JP2006298838A - X-ray contrast medium containing liposome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a X-ray contrast medium excellent in imaging potential with a high safety. <P>SOLUTION: The X-ray contrast medium is prepared by a super critical carbon dioxide method completely free from organic solvents including ethanol. The X-ray contrast medium comprises liposome which comprises a water soluble nonionic iodine based compound in the internal and external water phase of the lipid membrane as the imaging material and a physiologically acceptable auxiliary agent for preparation. The lipid membrane of the liposome comprises at least a phospholipid, a lipid having a polyethylene glycol group and sterols as the constituting components, and has 0.05-0.8 μm of particle diameter and at least 80% of the lipid membrane are composed of 2-10 sheets of liposome, and the contrast medium contains 100-250 mgI/mL of iodine atom in the iodine based compound and 60-150 mg/mL of total lipids in the contrast medium. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for producing a liposome-containing contrast agent comprising a liposome having excellent in vivo stability and a high encapsulation rate of a contrast substance, and a liposome-containing contrast agent obtained by the method.

  Most of the X-ray contrast agents currently in practical use use a compound containing a triiodophenyl group and water-solubilized as a contrast substance. When such a contrast agent is administered, most of the iodo compounds are rapidly discharged from the luminal site without interacting with the tissue or diseased site. It is obviously not useful for the purpose of observing tissues and diseased sites, especially cancer lesions, in more detail. Therefore, there is a demand for an X-ray contrast agent that selectively accumulates in a target tissue or diseased site, particularly a cancer tissue, and provides an image that can be distinguished from the surroundings of a lesion site or other sites with a clear contrast.

In order to solve the problem, a method of selectively delivering to a target tissue by making the contrast agent into a fine particle and improving its blood half-life is effective. Conventionally, a method of encapsulating a contrast material in a liposome composed of lipids similar to biological membranes and having low antigenicity has been studied. For example, International Publications WO88 / 09165, WO89 / 00988, WO90 / 07491, JP07-316079, and JP2003-5596 propose liposomes containing nonionic contrast substances. These contrast agents still have many problems despite using liposomes that are composed of highly safe lipids and have moderate degradability in vivo. In the production process by the Bangham method or the reverse phase evaporation method (REV method), an organic solvent, in particular, a chloro solvent such as chloroform or dichloromethane is used as a solvent for the phospholipid constituting the liposome membrane (for example, Patent Document 1). reference). Therefore, at present, none of the liposome-containing contrast agents has been put into practical use because of the toxic nature of the remaining solvent (Patent Document 2).

  Furthermore, in the conventional method, the drug cannot be sufficiently encapsulated in the liposome, and it is necessary to administer a large amount of the liposome-containing preparation, resulting in an excessive burden on the patient. Thus, when considering application to a diagnostic contrast medium in which the dose is higher than that of a therapeutic drug, a liposome-containing preparation with high contrast substance retention efficiency (encapsulation rate) has been demanded.

  Although the method using supercritical carbon dioxide described in Patent Document 3 does not use such a harmful organic solvent, there is room for improvement in the encapsulation rate. In order to increase the encapsulation rate of contrast material for the reason of contrast performance, the use of a co-solvent such as ethanol is desired, and liposomes with a high encapsulation rate cannot be produced without using an organic solvent (non-patented). Reference 1).

Even if the contrast material is successfully encapsulated inside the liposome, the problem of leakage to the outside over time or the situation where the liposome itself becomes unstable must be considered. Therefore, in addition to a method of efficiently encapsulating a nonionic iodine-based compound with excellent imaging ability in a liposome, it is possible to maintain it stably over time, improve the blood retention, improve the formulation composition, There are various demands for improving the safety of contrast media.
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)

  Since liposomes have a wide variety of forms, the present inventors have solved the problem of establishing the optimal liposome structure and form for an X-ray contrast agent and producing stable and safe liposomes. Based on the recognition that this is important, the present invention has been completed through intensive research. The present invention addresses the above-described requirements, and provides an X-ray contrast agent in which a nonionic iodine-based compound that is a water-soluble non-electrolyte is efficiently encapsulated in a liposome produced without using an organic solvent. suggest. This preparation is a highly safe X-ray contrast agent having good visualization of cancer tissue, easy excretion, and low side effects, and can be used for both systemic administration and local administration.

The X-ray contrast agent of the present invention comprises:
A contrast agent comprising a liposome containing a water-soluble nonionic iodine-based compound as a contrast medium and one or more physiologically acceptable formulation aids in an aqueous phase inside or outside a lipid membrane;
The lipid membrane of the liposome contains at least a phospholipid, a lipid having a polyethylene glycol (PEG) group and a sterol as a constituent component, and a molar ratio of phospholipid (not including a lipid having a PEG group) / sterol is 100 / It is a liposome having a molar ratio of phospholipid (excluding lipids having PEG groups) / lipid having PEG groups of 100/5 to 100/25 and an average particle size of 0.05 to 0.8 μm. And at least 70% of the liposomes composed of 2 to 10 lipid membranes,
100-250 mg I / mL contrast agent with iodine compound as iodine atom, 60-150 mg / mL total lipid
It is characterized by containing at the concentration of contrast agent.

Desirably, the phospholipid includes a phospholipid having a transition temperature in the range of 22 to 60 ° C.
The liposome comprises supercritical or subcritical carbon dioxide, 1 part by weight, at least a phospholipid, a lipid having a polyethylene glycol (PEG) group and a lipid containing sterols as a lipid membrane component, 0.075 to 0.125 wt. A liposome substantially free of organic solvent, prepared by adding a part and mixing under strong stirring,
As a stirring condition in the strong stirring, the stirrer has the following formula:
C = N x V ^-0.15
C: Rotation speed (rpm) of stirring bar or stirring blade
V: Volume of mixed solution (L)
N: 300-3000
It is characterized by satisfying.

It is preferable that the weight ratio with respect to the quantity of the said lipid of the part included in the liposome among the said iodo-type compounds is 1-7 (g / g). In addition, 65 to 85% by mass of the iodine compound is
They may be in a form not encapsulated in liposomes and present in an aqueous medium in which the liposomes are dispersed.

In the aqueous phase inside and outside the lipid membrane, cations are contained as chlorides, phosphates or hydrogen carbonates, and the amount of sodium ions, calcium ions and potassium ions is represented by the following formula:
[Na ⁺ ] +120 ・ [Ca ²⁺ ] +50 ・ [K ⁺ ] ≦ 130
(However, the amount of sodium ions is 20 to 70 mM, the amount of calcium ions is 0.1 to 0.6 mM, and the amount of potassium ions is 0.4 to 0.9 mM.)
And the amount of magnesium ions is 0.05 to 0.8 mM.

