JP4715133B2 - Anti-tumor liposome preparation and production method thereof - Google Patents

Description

  The present invention relates to an antitumor liposome preparation and a method for producing the same, and more specifically, a liposome in which an anticancer compound or the like is encapsulated inside a phospholipid membrane using a supercritical carbon dioxide method, an antitumor liposome preparation containing the liposome, and It relates to the manufacturing method.

  Cancer chemotherapy, as well as surgical treatment and radiation therapy, is evaluated as an effective treatment method for cancer and is based on the administration of anticancer drugs. Administration of anticancer agents is roughly divided into systemic chemotherapy and local injection therapy according to the administration form. Systemic chemotherapy should be applied especially when cancer in the blood, cancer that tends to metastasize throughout the body via blood or lymph, or when the lesion is located throughout the body. Anticancer drugs are distributed throughout the body in circulating blood flow, and only a part of them reaches the tumor, so side effects and useless administration often become problems. Various methods of administration have been proposed for local administration methods for systemic chemotherapy, and the aim is to enhance the direct administration effect of anticancer agents and avoid side effects as much as possible.

  The demand for intensive accumulation of anticancer agents in cancer lesions and the reduction of strong side effects as much as possible is particularly acute in anticancer agents. In the field of anticancer agents and chemotherapy using the same, development of dosage forms of anticancer agents that embody the concept of DDS (drug delivery system) and methods for their administration has been vigorously advanced. The central dosage form is a DDS-like improved preparation using liposomes. This is because the expectation for liposomes and their utilization was particularly high as a technology that can sufficiently meet the above requirements. However, despite many disclosures and proposals related to liposomes, liposome preparations containing anticancer agents cannot be obtained on the market. Stability, inclusion efficiency, cost, and the like were cited as reasons for hindering practical use (Patent Document 1).

  Lipid-soluble drugs are easily encapsulated in liposomes, but the encapsulated amount depends on other factors and is not necessarily large. A drug that is a water-soluble electrolyte can be encapsulated in the aqueous phase inside the liposome through the interaction between the charge of the drug and the charge of the charged lipid, but if the drug is a water-soluble non-electrolyte, such means should be used. It cannot be taken.

In JP-A-2003-119120 (Patent Document 2), a cosmetic containing a liposome and an external preparation for skin are disclosed.
A method of producing using supercritical carbon dioxide is disclosed, and an example of producing an external preparation for skin in which a hydrophilic medicinal component or a lipophilic medicinal component is encapsulated in liposomes is shown. However, although examples of water-soluble electrolytes have been shown as hydrophilic medicinal ingredients, it was unclear whether water-soluble non-electrolytes could be efficiently encapsulated in liposomes by this method.

Even if the anticancer drug is successfully encapsulated inside the liposome, the problem of leakage to the outside over time or the situation where the liposome itself becomes unstable must be considered. Furthermore, it has been pointed out that even when liposomes are administered in vivo, many of them are trapped in the reticuloendothelial tissues such as the liver and spleen, so the desired effect cannot be obtained (Cancer Res., 43, 5328 (1983))
.

Lastly, the safety of the drug product can be pointed out as the biggest challenge for practical application. In fact, liposomes are composed of lipids similar to biological membranes and have low antigenicity, so that they are not toxic even when intravenously injected into the human body (Patent Document 1). In the conventional dispensing method, despite the use of liposomes with high safety as a raw material and appropriate degradability in vivo, there is no exception as a solvent for phospholipids constituting the liposome membrane in the liposome production process. Organic solvents,
In particular, chloro solvents such as chloroform and dichloromethane are used (for example, Patent Document 3). Therefore, the toxicity of the remaining solvent inevitably remains. Accordingly, there is a demand for a method for preparing liposomes that can efficiently encapsulate and stably hold liposome preparations or contrast substances, and that does not pose a safety problem.
Patent No. 2882607 Japanese Patent Laid-Open No. 2003-119120 Japanese Patent Laid-Open No. 5-194191

  In order to solve the above-mentioned problems, the present inventors have conducted intensive research. As a result, in contrast to the use of an organic solvent that hinders the practical application of liposome preparations, the present invention relating to a method for preparing liposomes that do not use an organic solvent, stabilization of liposome structure, and stabilization of drug encapsulation has been completed.

  It is an object of the present invention to provide a preparation of a liposome having high tumor efficiency and high delivery efficiency by encapsulating a drug such as an anticancer compound in the liposome, and a method for producing the same. The liposome is produced using a supercritical carbon dioxide method instead of a toxic organic solvent, and is characterized in that it can stably hold the drug.

The present invention is prepared by mixing a lipid membrane component constituting a phospholipid membrane and carbon dioxide in a supercritical or subcritical state under conditions where at least one compound having a polyethylene glycol (PEG) group is present. And an average particle size of 0.05 to 0.7 μm, and at least one hydrophilic anticancer compound in the phospholipid membrane or in its internal aqueous phase is in a weight ratio of 3 to 8 with respect to the lipid membrane component. in contained, the upper limit of the concentration of the residual organic solvent medium is a liposome, which is a 10 [mu] g / L.

The phospholipid membrane is preferably composed of a single membrane or two or three membranes.
Furthermore, it is desirable that the average particle size is 0.1 to 0.2 μm.
It is desirable that the phospholipid membrane contains at least a phospholipid having a transition temperature.

The compound having a polyethylene glycol group is a PEG-phospholipid.
Before climate-cancerous compounds, cyclophosphamide, mechlorethamine, carbazyl quinone, melphalan, thiotepa, busulfan, nimustine, carmustine, procarbazine, dacarbazine, methotrexate, 6-mercaptopurine, 6-thioguanine, azathioprine, fluorouracil, Ftorafur, floxuridine, cytarabine, ancitabine, tegafur, doxyfluridine, actinomycin D, bleomycin, mitomycin C, chromomycin A3, sinerbin A, aclacinomycin A, adriamycin, pepomycin, doxorubicin, cisplatin, mitoxantrone, epirubicin, pirarubicin Vinblastine, vincristine, vindesine, etoposide, cisplatin, carboplatin, est phosphate At least one selected from ramustine, mitotane, porphyrin, and taxol.

The anticancer compound is preferably at least one selected from adriamycin, pirarubicin , vincristine, taxol, cisplatin, mitomycin, and 5-fluorouracil.

The said anti-cancer compound is a porphyrin, hematoporphyrin IX, Photofrin II, verteporfin (verteporfin), a Pururichin (purlytin) or lutetium texaphyrin (lutetium texaphyrin), be used in photodynamic therapy of cancer Good.

