JP2006298837A - Preparation containing liposome including polyhydric alcohol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a preparation containing liposome effectively including polyhydric alcohol. <P>SOLUTION: The preparation containing liposome includes liposome containing polyhydric alcohols with 500-950 of molecular weight in internal and external water phase of a lipid membrane. The liposome is produced in a super critical carbon dioxide method and substantially comprises 1-10 sheets of lipid membrane and 70% of the liposome is composed of 2-some sheets of lipid membrane. The liposome membrane in the preparation comprises lipid having transition temperature and the including content of the polyhydric alcohols are increased 25-35% and substantially free from a chlorine based solvents. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a liposome-containing preparation containing liposomes having a high polyhydric alcohol encapsulation rate and a method for producing the same.

  Liposomes are capable of constructing a capsule structure in which a water-soluble encapsulating substance is held in an aqueous phase contained in the liposome, and thus are actively studied for application to a drug delivery system (DDS).

  Conventionally, the Bangham method, the reverse phase evaporation method (REV method) and the like are used to prepare liposomes encapsulating encapsulated substances. In these methods, an organic solvent is used as a phospholipid solvent in the preparation process, even though the encapsulated substance is encapsulated in liposomes that are highly safe as materials and have appropriate degradability in vivo. To do. 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 there is a problem that an excessive burden is imposed on the patient particularly when it is necessary to administer a large amount of the liposome-containing preparation. 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. When the encapsulation rate of the encapsulated substance is high, the desired effect can be obtained even if the amount of liposomes taken into the target site in the body is small. Therefore, it is expected as a method for preparing a DDS preparation. 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 a method for efficiently encapsulating a drug in a liposome, a dosage form that can stably retain it over time and improve retention in blood, an improvement in the formulation composition, and an improvement in the safety of the formulation will continue. There are special requirements.
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 method for producing a liposome containing a polyhydric alcohol efficiently without using any organic solvent and a preparation containing the liposome. In particular, a liposome-containing preparation in which the polyhydric alcohol is a nonionic iodine-based compound is a highly safe contrast medium for X-ray examination having good imaging ability and easy excretion of cancer tissue.

  The liposome-containing preparation of the present invention comprises a liposome containing a polyhydric alcohol having a molecular weight of 500 to 950 in the aqueous phase inside and outside the lipid membrane, and the liposome membrane constituent contains at least a lipid having a transition temperature, It is characterized by not containing any chlorinated solvent. Said transition temperature is a lipid in the range of 22-60 ° C.

The liposome is a liposome prepared by a supercritical carbon dioxide method.
It is desirable that the liposome is substantially composed of one to ten lipid membranes, and that liposomes composed of two to several membranes occupy at least 70%.

The encapsulation rate of polyhydric alcohol in the liposome is increased to 25-35%.
The liposome membrane component includes at least a phospholipid, a lipid having a polyethylene glycol (PEG) group, and a lipid including a sterol, and a molar ratio of phospholipid (excluding PEG-phospholipid) / sterol is 100/60. It is a liposome-containing preparation characterized in that the molar ratio of ˜100 / 90, phospholipid (excluding PEG-phospholipid) / lipid having a polyethylene glycol group is 100/5 to 100/25.

The polyhydric alcohol is a water-soluble iodine compound and is desirably used as a contrast agent for X-ray examination.
It is preferable that the concentration of the polyhydric alcohol in the aqueous phase inside and outside the lipid membrane is substantially the same.

The method for producing a liposome-containing preparation according to the present invention comprises:
(I) a first step of mixing a liposome membrane component and supercritical carbon dioxide in a pressure vessel at 33 to 65 ° C. to obtain a suspension;
(Ii) Next, a second step of adding and mixing an aqueous polyhydric alcohol solution to the suspension,
(Iii) a third step of preparing an aqueous dispersion of liposome encapsulating the polyhydric alcohol by depressurizing the inside of the pressure vessel and discharging carbon dioxide; and (iii) the polyhydric alcohol. A fourth step of filtering an aqueous dispersion of liposomes encapsulated in a liposome 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
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.

