JP5382567B2 - Temperature sensitive liposome - Google Patents

Description

  The present invention comprises a temperature-sensitive liposome that contains a polymer compound that can impart temperature sensitivity to liposomes and whose membrane structure collapses at a specific temperature, a polymer compound that can impart temperature sensitivity to liposomes, and the temperature-sensitive liposome and drug. It relates to a temperature sensitive drug release system.

  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. The liposome used for such an application can 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.

  As liposomes that can release inclusions at a specific site, liposomes that have been disintegrated at a specific temperature to release inclusions have been studied, and by combining such liposomes encapsulating anticancer agents with thermotherapy for cancer, It is known that an excellent therapeutic effect can be obtained.

  Japanese Patent Application Laid-Open No. 2003-221755 (Patent Document 1) discloses a liposome in which a polymer compound having a thermosensitive portion and a hydrophobic portion is held on a liposome membrane via the hydrophobic portion. 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. Have been described.

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

Japanese Patent Application Laid-Open No. 2006-306794 (Patent Document 2) discloses a temperature-sensitive liposome in which a polymer compound having a thermosensitive portion and a hydrophobic portion and polyethylene glycol (PEG) are supported on a liposome membrane. ing.
Japanese Patent Laid-Open No. 2003-221755 JP 2006-306794 A

  The inventors of the present invention have made extensive studies on a polymer compound capable of imparting temperature sensitivity and long-term blood retention in vivo to liposomes, and have a high molecular weight having a polyethylene glycol portion, a heat-sensitive responsive portion, and a hydrophobic portion. The present invention was completed by finding that the above-mentioned properties can be imparted to the liposome by supporting the molecular compound on the liposome membrane.

The present invention provides a temperature sensitive liposome in which a polymer compound having a polyethylene glycol moiety, a thermosensitive responsive moiety, and a hydrophobic moiety is supported on a liposome membrane.
The present invention also provides a polymer compound having a polyethylene glycol moiety, a thermosensitive responsive moiety, and a hydrophobic moiety.
Furthermore, the present invention also provides a temperature sensitive drug release system comprising the above temperature sensitive liposome and a drug.

The temperature-sensitive liposome of the present invention can provide a temperature-sensitive drug release system capable of releasing a drug retained by the liposome by disrupting the liposome membrane structure in a specific temperature range. Moreover, since the temperature sensitive liposome of this invention is comprised from polyethyleneglycol, it can also show longer blood retention.
For example, the above-described temperature-sensitive drug release system in which the temperature-sensitive liposome of the present invention contains an anticancer drug as a drug is administered to subjects including humans, and the affected area is heated to a specific temperature at an appropriate time after administration. By applying thermotherapy, the anticancer agent can be specifically and efficiently released at the affected area, and a more preferable DDS can be obtained.

  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.

  The liposome of the present invention is composed of a liposome membrane-constituting lipid and a polymer compound having a polyethylene glycol (PEG) portion, a heat-responsive portion and a hydrophobic portion. Specifically, the above polymer compound is supported on a liposome membrane. In the present specification, the substance is “supported by the liposome membrane” means that the substance may be bound to the liposome membrane, or a part or the whole of the substance is associated with the lipid bilayer constituting the liposome membrane. It may be embedded by.

Regarding the high molecular compound The above high molecular compound is preferably a copolymer of a PEG moiety, a thermosensitive vinyl monomer containing at least one hydratable heteroatom, and a hydrophobic vinyl monomer. Examples of the copolymer include a block copolymer and a random copolymer.

In the present specification, the “PEG moiety” means a moiety derived from polyethylene glycol and its derivative and including a structure in which oxyethylene groups are polymerized. Preferably, the PEG moiety is derived from a polymerization initiating species having polyoxyethylene as a side chain for the production of a polymer compound.
Examples of the polyethylene glycol derivative include compounds in which the hydroxy group of polyethylene glycol is substituted with an alkoxy group such as a methoxy group or a halogen-containing group such as chloro.

  The molecular weight of the PEG moiety is preferably 120 to 12000, more preferably 350 to 12000, and still more preferably 350 to 5000.

The heat-sensitive moiety is preferably derived from a heat-responsive vinyl monomer containing one or more hydratable heteroatoms.
Examples of hydratable heteroatoms possessed by the thermosensitive vinyl monomer include oxygen atoms and nitrogen atoms. 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.