A contrast agent having an average particle size of the liposome of 0.2 μm to 0.8 μm is preferably used for liver imaging.
The method for producing an X-ray contrast medium according to the present invention includes:
(I) Supplying and mixing liquefied carbon dioxide to the liposome membrane components in the pressure vessel, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state, and finally, in a supercritical or subcritical state under strong stirring A lipid comprising at least a phospholipid, a lipid having a polyethylene glycol (PEG) group and a sterol as a liposome membrane component with respect to 1 part by weight of carbon dioxide of
A step of mixing and dispersing at a ratio of ~ 0.125 parts by weight;
(Ii) Next, a step of introducing a solution or suspension containing an iodo compound and a formulation aid, and mixing with vigorous stirring to form micelles;
(Iii) thereafter, depressurizing the inside of the pressure vessel to discharge carbon dioxide, and preparing an aqueous dispersion of liposomes containing the iodine compound and formulation aid therein;
(Iv) passing the aqueous dispersion of liposomes through a hydrostatic extrusion apparatus equipped with a filtration membrane having a pore size of 0.1 to 1 μm, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm; v) further concentrating the obtained filtrate by ultrafiltration;
Including at least
By these steps, the molar ratio of phospholipid (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, phospholipids (excluding lipids having PEG groups) / lipids having polyethylene glycol groups Is a molar ratio of 100/5 to 100/25,
Liposomes composed of 2 to 10 lipid membranes occupy at least 70%, and 100 to 250 mg I / mL contrast agent with iodine compound as iodine atom, and 60 to 150 mg / mL contrast agent with total lipid It is characterized by containing at a concentration of.

As another method for producing an X-ray contrast agent,
(I) As a liposome membrane component, at least phospholipid, polyethylene glycol (PE
G) Liquefied carbon dioxide is supplied into a pressure vessel containing a suspension obtained by mixing a lipid containing a group-containing lipid and a sterol, and an aqueous solution containing an iodine-based compound and a formulation aid, and vigorously stirred. Below, the step of finally mixing and dispersing lipid in a ratio of 0.075 to 0.125 parts by weight with respect to 1 part by weight of carbon dioxide,
(Ii) Then, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state, and mixing under strong stirring to form micelles;
(Iii) thereafter, depressurizing the inside of the pressure vessel to discharge carbon dioxide, and preparing an aqueous dispersion of liposomes containing the iodine compound and formulation aid therein;
(Iv) passing the aqueous dispersion of liposomes through a hydrostatic extrusion apparatus equipped with a filtration membrane having a pore size of 0.1 to 1 μm, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm; v) further concentrating the obtained filtrate by ultrafiltration;
Including at least
By these steps, the molar ratio of phospholipid (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, phospholipids (excluding lipids having PEG groups) / lipids having polyethylene glycol groups Is a molar ratio of 100/5 to 100/25,
Liposomes composed of 2 to 10 lipid membranes occupy at least 70%, and 100 to 250 mg I / mL contrast agent with iodine compound as iodine atom, and 60 to 150 mg / mL contrast agent with total lipid It may be a method of containing at a concentration of

As a stirring condition in the strong stirring, the stirrer has the following formula:
C = N x V ^-0.15
C: Rotation speed (rpm) of stirring bar or stirring blade
V: Volume of mixed solution (L)
N: 300-3000
Is to satisfy.

The phospholipid includes a phospholipid having a transition temperature in the range of 22 to 60 ° C., and is desirably mixed with supercritical carbon dioxide under a temperature condition of 32 to 55 ° C.
Further, as the iodo compound, iohexol, iopamidol, iomeprol, iopromide, ioxirane, iotasulfur, iotrolane or iodixanol is preferable.
Detailed Description of the Invention
In the present specification, a “liposome” is usually a structure formed from a lipid membrane, that is, a lipid bilayer membrane. In the present specification, the liposome membrane may also be referred to as “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. “Cancer” refers to a malignant tumor, and is sometimes simply referred to as “tumor”.
Contrast Material The contrast material used in the X-ray contrast medium of the present invention is a water-soluble nonionic iodine-based compound. The reason for making this a more desirable contrast material than ionic iodine compounds is that ionic functional groups dissociate into ions in solution and show high osmotic pressure, which is the main cause of side effects of contrast agents containing ionic iodine compounds. By focusing on being.

Examples of water-soluble nonionic iodide compounds include phenyl iodide, for example 2,4,6
-Nonionic iodo compounds having at least one triiodophenyl group are preferred.

In view of the above-mentioned criteria, production cost, and physiological and use restrictions, iodoprols, iopamidol, iohexol, iopentol, ioxirane, ioxirane, iocimid, iobenzol are particularly preferred as the contrast material of the X-ray contrast agent of the present invention. , Iotrolane, iodixanol, iodesimol, iotasulfur, metrizamide, 1,3-bis- (N-3,5-bis- (2,3-dihydroxypropylaminocarbonyl) -2,4,6-
And triiodophenyl) -N-hydroxyacetyl-amino) -propane.
These compounds may be used alone or in combination of two or more. Moreover, it is not limited to the illustration. Among the iodo compounds, iomeprol, iopamidol, iohexol, iopromide, ioxirane, iotasulfur, iotrolane or iodixanol is particularly preferable.

  The above-described contrast material has little or no binding to plasma proteins. However, the risk of side effects is not completely eliminated even with nonionic iodine compounds. Although there are certainly few compared to ion-type contrast agents, there is still concern over the occurrence of anaphylaxis-like systemic reactions induced by immune stimulation caused by contrast agents (Japanese Patent Laid-Open No. 5-208921). Therefore, encapsulating nonionic iodine compounds in liposomes reduces the possibility of stimulating the immune system and reduces the risk of iodine hypersensitivity. In particular, it is desirable to encapsulate the above-mentioned nonionic iodine-based compound in liposomes (described later) whose outer surface is stealthed by polyethylene glycol (PEG), because the safety of the contrast agent is enhanced from this aspect as well.

  The X-ray contrast medium of the present invention usually also contains an iodine-based compound that is not encapsulated in liposomes. In such a contrast agent, the ratio (encapsulation rate) of the contrast material encapsulated in the liposome must also be considered. The concentration of the iodine-based compound inside and outside the liposome can be arbitrarily set based on various factors such as the nature of the contrast medium, the intended administration route of the preparation, and clinical indicators.

