JP4649841B2 - Method for producing liposome-containing preparation, and liposome-containing preparation - Google Patents

Description

  The present invention relates to a method for producing a liposome-containing preparation having a high encapsulation rate of an encapsulated substance and containing finely divided liposomes, and a liposome-containing preparation obtained by the method.

  Liposomes are closed vesicles of bilayer membranes (liposome membranes) formed mainly by phospholipids and have similar structures and functions as biological membranes, and thus have been used as various research materials. Since this liposome can construct a so-called capsule structure that holds a water-soluble encapsulating substance in the aqueous phase inside and an oil-soluble encapsulating substance in the bilayer membrane, it can be used for diagnosis, treatment, makeup, etc. It has been used in various fields. Furthermore, in recent years, application to drug delivery systems (DDS) has been actively studied.

  Liposomes encapsulating such encapsulated substances are usually used as preparations in the form of dispersions, but obtaining liposomes with high retention efficiency of encapsulated substances (inclusion ratio of encapsulated substances relative to liposome membrane constituents). However, in order to obtain a desired effect, it is necessary to use a liposome-containing preparation in which liposomes are dispersed at a high concentration or to use a large amount of a liposome-containing preparation. When the concentration of liposome-containing preparation is high, the viscosity of the preparation increases, so when administering the liposome-containing preparation in vivo, the burden on the patient is large, and the same applies when administering a large amount of liposome-containing preparation. There was a problem. In particular, since a contrast agent using a drug-encapsulating function possessed by liposomes is administered (injected) into a living body via blood vessels or the like, the liposome-containing preparation to be administered is administered at a high concentration or in a large amount. When necessary, there were concerns about side effects as well as burdening patients. Therefore, when considering application to a contrast agent, a liposome-containing preparation having a high contrast substance retention efficiency (encapsulation rate) has been demanded.

In this drug delivery system (DDS), it is necessary to efficiently bring the encapsulated substance to a desired site in the body, and it is important to make the liposome finer. Therefore, conventionally, a liposome suspension prepared by the Bangham method, the reverse phase evaporation method (REV method) or the like is refined using ultrasonic irradiation or mechanical shearing force, or a liposome suspension. An extrusion method is used in which the liquid is passed through a membrane filter under high pressure. However, in the conventional method, since the organic solvent used for dissolving the membrane substance always remains in a very small amount, the strength of the liposome membrane tends to decrease. Therefore, there is a problem that ultrasonic treatment or extruder treatment destroys part of the membrane and lowers the encapsulation rate, and there is a concern that the broken membrane material will form micelles and cause unexpected action in vivo. was there.

In addition, a method for producing cosmetics and external preparations for skin containing liposomes using supercritical carbon dioxide has been disclosed (for example, see Patent Document 1). In this method, production conditions can be set in various ways, and liposomes having a uniform particle size distribution can be prepared relatively easily as compared with conventional methods for producing liposome suspensions. Therefore, the method using supercritical carbon dioxide is an effective method for preparing a DDS preparation. In this method, although supercritical carbon dioxide is used as a main solvent, mixing of an organic solvent used as a solvent auxiliary is inevitable, and the film strength tends to decrease due to the organic solvent. Therefore, similarly to the above, there is a problem that the encapsulation rate is lowered and there is a concern that an unexpected action is caused in the living body.
JP 2003-119120 A

  An object of the present invention is to provide a method for producing a liposome-containing preparation containing liposomes having a high encapsulation rate of encapsulated substances and fine particles, and a liposome-containing preparation obtained by the method.

Method for producing a liposome-containing formulation is an aqueous solution containing at least a water-soluble agent, at least a phospholipid phospholipids with transition temperatures with a solution comprising carbon dioxide in a supercritical state or subcritical state, A method of mixing while irradiating with ultrasonic waves,
The liposome contained in the liposome suspension is formed by encapsulating an aqueous phase containing a water-soluble drug in a phospholipid membrane composed of a phospholipid having at least the phospholipid having the transition temperature.
The temperature at the time of ultrasonic irradiation is the higher one of the critical temperature of carbon dioxide or the transition temperature of phospholipid to the transition temperature + 50 ° C.,
The water-soluble drug is encapsulated in an amount of 1 to 10 by weight with respect to the phospholipid having at least the phospholipid having the transition temperature.