The antitumor liposome preparation of the present invention is a preparation comprising the above-mentioned liposome.
The anti-tumor liposome preparation may further contain a contrast agent.
The antitumor liposome preparation is characterized by applying hyperthermia after administration to a patient.

The method of producing liposomes of the present invention, under conditions at least one compound having a polyethylene glycol group is present, as the lipid membrane component, together with the phosphorus lipid having at least the transition temperature, the cationic lipids and cholesterol, dihydrocholesterol , Cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol, 2,4-dihydrolanosterol, 1-O-sterol glycoside, 1-O-sterol maltoside and 1-O-sterol galactoside after dissolving or dispersing one compound selected in the carbon dioxide in the supercritical state or subcritical state, in introducing a solution or suspension containing at least one hydrophilic anticancer compounds Ri micelles to form and then emit carbon dioxide by adding water, a manufacturing method comprising preparing the liposome containing the compound therein.

The compound having a polyethylene glycol group may be used as a solubilizing agent at a ratio of 0.01 to 1% by mass with respect to carbon dioxide to be in a supercritical state or a subcritical state.
The solution or suspension of the anticancer compound may contain a formulation aid.
[Detailed Description of the Invention]
The antitumor liposome preparation of the present invention has an average particle size of 0.05 to 0.7 μm, contains at least one kind of drug in the phospholipid membrane or in its internal aqueous phase, and is substantially free of an organic solvent. Contains characteristic liposomes.

  A “liposome” is a structure usually formed by a liposome membrane composed of a lipid bilayer membrane. In the present specification, the liposome membrane may be referred to as “phospholipid membrane” or simply “lipid membrane”. “Encapsulated” in a liposome means that it is encapsulated in the liposome and associated with the phospholipid membrane, or is present in an aqueous phase (internal aqueous phase) confined inside the phospholipid membrane. Includes both states.

  A “drug” is a drug substance to be encapsulated in liposomes. “Drugs” contained in the phospholipid membrane or in its internal aqueous phase include anticancer compounds, contrast agents, neutron capture therapy substances, photodynamic therapy substances, formulation aids and the like. In this specification, “anticancer compound” is used to mean a single substance anticancer agent. “Cancer” refers to a malignant tumor, sometimes simply “tumor”.

“Substantially” means that the upper limit of the concentration of the residual organic solvent in the preparation is 10 μg / L.
Anticancer compound The anticancer compound contained in the liposome of the present invention is not particularly limited as long as it is a chemical substance used in cancer chemotherapy. Specific examples of such anticancer compounds include the following chemical substances:
Alkylating agents such as cyclophosphamide, melphalan, chlorambutyl, mechloretamine, carbazylquinone, thiotepa, busulfan, nimustine, carmustine, procarbazine, dacarbazine; methotrexate, 6-mercaptopurine, 6-thioguanine, azathioprine, 5-fluorouracil, Antimetabolites such as ftorafur, floxuridine, cytarabine, ancitabine, gemcitabine, tegafur, carmofur, UFT, doxyfluridine; actinomycin D, bleomycin, mitomycin C, chromomycin A3, synerbin A, aclacinomycin A, adriamycin, pepromycin, Antibiotics such as mitoxantrone, epirubicin, daunorubicin, idarubicin, pirarubicin; vinorelbine, Microtubule agents such as paclitaxel and docetaxel; plant components such as vinblastine, vincristine, vindesine, etoposide, taxol; irinotecan, cisplatin, carboplatin, etoposide, nedaplatin, mitotane, porphyrin, etc.

  Anticancer compounds include drugs such as hormone drugs such as mepithiostan, tamoxifen, phosfestol, medroxyprogesterone acetate, estramustine phosphate, and immunostimulants that are anticancer drugs in a broad sense.

These compounds may be used alone or in combination of two or more. Moreover, it is not limited to said illustration. In the present specification, the compound may be referred to in a form including a salt, a hydrate and the like in addition to the free form. Among the above anticancer compounds, a compound selected from at least one of adriamycin, pirarubicin , vincristine, taxol, cisplatin, mitomycin, and 5-fluorouracil is preferably used. Usually, it is considered that the water-soluble anticancer compound is encapsulated in the aqueous phase in the lipid membrane, and the lipophilic anticancer compound is encapsulated in the lipid membrane bilayer.

In the liposome of the present invention, the total amount of the anticancer compound encapsulated in the liposome can be arbitrarily set based on various factors such as the properties of the liposome, the intended administration route of the preparation and clinical indicators. The amount of anticancer compound encapsulated in the liposome is typically 65-98% by weight of the total anticancer compound in the liposome, preferably 75-95% by weight, more preferably 85-90% by weight. It is.
Liposome In the antitumor liposome preparation of the present invention, the above anticancer compound is used in a form encapsulated in a liposome as a microcarrier so as to be selectively and efficiently delivered to a target organ or tissue cancer lesion. The The liposome of the present invention has an average particle size of 0.05 to 0.7 μm and contains an anticancer compound or other drug in the phospholipid membrane or in its internal aqueous phase, preferably the phospholipid membrane is substantially It is a liposome composed of one or several membranes.

  The liposome of the present invention can realize the targeting function by appropriately designing the particle size of the liposome encapsulating an anticancer compound and the like and its bilayer membrane. Both passive targeting and active targeting are considered. Passive targeting is based on controlling in vivo behavior through adjustment of liposome particle size, lipid composition, charge, and the like. Adjustment to align the liposome particle size distribution within a narrow range is easily performed based on the method described below. The design of the liposome membrane surface can impart desired properties by changing the type and composition of phospholipids and coexisting substances.

  Employment of active targeting that allows for higher delivery selectivity and accumulation with respect to the internalization of administered liposomes should also be considered. As an example, introducing a polyalkylene oxide polymer chain or polyethylene glycol (PEG) on the surface of the liposome membrane is extremely beneficial because the induction process to the target site can be controlled. Therefore, the liposome suitable for the liposome preparation of the present invention has a higher retention in the blood by adding polyalkylene oxide or polyethylene glycol to its surface, and is less likely to be phagocytosed by reticuloendothelial cells such as the liver. Liposome. Liposomes that have not reached cancer tissue or the like are rapidly accumulated and excreted outside the body without accumulating at normal sites. This can be achieved by appropriately controlling the stability in relation to the extracorporeal discharge time when designing the liposome. If an anticancer compound or the like is water-soluble, it is rapidly excreted in the urine via the kidney. Therefore, adverse effects caused by staying in the body and delayed side effects can be prevented.

