JP2009143879A - Liposome and x-ray contrast medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liposome (1) being less toxic to the living body, (2) being high in encapsulation efficiency, (3) being excellent in capability of imaging systemic blood vessels and the liver, (4) being excellent also in capability of imaging the pancreas, and (5) being safe such that it is mostly excreted from the body within 24 hr and to provide an X-ray contrast medium containing the liposome. <P>SOLUTION: The liposome contains at least one glycolipid derivative represented by general formula (1), a nonionic iodine compound, and a phospholipid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

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

  In recent years, lipid derivatives modified with sugars have been actively studied as materials for binding sugar chains to the surface of liposomes that are attracting attention as drug carriers.

  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 are expected to be able to encapsulate nucleic acid components (DNA, RNA fragments) and to realize effective gene therapy by sending the target gene into target cells ( For example, see Patent Documents 1 and 2).

  However, all of the above sugar-modified cholesterols are cationic, so there is a concern about toxicity. Especially when the use in the contrast agent which is the research object of the present inventors is taken into consideration, it is not suitable for gene therapy. There was a problem which became a fatal fault because the amount used 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 mannotri Aose trisaccharide, oligomannose 3 pentasaccharide, oligomannose 4b hexasaccharide, oligomannose 5 heptasaccharide, oligomannose 6 octasaccharide, oligomannose 79 sugar, 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 Liposomes modified with trisaccharide, difucosyl lactose tetrasaccharide and 3-fucosyl lactose trisaccharide Beam is known (for example, 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.

  Furthermore, liposomes using lipids modified with specific oligosaccharide chains are known (see, for example, Patent Document 4). The liposome has excellent stability in blood and can selectively deliver a drug to an organ such as the liver. 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 shampoo oil is dispersed with lecithin have been studied about 20 years ago (for example, see 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 liposomes having high directivity to various selectins present on the surface of blood vessel, lung, brain, cancer tissue, heart, small intestine, liver, spleen, lymph node, thymus, and vascular endothelial cells in lesions. Is described. Patent Document 4 describes a liposome having high directivity to the liver.

However, the above-mentioned known liposomes have low accumulation at the pancreatic disease site, and it is currently impossible to expect application to a contrast agent for pancreatic disease. 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 JP 2007-153787 A 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) To provide a novel liposome that is excellent in pancreatic imaging and (5) is safe to be excreted most of the body within 24 hours, and an X-ray contrast medium containing the liposome. .

  The above object of the present invention is achieved by the following configurations.

  1. A liposome comprising at least one glycolipid derivative represented by the following general formula (1), a nonionic iodo compound, and a phospholipid.

(In the formula, R ¹ and R ² represent a hydrogen atom or a group selected from the following formulas (Q1) to (Q5), and R ³ represents a group selected from the following formulas (A1) to (A4). )

(Wherein R ⁴ and R ⁵ represent the same or different alkyl group having 5 to 20 carbon atoms.)
2. 2. An X-ray contrast medium comprising the liposome according to 1 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, the novel liposome which has the safety | security which most excretes outside a body within 24 hours, and the contrast agent for X-rays containing this liposome can be provided.

  As a result of intensive studies, the present inventors have found that a contrast agent containing a liposome containing at least one glycolipid derivative represented by the general formula (1), a nonionic iodo compound, and a phospholipid is (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, most of it outside the body within 24 hours It has been found that a novel contrast medium for X-rays (hereinafter also simply referred to as a contrast medium) having the safety of excreting is an excellent contrast medium.

  Hereinafter, the present invention will be described in detail.

<< Glycolipid derivative represented by general formula (1) >>
In the present specification, sugar residues constituting a sugar chain are expressed as Mannose residue as Man, Glucose residue as Glc, N-acylglucosamine residue as GlcNAc, Galactose residue as Gal, and Sialic acid residue as NeuAc. There is a case.

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

R ¹ and R ² in the general formula (1) may be the same or different and are appropriately selected from the group consisting of a hydrogen atom and formulas (Q1) to (Q5) according to the application. In particular, R ¹ and R ² are preferably the formula (Q1) or (Q2), particularly (Q1).

R ⁴ and R ⁵ in the general formula (1) are carbons such as linear or branched pentyl, hexyl, octyl, decanyl, isooctyl, dodecanyl, tetradecyl, 2-methyltetradecyl, pentadecyl, hexadecyl, octadecyl, nonadecyl, icosyl, etc. It is a C5-C20 alkyl group, Preferably it is a C8-C20 alkyl group, More preferably, it is a C10-C20 alkyl group. When the number of carbon atoms is less than 5, it becomes difficult to form a liposome structure, and when the number of carbon atoms exceeds 20, the handleability is poor.

  As a typical example of the glycolipid derivative according to the present invention, a compound in which the N-binding site is asparagine is shown below.

  Hereinafter, these glycolipid derivatives may be simply expressed. For example, the formula (i) is represented by “Asn-linked 2,6-disialo-glycan C18 lipid derivative”, and the formula (ii) is represented by “Asn-linked 2, “3-disialo-sugar chain C18 lipid derivative”, formula (iii) as “Asn-linked 2,6-monosialo-sugar chain C18 lipid derivative”, and formula (iv) as “Asn-linked 2,3-monosialo-sugar chain C18 lipid derivative” ”, Formula (v) as“ Asn-linked asialo sugar chain C18 lipid derivative ”, formula (vi) as“ Asn-linked diMan-GlcNAcC18 lipid derivative ”, and formula (vii) as“ Asn-linked diMan sugar chain C18 ”. The “lipid attractant”, the formula (viii) are shown as “Asn-linked 2,6-disialo sugar chain C14 lipid derivatives”, and the formula (xi) is shown as “Asn-linked 2,6-disialo sugar chain C14 lipid derivatives”.

  The glycolipid derivative according to the present invention can be produced by a method in which a sugar chain derivative having at least an amino group in the molecule and a bibranched carboxylic acid compound are reacted in the presence of a reaction activator. Specifically, the sugar chain derivative represented by the following general formula (2) is reacted with the carboxylic acid compound represented by the following general formula (3) or (4) in the presence of a reaction activator. Thus, the glycolipid derivative represented by the general formula (1) can be produced.

In the formula, R ¹ and R ² represent a hydrogen atom or a group selected from the above formulas (Q1) to (Q5), and R ⁶ represents an amino group or an asparagine residue.

Wherein, R ^4, R ⁵ may be the same or different, represents an alkyl group having 5 to 20 carbon atoms.

The sugar chain derivative represented by the general formula (2) includes a compound in which R ⁶ is an amino group (hereinafter sometimes referred to as “sugar chain amine”) and a compound in which R ⁶ is an asparagine residue (hereinafter referred to as “asparagine residue”). , Sometimes referred to as “sugar chain asparagine”). These sugar chain derivatives may be used alone or in combination of two or more. As a typical example of the compound represented by the general formula (2), sugar chain asparagine is shown below.

  Hereinafter, these sugar chain derivatives may be simply expressed. For example, the formula (ia) is indicated as “2,6-disialo sugar chain asparagine” or the like.

  The sugar chain asparagine can be prepared, for example, according to the method disclosed in International Publication No. 03/008431. This method is characterized in that individual sugar chain asparagine derivatives can be separated from a natural product-derived sugar chain asparagine mixture by introducing a fat-soluble protective group into the sugar chain asparagine and then derivatizing. . Examples of the fat-soluble protecting group include an Fmoc group, a t-butyloxycarbonyl (Boc) group, and a benzyl group, and among them, a functional group having high fat solubility is preferably used. In particular, the Fmoc group can be efficiently separated using a reverse phase column such as an ODS column, and is suitable even when sugars that are unstable to relatively acidic conditions such as sialic acid are present in the sugar chain. Available.

  The sugar chain asparagine into which the protecting group has been introduced can be separated and recovered using a gel filtration column, an HPLC column or the like, and further a sugar hydrolase such as galactose hydrolase, mannose hydrolase, N-acetylglucosamine hydrolase or the like. The target sugar chain asparagine can be obtained by adjusting the sugar chain structure and deprotecting the protecting group by a conventional method.

  The sugar chain amine can be prepared, for example, using the sugar chain asparagine as a starting material according to the methods disclosed in International Publication Nos. WO03 / 008431 and International Publication No. 05/010053.

  The carboxylic acid compound represented by the general formula (3) or (4) may be any compound of the general formula (3) and the formula (4), and may be used in combination.

In the present invention, an alkyl group having 12 to 20 carbon atoms in R ⁴ and R ⁵ is preferable in that the glycolipid derivative is a constituent component of the liposome. The usage-amount of a carboxylic acid compound is about 0.1-10 mol with respect to 1 mol of sugar chain derivatives.

  Examples of the reaction activator include N-hydroxysuccinimide and / or N-hydroxybenzotriazole (HOBT). The usage-amount of a reaction activator is about 0.1-10 mol with respect to 1 mol of sugar chain derivatives.

  The reaction is carried out, for example, at a temperature of about 0 to 80 ° C., preferably about 10 to 60 ° C., for about 10 minutes to 5 hours. The glycolipid derivative produced by the reaction can be separated and purified by a known method such as filtration or high performance liquid chromatography (HPLC). Identification of a compound can be performed by NMR and HPLC (ODS column) based on detection time in UV.

《Phospholipid》
Examples of the phospholipid according to the present invention include diacylphosphatidylcholines such as dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), and disteroylphosphatidylcholine (DSPC); Phosphatidines such as dimyristoyl phosphatidic acid; gangliosides such as ganglioside GM1; glycolipids such as glucosylceramide; dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), disteroyl phosphatidylglycerol (DSPG) ) Diacylphosphatidylglycerols; polyethylene glycol palmitate, polyethylene Polyethylene glycol (PEG) lipids such as glycol myristate; and cholesterol, and the like. Of these, DSPC, DPPC, DPPG, cholesterol and the like are preferable, and DPPG and the like are more preferably used. These liposome-constituting lipids can be used alone or in combination of two or more.

  The liposome-constituting lipids in the present invention are preferably used in combination of two or more.

  Examples of such combinations of liposome-constituting lipids include diacylphosphatidylcholines and cholesterol; diacylphosphatidylglycerols and / or PEG lipids and cholesterol; phosphatidylcholines and cholesterol and phosphatidylglycerols and / or PEG lipids. included. Specific examples of preferable liposome-constituting lipids include, for example, combinations of DSPC and cholesterol; DSPC, cholesterol and DPPG; PEG lipids having a molecular weight of DSPC, cholesterol and PEG of 500 or less.

  The ratio of the molecules constituting the liposome-constituting lipid is, in descending order, diacylphosphatidylcholines, cholesterol, diacylphosphatidylglycerols and / or PEG lipids. In particular, by using a small amount of diacylphosphatidylglycerols and / or PEG lipids (for example, about 0.1 to 30 mol%, preferably about 1 to 20 mol% based on the total amount of liposome-constituting lipid), It is preferable in terms of obtaining sugar structure-specific organ transferability.

Moreover, the liposome structure which has a hydrophilic group part which is 0.1 to 0.8, preferably 0.2 to 0.6 of the length of the sugar chain part of the glycolipid derivative represented by the general formula (1) It is preferably composed of lipids. The “length of the sugar chain portion of the glycolipid derivative represented by the formula (1)” means the length of the structural portion excluding the substituent R ³ in the general formula (1). In view of the above, the liposome-constituting lipids constituting the sugar chain-modified liposome include, for example, diacyl such as dimyristoyl phosphatidylglycerol (DMPG), dipalmitoyl phosphatidylglycerol (DPPG), and disteroylphosphatidylglycerol (DSPG). Preferred are phosphatidylglycerols; PEG lipids such as polyethylene glycol palmitate and polyethylene glycol myristate (PEG molecular weight of 500 or less, for example).

  When the liposome of the present invention is composed of a glycolipid derivative and a phospholipid, the rephospholipid (total amount) is, for example, 0.1 to 100 mol, preferably 1 to 80 mol, more preferably 1 mol with respect to 1 mol of the glycolipid derivative. Is used in an amount of about 2 to 50 mol.

  The liposome of the present invention may contain components other than the glycolipid derivative represented by the general formula (1) and the liposome-constituting phospholipid within a range not impairing the effects of the present invention. In addition, the liposome of the present invention may have a liposome surface subjected to a known treatment such as hydrophilization. As a method for making the liposome surface hydrophilic, for example, a method using polyethylene glycol (for example, having a molecular weight of 500 or less), polyvinyl alcohol, maleic anhydride copolymer or the like (JP 2001-302686 A), tris (hydroxy) A known method such as a method using methyl) aminomethane (JP-A-2003-226638, etc.) can be used.

《Nonionic iodine compound》
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. In order to bring the concentration of the nonionic iodine compound within this range, the contrast agent solution containing the nonionic iodine compound (for example, 306.2 mg / ml of JP iopamidol solution (150 mg / ml of iodine content)) Adjust the amount added.

  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. Constituent components such as glycolipid derivatives, phospholipids, stabilizers, and antioxidants are mixed in an organic solvent such as chloroform in the flask, and after the organic solvent is distilled off, vacuum drying is performed on the inner wall of the flask. A thin film is formed. 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, the glycolipid derivative 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 mmol, preferably in the range of 30 to 120 mmol, more preferably in the range of 50 to 100 mmol. 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. 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 lower than contrast agents containing a paramagnetic material having a lower magnetic moment, eg, iron (III). Requires medication. 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, the present invention will be specifically described with reference to examples, but the present invention is not limited thereto. Unless otherwise specified, “%” in the examples represents “mass%”.

(Synthesis example)
The glycolipid derivative according to the present invention was produced by the method described in JP-A-2007-153787. The structure is shown in the above formulas (i) to (ix).

Example 1
[Method A for Forming Liposomes Containing Nonionic Iodo Compound]
Dipalmitoyl phosphatidylcholine (50 nmol) (manufactured by NOF Corporation, COATSOME MC-6060), glycolipid derivative (10 nmol) and PEG-phospholipid (10 nmol) according to the present invention represented by the above formulas (i) to (ix) (Japan) Manufactured by Yushi Co., Ltd., 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.

[Formation Method B for Liposomes Containing Nonionic Iodo Compound]
Dipalmitoylphosphatidylcholine (50 nmol) (manufactured by NOF Corporation, COATSOME MC-6060), glycolipid derivative (10 nmol) and PEG-phospholipid (10 nmol) according to the present invention represented by the above formulas (i) to (ix) (Japan) A mixture of oil and fat Co., Ltd., 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.

[Test 1]
Measurement of Liposome Particle Size The obtained sample (liposome) was determined for the average particle size of the liposome with a WBC analyzer (Nihon Kohden 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 encapsulation rate of iodo compound The obtained sample (liposome) was dialyzed with isotonic saline, ethanol was added after the dialysis was completed to destroy the liposome, and the amount of iodo compound in the liposome was determined by measuring absorbance. . 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 1.

  As can be seen from Table 1, the liposome-containing contrast agent containing the glycolipid derivative according to the present invention represented by the above formulas (i) to (ix) of the present invention is a good average particle in any of the forming methods A and B. The diameter is shown.

Example 2
Using the liposomes (formation methods A and B) prepared in Example 1 and physiological saline, a contrast agent was prepared that gave an iodine amount of 2500 mg I / kg 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. The results obtained with 20 specimens are shown in Table 2.

  As shown in Table 2, it can be seen that the contrast agent of the present invention has low toxicity to the living body.

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

[Test 4]
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). Table 3 shows the average value 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 Table 3, it can be seen that the contrast medium of the present invention is excellent in contrasting systemic blood vessels and livers, is excellent in contrasting pancreas, and is safe to be excreted mostly outside the body within 24 hours.

Claims (2)

A liposome comprising at least one glycolipid derivative represented by the following general formula (1), a nonionic iodo compound, and a phospholipid.

(In the formula, R ¹ and R ² represent a hydrogen atom or a group selected from the following formulas (Q1) to (Q5), and R ³ represents a group selected from the following formulas (A1) to (A4). )

(Wherein R ⁴ and R ⁵ represent the same or different alkyl group having 5 to 20 carbon atoms.) An X-ray contrast agent comprising the liposome according to claim 1.