JP2006298844A - Liposome-containing preparation given by including pharmaceutical compound in liposome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liposome-containing preparation having a high inclusion ratio of a pharmaceutical compound. <P>SOLUTION: This liposome-containing preparation contains a liposome which contains the pharmaceutical compound and one or more kinds of physiologically acceptable preparation auxiliary agents inside and outside a lipid membrane and does not substantially contain an organic solvent, wherein the liposome substantially comprises the lipid membrane composed of one sheet to ten sheets of membranes of a lipid and at least one kind of phospholipid having a transition temperature in a range of 40-65°C is contained in a lipid membrane constituent thereof. Further, the preparation is prepared by mixing supercritical carbon dioxide with a liposome membrane constituent, the pharmaceutical compound, and the preparation auxiliary agents, then discharging the carbon dioxide by reducing pressure inside a pressure vessel, so as to obtain an aqueous liposome dispersion in which the pharmaceutical compound is included, incubating the dispersion for 0.1-3 hr at a temperature range from the transition temperature of the phospholipid to a temperature higher by 10°C than the transition temperature, and further subjecting the incubated dispersion to filtration operation, so as to be formed into the preparation having a prescribed particle dispersion. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a liposome-containing preparation containing a liposome having a high encapsulation rate of a pharmaceutical compound and a method for producing the same.

Liposomes are capable of constructing a capsule structure that retains a lipid membrane or a substance encapsulated inside the membrane, and are therefore actively studied for application to drug delivery systems (DDS).
The Bangham method, the reverse phase evaporation method (REV method), etc. are used to prepare liposomes encapsulating encapsulated substances. In these methods, although the encapsulated substance is encapsulated in liposomes having high safety as a raw material and appropriate degradability in vivo, an organic solvent is used as a solvent such as phospholipid in the preparation process. use. For this reason, in the liposome-containing preparation obtained by the above method, the remaining organic solvent is unavoidable, and problems remain in the characteristics and stability of the liposome (for example, see Patent Document 1). Furthermore, the present situation is that it has not been put into practical use because of the toxicity of the remaining solvent (Patent Document 2).

  In addition, the conventional method cannot sufficiently encapsulate the drug in the liposome, and in particular, when it is necessary to administer a large amount of the preparation, not only an excessive burden is imposed on the patient but also there is a fear of side effects. It was. In consideration of application to a contrast medium for diagnosis in which the dose is larger than that of a therapeutic drug, a liposome-containing preparation having a high encapsulating rate of contrast material has been demanded.

On the other hand, Patent Document 3 discloses a method for producing liposomes using supercritical carbon dioxide instead of an organic solvent. In this method, various production conditions can be set, and the particle size, structure, and the like can be adjusted relatively easily as compared with conventional liposome production methods. Even if the amount of liposomes taken into the target site in the body is small, this method is expected as a method for preparing a DDS preparation because a desired effect can be obtained if the encapsulation rate of the encapsulated substance is high. However, it is desired to use a solubilizing agent such as ethanol at the time of mixing supercritical carbon dioxide, lipid, and encapsulated material, and liposomes having a high encapsulation rate cannot be prepared without using any organic solvent (see Non-Patent Document 1). ). Even if a drug or the like is encapsulated in the liposome, the strength of the liposome membrane decreases due to the remaining dissolution aid, and the possibility of leakage to the outside over time must also be considered. Therefore, in addition to the method of efficiently encapsulating drugs in liposomes, we continue to specialize in dosage forms that can be retained stably over time and the retention in blood can be improved, the formulation composition is improved and the safety of the formulation is improved. There is a request.
Japanese Patent No. 2619037 Japanese Patent Laid-Open No. 7-316079 Japanese Patent Laid-Open No. 2003-119120 Pharm Tech Japan Vol. 19, No. 5, 91-100 (2003)

  The present invention addresses the above-mentioned demands, and proposes a liposome-containing preparation containing liposomes in which a pharmaceutical compound is efficiently encapsulated without using any organic solvent and a method for producing the same. In particular, the present liposome-containing preparation is suitable as a contrast medium for X-ray examination with good visualization and high safety of cancer tissue.

The liposome-containing preparation of the present invention comprises
Including liposomes containing a pharmaceutical compound and one or more physiologically acceptable formulation aids inside and outside the lipid membrane,
The liposome is a liposome substantially composed of 1 to 10 membranes, of which at least 70% are composed of 2 to several membranes, and the lipid membrane components include Contains at least one phospholipid having a transition temperature in the range of 40 to 65 ° C.,
It is characterized by substantially not containing an organic solvent.

The liposome is prepared by mixing supercritical carbon dioxide with the liposome membrane constituent, pharmaceutical compound and formulation aid under conditions of 40 to 65 ° C. and 10 to 30 MPa in a pressure vessel, and reducing the pressure in the pressure vessel. An aqueous dispersion of liposomes encapsulating the pharmaceutical compound is obtained by discharging carbon dioxide, and then prepared by incubating at the transition temperature of the phospholipid to (the transition temperature +10) ° C. for 0.1 to 3 hours. It is desirable to be a liposome.
Regarding the particle size of the liposome, it is preferable that the proportion of particles having a range of (average particle size−5%) to (average particle size + 5%) accounts for 50 to 90% of the total liposome particles.

  The phospholipid is preferably at least one selected from the group consisting of dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, hydrogenated soybean lecithin, hydrogenated soybean phosphatidylcholine, and distearoylphosphatidylcholine.

The phospholipid may include a phospholipid having a polyethylene glycol (PEG) group.
The phospholipid is at least one of PEG-dimyristoyl phosphatidylcholine and PEG-dipalmitoyl phosphatidylcholine, and further includes at least one of a nonionic iodine-based compound and an anticancer compound in the liposome. Such liposome-containing preparations are also included in the present invention.

  The liposome membrane component preferably includes at least a phospholipid, a lipid having a PEG group, and a lipid containing a sterol, and a molar ratio of phospholipid (excluding PEG-phospholipid) / sterol is 100/60 to 100 The molar ratio of phospholipid (not including PEG-phospholipid) / phospholipid having a polyethylene glycol group is 100/5 to 100/25.

It is desirable that the concentrations of the pharmaceutical compound and the formulation aid inside and outside the lipid membrane are substantially the same.
The method for producing the liposome preparation according to the present invention comprises:
(I) a step of mixing a supercritical carbon dioxide, a liposome membrane constituent, a pharmaceutical compound and a formulation aid in a pressure vessel at 40 to 65 ° C. and 10 to 30 MPa to obtain a suspension;
(Ii) Next, a step of preparing an aqueous dispersion of liposomes encapsulating the pharmaceutical compound by depressurizing the system and discharging carbon dioxide,
(Iii) Thereafter, the aqueous dispersion of liposomes encapsulating the pharmaceutical compound is incubated at the transition temperature of the phospholipid to (the transition temperature + 10) ° C. for 0.1 to 3 hours, and then the pharmaceutical compound is encapsulated. Filtering the aqueous dispersion of liposomes at 50 to 90 ° C. under a pressure of 0.01 to 0.8 MPa, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm;
It is characterized by having.

Detailed Description of the Invention
The liposome-containing preparation of the present invention comprises
Including liposomes containing a pharmaceutical compound and one or more physiologically acceptable formulation aids inside and outside the lipid membrane,
The liposome is a liposome substantially composed of 1 to 10 membranes, of which at least 70% are composed of 2 to several membranes, and the lipid membrane components include Contains at least one phospholipid having a transition temperature in the range of 40 to 65 ° C.,
It is a liposome-containing preparation characterized by being substantially free of an organic solvent.

The liposome is characterized in that the average particle size and the proportion of particles having a particle size of ± 5% occupy 50 to 90% of the whole particles.
In this specification, a supercritical state including a subcritical state is set. The liposome membrane is sometimes referred to as a “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. PEG-phospholipid refers to a phospholipid having a PEG group. "Anticancer compound" is used to mean a single substance anticancer agent. “Cancer” refers to a malignant tumor, sometimes simply “tumor”.