The liposome contained in the antitumor liposome preparation of the present invention comprises a lipid membrane component constituting the phospholipid membrane and a supercritical or subcritical state under the conditions in which at least one compound having a polyethylene glycol group is present as described later. It is produced by mixing with carbon dioxide. In general, phospholipids and / or glycolipids are preferably used as lipid membrane components constituting the phospholipid membrane of liposomes. Furthermore, the following lipid membrane stabilizing substances are also included as components. Examples of the main component of the lipid membrane component constituting the liposome of the present invention include neutral phospholipids such as lecithin, lysolecithin and / or hydrogenated products thereof, and hydroxide derivatives obtained from soybeans, egg yolks, and the like. .

Other phospholipids may be derived from egg yolk, soybeans or other animals or plants, or semi-synthetic phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, synthetically obtained phosphatidic acid, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristolphosphatidylcholine (DMPC), dioleylphosphatidylcholine (DOPC), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylserine (DSPS), distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylinositol (DPPI),
Examples include distearoyl phosphatidylinositol (DSPI), dipalmitoyl phosphatidic acid (DPPA), and distearoyl phosphatidic acid (DSPA).

  The phospholipid of the lipid membrane constituting the liposome of the present invention desirably contains at least a phospholipid having a transition temperature. The “(phase) transition temperature” of a phospholipid is a temperature that causes a phase transition between both the gel and liquid crystal states that the phospholipid can take. The measurement is by differential thermal analysis using a differential scanning calorimeter (DSC). As phospholipids having a phase transition point, dimyristoyl phosphatidylcholine (transition temperature, the same below, 23-24 ° C), dipalmitoylphosphatidylcholine (41.0-41.5 ° C), hydrogenated soybean lecithin (53 ° C), hydrogenated soybean phosphatidylcholine (54 ° C), distearoylphosphatidylcholine (54.1-58.0 ° C), and the like.

Cationic lipids used in the present invention are 1,2-dioleoyloxy-3- (trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS), dimethyldioctadecylammonium bromide ( DDAB), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (spermine-carboxamide) Ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxy And ethyl) ammonium bromide (DMRIE).

  Cationic phospholipids include esters of phosphatidic acid and amino alcohol, such as dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA) and hydroxyethylenediamine. These cationic lipids are contained in an amount of 0.1 to 5% by mass with respect to the total lipid amount, preferably 0.3 to 3% by mass with respect to the total lipid amount, and more preferably 0.5 to 2% by mass with respect to the total lipid amount. What is necessary is just to add.

  These phospholipids may be used alone or in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing aggregation of liposomes. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1.

Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, and sphingoglycolipids such as galactosylceramide, galactosylceramide sulfate, lactosylceramide, ganglioside G7, ganglioside G6, and ganglioside G4.

  As a lipid membrane component constituting the phospholipid membrane of the liposome, other substances can be added as needed in addition to the lipid. For example, sterols acting as a lipid membrane stabilizing substance such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol or 2,4-dihydrolanosterol can be mentioned. Further, sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside have been shown to be effective in stabilizing liposomes (Japanese Patent Laid-Open No. 5-245357). Of these, cholesterol is particularly preferred.

The amount of sterols used is desirably 0.05 to 1.5 parts by weight, preferably 0.2 to 1 part by weight, and more preferably 0.3 to 0.8 parts by weight with respect to 1 part by weight of phospholipid. If the amount is less than 0.05 parts by weight, the stabilization by the sterols that improve the dispersibility of the mixed lipid is not exhibited, and if it is more than 2 parts by weight, the formation of liposomes is inhibited, or even if formed, it becomes unstable.

  Cholesterol in the liposome membrane can also serve as an anchor for introducing polyalkylene oxide. Specifically, a cholesterol to be included in the membrane as a liposome membrane constituent may be bonded with a polyalkylene oxide group at the end thereof via a linker as necessary. As the linker, a short-chain alkylene group, oxyalkylene group, or the like is used. Japanese Patent Application Laid-Open No. 09-3093 discloses a novel cholesterol that can efficiently immobilize various functional substances at the end of a polyoxyalkylene chain by a covalent bond and can be used as a component for liposome formation. Derivatives are disclosed.

In addition to the sterols, glycols may be added as a constituent of the liposome membrane. When preparing a liposome, if glycols, such as phospholipids, are added as a lipid membrane stabilizing substance, the retention efficiency of the water-soluble anticancer compound in the liposome is increased. Glycols include ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, trimethylene glycol, 1,4-butanediol, 1,5-pentanediol, neopentyl glycol, 1,6-hexanediol, 1,7-heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol,
Examples include pinacol. The amount of glycols used is desirably 0.01 to 20% by mass, preferably 0.5 to 10% by mass, based on the total mass of lipid.

  Other examples of compounds that can be added include dialkyl phosphates such as dicetyl phosphate, which is a negatively charged substance, and aliphatic amines such as stearylamine, as compounds that give a positive charge.

In the present invention, a phospholipid or compound having a polyalkylene oxide (PAO) group or a similar group may be used as one component of the liposome membrane depending on the intended purpose of the liposome preparation. As a method for solving the problem of liposome being trapped by reticuloendothelial cells and the instability of liposome itself such as disintegration and aggregation, polyethylene glycol (PEG) chain, which is a polymer chain on the surface of liposome membrane, Attempts have been made to introduce — (CH ₂ CH ₂ O) _n —H (for example, JP-A-1-249717, FEBS letters, 268, 235 (1990)).

A new function can be imparted to the liposome by attaching a polyalkylene oxide group (polyoxyalkylene chain) or PEG chain to the surface of the liposome membrane. For example, P
The effect of becoming difficult to be recognized by the immune system can be expected from the EG liposome. Furthermore, it has been clarified that liposomes have a tendency to be hydrophilic, thereby increasing blood stability and maintaining the blood concentration over a long period of time (Biochim. Biophys. Acta., 1066, 29-36 (1991)). ). In order to improve the blood retention of liposomes, a method of incorporating polyalkylene oxide-modified phospholipids into the lipid membrane of liposomes has been disclosed (Japanese Patent Laid-Open No. 2002-37883). Such liposomes have also been shown to have improved stability over time. Alternatively, as will be described later, a functional substance that imparts tumorigenicity or cancer cell recognizability may be introduced into the liposome membrane.

  The above properties can be used to impart organ orientation to the liposome. As an example, when a liposome preparation is selectively delivered to cancer lesions scattered in the liver, it is desirable to use a liposome having a low PEG group content or no PEG group because the lipid component tends to accumulate in the liver. . When delivering to other organs, the introduction of PEG groups can make the liposomes stealthy and less likely to collect in the liver, etc., so the use of PEGylated liposomes is recommended.

The introduction of PEG groups forms a hydrated layer, stabilizes the liposomes, and improves blood retention. The function can be adjusted by appropriately changing the length of the oxyethylene unit of the PEG group and the ratio of introduction. As the PEG group, polyethylene glycol having oxyethylene units of 10 to 3500, preferably 100 to 2000 is suitable. When polyethylene glycol is used, the amount used is 0.1 to 30% by mass, preferably about 1 to 15% by mass, based on the lipid constituting the liposome.

