JP2005220034A - Method for producing contrast medium for roentgenologic examination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing a highly safe contrast medium for roentgenologic examination by which stabilization of a liposome and retention stability of an iodine compound are carried out to raise retentivity in blood of a contrastradiographic compound, and efficient delivery and good targeting of the contrast medium are achieved. <P>SOLUTION: This method for producing the roentgenologic contrast medium is carried out as follows. A supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizer is mixed with an aqueous solution containing a nonionic iodine compound and a pharmaceutical aid to form a liposome suspension, which is then permeated through a filtration membrane having 0.1-0.4 μm pore diameter. The contrast medium for the roentgenologic examination produced by the method contains preferably sterols and/or cationic phospholipids and/or glycols in the lipid membrane of the liposome prepared by a supercritical carbon dioxide method and a water-soluble iodine compound is included in the aqueous phase. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing a contrast medium for X-ray examination, and more particularly to a method for producing a contrast medium for X-ray examination including a liposome encapsulating a contrast substance therein.

  Simple imaging using CT and CT imaging (computer tomography) form the core of today's diagnostic imaging. Since so-called hard tissues such as bones and teeth absorb X-rays well, a high-contrast image can be easily obtained. On the other hand, since a difference in X-ray absorption is small between soft tissues, it is difficult to obtain a high contrast image. In such a case, a contrast agent is generally used to obtain an image with high contrast.

  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. These contrast agents are administered to luminal sites such as blood vessels, ureters, oviducts, and the like, and are used for diagnosis of luminal shape, stenosis, and the like. However, since these compounds are rapidly excreted from the luminal site without interacting with the tissue or diseased site, they are not useful for the purpose of diagnosing the tissue or diseased site, particularly cancer tissue, in more detail. Therefore, there is a demand for an X-ray contrast agent that selectively accumulates in a target tissue or diseased site and provides an image that can be distinguished from the surrounding or other sites with a clear contrast. With such a contrast medium for X-ray examination, even a fine cancer tissue can be detected with high accuracy.

  International publications WO98 / 46275, WO95 / 31181, WO94 / 19025, WO96 / 28414, WO96 / 00089, US Pat. A method for imaging a tumor, a liver, a spleen image, an adrenal cortex, an arteriosclerotic lesion, a blood vessel pool, a lymphatic system, etc. by dispersing in water under water is disclosed. In these methods, a contrast agent is atomized to increase the residence time in the body and selectively contrast the diseased part. However, the formulation methods proposed for that purpose are not sufficient in contrast efficiency and selectivity. Furthermore, there is a problem in that the iodine compound to be used is discharged at a low rate after imaging and the burden on the patient is large.

  On the other hand, as a method for making a contrast agent into a fine particle form, a method of encapsulating a contrast-enhancing compound in a liposome composed of a lipid similar to a biological membrane and considered to be highly safe due to its low antigenicity has been studied. For example, International Publications WO 88/09165, WO 89/00988, WO 90/07491, JP 07-316079 and JP 2003-5596 propose liposomes containing ionic or nonionic contrast agents. In these methods, despite the use of liposomes that are highly safe as materials and have appropriate degradability in vivo, organic solvents such as chloroform, Use a chlorinated solvent such as dichloromethane. Therefore, it has not been put into practical use because the remaining solvent is toxic (see, for example, Patent Document 1).

On the other hand, lipid-soluble drugs are easily encapsulated in liposomes, but the encapsulated amount depends on other factors and is not necessarily so much. A drug that is a water-soluble electrolyte can be encapsulated in the aqueous phase inside the liposome through the interaction between the charge of the drug and the charge of the charged lipid, but if the drug is a water-soluble non-electrolyte, such means should be used. It cannot be taken. Regarding X-ray contrast agents, it is generally desirable to encapsulate nonionic iodine compounds that are substantially less toxic than ionic contrast compounds in liposomes, but this is not easy for the reasons described above. Furthermore, the formed liposome is likely to be multi-layered, and the efficiency is poor because the encapsulation rate of the iodine compound is low. As means for efficiently encapsulating such a water-soluble non-electrolyte in the liposome, there are a reverse phase evaporation method and an ether injection method. However, since an organic solvent is used, there still remains a safety problem.

In JP-A-2003-119120 (Patent Document 2), a cosmetic containing a liposome and an external preparation for skin are disclosed.
A method of producing using supercritical carbon dioxide is disclosed, and an example of producing an external preparation for skin in which a hydrophilic medicinal component or a lipophilic medicinal component is encapsulated in liposomes is shown. However, although examples of water-soluble electrolytes have been shown as hydrophilic medicinal ingredients, it was unclear whether water-soluble non-electrolytes could be efficiently encapsulated in liposomes by this method. Therefore, there is a need for a method for producing a contrast agent that can efficiently deliver a contrast substance well retained in liposomes to a target site without using an organic solvent.
Japanese Patent Laid-Open No. 7-316079 Japanese Patent Laid-Open No. 2003-119120

  An object of the present invention is to provide a method for producing a contrast medium for X-ray examination that is excellent in contrast efficiency and selectivity and uses a liposome as a microcarrier of a contrast material and that is highly safe. Specifically, an object of the present invention is to provide a method for producing an X-ray contrast medium in which a water-soluble iodine compound is efficiently encapsulated in a liposome having a narrow particle size range without using a toxic organic solvent.

  The present invention for solving the above problems has the following configuration.

The method for producing a contrast medium for X-ray examination according to the present invention comprises mixing supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance with an aqueous solution containing a nonionic iodine compound and a formulation aid. The formed liposome suspension is further permeated through a filtration membrane having a pore size of 0.1 to 0.4 μm.

The supercritical carbon dioxide is preferably generated under a pressure of 90 to 150 kg / cm ² .
The phospholipid preferably contains at least a phospholipid having a transition temperature.

  Furthermore, a cationic substance may be included in the lipid membrane of the liposome at a ratio of 0.1 to 5% by mass with respect to the amount of phospholipid.

The lipid membrane stabilizing substance is preferably sterols and / or glycols. The sterols are desirably contained in a proportion of 0.05 to 1.5 parts by weight with respect to 1 part by weight of phospholipid. Moreover, it is desirable that the glycols be contained at a ratio of 0.5 to 20% by mass with respect to the amount of phospholipid.