Encapsulated substance In the liposome-containing preparation of the present invention, the medicinal compound to be encapsulated is not particularly limited, and examples thereof include substances widely used in pharmaceuticals. For example, contrast compounds, anticancer compounds, antioxidant compounds, antibacterial compounds, anti-inflammatory compounds, blood circulation promoting compounds, whitening compounds, rough skin prevention compounds, anti-aging compounds, hair growth promoting compounds, moisturizing compounds, hormone agents, vitamins , Dyes, and proteins.

  The above-mentioned pharmaceutical compound to be encapsulated is desirably a water-soluble compound, or a water-soluble salt or a water-soluble form by the addition of a solubilizing agent. It may be a hydrophobic or fat-soluble compound.

  The liposome-containing preparation of the present invention is particularly preferably used as a contrast agent or an anticancer agent. Among these, contrast materials are preferable, and particularly preferable contrast materials are water-soluble nonionic iodine-based compounds.

In the liposome-containing preparation of the present invention, a water-soluble iodine compound is encapsulated in the lipid membrane of the liposome as a contrast substance. The water-soluble iodine compound is not particularly defined as long as it has contrast properties, regardless of whether it is ionic or nonionic. In general, a nonionic iodine compound is more desirable because it has a lower osmotic pressure than an ionic iodine compound and a smaller burden on the administered human body. Contains phenyl iodide as a water-soluble nonionic iodide compound, for example 2,4,6
-Nonionic iodo compounds having at least one triiodophenyl group are preferred.

As such nonionic iodine compounds, specifically, iohexol,
Iopamidol, Iomeprol, Iopentol
Iopromide, Iosimide, Ioversol, Iotrolan, Iotasul, Iodixanol, Iodecimol, Ioxilan and the like. Particularly preferred nonionic iodide compounds that are highly hydrophilic and do not increase osmotic pressure even at high concentrations are iohexol, iomeprol, iopamidol, ioxirane, iotrolane, and iodixanol. When such a non-electrolyte substance is also prepared by the supercritical carbon dioxide method according to the present invention, it can be efficiently encapsulated in the liposome. These compounds may be used alone or in combination of two or more. Moreover, it is not limited to the illustration. In addition, in this specification, a compound may be mentioned including the salt, hydrate, etc. other than a free form compound.

As an encapsulating substance other than the contrast substance, an anticancer compound, a photodynamic therapy substance or the like may be included depending on the use of the contrast agent. The anticancer compound is not particularly limited as long as it is a chemical substance used for cancer chemotherapy. A compound selected from at least one of adriamycin, biralubicin, vincristine, taxol, cisplatin, mitomycin, and 5-fluorouracil is preferably used, but is not limited thereto.

Liposome membrane constituent The lipid membrane constituent of the liposome of the present invention is not particularly limited as long as it is generally used as a membrane constituent. Usually, as a lipid for liposomes, there are substances that have at least one essential component among phospholipids and glycolipids having liposome-forming ability and coexist as optional components. Optional components include sterols and glycols as lipid membrane stabilizing substances, fatty acids or salts thereof as charged substances, and lipid mixed emulsions that have been conventionally used as components for the formation of endoplasmic reticulum. included.

The lipid membrane components constituting the lipid membrane of the liposome of the present invention include at least phospholipids, glycolipids, sterols, glycols, lipids having a polyethylene glycol (PEG) group (for example, PEG-phospholipid) and the like. In general, phospholipids and / or glycolipids are preferably used as the lipid membrane component of the liposomes contained in the liposome-containing preparation of the present invention. Preferred neutral phospholipids include lecithin, lysolecithin and / or hydrogenated products 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 phospholipids of the lipid membrane constituting the liposome of the present invention have a PEG group as necessary,
In addition, at least a phospholipid having a transition temperature in the range of 22 to 70 ° C., preferably 40 to 65 ° C. is contained. 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 in the range of 22 to 70 ° C, dimyristoyl phosphatidylcholine (transition temperature, the same applies hereinafter, 23 to 24 ° C), dipalmitoyl phosphatidylcholine (41.0 to 41.5 ° C), hydrogenated soybean lecithin (53 ° C) ), Hydrogenated soybean phosphatidylcholine (54 ° C.), distearoyl phosphatidylcholine (54.1-58.0 ° C.), and the like. In particular, phospholipids obtained by binding PEG groups to these phospholipids can be used in the present invention.

  The phospholipid having these phase transition temperatures is desirably used in an amount of 20 to 100% by mass, preferably 30 to 90% by mass, and more preferably 40 to 80% by mass with respect to the total mass of the lipid. By using the phospholipid having this phase transition temperature in the above-mentioned amount, it is possible to prepare a liposome having a high encapsulation rate of a pharmaceutical substance to be encapsulated at a mixing temperature described later and excellent in in vivo stability.

  The phospholipid used in the present invention usually contains at least a phospholipid having a transition temperature, but one or more other phospholipids may be used in combination. 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 liposome aggregation.

  These phospholipids are usually used alone, but may be used 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 liposome aggregation. 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 constituent component of the liposome membrane, other substances can be added as necessary in addition to the lipid. Examples include sterols that act as lipid membrane stabilizers, such as cholesterol, dihydrocholesterol, cholesterol esters, phytosterols, sitosterol, stigmasterol, campesterol, cholestanol, or lanosterol. It has also been shown that sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective in stabilizing liposomes (JP-A-5-245357). Among these, cholesterol is particularly preferable.

  Cholesterol in the liposome membrane can also serve as an anchor for introducing polyalkylene oxide. Japanese Patent Application Laid-Open No. 09-3093 discloses a novel cholesterol derivative that can immobilize various functional substances at the end of a polyoxyalkylene chain by covalent bonds and can be used as a component for liposome formation. It is disclosed.

The amount of sterols used is such that the molar ratio of phospholipid (without PEG-phospholipid) / sterols is 100/60 to 100/90, preferably 100/70 to 100/85. This molar ratio is based on the amount of phospholipid excluding PEG-phospholipid. When the molar ratio is less than 100/60, stabilization by sterols that improve the dispersibility of the mixed lipid is not sufficiently exhibited.

  In addition to the sterols, glycols may be added as a constituent of the liposome membrane. When preparing a liposome, if glycols are added together with phospholipid and the like, the retention efficiency of the water-soluble iodine-based compound in the liposome is increased. Examples of glycols include ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, trimethylene glycol, and 1,4-butanediol. 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.

Depending on the intended purpose of the liposome-containing preparation of the present invention, a phospholipid or compound having a polyalkylene oxide (polyoxyalkylene chain) (PAO) group which is a polymer chain or a similar group is used as a component of the liposome membrane. May be used. By attaching a polyalkylene oxide group or a PEG group to the surface of the liposome membrane, instability of the liposome itself such as disintegration and aggregation is solved, and stability with time in an aqueous dispersion is also improved. Furthermore, a new function can be imparted to the liposome. For example, PEGylated liposomes can be expected to have an effect of being hardly recognized by the immune system. Furthermore, the liposome exhibits a hydrophilic tendency because the water molecule of the solvent interacts with the PEG group due to the introduction of the PEG group to form a hydrated layer. From this, it has been clarified that the stability in the aqueous dispersion of liposomes is increased, and the stability in blood is also increased so that the concentration in blood can be maintained for a long time (Biochim. Biophys. Acta.,
1066, 29-36 (1991)). Therefore - by varying (CH ₂ CH ₂ O) _n of oxyethylene units of the PEG group represented by -H length and rate of introduction can be appropriately modulate its function. As the PEG group, polyethylene glycol having oxyethylene units of 10 to 3,500, preferably 100 to 2,000 is suitable. The amount of polyethylene glycol used is 1 to 40% by mass, preferably 5 to 25% by mass, based on the lipid constituting the liposome. A known technique can be used for PEGylation of liposomes (for example, JP-A-1-249717, FEBS letters, 268 , 235 (1990)). Alternatively, an anchor (for example, cholesterol) to which PEG is bound may be mixed with a phospholipid that is a membrane component to prepare a liposome, and activated PEG may be bound to the anchor.
More preferably, PEG-phospholipid is used as a lipid having a polyethylene glycol group. This is because PEG-phospholipid also acts as a solubilizing agent when phospholipids are mixed with supercritical carbon dioxide. It is further desirable to add a PEG group to a phospholipid having a transition temperature in the range of 20 to 70 ° C, preferably 40 to 65 ° C.