  A known technique can be used for PEGylation of the liposome. Liposomes may be prepared by mixing an anchor to which a PEG group is bonded (for example, cholesterol) with a phospholipid that is a membrane component, and an activated PEG group may be bonded to the anchor. In addition, since the PEG group introduced on the liposome surface does not react with the “functional substance” described later, it is difficult to immobilize the “functional substance” on the group on the liposome surface. Alternatively, a liposome can be prepared by binding polyethylene glycol, which is further modified at the PEG tip, to a phospholipid and including this as a liposome constituent.

Instead of the polyethylene glycol, various known polyalkylene oxide groups,-(AO) _n -Y, may be introduced on the surface of the liposome membrane. Here, AO represents an oxyalkylene group having 2 to 4 carbon atoms, and n is an average added mole number of the oxyalkylene group. Y represents a hydrogen atom, an alkyl group or a functional functional group.

  Examples of the oxyalkylene group having 2 to 4 carbon atoms (represented by AO) include, for example, oxyethylene group, oxypropylene group, oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylene group, oxy-1,2 -A dimethylethylene group etc. are mentioned. n is a positive integer of 1 to 2000, preferably 10 to 500, more preferably 20 to 200.

  When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, it may be added in a random manner or a block shape. In the case of imparting hydrophilicity to the polyalkylene oxide chain, the oxyalkylene group is preferably an ethylene oxide added alone, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of added moles of oxyalkylene groups other than ethylene oxide is increased. For example, a liposome containing a block copolymer of polyethylene oxide and polypropylene oxide is a preferred embodiment of the present invention.

  Y is a hydrogen atom, an alkyl group or a functional functional group. Examples of the alkyl group include an aliphatic hydrocarbon group having 1 to 5 carbon atoms which may be branched. The above functional functional group is for attaching a “functional substance” such as sugar, glycoprotein, antibody, lectin, and cell adhesion factor to the end of the polyalkylene oxide chain. For example, an amino group, an oxycarbonylimidazole group, A reactive functional group such as an N-hydroxysuccinimide group may be mentioned.

  In addition to the effect of introducing a polyalkylene oxide chain, a liposome having a polyalkylene oxide chain immobilized with a “functional substance” at the tip is not disturbed by the polyalkylene oxide chain. Functions such as specific organ directivity and tumor tissue directivity are sufficiently exerted as a “recognition element”. In order to impart tumor tissue directivity, anti-tumor monoclonal antibodies corresponding to tumor-specific antigens present only in tumor cells are bound to liposome membranes as functional substances, resulting in liposomes with higher target selectivity (for example, JP-A-11-28087).

A phospholipid or compound having a polyalkylene oxide group can be used alone or in combination of two or more. The content thereof is 0.001 to 50 mol%, preferably 0.01 to 25 mol%, more preferably 0.1 to 10 mol%, based on the total amount of the liposome membrane constituents. If it is less than 0.001 mol%, the expected effect becomes small.

A known technique can be used for introducing the polyalkylene oxide chain into the liposome membrane. For example, a phospholipid polyalkylene oxide derivative or the like may be included in the raw material phospholipids in advance to prepare liposomes. Specific examples include polyethylene oxide (PEO) derivatives such as phosphatidylethanolamine, such as distearoyl phosphatidylethanolamine polyethylene oxide (DSPE-PEO). Furthermore, Japanese Patent Application Laid-Open No. 2002-37883 discloses a high-purity polyalkylene oxide-modified phospholipid for producing a water-solubilized polymer-modified liposome with improved blood retention. It is described that when a polyalkylene oxide-modified phospholipid having a low monoacyl content is used in preparing such a liposome, the temporal stability of the liposome dispersion is good.

As described above, in order to give the liposome a passive targeting ability, it is important to adjust the size of the particle size appropriately when preparing the liposome. Japanese Patent No. 2619037 describes that excluding liposomes having a particle size of 3 μm or more avoids inconvenient retention of liposomes in lung capillaries. However, liposomes in the 0.05-3 μm particle size range do not necessarily become tumorigenic in nature.

Normal cells and cancer cells differ in the degree of vascular system development, blood vessel properties, lymphatic vessel distribution, and the like. This affects the biodistribution of anticancer compounds through hemodynamic and perfusion conditions. In order to make the liposome encapsulating an anticancer compound to be tumorigenic, the average particle size is set to 0.1 to 0.2 in order to utilize the “EPR effect (Enhanced permeability and retention)”. It is desirable that the thickness be μm, more preferably 0.11 to 0.13 μm.
The pores of the neovascular wall in solid cancer tissue are abnormally large compared to the pore size of the capillary wall window (fenestra) of normal tissue, 0.03 to 0.08 μm, and even molecules with a size of approximately 0.1 μm to 0.2 μm Leaks from the vessel wall. The EPR effect is due to the higher permeability of neovascular walls in cancer tissues than microvascular walls of normal tissues.

Liposomes that have leaked from the pores of the blood vessel wall do not return to the blood vessels again because the lymphatic vessels are not sufficiently developed around the cancer cells, and remain in place for a long time. Since the EPR effect is a passive transport using the bloodstream, the retention in the blood must be improved as a requirement for its effective expression. That is, it is necessary that the liposome particles stay in the blood for a long time and pass through the blood vessels near the cancer cells many times. In order to produce the above EPR effect, which is effective for obtaining excellent drug delivery properties, liposomes have improved the loading efficiency as a carrier, that is, the stabilization of the liposome structure and the retention stability of the encapsulated material. In addition, it is required to have characteristics such as blood stability and blood retention. When the average particle size is increased to 0.2 μm or more, the possibility of being taken up by phagocytosis of liver Kupffer cells increases and accumulates at the cell site of the liver. For this reason, it is possible to concentrate the liposome preparation selectively on the cancer tissue by adjusting the average particle diameter of the liposome to the range of 0.11 to 0.13 μm.
Method for Producing Liposomes The method for producing liposomes according to the present invention comprises a phospholipid containing a phospholipid having at least a transition temperature as a lipid membrane component under the condition that at least one compound having a polyethylene glycol group may be present. And at least one of cationic lipids and sterols
A compound selected as a species, and if necessary, a lipophilic drug is dissolved or dispersed in supercritical or subcritical carbon dioxide, and then a solution or suspension containing at least one drug is introduced. Thus, micelles are formed, and then water is added to discharge carbon dioxide to prepare liposomes containing the compound therein.
Various methods have been proposed so far for preparing liposomes. When the production method is different, the shape and characteristics of the liposome finally produced are also significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is appropriately selected according to the desired form and characteristics of the liposome. In general, liposomes mix and dissolve lipid membrane components such as phospholipids and sterols in containers together with organic solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, and THF with almost no exception. Prepared for the first time. In particular, chlorinated solvents are often used. Such preparations of liposomes always contain an organic solvent. At present, in order to remove these remaining organic solvents, a multi-step process and a long-time treatment are required. Such residual organic solvents, especially chlorinated organic solvents, are concerned about adverse effects on living bodies, such as side effects.

