JP2009096749A - Cholesterol derivative, liposome, and x-ray contrast agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new cholesterol derivative (1) having low toxicity against a living body, (2) having a high inclusion rate, (3) being excellent in the imaging of systemic blood vessels and liver, and (4) being excellent in imaging pancreas and further (5) being safe since most of the derivative is excreted to the outside of the body within 24 hr, and to provide a liposome and an X-ray contrast agent containing the liposome. <P>SOLUTION: This cholesterol derivative is expressed by general formula (1) [wherein, G represents a saccharide bonded through a monovalent residue forming an ether bond at an anomeric position of the saccharide]. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a novel cholesterol derivative, a liposome, and an X-ray contrast medium containing the liposome.

  In recent years, a cholesterol derivative modified with a sugar has been actively studied as a material for binding a sugar chain to the surface of a liposome attracting attention as a drug carrier.

  Hashida et al. Of Kyoto University have found that galactose, mannose, and fucose are modified with cholesterol, and liposomes containing this as a constituent component accumulate specifically in the liver (see, for example, Non-Patent Document 1). In addition, Mitsubishi Chemical Corporation has proposed a new compound in which sugar and cholesterol are combined with spermidine as a joint. Liposomes comprising the cholesterol derivative as a constituent component can encapsulate nucleic acid components (DNA, RNA fragments), and are expected to realize effective gene therapy by sending a target gene into target cells (for example, Patent Documents 1 and 2).

  However, all of the above sugar-modified cholesterols are cationic, so there are concerns about toxicity, and they are used for gene therapy, especially when they are intended for use in the contrast agent that is the subject of the present study. There was a problem which became a fatal fault because the quantity was remarkably large. Further, when preparing liposomes encapsulating a nonionic iodo compound, the cationic material has a drawback that the encapsulation rate is very low.

  Further, as sugars in the sugar chain, alpha 1,2 mannobiose disaccharide, alpha 1,3 mannobiose disaccharide, alpha 1,4 mannobiose disaccharide, alpha 1,6 mannobiose disaccharide, alpha 1,3 alpha 1,6 man Notoliose trisaccharide, oligomannose 3 pentasaccharide, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 6 octasaccharide, oligomannose 7 hemisaccharide, oligomannose 80 sugar chain, oligomannose 91 sugar, 3 '-Sialyl lactose trisaccharide, 6'-sialyl lactose trisaccharide, 3'-sialyl lactosamine trisaccharide chain, 6'-sialyl lactosamine trisaccharide, Lewis X type trisaccharide, sialyl Lewis X type tetrasaccharide, 2'-fucosyl Lactose trisaccharide, difucosyl lactose tetrasaccharide and 3-fucosyl lactose trisaccharide were used to modify the sugar on the surface Liposome is known (e.g., see Patent Document 3). The liposome is used as a therapeutic drug delivery for injecting a drug or gene, recognizing a target cell or tissue such as cancer, and locally delivering the drug or gene to the affected area. However, there is no known example in which the liposome also contains a nonionic iodo compound, and in the study by the present inventors, there was a drawback that the encapsulation rate and stability over time were extremely low.

  On the other hand, a contrast agent containing an iodine atom is generally provided as a low molecular weight organic iodine agent and is soluble in water. Therefore, as in urography, angiography, and CT contrast, it is usually administered intravenously into blood vessels. Used in Iodine contrast media stays in the blood vessels for several minutes after being administered, but rapidly distributes throughout the body, and is usually excreted by the kidneys into the urine. The low molecular weight, water-soluble iodinated contrast agent currently used has a rapid transition from a blood vessel to an organ or tissue, and it has been difficult to clearly image a vein, particularly among blood vessels.

  In order to increase the blood retention, some oil emulsion type contrast agents in which iodized garlic oil is dispersed with lecithin have been studied about 20 years ago (see, for example, Non-Patent Document 2). These have improved the retention in the blood as expected, and the imaging ability of the whole body blood vessels has been remarkably improved, but the problems causing serious side effects such as fever, nausea and anaphylactic shock cannot be solved and have not been put into practical use.

  On the other hand, in recent years, imaging of cancer diseases in specific organs has been strongly demanded. However, since the above-mentioned contrast agent does not have organ-specific accumulation, it is difficult to satisfy the demand. Liposomes using the above-mentioned cationic cholesterol are known to have high accumulation in the liver (see, for example, Non-Patent Document 1), and can be expected to be applied to contrast agents for liver diseases. Patent Document 3 discloses that alpha 1,2 mannobiose disaccharide chain, alpha 1,3 mannobiose disaccharide chain, alpha 1,4 mannobiose disaccharide chain, alpha 1,6 mannobiose disaccharide chain, alpha 1,3 alpha 1 , 6 Mannotriose trisaccharide chain, oligomannose 3 pentasaccharide chain, oligomannose 4b hexasaccharide chain, oligomannose 5 heptosaccharide chain, oligomannose 6 octasaccharide chain, oligomannose 7-9 sugar chain, oligomannose 80 sugar chain In addition, all liposomes bound with oligomannose 91 sugar chains have high directivity to blood, lung, brain, cancer tissue, heart and small intestine. Moreover, the liposome which couple | bonded the alpha 1,2 mannobiose disaccharide chain, the oligomannose 3 pentasaccharide chain, and the oligomannose 4b hexasaccharide chain has the high directivity to a liver. In addition, liposomes bound with alpha 1,2 mannobiose disaccharide chain, alpha 1,3 mannobiose disaccharide chain, alpha 1,3 alpha 1,6 mannotriose trisaccharide chain have high directivity to the spleen. Moreover, the liposome which couple | bonded the alpha 1,2 mannobiose disaccharide chain, the alpha 1,4 mannobiose disaccharide chain, and the alpha 1,6 mannobiose disaccharide chain has high directivity to a lymph node. Furthermore, liposomes bound with oligomannose 6 octasaccharide have high directivity to the thymus.