The hydrophobic portion is preferably derived from a hydrophobic vinyl monomer.
As the hydrophobic vinyl monomer, a vinyl monomer 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 above polymer compound, the copolymerization ratio of the thermosensitive 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: 1. A degree, particularly 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 may be on the order of 1,000 to 30,000, more particularly on the order of 10,000 to 20,000.

  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, the number average molecular weight is about 2,000 to 50,000, more preferably about 10,000 to 25,000, and particularly preferably about 11,000 to 22,000. When the number average molecular weight is in this range, the collapse temperature of the liposome membrane structure can be set to a narrower temperature range.

The molecular weight distribution (weight average molecular weight (Mw) / number average molecular weight (Mn)) of the polymer compound is usually about 1 to 2, particularly about 1.0 to 1.3.
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 above polymer compound is more preferably the following formula:
R ^1- (A) _m- (B) _n -R ⁴
(Wherein R ¹ is a PEG moiety, A is a group derived from a thermosensitive vinyl monomer containing one or more hydratable heteroatoms, and B is a group derived from a hydrophobic vinyl monomer) R ⁴ is a hydrogen atom, a group derived from an alkoxy group having 1 to 10 carbon atoms or a polymerization terminator, m is 5 to 500, and n is 1 to 10). is there.
Preferable specific examples of the polymer compound include the following formula (I):

Where R ¹ is a PEG moiety;
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 to 10, and y is 0 to 4. ]
The compound represented by these is mentioned.

R ¹ in formula (I) above is a PEG moiety having a molecular weight in the above range. The PEG moiety is preferably of the following formula:

（式中、Ｒ⁵は、炭素数１〜３のアルキル基であり、Ｒ⁶は、水素原子又は炭素数１〜３のアルキル基であり、ｌは上記の範囲のＰＥＧ部分の分子量を与える数である）で表される。ＰＥＧ部分は、より好ましくは、以下の式：

Wherein R ⁵ is an alkyl group having 1 to 3 carbon atoms, R ⁶ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and l is a number that gives the molecular weight of the PEG moiety in the above range. ). The PEG moiety is more preferably of the following formula:

で表される。

It is represented by

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. Examples of such a group include a linear or branched alkyl group, an aryl group, an aralkyl group, and the like. Is mentioned. 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 types 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 4, and no oxyethylene exists when y = 0. When the oxyethylene chain is long, the collapse temperature of the liposome membrane structure can be relatively high.

  Preferred examples of the polymer compound are as follows.

(In each of the above formulas, R ¹ is the above PEG moiety, R ² is CH ₃ , C ₂ H ₅ , n- or iso-C ₃ H ₇ or n- or iso-C ₄ H ₉ . , R ⁴ is a group derived from a polymerization terminator such as a hydrogen atom or a methoxy group, and m and n are polymer compounds having a number average molecular weight (Mn) of about 11,000 to 50,000, (This value is such that the distribution (Mw / Mn) is about 1.0 to 1.3.)
In the compounds of the above formulas (II) to (IV), the octadecyloxy group as a side chain in the formulas (II) to (IV) is substituted with a dodecyloxy group, a tetradecyloxy group or a hexadecyloxy group. A small amount of the compound may be mixed.

  In the compounds of the above formulas (II) to (IV), the octadecyloxy group as a side chain in the formulas (II) to (IV) is substituted with a dodecyloxy group, a tetradecyloxy group or a hexadecyloxy group. Compounds can also be mentioned as preferred examples.

  The polymer compound of the above formula (I) is prepared by mixing a polymerizable monomer having a PEG moiety with a protonic acid to prepare a polymerization initiating species, and hydratable heteroatoms described above in a suitable solvent. Can be obtained by polymerizing a heat-sensitive vinyl monomer and a hydrophobic vinyl monomer containing at least one.

  As the proton acid, carboxylic acid such as trifluoroacetic acid, HCl and acetic acid, sulfonic acid such as methanesulfonic acid, and the like can be used.

The above polymer compound is polymerized by using a polymerizable monomer having a group containing polyoxyethylene represented by R ¹ in the above side chain (hereinafter referred to as PEG-containing monomer) and a protonic acid. It is more preferable to prepare seeds, polymerize the thermosensitive vinyl monomer in an appropriate solvent by cationic polymerization, and then copolymerize the hydrophobic vinyl monomer.