  The permeation of the filtration membrane is preferably performed using an extruder. The treatment with an extruder is desirably performed at a temperature equal to or higher than the transition temperature of a phospholipid having a transition temperature.

  The above-mentioned method for producing a contrast medium for X-ray examination is obtained by dividing a supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance and an aqueous solution containing a nonionic iodine compound and a formulation aid into two or more times. It is desirable that the liposomes formed by mixing or adding small amounts continuously and mixing contain 5 to 35% by mass of the iodine compound of the total iodine compound.

Since the method for producing a contrast medium for X-ray examination according to the present invention employs a method of preparing liposomes by mixing phospholipids or the like with supercritical carbon dioxide, a toxic solvent, particularly a highly toxic chlorinated solvent is used. There is no need. Furthermore, the encapsulation efficiency of the iodine compound encapsulated in the liposome is increased. Thus, the X-ray contrast medium according to the method of the present invention improves the stability of the liposome structure and the retention stability of the encapsulated substance.
[Detailed Description of the Invention]
The method for producing a contrast medium for X-ray examination according to the present invention comprises mixing supercritical carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance with an aqueous solution containing a nonionic iodine compound and a formulation aid. The formed liposome suspension is further permeated through a filtration membrane having a pore size of 0.1 to 0.4 μm. The lipid membrane of the liposome preferably contains sterols and / or cationic phospholipids and / or glycols. Hereinafter, the present invention will be described in detail in the order of the contrast compound used for the production of the contrast agent, the material and design of the liposome, and the method for producing the contrast agent for X-ray examination.
Contrast compounds used in the present invention include water-soluble nonionic iodide compounds including phenyl iodide, such as 2,4,6-
Nonionic iodo compounds having at least one triiodophenyl group are preferred.

Specifically, such nonionic iodine compounds include iohexol, iopentol, iodixanol, iopromide, iotrolane, iomeprol, N, N′-bis [2-hydroxy-1- (hydroxymethyl) -ethyl] -5 [[(2-hydroxy-1-oxopropyl) -amino] -2,4,6-triiodo-1,3-benzene-dicarboxamide (iopamidol); metrizamide and the like.

  These compounds may be used alone or in combination of two or more. Moreover, it is not limited to the illustration. In addition, in this specification, a compound may be mentioned including the salt, hydrate, etc. other than a free form.

  Examples of the iodine compound suitable for the contrast agent for X-ray examination of the present invention include iomeprol, iopamidol, iotrolan and iodixanol which are highly hydrophilic and 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 contrast agent 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, 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.
Liposome Material and Design A method for producing a contrast medium for X-ray examination according to the present invention comprises a method for selectively delivering the contrast compound to a target site such as a target organ or tissue. Including the step of encapsulating. The contrast agent thus produced is used in a form in which a nonionic iodide compound is encapsulated in the liposome. In order to produce an enhanced EPR (enhanced permeability and retention) effect that is effective for obtaining excellent tumor visualization, it is necessary to stabilize the liposome structure and improve the encapsulation rate of the encapsulated substance. A contrast agent is required to have characteristics such as stability in blood and retention in blood while improving efficiency. In this contrast agent, the retention in blood is improved by using liposomes with improved stability over time and in blood, and efficient drug delivery and targeting are achieved.

  In the production method of the present invention, the particle size of the liposome encapsulating the contrast compound and its bilayer are designed so that the produced X-ray contrast agent can realize the targeting function. Both passive targeting and active targeting are considered. The former can control the in vivo behavior through adjustment of liposome particle size, lipid composition, charge, and the like. Adjustment to align the liposome particle size in a narrow range is easily performed based on the method described later. Furthermore, in the design of the liposome membrane surface, by changing the type and composition of phospholipids and coexisting substances as described later, it is possible to satisfy the requirements of improving the retention efficiency of the encapsulated substance and stabilizing the liposome.

  Employment of active targeting that allows for higher delivery and selectivity of contrast agents should also be considered. For example, introduction of a polyalkylene oxide polymer chain or polyethylene glycol (PEG) on the liposome surface is a very useful technique because the induction process to the target site can be controlled. X-ray contrast agents that have not reached cancerous tissues, diseased sites, etc. are not accumulated in normal sites, but are decomposed and excreted from the body before the side effects occur. This can be achieved by appropriately controlling the stability of the liposome in relation to the extravasation time. Since the contrast material uses a water-soluble iodine compound, it is rapidly excreted in the urine via the kidney. Therefore, adverse effects caused by staying in the body and delayed side effects can be prevented.

  The design and production method of liposomes used in the delivery system of the present X-ray contrast agent will be described in detail below.

  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, there are substances in which phospholipids and / or glycolipids having liposome-forming ability, which are lipids for liposomes, are essential main components and other components are allowed to 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.

  The phospholipid used in the present invention is a phospholipid 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 dipalmitoylphosphatidylglycerol (DPPG), dimyristoylphosphatidylglycerol, distearoylphosphatidylglycerol (DSPG), dipalmitoylphosphatidylserine (DPPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidylinositol ( DPPI), distearoyl phosphatidylinositol (DSPI), distearoyl phosphatidic acid (DSPA), dipalmitoyl phosphatidic acid (DPPA), dimyristoyl phosphatidic acid and the like.

  The liposome lipid in the production method of the present invention desirably contains at least a phospholipid having a transition temperature. As phospholipids having a phase transition point, dimyristoyl phosphatidylcholine (transition temperature, the same below, 23-24 ° C), dipalmitoylphosphatidylcholine (41.0-41.5 ° C), hydrogenated soybean lecithin (53 ° C), hydrogenated soybean phosphatidylcholine (54 ° C), distearoylphosphatidylcholine (54.1-58.0 ° C) and the like. 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).

  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.

  These phospholipids are usually used alone, but may be used in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1. Furthermore, it has been found from our research that the presence of cationic substances including cationic phospholipids, cationic lipids and the like as membrane constituents of liposomes also improves the retention efficiency of water-soluble iodine compounds in liposomes. Presumably, when the constituent phospholipid molecules assemble to form a liposome membrane, the cationic substance promotes the rearrangement of phospholipid molecules within the lipid membrane based on the attraction and repulsion between charges, resulting in In addition, the membrane structure is strengthened, and the entire liposome structure is also stabilized. This is considered to result in an increase in the efficiency of uptake of the contrast material and conversely a decrease in its loss from the liposomes.