Encapsulated substance In the liposome-containing preparation of the present invention, a polyhydric alcohol is encapsulated in the lipid membrane of the liposome. The polyhydric alcohol used in the present invention has at least a plurality of hydroxyl groups in the molecule and has a molecular weight of 500 to 10,000, preferably 500 to 950, more preferably 600.
~ 900 compounds. Particularly preferred are water-soluble compounds. When preparing liposomes by the supercritical carbon dioxide method, the present inventors have clarified that polyhydric alcohol compounds containing a plurality of hydroxyl groups are more easily encapsulated in liposomes than analogs having a structure not containing hydroxyl groups. became. When liposomes are produced by using a supercritical carbon dioxide and undergoing a predetermined process under the conditions described below, if the encapsulated substance is a polyhydric alcohol having a molecular weight in the above range, it is efficiently encapsulated in the liposomes. The That is, when the number of hydroxyl groups in the molecule is large, it is easy to stably encapsulate in the liposome, so that there is an advantage that the encapsulation rate is increased and the amount of drug used is reduced.

  The number of hydroxyl groups in the polyhydric alcohol is at least 2, preferably 2-6, more preferably 3-5. Moreover, it is desirable that it is not a macromolecule but a medium molecule having a molecular weight in the above range. The reason for this is not clear, but the size of the polyhydric alcohol molecule and the presence of the hydroxyl group may have an advantageous effect on the inclusion in the lipid membrane.

Any polyhydric alcohol having such a molecular weight is not particularly limited, and examples thereof include substances widely used in pharmaceuticals. For example, specific examples of polyhydric alcohol-based pharmaceutical substances that can be suitably used in the present invention include nonionic iodine compounds, ascorbic acid dipalmitate, streptomycin, rifampicin, amphotericin B, nystatin, midecamycin, hesperidin, rutin, naringin, digoxin, etc. Is mentioned. However, the present invention is not limited to these exemplified compounds.

  The liposome-containing preparation of the present invention is particularly preferably used as a contrast agent using a polyhydric alcohol type iodine compound as a contrast medium. A preferred contrast material is a water-soluble nonionic iodine-based compound having a plurality of hydroxyl groups. As a specific water-soluble nonionic iodide compound, a nonionic iodide compound containing phenyl iodide and having at least one 2,4,6-triiodophenyl group is preferable.

  Specifically, examples of such nonionic iodo compounds include iohexol, iopentol, iodixanol, iotasulf, iopromide, iomeprol, iopamidol, ioxirane, and metrizamide.

  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.

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 a unique use of the contrast agent, and is distinguished from a position in which a pharmaceutical compound that is not encapsulated in liposomes is useless.

  The X-ray contrast medium which is one embodiment of the liposome-containing preparation of the present invention usually also contains an iodine 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 X-ray contrast medium 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 liposomes and exists in an aqueous medium in which the liposomes are suspended.

In the liposome-containing X-ray contrast medium of the present invention, the amount of the iodine compound encapsulated in the liposome in order to efficiently encapsulate the iodine compound and prevent the destabilization of the liposome carrying the compound over time is as follows: It is rather limited. That is, it is desirable that the total iodine compound in the X-ray contrast medium is 10 to 35% by mass, preferably 10 to 30% by mass, more preferably 15 to 25% by mass. 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 with liposome-containing X-ray contrast media, 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. Is also preferable.