  As such a phospholipid, for example, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, or distearoylphosphatidylcholine added with a PEG group is most preferable. In particular, the use of dimyristoylphosphatidylcholine (transition temperature, 23-24 ° C.), dipalmitoyl phosphatidylcholine (transition temperature, 41.0-41.5 ° C.) can form temperature-sensitive liposomes. In thermotherapy for cancer in which the affected area is heated to around 42 ° C., intensive delivery can be achieved by encapsulating an anticancer compound and a contrast substance in such a temperature-sensitive liposome.

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.
-Liposome The liposome-containing preparation of the present invention is intended to be efficiently delivered to a target organ or tissue lesion by using the pharmaceutical compound in a form encapsulated in a liposome as a microcarrier. That is, the targeting function can be imparted by appropriately designing the particle size of the liposome encapsulating the pharmaceutical compound and its lipid membrane. Passive targeting can control its in vivo behavior through adjustment of liposome particle size, lipid composition, charge, and the like. Adjustment of the liposome particle size to a narrow range can also be easily performed. In the design of the liposome membrane surface, desired properties can be imparted by changing the type and composition of phospholipids and coexisting substances. In addition, the adoption of active targeting, which allows for a higher delivery selectivity and accumulation, with regard to the internalization and distribution of administered liposomes should also be considered. As an example, introduction of the above-described polyalkylene oxide polymer chain or PEG group of the polymer chain on the surface of the liposome membrane is beneficial because the induction to the target site can be controlled.

  On the other hand, liposomes that have not reached the affected area, such as cancer tissue, are decomposed relatively quickly and excreted outside the body without accumulating in normal tissues. This can be achieved by appropriately controlling the stability of the liposome in relation to the extravasation time. By controlling such clearance, another effect of the DDS dosage form in which a liposome is encapsulated with a pharmaceutical compound that has no side effects in the free form can be expected. For example, when a water-soluble nonionic iodine-based compound is encapsulated in liposomes, the iodine-based compound deposits nonspecifically in the liver, kidney, etc., and it is difficult to fall into a situation where it takes time to decompose and excrete. For this reason, it is possible to prevent harmful effects caused by staying in the body and delayed side effects.

In the liposome-containing preparation of the present invention, the adjustment of the size and distribution of liposomes as fine particles is closely related to a high drug encapsulation rate, targeting property, and delivery efficiency. The particle size (particle size) can be measured by freezing a dispersion containing liposomes encapsulating a pharmaceutical compound, then depositing carbon on the crushed interface, and observing this carbon with an electron microscope (freeze crushing TEM method). it can. 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.

In order to provide the liposome with passive targeting ability, it is necessary to prepare the liposome with the particle size appropriately adjusted when the liposome is produced. In Japanese Patent No. 2619037,
It has been described that eliminating liposomes with a particle size of 3 μm or more avoids inconvenient retention of liposomes in lung capillaries. However, liposomes with a particle size range of 0.5-3 μm are not necessarily tumorigenic in nature. In the liposome-containing preparation of the present invention, liposomes are prepared by appropriately adjusting the average particle size according to the use, preferably within the range of 0.05 to 0.8 μm.

For example, in the case of a liposome-containing preparation for X-ray contrast imaging for the purpose of imaging the liver, the preferred average particle size of the liposome is 0.2 μm to 0.8 μm. This is because if the liposome encapsulating the water-soluble iodine compound is in this range, it becomes a target of capture phagocytosis by reticuloendothelial cells that are common in non-tumor tissues, and the contrast with tumor tissues becomes clear. In addition, it is desirable that the liposome used for the contrast medium for the liver has as little PEG on the surface as possible.

In order to make a liposome proneoplastic based on the “EPR effect (Enhanced permeability and retention)” utilizing the bloodstream, its average particle size is 0.1 to 0.2 μm, more preferably 0.11. It is desirable that the thickness is about 0.13 μm. For example, the liposome-containing preparation can be selectively concentrated on the cancer tissue by adjusting the average particle diameter of the liposome to the range of 0.11 to 0.13 μm.

Liposome Production Methods Various methods have been proposed for producing liposomes. When the production methods are different, the shape and characteristics of the final liposome are often significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is selected according to the desired form and characteristics of the liposome. In general, liposomes are prepared by first placing lipid membrane components such as phospholipids, cationic lipids, and sterols in containers together with organic solvents (eg chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, THF, etc.) almost without exception. Start with mixing and dissolving. In particular, chlorinated solvents are often used. Such preparations of liposomes always contain an organic solvent.

On the other hand, the method of preparing liposomes using supercritical carbon dioxide is relatively easy to handle with a critical temperature of carbon dioxide of 31.1 ° C and a critical pressure of 7.38 MPa. It can be said that this is an attractive production method because high-purity fluid is inexpensive and easily available. However, even in the conventional supercritical carbon dioxide method in which no organic solvent is used, use of ethanol or the like has been recommended in order to efficiently disperse lipids in supercritical carbon dioxide (see Non-Patent Document 1). In order to remove these remaining organic solvents, a plurality of steps and a long time treatment are required at present. Such residual organic solvents, especially chlorinated organic solvents, are concerned about adverse effects on living bodies, such as side effects.

The production method of the present invention is a method capable of producing liposomes having the optimum form and structure for contrast agents that have a particularly large dosage compared to ordinary pharmaceuticals. That is, the liposome produced by the method using supercritical carbon dioxide is substantially free of chlorinated solvents, ethanol and other organic solvents, and has various desirable characteristics for encapsulating pharmaceutical compounds, that is, conventional methods. It is shown that the encapsulation rate of the drug to be encapsulated and the retention rate of the encapsulated drug in the liposome are higher than those in the above. “Substantially” means that the upper limit of the concentration of the residual organic solvent in the liposome-containing preparation is 10 μg / L.

In the method for producing a liposome-containing preparation according to the present invention, the liposome is prepared by a method using substantially no solubilizing agent based on a method for preparing a liposome using supercritical carbon dioxide as an admixture (hereinafter referred to as supercritical carbon dioxide method). Is done. At that time, the pharmaceutical compound and the formulation aid are encapsulated in the liposome membrane. The component of the liposome membrane is characterized by containing a phospholipid having a transition temperature of at least 40 to 65 ° C. That is, the liposome is used in a pressure vessel under conditions of 40 to 65 ° C. and 10 to 30 MPa (preferably under the condition that a phospholipid having a transition temperature is melted as a liposome membrane constituent), supercritical carbon dioxide and the A liposome membrane constituent, a pharmaceutical compound, and a formulation aid are mixed preferably under strong stirring, and the pressure vessel is depressurized to discharge carbon dioxide, whereby an aqueous dispersion of liposomes encapsulating the pharmaceutical compound is obtained. In the method for producing the liposome according to the present invention, the liposome is prepared by incubating at a transition temperature of the phospholipid to (the transition temperature + 10) ° C. for 0.1 to 3 hours. The process is as follows.
(I) a first step of mixing a liposome membrane component and supercritical carbon dioxide in a pressure vessel at 40 to 65 ° C. to obtain a suspension;
(Ii) Next, a second step of adding a solution or dispersion containing a pharmaceutical compound and a formulation aid to the suspension and mixing, or the following order may be employed. It is suitable when it is a soluble compound. Depending on the nature of the pharmaceutical compound to be encapsulated, these mixing sequences may be used properly.
(I) A suspension obtained by mixing a liposome membrane component and a solution or dispersion containing a pharmaceutical compound and a formulation aid is contained in a pressure vessel, and then liquefied carbon dioxide is supplied. 1 process,
(Ii) Next, while mixing the suspension and liquefied carbon dioxide, the pressure vessel is pressurized under 30 to 65 ° C. to convert the liquefied carbon dioxide into supercritical carbon dioxide. The third step and the fourth step are continued.
(Iii) a third step of depressurizing the inside of the pressure vessel and discharging carbon dioxide to prepare an aqueous dispersion of liposomes containing a pharmaceutical compound and a formulation aid inside;
(Iv) After the third step, the transition temperature of the phospholipid to (the transition temperature + 10) ° C. is 0.1
An aqueous dispersion of liposomes encapsulating the pharmaceutical compound at 50-90 ° C. under a pressure of 0.01-0.8 MPa in a hydrostatic extruder equipped with a filtration membrane having a pore size of 0.1-1 μm, incubated for ˜3 hours The 4th process of filtering and sizing into a liposome with an average particle diameter of 0.05-0.8 micrometer.