  These cationic substances are contained in an amount of 0.1 to 5% by mass with respect to the amount of phospholipid, preferably 0.3 to 3% by mass with respect to the amount of phospholipid, and more preferably 0.5 to 1% by mass with respect to the amount of phospholipid. It may be added to.

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, galactosylceramide, galactosylceramide sulfate, lactosylceramide, sphingoglycolipids such as ganglioside G7, ganglioside G6, and ganglioside G4.

  In addition to the above lipids, other substances can be added as necessary as membrane constituents of the liposome. Examples thereof include sterols such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholesterol, lanosterol or 2,4-dihydrolanosterol which act as a lipid membrane stabilizing substance. 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 (Japanese Patent Laid-Open No. 5-245357). Of these, cholesterol is particularly preferred. Furthermore, when sterols are present as membrane constituents of the liposome, the retention efficiency of the water-soluble iodine compound in the liposome is also improved. It is thought that the binding of the molecules constituting the liposome membrane is strengthened by sterols and the entire liposome structure is stabilized, so that the efficiency of contrast medium uptake increases and at the same time the loss from the liposomes decreases. .

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

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

  In addition to the sterols, glycols may be added as a lipid membrane stabilizing substance for liposomes. 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. This is thought to be due to stabilization of the liposome structure, similar to the effect of improving retention efficiency by sterols. In other words, glycols are responsible for interaction with the aqueous medium surrounding liposomes based on their hydrophilicity, and promote the rearrangement of phospholipids and other molecules in the liposome lipid membrane, strengthening the intermolecular bonds in the membrane structure. It seems to be.

As glycols, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, trimethylene glycol, 1,4-butanediol, 1,5-pentanediol, neopentyl glycol, 1,6-hexanediol, 1 , 7-Heptanediol, 1,8-octanediol, 1,9-nonanediol, 1,10-decanediol, pinacol, hydrobenzoin, benzopinacol, cyclopentane-1,2-
Examples include diol, cyclohexane-1,2-diol, and cyclohexane-1,4-diol.

The amount of glycols used in the contrast agent of the present invention is desirably 0.5 to 20% by mass, preferably 2 to 10% by mass, based on the amount of phospholipid. If the amount is less than 0.5% by mass, stabilization by glycols that improve the dispersibility of the mixed lipid is not exhibited. If the amount is more than 20% by mass, the formation of liposomes is inhibited or unstable even if formed.

The above-mentioned compounds such as cationic phospholipids, sterols and glycols may be used alone or in combination of two or more. That is, when preparing liposomes, sterols and glycols may be added independently together with phospholipids, and sterols and glycols may be used together. Further, in any of the above cases, as the phospholipid, in addition to the neutral phospholipid, the cationic phospholipid may or may not be included. When these substances are used in combination, the amount of each added is the same as when used alone.

  By using these substances as the lipid membrane stabilizing substance, the liposome membrane structure is stably formed, the encapsulation of the water-soluble iodine compound in the liposome is improved, and the retention efficiency is improved. Other examples of compounds that can be added include dialkyl phosphates such as dicetyl phosphate, which are negatively charged substances, and examples of compounds that impart positive charges include aliphatic amines such as stearylamine and DOTAP.

In the X-ray contrast medium of the present invention, the following method is also considered in order to improve the retention efficiency of the contrast compound in the liposome. For such intended purposes, phospholipids or compounds having polyalkylene oxide (PAO) groups or similar groups may be used as a component of the liposome membrane. As a method for solving the problem of being trapped by reticuloendothelial cells and the instability of liposome itself such as disintegration and aggregation, polyethylene glycol (PEG) chain, which is a polymer chain on the surface of the liposome, -(CH ₂
Attempts have been made to introduce CH ₂ O) _n —H (for example, JP-A-1-249717, FEBS letters, 268, 235 (1990)).

A new function can be imparted to the liposome by attaching a polyalkylene oxide chain (polyoxyalkylene chain) or a PEG chain to the surface of the liposome. For example, PEGylated liposomes can be expected to have an effect of being hardly recognized by the immune system (in a so-called “stealthed” state). Alternatively, it has been clarified that liposomes have a tendency to be hydrophilic, thereby increasing blood stability and maintaining the concentration in blood over a long period of time (Biochim. Biophys. Acta., 1066, 29-36 (1991) ). In addition, a method of incorporating a polyalkylene oxide-modified phospholipid into a liposome lipid membrane in order to improve the blood retention of liposomes has been disclosed (Japanese Patent Laid-Open No. 2002-37883). Such liposomes have also been shown to have improved stability over time.

Using these properties, organ specificity can be imparted to the X-ray contrast medium. Specifically, since lipid components are easily stored in the liver, it is desirable not to use PEG or to use liposomes having a low PEG content when selective imaging of the liver is intended. Further, when the particle size is increased to 200 nm or more, the possibility of rapid uptake by the phagocytosis of liver Kupffer cells increases, and it accumulates at the site of the liver. In imaging of liver cancer, since the cancer tissue has fewer Kupffer cells than the normal tissue, the amount of contrast agent liposome taken up is relatively small, and the contrast becomes clear. The same applies to the spleen.

  On the other hand, in the case of contrast imaging of other organs, the introduction of PEG makes it possible to steal the liposome and make it difficult to collect in the liver and the like, and therefore the use of PEGylated liposome is recommended. Since the hydration layer is formed by the introduction of PEG, the liposome is stabilized and the retention in blood is also improved. By appropriately changing the length of the oxyethylene unit of PEG and the ratio of introduction, the function can be adjusted. As PEG, polyethylene glycol having 10 to 3500 oxyethylene units is preferred. The amount of PEG used is 0.1 to 30% by mass, preferably about 1 to 15% by mass, based on the lipid constituting the liposome.

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

Instead of the PEG, various known polyalkylene oxide groups,-(AO) _n -Y, may be introduced on the liposome surface. Here, AO represents an oxyalkylene group having 2 to 4 carbon atoms, n is an average added mole number of the oxyalkylene group, and is a positive number of 1 to 2000. Y represents a hydrogen atom, an alkyl group or a functional functional group.