Even with liposome-containing preparations other than contrast agents, sustained release pharmacokinetics or sustained mode of action are not the usual bursty increase in blood concentration and transient effects seen in the early days after administration. In order to realize the above, it is only necessary to include a polyhydric alcohol based pharmaceutical substance in the aqueous phase other than the liposome. The polyhydric alcohol-based drug substance encapsulated in the liposome membrane is gradually released into the body after being administered to the body after the release of the free drug substance.
Liposome membrane component The liposome-containing preparation of the present invention is intended to be efficiently delivered to the target organ or tissue lesion by using polyalcohol in a form encapsulated in liposome as a microcarrier. That is, a targeting function can be imparted by appropriately designing the particle size of the liposome encapsulating the polyhydric alcohol 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, introducing a polyalkylene oxide polymer chain or a polyethylene glycol group 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 rapidly without being accumulated in normal tissue and excreted outside the body. 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 polyhydric alcohol that has no side effects in the free form is encapsulated in the liposome 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, harmful effects caused by staying in the body, delayed side effects, etc. can be prevented.

Lipid membrane components constituting the lipid membrane of the liposome include at least phospholipids, glycolipids, sterols, glycols, lipids having a polyethylene glycol 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).

  It is desirable that the phospholipids of the lipid membrane constituting the liposome of the present invention contain 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 in the range of 22 to 60 ° 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.

  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 polyethylene glycol (PEG) group to the surface of the liposome membrane, instability of the liposome itself such as disintegration and aggregation is solved, and stability over time 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, it has been clarified that liposomes can increase the stability in blood and maintain the concentration in blood for a long time by forming a hydration layer by introducing PEG groups and exhibiting a hydrophilic tendency (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 the liposome. Preferably, PEG-phospholipid may be used as a lipid having a polyethylene glycol group. This is because when a phospholipid or the like is mixed with supercritical carbon dioxide, it also acts as a dissolution aid.

  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 liposome-containing preparation of the present invention, the adjustment of the size and distribution of liposomes as fine particles is closely related to the high drug encapsulation rate, targeting property, and delivery efficiency. The particle size (particle size) is measured by freezing a dispersion containing liposomes encapsulating polyhydric alcohol, then depositing carbon on the crushed interface, and observing this carbon with an electron microscope (freeze-crush TEM method). Can do. 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 an X-ray contrast agent intended for imaging of 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, for the preparation of liposomes, lipid membrane components such as phospholipids and sterols are mixed and dissolved in containers together with organic solvents (eg, chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, THF, etc.) almost without exception. To start with. 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 that has been developed so that liposomes having the optimum form and structure can be produced for contrast agents that have a particularly large dosage compared to conventional 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 polyhydric alcohols, It is shown that the encapsulation rate of the encapsulated drug and the retention rate of the encapsulated drug in the liposome are higher than those of the method. “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 polyhydric alcohol and the formulation aid are encapsulated in the liposome membrane. The component of the liposome membrane is characterized by containing at least a phospholipid having a transition temperature.

In the liposome production method according to the present invention, the first step and the second step are as follows.
(I) a first step of mixing a liposome membrane component and supercritical carbon dioxide in a pressure vessel at 33 to 65 ° C. to obtain a suspension;
(Ii) Next, the second step of adding and mixing an aqueous polyhydric alcohol solution to the suspension or the following order may be used.
(I) a first step of supplying liquefied carbon dioxide into a pressure vessel containing a suspension obtained by mixing a liposome membrane constituent and a polyhydric alcohol aqueous solution;
(Ii) Next, while mixing the suspension and liquefied carbon dioxide, the pressure vessel is pressurized under 33 to 65 ° C. to make the liquefied carbon dioxide supercritical carbon dioxide. The third step and the fourth step are continued.
(Iii) a third step of producing an aqueous dispersion of liposomes containing a polyhydric alcohol and a preparation auxiliary agent inside after depressurizing the inside of the pressure vessel and discharging carbon dioxide;
(Iv) After the third step, the polyhydric alcohol is encapsulated under a pressure of 50 to 90 ° C. and 0.01 to 0.8 MPa in a hydrostatic extruder equipped with a filtration membrane having a pore size of 0.1 to 1 μm. The 4th process which filters the aqueous dispersion liquid of the made liposome, and sizes in 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 10
0 / 60-100 / 90, molar ratio of phospholipid (without PEG-phospholipid) / lipid having polyethylene glycol group is 100 / 5-100 / 25, and lipid membrane is composed of 2-10 membranes A liposome-containing preparation is obtained, characterized in that at least 70% of the liposomes are made up.
-1st process and 2nd process In a 1st and 2nd process, a liposome membrane component, a liquefied carbon dioxide, and a chemical | medical-agent aqueous solution (a polyhydric alcohol and a formulation aid are contained) are mixed in a pressure vessel. 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 22 to 60 ° 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. Subsequently, micelles were introduced by introducing a pharmaceutical aqueous solution containing a polyhydric alcohol to be encapsulated, such as a nonionic iodine-based compound and a formulation aid, into supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance. Let it form.

