane-constituting lipid, polymer compound and PEG is formed on the container wall. This film is preferably further vacuum-dried for about 3 to 12 hours. Next, a suitable solution such as a buffer solution is put into the container, and liposomes can be formed by vigorous stirring using an ultrasonic treatment or a vortex mixer. By passing the obtained liposome dispersion through an extruder and appropriately setting the filter pore size, the particle size of the liposome can be adjusted.

  From the liposome dispersion obtained as described above, the unsupported polymer compound, PEG derivative, and the like can be removed by gel filtration, ultracentrifugation, dialysis, and the like. If the substance to be removed has a charge, ion exchange chromatography can be used.

  In the above production method, the order of mixing the liposome membrane-constituting lipid, the polymer compound and the PEG derivative is not particularly limited. For example, the liposome membrane-constituting lipid, the polymer compound and the PEG derivative may be mixed at once, or after the liposome membrane-constituting lipid is used to form liposomes in advance by the extruder method as described above, the polymer compound And PEG derivatives may be added simultaneously or separately to support these components on the liposome membrane.

  In the temperature-sensitive liposome of the present invention produced by the method as described above, the thermosensitive portion of the polymer compound and PEG may be carried on the outer surface of the liposome membrane, or the inner side of the liposome membrane. It may be supported on the surface, or may be supported on both the outer surface and the inner surface of the liposome membrane. In the production method described above, when the liposome membrane-constituting lipid, the polymer compound and the PEG derivative are mixed at once, the thermosensitive part of the polymer compound and PEG are supported on both the outer surface and the inner surface of the liposome membrane. However, when the polymer compound and the PEG derivative are added simultaneously or separately after forming the liposome, the thermosensitive part of the polymer compound and PEG are supported only on the outer surface of the liposome membrane.

The temperature sensitive drug release system comprising the above temperature sensitive liposome and drug is also one aspect of the present invention.
The drug may be a hydrophilic substance or a hydrophobic substance. When it is a hydrophilic substance, it is contained in the hydrophilic region of the closed space inside the temperature-sensitive liposome, and when it is a hydrophobic substance, it is supported on the temperature-sensitive liposome membrane.

  Although it does not specifically limit as said chemical | medical agent, For example, the anticancer agent used together with a thermotherapy, an anti-inflammatory agent, etc. are mentioned. Anticancer agents include metal complexes such as cisplatin, carboplatin, tetraplatin, and iproplatin; anticancer antibiotics such as adriamycin (ADR), mitomycin, actinomycin, ansamitocin, bleomycin, Ara-C, and daunomycin; 5-FU, methotrexate And antimetabolites such as TAC-788; alkylating agents such as BCNU and CCNU; interferons (α, β, γ), and lymphokines such as various interleukins. Examples of anti-inflammatory agents include predonin, Linderon, and Celestamine.

The agent can also include a gene for treatment of a disease. Examples of such genes include, but are not limited to, for example, an adenosine deaminase gene for the treatment of severe combined immunodeficiency, an LDL receptor gene for the treatment of familial hypercholesterolemia, and a cancer treatment. Interferon (IFN) -α, β or γ gene, granulocyte macrophage colony stimulating factor (GM-CSF) gene, various interleukin (IL) genes, tumor necrosis factor (TNF) -α gene, lymphotoxin (LT) -β gene , Granulocyte colony stimulating factor (G-CSF) gene, T cell activation costimulatory gene and the like. In addition, genes for the treatment of Alzheimer's disease, spinal cord injury, Parkinson's disease, arteriosclerosis, diabetes, hypertension and the like can be mentioned.
The amount of the drug is not particularly limited, and can be appropriately selected depending on the type of drug.

