JP2005225791A - Contrast medium containing liposome and used for x-ray inspection - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To prepare a contrast medium which is used for X-ray inspections, improves the stability of liposomes and the retention stability of an iodine compound, enhances the retainability of a contrast compound in blood, achieves the efficient delivery and good targeting of the contrast agent, and especially has an excellent tumor-imaging property. <P>SOLUTION: This contrast medium for the X-ray inspections contains liposomes made by a supercritical carbon dioxide method. The liposome contains at least one of cationic lipids, sterol compounds, glycol lipids, and glycerol lipids in the lipid membrane of the liposome, and contains the water-soluble iodine-based compound in the aqueous phase of the liposome. Thereby, the stability of the liposome and the retention stability of the iodine compound are improved. The contrast medium for the X-ray inspections does not contain a poisonous solvent such as a chlorine-based solvent, because of being produced by the supercritical carbon dioxide method, and has high safety. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

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

  X-ray simple imaging and CT imaging (computer tomography) occupy 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. In addition to X-ray imaging methods, other methods with different measurement principles have been developed as examination methods for tumor detection. For example, in MRI (magnetic resonance imaging), there is a limit in terms of sensitivity to accurately image cancer tissues, especially minute cancer tissues, and in PET (positron emission tomography), in terms of exposure and operating costs. None of them are general, and both require large-scale equipment and expensive equipment, so far they are not widely used inspection methods.

  International publications WO98 / 46275, WO95 / 31181, WO94 / 19025, WO96 / 28414, WO96 / 00089, US Pat. Nos. 4873075, 4567734, etc. include hydrophobic iodine compounds containing surfactants and oils and fats. A method of imaging a liver, a spleen image, an adrenal cortex, a tumor, an arteriosclerotic lesion, a blood pool, a lymph system, etc. by administering a substance dispersed in water under is disclosed. These methods are intended to selectively contrast a diseased site by making the contrast agent fine particles to increase the residence time in the body. The formulation method proposed for that purpose is not sufficient in contrast efficiency and selectivity. Furthermore, since the iodine compound to be used is hydrophobic, there is also a problem that the rate of discharge from the body after imaging is slow and the burden on the patient is large.

  On the other hand, as another method for making a contrast medium into a fine particle form, a technique of encapsulating a contrast compound in a liposome composed of a lipid similar to a biological membrane and considered to be highly safe due to 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. These methods have not yet been put into practical use despite the use of liposomes that are highly safe as materials and have appropriate degradability in vivo (for example, see Patent Document 1). This is because an organic solvent, particularly a chloro solvent such as chloroform or dichloromethane, is used as a solvent for the phospholipid constituting the liposome membrane in the production process, and there is a concern about the toxicity of the remaining solvent.

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.

Furthermore, even if the contrast medium is successfully encapsulated in the liposome, it is necessary to consider the problem of leakage to the outside over time, or the situation that the liposome itself becomes unstable during processing or storage. Furthermore, it has been pointed out that even when liposomes are administered in vivo, many of them are trapped in the reticuloendothelial tissues such as the liver and spleen, so the desired effect cannot be obtained (Cancer Res., 43, 5328 (1983)).
Japanese Patent Laid-Open No. 7-316079 Japanese Patent Laid-Open No. 2003-119120

  An object of the present invention is to provide a contrast medium for X-ray examination in which the contrast substance is favorably retained in a liposome and the delivery efficiency is improved. That is, an object of the present invention is to provide an X-ray contrast agent in which a water-soluble iodine-based compound encapsulated in a liposome without using a toxic organic solvent is well retained and has excellent contrast power.

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

(1) The present invention includes a liposome having a lipid membrane, the liposome containing 0.2 to 1 part by weight of sterols in the lipid membrane with respect to 1 part by weight of phospholipid, and water in the lipid membrane. A contrast agent for X-ray examination, characterized in that the phase contains an iodine compound and the liposome is produced by a supercritical carbon dioxide method.

(2) The present invention also includes a liposome having a lipid membrane, the liposome containing a cationic lipid in a proportion of 0.3 to 3% by mass with respect to the total lipid amount in the lipid membrane, A contrast agent for X-ray examination, wherein the aqueous phase contains an iodine compound and the liposome is produced by a supercritical carbon dioxide method.