  Alternatively, in a pressure vessel containing a suspension obtained by mixing a lipid solution having at least a phospholipid, a polyethylene glycol group and a sterol as a liposome membrane component, and an aqueous drug solution containing a polyhydric alcohol and a formulation aid, Micelle may be formed by supplying liquefied carbon dioxide, preferably mixing and dispersing under strong stirring, 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 aqueous drug solution in a pressure vessel. Alternatively, such a suspension may be prepared separately and then supplied into the pressure vessel.

The efficiency of the encapsulation 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 aqueous solution 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 be formed as several membranes rather than a single membrane, the amount of lipid added may be 1.5 to 2.5 times greater than the amount of lipids conventionally used. Specifically, under strong stirring, the carbon dioxide is finally mixed and dispersed at a ratio of 0.075 to 0.125 parts by weight, preferably 0.08 to 0.1 parts by weight, with respect to 1 part by weight of carbon dioxide. 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, a lipid phase containing the above-mentioned lipid may exist at the beginning of stirring, and the following strong stirring generates many CO ₂ / water interfaces where lipid molecules are arranged to form many small micelles of lipid. If so, the 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. 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. Moreover, it is desirable that the time of strong stirring is 1 to 120 minutes, preferably 5 to 60 minutes, and the time of strong stirring includes the time for supplying the aqueous drug solution.