The suspension of the liposome which the ultrasonic waves are irradiated is preferably filtered is passed through a filtration membrane having a pore size of 0.05～0.4Myuemu.
It is preferred that substantially no dissolution aid is used.
At least one lipid membrane stabilizing substance selected from the group consisting of cationic phospholipids, polyalkylene oxide-modified phospholipids, compounds having a polyalkylene oxide group, compounds having a polyethylene glycol group, sterols and glycols, It is preferable to mix with the phospholipid which has at least the phospholipid which has the said transition temperature.

  The liposome-containing preparation according to the present invention is manufactured by the above-described method, and the aqueous phase encapsulated in the phospholipid membrane preferably contains at least a nonionic iodine compound and a preparation aid. .

  It is also preferable that 5 to 35% by mass of the nonionic iodide compound is encapsulated in liposomes, and the formulation aid is selected from the group consisting of a buffer, a chelating agent, a viscosity modifier, and a stabilizer. It is also preferable that it is at least one selected.

According to the production method of liposome-containing formulations, in preparing the suspension of the liposome, by irradiating ultrasonic waves at a predetermined condition, liposomes is improved and inclusion rate of the encapsulated substance liposome fine A liposome-containing preparation can be produced. Furthermore, the liposome-containing preparation obtained by such a production method can be suitably used as a drug carrier or an artificial oxygen carrier.

  Hereinafter, the production method of the liposome-containing preparation of the present invention will be described in detail.

Method for producing a liposome-containing formulations, process for the preparation of liposomes with the reaction medium of supercritical carbon dioxide (hereinafter, supercritical carbon dioxide method) method applying ultrasonic waves in preparing a suspension of liposomes by in, is there.

  The liposome-containing preparation produced by such a method is obtained as an aqueous dispersion in which liposomes encapsulating a water-soluble encapsulating substance are dispersed in an aqueous solvent.

As liposomes, liposomes consisting of multilamellar vesicles (MLV), liposomes consisting of single-layer LUV with large particle size (Large unilamellar veislcles)
Examples thereof include small unilamellar vesicles composed of small single-film SUVs having a particle size of less than 50 nm, and these may be mixed.

  Liposomes are bilayer vesicles and are generally composed of lipid bilayers. The liposome membrane constituent material used in the production method of the present invention is not particularly limited as long as it is generally used as a membrane constituent material. Usually, a phospholipid and / or glycolipid having liposome-forming ability, which is a lipid for liposomes, is an essential main component, and the following substances may coexist as optional components. Optional components include sterols as lipid membrane stabilizers, fatty acids or salts thereof as charged substances, lipid mixed emulsions, and the like conventionally used as components for forming endoplasmic reticulum.

  Hereinafter, each component used when preparing a liposome containing formulation is demonstrated.

(Water-soluble drug)
Examples of the water-soluble drug used in the present invention include an imaging substance, an anticancer substance, an antioxidant substance, an antibacterial substance, an anti-inflammatory substance, a blood circulation promoting substance, a whitening substance, a rough skin preventing substance, and an anti-aging substance. , Hair growth-promoting substances, moisturizing substances, hormone agents, vitamins, pigments, and water-soluble drugs such as proteins. The liposome-containing preparation of the present invention preferably uses a contrast-enhancing substance or an anticancer substance as a water-soluble drug.

  As the contrast-enhancing substance, a water-soluble iodine compound can be used. The water-soluble iodine-based 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. As the water-soluble nonionic iodide compound, a nonionic iodide compound containing phenyl iodide, for example, having at least one 2,4,6-triiodophenyl group is preferable.