(3) Further, the present invention includes a liposome having a lipid membrane, the liposome containing glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount, and water in the lipid membrane. A contrast agent for X-ray examination, characterized in that the phase contains an iodine compound and the liposome is produced by a supercritical carbon dioxide method.

(4) The present invention includes a liposome having a lipid membrane, the liposome containing glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount, and in the aqueous phase in the lipid membrane. A contrast agent for X-ray examination, characterized in that it contains an iodine-based compound and the liposome is produced by a supercritical carbon dioxide method.

In the liposome of (1), a cationic lipid is further added to the lipid membrane in an amount of 0.3 to 3 with respect to the total lipid amount.
You may contain in the ratio of the mass%.

  The liposome of (1) may further contain glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.

  The liposome of (1) may further contain glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.

  The liposome of (2) may further contain glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.

  The liposome of (2) may further contain glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.

  The liposome of (3) may further contain glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.

The liposome of (3) may further contain any two of sterols, cationic lipids, and glycerol lipids in its lipid membrane.

The liposome of (4) may further contain a sterol and a cationic lipid in its lipid membrane.

The liposome (4) may further contain a sterol, a cationic lipid, and a glycol lipid in its lipid membrane.

[Detailed Description of the Invention]
The contrast agent for X-ray examination of the present invention contains a liposome produced by the supercritical carbon dioxide method. In the lipid membrane of the liposome, sterols and / or cationic lipids,
And / or glycol lipid is contained, and a water-soluble iodo compound is encapsulated in the aqueous phase. With this configuration, stabilization of the liposome and retention stability of the iodine compound are achieved. At the same time, the retention of the contrast compound in the blood is also increased, so that efficient delivery of the contrast medium and good targeting can be achieved. As a result, it was possible to achieve excellent tumor visualization and to improve the accuracy of diagnostic examination by X-ray contrast. Hereinafter, the present invention will be described in detail in the order of contrast compound, liposome, and contrast medium for X-ray examination.

Contrast Compound The water-soluble iodine-based compound in the present invention is not particularly defined as long as it has a contrast property, regardless of whether it is ionic or non-ionic. In general, a nonionic iodine compound is more desirable because it has a lower osmotic pressure than an ionic iodine compound. As the water-soluble nonionic iodide compound, a nonionic iodide compound containing phenyl iodide and having at least one 2,4,6-triiodophenyl group is preferable.

Specifically, 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.

Other iodo compounds include: diatrizoic acid; diatrizoate sodium; meglumine diatrizoate; acetolizoic acid and its soluble salts; diprotrizoic acid; iodamide, sodium iodipamide, meglumineiodipamide, iodohippuric acid and its solubles Salt; iodomethamic acid; iodopyrracetoiodo-2-pyridone-N-acetic acid, 3,5-
Diiodo-4-pyridone-N-acetic acid (iodopyracet); diethylammonium salt of said acid; iotalamic acid; metrizoic acid and its salts; Iopodate sodium and other similar iodinated compounds.

  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 30% 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 fine packing of the liposome particles, the retention stability of the contrast medium in the liposome is not impaired.

Liposome The contrast agent for X-ray examination of the present invention is used in a form encapsulated in a liposome as a microcarrier in order to selectively and efficiently deliver the contrast compound to a target site such as a target organ or tissue. . The contrast agent of the present invention improves the retention in blood by using liposomes with improved blood stability, and achieves efficient drug delivery and targeting. In order to produce an EPR (Enhanced permeability and retention) effect that is particularly effective for obtaining excellent tumor visualization, it is necessary to stabilize the liposome structure and retain the encapsulated substance. Contrast agents are required to have characteristics such as stability in blood and retention in blood after improving retention efficiency on the front and back sides.

  The X-ray contrast agent of the present invention can realize the targeting function by appropriately designing the particle size of the liposome encapsulating the contrast compound and its bilayer. 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.

The adoption of active targeting that allows for higher levels of accumulation and selectivity of contrast agents should also be considered. As an example, introducing 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. As a liposome suitable for the X-ray contrast medium of the present invention, for example, as a result of modification with addition of polyalkylene oxide or PEG on the surface, stability and retention in blood are further improved, and reticuloendothelial system such as liver Examples include liposomes that are not phagocytosed by cells. 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 in relation to the extracorporeal discharge time when designing the liposome. 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 usually formed from lipid bilayer membranes. In general, phospholipids and / or glycolipids are preferably used as components of the lipid membrane.

  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.

  As the sterols used in the present invention, cholesterol is preferably used, and examples of the sterol derivatives include dihydrocholesterol, cholesteryl hydroxystearate, lanolin fatty acid cholesteryl, cholesteryl oleate, and cholesteryl nonanoate. These sterols may be added so as to be contained at a ratio of 0.1 to 2 parts by weight, preferably 0.2 to 1 part by weight, more preferably 0.4 to 1 part by weight, relative to 1 part by weight of the phospholipid.

The cationic lipid used in the present invention is 1,2-dioleoyloxy-3- (trimethylammonium) propane (DOTAP), N, N-dioctadecylamide glycylspermine (DOGS), dimethyldioctadecylammonium bromide ( DDAB), N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N- [2 (spermine-carboxamide) Ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and N- [1- (2,3-dimyristyloxy) propyl] -N, N-dimethyl-N- (2-hydroxy And ethyl) ammonium bromide (DMRIE).

Cationic phospholipids include esters of phosphatidic acid and amino alcohol, such as dipalmitoyl phosphatidic acid (DPPA) or distearoyl phosphatidic acid (DSPA) and hydroxyethylenediamine. These cationic lipids are contained in an amount of 0.1 to 5% by mass with respect to the total lipid amount, preferably 0.3 to 3% by mass with respect to the total lipid amount, and more preferably 0.5 to 2% by mass with respect to the total lipid amount. What is necessary is just to add.

Cationic lipids can be used alone, but can also be used in combination with other lipids. For example, DOTMA can be used in a formulation (1: 1) with dioleoylphosphatidylethanolamine (DOPE), and Lipofectin ™ (GIBCO / BRL, Life Technologies Inc., Gay Serag, Mary Land, USA).

As the glycol lipid in the present invention, a lipid modified mainly with polyethylene glycol is used. Nippon Oil & Fats has been commercialized as a PEG-modified lipid, and 1,2-distearoyl-3-phosphatidylethanolamine-PEG2000 (product names SUNBRIGHT DSPE-020C, DSPE-020G, DSPE-020CN, DSPE-020PA, DSPE -020MA (manufactured by NOF), 1,2-distearoyl-3-phosphatidylethanolamine-PEG5000 (SUNBRIGHT DSPE-050C, DSPE-050G, DSPE-050CN
), Distearoylglycerol-PEG2000 (SUNBRIGHT GS-020), dipalmitoylglycerol-PEG2000 (SUNBRIGHT GP-020), dimyristoylglycerol-PEG2000 (SUNBRIGHT GM-020), and the like. These glycol lipids are 0.3 to 30% by mass,
Preferably, it may be added so as to contain 0.5 to 20% by mass with respect to the total amount of lipid, more preferably 2 to 15% by mass with respect to the total amount of lipid.

Examples of the glycerol lipid in the present invention include distearoyl phosphatidyl glycerol, dioleoyl phosphatidyl glycerol, dimyristoyl phosphatidyl glycerol, and dipalmitoyl phosphatidyl glycerol (product name: COATSOME MG-6060LS (manufactured by NOF Corporation)). These glycerol lipids may be added so as to contain 0.3 to 30% by mass, preferably 0.5 to 20% by mass, more preferably 1 to 15% by mass with respect to the total lipid amount. Good.

Various methods have been proposed so far for preparing 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 general, liposomes are prepared by dissolving and mixing 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. 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 (this causes the formation of liposomes) and a portion of the dispersion (including the encapsulating material) is encapsulated within the liposome vesicles so 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. Such preparations of liposomes always contain an organic solvent. Such residual organic solvents, particularly chlorinated organic solvents, are difficult to remove completely, and there are concerns about adverse effects on living bodies.

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. Suitable pressure of carbon dioxide in the supercritical state used in the production method of the present invention (including semi-critical state), 75~500kg / cm ^2, preferably 100~400 kg / cm ^2. The temperature of the carbon dioxide gas in a suitable critical state is 31 to 200 ° C, preferably 32 to 100 ° C, more preferably 35 to 80 ° C. Within these ranges, it is preferable to establish a supercritical state (including a subcritical state) by appropriately selecting and combining temperature and pressure.

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. Liposome membrane components are dissolved in carbon dioxide in a supercritical (or subcritical) state. As the membrane lipid component, the phospholipid is preferably combined with at least one compound selected from cationic lipids, polyalkylene oxide-modified phospholipids (preferably phospholipids having a polyethylene glycol group), sterols, glycol lipids and glycerol lipids. Mix 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 to dissolve in a supercritical state. Subsequently, an aqueous solution containing an iodine-based compound and, if necessary, a formulation aid described later (preferably a water-soluble amine buffer and a chelating agent) is continuously added to the resulting lipid mixture. A carbon emulsion is formed. 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. Further, the liposome is passed through a filtration membrane having a pore size of 0.1 to 0.4 μm. Subsequently, the contrast medium for X-ray examination of the present invention is prepared through a preparation process such as sterilization and packaging.

  It has been shown that the liposome preparation method using supercritical or subcritical carbon dioxide has higher liposome production rate, encapsulation rate of the encapsulated substance, and remaining rate of encapsulated substance in the liposome than the conventional method. Reference 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 particle trace on the carbon film is determined as a single film when there is no level difference, and the case where two or more levels are recognized is determined as a “multilayer film”. In the case of liposomes composed of multilamellar vesicles (MLV), it is desirable to peel off the bilayer of the liposome membrane to make the multilayer as thin as possible. For that purpose, after the production, an operation such as appropriate application of ultrasonic waves or passing through a filter having a constant pore size is further performed. It is also desirable to increase the proportion of unilamellar liposomes. Single membrane liposomes, especially large unilamellar veislcles (LUV), provide a larger entrapment capacity than multilamellar liposomes and do not increase the dose of liposomes, in other words, the dose of lipids. There are also advantages.

  When the weight of the encapsulated iodine compound is relatively large, the stability of the liposome is lowered. In particular, a weak tendency was observed for sudden changes in ionic strength. The liposome of the contrast agent of the present invention is adjusted to a relatively small particle size, and the lipid membrane is stabilized by adding a sterol, a cationic lipid, a glycol lipid, or a glycerol lipid alone or in combination of several kinds to the liposome membrane. I am trying. As a result, it has been found that such liposomes are also 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 agent of the present invention. The particle size can be measured by freezing a dispersion containing liposomes containing an iodine compound, then depositing carbon on the crushed interface, and observing the carbon with an electron microscope (freeze-crush TEM method). Here, the “center particle size” refers to the particle size 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 make the particle size distribution of the liposome to be produced in a narrower range, it may be filtered through a polycarbonate membrane. In this case, by passing through an extruder equipped with a filter having a pore size of 0.1 to 0.4 μm as a filtration membrane, it is possible to efficiently prepare liposomes having an optimum size having a central particle size of the liposome membrane of 100 to 300 nm or less. The extrusion filtration method is described in, for example, Biochim. Biophys. Acta 557, 9 (1979). By incorporating such an “extrusion” operation, in addition to the above sizing, it is possible to adjust the concentration of iodine compounds existing outside the liposome, exchange the liposome dispersion, and remove undesirable substances. There is also.

  As described above, sizing of the particle size is important for imparting passive targeting ability to the liposome. 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.

Contrast agent for X-ray examination Since the contrast agent for X-ray examination of the present invention has improved stability of the liposome itself as described above, the contrast agent for X-ray examination is also contained in the liposome during the preparation process, during storage and storage. Contrast material is stably held. Furthermore, the stability of the contrast medium in the blood is also achieved by inclusion in the liposome. The required amount of iodine delivered to the target organ that defines the contrast performance of X-ray contrast has been clarified (eg, 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. 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, relative to the liposome membrane lipid weight,
Preferably, it is contained in a weight ratio of 2 to 8, more preferably 3 to 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 viscosity of the preparation solution of the present invention is 6 cPa or less, preferably 0.9 to 3 cPa at 37 ° C.

  The contrast agent for X-ray examination of the present invention is encapsulated in the liposome in the form of an isotonic solution or suspension with respect to the osmotic pressure in the body so that the liposome is stably maintained in the body after administration. As a medium for such a solution or suspension, water, a buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, citrate buffer solution and the like can be used.

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 is good for liposomes to which autoclaving 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. Salts such as mannitol, glucose, sucrose, sorbitol and the like may be added to the medium.

When the X-ray contrast agent of the present invention is formulated, if necessary as a formulation aid, a physiologically acceptable stabilizer, EDTANa ₂ -Ca, EDTANa ₂ or the like as a chelating agent, α- Tocopherols, viscosity modifiers and the like may also be included.

  The contrast medium for X-ray examination of the present invention is administered to a subject parenterally, specifically intravascularly, preferably intravenously, as an injection or infusion, and imaged by X-ray irradiation. 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. In actual diagnostic examinations, the contrast agent for X-ray examination of the present invention is used in an X-ray imaging apparatus combined with a computed tomography apparatus (CT), thereby further effectively exhibiting the contrast agent performance. Is also expected.

  The X-ray contrast medium according to the present invention includes a liposome prepared by the supercritical carbon dioxide method, and in the lipid membrane, in addition to neutral or anionic phospholipid, cationic phospholipid and / or sterols and / or Or it contains glycol lipid. The water phase contains a water-soluble iodine compound. As a result, the stability of the liposome structure and the retention stability of the encapsulated material are improved.

  The contrast medium of the contrast agent of the present invention has good retention in the blood, exhibits an EPR effect, and selectively concentrates and accumulates on the target diseased site or tissue, particularly cancer tissue. After imaging, the iodine compound is water-soluble and will eventually be excreted outside the body.

  Since the X-ray contrast medium according to the present invention does not use a toxic solvent, particularly a highly toxic chlorinated solvent, compared to the conventional X-ray contrast medium, toxicity and side effects are much reduced. Therefore, the burden on the patient receiving the administration is small.

〔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.

Preparation of liposome sample <Sample 1>
200 mg of dipalmitoyl phosphatidylcholine (DPPC; manufactured by NOF Corporation), 80 mg of cholesterol HP (manufactured by NOF Corporation), Lipofectin ™ 5 mg as a cationic lipid, 4 mg of SUNBRIGHT DSPE-020CN (manufactured by NOF Corporation) as a glycerol lipid A mixture of 4 mg of coatsome MG-6060LS (manufactured by NOF Corporation) was charged into a stainless steel autoclave, the inside of the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. The pressure in the autoclave was increased from 50 kg / cm ² to 120 kg / cm ² and the autoclave was stirred to dissolve the DPPC mixture in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iopamidol 306.2 mg / mL (iodine content 150 mg / mL), trometamol 1 mg / mL, calcium edetate disodium 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH adjustment) Agent) Containing appropriate amount) 10 g of solution was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iopamidol was obtained. The obtained dispersion was heated to 60 ° C. and irradiated with ultrasonic waves for 3 minutes, heated to 80 ° C. and filtered under pressure in the order of 0.45 μm, 0.3 μm and 0.1 μm with a cellulose filter manufactured by Advantech. The obtained contrast agent was designated as Sample 1.

<Sample 2>
Dipalmitoylphosphatidylcholine (DPPC; manufactured by NOF Corporation) 80 mg, Cholesterol HP (manufactured by NOF Corporation) 80 mg, SUNBRIGHT DSPE-020CN (manufactured by NOF Corporation) as a glycol lipid, 8 mg, Coatsome MG-6060LS (Nippon Oil & Fats Co., Ltd.) 4 mg of the mixture was charged into a stainless steel autoclave, the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. The pressure in the autoclave was increased from 50 kg / cm ² to 200 kg / cm ² , and the inside of the autoclave was stirred to dissolve the DPPC mixture in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iopamidol 306.2 mg / mL (iodine content 150 mg / mL), trometamol as an additive 1 mg / mL, disodium calcium edetate 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH 10 g of the solution containing the appropriate amount of the adjusting agent) was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iopamidol was obtained. The obtained dispersion was heated to 80 ° C. and filtered under pressure in the order of 0.45 μm, 0.3 μm, and 0.1 μm with a cellulose filter manufactured by Advantech. The obtained contrast agent was designated as Sample 2.

<Sample 3>
200 mg of dipalmitoylphosphatidylcholine (DPPC; manufactured by NOF Corporation), Lipofectin ™ 10 mg as a cationic lipid, SUNBRIGHT DSPE-020CN (manufactured by NOF Corporation) as a glycol lipid, 4 mg, Coatsome MG-6060LS (manufactured by NOF Corporation) ) 40 mg of the mixture was charged into a stainless steel autoclave, the inside of the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. The pressure in the autoclave was increased from 50 kg / cm ² to 200 kg / cm ² , and the inside of the autoclave was stirred to dissolve the DPPC mixture in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iopamidol 306.2 mg / mL (iodine content 150 mg / mL), trometamol as an additive 1 mg / mL, disodium calcium edetate 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH 10 g of the solution containing the appropriate amount of the adjusting agent) was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iopamidol was obtained. The obtained dispersion was heated to 60 ° C. and irradiated with ultrasonic waves for 3 minutes, heated to 80 ° C., and filtered under pressure in the order of 0.45 μm, 0.3 μm, and 0.1 μm with a cellulose filter manufactured by Advantech. The obtained contrast agent was designated as Sample 3.

<Sample 4>
A mixture of 80 mg of dipalmitoyl phosphatidylcholine (DPPC; manufactured by NOF Corporation), 80 mg of cholesterol HP (manufactured by NOF Corporation), and 38 mg of SUNBRIGHT DSPE-020CN (manufactured by NOF Corporation) as a glycol lipid is charged into a stainless steel autoclave. Heat to 60 ° C., then add 13 g of liquid carbon dioxide. The pressure in the autoclave was increased from 50 kg / cm ² to 120 kg / cm ² and the autoclave was stirred to dissolve the DPPC mixture in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iopamidol 306.2 mg / mL (iodine content 150 mg / mL), trometamol as an additive 1 mg / mL, disodium calcium edetate 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH 10 g of the solution containing the appropriate amount of the adjusting agent) was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iopamidol was obtained. The obtained dispersion was heated to 80 ° C. and filtered under pressure in the order of 0.45 μm, 0.3 μm and 0.1 μm with a cellulose filter manufactured by Advantech. The obtained contrast agent was designated as sample 4.

<Sample 5>
A stainless steel mixture of 200 mg of dipalmitoylphosphatidylcholine (DPPC; manufactured by NOF Corporation), 40 mg of cholesterol HP (produced by NOF Corporation), 15 mg of Lipofectin TM as a cationic lipid, and 38 mg of SUNBRIGHT DSPE-020CN (manufactured by NOF Corporation) as a glycol lipid The autoclave was charged, the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. The pressure in the autoclave was increased from 50 kg / cm ² to 120 kg / cm ² and the autoclave was stirred to dissolve the DPPC mixture in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iohexol 647 mg / mL (iodine content 300 mg / mL), trometamol 1.21 mg / mL as additive, calcium edetate disodium 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH 10 g of the solution containing the appropriate amount of the adjusting agent) was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iohexol was obtained. The obtained dispersion was heated to 60 ° C. and irradiated with ultrasonic waves for 3 minutes, heated to 80 ° C. and filtered under pressure in the order of 0.45 μm, 0.3 μm and 0.1 μm with a cellulose filter manufactured by Advantech. The obtained contrast agent was designated as Sample 5.

<Sample 6>
100 mg of dipalmitoyl phosphatidylcholine (DPPC; manufactured by NOF Corporation) was charged into a stainless steel autoclave, the inside of the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. The pressure in the autoclave was increased from 50 kg / cm ² to 200 kg / cm ² and the autoclave was stirred to dissolve DPPC in supercritical carbon dioxide. While stirring this supercritical carbon dioxide, iopamidol 306.2 mg / mL (iodine content 150 mg / mL), trometamol as an additive 1 mg / mL, disodium calcium edetate 0.1 mg / mL, hydrochloric acid and sodium hydroxide (pH 10 g of the solution containing the appropriate amount of the adjusting agent) was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a liposome dispersion containing iopamidol was obtained. The obtained dispersion was heated to 60 ° C. and irradiated with ultrasonic waves for 3 minutes, heated to 80 ° C., and filtered under pressure in the order of 0.45 μm, 0.3 μm, and 0.1 μm with an Advantech cellulosic filter. The obtained contrast agent was designated as Sample 6. Further, a contrast agent obtained by performing only the pressure filtration similar to the sample 5 without ultrasonic irradiation was used as a sample 7.

Liposome morphology and particle size The liposome particle size and structure in the prepared contrast medium were examined with a transmission electron microscope (TEM) by freeze-fracturing. 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.
Quantitative determination of the amount of contrast medium encapsulated in liposomes Dialyze the liposome dispersion with isotonic saline, add ethanol after the dialysis to break the liposomes, and determine the amount of iodine compound in the liposomes by measuring absorbance. It was.
Table 1 shows the measurement results such as the particle size of the liposomes in each sample.

Stability samples in physiological saline Samples 1 to 7 were added to physiological saline, and after sealing, heated to 42 ° C and left for 3 days. The obtained sample and the sample before heating were observed with an optical microscope.

In all of the samples before heating, fine particles were not observed with an optical microscope. However, after heating, for Comparative Samples 6 and 7, fine particles of several μm that were considered to be a coalescence of liposome particles were observed. In Samples 2 and 4, fine particles that seemed to be a combination of liposome particles were slightly observed. However, no particular change was observed for samples 1, 3 and 5.

Samples 1-7 prepared in Example 1 were diluted with isotonic glucose solution to a concentration of 50 mg iodine / ml. When this solution was intravenously injected into rats, it was observed by X-ray images that Samples 1 and 2 were particularly concentrated in the liver.

  A cell suspension of VX2 carcinoma was implanted subcutaneously in the rabbit. Two weeks after transplantation, samples 1 to 7 obtained in Example 1 were intravenously injected into rabbits and observed by X-ray images after injection. When Samples 3, 4, and 5 were used, the contrast level of the transplanted part of the rabbit was high, and the rate of decrease of the contrast level was slow.

Claims (13)

Including liposomes having a lipid membrane;
The liposome contains sterols in the lipid membrane at a ratio of 0.2 to 1 part by weight with respect to 1 part by weight of phospholipid, and an iodine compound in the aqueous phase in the lipid membrane,
Moreover, a contrast agent for X-ray examination, wherein the liposome is produced by a supercritical carbon dioxide method. Including liposomes having a lipid membrane;
The liposome contains a cationic lipid in the lipid membrane at a ratio of 0.3 to 3% by mass with respect to the total lipid amount, and an iodine compound in the aqueous phase in the lipid membrane,
Moreover, a contrast agent for X-ray examination, wherein the liposome is produced by a supercritical carbon dioxide method. Including liposomes having a lipid membrane;
The liposome contains glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount, and contains an iodo compound in the aqueous phase in the lipid membrane,
Moreover, a contrast agent for X-ray examination, wherein the liposome is produced by a supercritical carbon dioxide method. Including liposomes having a lipid membrane;
The liposome contains glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total amount of lipid, and contains an iodo compound in the aqueous phase in the lipid membrane,
Moreover, a contrast agent for X-ray examination, wherein the liposome is produced by a supercritical carbon dioxide method.   The contrast medium for X-ray examination according to claim 1, wherein the liposome further contains a cationic lipid in a ratio of 0.3 to 3% by mass with respect to the total lipid amount in the lipid membrane. The contrast medium for X-ray examination according to claim 1, wherein the liposome further contains glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount. The contrast medium for X-ray examination according to claim 1, wherein the liposome further contains glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount. The contrast medium for X-ray examination according to claim 2, wherein the liposome further contains glycol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount. The contrast medium for X-ray examination according to claim 2, wherein the liposome further contains glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount. The contrast medium for X-ray examination according to claim 3, wherein the liposome further contains glycerol lipid in the lipid membrane at a ratio of 0.5 to 20% by mass with respect to the total lipid amount.   The contrast medium for X-ray examination according to claim 3, wherein the liposome further contains any two of sterols, cationic lipids, and glycerol lipids in its lipid membrane.   The contrast agent for X-ray examination according to claim 4, wherein the liposome further contains a sterol and a cationic lipid in its lipid membrane.   The contrast agent for X-ray examination according to claim 4, wherein the liposome further contains a sterol, a cationic lipid, and a glycol lipid in its lipid membrane.