  By mixing the liposome membrane component and supercritical carbon dioxide for a predetermined time under strong stirring that satisfies such conditions, liposome production efficiency is improved, and liposomes with a higher polyhydric alcohol encapsulation rate are obtained. The aqueous dispersion containing 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 or the like from the viewpoint of safety. Therefore, a more preferable solubilizing agent in view of efficacy and safety is a polyethylene glycol or a compound having a polyethylene glycol group. Specifically, the compound having a polyethylene glycol group is preferably a lipid having a polyethylene glycol 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 polyethylene glycol group, the molar ratio of phospholipid (excluding PEG-phospholipid) / lipid 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 polyhydric alcohol 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 polyethylene glycol group (for example, PEG-phospholipid) as a liposome membrane constituent acts to promote emulsification instead of a solubilizing agent such as ethanol. Supercritical carbon dioxide containing almost no lipids, as polyhydric alcohols 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 It becomes the state of a three-phase system with the phase of. Although emulsification of supercritical carbon dioxide proceeds by stirring, more interfaces are formed than in the conventional method, and lipids 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, etc., but uniform micro micelles are efficiently produced especially in the method with strong stirring. Thus, the polyhydric alcohol encapsulation rate increases from, for example, 17% to 21% as the formation of the lipid bilayer from the lipid monolayer, which is induced by the subsequent discharge of carbon dioxide. 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. The main body is formed. In 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 32 to 70 ° C, preferably 33 to 65 ° C, more preferably 45 to 65 ° C. ° C. When the phospholipid having a transition temperature is contained in the liposome membrane constituent component, it may be set to be “transition temperature + 10 ° C.” or less, preferably “transition temperature + 5 ° C.” or less, more preferably “almost transition temperature”. desirable. Conventionally, when heated to a temperature higher than the transition temperature of the phospholipid, the phospholipid having the transition temperature becomes a liquid crystal state and the fluidity is increased, and the phospholipid is efficiently mixed with supercritical carbon dioxide. Was prepared at 50-80 ° C. However, even if the temperature of carbon dioxide in the supercritical state is near the transition temperature of the phospholipid as described above, the present inventors do not denature the phospholipid because excessive heat is not applied. Was found to be regularly arranged to form a liposome membrane. In the situation where a strong stirring operation is performed to promote emulsification, it is desirable to employ the above temperature range from the viewpoint of reducing local overheating. The 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.
-3rd process In a 3rd process, after mixing a liposome membrane component, supercritical carbon dioxide, and chemical | medical agent aqueous solution fully, if necessary, water is added in the system and the inside of a pressure vessel is pressure-reduced. By discharging carbon dioxide from this mixed solution, an aqueous dispersion of liposomes encapsulating polyhydric alcohol and the like is prepared. In this process, it is presumed that the liposomes are phase-inverted into the aqueous phase, so that only by discharging carbon dioxide, an aqueous dispersion in which the liposomes containing the polyhydric alcohol are dispersed is generated. Since the aqueous solution is also enclosed inside the liposome, the polyhydric alcohol exists in the aqueous phase inside the liposome in addition to the external aqueous phase of the liposome, and is in an “encapsulated” state.
-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 pressure extrusion filtration operation 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 pressure filtration operation is repeatedly performed if necessary. This extrusion filtration method is described, for example, in Biochim. Biophys. Acta 557, 9 (1979). By incorporating such an “extrusion” operation, the present inventors have found that in addition to the above sizing, the inclusion rate of the polyhydric alcohol increases. 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 pressure extrusion operation described above, even in liposomes composed of multilamellar membranes, reorganization and sizing of the membrane structure including reconstitution of the lipid membrane occurs, and liposomes composed of 2 to 10 membranes are produced. Presumed to be because. Furthermore, by carrying out such a treatment, the encapsulation rate of the encapsulated substance in the liposome is improved and the liposome is made finer, substantially without using an organic solvent-based solubilizing agent such as ethanol. Can do. 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 liposome encapsulating polyhydric alcohol 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 carrying out these filtration operations, the encapsulation rate of polyhydric alcohol 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 liposomes consisting essentially of 1 to 10 membranes, preferably several membranes (eg, 3, 4, 5 or 6 membranes). 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 70%, preferably 80% or more of the total liposomes contained in the contrast medium. More preferably 85 to 98%.

On the other hand, a single-film liposome is a liposome composed of a unilamellar vesicle having a single phospholipid bilayer. This is composed of a phospholipid bilayer in which a replica is generally recognized as one layer in observation by 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 are stronger than single membrane liposomes, and are not broken even 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 method for producing liposomes using the Bangham method or the reverse phase evaporation method (REV method), liposomes composed of multilamellar vesicles (MLV) of various sizes and forms are present in a considerable proportion. There are many cases. 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 polyhydric alcohol encapsulation rate, 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 preparation of liposomes for use in the preparation of the present invention, the supercritical carbon dioxide method and the subsequent sizing process are improved so that liposomes having a bilayer to 10 membrane structure, preferably several membrane liposomes, are efficiently formed. is doing. Furthermore, the lipid membrane is stabilized by containing a compound having a polyalkylene oxide group (for example, phospholipid), sterols, glycol, etc. in the liposome membrane. As a result, it was found that the prepared liposome was 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 in the aqueous phase inside the lipid membrane of the liposome and the aqueous medium in which the liposome is suspended. . This formulation aid is a substance that is added together with the polyhydric alcohol when the liposome is formulated, and various substances are used as necessary based on the conventional preparation manufacturing technology. Specifically, various physiologically acceptable buffers, edetic acid chelating agents such as EDTANa ₂ -Ca and EDTANa ₂ , inorganic salts, pharmacologically active substances (eg, vasodilators, coagulation inhibitors, etc.) Furthermore, osmotic pressure regulators, stabilizers, antioxidants (for example, α-tocopherol, ascorbic acid), viscosity regulators, preservatives and the like can also be mentioned. 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 a polyhydric alcohol, a nonionic iodine-based compound, a formulation aid, or the like. As the water, use sterilized pyrogen-free water. When the above polyhydric alcohol and formulation aid are also contained in an aqueous solution other than the aqueous phase inside the liposomal lipid membrane (that is, the aqueous medium in which the liposome is suspended), the polyhydric alcohol and the aqueous phase inside and outside the lipid membrane are contained in the aqueous phase. An embodiment in which the formulation aid is contained at substantially the same concentration is preferable. In such a case, a significant osmotic pressure difference does not occur inside and outside 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 polyhydric alcohol 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 efficiency of encapsulation of polyhydric alcohol and the stability of encapsulation, the weight of liposome lipid in the liposome must be considered. As the membrane lipid weight of the liposome increases, the viscosity of the preparation increases. The amount of drug encapsulated in the liposome is 1 to 8, preferably all of the drug (including nonionic iodine-based compound and formulation aid) in the aqueous solution encapsulated in the liposome, relative to the liposome membrane lipid. It is desirable that it is contained in a weight ratio (g / g) of 3 to 8, more preferably 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.