  Examples of the oxyalkylene group having 2 to 4 carbon atoms (represented by AO) include, for example, oxyethylene group, oxypropylene group, oxytrimethylene group, oxytetramethylene group, oxy-1-ethylethylene group, oxy-1,2 -A dimethylethylene group etc. are mentioned. These oxyalkylene groups are groups obtained by addition polymerization of alkylene oxides such as ethylene oxide, propylene oxide, oxetane, 1-butene oxide, 2-butene oxide, and tetrahydrofuran.

  n is a positive number of 1 to 2000, preferably 10 to 500, and more preferably 20 to 200.

  When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, it may be added in a random manner or a block shape. In the case of imparting hydrophilicity to the polyalkylene oxide chain, AO 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 other than ethylene oxide is increased. For example, a liposome containing a block copolymer of polyethylene oxide and polypropylene oxide is preferred.

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

  In addition to the effect of introducing a polyalkylene oxide chain, a liposome with a polyalkylene oxide chain immobilized with a “functional substance” attached to the tip is a “functional substance” that is not obstructed by the polyalkylene oxide chain. Functions such as specific organ directivity, cancer tissue directivity, etc. are sufficiently exerted as a “recognition element”.

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

A known technique can be used to introduce the polyalkylene oxide chain into the liposome. An anchor (for example, cholesterol) to which polyalkylene oxide is bonded may be mixed with a phospholipid that is a membrane component to prepare a liposome, and the activated polyalkylene oxide may be bonded to the anchor. In such a method, it is necessary to carry out a multi-step chemical reaction on the surface of the liposome membrane after preparing the liposome, which limits the introduction amount of the intended “functional substance” and lowers the by-products produced by the reaction. There are problems such as contamination of impurities and large damage to the liposome membrane.

As a preferable alternative to this, it is preferable to prepare liposomes in advance by including a phospholipid polyalkylene oxide (PEO) derivative in the raw material phospholipids. For this purpose, modified phospholipids such as polyethylene oxide (PEO) derivatives such as phosphatidylethanolamine, such as distearoyl phosphatidylethanolamine polyethylene oxide (DSPE-PEO) have been proposed (Japanese Patent Laid-Open No. 7-165770). . Furthermore, JP 2002-37883 A discloses high-purity polyalkylene oxide-modified phospholipids for producing water-soluble polymer-modified liposomes with increased blood retention. It is described that when a polyalkylene oxide-modified phospholipid having a low monoacyl body content is used in producing such a liposome, the temporal stability of the liposome dispersion is good.
Production Method of Contrast Agent for X-Ray Examination / Production of Liposomes Various methods have been proposed so far for producing liposomes. When the production method is different, the shape and properties of the final liposome are also often significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is appropriately selected according to the desired form and characteristics of the liposome.

In the prior art, liposomes dissolve and mix lipid components such as phospholipids, sterols, and lecithins in containers together with organic solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, and THF, with almost no exception. It is prepared by. After evaporating the volatile material under reduced pressure, the lipid mixture is dispersed in a buffer containing a predetermined amount of encapsulated material. The whole is then stirred for several hours, and a portion of the dispersion (including the encapsulating material) is encapsulated in the liposome vesicles thus formed. Next, the size of the liposomes and the viscosity of the dispersion are reduced by sonicating the dispersion, using a surfactant, or other methods. At present, in order to remove these remaining organic solvents, a multi-step process and a long-time treatment are required. Such residual organic solvents, particularly chlorinated organic solvents, are difficult to remove completely, and there are concerns about adverse effects on living bodies, such as side effects.

  In the production method of the present invention, a solvent (preferably supercritical carbon dioxide) in which a phospholipid and a lipid membrane stabilizing substance are dissolved and dispersed is mixed with an aqueous solution containing an iodine-based compound and, if necessary, a formulation aid. The liposome suspension formed by the above is further permeated through a filtration membrane having a pore size of a certain size (for example, 0.1 to 0.4 μm).

In order to prepare the liposome used in the present invention, a liposome preparation method using supercritical or subcritical carbon dioxide that can avoid the above-mentioned problems is utilized. Carbon dioxide has a critical temperature of 31.1 ° C and a critical pressure of 75.3 kg / cm ^{2, which} is relatively easy to handle. It is preferable for the reason. 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. Among such temperature ranges, particularly suitable temperatures are
It 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 of forcibly aggregating lipids in water by applying high heat, lipids are denatured by high heat, for example, phospholipids are hydrolyzed to give lyso forms, and unsaturated components are peroxidized. There is no such problem.

A preferred method for preparing liposomes used in the X-ray contrast medium of the present invention is specifically performed as follows. Liquid carbon dioxide is added to the pressure vessel to bring it to the supercritical or subcritical state under the preferred pressure and temperature described above. Phospholipids and lipid membrane stabilizing substances are dissolved or dispersed as liposome membrane components in supercritical (or subcritical) carbon dioxide. As the membrane lipid component, the phospholipid is preferably selected from 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 with compound and dissolve. Alternatively, liquid carbon dioxide is added to a pressure vessel to which these compounds have been added in advance, and then the temperature and pressure are adjusted and mixed in a supercritical state. Subsequently, in the supercritical carbon dioxide containing the phospholipid and lipid membrane stabilizing substance produced, an aqueous solution containing a nonionic iodine compound and, if necessary, a formulation aid described later is added in two or more times, or Add in small portions continuously and mix. 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 side to be added and the side to be added may be reversed.

The efficiency of the encapsulation of the iodine compound in the liposome also depends on the ratio between the total amount of the lipid for the liposome and the aqueous solution containing the iodine compound. The total amount of lipid here is the total mass of all phospholipids, sterols and other added lipids constituting the liposome membrane. The total amount of lipid is 15 to 150 mmoles per liter (L) of the above aqueous solution.
When mixed at a ratio of preferably 30 to 120 mmoles, more preferably 50 to 100 mmoles, the incorporation of the water-soluble iodine compound into the liposome proceeds well, and as a result, the retention efficiency of the iodine compound is also improved. improves. If the amount of lipid is less than 15 mMoles, the ratio of water as a medium is relatively large, and the formation of liposomes is delayed, and the inclusion of a water-soluble nonionic iodide compound therein does not occur efficiently. On the other hand, when the amount of lipid exceeds 150 mmoles, mixing with an aqueous solution becomes insufficient, and aggregation of lipids or the like occurs, so that it takes time to disperse the lipid, and inclusion of iodine compounds hardly occurs.