The X-ray contrast medium of the present invention is characterized in that 65 to 85% 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 dispersed. A formulation in which substantially or most of the iodine compound is encapsulated in the liposome is also possible, but such a formulation has a difference in osmotic pressure, liposome shape and stability, efficiency of encapsulation, imaging ability of the formulation, etc. However, it is not particularly excellent in practical use as an actual preparation. In the drug delivery system (DDS) of drug substances, side effects are reduced by encapsulating them in liposomes, and in order to further increase the benefits obtained by encapsulation, preparations are prepared by separating and removing drug substances that are not encapsulated There is also. In fact, immediately after the liposome-suspended drug is manufactured and separated, there has been reported an example in which the encapsulation component leaks with time after a short period of time even though almost 100% encapsulation is achieved (Betageri, GV Drug Devel. Ind. Pharm. 19, 531-539 (1993)). This phenomenon is based on the destabilization of the liposome structure due to the osmotic pressure effect. 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 medium not encapsulated was discussed (Japanese Patent Publication No. 9-505821). This is rooted in the mode of use unique to the contrast agent, and the situation that should be separated from the standpoint of making the pharmaceutical compound not encapsulated in the liposome unnecessary for the intended purpose has already been described above.

  In particular, in order to efficiently encapsulate the iodine compound in the liposome and sufficiently prevent the time-dependent destabilization of the liposome carrying the iodine compound, the amount of the iodine compound encapsulated in the liposome is It is desirable that it is 10 to 40% by mass, preferably 15 to 35% by mass, and more preferably 15 to 30% by mass of the total iodine compound in the contrast agent. In the X-ray contrast medium, if the ratio of the iodine compound encapsulated in the liposome is 10 to 40% by mass (preferably 15 to 35% by mass) of the whole, the remaining 60 to 90% by mass (preferably 65 to The amount flowing out to the aqueous dispersion outside the liposome containing 85% by mass) is virtually negligible. 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 in the liposome-containing X-ray contrast medium of the present invention, the encapsulation rate of the iodine compound at the preparation preparation and the encapsulation rate at the time of use are kept substantially the same, and the quality of the contrast media It is also preferable from the viewpoint of management. As a result of the decrease in the encapsulation rate in the liposome during storage and storage, the contrast performance is affected depending on the preparation or storage period.

From the above, the weight of iodine atoms encapsulated in the aqueous phase (aqueous solution enclosed by the lipid membrane) inside the liposome membrane in the X-ray contrast medium of the present invention is 10 to 200 mg / mL, preferably 20 to 180 mgI / mL. More preferably, it is 70-110 mg / mL. This is a parameter defined by the inclusion rate and the total iodine content.
Liposomes The X-ray contrast medium of the present invention contains liposomes that contain a water-soluble non-electrolyte nonionic iodine compound as a contrast medium in the aqueous phase inside and outside the lipid membrane and are substantially free of organic solvents. It is characterized by that. That is, by using a water-soluble iodine compound or the like encapsulated in a liposome as a microcarrier, it is intended to be efficiently delivered to the target organ or tissue lesion. The liposome used in the present invention has a form and structure designed as an optimum liposome for an X-ray contrast medium. That is, the liposome used in the present invention is substantially composed of several lipid membranes, so that the stability over time and the stability in blood are improved, and the encapsulation rate of the contrast medium is increased. This enhances the contrast ability of the contrast agent.

In the X-ray contrast medium of the present invention, a targeting function can be imparted by appropriately designing the particle size of the liposome encapsulating the water-soluble iodine compound and its lipid membrane.
Particularly in the case of systemic administration, it is desirable to consider both passive targeting and active targeting. The former can control the 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 cancer tissue or the like are decomposed relatively quickly and excreted outside the body without accumulating in normal tissues. This can be achieved by appropriately controlling the stability of the liposome in relation to the extravasation time. By controlling such clearance, another effect of a DDS dosage form in which a nonionic iodine-based compound having no side effects in the free form is encapsulated in the liposome can be expected. That is, when a water-soluble nonionic iodine-based compound is encapsulated in liposomes, the iodine-based compound is non-specifically deposited in the liver, spleen, kidney, etc., and it is difficult to fall into a situation where it takes time to decompose and excrete. For this reason, it is possible to prevent harmful effects caused by staying in the body and delayed side effects.

  Lipid membrane components constituting the lipid membrane of the liposome include at least phospholipids, glycolipids, sterols, glycols, cationic lipids, lipids having a polyethylene glycol group (for example, lipids having a PEG group), and the like. In general, phospholipids and / or glycolipids are preferably used as the lipid membrane component of the liposome contained in the X-ray contrast medium of the present invention. Preferred neutral phospholipids include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like.

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

  The phospholipids of the lipid membrane constituting the liposome of the present invention desirably 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.
More preferably, it is 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 (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, preferably 100/70 to 100/85. This molar ratio is based on the amount of phospholipid excluding lipids having PEG groups. 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 X-ray contrast agent of the present invention, which is a liposome preparation, a phospholipid or compound having a polyalkylene oxide (polyoxyalkylene chain) (PAO) group or a similar group, which is a polymer chain, is added to the liposome. It may be used as a component of the membrane. 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 3500, preferably 100 to 2000 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, a lipid having a PEG group may be used as a lipid having a polyethylene glycol group.
This is because, as will be described later, when a phospholipid or the like is mixed with supercritical carbon dioxide, it also acts as a dissolution aid.

Instead of the polyethylene glycol, various known polyalkylene oxide groups,-(AO) _n -Y, may be introduced on the surface of the liposome membrane. Here, AO represents an oxyalkylene group having 2 to 4 carbon atoms, n is an average addition mole number of the oxyalkylene group, and is a positive integer of 1 to 2000, preferably 10 to 500, more preferably 20 to 200. is there. Y represents a hydrogen atom, an alkyl group (for example, an aliphatic hydrocarbon group having 1 to 5 carbon atoms which may be branched) or a functional functional group.

  Examples of the oxyalkylene group having 2 to 4 carbon atoms (represented by AO) include oxyethylene group, oxypropylene group, oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylene group, oxy-1,2 -A dimethylethylene group etc. are mentioned.

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

The functional functional group represented by Y is for attaching a “functional substance” such as sugar, glycoprotein, antibody, lectin, and cell adhesion factor to the end of the polyalkylene oxide chain. Examples thereof include functional groups rich in reactivity such as an oxycarbonylimidazole group and an N-hydroxysuccinimide group.

  Liposomes with a polyalkylene oxide chain immobilized with a “functional substance” at the tip are identified as functions of the “functional substance”, eg, “recognition element”, in addition to the effect of introducing the polyalkylene oxide chain. Effects such as organ directivity and tumor tissue directivity are sufficiently exerted. In order to impart tumor tissue directivity, anti-tumor monoclonal antibodies corresponding to tumor-specific antigens present only in tumor cells are bound to liposome membranes as functional substances, resulting in liposomes with higher target selectivity (for example, JP-A-11-28087).

A known technique can be used for introducing the polyalkylene oxide chain into the liposome membrane. A phospholipid or compound having a polyalkylene oxide group can be used alone or in combination of two or more. The content thereof is 0.001 to 50 mol%, preferably 0.01 to 25%, based on the total amount of the liposome membrane constituents.
It is mol%, More preferably, it is 0.1-10 mol%.