X-ray contrast agent In a preferred embodiment of the liposome-containing preparation of the present invention, the polyhydric alcohol is a nonionic iodine-based compound and contains one or more types of physiologically acceptable preparation aids. It is a preparation used as an agent. The liposome used has a form and structure designed as an optimal liposome for an X-ray contrast medium. Such liposomes have substantially improved stability over time and stability in blood because the lipid membrane is composed of several membranes, and by increasing the encapsulation rate of contrast media The contrast performance of the contrast agent is improved. The X-ray contrast medium of the present invention is usually 40 to 450 mgI / mL, preferably 70 to 400 mgI / mL, as the iodine content, in the assumed dosage of 10 to 300 mL of the preparation solution, preferably into the liposome. From the viewpoint of the efficiency of encapsulating the contrast material, it is in the range of 100 to 350 mgI / mL, particularly preferably 150 to 300 mgI / mL. In addition, it is preferable that the aqueous phase inside and outside the lipid membrane contains the iodine compound and the formulation aid at substantially the same concentration.

The X-ray contrast medium 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 iodine type 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 practical problem (Patent Document 1).

[The invention's effect]
In the liposome-containing preparation of the present invention, a polyhydric alcohol having a molecular weight of 500 to 950, preferably a nonionic iodine compound, is encapsulated in the liposome at a high encapsulation rate of 25 to 35% to impart targeting properties. In particular, a liposome-containing preparation encapsulating a nonionic iodine-based compound is excellent in contrast performance as an X-ray contrast agent, and enables a low dose.

  Since the preparation of the present invention is produced without using any highly toxic chlorinated solvent and other organic solvents, toxicity and side effects are reduced as compared with 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. [Example 1]
<Creation of contrast medium>
A mixture of 552.6 mg of dipalmitoyl phosphatidylcholine (DPPC), 221.1 mg of cholesterol, and 167.3 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) is charged into a special stainless steel autoclave and heated to 50 ° C in the autoclave. Then 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 5.0 MPa, was increased to 12 MPa by reducing the volume in the autoclave to bring carbon dioxide into a supercritical state, and lipids were dispersed and dissolved while stirring. It was. Contain a contrast medium solution (517.7 mg / mL (iodine content: 240 mg / mL), trometamol 1 mg / mL, calcium edetate disodium (EDTANa ₂ -Ca) 0.1 mg / mL while stirring, with an appropriate amount of hydrochloric acid. The pH was adjusted to around 7 with sodium hydroxide and 13 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. This sample was heated to 60 ° C. and filtered under pressure with a cellulosic filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 60 ° C., and pressure filtered through a cellulosic filter manufactured by Advantech, 0.40 μm to obtain a liposome-containing contrast agent. About this sample, the average particle diameter and the encapsulation rate were measured.
[Examples 2-3]
A liposome-containing contrast agent was obtained in the same manner as in Example 1 except that the lipid composition, the contrast agent concentration, and the total lipid amount were changed as described in Table 1.
[Comparative Example 1]
Dipalmitoyl phosphatidylcholine (DPPC) 552.6 mg, cholesterol 221.1 mg, PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) 167.3 mg 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. This was further mixed with 13 mL of the contrast agent solution, and the mixture was stirred with a mixer for about 10 minutes while being heated to 65 ° C. By further stirring, a liposome dispersion containing a contrast medium solution was obtained. This mixture was heated to 60 ° C. and pressure filtered through a cellulose filter manufactured by Advantech, 1.0 μm and 0.4 μm to obtain a liposome-containing contrast agent.
[Comparative Examples 2 to 4]
A liposome-containing contrast agent was obtained in the same manner as in Comparative Example 1 except that the contrast agent was changed as shown in Table 1.