In the production method according to the present invention, a liposome preparation method using supercritical carbon dioxide or subcritical carbon dioxide is used in order to produce the above-mentioned liposome without using an organic solvent, particularly a chlorinated organic solvent. Carbon dioxide has a critical temperature of 31.1 ° C and a critical pressure of 75.3 kg / cm ^{2, which} is relatively easy to handle. Even if it remains inert, it is harmless to the human body, and high-purity fluids are easily available at low cost. It is preferable for the reason. Liposomes produced by this method have various favorable properties and advantages for encapsulating anticancer compounds in liposomes as described below.

The temperature of carbon dioxide in the supercritical state (including subcritical state) used in the production method of the present invention is usually 25 to 200 ° C, preferably 31 to 100 ° C, and more preferably 35 to 80 ° C. Suitable pressure is usually 50 to 500 kg / cm ^2, preferably 100 to 400 kg / cm ^2, particularly preferably in the range of 90~150 kg / cm ^2.

When producing liposomes using supercritical or subcritical carbon dioxide, it is necessary to dissolve, disperse or mix the lipid membrane components in carbon dioxide in the supercritical state (including subcritical state). It becomes. At that time, it is preferable to dissolve, disperse or mix in the presence of at least one compound having a hydroxyl group as a dissolution aid (or cosolvent).
As hydroxyl group-containing compounds that can actually be used as dissolution aids, polyethylene glycol, compounds having a polyethylene glycol group, glycols, glycol ethers (including polyalkylene glycol monoalkyl ethers), block copolymer of polyethylene oxide and polypropylene oxide Or a compound selected from polyhydric alcohols other than glycols or lower alcohols. It is desirable to have an affinity for and easily mix with lipid membrane components such as phospholipids and cholesterol. Furthermore, in order to disperse and dissolve the lipid membrane component well in the polar liquid carbon dioxide, an amphiphilic material having both appropriate hydrophilicity and hydrophobicity is preferable.

In the case of the above-mentioned compound having a hydroxyl group, it is desirable not to use a lower alcohol or the like from the viewpoint of safety when there is a concern about the toxicity of the remaining dissolution aid. Therefore, a more preferable solubilizing agent in view of efficacy and safety is polyethylene glycol or a compound having a polyethylene glycol group. In particular, a lipid having a polyethylene glycol group, for example, a phospholipid having a polyethylene glycol group (PEG-phospholipid)
Is preferred. Polyethylene glycol having 10 to 3500, preferably 100 to 2000, oxyethylene units is suitable.

It is desirable to use one or more solubilizing agents in combination in order to further improve the solubility of the lipid membrane component in supercritical carbon dioxide and accelerate the dissolution or dispersion. 0.01 to 1 carbon dioxide to bring a compound having a hydroxyl group into a supercritical or subcritical state
It is good to use as a solubilizing agent in the ratio of 0.1 mass%, preferably 0.1-0.8 mass%.

The production method of the liposome used for the antitumor liposome preparation of the present invention is specifically performed as follows. Liquid carbon dioxide is added to the pressure vessel to bring it to the supercritical or subcritical state under the preferred pressure and temperature described above. Phospholipids, lipid membrane stabilizing substances, and poorly water-soluble drugs are dissolved or dispersed as membrane lipid components of liposomes in supercritical (or subcritical) carbon dioxide. The phospholipid desirably contains at least the phospholipid having a transition temperature. As the membrane lipid component, at least one compound selected from the group consisting of a cationic phospholipid, a compound having a polyalkylene oxide group (for example, a polyalkylene oxide-modified phospholipid), a compound having a polyethylene glycol group, a sterol, and a glycol is used as the phospholipid. Mix together and dissolve and disperse. Alternatively, liquid carbon dioxide may be added to a pressure vessel to which these compounds have been added in advance, and then the temperature and pressure are adjusted to obtain a supercritical state and mixed.

In the supercritical carbon dioxide containing phospholipids and lipid membrane stabilizing substances that are subsequently produced, water-soluble anticancer compounds, water-soluble iodine-based contrast agents, drugs for neutron capture therapy, If necessary, micelles are formed by introducing an aqueous solution containing the formulation aid. The side to be added and the side to be added may be reversed. After thoroughly mixing, when water is added to the system and the pressure is reduced to discharge carbon dioxide, an aqueous dispersion in which liposomes encapsulating anticancer compounds and the like are dispersed is generated. In this case, an anticancer compound may be contained in the aqueous phase inside and outside the liposome membrane. Since the aqueous solution is also encapsulated inside the liposome, the anticancer compound exists mainly in the aqueous phase inside the liposome in addition to the external aqueous phase (aqueous medium) of the liposome, and is in a so-called “encapsulation” state. Further, the liposome is passed through a filtration membrane having a pore size of 0.1 to 1.0 μm, preferably 0.5 to 1.0 μm. Liposomes can be ultrafiltered, centrifuged,
They can be separated by conventional techniques such as gel chromatography and dialysis. The preparation may then be lyophilized for storage. Such a dry preparation is resuspended in an aqueous medium immediately before use to form a dispersion. The osmolarity of liposomes after reconstitution is typically 250.
-500 mosmol / L, preferably 290-350 mosmol / L.