By these steps, the molar ratio of phospholipid (without PEG-phospholipid) / sterols is 100/60 to 100/90, phospholipid (without PEG-phospholipid) / phospholipid having polyethylene glycol group A liposome-containing preparation characterized by having a molar ratio of 100/5 to 100/25 and having at least 70% of liposomes composed of 2 to 10 lipid membranes is obtained.
-1st process and 2nd process In a 1st and 2nd process, a liposome membrane structural component, a liquefied carbon dioxide, and a chemical | medical solution or dispersion liquid (a pharmaceutical compound and a formulation aid are contained) are contained in a pressure vessel. Mixing is divided into two methods based on the order of addition.

Phospholipids and substances having a lipid membrane stabilizing action are added to the pressure vessel as lipid components constituting the liposome membrane, and liquefied carbon dioxide is further added, preferably mixed under strong stirring conditions. A lipid having a polyethylene glycol group, a sterol or the like is added as a substance having a lipid membrane stabilizing action. As the phospholipids, various phospholipids including at least one phospholipid having a transition temperature in the range of 40 to 65 ° C. are preferable. The liquefied carbon dioxide is brought into a supercritical state or a subcritical state under the pressure and temperature in the range described later. Liposome membrane components and supercritical (or subcritical) carbon dioxide are thoroughly mixed and dissolved or dispersed. Alternatively, these compounds may be added to liquefied carbon dioxide in the pressure vessel in advance and mixed, and then the temperature and pressure may be adjusted to achieve a supercritical state and mixed. By introducing a drug solution or dispersion containing a pharmaceutical compound to be encapsulated, for example, a nonionic iodine-based compound and a formulation aid, into supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance, etc. Allow micelles to form.

  Alternatively, liquefaction is carried out in a pressure vessel containing a suspension obtained by mixing at least a phospholipid, a lipid having a polyethylene glycol group and a sterol as a liposome membrane component, and a pharmaceutical aqueous solution containing a pharmaceutical compound and a formulation aid. The micelles may be formed by supplying carbon dioxide, mixing and dispersing, preferably under strong stirring, and then heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state and further mixing under strong stirring. The suspension is prepared by mixing the liposome membrane component and the drug solution in a pressure vessel. Alternatively, such a suspension may be prepared separately and then supplied into the pressure vessel.

The encapsulation efficiency of the encapsulated substance in the liposome also depends on the ratio of the total lipid amount of the lipid for the liposome membrane to the supercritical carbon dioxide, and the ratio of the solution or dispersion containing the encapsulated substance. The total lipid amount here is the total mass of all phospholipids, sterols and other added lipids constituting the liposome membrane. In order for most liposomes to form as a few membranes rather than a single membrane, the amount of lipid added should be 1.5 to 2.5 times greater than the amount of lipid used in the conventional supercritical carbon dioxide method. May be. Specifically, it is desirable to mix and disperse in a ratio of 0.075 to 0.125 parts by weight, preferably 0.08 to 0.1 parts by weight of lipid with respect to 1 part by weight of carbon dioxide under strong stirring. In the production method according to the present invention, liposomes were formed efficiently, and up to a certain amount, the encapsulation rate tended to increase as the amount of lipid increased.

If the amount of lipid is too large, there is a possibility that undissolved lipid may be produced when preparing liposomes. However, at the beginning of stirring, a lipid phase containing the above-mentioned lipid may exist. When strong stirring as described below, a large number of small micelles of lipid are formed by generating a large number of CO ₂ / water interfaces where lipid molecules are arranged. Inclusion rate will also increase. Emulsification of lipid and supercritical carbon dioxide is promoted by the presence of a lipid having a PEG group as described below without adding ethanol or the like.

“Strong agitation” differs in a preferable range depending on the volume of the mixed solution and the agitation means. For example, when the volume of the mixed solution is about 10 to 100 mL, using a substantially cylindrical stirrer having a length of 15 mm and a diameter of 5 mm, a rotational speed of 400 to 4000 rpm, preferably 1000 to 1500 rpm, particularly preferably using a magnetic stirrer. Means stirring at 1200-1400 rpm. In addition,
Even when the volume of the mixed solution and the stirring means are different from those described above, the volume is appropriately set according to the amount of the mixed solution and the amount of lipid. Further, the strong stirring time is desirably 1 to 120 minutes, preferably 5 to 60 minutes, and the strong stirring time includes the time for supplying the drug solution.

  By mixing the liposome membrane component and supercritical carbon dioxide for a predetermined time under strong stirring under such conditions, the production efficiency of the liposome is improved and the inclusion rate of the pharmaceutical compound is higher. An aqueous dispersion can be obtained.

Lipids including phospholipids and cholesterol are hardly soluble in supercritical carbon dioxide and water in the first place, and difficult to disperse. In order for liposomes of the desired form to be formed efficiently, it is important that lipids are well dispersed in supercritical carbon dioxide, and emulsification between them proceeds to form a homogeneous state. . For this purpose, it is preferable to dissolve, disperse or mix using at least one compound having a hydroxyl group as a solubilizing agent (or cosolvent or dispersion accelerator).

  A compound having a hydroxyl group (ie, a hydroxyl group-containing compound) includes a compound having a hydrophilic group, for example, a hydroxyl group, a polyol group, a polyalkylene glycol ether group, or a combination of a polyol / polyglycol ether group. As a hydroxyl group-containing compound that can actually be used as a solubilizing agent, a compound that exhibits affinity with lipid membrane components such as phospholipids and cholesterol and is sufficiently mixed with these components is desirable.

  In the above compound having a hydroxyl group, when there is a concern about the toxicity of the remaining dissolution aid, it is desirable not to use a lower alcohol such as ethanol from the viewpoint of safety. Therefore, more preferable solubilizers in view of efficacy and safety are polyethylene glycol or a compound having a PEG group. Specifically, the compound having a PEG group is preferably a lipid having a PEG group, such as PEG-phospholipid. Polyethylene glycol having an oxyethylene unit of 10 to 3,500, preferably 100 to 2,000 is suitable.

Use of one or more such compounds having a hydroxyl group is desirable in order to improve the encapsulation rate. The compound having a hydroxyl group may be used as a solubilizing agent at a ratio of 0.01 to 1% by mass, preferably 0.1 to 0.8% by mass, of carbon dioxide to be in a supercritical state or a subcritical state. When the compound having a hydroxyl group is a lipid having a PEG group, the molar ratio of phospholipid (excluding PEG-phospholipid) / phospholipid having a polyethylene glycol group is 100/5 to 100/25, preferably 100 / 5 to 100/10.

  In the conventional supercritical carbon dioxide method using ethanol or the like as a dissolution aid, ethanol promotes dissolution or dispersion of lipids in supercritical carbon dioxide. As a result, a two-phase system consisting of a carbon dioxide region containing cholesterol and phospholipid and a water region mainly containing PEGylated phospholipid and a pharmaceutical compound is formed, and when this is stirred, supercritical carbon dioxide Emulsification proceeds and lipid micelles are formed. Finally, liposomes having a single lipid membrane are mainly prepared.

  In contrast, in the method of the present invention, a lipid having a PEG group as a liposome membrane constituent (for example, PEG-phospholipid) acts to promote emulsification instead of a solubilizing agent such as ethanol. Drug compounds and PEGylated phospholipids dissolve or disperse in the aqueous phase to lower the surface tension of the water, and a lipid phase in which phospholipids and cholesterol are collected is also formed, which is a supercritical carbon dioxide that contains almost no lipids. It becomes a three-phase system state with the phase. Although emulsification of supercritical carbon dioxide proceeds by stirring, more interfaces are formed than in the conventional method, and lipid molecules are arranged on such many interfaces to form a lipid monolayer. In particular, under conditions where the amount of lipid is increased and the above-described strong stirring is performed, a large number of micelles that are uniform and small are formed.

  In the end, supercritical carbon dioxide is emulsified and lipid micelles are formed regardless of the presence or absence of ethanol. However, particularly in the method with strong stirring, uniform micro micelles are formed efficiently, so that in the process of being induced by the subsequent discharge of carbon dioxide and forming a lipid bilayer from a lipid monolayer, The rate of inclusion increases from 17% to 21%, for example. In the conventional supercritical carbon dioxide method using ethanol or the like, single membrane liposomes are mainly formed, whereas in the method of the present invention, when the amount of lipid is increased, multilamellar liposomes from 2 to 10 membranes are formed. Is formed mainly. For such multilamellar liposomes, an increase in the encapsulation rate is observed again in the sizing process, suggesting that membrane reorganization has occurred.

The temperature of carbon dioxide in the supercritical state (including subcritical state) used in the production method of the present invention is generally set to 33 to 70 ° C, preferably 40 to 65 ° C. When the phospholipid having a transition temperature in the above range is contained in the liposome membrane component, “the transition temperature + 10 ° C.” or less, preferably “the transition temperature + 5 ° C.” or less, more preferably “approximately the transition temperature”. It is desirable to set as follows. When the phospholipid is heated above the transition temperature of the phospholipid, the phospholipid having the transition temperature “melts”, specifically, a phase transition from a gel state to a liquid crystal state. This increases the fluidity of the lipid, and the phospholipid is efficiently mixed with the supercritical carbon dioxide. Therefore, the liposome membrane is formed smoothly, and the incorporation of a pharmaceutical compound or the like into the interior is also promoted, and the encapsulation rate is improved. In the production method of the present invention, the encapsulation rate of the pharmaceutical compound in the liposome is increased to 25 to 35% by setting to suitable conditions. Since the mixture with supercritical carbon dioxide becomes better and the phospholipids with improved fluidity are regularly arranged to form a liposome membrane, the formed liposomes have a uniform particle size. The width of the diameter distribution is relatively small. In particular, this tendency is further enhanced by adding an incubation process.

Since the temperature of the carbon dioxide in the supercritical state is set near the phospholipid transition temperature or a relatively low temperature as described above, the phospholipid is not excessively heated so that it is not denatured, especially emulsified. In the situation where a strong stirring operation is performed for promotion, it is desirable to adopt the above temperature range from the viewpoint of reducing local overheating. A suitable pressure of carbon dioxide in the supercritical state is appropriately selected according to the above temperature range, but is 5 to 50 MPa, preferably 10 to 30 MPa.
-Third step In the third step, after sufficiently mixing the liposome membrane constituent, supercritical carbon dioxide, and a drug solution (containing a pharmaceutical compound and a formulation aid), water is added to the system if necessary. To depressurize the inside of the pressure vessel. By discharging carbon dioxide from this mixed solution, an aqueous dispersion of liposomes encapsulating a pharmaceutical compound and the like is prepared. In this process, it is presumed that the liposomes are phase-inverted to the aqueous phase, so that only by discharging carbon dioxide, an aqueous dispersion in which the liposomes containing the pharmaceutical compound are dispersed is generated. Since the aqueous solution is also encapsulated inside the liposome, the pharmaceutical compound exists in the aqueous phase inside the liposome in addition to the external aqueous phase of the liposome, and is in an “encapsulated” state.

  An aqueous dispersion of liposomes obtained as described above and encapsulating a pharmaceutical compound is then transferred from the phospholipid transition temperature to (the transition temperature + 10) ° C., ie, the phospholipid transition temperature or higher. It is preferable to heat to a temperature of “+ 10 ° C.” for a certain period of time. This “transition temperature + 10 ° C.” is about 35 ° C. to 65 ° C., depending on the type and composition of the phospholipid used. As the heating time, it is desirable to incubate for 0.1 to 3 hours, preferably 10 to 60 minutes.

  Incubation of warming the aqueous dispersion of liposomes prepared as described above is preferably maintained at a constant temperature for a predetermined time with stirring. Specifically, it may be carried out in a pressure vessel having a liposome dispersion before the next filtration step, heated to a predetermined temperature by a heating device provided in the pressure vessel, and further maintained for a predetermined time. In this way, the aqueous dispersion of liposomes is incubated. Alternatively, after the liposome dispersion liquid is transferred to another container, the same treatment may be performed under the above conditions. For example, prior to filtration of the liposome dispersion liquid, the filtration container may be heated to the above temperature range. Good.

  By incubating at a temperature above the transition temperature of the phospholipid under the above conditions, the phospholipid having the transition temperature becomes a liquid crystal state, the phospholipid molecule becomes extremely flexible, and thus the fluidity of the lipid membrane is increased. . By such additional operation, separation and dispersion of the aggregated aggregated liposome are promoted. As a result, a situation is formed in which the liposomes that are produced tend to become a particle population having a relatively uniform distribution as a whole. As described below, uniform distribution refers to both the size (ie, particle size) and structure (eg, number of lipid membranes) of liposome particles.

Therefore, during the filtration operation of the next step, rearrangement of lipid molecules with increased fluidity occurs in the lipid membrane, so that a more stable membrane structure is efficiently formed and the encapsulation of the pharmaceutical compound is promoted. Expected to improve the inclusion rate. The reason why such an effect is obtained is not clear, but the phospholipid membrane is reconstructed in the liposome composed of the multi-layer membrane, and the liposome composed of a plurality of membranes and the SUV (single unilamellar vesicle) type liposome are generated. Presumed to be because. Furthermore, rearrangement of constituent molecules also occurs in the lipid membrane, and a more stable membrane structure is formed. These are thought to lead to an improvement in the encapsulation rate of the encapsulated material.
-4th process Adjustment of the particle size of a liposome can be performed by changing prescription or process conditions. For example, when the pressure in the supercritical state is increased, the particle size of the liposome formed 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 this reason, in the fourth step, an aqueous dispersion of liposomes obtained by adjusting the pressure vessel to atmospheric pressure by introducing air or the like (third step) is converted into a plurality of filtration membranes having a pore size of 0.1 to 1.0 μm. Through. It is preferable to gradually reduce the pore size of the filtration membrane from large to small, and finally 0.05 to 0.4 μm
Preferably, the pore diameter is reduced to a range of 0.1 to 0.4 μm, more preferably 0.15 to 0.2 μm. The operation of extrusion filtration is performed at 50 to 90 ° C., preferably 55 to 85 ° C., under a pressure of 0.01 to 1.0 MPa, preferably 0.01 to 0.8 MPa. In the production method of the present invention, the phospholipids of the lipid membrane constituting the liposome contain at least a phospholipid having a transition temperature. Lipids are in a liquid crystal state and fluidity increases.

For the operation for sizing, for example, various hydrostatic extrusion apparatuses equipped with a filtration membrane, for example, “Extruder” (trade name, manufactured by NOF liposome) and the like are forcibly passed through the filter. By passing through a hydrostatic extruder, liposomes with a uniform particle size distribution can be efficiently prepared. The filtration operation is repeated if necessary. This extrusion filtration method is described, for example, in Biochim. Biophys. Acta 557, 9 (1979).

  By incorporating such an “extrusion” operation, the particle size of the liposomes is aligned so that its distribution settles in a relatively narrow range, coupled with the effects of previous incubations. Regarding the particle size of the liposome, the particle size distribution within a range of ± 5% of the average particle size, that is, the proportion of particles having a range of (average particle size−5%) to (average particle size + 5%) is liposome particles. It is desirable to occupy 30 to 95% of the whole, preferably 50 to 90%, more preferably 70 to 90%.

  In fact, the state in which the average particle size of liposomes and the proportion of particles having a particle size of ± 5% of the final particle finally occupy 80% of the total particles incorporate three incubation steps. However, without incubation, it cannot be achieved by repeating the filtration operation 9 times.

  The present inventors have found that in addition to the above sizing, the encapsulation rate of the pharmaceutical compound increases. Furthermore, in a preferred embodiment in which the lipid for liposome constitutes a uniform lipid membrane having a regular orientation, liposomes having a uniform particle size can be obtained without causing clogging of the filter even in a liposome suspension having a relatively high viscosity. Can be produced.

When a larger amount of lipid is used than usual in the first step, the formed liposomes are not a single membrane (Non-patent Document 1) as already reported, but a multilamellar membrane. As a result of the extrusion filtration described above, even in liposomes composed of multilamellar membranes, reorganization and sizing of the membrane structure, including reconstitution of the lipid membrane, has occurred, resulting in the formation of liposomes composed of 2 to 10 membranes. It is guessed.

  Liposomes produced under the conditions of the present invention have a large number of multilayer membranes, and during the filtration operation of this step, the outer shell layer of the multilayer membrane is broken and peeled off, or the liposome membrane is broken. It is considered that the membrane fragments are joined together to form the liposome structure again. Even if the whole liposome collapses at the time of filtration, the structural fragments that formed the membrane structure may recombine to form new liposomes.

  Therefore, by incorporating the above incubation and maintaining the temperature at or above the phospholipid transition temperature for a certain period of time, the fluidity of the lipid membrane is enhanced, promoting the dispersion of liposomes and lipids, and the reconstruction of the membrane structure as described above. It is thought to have a positive impact.

By performing such a treatment, the encapsulation rate of the encapsulated substance in the liposome can be improved and the liposome can be made into fine particles without substantially using an organic solvent-based solubilizing agent such as ethanol. it can. Furthermore, since there is no possibility that the membrane strength is lowered by the remaining dissolution aid, the storage stability of the liposome is excellent.
-Concentration step The aqueous dispersion of liposomes is filtered through a filtration membrane as described above, and if necessary, the drug not encapsulated in the liposomes is removed by methods such as ultrafiltration, centrifugation, and gel filtration. It may be purified. In the method of the present invention, it is preferable to concentrate the aqueous dispersion of liposomes encapsulating a pharmaceutical compound by performing ultrafiltration after the third step or the fourth step. Ultrafiltration can be performed using conventional ultrafiltration membranes and equipment. In the production method according to the present invention, by performing these filtration operations, the encapsulation rate of the pharmaceutical compound in the liposome can be increased to 25 to 35%. Concentration by the ultrafiltration method may be carried out after the third step, and in this case, it has the meaning of the concentration operation for extrusion filtration which is the fourth step using an extruder or the like to be performed subsequently. It is also possible to efficiently obtain liposomes in powder form by concentrating the aqueous dispersion of liposomes to reduce the volume and then freeze-drying the liposomes. When liposomes are lyophilized, they are resuspended in an aqueous medium immediately before use. Further, after the ultrafiltration, steam sterilization may be performed at 115 to 125 ° C, preferably 118 to 123 ° C, more preferably 121 ° C. This gives a sterile preparation.

Liposomes produced by the method of the present invention are substantially composed of 1 to 10 membranes, preferably 2 to several membranes (eg, 3, 4, 5 or 6 membranes). It is. Such liposome is produced as a main component in the production method according to the above steps (i) to (v). “Substantially” means that in the liposome-containing preparation of the present invention, the liposome composed of 2 to 10 membranes is at least 60%, preferably 70% or more of the total liposomes contained in the contrast medium. More preferably 85 to 98%. Particularly preferably, liposomes composed of two to several membranes occupy at least 70%, preferably 75 to 90%.

On the other hand, a single membrane liposome is a liposome composed of a membrane having a single phospholipid bilayer. This is composed of a phospholipid bilayer in which a replica is generally recognized as one layer in observation with a transmission electron microscope (TEM) by a freeze fracture replica method. That is, the observed particles of the carbon film having no step are determined as a single film, and those having two or more steps are determined as a “multilayer film”. Liposomes composed of two or three membranes have a relatively higher strength than single membrane liposomes, and are not broken by the sizing treatment performed in the fourth step.

  Single-membrane liposomes are known to be produced by supercritical carbon dioxide as a solvent for lipid membrane components and by a phase separation method using water, and can be efficiently produced especially when ethanol is used as a dissolution aid. On the other hand, according to the conventional liposome preparation methods such as the Bangham method and the reverse phase evaporation method (REV method), liposomes consisting of multilamellar vesicles (MLV) of various sizes and forms are unevenly distributed. Tend to be formed. Single-membrane or several-membrane liposomes have the advantage that the dose of liposomes, in other words, the amount of lipids to be administered, does not increase compared to MLV.

Liposome membranes with a small number of lipid membranes, especially large unilamellar vesicles (LUVs), have the advantage of providing a larger encapsulation capacity than multilamellar liposomes. is there. On the other hand, even in the case of single-film or several-film liposomes with a good encapsulation rate of the pharmaceutical compound, the stability of the liposomes decreases if the encapsulated compound is too heavy. In particular, a tendency to be vulnerable to rapid changes in ionic strength was observed. In the production of liposomes used in the preparation of the present invention, liposomes having a bilayer to 10 membrane structure, preferably 2 to several membranes (for example, 3, 4, 5, or 6 membranes) are used. The supercritical carbon dioxide method and the subsequent sizing process are improved so that liposomes can be formed efficiently. Furthermore, phospholipids having a polyalkylene oxide group, sterols, glycols and the like are contained in the liposome membrane to stabilize the lipid membrane. As a result, the prepared liposome was found to be stable against salt shock.

Production of liposome-containing preparation The liposome-containing preparation of the present invention contains one or more physiologically acceptable formulation aids together with a pharmaceutical compound in an aqueous phase inside the lipid membrane of the liposome and an aqueous medium in which the liposome is suspended. is doing. This formulation aid is a substance that is added together with a pharmaceutical compound when formulating liposomes, and various substances are used as necessary based on conventional formulation techniques. Specifically, various physiologically acceptable buffers, edetic acid chelating agents (eg EDTANa ₂ -Ca, EDTANa ₂ etc.), inorganic salts, pharmacologically active substances (eg vasodilators, coagulation inhibitors etc.) ), An osmotic pressure regulator, a stabilizer, an antioxidant (for example, α-tocopherol, ascorbic acid), a viscosity regulator, a preservative, and the like. Preferably, both an amine buffer and a chelating agent are included. As a pH buffer, a water-soluble amine buffer and a carbonate buffer are preferably used. Particularly preferred is an amine buffer, with trometamol being preferred. The chelating agent is preferably EDTANa ₂ -Ca (calcium edetate disodium).