Liposomes bound with 3'-sialyl lactose trisaccharide chain, 6'-sialyl lactose trisaccharide chain, 3'-sialyl lactosamine trisaccharide chain and 6'-sialyl lactosamine trisaccharide chain are generally absorbed in the intestinal tract. The nature is extremely high. In addition, a liposome combined with a Lewis X-type trisaccharide chain, a sialyl Lewis X-type tetrasaccharide chain, a 2'-fucosyl lactose trisaccharide chain, a difucosyl lactose tetrasaccharide chain and a 3-fucosyl lactose trisaccharide chain is used as a vascular endothelium in a lesion. High directivity to various selectins existing on the cell surface. However, known liposomes have low accumulation at pancreatic disease sites, and it is currently impossible to expect application to contrast media for pancreatic diseases. Currently, pancreatic diseases, particularly metastatic cancers of the pancreas are difficult to detect at an early stage and are known as diseases with a high mortality rate, and the development of new contrast agents is eagerly desired.
JP-T-2004-522722 Special table 2004-520301 gazette International Publication No. 05/011632 Pamphlet Chem. Pharm. Bull. 53 (8), 871-880 (2005) Radiology, 216, 154-162 (2000)

  The present invention has been made in view of the above problems, and has as its purpose: (1) low toxicity to the living body, (2) high inclusion rate, (3) excellent systemic blood vessel and liver imaging, and ( 4) Providing a novel cholesterol derivative, liposome, and X-ray contrast medium containing the liposome, which are excellent in pancreatic imaging, and (5) are safe to be largely excreted outside the body within 24 hours. That is.

1. The above object of the present invention is achieved by the following configurations.
A cholesterol derivative represented by the following general formula (1):

(In the formula, G represents a sugar bonded via a monovalent residue that formed an ether bond at the anomeric position of the sugar;

Represents that the steric structure of the anomeric position of the sugar is α-form and / or β-form, and J represents a divalent alkylene group or alkyleneoxyalkylene group. The sugars are alpha 1,2 mannobiose disaccharide, alpha 1,3 mannobiose disaccharide, alpha 1,4 mannobiose disaccharide, alpha 1,6 mannobiose disaccharide, alpha 1,3 alpha 1,6 mannotriose trisaccharide, oligomannose 3 pentasaccharides, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 6 octasaccharide, oligomannose 79 saccharide, oligomannose 8 saccharide, oligomannose 91 saccharide, 3'-sialyl lactose trisaccharide 6'-sialyl lactose trisaccharide, 3'-sialyl lactosamine trisaccharide, 6'-sialyl lactosamine trisaccharide, Lewis X type trisaccharide, sialyl Lewis X type tetrasaccharide, 2'-fucosyl lactose trisaccharide, difucosyl It represents any one of lactose tetrasaccharide and 3-fucosyl lactose trisaccharide. )
2. 2. A liposome comprising at least one cholesterol derivative as described in 1 above, a nonionic iodo compound and a phospholipid.

  3. 3. An X-ray contrast medium comprising the liposome according to 2 above.

  According to the present invention, (1) low toxicity to the living body, (2) high inclusion rate, (3) excellent systemic blood vessel and liver imaging, (4) excellent pancreatic imaging, (5) Furthermore, it is possible to provide a novel cholesterol derivative, a liposome, and a contrast medium for X-rays containing the liposome, which have the safety of being largely excreted outside the body within 24 hours.

  As a result of intensive studies, the present inventors have found that the cholesterol derivative having the specific structure represented by the general formula (1) has (1) low toxicity to the living body, (2) high inclusion rate, (3) systemic blood vessels and X-rays that are excellent in imaging of the liver, (4) excellent in imaging of pancreas, and (5) a novel cholesterol derivative that has the safety of being largely excreted outside the body within 24 hours. It has been found that a contrast medium for imaging (hereinafter also referred to as a contrast medium) is an excellent contrast medium.

  Hereinafter, the present invention will be described in detail.

<< Cholesterol derivative represented by the general formula (1) >>
First, the cholesterol derivative represented by the general formula (1) of the present invention will be described in detail.

  In the general formula (1), G represents a sugar bonded through a monovalent residue that forms an ether bond at the anomeric position of the sugar, and the sugar is an alpha 1,2 mannobiose disaccharide, an alpha 1,3 mannobiose disaccharide. Sugar, alpha 1,4 mannobiose disaccharide, alpha 1,6 mannobiose disaccharide, alpha 1,3 alpha 1,6 mannotriose trisaccharide, oligomannose 3 pentasaccharide, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, Oligomannose 6-octasaccharide, oligomannose-79 kucrose, oligomannose-eight sugar chain, oligomannose-nine sugar, 3'-sialyl lactose trisaccharide chain, 6'-sialyl lactose trisaccharide, 3'-sialyl lactosamine tri Sugar chain, 6'-sialyl lactosamine trisaccharide, Lewis X type trisaccharide, sialyl Lewis X type tetrasaccharide, 2'-fucosyl lactose trisaccharide, di It represents a cosyl lactose tetrasaccharide and 3- fucosyl any one of the sugar sill lactose trisaccharide.