As such a nonionic iodine compound, specifically, iopamidol (Iopamidol)
), Iomeprol, Iohexol, Iopentol
, Iopromide, Iosimide, Ioversol, Iotrolan, Iotasul, Iodixanol, Iodecimol, (1,3-bis- (N-3,5-) Bis- [2,3-dihydroxypropylaminocarbonyl] -2,4,6-triiodophenyl) -N-hydroxyacetylamino) propane. These compounds may be used alone or in combination of two or more.

  Specific examples of the anticancer compound include methotrexate, doxorubicin, ebirubicin, daunorubicin, vincristine, vinblastine, etovoside, eribucine, captodecin, paclitaxel, docetaxol, cisbratine, and bradnisone. These compounds may be used alone or in combination of two or more.

  In addition, in this specification, the said compound may be mentioned including the salt, hydrate, etc. other than a free form.

  When the liposome-containing preparation of the present invention is used as a contrast medium for X-ray examination, preferred iodo compounds are highly hydrophilic and iomeprol, iopamidol, iotrolan, and iodixanol, which do not increase osmotic pressure even at high concentrations. . In particular, dimer nonionic iodine compounds such as iotrane and iodixanol have the advantage of further reducing the osmotic pressure because the total number of moles is low even when a contrast agent having the same iodine concentration is prepared.

  The concentration of the water-soluble iodo compound in the liposome-containing preparation of the present invention can be arbitrarily set based on factors such as the nature of the contrast compound, the intended route of administration of the preparation, and clinical indicators. The amount of the iodine compound encapsulated in the liposome is typically 5 to 40% by mass, preferably 5 to 35% by mass, and more preferably 10 to 25% by mass of the total iodine compound in the X-ray contrast medium. It is desirable. 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.

(Phospholipid)
Examples of phospholipids include phospholipids represented by phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, sphingomyelin and the like. Phospholipids derived from egg yolk, soybeans and other animal and plant materials, hydrogenated products thereof, semi-synthetic phospholipids such as hydroxide derivatives, or synthetic processed products are used without limitation. The constituent fatty acid of the phospholipid is not particularly limited, and may be either a saturated fatty acid or an unsaturated fatty acid.

  Specific examples of neutral phospholipids include dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristol phosphatidylcholine (DMPC), dioleyl phosphatidylcholine (DOPC), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanol Examples include amines and distearoyl phosphatidylethanolamine.

  In addition to the neutral phospholipid, a charged phospholipid such as an anionic phospholipid and a cationic phospholipid, a polymerizable phospholipid, and a cationic (positively charged) lipid may be included.

Negatively charged phospholipids include dipalmitoyl phosphatidylglycerol (DPPG), dimyristoyl phosphatidylglycerol, distearoyl phosphatidylglycerol (DSPG), dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol ( DPPI), distearoyl phosphatidylinositol (DSPI), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidic acid and the like.

  Cationic phospholipids include esters of phosphatidic acid and amino alcohol, such as esters of dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA) and hydroxyethylenediamine.

  The phospholipid used in the present invention desirably contains at least a phospholipid having a transition temperature. The “(phase) transition temperature” of a phospholipid is a temperature that causes a phase transition between both the gel and liquid crystal states that the phospholipid can take. The measurement is by differential thermal analysis using a differential scanning calorimeter (DSC).

Phospholipids having a phase transition point include dimyristoyl phosphatidylcholine (transition temperature (same below): 23-24 ° C), dipalmitoylphosphatidylcholine (41.0-41.5 ° C), hydrogenated soybean lecithin (53 ° C), hydrogenated soybean phosphatidylcholine. (54 ° C.), distearoyl phosphatidyl choline (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.

Examples of cationic lipids include 1,2-dioleoyloxy-3- (trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS).
), Dimethyl dioctadecyl ammonium bromide (DDAB), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA),
2,3-dioleoyloxy-N- [2 (spermine-carboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and N- [1
-(2,3-Dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxyethyl) ammonium bromide (DMRIE) and the like.

  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.