When the inside of the system is decompressed and carbon dioxide is discharged, an aqueous dispersion in which liposomes encapsulating an iodine compound are dispersed is generated. The liposome is then passed through a filtration membrane having a pore size of 0.1 to 0.4 μm.

  Liposome preparation methods using supercritical or subcritical carbon dioxide have been shown to have higher liposome production rates, encapsulation rates of encapsulated substances, and retention rates of encapsulated substances in liposomes than the conventional methods (see above). Patent Document 2). 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 the production of the X-ray contrast medium of the present invention. It 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. For that purpose, after the production, an operation such as an appropriate application of ultrasonic waves or a filter having a fixed pore size is further performed. Alternatively, it is also desirable to increase the ratio of unilamellar liposomes. Single-membrane liposomes, especially LUV, which are large single-membrane liposomes
This is because (Large unilamellar veislcles) provides a larger encapsulation capacity than multilamellar liposomes, and has the advantage that the dosage of liposomes, in other words, the dosage of lipids does not increase.

When the weight of the encapsulated iodine compound is relatively large, the stability of the liposome decreases. In particular, a weak tendency was observed for a sudden change in ionic strength. The liposome of the contrast agent of the present invention is adjusted to a relatively small particle size, and the liposome membrane contains sterols and / or glycols, or contains a cationic phospholipid, a phospholipid or compound having a polyalkylene oxide group. Therefore, stabilization of the lipid membrane is aimed at. As a result, such liposomes have been found to be stable, for example, against salt shock.

The size of the liposome particles and their distribution are closely related to the high blood retention, targeting and delivery efficiency aimed at by the X-ray contrast medium produced by the method of the present invention. Here, the “center particle diameter” refers to the particle diameter having the highest appearance frequency in the particle distribution. The adjustment of the particle size can be carried out according to the formulation or process conditions. For example, when the above supercritical pressure is increased, the liposome particle size formed is reduced. In order to improve the retention stability of the contrast medium by the liposome and to make the particle size distribution of the liposome uniform within a narrower range, the prepared liposome suspension is a filtration membrane having a fixed pore size, preferably a polycarbonate membrane. You may forcibly permeate. In this case, the filtration membrane is 0.1 to 0.4 μm, preferably 0.15
By passing through a hydrostatic extruder equipped with a filter with a pore size of ~ 0.2μm, the number of lipid membranes in the liposome multilayer membrane is reduced, and uniform liposomes with an optimal size of 100-300nm as the center particle size are efficiently prepared. can do. Specifically, various types of hydrostatic extrusion equipment, such as “Extruder” (trade name, manufactured by NOF Liposome), “Liponizer” (trade name, manufactured by Nomura Microscience), etc. are used to force the filter. Make it transparent. As the filter, a polycarbonate type, a cellulose type or the like can be appropriately used. 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 a desirable embodiment of the present invention, the extrusion filtration is carried out by heating the aqueous suspension to a temperature not lower than the transition temperature of a phospholipid having a transition temperature, preferably not lower than “transition temperature + 10” ° C., and then forming a membrane having pores. It is preferable to pass through. This “transition temperature + 10” ° C. or higher is generally from 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 is in a liquid crystal state and the fluidity is increased. Since the lipid for liposomes according to the production method of the present invention has a uniform lipid membrane configuration with a regular orientation, even a highly viscous liposome suspension encapsulating a water-soluble drug may cause clogging of the filter. It is possible to prepare liposomes having a uniform particle size.

  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 drug by a method such as centrifugation, ultrafiltration, or gel filtration. Operations such as concentration, dilution, and lyophilization may be optionally performed. Subsequently, the contrast medium for X-ray examination of the present invention is prepared through a preparation process such as sterilization and packaging.

  As described above, sizing of the particle size is important for imparting passive targeting ability to liposomes. Japanese Patent No. 2619037 describes that exclusion of liposomes having a particle size of 3000 nm or more avoids inconvenient retention in the pulmonary capillaries. However, liposomes with a particle size range of 150-3000 nm are not necessarily tumorigenic.

The center particle diameter of the contrast medium liposome of the present invention is usually 50 to 300 nm, preferably 50 to 200 nm, more preferably 50 to 130 nm. The particle size can be set appropriately according to the purpose of X-ray imaging. For example, for selective imaging of a tumor part, 110 to 130 nm is particularly preferable. By aligning the particle size of the liposome in the range of 100 to 200 nm, more preferably 110 to 130 nm, the X-ray contrast agent can be selectively concentrated on the cancer tissue. This is known as the “EPR effect”. The pores of the neovascular wall in solid cancer tissue are abnormally larger than the pore size of the capillary wall window (fenestra) of normal tissue, less than 30-80 nm, even with molecules of about 100 nm to about 200 nm in size Leaks from. That is, the EPR effect is due to the fact that the neovascular wall in cancer tissue is more permeable than the microvascular wall in normal tissue.