  In the production of the above-described temperature-sensitive drug release system, a known method can be used as a method for containing a drug in the temperature-sensitive liposome according to the type of drug. The method is not limited, for example, a method in which a temperature-sensitive liposome is formed according to the above-described method for producing a temperature-sensitive liposome, and then the liposome is immersed in a solution containing the drug so that the drug is taken into the liposome. Examples of methods for producing temperature-sensitive liposomes include a method in which a solution containing a drug is placed in a container in which a thin film is formed, and then a liposome membrane structure is formed to encapsulate the drug.

  The temperature sensitive drug delivery system of the present invention preferably further comprises at least one pharmaceutical additive. The temperature sensitive drug release system may be in the form of a solid preparation such as a tablet or powder, but is preferably in the form of a liquid preparation such as an injection preparation.

  When the above temperature sensitive drug release system is a liquid formulation, the pharmaceutical additive can be a carrier (eg, saline, sterile water, buffer, etc.), a membrane stabilizer (eg, cholesterol), an isotonic agent (eg, chloride). Sodium, glucose, glycerin, etc.), antioxidants (eg, tocopherol, ascorbic acid, glutathione, etc.), preservatives (eg, chlorbutanol, parabens, etc.), and the like. The carrier can be a solvent used in producing temperature-sensitive liposomes.

  When the above temperature sensitive drug release system is a solid formulation, the pharmaceutical additive may be an excipient (e.g., sugars such as lactose, sucrose, starches such as corn starch, celluloses such as crystalline cellulose, Arabic Rubber, magnesium aluminate metasilicate, calcium phosphate, etc.), lubricant (eg, magnesium stearate, talc, polyethylene glycol, etc.), binder (eg, saccharides such as mannitol, sucrose, crystalline cellulose, polyvinylpyrrolidone, hydroxypropylmethylcellulose) Etc.), disintegrating agents (eg, starches such as potato starch, celluloses such as carboxymethyl cellulose, cross-linked polyvinyl pyrrolidone, etc.), coloring agents, flavoring agents and the like.

  The above temperature sensitive drug release system can be produced by mixing the temperature sensitive liposome containing the above drug as it is or freeze-dried with the above pharmaceutical additive. When freeze-drying temperature-sensitive liposomes containing a drug, an appropriate excipient should be added before freeze-drying.

The temperature-sensitive drug release system of the present invention is administered to a subject, and the temperature of the affected area is heated to 40 ° C. or higher, preferably 40 to 45 ° C., thereby disrupting the membrane structure of the temperature-sensitive liposome and Can be released at the affected area. By such a method, the drug can be specifically and efficiently released from the liposome that has reached the affected area.
As said object, a mammal is preferable, Especially preferably, it is a human.

  The method of raising the temperature of the affected area to 40 ° C. or higher is not particularly limited as long as it is a method used in normal cancer thermotherapy, and examples thereof include a method of irradiating an electromagnetic wave such as a laser or microwave.

  The temperature sensitive drug release system described above can be administered by either parenteral or oral routes. For example, when an anticancer agent is used as a drug, parenteral route, particularly administration by intravenous injection is preferable.

  The dosage of the above temperature sensitive drug release system can be appropriately selected according to the severity of the subject and the amount of drug contained in the liposome.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the following examples.
<Measurement method of molecular weight>
The weight average molecular weight (Mw) and number average molecular weight (Mn) of the polymer compound were calculated from the results of gel filtration chromatography (GPC), respectively. GPC is performed using a high performance liquid chromatograph (TSKgel column G-200HXL + G-3000 HXL + G-4000HXL (Tosoh Corp.), eluent: chloroform), and the molecular weight of the test compound is determined from the chromatographic results. By calculating polystyrene as a standard substance, Mw and Mn in terms of polystyrene were obtained.

<Adriamycin release test>
Using adriamycin (ADR), which is a fluorescent substance and known as an anticancer agent, as a drug, the release of ADR from liposomes was examined by the following method.
Liposomes containing ADR were dispersed in HBS buffer (20 mM Hepes, 150 mM NaCl, pH 7.4), and the temperature of the liposome dispersion was set to 10 ° C. The HBS buffer solution containing fetal calf serum (FCS) at various concentrations (0 to 50%) is brought to a predetermined temperature, and the liposome dispersion is added to the FCS-containing HBS buffer solution so that the liposome lipid concentration becomes 5 μM. In addition, the fluorescence intensity was measured after a predetermined time.