  In addition to the above lipid components, other components can be added as membrane constituents of the liposome as necessary. Examples thereof include sterols such as cholesterol and cholesterol esters as membrane stabilizers or anchors for introducing polyalkylene oxide groups, and dialkyl phosphates such as dicetyl phosphate which is a charged substance.

  As another preferred embodiment of the liposome in the present invention, a phospholipid selected from the group consisting of phosphatidylcholine and phosphatidylserine can be used as a membrane component, and both may be combined.

(Formulation aid)
Further, it is preferable that the formulation aid is dissolved or dispersed in the liposome suspension. The “formulation aid” is added together with the encapsulated substance during formulation, and various substances are used as appropriate. Specific examples include various physiologically acceptable buffers, chelating agents, viscosity modifiers, stabilizers, osmotic pressure regulators, antioxidants such as α-tocopherol, and preservatives such as methyl parahydroxybenzoate. It is done. In the present invention, it is preferable to use at least one selected from the group consisting of a buffer, a chelating agent, a viscosity modifier, and a stabilizer.

  Using each of these components, the liposome-containing preparation of the present invention is prepared as follows.

Method for producing liposome-containing preparation
As a method for producing liposome-containing preparations may be mentioned embodiment 1 and embodiment 2. Embodiment 1 is a method of producing a liposome-containing preparation by irradiating the prepared liposome suspension with ultrasonic waves under predetermined conditions, and Embodiment 2 is a method of producing a liposome suspension by supercritical carbon dioxide method. It is a method for producing a liposome-containing preparation by irradiating ultrasonic waves during preparation. First, Embodiment 1 will be described.

(Embodiment 1)
Method for producing a liposome-containing formulations, in that embodiment 1, first, preparing a suspension of liposomes.

In Embodiment 1, the suspension of liposomes is mixed with supercritical carbon dioxide containing a lipid membrane constituent having at least a phospholipid having a transition temperature and an aqueous solution containing an encapsulating substance such as a water-soluble drug. Is formed. Carbon dioxide is relatively easy to handle with a critical temperature of 31.1 ° C. and a critical pressure of 75.3 kg / cm ^2, and the gas is inert so it is harmless to the human body even if it remains. Is preferable because it is inexpensive and easily available. The temperature of carbon dioxide in the supercritical state (including subcritical state) used in the production method of the present invention is usually 32 to 100 ° C, preferably 32 to 80 ° C. A particularly suitable temperature within such a temperature range is at or above the phase transition temperature of the phospholipid used. The pressure is usually in the range of 50 to 500 kg / cm ² , preferably 90 to 150 kg / cm ² , particularly preferably 100 to 120 kg / cm ² . In addition, because it is not a method for compulsorily compatibilizing lipids in water at high temperatures, modification of lipids at high temperatures, for example, phospholipids are hydrolyzed to form lyso forms, and unsaturated components are peroxidized. There is no problem.

  The preparation of the liposome suspension is specifically performed as follows.

  First, a phospholipid, a lipid membrane stabilizing substance, and the like are charged in a pressure vessel as a lipid membrane constituent material of a liposome. The above phospholipid as a lipid membrane constituent is preferably at least one selected from cationic phospholipids, polyalkylene oxide modified phospholipids, compounds having a polyalkylene oxide group, compounds having a polyethylene glycol group, sterols, and glycols. Mix together with the compound. After the lipid membrane constituent material is charged, the whole is melted by heating to a predetermined temperature by a heating means provided in the pressure vessel, and then liquid carbon dioxide is added while stirring by a stirring means such as a magnetic stirrer. Further, the liquid carbon dioxide is brought into a supercritical state or a subcritical state with the above pressure and temperature inside the pressure vessel. Next, a phospholipid, a lipid membrane stabilizing substance, and the like are mixed with the carbon dioxide in a supercritical (or subcritical) state as a lipid membrane constituent material of the liposome.

  Next, while stirring the supercritical carbon dioxide, the encapsulated substance and, if necessary, the aqueous solution containing the above-mentioned formulation aid are added in two or more times, or added in small portions continuously and mixed. To do. Specifically, the aqueous solution is 1/1000 to 1/5 volume, preferably 1/500 to 1/10 volume, more preferably 1/200 to 1/20 volume of the supercritical carbon dioxide capacity per minute. It is better to add in proportions.

  The efficiency of the encapsulation of the encapsulated substance in the liposome also depends on the ratio between the total lipid amount of the lipid for liposome and the aqueous solution containing the encapsulated substance. The total amount of lipid here is the total mass of all phospholipids, sterols and other added lipids constituting the liposome membrane. When the total amount of lipid is mixed in a ratio of 15 to 150 mmoles, preferably 30 to 120 mmoles, more preferably 50 to 100 mmoles per 1 liter (L) of the aqueous solution, the water-soluble encapsulating substance is encapsulated in the liposome. The process proceeds well, and as a result, the retention efficiency of the encapsulated material is improved.

  After preparing a mixed solution in which supercritical carbon dioxide and an aqueous solution are mixed as described above, when the pressure vessel is depressurized and carbon dioxide is discharged, the suspension of liposomes in which the liposomes encapsulating the encapsulated material are dispersed is dispersed. A turbid liquid is formed.

The liposome preparation method using supercritical or subcritical carbon dioxide has higher liposome production rate, encapsulation rate of the encapsulated substance, and retention rate of the encapsulated substance in the liposome than the conventional method. Furthermore, this method, which can be applied on an industrial scale, can efficiently encapsulate a nonionic and water-soluble substance in a liposome without using an organic solvent, is useful for producing the liposome-containing preparation of the present invention. Is a useful method.

Many of the liposomes in the present invention are usually present as multilamellar liposomes, but single membrane liposomes may also be present. The single membrane liposome here is a liposome composed of a membrane (unilamellar vesicle) in which a phospholipid bilayer substantially forms one layer. Here, “substantially” means that the liposome is composed of a phospholipid bilayer in which the replica is generally recognized as one layer in the following transmission electron microscope (TEM) observation by the Freeze fracture replica method. It means that 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”. As for the liposome (multilamellar vesicles; MLV) which consists of a multilayer film, it is desirable to peel the bilayer membrane of a liposome film and to make the multilayer as thin as possible. Single membrane liposomes have a larger encapsulation capacity and higher encapsulation efficiency than multilamellar liposomes. Therefore, since the amount of lipid to be administered does not increase, it is desirable that the ratio of single membrane liposomes is large.

  A liposome-containing preparation is produced by irradiating the thus prepared liposome suspension with ultrasonic waves under predetermined conditions.

(Ultrasonic irradiation)
After the liposome suspension thus prepared is heated to the transition temperature of the phospholipid to the transition temperature + 50 ° C. (but not exceeding 100 ° C.), the temperature ± 10 ° C. (however, not exceeding 100 ° C.) And irradiating with ultrasonic waves under predetermined conditions.

Irradiation conditions, the form of liposomes contained in the liposome suspension, the particle size distribution, Ru is selected appropriately depending encapsulated rate and the like.

  When heated above the transition temperature of the phospholipid in this way, the phospholipid having the transition temperature becomes a liquid crystal state and the fluidity is increased. Further, by performing ultrasonic treatment in that state, the organic solvent, etc. Even if the solubilizing agent is not used, the encapsulation rate of the encapsulated substance of the liposome can be improved efficiently and the liposome can be miniaturized. Furthermore, since the film strength is not reduced by the dissolution aid, the encapsulation rate is not reduced in the subsequent steps.

  As the liposome suspension used in the present invention, a liposome suspension prepared by the supercritical carbon dioxide method as described above is preferably used. Since the liposome suspension prepared by this method contains liposomes having a uniform particle size distribution, the above-described ultrasonic treatment of the present invention improves the encapsulation rate of the encapsulated substance of the liposome as described above. At the same time, the liposome is further refined, and a liposome-containing preparation having a uniform particle size distribution is obtained.

  Specifically, the ultrasonic treatment of the liposome suspension can be performed as follows.

After preparing a suspension of liposomes as described above,
(1) A method of irradiating ultrasonic waves under the above conditions while stirring the obtained liposome suspension in a container,
(2) A method of circulating a suspension of the obtained liposome and passing a portion irradiated with ultrasonic waves under the above conditions,
(3) The liposome suspension can be sonicated by a method of irradiating ultrasonic waves under the above conditions without stirring the obtained liposome suspension in a container. In the case of performing the method (2) above, the ultrasonic treatment time is obtained from the accumulated time during which the ultrasonic wave was irradiated. In the present invention, it is preferable to perform ultrasonic treatment by the method (1) above, which has a simple manufacturing process.

The suspension of liposomes to which the ultrasonic waves are irradiated as above SL, it is preferable to further filtration.

(filtration)
Filtration of the suspension of liposomes irradiated with ultrasonic waves is preferably carried out through a filtration membrane having a predetermined pore size, and is desirably carried out using an extruder. As the filtration membrane, a polycarbonate type, a cellulose type or the like can be used as appropriate, and the pore diameter is 0.05 to 0.4 μm, preferably 0.1 to 0.4 μm, more preferably 0.15. A range of 0.2 μm is desirable.

  Specifically, uniform liposomes having optimum dimensions can be efficiently prepared by passing through a hydrostatic extrusion apparatus equipped with a filter having the above pore diameter. Examples of the hydrostatic extrusion apparatus include various hydrostatic extrusion apparatuses such as “Extruder” (trade name, manufactured by NOF Liposome), “Liponizer” (trade name, manufactured by Nomura Microscience) and the like. As a result, a lipid for liposomes having a uniform orientation of lipid molecules and good water dispersibility can be obtained. The extrusion filtration method is described in, for example, Biochim. Biophys. Acta 557, 9 (1979).

As desirable embodiment, the extrusion filtration, the suspension of liposomes phospholipid transition temperature or more with a transition temperature, preferably warmed to above "transition temperature + 10 ° C.", then a film having fine pores It is preferable to pass through. The “transition temperature + 10 ° C. or higher” is about 35 ° C. to 65 ° C., depending on the type and composition of the phospholipid used.

  In the operation of pressure extrusion filtration, when the temperature is higher than the transition temperature of the phospholipid as described above, the phospholipid having the transition temperature becomes a liquid crystal state and the fluidity is increased. Since the lipid for liposomes according to the production method of the present invention constitutes a uniform lipid membrane having a regular orientation, even a highly viscous liposome suspension encapsulating a water-soluble encapsulating substance can clog the filter. Liposomes with a uniform particle size can be produced without waking up.

Incorporation of such an “extrusion” operation step has the advantage that, in addition to the above sizing, it is possible to exchange liposome dispersions, remove undesirable substances, and filter sterilization. Subsequently, the liposome may be purified by removing unretained encapsulated material by a method such as centrifugation, ultrafiltration, or gel filtration. Operations such as concentration, dilution, and lyophilization may be optionally performed. Then, sterilization, through the preparation process, such as packaging, liposome-containing formulation is prepared.

  Next, Embodiment 2 of the method for producing a liposome-containing preparation according to the present invention will be described.

(Embodiment 2)
Embodiment 2 of the present invention is a phospholipid having at least an aqueous solution containing at least a water-soluble drug and a phospholipid having a transition temperature in the method for preparing a liposome suspension by the supercritical carbon dioxide method of Embodiment 1 described above. Is a method for producing a liposome-containing preparation in which an ultrasonic wave is irradiated under a predetermined condition when mixing with a solution comprising carbon dioxide in a supercritical state. The production of the liposome-containing preparation in Embodiment 2 is basically performed under the same conditions as in Embodiment 1 and substantially no dissolution aid is used.

In Embodiment 2, the temperature at which the ultrasonic irradiation is performed (that is, the temperature at which supercritical carbon dioxide is generated) is the higher of the critical temperature of carbon dioxide or the transition temperature of phospholipid, which is higher than the transition temperature + 50 ° C. A range is preferable. The ultrasonic irradiation conditions are appropriately selected depending on the form of the liposome, the particle size distribution, the encapsulation rate, and the like .

By ultrasonic treatment, even without using a dissolution aid such as substantially organic solvent, it causes efficiently improve the inclusion rate of the enclosed substances liposomes can be miniaturized liposomes. Furthermore, since the film strength is not reduced by the dissolution aid, the encapsulation rate is not reduced in the subsequent steps.

  In the present invention, after preparing a liposome suspension by irradiation with ultrasonic waves as described above, it is preferable to filter in the same manner as in Embodiment 1, and further operations such as concentration, dilution, and lyophilization are optional. You may go to Subsequently, the liposome-containing preparation of the present invention is prepared through preparation processes such as sterilization and packaging.

Liposome-containing preparation The liposome-containing preparation of the present invention preferably has an average particle size of the liposome of 50 to 800 nm. The liposome particle size is preferably smaller than 200 nm when incorporated into a specific site in the body, and preferably 200 nm or more when incorporated into predatory cells in the body.

  Furthermore, the liposome in the liposome-containing preparation is encapsulated in an amount such that the encapsulated substance is in a weight ratio of 1 to 10, preferably 2 to 8, more preferably 3 to 6 with respect to the liposome membrane constituent. desirable.

  Thus, since the liposome-containing preparation has an improved encapsulation rate of the encapsulated substance in the liposome, the amount of the liposome-containing preparation to be administered into the living body is small, and the burden on the patient is small. When applied to a contrast medium using DDS), the burden on the patient is further reduced.

  Such a liposome-containing preparation of the present invention is not particularly limited, and can be applied to cosmetics, external preparations for skin, internal medicines, contrast agents and the like. In this invention, since the said effect is acquired, it can use suitably for a contrast agent.

[Example]
EXAMPLES Hereinafter, although this invention is demonstrated further more concretely based on an Example, this invention is not limited to these Examples.

  In addition, the following was used as a nonionic iodine compound.

Nonionic iodine compound trade name: Oivaromin 150 (X-ray contrast agent manufactured by Konica Minolta MG Co., Ltd .: 306.2 mg / ml of iopamidol (iodine content 150 mg / ml) )

[Example 1 ]
A 28 kHz vibrator (4392C. Kaijo Co., Ltd.) was placed in a stainless steel pressure vessel, and 860 mg of dipalmitoylphosphatidylcholine (DPPC) and 384 mg of cholesterol were placed on it, and the lid was capped. Thereafter, the entire pressure vessel was heated to 60 ° C. with an electric heater, and then 130 g of liquefied carbon dioxide at a pressure of 50 kg / cm ² was injected into the pressure vessel while stirring the inside of the pressure vessel with a magnetic stirrer. After the injection, the ultrasonic transmitter (4021 type, Kaijo Corporation) was turned on. Furthermore, by reducing the volume of the pressure vessel, carbon dioxide was brought into a supercritical state and further pressurized to 120 kg / cm ² . Thereafter, 100 ml of euvalomin 150 was injected into the pressure vessel at a flow rate of 1 ml / min using an HPLC liquid feed pump.

After completion of the injection, the ultrasonic vibrator was turned off, and the carbon dioxide in the pressure vessel was gradually removed over about 20 minutes to return to atmospheric pressure, and then the solution inside was taken out. A portion of the resulting solution was dialyzed 3 times with 1 L of physiological saline, a small amount of ethanol was added to break the liposomes, and after centrifugation, the absorption of 242 nm of iopamidol was measured with a spectrophotometer. The amount of iopamidol encapsulated inside was quantified. As a result, the encapsulation rate was 16%. Further, the obtained solution was heated with an extruder heated to 80 ° C. three times with a mixed cellulose membrane filter having a pore size of 1 μm, three times with a mixed cellulose membrane filter having a pore size of 0.45 μm, and with a mixed cellulose membrane filter having a pore size of 0.3 μm. Filtration was performed 6 times to prepare a liposome-containing preparation (contrast agent). When the encapsulation rate of this contrast medium was measured, there was no change at 16%. Further, when the particle size of the obtained liposome-containing preparation was measured with a light scattering particle size measuring device (Malvern, Zetasizer 1000), the average particle size was 220 nm.