  The “aqueous medium” is a water-based solvent that dissolves or suspends pharmaceutical compounds (for example, nonionic iodine compounds), formulation aids, and the like. As the water, use sterilized pyrogen-free water. When an aqueous solution other than the aqueous phase inside the liposome lipid membrane (that is, an aqueous medium in which the liposome is suspended) also contains the above-mentioned pharmaceutical compound and formulation aid, the pharmaceutical compound and formulation aid are contained in the aqueous phase inside and outside the lipid membrane. An embodiment in which the agent is contained at substantially the same concentration is preferable. In such a case, no significant osmotic pressure difference occurs between the inside and outside of the membrane, and the structural stability of the liposome is maintained.

The pH range of the solution or suspension is preferably 6.5 to 8.5, more preferably 6.8 to 7.8 at room temperature. When the pharmaceutical compound is a water-soluble iodo compound, a preferred buffer is a buffer having a negative temperature coefficient as described in US Pat. No. 4,278,654. This type of buffer has a low pH at the autoclave temperature, which increases the stability of the liposome-containing formulation in the autoclave, while returning to a physiologically acceptable pH at room temperature. The amine-based buffer has a property that satisfies such a requirement. Therefore, it is advantageous that the liposome preparation can be sterilized by autoclave in order to produce a sterile preparation for injection, and storage stability and the like can be ensured. The preparation of the present invention is preferably provided in a sterilized form, in which case the sterile preparation is obtained by sterile filtration, autoclaving or heat sterilization.

In the present invention, in addition to the encapsulation efficiency of the pharmaceutical compound and the stability of the encapsulation, the weight of the membrane lipid of the liposome must be considered. As the membrane lipid weight increases, the viscosity of the preparation increases. As the amount of drug (pharmaceutical compound and formulation aid) encapsulated in the liposome, all of the drug in the aqueous solution encapsulated in the liposome is 1 to 8, preferably 3 to 8, relative to the liposome membrane lipid. Preferably, it is contained in a weight ratio (g / g) of 5 to 8. When the weight ratio of all the drugs encapsulated in the liposome is less than 1, a relatively large amount of lipid is contained, and the viscosity of the preparation increases, resulting in poor drug delivery efficiency. Single-membrane or several-membrane liposomes are advantageous because they have excellent encapsulated volume and delivery efficiency. On the other hand, when the encapsulated weight ratio of all drugs to liposome membrane lipids exceeds 8, liposomes are also structurally unstable, and drug diffusion and leakage outside the liposome membrane were injected during storage or in vivo. Inevitable later.

Liposome-containing preparation The liposome-containing preparation of the present invention is not particularly limited, and can be applied to contrast agents, internal medicines, external preparations for skin, cosmetics, and the like. In particular, it can be suitably used for an X-ray contrast medium.

  A preferred embodiment of the liposome-containing preparation of the present invention is a liposome-containing contrast agent in which the contrast material is a nonionic iodine-based compound and contains at least one physiologically acceptable preparation aid. The liposome used for encapsulating the contrast medium and the formulation aid has a form and structure designed as a liposome suitable for the liposome-containing formulation. In such a liposome, since the lipid membrane is substantially composed of several membranes, stability over time and stability in blood are improved. Moreover, since the encapsulation rate of the contrast material is increased, the contrast performance as a contrast agent is also improved.

In the DDS of a pharmaceutical substance, side effects are reduced by encapsulating in a liposome, and in order to further increase the benefits obtained by encapsulation, a pharmaceutical preparation is often prepared by separating and removing the pharmaceutical substance that is not encapsulated. In fact, there have been reports of cases where liposomes that have achieved almost 100% encapsulation have been separated, but thereafter, the encapsulated components of the liposome suspension formulation have been lost over time (Betageri, GV Drug Devel. Ind. Pharm 19, 531-539 (1993)). Further, it has been reported that when a contrast agent having an X-ray contrast material only inside the liposome is autoclaved like the liposome preparation of WO88 / 09165, the contrast material leaks out of the liposome (Patent Document 2). . On the other hand, the diagnostic significance of a preparation containing a free contrast material not encapsulated was discussed (Japanese Patent Publication No. 9-505821). This is based on the specific use of the contrast agent.

  The above-mentioned liposome-containing contrast agent usually also contains an iodine-based compound that is not encapsulated in the liposome. In such a contrast agent, the ratio (encapsulation rate) of the contrast material encapsulated in the liposome must also be considered. The liposome-containing preparation of the present invention is characterized in that 65 to 80% by mass of the water-soluble iodine compound is in a form not encapsulated in the liposome and exists in an aqueous medium in which the liposome is suspended.

In the liposome-containing preparation of the present invention, in order to efficiently encapsulate the iodine compound and to prevent the time-dependent destabilization of the liposome carrying the compound, the amount of the iodine compound encapsulated in the liposome is rather Limited. The amount of the iodine compound encapsulated in the liposome is 5 to 40% by mass, preferably 10% of the total iodine compound in the liposome-containing preparation.
It is -35 mass%, More preferably, it is 10-30 mass%, Most preferably, it is 15-25 mass%. Since the encapsulation rate is below the limit of the dense packing of the liposome particles, the retention stability of the contrast medium in the liposome is not impaired. Therefore, instability due to the osmotic pressure effect of the liposome encapsulating the iodine compound can be prevented, and the retention stability of the contrast medium in the liposome over time is improved. This means that even in liposome-containing preparations, the encapsulation rate of the iodine compound at the time of preparation preparation and the encapsulation rate at the time of use are kept substantially the same, and this is preferable from the viewpoint of contrast performance and quality control.

In the liposome-containing preparation of the present invention, the iodine content is usually 40 to 450 mg I / mL, preferably 70 to 400 mg I / mL, preferably 10 to 300 mL, and the contrast medium in the liposome. From the viewpoint of the efficiency of encapsulating, 100 to 350 mgI / mL, particularly preferably 150 to 300 mgI / mL. Moreover, it is preferable that the pharmaceutical compound and the formulation aid are contained in substantially the same concentration in the aqueous phase inside and outside the lipid membrane.

  The liposome-containing preparation according to the present invention is used for either systemic or local administration. Preferably, it is intravenously administered systemically as an injection or infusion. The dosage and concentration depend on the purpose of the imaging, the site, the nature of the compound in the contrast agent and the patient's condition and can be adjusted as needed. For this reason, you may make it the total amount of the pharmaceutical compound inside and outside a liposome become comparable as the conventional dosage. The viscosity of the liposome dispersion of the present invention (when measured by the Ostwald method) is 30 mPa at 37 ° C. in order to reduce the infusion resistance at the time of administration and reduce patient pain and avoid the risk of extravasation. S or less, preferably 25 mPa · s or less, more preferably 20 mPa · s or less. Within such a range, there is no particular problem in practical use (Patent Document 1).

As an example of the above-mentioned topical administration, an embodiment in which the above-mentioned temperature-sensitive liposome is used for thermotherapy of cancer can be mentioned. In this case, a photodynamic therapy substance can be used in combination. There are many known compounds as photodynamic therapy substances.
[The invention's effect]
The liposome-containing preparation of the present invention contains a liposome encapsulating a pharmaceutical compound therein, and the liposome membrane component contains at least a PEG-phospholipid having a transition temperature in the range of 40 to 65 ° C. . Since it is produced by the supercritical carbon dioxide method at a temperature near the transition temperature at which the phospholipid melts, the contrast material is stably encapsulated at a high encapsulation rate of 25 to 35%, enabling a lower dose. And

  Since the contrast agent of the present invention is produced without using any highly toxic chlorinated solvent and other organic solvents, toxicity and side effects are reduced compared to conventional liposome-containing preparations.