  Since ADR is quenched at a high concentration, it does not fluoresce when encapsulated inside the liposome, but fluoresces when released from the liposome into a buffer outside the liposome. Therefore, by monitoring the fluorescence of the liposome dispersion while raising the temperature of the buffer, the degree of ADR release from the liposome can be examined. The excitation wavelength and fluorescence wavelength of ADR were 470 nm and 590 nm, respectively.

The release rate (%) of ADR from the liposome was calculated using the following formula.
Release rate (%) = (F ^t × F ^f / F ^{′ f} −F ⁱ ) / (F ^f −F ⁱ ) × 100
Here, F ^t is the fluorescence intensity when incubated at a predetermined temperature for a predetermined time;
^F′f is the fluorescence intensity when Triton X-100 is added and the liposome is destroyed after 30 minutes at a predetermined temperature;
F ⁱ is the initial fluorescence intensity at 10 ° C .;
F ^f represents the fluorescence intensity when Triton X-100 is added and the liposome is broken after 30 minutes at 10 ° C.

<Manufacture of polymer compounds>
Toluene (6.2 ml) as solvent, ethyl acetate (10 mmol) as added base, (2-ethoxy) ethoxyethyl vinyl ether (EOEOVE) (4 mmol) as monomer, 1-methoxyethoxyethyl acetate (0.04 mmol) as polymerization initiator Was used. These were collected in a Schlenk tube and cooled to 0 ° C., and polymerization was started by adding a similarly cooled ethylaluminum sesquichloride solution (0.2 mmol). Furthermore, in order to synthesize a block copolymer, octadecyl vinyl ether (ODVE) (0.2 mmol) diluted with toluene was sequentially added at the end of polymerization of the EOEOVE monomer. At the end of the polymerization of the ODVE monomer, the polymerization reaction was stopped by adding ammoniacal methanol to the polymerization system.
Next, the block copolymer solution diluted with dichloromethane was washed with dilute hydrochloric acid to remove the residue of the polymerization initiator, thereby purifying the block copolymer. The system was then returned to neutral and evaporated to remove solvent, unreacted monomer seed residues, and the like. In order to completely remove the unreacted monomer, it was dried in a vacuum dryer for 8 hours (80 ° C., 0.1 mmHg). The molecular weight distribution (Mw / Mn) and Mn of the obtained block copolymer (polymer compound) were GPC, and the composition ratio was determined by NMR. As a result, a block copolymer having Mn = 15000 and Mw / Mn = 1.24 was obtained. The copolymer obtained had an ODVE / EOEOVE copolymerization ratio of approximately 100/5.

Example 1 Production of Temperature Sensitive Liposomes As liposome membrane-constituting lipids, 0.5 mg of 10 mg / ml egg yolk phosphatidylcholine (EYPC) in chloroform and 0.218 mL of 10 mg / ml cholesterol in chloroform were used. 0.281 mL of a chloroform solution of the polymer compound (Mn = 15000) produced in 1) and 5 mg / mL PEG5000-distearoylphosphatidylethanolamine (PE) complex (1,2-distearoyl-sn-glycero-3-phospho Ethanolamine-N- [poly (ethylene glycol) 5000] (available from Avanti Polar Lipids) in a 10 mL volume flask together with 290 μL of chloroform solution, and using a rotary evaporator to remove chloroform, liposomes are added to the inner wall of the flask. A thin film was formed of lipids, polymer compounds, and PEG (EYPC: Cholestero) Lu: polymer compound: PEG = 50: 45: 3: 2 (molar ratio)).

  Next, 1 mL of an ammonium sulfate solution (125 mM ammonium sulfate, 10 mM HEPES, 2 mM EDTA, pH 5.2) is added to the container, and ultrasonic waves are irradiated for 2 minutes to form liposomes. Further, an extruder (manufactured by Avestin) The particle size of the liposomes was adjusted using a filter pore size of 100 nm). The liposome dispersion was passed through a Sepharose 4B column (Amersham Biosciences) equilibrated with HBS buffer to make the inside of the liposome acidic and the outside neutral. Subsequently, the lipid concentration of the liposome dispersion was measured according to the instruction manual of the phospholipid C test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) kit. From the obtained results, ADR was added so as to have a ratio of 100 μg with respect to 1 μmol of lipid, and allowed to stand at 30 ° C. for 1 hour to incorporate ADR into the liposome.

  5 μL of the obtained ADR-containing liposome dispersion was diluted 200-fold with methanol, and the absorbance was measured at a wavelength of 477 nm. The remaining ADR-containing liposome dispersion was passed through a Sepharose 4B column equilibrated with HBS, and the lipid concentration of the recovered dispersion was measured with a phospholipid-Test Wako kit. Further, 5 μL of the recovered dispersion was diluted 200 times with methanol, and the absorbance was measured at a wavelength of 477 nm. From these values, the encapsulation efficiency of ADR in liposomes was calculated.

Example 2
PEG is a ratio of EYPC: cholesterol: polymer compound: PEG = 50: 45: 3: 3 (molar ratio) in the same manner as in Example 1 except that the amount of the PEG-PE complex is 435 μL in Example 1. Temperature-sensitive liposomes were prepared.

Comparative Example 1
Liposomes not retaining PEG were produced in the same manner as in Example 1 except that the PEG-PE complex was not added in Example 1.
The results of measuring the encapsulation rate of ADR for each of the liposomes of Examples 1 and 2 and Comparative Example 1 are shown in Table 1 below.

  From the results in Table 1, it is considered that there is almost no influence on the encapsulation efficiency of ADR in liposomes by retaining PEG.

Test example 1
For the liposomes of Examples 1 and 2 and Comparative Example 1, using the ADR release test described above, the time change in the release rate of ADR from the liposomes was 30 ° C., 37 ° C., 39 ° C., 42 ° C., 43 ° C., 45 Measured at 0 ° C and 50 ° C, respectively.
The results are shown in FIG. FIG. 1 shows the results for A: the liposome of Example 1; B: the liposome of Example 2; C: the liposome of Comparative Example 1.
From the results of FIG. 1, it can be seen that by introducing PEG, the release rate of ADR at a temperature of 39 ° C. or lower is reduced.

In the result obtained in Test Example 1, a graph summarizing the relationship between each temperature and the release rate using the value of the release rate 3 minutes after reaching each temperature is shown in FIG.
From the results in FIG. 2, it can be seen that the temperature-sensitive liposome of the present invention has a higher temperature at which ADR is released, compared to liposomes that do not retain PEG. Moreover, it turns out that the release rate of ADR increases rapidly in the temperature sensitive liposome of Example 1 especially at the temperature exceeding 40 degreeC.

Test example 2
This study was conducted to investigate the release behavior of ADR from temperature-sensitive liposomes under conditions closer to the living body. The release rate of ADR from the temperature-sensitive liposome of Example 1 in HBS buffer containing fetal calf serum (FCS) in an amount of 0, 10, 30 or 50% by weight is determined according to the ADR release test procedure described above. Examined.
The results are shown in FIG.
FIG. 3 is a graph showing the ADR release rate 3 minutes after the liposome of Example 1 was incubated at 37 ° C. or 45 ° C.
From the results of FIG. 3, it can be seen that the release of ADR from the temperature-sensitive liposome of the present invention is not significantly affected by serum.

Example 3
In Example 1, except that the amount of the polymer compound was 0.094 mL, PEG was used in the ratio of EYPC: cholesterol: polymer compound: PEG = 50: 45: 1: 2 (molar ratio) except that the amount of the polymer compound was 0.094 mL. Containing temperature sensitive liposomes were prepared.

Comparative Example 2
Liposomes not retaining the polymer compound were produced in the same manner as in Example 1 except that the polymer compound was not added in Example 1.

Test example 3
For the liposomes of Examples 1 and 3 and Comparative Example 2, the ADR release rate 3 minutes after reaching each temperature was measured in the same manner as in Test Example 1. The results are shown in FIG.

  According to the present invention, it is possible to obtain temperature-sensitive liposomes that can release a drug at a target site and have a long residence time in blood when administered into human blood.

FIG. 1 is a graph showing changes over time in the release rate of ADR from the liposomes of A: Example 1; B: Example 2; and C: Comparative Example 1. FIG. 2 is a graph showing the temperature dependence of the release rate of ADR from the liposomes of Examples 1 and 2 and Comparative Example 1. FIG. 3 is a graph showing the ADR release rate when the temperature-sensitive liposome of the present invention is incubated at 37 ° C. or 45 ° C. under conditions containing fetal calf serum. FIG. 4 is a graph showing the temperature dependence of the release rate of ADR from the liposomes of Examples 1 and 3 and Comparative Example 2.

Claims (11)

  A temperature-sensitive liposome in which a polymer compound having a thermosensitive portion and a hydrophobic portion and polyethylene glycol are supported on a liposome membrane. It is composed of a liposome membrane-constituting lipid, a polymer compound having a heat-responsive part and a hydrophobic part, and polyethylene glycol,
Liposome membrane-constituting lipid: polymer compound: polyethylene glycol is used at a molar ratio of 1: 0.003-0.2: 0.001-0.2, using liposome membrane-constituting lipid, polymer compound and polyethylene glycol. Temperature-sensitive liposomes.   The temperature-sensitive liposome according to claim 1 or 2, which is obtained by bringing a polymer compound and a polyethylene glycol derivative into contact with a liposome membrane-constituting lipid.   The temperature-sensitive liposome according to claim 3, wherein the polyethylene glycol derivative is a polyethylene glycol-lipid complex, and the lipid constituting the polyethylene glycol-lipid complex is selected from the group consisting of phosphatidylethanolamine, diacylglycerol and cholesterol. .   The molecular weight of polyethylene glycol is 350-12000, The temperature sensitive liposome as described in any one of Claims 1-4.   The polymer compound is a block copolymer of a thermosensitive vinyl monomer containing at least one hydratable heteroatom and a hydrophobic vinyl monomer. Temperature sensitive liposomes. The polymer compound has the following formula (I):

[Wherein R ¹ is a hydrogen atom, the following formula (II):

(In the formula, X is the same or different and is a hydrogen atom or a halogen atom, and R ¹¹ is a hydrogen atom, an alkyl group having 1 to 20 carbon atoms or an aryl group.)
Or a group derived from a polymerization initiator;
R ² is an alkyl group having 1 to 8 carbon atoms, a halogenated alkyl group having 1 to 8 carbon atoms, an aldehyde group, a phenyl group, a biphenyl group, or an aralkyl group having 7 to 14 carbon atoms;
R ³ is a hydrocarbon group having 4 to 30 carbon atoms;
R ⁴ is a hydrogen atom, a C 1-10 alkoxy group or a group derived from a polymerization terminator;
m is 5 to 500;
n is 1-10;
y is 0-100. ]
The temperature-sensitive liposome according to any one of claims 1 to 6, which is a compound represented by the formula:   The temperature-sensitive liposome according to any one of claims 1 to 7, wherein the polymer compound is obtained by living cationic polymerization.   A temperature-sensitive drug release system comprising the temperature-sensitive liposome according to claim 1 and a drug.   The temperature sensitive drug release system according to claim 9, wherein the drug is an anticancer drug.   11. A temperature sensitive drug release system according to claim 9 or 10, further comprising at least one pharmaceutical additive.

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