Represents that the steric structure of the anomeric position of the sugar is α-form and / or beta-form, but each of α-form and β-form may be used alone, or in any ratio of α-form and β-form. It may be a mixture. J represents a divalent alkylene group or alkyleneoxyalkylene group, which may be linear or branched, and is preferably a linear alkylene group or alkyleneoxyalkylene group. The alkyleneoxyalkylene group is preferably an ethyleneoxyethylene group or a polyethyleneoxyethylene group. The total number of carbon atoms is preferably 2 to 12, and particularly preferably 2 to 8. The alkylene group and alkyleneoxyalkylene group may have a substituent, but are preferably unsubstituted. Specific examples of the substituent include an alkoxy group, a carbamoyl group, an acylamino group, a sulfamoyl group, a sulfonamide group, and an ester group.

  Next, specific examples of the compound represented by the general formula (1) of the present invention are shown.

  However, the present invention is not limited to these. The synthesis method of these exemplary compounds is shown in the Examples.

<< Liposome containing cholesterol derivative, nonionic iodine compound, and phospholipid >>
The liposome of the present invention is formed from a lipid bilayer membrane in the same manner as a generally known liposome. In general, phospholipids are preferably used as components of the lipid membrane. The phospholipid used in the present invention is generally known and is not limited. Phospholipids such as phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, cardiolipin, and sphingomyelin. Phospholipids derived from egg yolk, soybean 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 phosphatidylenolamine, dipalmitoyl phosphatidylethanol Examples include amines and distearoyl phosphatidylethanolamine.

  These phospholipids may be used alone or in combination of two or more.

  The cholesterol derivative of the present invention is a component constituting the above-mentioned lipid bilayer, and the amount used is 0.05 to 1.5 parts by mass, preferably 0.2 to 1 part by mass with respect to 1 part by mass of phospholipid. A ratio of 1 part by mass, more preferably 0.3 to 0.8 part by mass is desirable.

  The nonionic iodo compound according to the present invention is a component encapsulated in the above-mentioned liposome, but is preferably water-soluble, specifically an iodinated phenyl group such as 2,4,6-triiodophenyl. Nonionic iodo compounds having at least one group are preferred. Specific preferred nonionic iodo 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.

  Nonionic iodo compounds are not limited to these examples. Moreover, a nonionic iodo compound may be used independently or may be used in combination of 2 or more type. In addition, in this specification, a compound may be mentioned including the salt, hydrate, etc. other than a free form. Particularly suitable iodine compounds include iomeprol, iopamidol, iotrolan, iodixanol, etc., which are highly hydrophilic and do not increase osmotic pressure even at high concentrations. Dimer nonionic iodo compounds such as iotrane and iodixanol have high iodine loading efficiency, and even if a contrast agent having the same iodine concentration is prepared, the total number of moles is low, and thus there is an advantage of lowering the osmotic pressure.

  The concentration of the nonionic iodo compound according to the present invention can be arbitrarily set based on factors such as the nature of the compound, the intended route of administration of the preparation, and clinical indicators. The amount of the iodine compound encapsulated in the liposome is typically 5 to 40% by mass, preferably 5 to 35% by mass, and more preferably 10 to 25% by mass of the total iodine compound added. To bring the concentration of the nonionic iodine compound within this range, the addition of a contrast agent solution containing the nonionic iodine compound (for example, 306.2 mg / ml of JP iopamidol solution (iodine content 150 mg / ml)) Adjust the amount.

  Since the encapsulation rate is below the limit of the fine packing of the liposome particles, the retention stability of the contrast medium in the liposome is not impaired. The component of the liposome of the present invention is not limited to the above, and other components can be added. As an example, FEBS Lett. 223, 42 (1987); Proc. Natl. Acad. Sci. , USA 85, 6949 (1988), and the like, Chem. Lett. 2145 (1989); Biochim. Biophys. Acta 1148, 77 (1992) and the like, glucuronic acid derivatives described in Biochim. Biophys. Acta 1029, 91 (1990); FEBS Lett. 268, 235 (1990) and the like, but are not limited thereto.

<Method for forming liposome>
The liposomes of the present invention may be formed by any method available to those skilled in the art. Examples of the forming method include Ann. Rev. Biophys. Bioeng. 9, 467 (1980), “Liposomes” (edited by MJ Ostro, MARCELL DEKER, INC.) And the like. Specific examples include sonication, ethanol injection, French press method, ether injection method, cholic acid method, calcium fusion method, freeze-thaw method, reverse phase evaporation method, etc. Absent. The structure of the liposome is not particularly limited, and may be any form such as unilamellar or multilamellar. Moreover, it is also possible to mix | blend the 1 type, or 2 or more types of an appropriate compound inside a liposome.

  An example of the forming method is shown below. Components such as phospholipids, stabilizers and antioxidants are mixed in an organic solvent such as chloroform in the flask, the organic solvent is distilled off, and then vacuum drying is performed to form a thin film on the inner wall of the flask. Next, an aqueous solution of a nonionic iodo compound is added to the flask and vigorously stirred to obtain a liposome dispersion. The obtained liposome dispersion is centrifuged, and the supernatant is decanted to remove the unencapsulated compounds and the like. If necessary, the liposome of the present invention can be obtained by adding a solution of a lipid derivative of a hydrophilic polymer and heating the solution.

  The liposome of the present invention can also be obtained by adding a solution of a lipid derivative of a hydrophilic polymer during mixing of the constituent components.

  As another method, the liposomes of the present invention can be obtained by mixing the above-described components and discharging them at high pressure using a high-pressure discharge type emulsifier.

The above-mentioned liposome-formed product always contains an organic solvent. 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 form the liposome used in the present invention, it is preferable to use a liposome forming method using supercritical or subcritical carbon dioxide as a method that can avoid the above problems. Carbon dioxide has a critical temperature of 31.1 ° C. and a critical pressure of 75.3 kgf / cm ² (7.38 MPa), and is relatively easy to handle. This is preferable because a high-purity fluid is inexpensive and easily available. A suitable pressure of carbon dioxide in the supercritical state (including subcritical state) used in the present invention is 80 to 500 kgf / cm ² (7.8 to 49 MPa), preferably 90 to 400 kgf / cm ² (8.8). -39 MPa), more preferably 100-200 kgf / cm ² (9.8-20 MPa). Moreover, as temperature of the carbon dioxide gas of a suitable critical state, it is 32-200 degreeC, Preferably it is 35-100 degreeC, More preferably, it is 40-80 degreeC. Within these ranges, a supercritical state (including a subcritical state) is preferably obtained by appropriately selecting and combining the temperature and pressure.

  The preparation method of liposome using supercritical carbon dioxide 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. The liposome membrane constituent material of the present invention is dissolved in supercritical (or subcritical) carbon dioxide, or 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 achieve super It may be dissolved in a critical state.

  At this time, when a solubilizing agent is added, the membrane lipid component is sufficiently dissolved in the supercritical carbon dioxide in a short time, so that the ratio of the single membrane liposome in the liposome to be formed increases. As a solubilizer, it is desirable to use one or more of lower alcohols, glycols, glycol ethers and the like in combination. For example, alcohols may be used as a co-solvent at a ratio of 0.1 to 10% by mass, preferably 1 to 8% by mass of supercritical carbon dioxide. A more preferred anti-proliferative agent is ethanol from the viewpoint of safety.

  Subsequently, an aqueous solution containing the nonionic iodo compound according to the present invention and, if necessary, a known formulation aid, is continuously added to the resulting lipid mixture to form an aqueous phase / carbon dioxide emulsion. When the system is depressurized and carbon dioxide is discharged, an aqueous dispersion in which liposomes encapsulating the nonionic iodine compound according to the present invention are dispersed is generated.

  The ratio of the iodo compound encapsulated in the liposome also depends on the ratio between the total amount of lipid for the liposome and the aqueous solution containing the iodo compound (and, if necessary, a formulation aid). For smooth formation of liposomes and efficient encapsulation of iodo compounds in the lipid membrane, the ratio between the total amount of lipid used and the aqueous solution containing the encapsulated substance must also be adjusted. The total amount of lipid is the total mass of all phospholipids constituting the liposome membrane, cholesterol derivatives represented by the general formula (1), other sterols, and other added lipids. A solvent containing the liposome membrane constituent material is mixed with 1 liter of the aqueous solution at a ratio of the total lipid amount in the range of 5 to 150 mmoles, preferably in the range of 30 to 120 mmoles, more preferably in the range of 50 to 100 mmoles. In such a case, the encapsulation of the nonionic iodo compound according to the present invention in the liposome proceeds well, and as a result, the retention efficiency of the iodo compound is also improved.

  Next, the size and distribution of liposome particles will be described in detail. The “center particle diameter” refers to the particle diameter having the highest frequency of appearance in the particle distribution. The particle size or particle size means the particle diameter. The adjustment of the particle size can be carried out according to the formulation or process conditions. For example, when the supercritical pressure is increased, the liposome particle size formed is decreased. In order to make the distribution of the liposome particle size in a narrower range, the prepared liposome suspension may be forcibly permeated through a filtration membrane having a fixed pore size, preferably a polycarbonate membrane. In this case, by passing through a hydrostatic extrusion apparatus equipped with a filter having a pore size of 0.05 to 0.4 μm, preferably 0.1 to 0.4 μm, more preferably 0.15 to 0.2 μm as a filtration membrane, In addition to reducing the number of lipid membranes in the liposome multilayer membrane, it is possible to efficiently prepare uniform liposomes having an optimal size of 100 to 300 nm as the central particle size. Specifically, the filter is forced to permeate using various hydrostatic extrusion apparatuses such as “Extruder” (trade name, manufactured by NOF Liposome). As the filter, a polycarbonate type, a cellulose type or the like can be appropriately used. By incorporating such an “extrusion” operation step, there is an advantage that, in addition to the above sizing, it is possible to exchange the liposome dispersion and filter sterilize together. Subsequently, the liposome dispersion may be purified by removing unretained drug by a method such as centrifugation or gel filtration, or may be optionally subjected to operations such as concentration, dilution, and lyophilization. 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.

<Contrast agent for X-ray>
Next, the contrast agent containing the liposome of the present invention will be described in detail.

A contrast agent of the invention formulated for injection should only have a moderate viscosity to allow for quick and convenient injection. The viscosity is less than 10.20 × 10 ⁻⁴ kgf · s / m ² (10 mPa · s, 10 cP), or preferably less than 5.10 × 10 ⁻⁴ kgf · s / m ² (5 mPa · s, 5 cP), Or more preferably, it is less than 2.04 × 10 ⁻⁴ kgf · s / m ² ( ² mPa · s, ² cP). Also, the contrast agent of the present invention formulated for injection should not have excessive osmotic pressure. This is because it can increase toxicity. The osmotic pressure is less than 3000 milliosmol / kg, or preferably less than 2500 milliosmol / kg, or most preferably less than 900 milliosmol / kg.

  The contrast agent of the present invention can contain salts derived from inorganic or organic acids and bases. Specific examples of the salt include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, Cyclopentanepropionate, digluconate, dodecyl sulfate, ethane sulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrogen bromide Acid salt, hydroiodide salt, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectate, Persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, toshi Acid salts, undecanoate salts, ammonium salts, alkali metal salts (eg sodium and potassium salts), alkaline earth metal salts (eg calcium, magnesium and zinc salts), salts with organic bases (eg dicyclohexylamine salts, N-methyl-D-glucamine), and salts having amino acids (for example, arginine, lysine). Basic nitrogen-containing groups also include lower alkyl halides (eg, methyl, ethyl, propyl and butyl chloride, bromide and iodide), dialkyl sulfates (eg, dimethyl, diethyl, dibutyl and diamyl sulfate), long chain halides (eg, Can be quaternized with agents such as decyl, lauryl, myristyl and stearyl chloride, bromide and iodide), aralkyl halides (eg, benzyl and phenethyl bromide) and others. Thereby, a water-soluble or oil-soluble or water-dispersible or oil-dispersible product is obtained. Preferred salts of the present invention are N-methyl-D-glucamine, calcium and sodium salts.

  The contrast agent of the present invention can contain any carrier, adjuvant or vehicle. Carriers, adjuvants and vehicles that can be used in the contrast agents of the present invention are ion exchange materials, alumina, aluminum stearate, lecithin, serum proteins (eg, human serum albumin), buffering materials (eg, phosphate), glycine, sorbine Acids, potassium sorbate, TRIS (tris (hydroxymethyl) aminomethane), partial glyceride mixtures of vegetable saturated fatty acids, water, salts or electrolytes (eg protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride , Zinc salt), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based material, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, wax, polyethylene-polyoxypropylene - block polymers, including polyethylene glycol and lanolin, and the like.

  The contrast agents of the invention may be in the form of sterile injectable pharmaceutical preparations (eg, sterile injectable aqueous or oleaginous suspension). This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Sterile injectable formulations are also sterile injectable solutions or suspensions (eg, as solutions in 1,3-butanediol) in non-toxic parenterally acceptable diluents or solvents. It may be. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic saline. In addition, conventionally, sterile, fixed oils are used as solvents or suspending media. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids (eg oleic acid and its glyceride derivatives) are similar to natural pharmaceutically acceptable oils (eg olive oil or castor oil), in the preparation of injectables, in particular their polyoxyethylated variants. Useful in the product. These oil solutions or suspensions may also contain a long chain alcohol diluent or dispersant (eg, Ph. Helv or similar alcohol).

  The contrast agent of the present invention can be administered orally, parenterally, by inhalation spray, topical, rectal, nasal, buccal, vaginal or conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Can be administered via a reservoir embedded in a dosage formulation containing As used herein, the term “parenteral” refers to subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. including.

  When administered orally, the pharmaceutical compositions of the invention can be administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions or solutions. When tablets are used orally, commonly used carriers include lactose and corn starch. Typically, a lubricant such as magnesium stearate is also added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring, or coloring agents may also be added. Alternatively, when administered in suppository form for rectal administration, the contrast agents of the invention are admixed with suitable nonirritating excipients that are solid at room temperature but liquid at rectal temperature. Can be prepared, so that it dissolves in the rectum and releases the drug. Such materials include cocoa butter, beeswax and polyethylene glycol.

  In addition, as described above, the contrast agent of the present invention is used particularly when the treatment target includes an area or organ (including the eye, skin, or lower intestinal tract) that is easily accessible by topical application. Can be administered topically. Appropriate topical formulations are readily prepared for each of these areas or organs.

  Topical application for the lower intestinal tract can be made with a rectal suppository formulation or a suitable enema formulation. Topically-transdermal patches may also be used. For topical application, the contrast agents of the invention may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the contrast agents of the present invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the contrast agents of the present invention may be formulated in a suitable lotion or cream containing the active ingredient suspended or dissolved in one or more carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

  For ophthalmic use, the contrast agents of the present invention may or may not contain a preservative (eg, benzylalkonium chloride). It can be formulated as a micronized suspension in pH adjusted isotonic sterile saline, or preferably as a solution in pH adjusted isotonic sterile saline. Alternatively, for ophthalmic use, the contrast agent of the invention can be formulated in an ointment such as petrolatum.

  For administration by nasal aerosol or inhalation, the contrast agents of the invention are prepared according to techniques well known in the pharmaceutical formulation art and are benzyl alcohol or other suitable preservatives, absorption enhancers that increase bioavailability. , Fluorocarbons, and / or other conventional solubilizers or dispersants may be prepared as a solution in saline.

  Dosing depends on the sensitivity of the diagnostic imaging device as well as the composition of the contrast agent. For example, for MRI imaging, contrast agents containing the cholesterol derivatives of the present invention are generally more effective than contrast agents containing paramagnetic materials having a lower magnetic moment, such as iron (III). Requires low dosing. Preferably, dosage is in the range of about 0.001-1 mmol / kg body weight of active metal-ligand complex per day. More preferably, dosage is in the range of about 0.005 to 0.05 mmol / kg body weight per day.

  However, the specific dosing regimen for any particular patient will also include various factors (including age, weight, health status, sex, therapeutic diet, administration time, excretion rate, combination of drugs, and the judgment of the treating physician) It should be understood that it depends on

  In the present invention, X-ray imaging is performed following administration of an appropriate dose of contrast agent. X-ray imaging of the present invention is based on cerebral angiography, selective angiography, limb angiography, venous angiography by digital X-ray imaging, contrast imaging in computer tomography, venous urography, etc. Imaging is preferred. Since the contrast agent of the present invention is excellent for the imaging of whole body blood vessels and the liver, particularly the pancreas, abdominal contrast imaging in selective angiography and computed tomography is the optimal imaging method.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these. Unless otherwise specified, “%” in the examples represents “mass%”.

(Synthesis example)
Exemplified compounds 1,2Manb-O-chol were synthesized according to the following scheme (Scheme 1).

  10 g of molecular sieves 4A obtained by dissolving 13 g (17 mmol) of acetyl-trichloroacetimidoyl-1,2-mannobiose (1) and 8.6 g (17 mmol) of intermediate (2) in 200 ml of methylene chloride and sufficiently drying under reduced pressure. In addition, the mixture was stirred for 1 hour. Thereafter, the internal temperature was cooled to −20 ° C., and a solution obtained by diluting 0.02 ml (0.2 mmol) of trimethylsilyl triflate (TMSOTf) in 3 ml of methylene chloride was added dropwise over 30 minutes while maintaining the internal temperature. After completion of the dropwise addition, the mixture was stirred at the same temperature for 30 minutes and then returned to room temperature. The reaction solution was washed with saturated aqueous sodium hydrogen carbonate and pure water, and the organic layer was dried over magnesium sulfate. After the desiccant was filtered off, the solvent was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography (developing solvent: ethyl acetate: normal hexane = 1: 1) to obtain 17 g of intermediate (3) (yield) 90%). Next, 10 g (9 mmol) of the intermediate (3) was dissolved in a mixed solvent of 50 ml of dioxane and 100 ml of methanol, 1.7 ml (1.7 mmol) of 1M sodium methoxide solution was added and stirred for 2 hours. Thereafter, 3 g of cation exchange resin Dowex50W was added for neutralization, the resin was filtered off, and the solvent was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (developing solvent: methylene chloride: ethanol: 8: 1) to obtain 9.1 g (yield 85%) of the target product 1,2 Manb-O-chol. Total yield 77%. Elemental analysis confirmed the desired product.

Elemental analysis value (calculated value%): C, 62.76; H, 8.98; O, 26.60
(Measured value%): C, 62.66; H, 8.88; O, 26.65
Hereinafter, the compound of the present invention was synthesized by the same method as above. The results are shown in Table 1.

Example 1
[Method A for Forming Contrast Agent Containing Liposomes]
Dipalmitoyl phosphatidylcholine (50 nmol) (manufactured by Nippon Oil & Fats Co., Ltd., COATSOME MC-6060), cholesterol derivatives of the present invention listed in Table 1 and comparative cholesterol derivatives (10 nmol) and PEG-phospholipids (10 nmol) (Nippon Oil & Fat Co., Ltd.) Manufactured by SUNBRIGHT DSPE-020CN). Med. Chem. , 25 (12), 1500 (1982), dissolved in chloroform in an eggplant-shaped flask to form a homogeneous solution, and then the solvent was distilled off under reduced pressure to form a thin film on the bottom of the flask. This thin film was dried in a vacuum, and then a contrast agent solution containing a nonionic iodo compound (Japanese iopamidol solution 306.2 mg / ml (iodine content 150 mg / ml), trometamol 1 mg / ml, edetate calcium disodium ( EDTANa2-Ca) 0.1 mg / ml, pH adjusted to around 7 with appropriate amounts of hydrochloric acid and sodium hydroxide) was injected 5 ml. Next, ultrasonic irradiation (Branson, No. 3542 probe type oscillator, 0.1 mW) was carried out for 5 minutes under ice cooling to obtain a dispersion of liposomes containing a nonionic iodine compound.

The obtained dispersion was heated to 60 ° C. and filtered under pressure with a cellulose filter manufactured by Advantech, 1.0 μm. The obtained sample was sealed in a Biotech RC dialysis tube manufactured by Spectrapro, and dialyzed by immersing it in a large amount of physiological saline (9.0 × 10 ⁻¹ %, 1000 g), and could not be taken into the liposome. Nonionic iodine compounds and the like were removed.

  The obtained liposome-containing contrast agent is shown in Table 2.

[Method B for Forming Contrast Agent Containing Liposomes]
Dipalmitoyl phosphatidylcholine (50 nmol) (manufactured by Nippon Oil & Fats Co., Ltd., COATSOME MC-6060), cholesterol derivatives of the present invention listed in Table 1 and comparative cholesterol derivatives (10 nmol) and PEG-phospholipids (10 nmol) (Nippon Oil & Fat Co., Ltd.) The mixture of SUNBRIGHT DSPE-020CN) was charged into a special stainless steel autoclave, the inside of the autoclave was heated to 60 ° C., and then 13 g of liquid carbon dioxide was added. While stirring, the pressure in the autoclave was 50kgf / cm ² (4.9MPa), by reducing the volume of the autoclave raised to the 120kgf / cm ² (12MPa), the carbon dioxide supercritical state The lipids were dispersed and dissolved with stirring. In addition, with contrast, the contrast agent solution (JPO iopamidol solution 306.2 mg / ml (iodine content 150 mg / ml), trometamol 1 mg / ml, edetate calcium disodium (EDTANa2-Ca) 0.1 mg / ml The pH was adjusted to around 7 with appropriate amounts of hydrochloric acid and sodium hydroxide), and 5 ml was continuously injected with a metering pump over 50 minutes. After completion of the injection, the system was depressurized to discharge carbon dioxide to obtain a liposome dispersion containing a nonionic iodo compound.

The obtained dispersion was heated to 60 ° C. and filtered under pressure with a cellulose filter manufactured by Advantech, 1.0 μm. The obtained sample was sealed in a Biotech RC dialysis tube manufactured by Spectrapro, and dialyzed by immersing it in a large amount of physiological saline (9.0 × 10 ⁻¹ %, 1000 g), and could not be taken into the liposome. Nonionic iodine compounds and the like were removed.

  The obtained liposome-containing contrast agent is shown in Table 2.

[Method for Forming Contrast Agent Containing Comparative Liposomes (see International Publication No. 05/011632 Pamphlet Examples 29-32)]
Liposomes were prepared using the cholic acid dialysis method. That is, dipalmitoyl phosphatidylcholine, cholesterol, dicetyl phosphate, ganglioside and dipalmitoyl phosphatidylethanolamine in a molar ratio of 35: 40: 5: 15: 5 to a total lipid amount of 45.6 mg, respectively. Then, 46.9 mg of sodium cholate was added and dissolved in 3 ml of chloroform / methanol solution. The solution was evaporated and the precipitate was dried in vacuo to obtain a lipid membrane. This thin film was dried in a vacuum, and then a contrast agent solution containing a nonionic iodo compound (Japanese iopamidol solution 306.2 mg / ml (iodine content 150 mg / ml), trometamol 1 mg / ml, edetate calcium disodium ( EDTANa2-Ca) 0.1 mg / ml, pH adjusted to around 7 with appropriate amounts of hydrochloric acid and sodium hydroxide) was injected 5 ml.

  Next, ultrasonic irradiation (manufactured by Branson, No. 3542 probe type oscillator, 0.1 mW) was carried out for 5 minutes under ice cooling to obtain a dispersion of liposomes containing nonionic iodine compounds.

The obtained dispersion was heated to 60 ° C. and filtered under pressure with a cellulose filter manufactured by Advantech, 1.0 μm. The obtained sample was sealed in a Biotech RC dialysis tube manufactured by Spectrapro, and dialyzed by immersing it in a large amount of physiological saline (9.0 × 10 ⁻¹ %, 1000 g), and could not be taken into the liposome. Nonionic iodine compounds and the like were removed. 10 ml of this liposome solution was subjected to ultrafiltration using an XM300 membrane (Amicon Co., USA) and a CBS buffer (pH 8.5) to adjust the pH of the solution to 8.5.

  Next, 10 ml of a crosslinking reagent bis (sulfocuccinimidyl) suberate (BS3; Pierce Co., USA) was added and stirred at 25 ° C. for 2 hours. Thereafter, the mixture was further stirred overnight at 7 ° C. to complete the chemical binding reaction between the lipid dipalmitoylphosphatidylethanolamine and BS3 on the liposome membrane. Then, this liposome solution was subjected to ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.5). Next, 40 mg of tris (hydroxymethyl) aminomethane dissolved in 1 ml of CBS buffer (pH 8.5) was added to 10 ml of the liposome solution, stirred at 25 ° C. for 2 hours, and then stirred overnight at 7 ° C. to obtain lipids on the liposome membrane. The chemical binding reaction between BS3 bound to tris and tris (hydroxymethyl) aminomethane was completed. As a result, the hydroxyl group of tris (hydroxymethyl) aminomethane was coordinated on the lipid dipalmitoylphosphatidylethanolamine of the anticancer drug doxorubicin-encapsulated liposome membrane to make it hydrated and hydrophilic.

further. It was carried out by the coupling reaction method according to a previously reported technique (Yamazaki, N., Kodama, M. and Gabis, H.-J. (1994) Methods Enzymol. 242, 56-65). That is, this reaction is performed in a two-step chemical reaction. First, 43 mg of sodium metaperiodate in which ganglioside present on the liposome membrane surface is dissolved in 1 ml of TAPS buffer (pH 8.4) is added, and then at room temperature for 2 hours. After stirring and periodate oxidation, 10 ml of oxidized liposomes were obtained by ultrafiltration with an XM300 membrane and PBS buffer (pH 8.0). To this liposome solution, 20 mg of human serum albumin (HSA) was added and stirred at 25 ° C. for 2 hours. Next, 100 μl of 2M NaBH ₃ CN was added to PBS (pH 8.0) and stirred overnight at 10 ° C. to prepare liposomes. HSA was bound by a coupling reaction between the above ganglioside and HSA. Then, after ultrafiltration with an XM300 membrane and a CBS buffer (pH 8.5), 10 ml of an HSA-conjugated nonionic iodine contrast agent-encapsulated liposome solution was obtained.

Further, 50 μg of an alpha 1,6 mannobiose disaccharide chain (Calbiochem Co., USA) was added to a 0.5 ml aqueous solution in which 0.25 g of NH ₄ HCO ₃ was dissolved. After stirring at 37 ° C. for 3 days, 0.45 μm Filtration through a filter completed the amination reaction at the reducing end of the sugar chain to obtain 50 μg of a glycosylamine compound having an alpha 1,6 mannobiose disaccharide chain.

  Next, 1 mg of a cross-linking reagent 3,3′-dithiobis (sulfocuccinimidyl) propionate (DTSSP; Pierce Co., USA) was added to 1 ml of a part of the liposome encapsulating non-ionic iodine contrast medium added with HSA, followed by 2 hours at 25 ° C. The mixture was stirred overnight at 7 ° C., and ultrafiltered with an XM300 membrane and a CBS buffer (pH 8.5) to obtain 1 ml of liposome in which DTSSP was bound to HSA on the liposome.

  Next, 50 μg of the above-mentioned alpha 1,6 mannobiose disaccharide glycosylamine compound is added to the liposome solution, and the mixture is stirred at 25 ° C. for 2 hours, and then stirred overnight at 7 ° C., and the XM300 membrane and PBS buffer solution ( Ultrafiltration was performed at pH 7.2), and alpha 1,6 mannobiose disaccharide chain was bound to DTSSP on liposome membrane-bound human serum albumin.

  Next, 13 mg of tris (hydroxymethyl) aminomethane (Wako Co., Japan) was added to the liposome solution, and the mixture was stirred at 25 ° C. for 2 hours, and then stirred at 7 ° C. overnight, and the XM300 membrane and PBS buffer solution (pH 7. After ultrafiltration in 2), tris (hydroxymethyl) aminomethane was bound to DTSSP on liposome membrane-bound human serum albumin. As a result, 2 ml of nonionic iodinated contrast agent-encapsulated liposome (abbreviation: IH-A6) obtained by hydrophilizing linker protein (HSA) in which alpha 1,6 mannobiose disaccharide chain, human serum albumin and liposome are bound ( 2 mg of total lipid and 200 μg of total protein) were obtained.

  The obtained liposome-containing contrast agent is shown in Table 2.

[Test 1]
Measurement of Liposome Particle Size The average particle size of liposomes was determined for the sample (liposome dispersion) in Table 1 using a WBC analyzer (Nihon Koden Co., Ltd., A-1042). Those having an average particle diameter of 100 to 400 nm are preferable, and those having an average particle diameter of 600 nm or more are not preferable because aggregation occurs.

[Test 2]
Measurement of the encapsulation rate of iodo compounds The sample (liposome dispersion) in Table 1 was dialyzed with isotonic saline, ethanol was added after the dialysis was completed to destroy the liposomes, and the amount of iodo compound in the liposomes was determined by measuring the absorbance. Asked. The ratio with respect to the total amount of iodine compounds in the sample is expressed as the encapsulation rate (%).

  The results of Tests 1 and 2 are shown in Table 2.

  As can be seen from Table 2, the liposomes containing the cholesterol derivative of the present invention showed a good average particle size in both of the forming methods A and B, but the comparative cholesterol derivatives were all aggregated particularly in the forming method B. The occurrence particle size exceeds 600 nm, which is not preferable. In addition, the liposome using the cholesterol derivative of the present invention shows a good encapsulation rate, but the encapsulation rate of the liposome using the comparative cholesterol derivative is clearly low, especially in the formation method B. . This is probably because the comparative cholesterol derivative is cationic and has low solubility in supercritical carbon dioxide.

Example 2
Using the liposome-containing contrast medium (formation methods A and B) prepared in Example 1 and physiological saline, a contrast medium having an iodine amount of 2500 mg I / kg was prepared by injecting 3 ml per mouse weighing 30 g. .

[Test 3]
Acute toxicity test By administering this contrast medium from the tail vein of ddy mice, the number of mice that died within 24 hours after the administration was counted. Table 3 shows the results obtained with 20 specimens.

  As shown in Table 3, it can be seen that the contrast agent of the present invention has low toxicity to the living body.

Example 3
Using the liposome-containing contrast medium (formation method A) prepared in Example 1 and physiological saline, a contrast medium having an iodine amount of 250 mg I / kg was prepared by injecting 0.3 ml per mouse having a body weight of 30 g. .

[Test 3]
Evaluation of tissue distribution Distribution of this contrast agent was evaluated from the tail vein of ddy mice. The concentration of iodine in the liver, pancreas, and blood at 5 minutes, 30 minutes, 2 hours, and 24 hours after the injection was measured with an inductively coupled plasma emission spectrometer (Urtima-2, manufactured by Jyobin Yvon). Tables 4 and 5 show the average values of the results obtained when the number of specimens was 20. There were no specimens that died within 24 hours after administration or had serious disability.

  From Tables 4 and 5, the contrast agent of the present invention is superior to the comparative contrast agent in contrasting systemic blood vessels and livers, and also excellent in contrasting pancreas, and most of it is excreted outside the body within 24 hours. It can be seen that it has safety.

Claims (3)

A cholesterol derivative represented by the following general formula (1):

(In the formula, G represents a sugar bonded via a monovalent residue that formed an ether bond at the anomeric position of the sugar;

Represents that the steric structure of the anomeric position of the sugar is α-form and / or β-form, and J represents a divalent alkylene group or alkyleneoxyalkylene group. The sugars are alpha 1,2 mannobiose disaccharide, alpha 1,3 mannobiose disaccharide, alpha 1,4 mannobiose disaccharide, alpha 1,6 mannobiose disaccharide, alpha 1,3 alpha 1,6 mannotriose trisaccharide, oligomannose 3 pentasaccharides, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 6 octasaccharide, oligomannose 79 saccharide, oligomannose 8 saccharide, oligomannose 91 saccharide, 3'-sialyl lactose trisaccharide 6'-sialyl lactose trisaccharide, 3'-sialyl lactosamine trisaccharide, 6'-sialyl lactosamine trisaccharide, Lewis X type trisaccharide, sialyl Lewis X type tetrasaccharide, 2'-fucosyl lactose trisaccharide, difucosyl It represents any one of lactose tetrasaccharide and 3-fucosyl lactose trisaccharide. ) A liposome comprising at least one cholesterol derivative according to claim 1, a nonionic iodo compound, and a phospholipid. An X-ray contrast medium comprising the liposome according to claim 2.