The contrast agent leaking from the hole in the blood vessel wall does not return to the blood vessel again because the lymphatic vessel is not sufficiently developed around the cancer cell, and remains in the place for a long time. Since the EPR effect is a passive transport using the bloodstream, the retention in the blood must be improved as a requirement for its effective expression. That is, it is required that the contrast agent particles (liposome particles containing the iodine compound) stay in the blood for a long time and pass through the blood vessels near the cancer cells many times. Since the X-ray contrast agent of the present invention is not particularly large particles, it is difficult to be captured by reticuloendothelial cells. Liposomes behave like erythrocytes, and are not rapidly excreted via the kidneys. If they are stealthed, they are phagocytosed by reticuloendothelial cells. But stays relatively long in the bloodstream. The EPR effect inevitably increases the transferability of the contrast compound to the target organ or tissue, and achieves selective concentration and accumulation of the contrast agent in the cancer tissue. An increase in the tumor cell / normal cell accumulation ratio of the contrast compound enhances the contrast performance of the X-ray contrast agent. Such improvement in tumor visibility makes it possible to even discover micrometastatic cancers that have been difficult to detect.
-Production of X-ray contrast medium The contrast medium for X-ray examination produced by the method of the present invention has improved stability of the liposome itself as described above. The contrast medium is stably retained in the liposome over time. Furthermore, the stability of the contrast medium in the blood is also achieved by inclusion in the liposome. In the X-ray contrast agent of the present invention, a formulation aid may or may not be included in an aqueous solution containing an iodine compound at the time of formulation. It is typically preferred to add formulation aids. The “formulation aid” is added together with the contrast material at the time of formulation, and various materials are appropriately used based on the conventional contrast agent production technique. Specifically, various physiologically acceptable buffers, chelating agents, and if necessary, osmotic pressure regulators, stabilizers, viscosity regulators, antioxidants such as α-tocopherol, paraoxybenzoic acid And preservatives such as methyl.

The required amount of iodine delivered to the target organ that defines the contrast performance of X-ray contrast has been clarified (for example, in the case of the liver in Japanese Patent No. 2619037). When the iodine compound is encapsulated in a microcarrier called liposome as in the present invention, the dose of lipid must be considered in addition to the retention stability and delivery efficiency of the contrast medium. As the amount of lipid increases, the viscosity of the contrast agent increases. The viscosity of the contrast medium solution for X-ray examination produced by the method of the present invention is 37 ° C. and 6 cPa or less, preferably 0.9 to 3 cPa. As the amount of the iodine compound encapsulated in the liposome, that is, in the aqueous solution encapsulated in the liposome, the iodine compound is 1 to 10, preferably 2 to 8, more preferably 3 to the liposome membrane lipid weight. It is desirable that it is contained in a weight ratio of 6.

When the weight ratio of the iodo-based compound encapsulated in the liposome aqueous phase is less than 1, it is necessary to inject a relatively large amount of lipid, resulting in poor contrast substance delivery efficiency. According to the description of Japanese Patent No. 2619037, even if the ratio is 1, it was a high value from the technical level at that time. Conversely, when the encapsulated weight ratio of the iodine compound to the liposome membrane lipid weight exceeds 10, the liposome also becomes structurally unstable, and the diffusion and leakage of the iodine compound outside the liposome membrane may occur during storage or in vivo. Inevitable even after being injected. 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).

The contrast agent for X-ray examination produced by the method of the present invention is usually 100 to 500 mg I / ml, preferably 150 to 150 mg in terms of the iodine content. 300 mg I / ml. The amount of free iodine ions present in the contrast medium for X-ray examination should be 0.001 mole / L contrast medium, preferably 0.0005 mole / L or less. It is preferable to add to the contrast agent a chelating agent that functions as a trapping agent for such free iodine ions that are liberated from or mixed with the iodine compound used as the contrast material. Examples of the chelating agent include EDTA, EDTANa ₂ -Ca (disodium calcium edetate), hexametaphosphoric acid, and the like that are approved for pharmaceutical use.

  Furthermore, the contrast agent of the present invention is encapsulated in the liposome with an isotonic solution with respect to the osmotic pressure in the body so that the liposome is stably maintained in the body after administration. As a solvent for such a solution, water, various buffers, and the like can be used. Buffering agents include water-soluble amine buffers, phosphate buffers, citrate buffers, and the like. Among these, a water-soluble amine buffer is preferable. Specific examples include tris, ethanolamine, diethanolamine, triethylamine, morpholine, trometamol, and the like.

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 X-ray contrast agent is a water-soluble iodine-based 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. Therefore, 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.

  To obtain an isotonic solution or suspension, the contrast agent is dissolved or suspended in the medium at a concentration that provides the isotonic solution. For example, if the contrast agent alone is unable to provide an isotonic solution due to the low solubility of the contrast agent compound, other non-toxic water-soluble materials such as sodium chloride will form an isotonic solution or suspension. Sugars such as mannitol, glucose, sucrose, and sorbitol may be added to the aqueous medium.

The contrast medium for X-ray examination produced by the method of the present invention is administered to a subject parenterally, specifically intravascularly, preferably intravenously, as an injection or infusion. Is imaged. The dose is in accordance with a conventional iodine-based contrast agent. The total iodine amount in the liposome or the sum of the iodine amount outside the liposome and the total iodine amount outside the liposome may be the same as the conventional dosage.
〔Example〕
Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited in any way by such examples.
〔test〕
The shape and particle size of the liposome are measured by freezing the dispersion containing the liposome containing the iodine compound, then depositing carbon on the fractured interface, and observing this carbon with an electron microscope (freeze fracture TEM method). be able to. Here, the “center particle size” refers to the particle size having the highest appearance frequency in the particle distribution. Specifically, the particle size and structure of liposomes in the prepared contrast agent were examined with a transmission electron microscope (TEM) by a freeze-fracturing method. That is, the liposome dispersion is rapidly frozen in liquid nitrogen and crushed in a frozen state to expose the internal structure of the liposome. The crushed surface was vapor-deposited with carbon, and the formed carbon film was observed with a transmission electron microscope.

The particle size was a simple average of the observed diameters of about 20 contrast agent particles. The structure of the liposome particles was determined as “single film” when there was no step in the traces of the particles left on the observed carbon film, and “multilayer film” when two or more steps were observed. About 20 particles were observed, and those having a single membrane structure of 80% or more were substantially determined as single membrane liposomes.
A sample for measuring the encapsulation rate of the iodine compound (liposome dispersion) was dialyzed against isotonic saline, ethanol was added after the dialysis was completed to destroy the liposome, and the amount of the iodine compound in the liposome was determined by measuring the absorbance. The ratio with respect to the total iodine compound amount in the sample was expressed as the encapsulation rate (mass%).
Generation of agglomerates The state of occurrence of agglomerates was examined visually on the sample before pressure filtration. It was investigated whether what was generated could be removed by pressure filtration. Items that can be removed are defined as “very slight occurrence” and “slightly occurring”, which are not a big problem, depending on the amount. ”And the problem of“ small outbreaks ”.