It has been shown that liposome preparation methods using supercritical carbon dioxide or subcritical carbon dioxide have higher liposome production rates, encapsulated drug encapsulation rates, and higher retention rates of encapsulated drugs in liposomes than conventional methods. (Patent Document 2). Furthermore, application on an industrial scale is also possible. This method capable of efficiently encapsulating a drug in liposomes substantially without using an organic solvent is a useful method for producing the antitumor liposome preparation of the present invention. “Substantially” means that the upper limit of the residual organic solvent concentration in the liposome preparation is 10 μg / L.
Anti-tumor liposome preparation The anti-tumor liposome preparation of the present invention improves the retention of a drug in the blood by using the above-described liposome with improved blood stability, and achieves efficient delivery and targeting. Yes. The liposome of the present invention has at least 1 in its lipid membrane or in its internal aqueous phase.
Contains more than one species of drug. Such drugs include anticancer compounds, contrast agents, neutron capture therapy substances, photodynamic therapy substances, formulation aids, and the like. In a preferred embodiment, a formulation aid is contained in an aqueous phase inside the lipid membrane of the liposome and an aqueous medium in which the liposome is suspended. This “preparation aid” is a substance that is added together with an anticancer compound or the like when formulating a liposomal preparation. Various substances based on the manufacturing technology of conventional anticancer agents and liposome preparations Used as desired. Specifically, various physiologically acceptable buffers, edetic acid-based chelating agents such as EDTANa ₂ -Ca, EDTANa ₂ and the like, pharmacologically active substances (for example, vasodilators, coagulation inhibitors, etc.) and further necessary Depending on the conditions, osmotic pressure regulators, stabilizers, antioxidants (for example, α-tocopherol, ascorbic acid), viscosity modifiers, preservatives and the like can be mentioned. Preferably, both a water-soluble amine buffer and a chelating agent are included. As the pH buffer, amine-based buffers and carbonate-based buffers are preferably used, and amine-based buffers are particularly preferable, and trometamol is particularly preferable. The chelating agent is preferably EDTANa ₂ -Ca (calcium edetate disodium).

  An “aqueous medium” is a water-based solvent that dissolves anticancer compounds, formulation aids, and the like. As the water, use sterilized pyrogen-free water. In addition to an anticancer compound, at least an aqueous solution other than the aqueous phase inside the lipid membrane of the liposome (an aqueous solution encapsulated by the lipid membrane) (ie an aqueous medium in which the liposome is dispersed) When a neutral amine buffer, a chelating agent, etc.) are contained, no significant osmotic pressure difference occurs between the inside and outside of the membrane, thereby maintaining the structural stability of the liposome.

The liposome of the present invention is preferably a liposome substantially consisting of a single membrane or several membranes. Single-membrane liposomes are liposomes composed of a membrane (unilamellar vesicle) having a single phospholipid bilayer. In the observation by a transmission electron microscope (TEM) by a freeze fracture replica method, a liposome composed of a phospholipid bilayer in which a replica is generally recognized as one layer is called a monolayer liposome. That is, the observed particle traces left on the carbon film are determined to be a single film, and those having two or more steps are determined to be “multilayer films”. Liposomes composed of two or three membranes are stronger than single membrane liposomes. “Substantially” means that in the antitumor liposome preparation of the present invention, such a single membrane liposome or a liposome composed of several membranes is at least 80% of the total liposomes contained in the liposome formulation. , Preferably 90% or more.

  The above-mentioned single membrane liposomes or liposomes composed of several membranes can be efficiently produced by a phase separation method using water, using the supercritical carbon dioxide or subcritical carbon dioxide as a solvent for lipids. On the other hand, according to the conventional method for preparing liposomes, liposomes composed of multilamellar vesicles (MLV) of various sizes and forms are often present in a considerable proportion. In order to increase the ratio of single-film or several-film liposomes, it was necessary to further irradiate ultrasonic waves or pass through a filter having a fixed pore size many times. Single-membrane or several-membrane liposomes also have the advantage that the dose of liposomes, in other words, the amount of lipid administered does not increase, compared to MLV.

Liposomes with a small number of lipid membranes, in particular LUV (Large unilamellar veislcles), which are monolayer liposomes with a large particle size, have the advantage of providing a large encapsulation capacity compared to multilamellar liposomes. The liposome preferably used in the antitumor liposome preparation of the present invention is located between LUV having a particle size of 0.7 to 1 μm and SUV (Small unilamellar vesicles), which is a small unilamellar liposome having a particle size of less than 0.05 μm. To do. For this reason, the holding volume is also larger than that of SUV, and the trapping efficiency of the water-soluble anticancer compound, in other words, the encapsulation efficiency, is remarkably excellent as described later. In addition, unlike MLV and LUV, they are not taken up by reticuloendothelial cells and rapidly disappear from the bloodstream. On the other hand, even in the case of a single-film or several-film liposome with good encapsulation efficiency of the anticancer compound, the stability of the liposome is lowered if the encapsulated anticancer compound is too heavy. In particular, a tendency to be vulnerable to rapid changes in ionic strength was observed. Liposomes used in the preparation of the present invention are adjusted to a relatively small particle size. Furthermore, the lipid membrane is stabilized by containing at least one compound selected from a compound having a polyalkylene oxide group (for example, phospholipid), sterols, and glycol in the liposome membrane. As a result, such liposomes were found to be stable against salt shock.

In the liposome of the present invention, the adjustment of the size and distribution of the liposome as fine particles is closely related to high blood retention, targeting and delivery efficiency. The particle size (particle size) can be measured by freezing a dispersion containing liposomes containing an anticancer compound, then depositing carbon on the crushed interface, and observing the carbon with an electron microscope (freezing) Crushing TEM method). Here, “average particle diameter” refers to a simple average of a certain number of observed liposome particles, for example, 20 diameters. This usually coincides with or is approximately similar to the “center particle size”, which refers to the particle size having the highest frequency in the particle size distribution. The particle size can be adjusted by changing the formulation or process conditions. For example, when the pressure in the supercritical state is increased, the formed liposome particle size is decreased. In order to make the particle size distribution of the liposome to be produced in a narrower range, filtration may be performed with a polycarbonate membrane, a cellulose membrane, or the like. For example, liposomes having an average particle size of 0.7 μm or less can be efficiently prepared by passing through an extruder equipped with a filter having a pore size of 0.1 to 0.8 μm as a filtration membrane. The extrusion filtration method is described in, for example, Biochim. Biophys. Acta 557, 9 (1979). By incorporating such an “extrusion” operation, in addition to the above sizing, it is possible to adjust the concentration of the anticancer compound existing outside the liposome, exchange the liposome dispersion, and remove undesirable substances. There is also an advantage.

  When a drug is encapsulated in a microcarrier called liposome as in the antitumor liposome preparation of the present invention, the weight of liposome lipid must be considered in addition to the delivery efficiency and retention stability of anticancer compounds and the like. . As the membrane lipid weight of the liposome increases, the viscosity of the preparation increases. As the amount of the drug enclosed in the liposome, the hydrophilic anticancer compound is 0.5 to 8, preferably 3 to 8, more preferably 5 to 5% of the liposome membrane lipid in the aqueous solution sealed in the liposome. It is desirable that it is contained in a weight ratio of 8.

When the weight ratio of the hydrophilic anticancer compound encapsulated in the liposome aqueous phase is less than 0.5, it is necessary to inject a relatively large amount of lipid, resulting in the delivery efficiency of the anticancer compound and the like. Becomes worse. In this respect, single-membrane or several-membrane liposomes are advantageous because they are excellent in retention volume and encapsulation efficiency relative to their lipid amount. On the other hand, when the encapsulated weight ratio of the hydrophilic anticancer compound to the liposome membrane lipid exceeds 8, the liposome is structurally unstable, and the diffusion and leakage of the anticancer compound outside the liposome membrane It is inevitable even during storage or after being injected into the living body. Moreover, when it is a hydrophobic anticancer compound, it is desirable that it is contained in a weight ratio of 0.2 to 2, preferably 0.5 to 1, with respect to the liposome membrane lipid.

In addition, even if 100% encapsulation is achieved immediately after the liposome suspension drug is manufactured and separated,
Based on the destabilization due to the osmotic pressure effect, it is described that the encapsulated component decreases as soon as possible (Japanese Patent Publication No. 9-505821).
Use of anti-tumor liposome preparation for cancer treatment The anti-tumor liposome preparation of the present invention is administered parenterally, specifically intravascularly, preferably intravenously, as an injection or infusion. Is done. When it is necessary to administer a relatively high concentration of liposome preparation in a large amount of time in a short time, the requirements for enabling such bolus injection are the fluidity and low viscosity of the preparation. The viscosity of the liposome dispersion of the present invention (when measured by the Ostwald method) is 20 mPa · s or less at 37 ° C. in order to reduce infusion resistance and reduce patient pain and avoid the risk of extravasation. , Preferably 18 mPa · s or less, more preferably 15 mPa · s or less.

  When the osmolality of the liposome preparation to be administered is high, the burden on the heart and circulatory system is large. To obtain a dispersion that is isotonic with blood, the liposomes are suspended in a medium at a concentration that provides the isotonic solution. For example, if an anti-cancer compound alone cannot provide an isotonic solution due to its low solubility, such as an anti-cancer compound, other non-toxic water solutions are formed so that an isotonic solution or suspension is formed. Substances such as salts such as sodium chloride, saccharides such as mannitol, glucose, sucrose and sorbitol may be added to the medium.

  The optimum dose of an anticancer drug is usually set individually taking into account the type, location, symptoms, and conditions of the patient. Also in the liposome preparation of the present invention, the anticancer agent in the liposome may be the same as the conventional dose. If the solution has an excessively high concentration, the disadvantageous situation of aggregation of liposomes and increase in viscosity must be taken into consideration.

  The liposome of the present invention and the antitumor liposome preparation containing the same are preferably used in a form in which photodynamic therapy and neutron capture therapy are combined in addition to normal systemic chemotherapy. The best mode for carrying out the invention will be described below.

When the anticancer compound encapsulated in the liposome is also a photosensitizer, it is preferably used for photodynamic therapy (PDT). Utilizing the action of free radicals and active oxygen generated by photosensitizers excited by laser irradiation after the liposome preparation containing the photosensitizer is administered to the patient, kills cancer cells Is. Examples of porphyrin-based photosensitizers for PDT include porphyrin, hematoporphyrin IX, photofrin II, verteporfin, purlytin, and lutetium texaphyrin. These are encapsulated in liposomes and administered to patients. A liposome preparation containing a photosensitizer is intravenously injected into the patient in advance, and laser light with a wavelength that matches the excitation wavelength of the photosensitizer is 48 to 72 hours after the drug concentration difference between the cancer tissue and normal tissue becomes maximum. To the affected area where liposomes are accumulated. The laser used in PDT has a low output of almost 1/100 that of a laser scalpel, and photosensitizers accumulate in many cancer tissues, minimizing damage to normal tissues. Can be selectively necrotic.

  The technology of encapsulating drugs such as anticancer compounds in liposomes reduces the side effects compared to drugs that exist in the free state, in addition to the inherent significance of DDS that reduces unnecessary administration by selective drug delivery. There is also a great advantage. However, it is rather rare that the drug efficacy is improved as a result of encapsulating the drug in liposomes. This is because even if liposomes accumulate in the vicinity of cancer cells due to the EPR effect or the like, anticancer compounds and the like are often not immediately released from the liposomes. In this respect, since the lipid membrane of the liposome of the present invention is a liposome substantially consisting of a single membrane or several membranes, an anticancer compound and the like are easily released as compared with a multilayer membrane liposome. As a technique for controlling the release of a drug from a liposome, a technique for causing a change in the structure of the liposome in response to pH, temperature and the like has been studied. Some achievements have been published and can be applied.

A combination of liposomes containing phospholipids having a transition temperature and cancer thermotherapy is also possible. Hyperthermia is a cancer treatment that uses the property that cancer cells are more susceptible to heat than normal cells. It is known that the effect on cancer appears at 41 ° C or higher, and is particularly strong at 42.5 ° C or higher. After the anti-tumor liposome preparation of the present invention is administered to cancer patients, this hyperthermia is performed. Cancer that is close to the surface of the body can be heated relatively easily to the target temperature, but cancer that is deep in the body is often difficult to warm enough because fat, air, and bones get in the way. . A method of inserting several electrode needles into a cancer tissue and heating it is also attempted.

The objective is achieved if the cancer cell killing action can be exerted by another mechanism without releasing the drug encapsulated by the liposome that has reached the vicinity of the cancer cell. One technique that makes this possible is “neutron capture therapy”. The compound to be encapsulated in the liposome is preferably a boron compound or a gadolinium compound. Boron compounds that are more likely to gather in cancer tissues than normal tissues undergo a nuclear reaction ¹⁰ B (n, α) ⁷ Li when irradiated with thermal neutrons. As a result, alpha particles are released, and the alpha particles kill nearby cancer cells. As is well known, α rays exhibit a remarkable cancer cell killing effect even in an extremely short range, and have been used for radiation therapy since ancient times. Examples of the tumor-accumulating boron compound include borocaptate (BSH) and paraboronophenylalanine (BPA). In the case of a gadolinium compound, it emits long-range γ rays and similarly exhibits cancer cell killing action. The intratumoral gadolinium concentration necessary for killing cancer cells is estimated to be 150 μg Gd / g wet tissue. Of the gadolinium compounds, gadopentetate is the only water-soluble chelate substance that has been put into practical use as an MRI (Magnetic Resonance Imaging) contrast agent. Therefore, when a liposome preparation encapsulating such a gadolinium compound is used for neutron capture therapy, it can be used in combination with MRI imaging. Basic data such as effective cancer types, required doses, and irradiation methods are collected.