As the PEG-containing monomer, a vinyl monomer having polyoxyethylene is preferable.
The PEG-containing monomer can be obtained by reacting polyethylene glycol having a desired molecular weight or a derivative thereof with acetylene. Alternatively, it can be obtained by reacting polyethylene glycol having a desired molecular weight or a derivative thereof with methanesulfonyl chloride and then reacting with ethylene glycol monovinyl ether.
In the cationic polymerization, an organoaluminum compound such as ethylaluminum sesquichloride or triethylaluminum is preferably used as a catalyst.
The cationic polymerization is preferably carried out in the presence of a Lewis base. Examples of the Lewis base include ethers such as 1,4-dioxane and esters such as ethyl acetate.
Examples of the solvent that can be used for the polymerization include toluene, hexane, and a mixed solution thereof.

  The copolymerization of the hydrophobic vinyl monomer can be stopped by adding ammoniacal methanol, ammonia or the like. Specifically, for example, Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 30, p.2407-2413 (1992); Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 43, p.1155-1165 (2005); Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 43, p.2712-2722 (2005).

  As the cationic 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 the 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 above polymerization, the thermosensitive vinyl monomer is preferably used in an amount of 50 to 300 mole equivalent, more preferably 60 to 250 mole equivalent, and still more preferably 75 to 1 mole of PEG-containing monomer. ~ 200 molar equivalents.
In the above polymerization, the hydrophobic vinyl monomer is preferably used in an amount of 1 to 30 mole equivalent, more preferably 1 to 20 mole equivalent, per mole of PEG-containing monomer.

The above-mentioned polymer compound having a PEG moiety, a heat-sensitive responsive moiety, and a hydrophobic moiety imparts characteristics of temperature sensitivity and extension of blood residence time to the liposome by preparing the liposome using the polymer compound. it can.
Therefore, the present invention also provides a polymer compound having a PEG portion, a thermosensitive response portion, and a hydrophobic portion, which can impart temperature-sensitive and blood retention time extension properties to the liposome.

Regarding the liposome membrane-constituting lipid 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.

About temperature sensitive liposome The particle diameter of the temperature sensitive liposome of this invention should just be about 0.05-10 micrometers, and can be made into various particle diameters according to a use purpose. 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 is within the above particle size range. .

  The above temperature-sensitive liposome is preferably obtained by using the above polymer compound in an amount of 0.01 to 30 mol% with respect to the total number of moles of the liposome membrane-constituting lipid and polymer compound, and more preferably 0.05 to 6 mol%.

The temperature-sensitive liposome of the present invention is produced by bringing the above-described liposome membrane-constituting lipid and the above-described polymer compound into contact with each other at a ratio that satisfies the above ratio in a known liposome production method. Can do.
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 Fairlake Tokyo). ing.

  For example, a method for producing the liposome of the present invention by the extruder method will be described. A solution is prepared by dissolving a predetermined amount of liposome membrane-constituting lipid and polymer compound in an appropriate organic solvent such as chloroform, and the mixture is placed in a container and mixed. Next, the solvent is removed using an evaporator, and a thin film composed of a liposome membrane-constituting lipid and a polymer compound 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, unsupported polymer compounds 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 liposome membrane-constituting lipid is used to form a liposome in advance by the extruder method as described above, and then a polymer compound is added to support the polymer compound on the liposome membrane. it can.

  In the temperature-sensitive liposome of the present invention produced by the method as described above, the thermosensitive responsive portion and the PEG portion of the polymer compound may be carried on the outer surface of the liposome membrane, It may be carried on the inner surface, or may be carried on both the outer surface and the inner surface of the liposome membrane.

  The temperature-sensitive liposome of the present invention may further contain another polymer compound having the heat-sensitive responsive part and the hydrophobic part. By including such a polymer compound, it is considered that the temperature at which the structure of the temperature-sensitive liposome is destroyed can be controlled more easily.

Regarding the temperature-sensitive drug release system, a temperature-sensitive drug release system comprising the above-described 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 temperature sensitive liposome may also contain a gene for treatment of a disease instead of the drug. 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, powder or capsule, but is preferably in the form of a liquid preparation such as an injection preparation. The liquid formulation may be provided as a dry product that is regenerated with water or other suitable excipients at the time of use.
The above tablets and capsules are desirably enteric-coated by a usual method. As the enteric coating, those normally used in this field can be used. Capsules can also contain either powder or liquid.

  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-mentioned drug as it is or freeze-dried with the above-mentioned pharmaceutical additive. When freeze-drying temperature-sensitive liposomes containing a drug, an appropriate excipient should be added before freeze-drying.

By administering the temperature-sensitive drug release system of the present invention to a subject and heating the affected area to a temperature of 40 ° C. or higher, preferably 40 to 47 ° C., more preferably 42 to 45 ° C., a temperature-sensitive liposome membrane The structure can be disrupted to release the drug 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.
Therefore, according to the present invention, an effective amount of the above temperature sensitive drug release system is orally or parenterally administered to a subject who needs treatment or prevention, and the skin above the affected area of the subject is heated after a certain period of time. A method of treating or preventing a disease is provided.

The subject is preferably a mammal, particularly preferably a human.
The fixed period is preferably at least 3 hours after administration of the temperature sensitive drug release system, more preferably 3 to 48 hours, even more preferably 6 to 24 hours, and even more preferably 6 to 12 hours.

In the treatment method of the present invention, the temperature for heating the skin above the affected area is such that at least a part of the administered temperature-sensitive drug release system is destroyed and the skin above the affected area is not burned. Good. For example, at least a part of the temperature-sensitive drug release system is heated by heating so that the temperature of the skin above the affected area is 38 to 50 ° C, more preferably 39 to 47 ° C, and still more preferably 42 to 45 ° C. Can be destroyed. At the time of the above heating, a protective cloth or the like may be applied to the skin to be heated so as not to cause a burn on the skin above the affected area.
The method of heating the skin above the affected area is not particularly limited as long as it is a method used in thermotherapy, and examples thereof include a method of irradiating an electromagnetic wave such as 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.
The materials produced in the following examples were characterized by the following measurement method.
<Measurement method of molecular weight>
The weight average molecular weight (Mw) and the 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.

<Measurement of lipid concentration>
Phospholipids were quantified using Phospholipid C-Test Wako (Wako Pure Chemical Industries, Ltd.). The sample solution (liposome dispersion) and the standard solution were mixed well with the luminescent solution, respectively, and heated at 37 ° C. for 5 minutes. The absorbance of the sample solution at a wavelength of 600 nm was measured using a V-520 type ultraviolet / visible spectrophotometer manufactured by JASCO Corporation, and the lipid concentration of the sample solution was determined from the obtained absorbance.

<Measurement of liposome particle size>
The particle size of the liposome was determined by a dynamic light scattering method. The liposome dispersion was added to HBS buffer (20 mM Hepes, 150 mM NaCl, pH 7.4) (2 ml) adjusted to 25 ° C. to a lipid concentration of 0.5 mM, and granulated using ELS-8000F manufactured by Otsuka Electronics Co., Ltd. The diameter was measured.

<Measurement of adriamycin encapsulation efficiency in liposome>
Adriamycin (ADR) was added to the liposomes, and allowed to stand at 30 ° C. for 1 hour, and 10 μl of the liposome dispersion was dissolved in methanol (total amount 2 ml). The absorbance of this methanol solution at 499 nm was measured using a V-560 type ultraviolet / visible spectrophotometer manufactured by JASCO Corporation. By dividing this value by the lipid concentration determined by Test Wako C, the ratio of ADR to lipid was determined, and this value was taken as the value of 100% ADR encapsulation efficiency. By dividing this value by the value measured in the same manner for the liposome dispersion from which ADR that was not encapsulated through the column was removed, the encapsulation efficiency of ADR in the liposome was determined.

In the following examples, 2- (2-ethoxy) ethoxyethyl vinyl ether (EOEOVE) as a thermosensitive vinyl monomer having PEG having a molecular weight of 750 as a PEG moiety and containing one or more hydratable heteroatoms A method for producing a polymer compound obtained by polymerizing octadecyl vinyl ether (ODVE) as a hydrophobic vinyl monomer will be described.
In the names of the following polymer compounds (PEG (750) -EOEOVE (X) -ODVE (Y)), “PEG (750)-” represents that the PEG moiety has a molecular weight of 750, and “-EOEOVE (X X of “)-” represents the degree of polymerization of EOEOVE, and Y of “-ODVE (Y)” represents the molar ratio of the reacted ODVE to the polymerization initiation species.

Example 1 Production of polymer compound of PEG (750) -EOEOVE (87) -ODVE (4.5) (1) Production of PEG-containing monomer

  In a dry glass apparatus, 63 g of methoxypolyethylene glycol (mPEG, Mw = 750, Aldrich) was added, azeotropically dried with toluene, cooled, and dissolved in 200 mL of dehydrated dichloromethane. At 0 ° C., methanesulfonyl chloride (6.9 mL, 1.06 equivalent, Nacalai Tesque) and triethylamine (12.4 mL, 1.06 equivalent, Aldrich) were added dropwise with stirring, and after 20 hours of reaction, the solution was filtered. After concentrating the solvent, it was added to diethyl ether at 0 ° C. for reprecipitation. Thereafter, the precipitated product was collected and dried under reduced pressure to obtain Compound (V) (Reference: Bioconjugate Chem. 1996, 7, 363-368).

  Sodium hydride (2.00 g, 60% in oil, 50 mmol, Nacalai Tesque) was placed in a dry glass apparatus under dry nitrogen. The oil was washed with dehydrated hexane, and dehydrated tetrahydrofuran (THF) (150 mL) was added. Thereto, ethylene glycol monovinyl ether (8.82 g, 100 mmol, Tokyo Chemical Industry) was slowly added dropwise to convert it into an alkoxide. After the reaction proceeded completely, the compound (V) (21.00 g, 25 mmol) produced as described above was added and reacted at room temperature with stirring for 24 hours. THF was evaporated, diluted with 100 mL of dichloromethane, and washed 4 times with about 10 mL of distilled water. Dichloromethane was evaporated, dissolved in 75 mL benzene and dried azeotropically for 4 hours. It was added to diethyl ether (0 ° C, 350 mL), and the precipitated product was dried under reduced pressure to obtain a vinyl monomer having methoxypolyoxyethylene in the side chain as a PEG-containing monomer (references: Polymer Bulletin 2002, 48, 221-224).

(2) Polymerization of PEG-containing monomer with thermosensitive vinyl monomer and hydrophobic vinyl monomer

  The next reaction was performed under dry nitrogen in a Schlenk with a three-way stopcock, which was baked with an industrial dryer and then cooled to room temperature. Toluene, 1,4-dioxane as an added base (final concentration: 1.2 mol / L), prepared by mixing equimolar amounts of the PEG-containing monomer prepared above and trifluoroacetic acid Polymerization initiating species (final concentration: 4 mmol / L each), 2- (2-ethoxy) ethoxyethyl vinyl ether (EOEOVE) as a thermosensitive vinyl monomer containing at least one hydratable heteroatom (final concentration: 0.35 mol / L) was placed in a Schlenk in order using a glass syringe and stirred, and cooled to 0 ° C. Polymerization was started by adding a toluene solution (1.0 mol / L) of commercially available ethylaluminum sesquichloride as a catalyst to 20 mmol / L.

  After 23 minutes, 4.5 eq. Of octadecyl vinyl ether (ODVE) as the second monomer (hydrophobic vinyl monomer) to the polymerization solution in which the first monomer (EOEOVE) was almost consumed added. After 12 minutes, the polymerization was terminated by adding a large excess of methanol containing 0.1% by volume of ammonia water relative to the number of moles of the polymerization starting species. The polymerization solution was diluted with dichloromethane and then separated 10 times with ion-exchanged water to remove the catalyst residue, and the solvent was evaporated to obtain PEG (750) -EOEOVE (87) -ODVE (4.5).

The molecular weight distribution (Mw / Mn) and Mn of the obtained polymer compound were determined by GPC, and the composition ratio was determined by proton nuclear magnetic resonance ( ¹ H-NMR) (JEOL JNM-LA 400). As a result, it was found that the obtained polymer compound had Mn = 9700, Mw / Mn = 1.04, EOEOVE polymerization degree = 87, and ODVE polymerization degree = 5.

Example 2 Production of Polymer Compound of PEG (750) -EOEOVE (131) -ODVE (4) In Example 1, the final concentration of EOEOVE was 0.53 mol / L, and ODVE was based on the number of moles of polymerization starting species. A polymer compound of PEG (750) -EOEOVE (131) -ODVE (4) was obtained in the same manner as in Example 1 except that 4 equivalents were added.
The obtained polymer compound had Mn = 10000, Mw / Mn = 1.08, EOEOVE polymerization degree = 131, and ODVE polymerization degree = 4.

Example 3 Production of Polymer Compound of PEG (750) -EOEOVE (174) -ODVE (3) In Example 1, the final concentration of EOEOVE was 0.70 mol / L, and ODVE was based on the number of moles of the polymerization starting species. A polymer compound of PEG (750) -EOEOVE (174) -ODVE (3) was obtained in the same manner as in Example 1 except that 3 equivalents were added.
The obtained polymer compound had Mn = 13000, Mw / Mn = 1.08, EOEOVE polymerization degree = 174, and ODVE polymerization degree = 3.

Comparative Example 1 Production of EOEOVE (87) -ODVE (4.5) Toluene (6.2 ml) as a solvent, ethyl acetate (10 mmol) as an added base, (2-ethoxy) ethoxyethyl vinyl ether (EOEOVE) (3.5 mmol) as a monomer, 1-methoxyethoxyethyl acetate (0.04 mmol) was used as a polymerization initiator. 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) (0.18 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 EOEOVE / ODVE copolymerization ratio of approximately 100/5.

Example 4 Production of Temperature Sensitive Liposomes (Production of Temperature Sensitive Liposomes Composed of Each Component in Ratio of EYPC / Cholesterol / PEG (750) -EOEOVE (174) -ODVE (3) = 51.6: 46.4: 2 (mol%))
Egg yolk phosphatidylcholine (EYPC, Nippon Oil & Fats) chloroform solution (10 mg / ml) 0.4 ml, cholesterol (SIGMA) chloroform solution (10 mg / ml) 0.174 ml and PEG (750) prepared in Example 3 -EOEOVE (174) -ODVE (3) in methanol solution (10 mg / ml) 0.582 ml (weight ratio, 1: 0.436: 1.46) is placed in a 10 ml eggplant flask and mixed, and the solvent is removed with a rotary evaporator to form a thin film. Formed. Thereafter, 0.8 ml of an aqueous ammonium sulfate solution (300 mM ammonium sulfate (pH 5.2)) was added, and ultrasonic irradiation was performed until the thin film was peeled off. Furthermore, liposomes were prepared by repeating freeze-thaw four times, and passed through a polycarbonate membrane with a pore size of 100 nm using an extruder to make the particle sizes of the liposomes uniform. The resulting liposome dispersion was passed through a Sepharose 4B column equilibrated with Hepes buffer (HBS) (20 mM Hepes, 150 mM NaCl (pH 7.4)) to remove the polymer compound not supported on the liposomes and A pH gradient of the layer was formed.

Then, add adriamycin (ADR) aqueous solution (10 mg / ml) to the liposome dispersion to a concentration of 100 μg / μmol lipid, leave it in the dark at 30 ° C for 1 hour, and then leave it in the refrigerator for 1 day. Thus, an ADR-containing liposome in which ADR was encapsulated in the liposome was obtained. This ADR-containing liposome dispersion was passed through a Sepharose 4B column equilibrated with HBS to remove ADR not encapsulated in the liposomes.
The encapsulation efficiency of ADR measured as described above was about 100%.

Example 5 Production of temperature-sensitive liposomes (Production of temperature-sensitive liposomes comprising EYPC / cholesterol / PEG (750) -EOEOVE (174) -ODVE (3) = 50.5: 45.5: 4 (mol%) components)
Example 4 was the same as Example 4 except that 1.189 ml of a methanol solution (10 mg / ml) of PEG (750) -EOEOVE (174) -ODVE (3) was used (weight ratio, 1: 0.436: 2.97). Thus, ADR-containing temperature-sensitive liposomes having the above composition were obtained.
The encapsulation efficiency of ADR measured as described above was about 100%.

Example 6 Production of Temperature Sensitive Liposomes (Production of Temperature Sensitive Liposomes Consisting of EYPC / Cholesterol / PEG (750) -EOEOVE (174) -ODVE (3) = 49.5: 44.5: 6 (mol%) Components)
Example 4 was the same as Example 4 except that 1.82 ml of a methanol solution (10 mg / ml) of PEG (750) -EOEOVE (174) -ODVE (3) was used (weight ratio, 1: 0.436: 4.55). Thus, ADR-containing temperature-sensitive liposomes having the above composition were obtained.
The encapsulation efficiency of ADR measured as described above was about 100%.

Example 7 Production of Temperature Sensitive Liposomes (Production of Temperature Sensitive Liposomes Consisting of EYPC / Cholesterol / PEG (750) -EOEOVE (87) -ODVE (4.5) = 51.6: 46.4: 2 (mol%))
In Example 4, instead of 0.582 ml of a methanol solution (10 mg / ml) of PEG (750) -EOEOVE (174) -ODVE (3), PEG (750) -EOEOVE (87) obtained in Example 1 was used. -ADR-containing temperature-sensitive liposomes were obtained in the same manner as in Example 4 except that 0.525 ml of methanol solution (6 mg / ml) of -ODVE (4.5) was used (weight ratio, 1: 0.436: 1.31). It was.
The encapsulation efficiency of ADR measured as described above was about 100%.

Example 8 Production of Temperature Sensitive Liposomes (Production of Temperature Sensitive Liposomes Composed of Each Component in Ratio of EYPC / Cholesterol / PEG (750) -EOEOVE (131) -ODVE (4) = 51.6: 46.4: 2 (mol%))
In Example 4, instead of 0.582 ml of a methanol solution (10 mg / ml) of PEG (750) -EOEOVE (174) -ODVE (3), PEG (750) -EOEOVE (131) obtained in Example 2 was used. -ADR-containing temperature-sensitive liposomes were obtained in the same manner as in Example 4 except that 0.446 ml of a methanol solution (10 mg / ml) of -ODVE (4) was used (weight ratio, 1: 0.436: 1.12). It was.
The encapsulation efficiency of ADR measured as described above was about 100%.

Experimental Example 1 Measurement of Phase Transition Temperature of Polymer Compound The phase transition temperatures of the polymer compounds of Examples 1 to 3 and the polymer of Comparative Example 1 were evaluated by differential scanning calorimetry (DSC) measurement. 1 ml of a methanol solution (10.0 mg / ml) of each polymer compound was placed in a dripping eggplant flask and the solvent was removed by a rotary evaporator to form a thin film. Thereafter, 100 μl of distilled water was added and the mixture was stirred and dispersed with a voltex. The solution (50 μl) was sealed in an aluminum pan for measurement, and the endothermic amount was measured using DSC-120 manufactured by Seiko Electronics. Differential scanning calorimetry was performed at a heating rate of 0.5 ° C./min.

  The results are shown in FIG. From the results of FIG. 1, it can be seen that the polymer compound of the present invention having a PEG moiety exhibits endotherm at around 40 ° C., and transitions from hydrophilic to hydrophobic in this temperature range. Since the phase transition temperature of the polymer of Comparative Example 1 having no PEG portion is around 37 ° C., it seems that the phase transition temperature slightly increased by having the PEG portion. Further, as the degree of polymerization of EOEOVE, which is a heat-sensitive part, increases, the endothermic peak becomes sharper and the amount of spherical heat increases. Therefore, it is considered that the higher the degree of polymerization of the thermosensitive responsive portion, the more efficient the phase transition and the formation of a highly hydrophobic domain in a narrow temperature range.

Experimental Example 2 Relationship between Cloud Point and Polymer Compound Concentration The absorbance at 700 nm of a dispersion of the polymer compound of Examples 1 to 3 in a predetermined concentration in HBS buffer was measured by JASCO Corporation V-560 type. The measurement was performed at a temperature rising rate of 0.5 ° C./min using an ultraviolet / visible spectrophotometer. Here, the clouding point was defined as the temperature at which the absorbance begins to rise with respect to the temperature rise.
FIG. 2 shows changes in cloud point at each concentration of the polymer compound.
The polymer compound of Example 1 has a low cloud concentration and a high cloud point, and at a concentration of 0.5 mg / ml or more, the cloud point is around 38 ° C. On the other hand, the high molecular compounds of Examples 2 and 3 have a cloud point of around 38 ° C. regardless of their concentrations. From these facts, it is suggested that when the degree of polymerization of the thermosensitive responsive portion is relatively short, the chain length becomes short, so that the interaction between molecules is suppressed and the transition is unlikely to occur.