As can be seen from Table 1, the liposome-containing contrast medium of the present invention had a high encapsulation rate and was satisfactory with little change after 3 months. The dispersion stability with time was also good.
(Evaluation)
<Evaluation of dispersion stability with time>
The obtained liposome contrast agent was put into a 20 mL small bottle, covered, and the periphery of the lid was sealed with a sealing tape. The sample was stored in a dark place in an atmosphere of 23 ° C. and 55% humidity for 1 month and 3 months for visual evaluation (ranking).
5: There is no precipitate at all, and there is no change from the time of preparation.
4: Although it feels slightly cloudy, there is no separation / precipitation.
3: Turbidity is rising, but separation / precipitation is not observed.
2: A precipitate is generated.
1: The clear liquid and the white precipitate are completely separated.

Claims (9)

  Liposomes containing polyhydric alcohol having a molecular weight of 500 to 950 are included in the aqueous phase inside and outside the lipid membrane, and the liposome membrane constituents contain at least a lipid having a transition temperature and are substantially free of chlorinated solvents. A liposome-containing preparation characterized by   The liposome-containing preparation according to claim 1, wherein the liposome is a liposome prepared by a supercritical carbon dioxide method.   The said liposome is a liposome which consists of a lipid membrane of 1 to 10 membranes substantially, The liposome comprised from 2 to several membranes occupies at least 70%. The liposome-containing preparation according to 1 or 2.   The liposome-containing preparation according to any one of claims 1 to 3, wherein the encapsulation rate of the polyhydric alcohol in the liposome is increased to 25 to 35%.   The said transition temperature exists in the range of 22-60 degreeC, The liposome containing formulation of Claim 1 characterized by the above-mentioned. The liposome membrane component includes at least a phospholipid, a lipid having a polyethylene glycol (PEG) group, and a lipid including a sterol, and a molar ratio of phospholipid (excluding PEG-phospholipid) / sterol is 100/60. The molar ratio of ˜100 / 90, phospholipid (without PEG-phospholipid) / lipid having a polyethylene glycol group is 100/5 to 100/25, The liposome-containing preparation described.   The liposome preparation according to any one of claims 1 to 6, wherein the polyhydric alcohol is a water-soluble iodine compound and is used as a contrast agent for X-ray examination.   The liposome-containing preparation according to any one of claims 1 to 7, wherein the concentration of the polyhydric alcohol in the aqueous phase inside and outside the lipid membrane is substantially the same. (I) a first step of mixing a liposome membrane component and supercritical carbon dioxide in a pressure vessel at 33 to 65 ° C. to obtain a suspension;
(Ii) Next, a second step of adding and mixing an aqueous polyhydric alcohol solution to the suspension,
(Iii) a third step of preparing an aqueous dispersion of liposome encapsulating the polyhydric alcohol by depressurizing the inside of the pressure vessel and discharging carbon dioxide; and (iii) the polyhydric alcohol. A fourth step of filtering an aqueous dispersion of liposomes encapsulated in a liposome 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: