JP2005179214A - Contrast medium for x-ray examination - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an X-ray contrast medium which is highly safe and has a proper half-life, and to provide a method for producing the same. <P>SOLUTION: This contrast medium for X-ray examination is characterized by containing liposomes containing a water-soluble and nonionic iodine compound and having a blood retention time of 0.25 to 4 hours as a half-life. The above-mentioned liposome is preferably a single membrane liposome. The central particle diameter of the liposome is preferably 50 to 500 nm. The liposome is especially preferably a PEG-reacted liposome containing the PEG having oxyethylene units of 10 to 3,500 in an amount of 0.1 to 30 mass % based on the lipid constituting the liposome. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

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

X-ray simple imaging 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. In contrast, it is difficult to obtain a high contrast image because the difference in X-ray absorption between soft tissues is small. 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 the contrast medium is rapidly discharged from the luminal site without interacting with the tissue or diseased site, it does not serve 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. As an examination method developed for tumor detection, not only an X-ray imaging method but also other methods with different measurement principles have been developed. 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. A method for imaging a tumor, liver, spleen, adrenal cortex, arteriosclerotic lesion, vascular pool, lymphatic system, etc. by dispersing in water underneath is disclosed. In these methods, by contrasting a contrast agent into a fine particle, the residence time in the body is lengthened to selectively contrast the diseased part. However, for that purpose, the proposed formulation method 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 discharge rate to the outside of the body is slow after imaging and the burden on the patient is large.
On the other hand, as a method for making a contrast medium 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 low antigenicity has been studied. For example, International Publications WO88 / 09165, WO89 / 00988, WO90 / 07491, JP07-316079 and JP2003-5596 propose liposomes containing ionic or nonionic contrast substances. Although these methods use liposomes that are highly safe as materials and have appropriate degradability in vivo, organic solvents, particularly chloroform and dichloromethane, are used as solvents for phospholipids constituting liposomes in the production process. Use chlorinated solvents such as Therefore, it has not yet 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 is not so much because it depends on other factors. 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.
JP-A-2003-119120 (Patent Document 2) discloses a method for producing cosmetics and skin external preparations containing liposomes using supercritical carbon dioxide, and includes hydrophilic medicinal ingredients and lipophilic medicinal ingredients. A production example of a skin external preparation 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.
Japanese Patent Laid-Open No. 7-316079 Japanese Patent Laid-Open No. 2003-119120

For high-accuracy X-ray examination and diagnosis, it is necessary that the contrast material stays in the blood for an appropriate time. That is, an extremely long half-life retention is not necessary for diagnostic imaging, and it is desirable that it be excreted quickly after diagnosis in view of the influence on the living body.
An object of the present invention is an X-ray contrast agent in which a water-soluble iodine-based compound that is nonionic is encapsulated in liposomes, and the contrast material has an adequate half-life retention in blood and is safe. It is to provide a high X-ray contrast medium and a method for producing the same.

  The configuration of the present invention for solving the above problems is as follows.

  The present invention includes a liposome containing a water-soluble and nonionic iodine-based compound, and the residence time of the contrast medium in blood is 0.25 to 4 hours as a half-life. Contrast agent.

The center particle size of the liposome of the contrast medium for X-ray examination is 50 to 500 nm.

  The liposome is substantially a monolayer liposome.

The liposome contained in the contrast medium for X-ray examination according to the present invention is a PEGylated liposome comprising 0.1 to 30% by mass of PEG having 10 to 3500 oxyethylene units with respect to the lipid constituting the liposome. preferable.

Furthermore, in the method for producing a contrast medium for X-ray examination according to the present invention, a phospholipid that forms a liposome is dissolved in supercritical carbon dioxide to produce a liposome containing a nonionic iodide compound therein. Is a manufacturing method characterized by
[Detailed Description of the Invention]
The contrast agent for X-ray examination of the present invention has a contrast substance that is convenient for diagnostic examination and exhibits a blood retention property that enables high-accuracy imaging. It is specified in the range of 25 to 4 hours.

In general, the blood concentration of a contrast medium administered to the circulatory system reaches a peak immediately after administration, and then the contrast medium extravasates in a short time, so that a rapid decrease occurs. In this case, since the contrast material is nonspecifically distributed in the cell gap of the tissue from within the blood vessel, there is no difference in concentration between the normal tissue and the abnormal tissue, and a sufficient contrast cannot be obtained. When a polymer carrier such as liposome is used, trapping by reticuloendothelial cells such as liver and spleen also contributes to disappearance in a short time. Thereafter, a slow decrease in the blood concentration of the contrast medium is observed depending on excretion.
In the contrast medium for X-ray examination of the present invention, since the contrast medium encapsulated in the liposome exists, the contrast medium in blood does not show a simple disappearance mode. As used herein, “retention in the blood” means that the X-ray contrast material in the blood vessel is excreted from the kidney or trapped in the liver or the like, but replaces the contrast material distributed in the organ tissue. However, it means circulating in the blood vessel for a certain time. The “half-life” as an index of the blood retention is the time until the peak concentration is halved with respect to the blood concentration of the contrast medium for X-ray examination. In the present specification, the contrast material of the contrast agent for X-ray examination does not distinguish between a contrast compound encapsulated in liposomes and a non-encapsulated contrast compound unless otherwise specified, and is referred to as a generic term thereof. To do. As will be described later, the contrast compound is a water-soluble and nonionic iodide compound.

In the contrast agent for X-ray examination of the present invention, a water-soluble nonionic iodide compound is encapsulated in liposomes having a relatively narrow particle size range. According to this configuration, the present invention defines a half-life in which the contrast compound is localized in the blood vessel for a certain period of time immediately after administration, resulting in blood kinetics resulting in improved contrast performance. That is, the liposome as the microcarrier behaves like an erythrocyte and has a suitable blood retention by appropriately standardizing its particle size. Furthermore, by making it non-antigenic, in addition to preventing capture loss by the immune system and efficiently delivering it to the target site, good targeting is possible. As a result, the X-ray contrast medium of the present invention can realize excellent tumor visualization and can improve the accuracy of diagnostic examination by X-ray contrast. As a result, the X-ray contrast medium according to the present invention can be used in a small amount as compared with the conventional X-ray contrast medium, and there is almost no accumulation in the body, so that toxicity and side effects are greatly reduced. 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 nonionic iodide compound in the present invention is not particularly defined as long as it has contrast properties. 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.

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

Preferred iodo compounds used in the contrast medium for X-ray examination of the present invention are iomeprol, iopamidol, iotrolan, iodixanol, etc., which are highly hydrophilic and do not increase osmotic pressure even at high concentrations. In particular, dimer iodine compounds such as iodolan and iodixanol have the advantage of 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 iodine compound in the contrast agent of the present invention can be arbitrarily set based on factors such as the nature of the contrast agent, the intended route of administration of the final preparation, and clinical indicators. The amount of the iodine compound encapsulated in the liposome is typically 5 to 90% by mass, preferably 5 to 70% by mass, and more preferably 20 to 70% by mass of the total iodine compound in the X-ray contrast medium. In particular, in order to sufficiently prevent destabilization due to the osmotic pressure effect of the liposome encapsulated with the iodine compound, the amount of the iodine compound encapsulated in the liposome is 5 to 25% by mass of the total iodine compound.
More preferably, the content is 5 to 20% by mass.
Liposomes The contrast agent for X-ray examination of the present invention is used by being encapsulated in liposomes in order to selectively and efficiently deliver the above-mentioned contrast compound to a target organ, tissue or the like, that is, a target site. The The contrast agent of the present invention uses liposome as a microcarrier to improve blood retention, achieve efficient drug delivery and targeting. In order to produce an EPR (Enhanced permeability and retention) effect that is effective for obtaining particularly excellent tumor visualization, it is essential that the liposome has these characteristics.

The X-ray contrast medium of the present invention can realize the targeting function through the particle size of the liposome encapsulating the contrast compound and the surface design thereof. In the contrast agent of the present invention, 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. The design of the liposome surface can obtain desired properties by changing the composition of membrane constituents including the type of phospholipid.
The adoption of active targeting that allows for higher levels of accumulation and selectivity of contrast agents should also be considered. As an example, introduction of polyethylene glycol (PEG) onto the liposome surface is extremely beneficial because the induction process to the target site can be controlled. In the X-ray contrast medium of the present invention, the retention in blood is further enhanced by adding PEG to the liposome surface, and it becomes difficult to be phagocytosed by reticuloendothelial cells such as the liver. X-ray contrast materials that have not reached cancerous tissues, diseased sites, etc. do not collect at normal sites, but are degraded and excreted outside the body before side effects appear. This can be achieved by appropriately controlling the stability in relation to the extracorporeal discharge time when designing the liposome. Since a water-soluble iodine-based compound is used as a contrast medium, it is possible to prevent harmful effects caused by being rapidly excreted in the urine via the kidney and staying in the body, delayed side effects, and the like.
The design and production method of the liposome used for the drug delivery system in the X-ray contrast medium of the present invention will be described in detail below.
Liposomes are usually formed from lipid bilayer membranes. As a component of the lipid membrane, usually phospholipid and / or glycolipid is preferably used.

Preferred neutral phospholipids in the liposome of the present invention include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like. These may be used alone or in combination.
Other phospholipids include egg yolk, soybean or other phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine, sphingomyelin, synthetically obtained phosphatidic acid, dipalmitoyl phosphatidylcholine (DPPC), distearoyl Phosphatidylcholine (DSPC), Dimyristolphosphatidylcholine (DMPC), Dioleylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylglycerol (DPPG), Distearoylphosphatidylserine (DSPS), Distearoylphosphatidylglycerol (DSPG), Dipalmitoylphosphatidylinositol (DPPI) ), Distearoyl phosphatidylinositol (DSPI), dipalmi Yl lysophosphatidic acid (DPPA), distearoyl lysophosphatidic acid (DSPA), and the like.

  These phospholipids are usually used alone, but may be used in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation. When neutral phospholipids and charged phospholipids are used in combination, the weight ratio is usually 200: 1 to 3: 1, preferably 100: 1 to 4: 1, and more preferably 40: 1 to 5: 1.

Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, galactosylceramide, galactosylceramide sulfate, lactosylceramide, sphingoglycolipids such as ganglioside G7, ganglioside G6, and ganglioside G4.
In addition to the above, other components can also be added as the membrane constituent of the liposome as necessary. Examples thereof include sterols such as cholesterol and cholesterol esters as membrane stabilizers or anchors for introducing polyalkylene oxide groups, and dialkyl phosphates such as dicetyl phosphate which is a charged substance.

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

In the present invention, polyethylene glycol (PEG) can be used as a component of liposomes for the intended purpose of the X-ray contrast agent. That is, a new function can be imparted by attaching PEG to the liposome. For example, the organ specificity of the contrast agent, the liposome has a hydrophilic tendency, or is difficult to be recognized by the immune system (so-called “stealthy” state), or increased in blood stability Can be expected. Specifically, since lipid components are easily stored in the liver, PEG is not used or liposomes with a low PEG content are used 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 Kupffer cells present in the capillary network of the liver 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. By introducing PEG, a hydrated layer is formed, so that the liposome is stabilized and the retention in blood is also improved. The function can be adjusted by changing the length of the oxyethylene unit of PEG, the ratio of introduction, or by further modifying the PEG. As PEG, polyethylene glycol having oxyethylene units of 10 to 3500, preferably 100 to 1000 is suitable. 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. Or you may introduce | transduce well-known various polyalkylene oxide groups on the liposome surface other than PEG as needed. Its length and amount used are the same as in the case of PEG.

PEGylation of liposomes can be prepared using known techniques. 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. Alternatively, in the liposome production method described later, a liposome is produced by including a polyethylene oxide (PEO) derivative such as phosphatidylethanolamine in the raw material phospholipids. Examples of this include DSPE-PEG. Japanese Patent Application Laid-Open No. 2002-37883 discloses a high-purity polyalkylene oxide-modified phospholipid for water-soluble polymer-modified liposomes with high safety and a method for producing the same. It is described that when a polyalkylene oxide-modified phospholipid having a low monoacyl content is used, the storage stability of the liposome dispersion is good.
Various methods have been proposed so far for preparing liposomes. Different preparation methods often result in significantly different morphology and properties of the final liposomes. The production method is appropriately selected according to the desired form and characteristics of the liposome. In general, liposomes are prepared by mixing lipid components such as phospholipids, sterols, and lecithins in containers with organic solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, THF, and the like. Yes. 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. The dispersions are then reduced by sonication or by the use of surfactants to reduce the size of the liposomes and the viscosity of the dispersion. 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. The critical temperature (31.1 ° C) and critical pressure (7.3 MPa) of carbon dioxide are relatively easy to handle, and because it is an inert gas, it remains harmless to the human body, and high purity fluids can be easily obtained at low cost. Is preferred. Furthermore, the liposome produced by this method has various preferable characteristics and advantages for encapsulating the iodine compound of the X-ray contrast agent as described later.
When liposomes are produced using supercritical or subcritical carbon dioxide, it is necessary to dissolve, disperse or mix the lipid component in carbon dioxide in a supercritical state (including a subcritical state). Such supercritical carbon dioxide is excellent in solubility because it easily penetrates into the substance. At that time, it is desirable to use one or two or more alcohols such as lower alcohols, glycols and glycol ethers as a co-solvent in order to further improve the solubility of the lipid component. For example, alcohol is 0.1 to 10% by mass of supercritical carbon dioxide, preferably 1 to 8%.
It is good to use it as a cosolvent in the ratio of the mass%. Among these, a more preferable dissolution aid is ethanol from the viewpoint of safety and ease of removal.

Suitable pressure of carbon dioxide in the supercritical state used in the production method of the present invention (including semi-critical state), 50~500kg / cm ^2, preferably 100~400 kg / cm ^2. Further, the temperature of the carbon dioxide gas in a suitable critical state is 25 to 200 ° C, preferably 31 to 100 ° C, and 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. A lipid component and, if necessary, PEG phospholipid are added to carbon dioxide in the supercritical state or subcritical state under the above-mentioned suitable pressure and temperature, and dissolved under stirring. In that case, the said dissolution aid of a lipid can be used together as needed. Subsequently, an aqueous solution of an iodine-based compound is continuously added to form an aqueous phase / carbon dioxide emulsion. In this emulsion system, it is presumed that the lipid component is in a micelle form and is separated and concentrated. After stirring for a while to stabilize the emulsion containing micelles, water is continuously added until the carbon dioxide phase and the aqueous phase are separated. As the aqueous phase increases, the phase transition of the system occurs, and excess carbon dioxide separates from the carbon dioxide / water emulsion via a two-phase system of water / carbon dioxide emulsion + carbon dioxide / water emulsion. Since liposomes are phase-inverted to the aqueous phase, when the system is depressurized and carbon dioxide is discharged, an aqueous dispersion in which the liposomes containing the iodine compound are dispersed is generated. In this case, an iodine compound may be contained in an aqueous phase other than the inside of the liposome. Since the aqueous solution is also encapsulated inside the liposome, the iodine compound exists mainly in the aqueous phase inside the liposome in addition to the external aqueous phase of the liposome, and is in a so-called “encapsulation” state. 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, the present invention, 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 X-ray imaging of the present invention. It is useful in the manufacture of agents.

The liposome of the present invention is substantially a single membrane liposome, that is, a liposome composed of a unilipidella vesicle in which a phospholipid bilayer is formed as one layer. Here, “substantially” means that the liposome is composed of a phospholipid bilayer generally recognized as one layer in an electron microscope observation by the following freeze replica method. In the X-ray contrast medium of the present invention, such a single membrane liposome contains at least 80%, preferably 90%, of the total amount of liposomes contained in the contrast medium.
Such a single membrane liposome can be efficiently produced by the above-described supercritical carbon dioxide and using a phase separation method with water. On the other hand, in the conventional method for preparing liposomes, liposomes composed of multilamellar vesicles (MLV) often exist in a considerable proportion. For this reason, in order to increase the ratio of single membrane liposomes, an operation such as irradiation with ultrasonic waves or passing through a filter having a constant pore size is further required. Single membrane liposomes are ML
Compared with V, there is also an advantage that the dose of liposome, in other words, the dose of lipid does not increase.
Single membrane liposomes, particularly large unilamellar veislcles (LUV), have the advantage of providing a large encapsulation capacity compared to multilamellar liposomes.
The liposome used in the contrast agent of the present invention is located between LUV having a particle size of 200 to 1000 nm and SUV (Small unilamellar vesicles) which are small unilamellar liposomes having a particle size of less than 50 nm. For this reason, the holding volume is also larger than that of SUV, and the trapping efficiency of iodine compounds, particularly water-soluble iodine compounds, in other words, the entrapment efficiency, will be remarkably excellent as will be described later. In addition, unlike MLV and LUV, they are not taken up by reticuloendothelial cells and rapidly disappear from the bloodstream.
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, the filtration membrane is 0.1
By passing through an extruder equipped with a filter having a pore diameter of ˜0.4 μm, it is possible to efficiently prepare liposomes having an optimal size having a central particle diameter 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 by excluding liposomes having a particle size of 3000 nm or more, inadequate retention in lung capillaries is avoided. However, liposomes with a particle size range of 150-3000 nm are not good tumor prone.

The center particle diameter of the contrast medium liposome of the present invention is usually 50 to 500 nm, preferably 50 to 300 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 making the particle size of the liposome in the range of 100 to 200 nm, more preferably 110 to 130 nm, the X-ray contrast material 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 medium 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 contrast material particles (liposome particles containing an iodine compound) stay in the blood for a long time and pass through blood vessels near 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, thereby achieving selective concentration and accumulation of the contrast material 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.
In contrast medium examination and diagnosis for X-ray examination, it is necessary that the contrast material stays in the blood for an appropriate time. When a contrast material of a conventional X-ray contrast agent is administered into a vein or the like, it quickly diffuses into the circulatory system, and the contrast material is nonspecifically distributed in the cell gap of the tissue from within the blood vessel. Such contrast materials are rapidly discharged soon after interacting with the target tissue site, and it is difficult to accurately grasp the state of the lesion. Conversely, an extremely long half-life is unnecessary for diagnostic imaging, and it is desirable that it be excreted rapidly outside the body after examination / diagnosis considering the effect on the living body. When the retention in the blood is 10 hours to several days, biotoxicity becomes a problem. Particularly in the case of a hydrophobic iodine compound, fat-soluble accumulation at a lipidic site becomes serious.
The X-ray contrast medium according to the present invention realizes blood retention that is convenient for diagnostic tests by encapsulating a water-soluble contrast compound in the liposome. The specific residence time in blood has a half-life of the contrast medium of 0.25 to 4 hours, more preferably 0.5 to 3 hours. If such a half-life is less than 0.25 hours, detailed examination / diagnosis becomes difficult, and a length exceeding 4 hours is not required for normal image diagnosis. Since the contrast material of the present invention in the body is a water-soluble contrast material encapsulated in a biocompatible and biodegradable carrier after imaging, its excretion is removed relatively quickly by urinary excretion via the kidney. Since the half-life is at most 4 hours, only 1.5% remains in the blood after 24 hours of administration (in the case of exponential decay).
It has low toxicity because there is almost no accumulation in the body.

The blood half-life can be calculated by measuring the blood radioactivity distribution rate over time after intravenous injection of an X-ray contrast medium, as shown in the Examples below.
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 delivery efficiency and retention stability 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, preferably 3 to 8, more preferably 5 to the liposome membrane lipid weight. It is desirable that it is contained in a weight ratio of 8.
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. Since the viscosity of the X-ray contrast medium depends on the amount of lipid in the liposome, the superiority of the single membrane liposome having excellent retention volume and encapsulation efficiency is clear. 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, 100% immediately after the liposome suspension drug is manufactured and separated
Even if encapsulation is achieved, it is described that the encapsulated components decrease as soon as possible based on destabilization due to the osmotic pressure effect (Japanese Patent Publication No. 9-505821).
The contrast agent 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, the buffer solution is a buffer solution 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. In this case, there is no restriction of miniaturizing the liposome particle size as in filtration sterilization. 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 material 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 as a chelating agent, α-tocopherol as an antioxidant, viscosity A regulator or the like may also be included. In order to localize within a blood vessel for a certain period of time immediately after administration, the lower the viscosity of the X-ray contrast agent, the easier it is to diffuse into the circulatory system from the blood vessel at the administration site.

  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 an actual diagnostic examination, 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. It is also expected.

A contrast agent having an appropriate half-life is prepared from the range of 0.25 to 4 hours as the blood half-life according to the purpose of X-ray examination, target organ, tissue characteristics, type of X-ray imaging apparatus, etc. be able to. This depends on the particle size of the liposome, the modification of the liposome surface, and the like as described above.

  In order to enhance the tumor visualization with a single administration through the EPR effect, it is necessary to increase the retention in the blood appropriately. On the other hand, a half-life of up to about 1 hour is preferred for the purpose of angiography for evaluating blood vessel distribution images, detecting blood vessel narrowing and stenosis.

The contrast medium of the X-ray contrast medium according to the present invention has a blood retention in the range of 0.25 to 4 hours as a blood half-life that is convenient for diagnostic examination. In addition, the EPR effect is exhibited because of its good retention in the blood, and as a result, it is possible to selectively concentrate and accumulate on the target diseased site or tissue, particularly cancer tissue, thereby enabling high-resolution imaging. . After imaging, since the iodine compound is water-soluble, it is excreted from the body relatively quickly.

  The X-ray contrast medium according to the present invention requires only a small amount of use compared to conventional X-ray contrast mediums and has little accumulation in the body, so that it has far less toxicity and side effects. Therefore, the burden on the patient receiving the administration is reduced.

〔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.

Dipalmitoylphosphatidylcholine (DPPC) and carbon dioxide were charged together with ethanol into a stainless steel autoclave, and the inside of the autoclave was stirred at 60 ° C. and 300 kg / cm ² to dissolve DPPC in supercritical carbon dioxide. While stirring this supercritical carbon dioxide solution, the iomeprol solution (816.5 mg of iomeprol was heated and dissolved in water for injection, dissolved in ascorbic acid to 20 mM, and further dissolved in 1 mg of trometamol. The pH was adjusted to physiological pH with dilute hydrochloric acid, and finally, a solution prepared by adding water for injection to 1.0 ml was continuously injected with a metering pump. Thereafter, the system was depressurized to discharge carbon dioxide, and a dispersion of liposomes containing iomeprol was obtained.

  Further, the obtained dispersion was put in a glass vial and autoclaved at 121 ° C. for 20 minutes to obtain a contrast agent.

The particle size of the liposome in the obtained contrast agent was measured by freeze-fracture TEM method, and the particle size with the highest appearance frequency in the particle size distribution was defined as the central particle size. The particle size of the liposomes in the contrast agent was 130 nm. Further, according to the morphology observation by the same method, the completed liposome was a monolayer liposome. This is designated as sample 1.

    The contrast medium of Sample 1 was diluted with isotonic glucose solution to a concentration of 50 mg iodine / ml. When this solution was intravenously injected into rats, X-ray images showed that the solution was concentrated in the liver, especially compared to a contrast agent solution containing the same amount of iodine not encapsulated in liposomes injected for comparison. Was observed. In addition, with the passage of time, the contrast level in the liver decreased in parallel with all other organ contrast levels, indicating that most of iomeprol was excreted in the urine.

The contrast medium sample 1 obtained in Example 1 was diluted with an isotonic glucose solution to a concentration of 50 mg iodine / ml. This solution was injected intravenously into rats with multiple liver cancer metastases. The tumor metastasis portion had a high contrast level, and a tumor with a diameter of about 5 mm was observed. Furthermore, the decrease in the level of contrast enhancement of the tumor was later than that of other organs over time.

A contrast agent was prepared in the same manner as in Example 1 except that liposomes having particle sizes of 70 nm, 180 nm, and 220 nm were prepared by controlling the pressure and temperature. Samples 2, 3, and 4 are arranged in ascending order of particle diameter.

A contrast agent was prepared in the same manner as in Example 1 except that 40 g of PEG having 2000 oxyethylene units was added. Its particle size was 120 nm. This is designated as Sample 5.

  Contrast agent samples 2 to 4 prepared in Example 4 were evaluated in the same manner as in Example 2. It was observed in the X-ray image that the contrast medium prepared in Example 4 was concentrated and distributed in the liver after injection, compared to the non-liposomal contrast medium, regardless of the particle diameter.

Contrast agent samples 2, 3, and 5 prepared in Examples 4 and 5 were evaluated in the same manner as in Example 3.
In contrast agent sample 2 containing liposomes having a particle size of 70 nm, the contrast level of tumor was higher than that of Example 3 in contrast to the contrast agent of Example 3.
In contrast medium sample 3 containing liposomes having a particle size of 180 nm, the contrast level of tumor was lower than that of Example 3 in contrast to the contrast medium of Example 3.
The contrast medium sample 5 prepared in Example 5 had a high tumor partial contrast level and a slow decrease in contrast level.

  The contrast media of Sample 1, Sample 2 and Sample 4 were administered into rat veins, and after a certain period of time, the iodine content in various organs was examined.

Sprague-Dawley female rats (body weight 145 to 171 g) were injected with a total iodine dose of 250 mg / kg of each contrast medium sample solution. For comparison, a contrast medium solution not encapsulated in liposomes was injected so that the amount of iodine per kg of rats was the same. At 0.15, 0.25, 0.5, 1, 4 and 24 hours after infusion, the animals were sacrificed by exsanguination and liver, spleen and lungs were collected in addition to blood. Their iodine content was examined using FEA (X-ray fluorescence spectroscopy). The results (% of dose) are shown in Tables 1 to 4 and FIG.

  As shown in FIG. 1, the blood half-life of the contrast medium of the X-ray contrast medium of the present invention is about 0.35 for sample 1, about 0.8 for sample 2, and about 3.5 for sample 4.

FIG. 1 shows the time course of blood iodine content after administration of an X-ray contrast medium.

Claims (5)

A contrast medium for X-ray examination, comprising a liposome containing a water-soluble and nonionic iodine-based compound, wherein the contrast substance has a blood residence time of 0.25 to 4 hours as a half-life.   2. The contrast medium for X-ray examination according to claim 1, wherein the liposome has a central particle size of 50 to 500 nm. 3. The liposome according to claim 1, wherein the liposome is substantially a single membrane liposome.
A contrast medium for X-ray examination as described in 1. The said liposome is a PEGylated liposome comprising PEG having 10 to 3500 oxyethylene units in an amount of 0.1 to 30% by mass with respect to the lipid constituting the liposome. A contrast medium for X-ray examination as described in 1. The phospholipid that forms the liposome is dissolved in supercritical carbon dioxide to produce a liposome containing a nonionic iodine-based compound therein. A method for producing a contrast medium for X-ray examination.

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