Experimental Example 3 Release of Adriamycin from Liposomes Using the ADR-containing liposomes of Examples 4, 7 and 8 above, the release rate of ADR from the liposomes was measured according to the following Adriamycin (ADR) release test.
<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. Bring 10% fetal bovine serum (FBS) -containing or non-FBS-containing HBS buffer solution to a specified temperature, and add the liposome dispersion to 3 ml of this FBS-containing or non-containing HBS buffer solution so that the liposome lipid concentration is 13 μM. After a predetermined time, the fluorescence intensity was measured using an FP-6200 type fluorimeter manufactured by JASCO Corporation.

  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 468 nm and 590 nm, respectively.

The release rate (%) of ADR from the liposome was calculated using the following formula.
Release rate ^{(%) = (F t -F} i) / {(F 'f × 1.07) -F i} × 100
Here, F ^t is the fluorescence intensity when incubated at a predetermined temperature for a predetermined time;
F ^'f, the fluorescence intensity when destroying liposome by adding 60μl of surfactant (Triton X-100) at a predetermined temperature;
^Fi is the initial fluorescence intensity at 10 ° C.

The results are shown in FIG. FIG. 3 (A) shows the liposomes of Example 4 (■), Example 7 (♦), and Example 8 (▲) when 5 minutes have passed since the surfactant was added in the FBS-free HBS buffer. Shows the ADR release rate.
FIG. 3 (B) shows the liposomes of Example 4 (■), Example 7 (♦), and Example 8 (▲) when a surfactant was added in a 10% FBS-containing HBS buffer for 5 minutes. The ADR release rate from is shown.
From these results, it can be seen that the temperature-sensitive liposome of the present invention can release inclusions even when serum is present when a certain temperature is reached.

Experimental Example 4 Release of Adriamycin from Liposomes Using the ADR-containing liposomes of Examples 4, 5 and 6 above, the release rate of ADR from the liposomes was measured in the same manner as in Experimental Example 1.
The results are shown in FIG. FIG. 4 (A) shows the liposomes of Example 4 (●), Example 5 (♦), and Example 6 (▲) when a surfactant was added in an FBS-free HBS buffer for 5 minutes. Shows the ADR release rate.
FIG. 4 (B) shows the liposomes of Example 4 (■), Example 5 (♦), and Example 6 (▲) when a surfactant was added in an HBS buffer containing 10% FBS for 5 minutes. The ADR release rate from is shown.

  From these results, it can be seen that the temperature-sensitive liposome of the present invention releases the contents more rapidly when the content of the polymer compound in the membrane component increases.

The result of the differential scanning calorimeter (DSC) measurement of the high molecular compound of Examples 1-3 is shown. The change of the cloud point in each density | concentration of a high molecular compound is shown. (A) ADR release rate from liposomes of Example 4 (■), Example 7 (♦), and Example 8 (▲) when 5 minutes have passed after adding a surfactant in an FBS-free HBS buffer Indicates. (B) ADR release from liposomes of Example 4 (■), Example 7 (♦), and Example 8 (▲) when 5 minutes have elapsed after adding a surfactant in an HBS buffer containing 10% FBS Indicates the rate. (A) ADR release rate from the liposomes of Example 4 (●), Example 5 (♦), and Example 6 (▲) when 5 minutes have passed since the surfactant was added in the FBS-free HBS buffer Indicates. (B) ADR release from liposomes of Example 4 (■), Example 4 (♦), and Example 6 (▲) when 5 minutes have elapsed after adding a surfactant in an HBS buffer containing 10% FBS Indicates the rate.

Claims (6)

  A temperature-sensitive liposome in which a polymer compound having a polyethylene glycol part, a thermosensitive part and a hydrophobic part is supported on a liposome membrane.   The temperature-sensitive liposome according to claim 1, obtained by bringing a polymer compound having a polyethylene glycol moiety, a thermosensitive responsive moiety, and a hydrophobic moiety into contact with a liposome membrane-constituting lipid.   The temperature-sensitive liposome according to claim 1 or 2, wherein the polyethylene glycol moiety has a molecular weight of 350 to 12,000.   A temperature-sensitive drug release system comprising the temperature-sensitive liposome according to any one of claims 1 to 3 and a drug.   6. The temperature sensitive drug release system according to claim 5, wherein the drug is an anticancer drug. The temperature sensitive drug release system according to claim 4 or 5 , further comprising at least one pharmaceutical additive.

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