[Contrast agent administration]
The contrast agent obtained in Example 1 was administered to the hepatocarcinogenic rat from the tail vein at a dose of 2 mg / kg. One hour after administration, the rat was anesthetized with diethyl ether, and when an X-ray image of the whole body of the rat was taken, all of the tumor portions could be clearly identified. 12 hours later, anesthesia with diethyl ether was performed again, and when an X-ray image of the whole body of the rat was taken, contrast enhancement by contrast medium was not observed, and it was barely discriminable (a level that could be discriminated without adding contrast medium). .

(Preparation of hepatocarcinogenic rat)
According to Japanese Patent Laid-Open No. 2002-95382, hepatocarcinogenic rats were prepared. F344 male rats (5 weeks old) were bred on CDAA diet for 1 week, then DEN 20mg / kg body weight was administered once intraperitoneally, and bred for 64 weeks on CDAA diet. Rats with hepatocarcinogenesis were prepared by rearing in 1) for at least 8 weeks.

According to the production method of liposome-containing formulations, in preparing the suspension of the liposome, by irradiating ultrasonic waves at a predetermined condition, liposomes is improved and inclusion rate of the encapsulated substance liposome fine A liposome-containing preparation can be produced. Furthermore, the liposome-containing preparation obtained by such a production method can be suitably used as a drug carrier or an artificial oxygen carrier.

Claims (8)

A liposome containing an aqueous solution containing a water-soluble drug and a solution containing phospholipids having at least a phospholipid having a transition temperature in carbon dioxide in a supercritical state or a subcritical state while being irradiated with ultrasonic waves. A method for producing a formulation comprising:
The liposome contained in the liposome suspension is formed by encapsulating an aqueous phase containing a water-soluble drug in a phospholipid membrane composed of a phospholipid having at least the phospholipid having the transition temperature.
The temperature at the time of ultrasonic irradiation is the higher one of the critical temperature of carbon dioxide or the transition temperature of phospholipid to the transition temperature + 50 ° C.,
A method for producing a liposome-containing preparation, wherein the water-soluble drug is encapsulated in an amount of 1 to 10 by weight with respect to a phospholipid having at least the phospholipid having the transition temperature. The method for producing a liposome-containing preparation according to claim 1 , wherein the suspension of the liposome irradiated with ultrasonic waves is filtered through a filtration membrane having a pore size of 0.05 to 0.4 µm. . The method for producing a liposome-containing preparation according to claim 1 or 2 , wherein substantially no solubilizing agent is used. At least one lipid membrane stabilizing substance selected from cationic phospholipids, polyalkylene oxide-modified phospholipids, compounds having a polyalkylene oxide group, compounds having a polyethylene glycol group, sterols and glycols, The method for producing a liposome-containing preparation according to any one of claims 1 to 3 , wherein a phospholipid having a temperature is mixed with at least a phospholipid having a temperature. A liposome-containing preparation produced by the method for producing a liposome-containing preparation according to any one of claims 1 to 4 . The liposome-containing preparation according to claim 5 , wherein the aqueous phase encapsulated in the phospholipid membrane contains at least a nonionic iodide compound and a preparation aid. Liposome-containing preparation according to claim 6, wherein 5 to 35% by weight of non-ionic iodine compounds, characterized in that it is encapsulated within the liposome. The liposome-containing preparation according to claim 6 , wherein the preparation aid is at least one selected from the group consisting of a buffer, a chelating agent, a viscosity modifier, and a stabilizer.