  The liposome preparation of the present invention may contain a contrast agent. Suitable contrast agents include contrast agents for X-ray examinations such as water-soluble nonionic iodine compounds and oily contrast agents. These contrast agents may be encapsulated in the liposome separately or together with the anticancer compound. In addition to water-soluble iodine compounds, lipiodol having tumor affinity can be further improved in delivery to a cancer lesion by being encapsulated in the liposome of the present invention. The contrast agent may also be present in an aqueous medium in which the liposomes are suspended. Preferred iodo compounds are iomeprol, iopamidol, iohexol, iopromide, ioxirane, iotasulfur, iotrolane or iodixanol. Examples of oil-based contrast agents include iodinated poppy oil fatty acid ethyl ester. Particularly for hepatocellular carcinoma and the like, lipiodol with high hepatic tumor accumulation is suitable.

By administering a liposome preparation containing an anticancer compound and a contrast agent, it is possible to perform contrast imaging and cancer treatment of a target cancer lesion. After the administration of the liposome preparation of the present invention to a patient, if the cancer lesion is depicted with a contrast agent encapsulated in the liposome, the anticancer compound encapsulated in the liposome is also included in the cancer lesion. It is thought that it reaches and exists, and the therapeutic effect by it is expected.
[The invention's effect]
The antitumor liposome preparation of the present invention imparts targeting properties by supporting an anticancer compound or the like on a microcarrier liposome, and by adjusting the average particle size of the liposome to 0.05 to 0.7 μm, The delivery effect is enhanced.

The liposome preparation of the present invention enables further dose reduction of an anticancer compound and the like, and does not substantially contain a chlorinated organic solvent as in the conventional liposome preparation. This further reduces the burden on the subject.
[Example]
Hereinafter, the present invention will be described in more detail with reference to specific examples. The apparatus names used in the following examples, the indicated materials used, the concentration, the amount used, the processing time, the processing conditions, the numerical conditions such as the processing temperature, the processing method, etc. are merely preferred examples within the scope of the present invention.
Liposomes of particle size diameter (particle size), the dispersion containing the liposome encapsulating the anticancer compounds rapidly frozen in liquid nitrogen, then crushed interface to the carbon deposition, formed the carbon Was observed with a transmission electron microscope (freeze-crushing TEM method).

  The particle size was a simple average of the observed 20 liposome particles.

Preparation of liposome preparation A mixture of 86 mg dipalmitoylphosphatidylcholine (DPPC), 38.4 mg cholesterol, PEG-phospholipid (Nippon Yushi Co., Ltd., SUNBRIGHT DSPE-020CN) 19.2 mg, and 45 mg adriamycin was charged into a special stainless steel autoclave and autoclaved. The inside was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are stirred while stirring. Dispersed and dissolved. While further stirring, 5 mL of physiological saline was continuously injected with a metering pump over 50 minutes. After completion of the injection, the system was depressurized to discharge carbon dioxide to obtain a liposome dispersion containing an anticancer compound. The resulting dispersion was heated to 60 ° C. and filtered under pressure using cellulose filters manufactured by Advantech, 1.0 μm and 0.45 μm.

As a result of quantifying adriamycin released outside the liposome and adriamycin contained in the liposome by gel filtration, 90% of adriamycin was encapsulated in the liposome with respect to the adriamycin charged. Further, when observed with a transmission electron microscope (TEM), no crystals or the like were found around the liposomes. The observed particle size of the liposome particles was 0.05 to 0.7 μm.

Claims (14)

  The average particle size is produced by mixing the lipid membrane components constituting the phospholipid membrane with carbon dioxide in a supercritical or subcritical state under the condition that at least one compound having a polyethylene glycol (PEG) group is present. A diameter of 0.05 to 0.7 μm and containing at least one hydrophilic anticancer compound in the phospholipid membrane or in its internal aqueous phase in a weight ratio of 3 to 8 with respect to the lipid membrane component; A liposome, wherein the upper limit value of the concentration of the residual organic solvent is 10 μg / L.   The liposome according to claim 1, wherein the phospholipid membrane is composed of a single membrane or two or three membranes.   The liposome according to claim 1 or 2, wherein the average particle size is 0.1 to 0.2 µm.   The liposome according to any one of claims 1 to 3, wherein the phospholipid membrane contains at least a phospholipid having a transition temperature.   The liposome according to any one of claims 1 to 4, wherein the compound having a polyethylene glycol group is PEG-phospholipid.   The anticancer compound is cyclophosphamide, mechloretamine, carbazylquinone, melphalan, thiotepa, busulfan, nimustine, carmustine, procarbazine, dacarbazine, methotrexate, 6-mercaptopurine, 6-thioguanine, azathioprine, 5-fluorouracil Ftorafur, floxuridine, cytarabine, ancitabine, tegafur, doxyfluridine, actinomycin D, bleomycin, mitomycin, chromomycin A3, synerbin A, aclacinomycin A, adriamycin, pepomycin, cisplatin, mitoxantrone, epirubicin, pirarubicin, vinblastine , Vincristine, vindesine, etoposide, cisplatin, carboplatin, estramustine phosphate, The liposome according to any one of claims 1 to 5, wherein at least one selected from the group consisting of tin, porphyrin, and taxol.   The liposome according to claim 6, wherein the anticancer compound is selected from at least one of adriamycin, pirarubidine, vincristine, taxol, cisplatin, mitomycin, and 5-fluorouracil. The anticancer compounds, porphyrin, hematoporphyrin IX, Photofrin II, is verteporfin (verteporfin), Pururichin (purlytin) or lutetium texaphyrin (lutetium texaphyrin), to be used in the photodynamic therapy of cancer The liposome according to any one of claims 1 to 5.   The antitumor liposome formulation which comprises the liposome in any one of Claims 1-7.   The antitumor liposome preparation according to claim 9, further comprising a contrast agent.   The antitumor liposome preparation according to claim 9, wherein hyperthermia is applied after administration to a patient.   Cationic lipids and cholesterol, dihydrocholesterol, cholesterol esters, phytosterols, sitosterols, stigmasterols as lipid membrane components, together with phospholipids having at least a transition temperature, under conditions where at least one compound having a polyethylene glycol group is present A compound selected from at least one selected from the group consisting of campesterol, cholestanol, lanosterol, 2,4-dihydrolanosterol, 1-O-sterol glycoside, 1-O-sterol maltoside and 1-O-sterol galactoside After dissolving or dispersing in carbon dioxide in a critical state, micelles are formed by introducing a solution or suspension containing at least one hydrophilic anticancer compound, and then water is added. Ete carbon dioxide was drained and method for producing liposome according to claim 1 which comprises preparing a liposome containing the compound therein.   13. The liposome according to claim 12, wherein the compound having a polyethylene glycol group is used as a solubilizing agent at a ratio of 0.01 to 1% by mass with respect to carbon dioxide in a supercritical state or a subcritical state. Production method.   14. The method for producing a liposome according to claim 12 or 13, wherein the solution or suspension of the anticancer compound contains a formulation aid.