A mixture of 86 mg of dipalmitoyl phosphatidylcholine (DPPC), 38.4 mg of cholesterol, and 19.2 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and the inside of the autoclave was heated to 60 ° C. Then, 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are dispersed and dissolved while stirring. I let you. While stirring, further contain contrast medium solution (Japan iopamidol 306.2 mg / mL, iodine content 150 mg / mL, trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, with appropriate amounts of hydrochloric acid and sodium hydroxide. pH was adjusted to around 7) 5 mL was continuously injected with a metering pump over 50 minutes. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was heated to 60 ° C. and filtered under pressure with a cellulose filter manufactured by Advantech, 1.0 μm. The obtained liposome-containing contrast agent was used as Sample 11.

After pressure filtration at 1.0 μm, the dispersion was heated to 60 ° C., and samples filtered with a 0.4 μm filter, a 0.2 μm filter, and a 0.1 μm filter were sample No. They were 12, 13, and 14. In addition, a sample obtained by pressure-filtering with a 0.2 μm filter at room temperature without heating the dispersion was designated as Sample No. It was set to 15. Further, a sample naturally filtered through a 0.4 μm filter at 60 ° C. was No. It was set to 16.
Table 1 shows the encapsulation rate of each sample (ratio of iopamidol encapsulated in liposomes).

As is apparent from Table 1, the inclusion rate can be increased when the filter size is 0.4 μm or less. Further, pressure filtration has a greater effect of improving the encapsulation rate than natural filtration. If the filtration temperature is higher than the transition temperature of the phospholipid used (DPPC: approximately 41 ° C.), the effect of improving the encapsulation rate is large.

  Samples 21 to 25 were prepared in the same manner as Sample 13 of Example 1 except that the pressure in the autoclave was changed as shown in Table 2, and the inclusion rate was measured and the state of occurrence of aggregates was observed.

As is apparent from Table 2, the inclusion rate increases when the internal pressure of the autoclave is 150 kg / cm ² or less, and at 80 kg / cm ² , a slight agglomeration occurs. ˜90 kg / cm ² is preferred.

A mixture of 86 mg of dipalmitoyl phosphatidylcholine (DPPC), 38.4 mg of cholesterol, and 19.2 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and the inside of the autoclave was heated to 60 ° C. Then, 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are dispersed and dissolved while stirring. I let you. In addition, it contains a contrast agent solution (Japan iopamidol 306.2 mg / mL, iodine content 150 mg / mL, trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, pH adjusted to around 7 with appropriate amounts of hydrochloric acid and sodium hydroxide. 5ml was pumped and injected at once. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was heated to 60 ° C., filtered under pressure with a cellulose filter manufactured by Advantech Co., Ltd., 1.0 μm, and further, the dispersion was kept at 60 ° C. and filtered under pressure with a 0.2 μm filter. This sample was designated as sample No. 31.

A mixture of 86 mg of dipalmitoyl phosphatidylcholine (DPPC), 38.4 mg of cholesterol, and 19.2 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and the inside of the autoclave was heated to 60 ° C. Then, 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are dispersed and dissolved while stirring. I let you. While stirring, further contain contrast medium solution (Japan iopamidol 306.2 mg / mL, iodine content 150 mg / mL, trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, and appropriate amounts of hydrochloric acid and sodium hydroxide. The pH was adjusted to around 7 with 5 mL of 2.5 mL divided into two portions and injected by a pump. After completion of the second injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The obtained dispersion was heated to 60 ° C., filtered under pressure using a cellulose filter manufactured by Advantech, 1.0 μm, and further, the dispersion was kept at 60 ° C. and filtered under pressure with a 0.2 μm filter. This sample was designated as sample No.32.

A mixture of 86 mg of dipalmitoyl phosphatidylcholine (DPPC), 38.4 mg of cholesterol, and 19.2 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, and the inside of the autoclave was heated to 60 ° C. Then, 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are dispersed and dissolved while stirring. I let you. While stirring, further contain contrast medium solution (Japan iopamidol 306.2 mg / mL, iodine content 150 mg / mL, trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, and appropriate amounts of hydrochloric acid and sodium hydroxide. The pH was adjusted to around 7 with 5 mL). After completion of the fifth injection, the system was depressurized to discharge carbon dioxide to obtain a liposome dispersion containing a contrast agent solution. The obtained dispersion was heated to 60 ° C., filtered under pressure with a cellulose filter manufactured by Advantech Co., Ltd., 1.0 μm, and further, the dispersion was kept at 60 ° C. and filtered under pressure with a 0.2 μm filter. This sample was designated as sample No. 33.

Samples prepared in the same manner as Sample 13 except that the continuous mixing time was 15 minutes and 5 minutes were designated as Sample Nos. 34 and 35, respectively.

  The measurement of the encapsulation rate of each sample and the observation of aggregates were evaluated in the same manner as in Example 2, and the results are shown in Table 3.

  As is apparent from Table 3, the mixing of carbon dioxide containing a phospholipid and a lipid membrane stabilizing substance with an aqueous solution containing a nonionic iodine compound and a formulation aid is divided into two or more times or in small portions. The continuous addition method is preferable from the viewpoint of not generating aggregates and increasing the encapsulation rate.

A mixture of 192 mg of dipalmitoyl phosphatidylcholine (DPPC), 78 mg of cholesterol, and 38.4 mg of PEG-phospholipid (manufactured by NOF Corporation, SUNBRIGHT DSPE-020CN) is charged into a special stainless steel autoclave, and the inside of the autoclave is heated to 60 ° C. Then 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave, which was 50 kg / cm ² , is increased to 120 kg / cm ² by reducing the volume in the autoclave, carbon dioxide is brought into a supercritical state, and lipids are dispersed and dissolved while stirring. I let you. While stirring, further contain contrast medium solution (Japan iopamidol 306.2 mg / mL, iodine content 150 mg / mL, trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, and appropriate amounts of hydrochloric acid and sodium hydroxide. PH was adjusted to around 7) 5 mL was continuously injected with a metering pump. After completion of the injection, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing a contrast agent solution was obtained. The resulting dispersion was heated to 60 ° C. and filtered under pressure with a cellulose filter manufactured by Advantech, 0.2 μm. The obtained liposome-containing contrast agent was used as Sample 41.

As a result of conducting the above test on the sample 41, the particle size was 220 nm, and about 70% was a single-membrane liposome. The encapsulation rate was 15% by mass of the total amount of iopamidol. Sample 41 has a total lipid amount of 95 mmol / L, and cholesterol is 0.4 parts by weight of the phospholipid amount.

  Samples 42 to 48 were prepared in the same manner as Sample 41 except that the total lipid amount was changed as shown in Table 4. Table 4 shows the measurement results for the liposomes in each sample.

As is apparent from Table 4, when the total lipid amount is less than 15 mmol / L, the encapsulation rate is low, and the delivery efficiency targeted by the present invention cannot be expected. When it exceeds 15 mmol / L, the inclusion rate suddenly increases. At 15 mmol / L or higher, the inclusion rate improves as the amount of lipid used increases, but at 100 mmol / L or higher, the inclusion rate is less likely to increase relative to the amount of lipid used. Occurs, and the filter is difficult to filter.

  Samples 51 to 58 were produced in the same manner as the sample 41 of Example 4 except that the amount of cholesterol was changed as shown in Table 5. Table 2 shows the measurement results such as the encapsulation rate of the liposomes in each sample.

As is apparent from Table 5, the inclusion rate is relatively low when the amount of cholesterol is less than 0.05 part by weight relative to 1 part by weight of phospholipid, and the inclusion rate increases when it exceeds 0.2 part by weight. When the amount is 0.3 parts by weight or more, the inclusion rate is improved with an increase in the amount of lipid used. If the amount exceeds 1.5 parts by weight, the mechanism is unknown, but the encapsulation rate decreases.

Example 4 except that DOTAP (1,2-dioleoyloxy-3- (trimethylammonium) propane) was used as the cationic phospholipid instead of cholesterol, and the amount used was changed as shown in Table 6. Samples 61 to 67 were prepared in the same manner as Sample 41. Table 6 shows the measurement results such as the encapsulation rate for the liposomes in each sample.

As is apparent from Table 6, the encapsulation rate is relatively low when the DOTAP amount is less than 0.1% by mass relative to the phospholipid, and the encapsulation rate increases when it exceeds 0.3% by mass. When the amount of lipid is 0.5% by mass or more, the encapsulation rate is improved with an increase in the amount of lipid used. If it exceeds 5% by mass, the mechanism is unknown, but the encapsulation rate decreases.

Samples 71 to 78 were produced in the same manner as the sample 41 of Example 4 except that propylene glycol was used instead of cholesterol and the amount used was changed as shown in Table 7. Table 7 shows the measurement results such as the encapsulation rate for the liposomes in each sample.

As apparent from Table 7, the inclusion rate is relatively low when the amount of propylene glycol is less than 0.5% by mass relative to the phospholipid, and the inclusion rate increases when it exceeds 0.5% by mass. When the content is 10% by mass or more, the encapsulation rate is hardly increased with respect to the amount of lipid used. If the amount exceeds 20% by mass, the mechanism is unknown, but the encapsulation rate decreases.

The sample of Example 4 except that the amount of dipalmitoylphosphatidylcholine (DPPC), cholesterol, DOTAP, propylene glycol, and PEG-phospholipid (DSPE-020CN) was changed as shown in Table 8. Samples 81 to 95 were produced in the same manner as in 41. Table 8 shows the measurement results such as the encapsulation rate for the liposomes in each sample.

  The obtained sample was evaluated in the same manner as in Example 4, and the sample was stored over time (40 ° C., 2 weeks) to examine changes in the encapsulation rate.

  When cholesterol and DOTAP are used in combination, the encapsulation rate is improved and the temporal stability is improved as compared with the case where cholesterol is not used. When DOTAP and propylene glycol are used in combination, the degree of improvement in the encapsulation rate is higher than in the case where each is used alone, and the temporal stability is also improved. When cholesterol, DOTAP, and propylene glycol are used in combination, even if the amount used is small, the effect of improving the encapsulation rate is high and the temporal stability is also improved.

  PEG-phospholipid also has an effect of improving the encapsulation rate, and particularly when it is 10% by mass or more with respect to phospholipid, the effect of improving the encapsulation rate is remarkable.

Claims (10)

Supercritical carbon dioxide containing phospholipids and lipid membrane stabilizing substances;
A suspension of liposomes formed by mixing an aqueous solution containing a nonionic iodine compound and a formulation aid,
Furthermore, the manufacturing method of the contrast agent for a X-ray test | inspection characterized by permeate | transmitting the filtration membrane which has a 0.1-0.4 micrometer hole diameter. ^2. The method for producing a contrast medium for X-ray examination according to claim 1, wherein the supercritical carbon dioxide is generated under a pressure of 90 to 150 kg / cm ² .   2. The method for producing a contrast agent for X-ray examination according to claim 1, wherein the phospholipid contains at least a phospholipid having a transition temperature.   2. The method for producing a contrast medium for X-ray examination according to claim 1, wherein the lipid membrane of the liposome contains a cationic substance in a proportion of 0.1 to 5% by mass with respect to the amount of phospholipid.   2. The method for producing a contrast agent for X-ray examination according to claim 1, wherein the lipid membrane stabilizing substance is a sterol and / or a glycol.   The method for producing a contrast medium for X-ray examination according to claim 5, wherein the sterols are contained in a proportion of 0.05 to 1.5 parts by weight with respect to 1 part by weight of phospholipid. The method for producing a contrast medium for X-ray examination according to claim 5, wherein the glycol is contained in a proportion of 0.5 to 20% by mass with respect to the amount of phospholipid. 2. The permeation of the filtration membrane is performed using an extruder.
A method for producing a contrast medium for X-ray examination as described in 1 above.   The method for producing a contrast medium for X-ray examination according to claim 8, wherein the processing by the extruder is performed at a temperature equal to or higher than a transition temperature of a phospholipid having a transition temperature. Mix supercritical carbon dioxide containing phospholipid and lipid membrane stabilizing substance with aqueous solution containing nonionic iodine compound and formulation aid in two or more times, or add in small portions continuously. The liposomes formed by mixing are 5 to 35 of the total iodine compound.
2. The method for producing a contrast medium for X-ray examination according to claim 1, which contains a mass% iodine compound.