  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 X-ray contrast medium of the present invention, the adjustment of the size and distribution of liposomes as fine particles is closely related to the high encapsulation rate of iodine compounds, targeting properties, and delivery efficiency. The particle size (particle size) is measured by freezing a dispersion containing liposomes encapsulating a water-soluble iodine compound, then depositing carbon on the crushed interface, and observing this carbon with an electron microscope (freeze-crushing TEM method). can do. Here, the “average particle diameter” refers to a simple average of a certain number of observed contrast agent 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 X-ray contrast medium of the present invention, liposomes are prepared by appropriately adjusting the average particle size in the range of 0.05 to 0.8 μm according to the application. 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. By aligning the average particle size of the liposome in the range of 0.11 to 0.13 μm, the liposome-containing contrast agent can be selectively concentrated on the cancer tissue. The pores of the neovascular wall in solid cancer tissue are abnormally large compared to the pore size of the capillary wall window (fenestra) of normal tissue, 0.03 to 0.08 μm, and even molecules with a size of approximately 0.1 μm to 0.2 μm Leaks from the vessel wall. Thus, the EPR effect is due to the higher permeability of the neovascular wall in cancer tissue than the microvascular wall of normal tissue.
Liposome Production Methods Various methods have been proposed for producing liposomes. When the production methods are different, the shape and characteristics of the final liposome are often significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is selected according to the desired form and characteristics of the liposome. In general, liposomes are prepared by first placing lipid membrane components such as phospholipids, cationic lipids, and sterols in containers together with organic solvents (eg chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, THF, etc.) almost without exception. Start with mixing and dissolving. In particular, chlorinated solvents are often used. Such preparations of liposomes always contain an organic solvent.

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

The liposome used in the contrast medium of the present invention is a method developed so that a liposome having an optimal form and structure for an X-ray contrast medium can be produced. 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 preferable characteristics for encapsulating water-soluble iodine-based compounds, It has been shown that the production rate of liposomes, the encapsulation rate of encapsulated drugs, and the retention rate of encapsulated drugs in liposomes are higher than those of conventional methods (see Patent Document 3 above). Furthermore, application on an industrial scale is also possible. “Substantially” means that the upper limit of the concentration of the remaining organic solvent in the contrast agent is 10 μg / L.

The first first and second steps may be any of the following two steps in which the order of addition of substances is different.
(I) Supplying and mixing liquefied carbon dioxide to the liposome membrane components in the pressure vessel, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state, and finally, in a supercritical or subcritical state under strong stirring A step of mixing and dispersing at a rate of 0.075 to 0.125 parts by weight of at least phospholipid, a lipid having a polyethylene glycol group and a lipid containing sterols as a liposome membrane constituent, with respect to 1 part by weight of carbon dioxide of
(Ii) Next, a step of introducing micelles by introducing a solution or suspension containing an iodine-based compound and a formulation aid and mixing under strong stirring,
Perform in this order. Alternatively, as another order, (i) a suspension obtained by mixing a lipid containing at least a phospholipid, a polyethylene glycol group-containing lipid and a sterol as a liposome membrane component, and an aqueous solution containing an iodine compound and a formulation aid. A step of supplying liquefied carbon dioxide into a pressure vessel accommodated therein, and finally mixing and dispersing the lipid in a ratio of 0.075 to 0.125 parts by weight with respect to 1 part by weight of carbon dioxide under strong stirring. ,
(Ii) Next, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state and mixing under strong stirring to form micelles,
The method may be performed in the following order.

Further, the following third to fifth steps are continued.
(Iii) a step of depressurizing the inside of the pressure vessel and discharging carbon dioxide to prepare an aqueous dispersion of liposomes containing the iodine compound and a formulation aid therein;
(Iv) passing the aqueous dispersion of liposomes through a hydrostatic extruder equipped with a filtration membrane having a pore size of 0.1 to 1 μm, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm, and (v ) A step of further concentrating the obtained filtrate by ultrafiltration.

By these steps, the molar ratio of phospholipid (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, phospholipids (excluding lipids having PEG groups) / lipids having polyethylene glycol groups The liposome has a molar ratio of 100/5 to 100/25, encapsulates the iodine compound and the formulation aid, and is composed of at least 70% of liposomes composed of 2 to 10 membranes.

Hereinafter, it demonstrates along each process.
-1st process (i) and 2nd process (ii)
The first and second steps are divided into two methods based on the order of addition of the liposome membrane constituent, liquefied carbon dioxide, and the aqueous drug solution containing the nonionic iodide compound and the formulation aid in the pressure vessel.

Liquefied carbon dioxide is added to the pressure vessel, and the liquefied carbon dioxide is brought into a supercritical state or a subcritical state under the pressure and temperature in the range described below. In the supercritical (or subcritical) carbon dioxide, phospholipids and substances having a lipid membrane stabilizing action are dissolved or dispersed as lipid components constituting the liposome membrane. As the phospholipid, various phospholipids including at least one phospholipid having a transition temperature in the range of 22 to 60 ° C. are preferable. As membrane lipid components other than phospholipids, sterols, which are substances having a lipid membrane stabilizing action, and lipids having polyethylene glycol groups are mixed and dissolved and dispersed together with the phospholipids. Alternatively, liquefied carbon dioxide may be added to a pressure vessel to which these compounds have been added in advance, and then the temperature and pressure are adjusted to obtain a supercritical state and mixed. Subsequently, micelles are formed by introducing an aqueous drug solution containing a nonionic iodine-based compound of a water-soluble drug to be encapsulated and a formulation aid into supercritical carbon dioxide containing phospholipids and lipid membrane stabilizing substances. Let

  Alternatively, in a pressure vessel containing a suspension obtained by mixing at least a phospholipid, a lipid having a polyethylene glycol group and a sterol as a liposome membrane component, and an aqueous drug solution containing an iodine compound and a formulation aid, The micelles may be formed by supplying liquefied carbon dioxide, 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.

These are desirably performed under the condition that at least one kind of lipid having a polyethylene glycol group is present. Under vigorous 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. This amount of lipid is 1.5 to 2.5 times greater than the amount of lipid conventionally used. This is to form several membranes of liposomes rather than a single membrane as will be described later. 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 may be present at the beginning of agitation, and by vigorous agitation as described below, a large number of CO ₂ / water interfaces where lipids are arranged can be generated, thereby increasing the encapsulation rate. Emulsification of lipid and supercritical carbon dioxide is promoted by the presence of a lipid having a PEG group as described below.

“Strong agitation” has a preferable range that varies depending on the volume of the mixed solution and the stirring means. For example, when the volume of the mixed solution is about 10 to 100 mL, a substantially cylindrical stirring bar having a length of 15 mm and a diameter of 5 mm Use a magnetic stirrer with a rotational speed of 400-4000rpm, preferably 1000-1500r
It means stirring at pm, particularly preferably 1200-1400 rpm. Even when the volume of the mixed solution and the stirring means are different from the above, it is preferable to appropriately determine the stirring conditions in consideration of the shearing force applied to the mixed solution under the stirring conditions. Specifically, it is particularly preferable that the stirring condition satisfies the following formula.

Formula: C = N × V− ^0.15
C: Rotation speed (rpm) of stirring bar or stirring blade
V: Volume of mixed solution (L)
N: 300-3000
Moreover, the time of strong stirring is 1 to 120 minutes, preferably 5 to 60 minutes, and is appropriately set according to the amount of the mixed solution and the amount of lipid. The time for the 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 these conditions, liposomes with higher liposome production rate and higher water-soluble drug encapsulation rate can be obtained. The containing formulation can be obtained. Although it does not specifically limit as a stirring means, You may use normal stirrers, such as a magnetic stirrer, a homogenizer, a homomixer, and an ultra mixer.

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).

The compound having a hydroxyl group (that is, a hydroxyl group-containing compound) includes, for example, a compound having a hydroxyl group, a polyol group, a polyalkylene glycol ether group, or a combination of a polyol / polyglycol ether group as a hydrophilic 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. Furthermore, in order to disperse and dissolve the lipid membrane component well in polar liquefied carbon dioxide, an amphiphilic compound having appropriate hydrophilicity and hydrophobicity is preferable.

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, for example, a lipid having a PEG group. Its oxyethylene unit is 10-3500, preferably 100-2000
Polyethylene glycol 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 (not including lipid having a PEG group) / 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 a water-soluble drug is formed, and when this is stirred, supercritical carbon dioxide Emulsification proceeds and lipid micelles are formed. Finally, liposomes having a single lipid membrane were mainly prepared.

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

  Eventually, even if ethanol is present or not, supercritical carbon dioxide is emulsified to form lipid micelles. However, in the method of the present invention in which strong stirring is performed, uniform micro micelles are efficient. In the process of formation of a lipid bilayer from a lipid monolayer, which is triggered by subsequent carbon dioxide emissions, the inclusion rate of water-soluble drugs increases, for example, from 17% to 21% . 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, the amount of lipid is increased, so that multiple membranes from two to ten membranes are formed. It is mainly composed of liposomes. Thus, it is noticed that by changing the lipid amount, stirring conditions and dissolution aid, the improvement in the encapsulation rate and the form of the generated liposomes are different. Furthermore, surprisingly, in such multilamellar liposomes, an increase in the encapsulation rate was observed again during the sizing process, suggesting that membrane rearrangement 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 40 to 65 ° C, more preferably 45 to 60 ° C. ° C. A suitable pressure of carbon dioxide in the supercritical state is appropriately selected from the above temperature range, but is 5 to 50 MPa, preferably 10 to 30 MPa. In the present invention, the supercritical state including the subcritical state is set.

  In the present invention, since the liposome is formed from phospholipids including phospholipids having a transition temperature with carbon dioxide being in a supercritical state at a temperature close to body temperature in this way, the liposome formation temperature and the dosage form are administered. After that, the temperature (body temperature) that the liposome receives in vivo is substantially equal. Therefore, even after the liposome preparation of the present invention is administered in vivo, even if the liposome is heated at body temperature in the living body, the temperature is substantially equal to the temperature when the liposome is formed. It is considered that the stability of liposomes in vivo is improved with little influence from temperature.

Such an effect is that the temperature of the carbon dioxide in the supercritical state becomes a transition temperature to “the transition temperature + 10 ° C.”, preferably the transition temperature to “the transition temperature + 5 ° C.” with respect to the phospholipid having the transition temperature. By making such a setting, it becomes more prominent. 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 liposome 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.
-Third step (iii)
In the third step, after the liposome membrane component, supercritical carbon dioxide, and aqueous drug solution are sufficiently mixed, water is added to the system if necessary to decompress the pressure vessel. By discharging carbon dioxide from this mixed solution, an aqueous dispersion of liposomes encapsulating iodine compounds and the like is prepared. In this process, it is presumed that the liposomes are phase-inverted to the aqueous phase, and therefore, only by discharging carbon dioxide, an aqueous dispersion in which liposomes encapsulating iodine compounds and the like are dispersed is generated. Since the aqueous solution is also enclosed inside the liposome, the contrast medium exists in the aqueous phase inside the liposome in addition to the external aqueous phase of the liposome, and is in a so-called “encapsulation” state. In the case of producing a contrast agent, a water-soluble drug may be contained in an aqueous phase other than the inside of the liposome.
-Fourth step (iv)
The particle size of the liposome can be adjusted by changing the formulation or process conditions. For example, when the pressure in the supercritical state is increased, the size of the liposome formed is reduced. 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. Therefore, in the fourth step, the pressure vessel is adjusted to atmospheric pressure by introducing air or the like, and the aqueous dispersion of liposomes obtained in the third step is 0.1 to 1.0 μm, and finally 0.1 to 0.45 μm in pore size. Through a filtration membrane having For example, it is carried out by passing through a hydrostatic extrusion apparatus equipped with a filter having a pore diameter of 0.6 to 1 μm as a filtration membrane. Specifically, various types of hydrostatic extrusion equipment such as “Extruder” (trade name, manufactured by NOF Liposome), “Riponizer” (trade name, manufactured by Nomura Micro Science), etc. are used to forcibly pass through the filter. Let By passing through an extruder, liposomes having an average particle size of 0.8 μm can be efficiently prepared. To make the average particle size even smaller, filter with a filter such as 0.45 μm. Such an operation is repeatedly performed if necessary. For example, Biochim. Biophys. Acta 557, 9
Page (1979). By incorporating such an “extrusion” operation, in addition to the above sizing, it is possible to adjust the concentration of the water-soluble iodine compound existing outside the liposome, exchange the liposome dispersion, and remove undesirable substances. There is also an advantage. In addition, before filtering with the filter membrane of the said hole diameter, it may filter with a filter membrane of about 1.0-2.0 micrometers, and may preliminarily perform sizing and removal of an undesirable substance.
・ Fifth step (v)
This step is a step of further concentrating the obtained liposome-containing filtrate by ultrafiltration. The aqueous dispersion of liposomes is filtered through a filtration membrane as described above, and may be purified by removing unretained drugs in the liposomes by methods such as ultrafiltration, centrifugation, and gel filtration as necessary. . Further, it may be concentrated by an ultrafiltration method so as to have a predetermined concentration, and further a formulation adjuvant such as a commonly used diluent may be appropriately mixed. The liposome-containing X-ray contrast medium of the present invention can also be obtained by freeze-drying liposomes from an aqueous liposome dispersion according to a conventional method. When liposomes are lyophilized, they are resuspended in an aqueous medium immediately before use.

  The liposome contained in the X-ray contrast medium of the present invention has substantially 2 to 10 membranes, preferably 2 to several membranes (eg, 3, 4, 5 or 6 membranes). A liposome consisting of 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 X-ray contrast medium of the present invention, the liposome composed of 2 to 10 membranes is at least 70%, preferably 80% of the total liposomes contained in the contrast medium. More preferably 80 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 in the sizing process.

  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 with few lipid membranes, especially LUV (Large unilamellar veislcles), which is a single membrane liposome with a large particle size, has the advantage of providing a larger encapsulation capacity than multilamellar liposomes. is there. On the other hand, even in the case of a single membrane or several membranes of liposomes having a good encapsulation rate of the water-soluble iodine-based compound, the stability of the liposome is lowered if the weight of the encapsulated compound is relatively excessive. In particular, a tendency to be vulnerable to rapid changes in ionic strength was observed. In the production of liposomes used in the contrast agent of the present invention, liposomes having a bilayer to 10 membrane structure, preferably 2 to several membranes (eg, 3, 4, 5 or 6 membranes) are used. The supercritical carbon dioxide method and the subsequent sizing process are improved so that liposomes can be formed efficiently. Furthermore, 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, the prepared liposome was found to be stable against salt shock. Production of X-ray contrast agent The X-ray contrast agent of the present invention can be produced by a technique known in the art using the liposome and one or more physiologically acceptable formulation aids. The preparation of the present invention contains a formulation aid in an aqueous phase in which the liposome is dispersed in an aqueous phase inside the lipid membrane of the liposome. This formulation aid is a substance that is added together with a water-soluble iodine-based compound and the like when formulating liposomes, and various substances are used as necessary based on conventional contrast agent production techniques. Specifically, various physiologically acceptable buffers, EDTANa ₂ -Ca
, EDTANa ₂ and other edetic acid chelating agents, inorganic salts, pharmacologically active substances (eg, vasodilators, coagulation inhibitors, etc.), osmotic pressure regulators, stabilizers, antioxidants (eg α- Tocopherols, ascorbic acid), viscosity modifiers, preservatives and the like are also 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). Preferably, both an amine buffer and a chelating agent are included.

  The above-mentioned “aqueous medium” is a water-based solvent that dissolves or suspends a water-soluble nonionic iodine-based compound, a formulation aid, and the like. As the water, use sterilized pyrogen-free water. As a preferred embodiment when the above formulation aid is also contained in an aqueous solution other than the aqueous phase inside the liposomal lipid membrane (that is, an aqueous medium in which the liposome is dispersed), the formulation aid is respectively present in the aqueous phase inside and outside the lipid membrane. Contained at substantially the same concentration. 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. It is possible to prevent destabilization due to the osmotic pressure effect of liposomes encapsulating, for example, during storage, and the retention stability of the water-soluble iodine-based compound in the liposomes is improved.

  Contrast agent cations (ie, sodium, potassium, magnesium and calcium) concentrations with non-ionic iodine compounds as contrast agents are not within or apart from normal plasma ion concentrations, for example, undesirable side effects such as It has been shown to cause effects on muscle contractility, erythrocyte aggregation, or decreased cardiac contractility. Furthermore, in the case of liposomal contrast agents, the present inventors have clarified that the type and composition of ions, particularly cations, also affect the encapsulation rate of liposomes and the stability and aggregation properties of liposomes. .

  The X-ray contrast medium of the present invention has cations, preferably four plasma cations, sodium, calcium, potassium, and magnesium, with an ion concentration that is not problematic from the viewpoint of safety, and has a liposome encapsulation rate and stability. Containing in an improved composition.

Usually the ratio of sodium to calcium should be above that in plasma. Calcium addition must also be in relatively small amounts to avoid undesired increases in cardiac contractility and to avoid the possibility of arrhythmias. Therefore, the ratio between the sodium ion concentration and the calcium ion concentration ([Na ⁺ ] / [Ca ²⁺ ]) is greater than 55, preferably greater than 60, more preferably 80 or more, and particularly 100 to 250.

The X-ray contrast agent of the present invention may further contain potassium ions and magnesium ions. This is because inclusion of a relatively small amount of potassium and / or magnesium can further reduce the occurrence of arrhythmia (atrial fibrillation). The concentration ratio of sodium ions to potassium ions ([Na ⁺ ] / [K ⁺ ]) is preferably greater than 15, more preferably greater than 20, especially greater than 30, for example 25-80. When calcium and magnesium salts are included, the calcium ion to magnesium ion concentration ratio ([Ca ²⁺ ] / [Mg ²⁺ ]) is at least about 1.4, preferably at least about 1.5, more preferably at least normal plasma. Ratio (about 2.9) or somewhat higher, for example 3-8.

In the X-ray contrast medium of the present invention, the relationship established between the amounts of sodium ions, calcium ions and potassium ions is preferably set to the following conditions.
The amount of sodium ion, calcium ion and potassium ion is represented by the following formula:
[Na ⁺ ] +120 ・ [Ca ²⁺ ] +50 ・ [K ⁺ ] ≦ 130
However, it is desirable that the sodium ion amount is 20 to 70 mM, the calcium ion amount is 0.1 to 0.6 mM, and the potassium ion amount is 0.4 to 0.9 mM. Sodium ion,
In contrast agents in which the amounts of calcium ions and potassium ions satisfy the above formula, the encapsulation rate of liposomes is high, the aggregation property is small, and the generation of aggregates tends not to be observed.

  Sodium, calcium, potassium and magnesium ions can be incorporated into the contrast agents of the present invention in the form of salts with physiologically acceptable counterions. Particularly suitable counter ions include plasma anions such as chloride, phosphate and bicarbonate ions.

When a drug such as a water-soluble iodine compound is encapsulated in a microcarrier called a liposome as in the X-ray contrast agent of the present invention, in addition to the encapsulation efficiency and encapsulation stability of the water-soluble iodine compound, the weight of the membrane lipid of the liposome is also determined. 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 7, preferably all of the drug (including the nonionic iodine compound and formulation aid) in the aqueous solution encapsulated in the liposome, relative to the liposome membrane lipid. 3-7, more preferably 5-7 weight ratio (g / g
) Is desirable.

When the weight ratio of all the drugs encapsulated in the liposome is less than 1, the delivery efficiency of the contrast medium is deteriorated, and the imaging ability is inevitably lowered. The contrast agent of the present invention in which a contrast medium is encapsulated in several membrane liposomes at a high encapsulation rate is advantageous for improving the contrast performance because it ensures the stability of the liposome and is excellent in encapsulation volume and delivery efficiency. . On the other hand, when the encapsulated weight ratio of all drugs to liposome membrane lipid exceeds 7, the liposome is structurally unstable, and the diffusion and leakage of the drug outside the liposome membrane was injected during storage or in vivo. Inevitable later. Also, it is described that even if 100% encapsulation is achieved immediately after the liposome suspension drug is manufactured and separated, the encapsulated components will decrease as soon as possible based on destabilization due to the osmotic pressure effect. (Japanese National Publication No. 9-505821).

X-Ray Contrast Agent The concentration of the X-ray contrast agent of the present invention depends on the purpose of imaging, the site, the nature of the compound in the contrast agent, and the patient's condition, and can be adjusted as necessary. The optimum dose of the X-ray contrast medium is usually set individually considering the above conditions. 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. If the solution is too high in concentration, an adverse situation such as aggregation of liposomes and increase in viscosity may occur. 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 total lipid concentration in the X-ray contrast medium of the present invention is 20 to 200 mg / mL contrast medium, preferably 60 to 150 mg / mL contrast medium, more preferably 60 to 100 mg / mL contrast medium. It is said. The “total lipid” in this case is meant to include all types of lipids such as phospholipid, sterol and glycol constituting the liposome. Such total lipids may be generally regarded as the amount of liposomes contained in the contrast agent. Due to the variety of liposome forms, the total lipid content simply does not correspond to the number of liposomes.

  The X-ray contrast medium of the present invention is used for either systemic administration or local administration. Preferably, it is intravenously administered systemically as an injection or infusion. For local administration to sites where direct administration is not possible, administration can be accomplished by techniques known in the art using, for example, a catheter or other suitable drug delivery system.

When it is necessary to administer a relatively high concentration X-ray contrast medium in a large amount in a short time, the requirements for enabling such bolus injection are the flowability and low viscosity of the X-ray contrast medium. In order to reduce patient resistance by reducing injection resistance and avoid the risk of extravasation, the viscosity of the liposome dispersion of the present invention (when measured by the Ostwald method) is 30 mPa · s or less at 37 ° C. , Preferably 25
mPa · s or less, more preferably 20 mPa · s or less. Within such a range, there is practically no problem (Patent Document 1), but water for injection can be used at the time of administration as necessary to dilute to a predetermined viscosity.
The osmolarity of the X-ray contrast agent is typically 250 to 500 mosmol / L, preferably 290 to 350 mosmol / L. When the osmolality of the contrast medium to be administered is high, the burden on the heart and circulatory system is large. To obtain a solution or suspension that is isotonic with blood, the contrast agent is dissolved or suspended in the medium at a concentration that provides the isotonic solution. For example, if the contrast material alone is not able to provide an isotonic solution due to the low solubility of the contrast material, other non-toxic water-soluble materials such as sodium chloride will form an isotonic solution or suspension. Sugars such as salts, mannitol, glucose, sucrose, and sorbitol may be added to the medium.

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 contrast material is a water-soluble iodo compound having multiple hydroxyl groups, a preferred buffer is a buffer having a negative temperature coefficient as described in US Pat. No. 4,278,654. The amine-based buffer has a property that satisfies such a requirement, and tris (TRIS) is particularly preferable. This type of buffer has a low pH at the autoclave temperature, which increases the stability of the X-ray contrast agent in the autoclave, while returning to a physiologically acceptable pH at room temperature. The preparation of the present invention is preferably marketed in a sterilized state, and the sterilization is performed by sterilization filtration, autoclave sterilization, heat sterilization or the like. For example, in order to produce a sterile contrast medium for injection, it is extremely convenient that the liposome preparation can be sterilized by autoclave, and storage stability and the like can be ensured. However, filtration sterilization should be performed on liposomes to which autoclave sterilization cannot be applied.
[The invention's effect]
The X-ray contrast agent of the present invention imparts targeting properties by efficiently and stably supporting a nonionic iodine-based compound, which is a water-soluble non-electrolyte, as a contrast medium on liposomes, which are microcarriers. Increases performance.

  A lipid having a polyethylene glycol group and a lipid containing a phospholipid having a transition temperature are used, and a large amount of several membrane liposomes are formed by mixing an increased amount of lipid with supercritical carbon dioxide under strong stirring. The inclusion rate of the iodine-type compound enclosed in this is high, and is stably included.

  Since the X-ray contrast medium of the present invention is produced without using any organic solvent, the toxicity and side effects are much reduced compared to the conventional X-ray contrast medium, and the burden on the patient receiving the administration is Few.

The present invention will be more specifically described by the following examples. However, the examples are intended to be described by way of examples, and are not intended to limit the scope of the present invention in any way.
[Example 1]
Preparation of contrast agent A mixture of 552.6 mg of dipalmitoylphosphatidylcholine (DPPC), 221.1 mg of cholesterol, and 167.3 mg of phospholipid having a PEG group (SUNBRIGHT DSPE-020CN), prepared in a stainless steel special autoclave The interior was heated to 50 ° C., and 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 the carbon dioxide into a supercritical state. Containing a contrast medium solution (iohexol solution 517.7 mg / mL (iodine content 240 mg / mL), trometamol 1 mg / mL, calcium disodium edetate (EDTANa ₂ -Ca) 0.1 mg / mL with strong stirring at 1400 rpm The pH was adjusted to around 7 with appropriate amounts of hydrochloric acid and 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 80 ° C., and filtered under pressure with a polycarbonate filter manufactured by Advantech, 0.80 μm. Subsequently, the mixture was similarly heated to 80 ° C., pressure-filtered with a polycarbonate filter manufactured by Advantech, 0.40 μm, and treated with an autoclave at 121 ° C. and a pressure of 0.21 MPa for 15 minutes to obtain a liposome-containing contrast agent.
[Comparative Example 1]
Dipalmitoylphosphatidylcholine (DPPC) 552.6mg, cholesterol 221.1mg, lipid with PEG group (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) 167.3mg mixture of chloroform, ethanol and water (weight ratio 100: 20 : 0.1) 13 mL was mixed in a volumetric flask. The mixture was heated on a hot water bath (50 ° C.) and the solution was evaporated on a rotary evaporator. The residue was further vacuum dried for 2 hours to form a lipid film. Further, 13 mL of the contrast agent solution was mixed therewith, and the mixture was stirred for about 10 minutes with a mixer while being heated to 50 ° C. By further stirring, a liposome dispersion containing a contrast medium solution was obtained. The mixture was heated to 60 ° C. and pressure filtered through a polycarbonate filter manufactured by Advantech, 0.80 μm and 0.40 μm to obtain a liposome-containing contrast agent.

[Examples 2-6, Comparative Examples 2-4]
Examples 2 to 6 and Comparative Examples 3 and 4 were the same as Example 1 except that the contrast medium type and concentration to be encapsulated in the liposome and the lipid content in the contrast medium were changed as shown in Table 1. A liposome-containing contrast agent was obtained. The contrast agent of Comparative Example 2 was obtained in the same manner as Comparative Example 1.
[Evaluation]
<Encapsulation rate of iodine-based compounds>
The sample (liposome dispersion) was dialyzed against isotonic saline, ethanol was added after the dialysis, the liposome was broken, and the amount of iodine compound in the liposome was determined by measuring the absorbance.

As can be seen from Table 1, according to the present invention, a liposome-containing contrast agent having a high encapsulation rate and a viscosity with no practical problems could be prepared.

Claims (12)

A contrast agent comprising a liposome containing a water-soluble nonionic iodine-based compound as a contrast medium and one or more physiologically acceptable formulation aids in an aqueous phase inside or outside a lipid membrane;
The lipid membrane of the liposome contains at least a phospholipid, a lipid having a polyethylene glycol (PEG) group and a sterol as a constituent component, and a molar ratio of phospholipid (not including a lipid having a PEG group) / sterol is 100 / It is a liposome having a molar ratio of phospholipid (excluding lipids having PEG groups) / lipid having PEG groups of 100/5 to 100/25 and an average particle size of 0.05 to 0.8 μm. And at least 70% of the liposomes composed of 2 to 10 lipid membranes,
100-250 mg I / mL contrast agent with iodine compound as iodine atom, 60-150 mg / mL total lipid
An X-ray contrast medium comprising a contrast medium concentration.   The X-ray contrast medium according to claim 1, wherein the phospholipid includes a phospholipid having a transition temperature in a range of 22 to 60 ° C. The liposome contains at least phospholipid, a lipid having a polyethylene glycol (PEG) group and a sterol, 0.075 to 0.125 part by weight, as a lipid membrane component, with respect to 1 part by weight of carbon dioxide in a supercritical or subcritical state. Is a liposome substantially free of organic solvent produced by mixing under strong stirring,
As a stirring condition in the strong stirring, the stirrer has the following formula:
C = N x V ^-0.15
C: Rotation speed (rpm) of stirring bar or stirring blade
V: Volume of mixed solution (L)
N: 300-3000
The X-ray contrast medium according to claim 1, wherein:   2. The X-ray contrast medium according to claim 1, wherein a weight ratio of a portion encapsulated in the liposome to the amount of the lipid in the iodine compound is 1 to 7 (g / g).   X-ray imaging according to claim 1 or 4, wherein 65 to 85% by mass of the iodine compound is in a form not encapsulated in liposomes and is present in an aqueous medium in which the liposomes are dispersed. Agent. In the aqueous phase inside and outside the lipid membrane, cations are contained as chlorides, phosphates or hydrogen carbonates, and the amount of sodium ions, calcium ions and potassium ions is represented by the following formula:
[Na ⁺ ] +120 ・ [Ca ²⁺ ] +50 ・ [K ⁺ ] ≦ 130
(However, the amount of sodium ions is 20 to 70 mM, the amount of calcium ions is 0.1 to 0.6 mM, and the amount of potassium ions is 0.4 to 0.9 mM.)
And the amount of magnesium ions is 0.05 to 0.8 mM.
5. The X-ray contrast medium according to any one of 5 above.   The X-ray contrast agent according to any one of claims 1 to 6, wherein the liposome has an average particle size of 0.2 to 0.8 µm and is used for liver imaging. (I) Supplying and mixing liquefied carbon dioxide to the liposome membrane components in the pressure vessel, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state, and finally, in a supercritical or subcritical state under strong stirring A lipid comprising at least a phospholipid, a lipid having a polyethylene glycol (PEG) group and a sterol as a liposome membrane component with respect to 1 part by weight of carbon dioxide of
A step of mixing and dispersing at a ratio of ~ 0.125 parts by weight;
(Ii) Next, a step of introducing a solution or suspension containing an iodo compound and a formulation aid, and mixing with vigorous stirring to form micelles;
(Iii) thereafter, depressurizing the inside of the pressure vessel to discharge carbon dioxide, and preparing an aqueous dispersion of liposomes containing the iodine compound and formulation aid therein;
(Iv) passing the aqueous dispersion of liposomes through a hydrostatic extrusion apparatus equipped with a filtration membrane having a pore size of 0.1 to 1 μm, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm; v) further concentrating the obtained filtrate by ultrafiltration;
Including at least
By these steps, the molar ratio of phospholipid (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, phospholipids (excluding lipids having PEG groups) / lipids having polyethylene glycol groups Is a molar ratio of 100/5 to 100/25,
Liposomes composed of 2 to 10 lipid membranes occupy at least 70%, and 100 to 250 mg I / mL contrast agent with iodine compound as iodine atom, and 60 to 150 mg / mL contrast agent with total lipid A method for producing an X-ray contrast agent, comprising: (I) As a liposome membrane component, at least phospholipid, polyethylene glycol (PE
G) Liquefied carbon dioxide is supplied into a pressure vessel containing a suspension obtained by mixing a lipid containing a group-containing lipid and a sterol, and an aqueous solution containing an iodine-based compound and a formulation aid, and vigorously stirred. Below, the step of finally mixing and dispersing lipid in a ratio of 0.075 to 0.125 parts by weight with respect to 1 part by weight of carbon dioxide,
(Ii) Then, heating and pressurizing to bring the liquefied carbon dioxide into a supercritical state, and mixing under strong stirring to form micelles;
(Iii) thereafter, depressurizing the inside of the pressure vessel to discharge carbon dioxide, and preparing an aqueous dispersion of liposomes containing the iodine compound and formulation aid therein;
(Iv) passing the aqueous dispersion of liposomes through a hydrostatic extrusion apparatus equipped with a filtration membrane having a pore size of 0.1 to 1 μm, and sizing into liposomes having an average particle size of 0.05 to 0.8 μm; v) further concentrating the obtained filtrate by ultrafiltration;
Including at least
By these steps, the molar ratio of phospholipid (excluding lipids having PEG groups) / sterols is 100/60 to 100/90, phospholipids (excluding lipids having PEG groups) / lipids having polyethylene glycol groups Is a molar ratio of 100/5 to 100/25,
Liposomes composed of 2 to 10 lipid membranes occupy at least 70%, and 100 to 250 mg I / mL contrast agent with iodine compound as iodine atom, and 60 to 150 mg / mL contrast agent with total lipid A method for producing an X-ray contrast agent, comprising: As a stirring condition in the strong stirring, the stirrer has the following formula:
C = N x V ^-0.15
C: Rotation speed (rpm) of stirring bar or stirring blade
V: Volume of mixed solution (L)
N: 300-3000
The method for producing an X-ray contrast agent according to claim 8 or 9, wherein:   The phospholipid includes a phospholipid having a transition temperature in a range of 22 to 60 ° C, and is mixed with supercritical carbon dioxide under a temperature condition of 32 to 55 ° C. A method for producing an X-ray contrast medium according to claim 1. 12. The method for producing an X-ray contrast agent according to claim 8, wherein the iodo compound is iohexol, iopamidol, iomeprol, iopromide, ioxirane, iotasulfur, iotrolan or iodixanol.