The present invention will be specifically described by the following examples. However, the examples are to be described by way of examples, and are not intended to limit the scope of the present invention. [Evaluation]
<Average size of liposome>
The particle size (particle size) is determined by rapidly freezing the dispersion containing liposomes encapsulating iodine compounds with liquid nitrogen, then depositing carbon on the crushed interface, and observing the formed carbon with a transmission electron microscope. (Freeze-crushing TEM method). The particle size was a simple average of the observed 20 liposome particles.
<Evaluation of drug permeability of liposome>
A 5 mL sample in which the iodine compound was present only in the liposomes was prepared by washing the iodo compound not encapsulated three times by centrifugation for 5 mL of the prepared sample and dispersing it in physiological saline. This sample was stored for 3 months in the dark at 23 ° C. and 55% relative humidity. Thereafter, the sample was dialyzed with physiological saline, ethanol was added after the dialysis was completed to destroy the liposome, and the amount of iodine compound in the liposome was determined by measuring the absorbance.

The drug permeability was expressed as (retention rate after 3 months) / (encapsulation rate at the time of preparation) as the retention rate.
<Evaluation of average liposome membrane number>
In the mean particle size measurement, it was also observed.
[Example 1]
A mixture of 368.4 mg of dipalmitoylphosphatidylcholine (DPPC), 147.4 mg of cholesterol, and 111.5 mg of phospholipid having a PEG group (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and then 13 g of liquid carbon dioxide Was added to obtain a supercritical state at 50 ° C. and 12 MPa. Next, while stirring the inside of the container, a contrast medium solution (JP iohexol solution (iodine concentration 300 mg / mL), trometamol 1 mg / mL, calcium disodium edetate 0.1 mg / mL), and appropriate amounts of hydrochloric acid and hydroxide The pH was adjusted to around 7 with sodium) and 10 mL was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was incubated at 44 ° C. for 2 hours, heated to 80 ° C., and filtered under pressure with a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.40 μm, to obtain a liposome-containing contrast agent.
[Example 2]
A mixture of 368.4 mg of dipalmitoylphosphatidylcholine (DPPC), 147.4 mg of cholesterol, and 111.5 mg of phospholipid having a PEG group (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and then 13 g of liquid carbon dioxide Was added to obtain a supercritical state at 50 ° C. and 12 MPa. Next, while stirring the inside of the container, a contrast medium solution (JP iohexol solution (iodine concentration 300 mg / mL), trometamol 1 mg / mL, calcium disodium edetate 0.1 mg / mL), and appropriate amounts of hydrochloric acid and hydroxide The pH was adjusted to around 7 with sodium) and 10 mL was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was incubated at 44 ° C. for 30 minutes, heated to 80 ° C., and filtered under pressure with a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.40 μm, to obtain a liposome-containing contrast agent.
[Example 3]
A mixture of 394.0 mg of hydrogenated soybean phosphatidylcholine (HSPC), 147.4 mg of cholesterol, and 111.5 mg of phospholipid having PEG group (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and then liquid carbon dioxide Add 13g, 60
A supercritical state at 12 ° C. was achieved. Next, while the inside of the container is stirred, the contrast medium solution (JP iohexol solution (iodine concentration 300 mg / mL), trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, and appropriate amounts of hydrochloric acid and hydroxylated) The pH was adjusted to around 7 with sodium) and 10 mL was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was incubated at 60 ° C. for 2 hours, heated to 80 ° C., and filtered under pressure with a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.40 μm, to obtain a liposome-containing contrast agent.
[Example 4]
A mixture of 368.4 mg of dipalmitoylphosphatidylcholine (DPPC), 147.4 mg of cholesterol, and 111.5 mg of phospholipid having a PEG group (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and then 13 g of liquid carbon dioxide Was added to achieve a supercritical state at 50 ° C. and 12 MPa. Next, while the inside of the container is stirred, the contrast medium solution (JP iohexol solution (iodine concentration 300 mg / mL), trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, and appropriate amounts of hydrochloric acid and hydroxylated) The pH was adjusted to around 7 with sodium) and 10 mL was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was heated to 80 ° C. and filtered under pressure with a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.40 μm, to obtain a liposome-containing contrast agent.
[Comparative Example 1]
(Bangham method)
A mixture of dipalmitoyl phosphatidylcholine (DPPC) 368.4 mg, cholesterol 147.4 mg, PEG-phospholipid (Nippon Yushi Co., Ltd., SUNBRIGHT DSPE-020CN) 111.5 mg is a mixture of chloroform, ethanol and water (weight ratio 100: 20: 0.1) 10 mL was mixed in a volumetric flask. The mixture was heated on a hot water bath (65 ° C.), and the solution was evaporated on a rotary evaporator. The residue was further vacuum dried for 2 hours to form a lipid film. Further, 10 mL of the contrast medium solution was mixed therein, and the mixture was stirred for about 10 minutes with a mixer while being heated to 65 ° C. By further stirring, a liposome dispersion containing a contrast medium solution was obtained. This mixture was heated to 80 ° C., and filtered under pressure through a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.40 μm, to obtain a liposome-containing contrast agent.

Claims (9)

Including liposomes containing a pharmaceutical compound and one or more physiologically acceptable formulation aids inside and outside the lipid membrane,
The liposome is a liposome substantially composed of 1 to 10 membranes, of which at least 70% are composed of 2 to several membranes, and the lipid membrane components include Contains at least one phospholipid having a transition temperature in the range of 40 to 65 ° C.,
A liposome-containing preparation characterized by being substantially free of an organic solvent. The liposome is prepared by mixing supercritical carbon dioxide with the liposome membrane constituent, pharmaceutical compound and formulation aid under conditions of 40 to 65 ° C. and 10 to 30 MPa in a pressure vessel, and reducing the pressure in the pressure vessel. An aqueous dispersion of liposomes encapsulating the pharmaceutical compound is obtained by discharging carbon dioxide, and then prepared by incubating at the transition temperature of the phospholipid to (the transition temperature +10) ° C. for 0.1 to 3 hours. The liposome-containing preparation according to claim 1, which is a liposome.   The proportion of particles having a range of (average particle size -5%) to (average particle size + 5%) occupies 50 to 90% of the total liposome particles. Or the liposome containing formulation of 2. The phospholipid is selected from the group consisting of dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, hydrogenated soybean lecithin, hydrogenated soybean phosphatidylcholine, and distearoylphosphatidylcholine.
4. The liposome-containing preparation according to any one of 3. The liposome-containing preparation according to any one of claims 1 to 4, wherein the phospholipid includes a phospholipid having a polyethylene glycol (PEG) group.   The phospholipid is at least one of PEG-dimyristoyl phosphatidylcholine and PEG-dipalmitoyl phosphatidylcholine, and further includes at least one of a nonionic iodine-based compound and an anticancer compound in the liposome. The liposome-containing preparation according to any one of claims 1 to 5, wherein:   The liposome membrane component includes at least a phospholipid, a lipid having a PEG group, and a lipid containing sterols, and a molar ratio of phospholipid (excluding PEG-phospholipid) / sterols is 100/60 to 100/90. The liposome according to any one of claims 1 to 6, wherein the molar ratio of phospholipid (excluding PEG-phospholipid) / phospholipid having a polyethylene glycol group is 100/5 to 100/25. Containing formulation.   The liposome-containing preparation according to any one of claims 1 to 7, wherein the concentration of the pharmaceutical compound and the preparation aid in and out of the lipid membrane is substantially the same. (I) a step of mixing a supercritical carbon dioxide, a liposome membrane constituent, a pharmaceutical compound and a formulation aid in a pressure vessel at 40 to 65 ° C. and 10 to 30 MPa to obtain a suspension;
(Ii) Next, a step of preparing an aqueous dispersion of liposomes encapsulating the pharmaceutical compound by depressurizing the system and discharging carbon dioxide,
(Iii) Thereafter, the aqueous dispersion of liposomes encapsulating the pharmaceutical compound is incubated at the transition temperature of the phospholipid to (the transition temperature + 10) ° C. for 0.1 to 3 hours, and then the pharmaceutical compound is encapsulated. Filtering the aqueous dispersion of liposomes at 50 to 90 ° C. under a pressure of 0.01 to 0.8 MPa, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm;
A method for producing a liposome-containing preparation, characterized by comprising: