JP2005220045A - Fluorescent contrast agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a fluorescent contrast agent exhibiting excellent contrasting power and comprising a fluorochrome hardly accumulating in a living organism. <P>SOLUTION: This fluorescent contrast agent preferably comprises a targeting microcarrier including a fluorochrome, particularly a cyanine-based near-infrared fluorochrome as a near-infrared fluorochrome. A liposome, particularly a liposome enhanced in targeting functions is exemplified as the suitable targeting microcarrier. Desirably, the liposome is prepared by a supercritical carbon dioxide method. Further preferably, the liposome has a polyalkylene oxide chain or a polyethylene glycol chain introduce into the surface of the lipid membrane thereof and has a median particle size of 50-200 nm. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a fluorescent contrast agent, and more particularly to a fluorescent contrast agent characterized by containing a fluorescent dye encapsulated in a microcarrier.

  In the treatment of illnesses, it is desired to detect the morphological changes of organs and tissues caused by the illness in the early stage of the illness in a precise and rapid manner and with a simpler method. Particularly in the early treatment of cancer, it is essential to identify a small lesion site in the early stage of carcinogenesis and determine its size. As diagnostic methods therefor, image diagnosis such as biopsy with an endoscope, X-ray imaging, MRI and ultrasonic imaging can be mentioned. Each of these diagnostic imaging methods has excellent characteristics, but the subject is forced to experience psychological pressure, pain, distress, and exposure, or the installation, operation, and maintenance of the device requires a great deal of labor and cost. Sometimes.

On the other hand, light is a means capable of non-invasively diagnosing a living body with a relatively simple device. For example, an autofluorescence observation method using an endoscope utilizing the fact that autofluorescence of tumor cells is smaller than autofluorescence of normal cells (excitation at 450 nm, generation of fluorescence at 520 nm) has been put into practical use. However, since there are many hemoglobins or the like having absorption in the visible light region in the living body, there is a barrier that only information on the very surface of the living body can be collected. In contrast, the near infrared region (700-1300nm)
Then, although there exists absorption of each substituent which has a hydrogen bond, since the absorption is comparatively small, near-infrared light tends to permeate | transmit a biological tissue. By utilizing such near-infrared light characteristics, it is considered possible to obtain in-vivo information without imposing unnecessary loads on the body. However, since light is strongly scattered by living tissue, it is usually not easy to know which part of the living body the detected light has passed and which part of the information is transmitted. Recently, it has become possible to obtain information on the deep part of the body by combining a highly sensitive sensor, a laser that generates an extremely short pulse, and a light scattering simulation method in the body using the Monte Carlo method.

  As a diagnostic method using near-infrared light, a near-infrared fluorescence imaging method in which near-infrared fluorescent dyes are collected in a tumor portion and the tumor portion is imaged is attracting attention. In this method, a compound having the property of emitting fluorescence in the near-infrared region when irradiated with excitation light is administered in vivo as a contrast agent. Next, excitation light having a near-infrared wavelength is irradiated from the outside of the body, and fluorescence emitted from the fluorescent contrast agent collected in the tumor portion is detected to determine the lesion site.

  An example of such a fluorescent contrast agent is indocyanine green, which has been confirmed to be safe in vivo. It is said that the blood vessels in the tumor part are opened and closed at random, and the blood flow is retained (so-called blood pool). When indocyanine green is administered to an animal with a tumor, blood in the normal part and the tumor part Because the medium residence time is different (it is immediately excreted from the tissue consisting of normal cells), the tumor part can be lifted by applying excitation light with a wavelength in the near infrared region (Ohata et al., In rat experimental tumors) Fundamental study of cancer diagnosis using indocyanine green and near-infrared light topography: Journal of Japan Medical Association. 62 (6) .284-286.2002).

Since the reporting of cyanine-based fluorescent contrast agents, a technology has been disclosed in which various peripheral cyanine-based compounds are used as contrast agents in order to modify the compounds with high hydrophilicity, molar extinction coefficient, and quantum yield (for example, Patent Documents 1 to 5). However, along with resolution performance (contrast power) that can distinguish normal tissue from diseased tissue, it is completely decomposed and harmless in the living body after contrasting from the living body, or completely discharged (non-accumulating) Although necessary, a safer cyanine compound and a contrast agent containing the compound have not been found so far.
JP 2000-95758 A JP-T-2002-526458 Special table 2003-517025 gazette JP 2003-160558 A JP 2003-261464 A

  An object of the present invention is to provide a fluorescent contrast agent, particularly a near-infrared fluorescent contrast agent, that is selectively delivered to a targeted lesion site in the body. For example, a near-red fluorescent dye that contains a targeting microcarrier that contains cyanine compounds that are difficult to accumulate in the body as a near-infrared fluorescent dye. It is to provide an external fluorescent contrast agent.

The above object of the present invention can be achieved by the following constitution.
(1) The fluorescent contrast agent of the present invention is characterized by containing a fluorescent dye encapsulated in a microcarrier.
(2) The fluorescent contrast agent, wherein the fluorescent dye is preferably a near-infrared fluorescent dye.
(3) A fluorescent contrast agent, wherein the microcarrier is preferably a liposome.
(4) The liposome is preferably a liposome produced by the supercritical carbon dioxide method.
(5) Further, the liposome preferably has a polyalkylene oxide chain or a polyethylene glycol chain introduced on the lipid membrane surface, and has a central particle size of 50 to 200 nm.
(6) In the fluorescent contrast agent according to (2) above, the near-infrared fluorescent dye is a cyanine near-infrared fluorescent dye.
(7) The cyanine near-infrared fluorescent dye is indocyanine green or a derivative thereof.
(8) Further, in the fluorescent contrast agent according to (6) above, the cyanine near-infrared fluorescent dye is preferably a compound represented by the following general formula (I);

[Wherein, R represents a hydrogen atom, a lower alkyl group or an aromatic group,
R ₁ and R ₂ may be the same or different and each represents an aliphatic group substituted with a water-solubilizing group;
R ₃ and R ₄ may be the same or different and each represents a lower alkyl group or an aromatic group, or R ₃ and R ₄ may combine to form a carbocycle, and n is 1 Alternatively, when it is 2, L ₆ and R ₃ or R ₄ may combine to form a carbocycle, and when n is 0, L ₄ and R ₃ or R ₄ are A nonmetallic atom group that may be bonded to form a carbocyclic ring.

L _{1 to} L ₆ each represent the same or different methine group.

Z ₁ and Z ₂ may be the same or different and each represents a group of nonmetallic atoms necessary to form a 5-membered or 6-membered fused ring by bonding to a hetero 5-membered ring.

  X represents a counter ion necessary for neutralizing the charge of the molecule, and p represents the number of X necessary for neutralizing the charge of the whole molecule.

m represents an integer of 2 to 4, and n represents an integer of 0 to 2. ].
(9) The water-solubilizing group in the general formula (I) is preferably a sulfonic acid group.
(10) In the fluorescent contrast agent according to (8) above, the cyanine near-infrared fluorescent dye is preferably represented by the following general formula (II) and has at least two water-solubilizing groups in the molecule:

[Wherein, J ₁ and J ₂ may be the same or different and each represents an alkylene group having 1 to 5 carbon atoms, and R, R ₃ , R ₄ , L _{1 to} L ₆ , Z ₁ , Z ₂ , m, n, p and X are the same as defined in general formula (I). ].
(11) In the fluorescent contrast agent according to any one of (8) to (10), it is desirable that the number of intramolecular sulfonic acid groups of the cyanine compound is at least four.
(12) In the fluorescent contrast agent according to (10) above, the cyanine near-infrared fluorescent dye is preferably represented by the following general formula (III):

[Wherein R ₅ and R ₆ each represent a sulfoalkyl group having 3 to 5 carbon atoms substituted with a water-solubilizing group, and R, R ₃ , R ₄ , L _{1 to} L ₇ , m , N, p and X are the same as defined in the general formula (I), and R _{10 to} R ₁₇ may be the same or different and each represents a hydrogen atom or a substituent having a π value of less than 0.3. The π value is represented by the following formula:
π = logP (PhX) −logP (PhH)
(In the formula, P means the partition coefficient of the compound with respect to octanol / water, logP (PhX) indicates the logP value of benzene (PhX) having the substituent X, and logP (PhH) indicates the logP value of benzene (PhH). Is shown.)
It is represented by ].
(13) The fluorescence imaging method of the present invention includes a step of introducing the fluorescent contrast agent according to any one of (2) to (12) into a living body, a step of irradiating the living body with excitation light, and the near red A fluorescence imaging method including a step of detecting near-infrared fluorescence from an outer fluorescent contrast agent.

  The fluorescent contrast agent according to the present invention preferably comprises a liposome with improved structural stability and retention stability of the encapsulated material. The fluorescent dye encapsulated in the liposome has good in vivo stability and retention in blood, and exhibits an EPR effect. As a result, it is selective for the target disease site or diseased tissue, particularly cancer tissue. Concentrate on and accumulate. Therefore, the fluorescent contrast agent is excellent in contrast power that realizes a resolution performance capable of distinguishing a diseased tissue from a normal tissue, and can detect a lesion in a living body. In particular, cyanine-based fluorescent dyes are water-soluble and can be used safely because they are highly discharged.

  The production of the liposome employs a method in which phospholipids are prepared by dissolving them in supercritical carbon dioxide, so that it is not necessary to use a toxic solvent, particularly a highly toxic chlorinated solvent.

  Hereinafter, the fluorescent contrast agent of the present invention, particularly the near-infrared fluorescent contrast agent, will be described in detail in the order of fluorescent dye, liposome, and fluorescent contrast agent using these. The fluorescent contrast agent is a contrast agent for fluorescent imaging in image diagnosis. The near-infrared fluorescent contrast agent means a contrast agent that emits fluorescence in the near-infrared region. In this specification, “fluorescent contrast agent” may be referred to as “near infrared fluorescent contrast agent”, and “fluorescent dye” may be referred to as including “near infrared fluorescent dye”.

As a fluorescent dye used as a contrast material in a fluorescent dye fluorescent contrast agent, a substance that emits fluorescence when irradiated with excitation light having a specific wavelength is used. For example, porphyrin compounds that accumulate in tumors are exemplified. Specific porphyrin compounds include hematoporphyrin, photofurin, benzoporphyrin and the like. Other examples include fluorescent dyes such as fluorescamine, carboxyfluorescene, fluorescein, fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin, riboflavin, and anilinonaphthalene fluorescent dye and aminostyryl fluorescent dye. .

  These fluorescent dyes emit fluorescence in the visible light region of 400 to 600 nm. Such fluorescence cannot be expected to be imaged in the deep part of the body because the permeability of living tissue is extremely low.

The near-infrared fluorescent dye contained as a contrast material in the near-infrared fluorescent contrast agent is not particularly limited as long as it is a compound that emits fluorescence in the near-infrared region, for example, 700 to 1300 nm, particularly 700 to 900 nm when irradiated with excitation light. Absent. The molar extinction coefficient is desirably at least 100,000. For example,
Indocyanine green derivatives such as indocyanine green (ICG) and various conventionally reported cyanine compounds may be used (WO96 / 17628, WO97 / 13490). Indocyanine green has been used in liver and circulatory dysfunction tests and is relatively safe. In particular, a group of cyanine compounds that have low toxicity and are sufficiently water-soluble and have good selectivity for an imaging target site have been proposed (Patent Documents 1 to 5, etc.). Any of these cyanine compounds can be used.

  In the near-infrared fluorescent contrast agent of the present invention, a cyanine compound represented by the following general formula (I) can also be used as a near-infrared fluorescent dye.

[Wherein, R represents a hydrogen atom, a lower alkyl group or an aromatic group,
R ₁ and R ₂ may be the same or different and each represents an aliphatic group substituted with a water-solubilizing group;
R ₃ and R ₄ may be the same or different and each represents a lower alkyl group or an aromatic group, or may be bonded between R ₃ and R ₄ to form a carbocycle, and n is 1 Or when it is 2, it may combine between L ₆ and R ₃ or R ₄ to form a carbocycle, and when n is 0, L ₄ and R ₃ or R ₄ Represents a group of nonmetallic atoms which may be bonded to each other to form a carbocycle.

L _{1 to} L ₆ each represent the same or different methine group.

Z ₁ and Z ₂ may be the same or different and each represents a group of nonmetallic atoms necessary to form a 5-membered or 6-membered fused ring by bonding to a hetero 5-membered ring.

  X represents a counter ion necessary for neutralizing the charge of the molecule, and p represents the number of X necessary for neutralizing the charge of the whole molecule.

m represents an integer of 2 to 4, and n represents an integer of 0 to 2. ]
In general formula (I), R represents a hydrogen atom, a lower alkyl group or an aromatic group.

  The lower alkyl group is a linear or branched alkyl group having 1 to 5 carbon atoms, and specific examples include a methyl group, an ethyl group, a propyl group, a butyl group, and an isobutyl group. These may have a substituent, and examples of the lower alkyl group having a substituent include 2-hydroxyethyl, 3-sulfamoylpropyl, 3-carboxypropyl, and the like.

  Aromatic groups include substituted and unsubstituted carbon aromatic ring groups and heteroaromatic ring groups, such as phenyl groups, m-hydroxyphenyl groups, p-methoxyphenyl groups, thienyl groups, pyridyl groups, pyrimidinyl groups. Etc.

In the general formula (I), R ₁ and R ₂ may be the same or different and each represents an aliphatic group substituted with a water-solubilizing group. Examples of the aliphatic group include an alkyl group, an alkenyl group, a cyclic alkyl group, and an alkynyl group.

The alkyl group is preferably a linear or branched lower alkyl group having 1 to 5 carbon atoms. Specifically, methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, 2-methylpropyl group, 1,1 -Examples include dimethylpropyl group.

  The alkenyl group is preferably a linear or branched lower alkenyl group having 3 to 5 carbon atoms, and specific examples include an allyl group, a 2-butenyl group, and an isobutenyl group.

  The cyclic alkyl group is preferably a lower cyclic alkyl group having 3 to 6 carbon atoms, and specific examples include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group.

  The alkynyl group is preferably a linear or branched lower alkynyl group having 3 to 5 carbon atoms, and specific examples include a 2-propynyl group and a 2-butynyl group.

  Examples of the water-soluble substituent include a carbamoyl group, a sulfonic acid group, a carboxyl group, a hydroxyl group, and a phosphoric acid group.

“Aliphatic group substituted with water-solubilizing group” includes, for example, 2-hydroxyethyl group, 2-hydroxy-3-sulfopropyl group, 3-hydroxypropyl group, carboxymethyl group, carboxyethyl group, carboxybutyl group, Examples include 2-phosphonoethyl group, 3-phosphonopropyl group, sulfomethyl group, 2-sulfoethyl group, 3-sulfopropyl group, 4-sulfobutyl group, 3-sulfobutyl group, 2-hydroxy-3-sulfopropyl group, and the like. It is done. Among these, R ¹ and R ² are preferably a lower alkyl group having 1 to 5 carbon atoms substituted with a sulfonic acid group, such as a 2-sulfoethyl group, a 3-sulfopropyl group, a 4-sulfobutyl group, and a 3-sulfobutyl group. 2-hydroxy-3-sulfopropyl group and the like are preferably used.

In the general formula (I), R ₃ and R ₄ may be the same or different and each represents a lower alkyl group or an aromatic group. Examples of the lower alkyl group and aromatic group are the same as the lower alkyl group and aromatic group.

Alternatively, R ₃ and R ₄ may be bonded between R ₃ and R ₄ to form a carbocycle, and when n is 1 or 2, between L ₆ and R ₃ or R ₄ To form a carbocyclic ring,
When n is 0, it represents a nonmetallic atom group that may be bonded between L ₄ and R ₃ or R ₄ to form a carbocycle.

Examples of the carbocycle formed by combining R ³ and R ⁴ include a cyclopropane ring, a cyclobutane ring, a cyclopentane ring, and a cyclohexane ring.

Examples of the carbocycle formed by bonding between L ₆ and R ₃ or R ₄ , or the carbocycle formed by bonding between L ₄ and R ₃ or R ₄ include, for example, a cyclobutene ring, cyclopentene Ring, cyclohexene ring and the like. These carbocycles are substituted or unsubstituted lower alkyl group, sulfonic acid group, carboxyl group, hydroxyl group, cyano group, amino group or substituted amino group (for example, dimethylamino group, ethyl-4-sulfobutylamino group, di ( 3-sulfopropyl) amino group and the like. Even in the pyrrole ring to which the nonmetallic atom group Z ₁ is bonded, R ₃ or R ₄ forms a carbocycle that is bonded to L ₆ or L ₄ to form a carbocycle. The formation of such a carbocycle can increase the wavelength of absorption without destabilizing the compound structure or introducing an undesirable hydrophobic conjugated structure that increases the affinity for tissues. There is an advantage. Furthermore, the introduction of such a carbocycle contributes to the realization of a compound that is inactive in biological systems and has good fluorescence characteristics.

In the general formula (I), Z ₁ and Z ₂ may be the same or different, and are a group of nonmetallic atoms necessary for bonding to a nitrogen-containing hetero five-membered ring to form a 5-membered or 6-membered condensed ring Represents.

  The ring formed by such a nonmetallic atom group is composed of a 5-membered ring, a 6-membered ring, a condensed ring composed of 2 or more rings, a hetero 5-membered ring, a hetero 6-membered ring, or 2 or more rings. Heterocondensed rings are exemplified. Any position in these rings may be substituted with a substituent.

Examples of such a substituent include a sulfonic acid group, a carboxyl group, a hydroxyl group, a cyano group, a substituted amino group (for example, a dimethylamino group, an ethyl-4-sulfobutylamino group, a di (3-sulfopropyl) amino group), Or the substituted or unsubstituted alkyl group couple | bonded with the ring directly or through the bivalent coupling group is mentioned. Examples of the divalent linking group include —O—, —NHCO—, —NHSO ₂ —, —NHCOO—, —NHCONH—, and —C.
OO—, —CO—, —SO ₂ — and the like are preferable. Examples of the unsubstituted alkyl group bonded to the ring directly or through a divalent linking group include a methyl group, an ethyl group, a propyl group, and a butyl group, and a methyl group and an ethyl group are preferable. In the substituted alkyl group, an arbitrary position of the alkyl group is substituted with a substituent, and examples of the substituent include a sulfonic acid group, a carboxyl group, and a hydroxyl group, and among them, a sulfonic acid group is preferable.

  The ring formed by the nonmetal atom group is particularly preferably a carbocyclic or nitrogen-containing heterocyclic ring substituted with a hydrophilic group.

In general formula (I), L < ₁ > -L < ₆ > respectively represents the same or different methine group, and the methine group may be substituted by arbitrary substituents. As such substituents, substituted or unsubstituted alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, 2-phenoxyethyl, 2-sulfoethyl;
Halogen atoms such as chlorine, fluorine, bromine, iodine;
A substituted or unsubstituted aryl group such as a phenyl group, a sulfonic acid group-substituted phenyl group, a methoxy group-substituted phenyl group, or a naphthyl group;
Heterocyclic groups such as furyl, thienyl, pyrrolyl, imidazolyl, pyrrolidino, morpholino;
Lower alkoxy groups such as methoxy group and ethoxy group;
Examples thereof include substituted amino groups such as dimethylamino group and 2-sulfoethylamino group, and amino groups. Among the above substituents of the methine group represented by L _{1 to} L ₆ , preferred are an alkyl group, an amino group, and a heterocyclic group.

Moreover, the substituents of the methine group represented by L _{1 to} L ₆ may be bonded to each other to form a ring containing three methine groups, and this ring further forms a condensed ring with a ring containing another methine group. It may be formed.

Specific examples of the ring containing three methine groups formed by bonding substituents of the methine group represented by L _{1 to} L ₆ include a 4-oxo-2-hydroxycyclobutene ring, a cyclopentene ring, Examples thereof include a cyclohexene ring and a 4,4-dimethylcyclohexene ring, and a cyclopentene ring is particularly preferable in the present invention.

  Of pX in the general formula (I), X represents a counter ion necessary for neutralizing the charge of the molecule, and p represents the number of X necessary for neutralizing the charge of the whole molecule. Although the value of p will not be specifically limited if the charge of the whole molecule | numerator can be neutralized, Usually, it is 1-10.

Examples of the counter ion include a cation and an anion, and any counter ion may be used as long as it forms a non-toxic salt. Specific examples of cations include ions of alkali metals such as sodium and potassium; ions of alkaline earth metals such as magnesium and calcium; ions of organic ammonium such as ammonium, triethylammonium, tributylammonium and pyridinium; lysine salts, arginine salts and the like Examples include ammonium ions of the amino acids. Specific examples of the anion include halogen ions such as chlorine, bromine and iodine; sulfate ions; organic carboxylic acid ions such as acetic acid and citric acid; toluenesulfonate ions and the like.

  Particularly preferred as the counter ion is sodium ion or chloro ion that can further reduce toxicity to the living body.

  Such a cyanine compound represented by the general formula (I) is preferably a cyanine compound represented by the following general formula (II) and having at least two water-solubilizing groups in the molecule.

[Wherein, J ₁ and J ₂ may be the same or different and each represents an alkylene group having 1 to 5 carbon atoms, and R, R ₃ , R ₄ , L _{1 to} L ₆ , Z ₁ , Z ₂ , m, n, p and X are each the same as defined in the general formula (I). ]
Examples of the alkylene group having 1 to 5 carbon atoms represented by J ₁ and J ₂ include a methylene group, an ethylene group, a propylene group, a butylene group, a pentene group, and a 2-methylpropylene group. In particular, an ethylene group is preferable.

Furthermore, as the cyanine compound represented by the general formula (II), a cyanine compound represented by the following general formula (III) is also preferable.

[Wherein R ₅ and R ₆ each represent a sulfoalkyl group having 3 to 5 carbon atoms substituted with a water-solubilizing group, and R, R ₃ , R ₄ , L _{1 to} L ₇ , m , N, p and X are the same as defined in the general formula (I), and R _{10 to} R ₁₇ may be the same or different and each represents a hydrogen atom or a substituent having a π value of less than 0.3. . ]
The group that R ₅ and R ₆ can take is a “sulfoalkyl group having 3 to 5 carbon atoms substituted with a water-solubilizing group”, and examples of the water-solubilizing group include those represented by the general formula (I). In addition to the groups defined in, hydrophilic nonionic groups are also indicated. Examples of the hydrophilic nonionic group include a carbamoyl group, a sulfamoyl group, an acetamide group, a sulfonamide group, and a methanesulfonamide group.

Specific examples of the “sulfoalkyl group having 3 to 5 carbon atoms substituted with a water-solubilizing group” include 2-hydroxy-3-sulfopropyl group, 2-carbamoylmethyl-4-sulfobutyl group, 2 -Acetamido-4-sulfobutyl group, 2-sulfamoyl-3-sulfopropyl group, 3-methanesulfonamido-5-sulfopentyl group, 3-methanesulfonyl-4-sulfobutyl group, 2-carboxy-4-sulfobutyl group, 3 -Phosphonoxy-5-sulfobutyl group and the like are mentioned, and 2-hydroxy-3-sulfopropyl group is particularly preferable.

In the general formula (III), R _{10 to} R ₁₇ may be the same or different and each represents a hydrogen atom,
Alternatively, it represents a substituent having a π value smaller than 0.3. The definition of the π value used for defining the substituents R _{10 to} R ₁₇ will be described.

  The π value is a parameter indicating the influence of a substituent on the hydrophilicity / hydrophobicity of a compound molecule, and is defined by the following formula.

Formula: π = logP (PhX) −logP (PhH)
In the above formula, P means the distribution coefficient of the compound with respect to the octanol / water system, and the difference between the logP value of benzene (PhX) having substituent X and the logP value of benzene (PhH) is the π value of substituent X Assigned as.

The logP value can be obtained by actual measurement by the method described in the following document (a), and can also be obtained by calculation using the fragment method described in document (a) or the software package described in document (b). If the measured value does not match the calculated value, the measured π value is used in principle.

(A) C. Hansch, AJ Leo, “Substituent Constants for Correlation Analysis in Chemistry and Biology”, John Wiley & Sons, New York, 1979 (b) Medichem software package (Pomona College, Claremont, California)
Developed and sold from version 3.54)
The π values for the respective substituents thus obtained are summarized as a list in the literature (a). Major substituents having a π value of 0.3 or less include OSO ₃ H, OH, CN, COCH ₃ , C
OOH, OCH ₃ , COOCH ₃ , H 2, F, N (CH ₃ ) ₂ , and the like. Preferred substituents having a π value less than 0.3 include phosphono groups, sulfonic acid groups, carboxyl groups, hydroxyl groups, cyano groups, substituted amino groups (eg, dimethylamino group, ethylamino group, etc.), or π values less than 0.3. Examples thereof include a substituted or unsubstituted methyl group or ethyl group in which the π value bonded to the ring through a divalent linking group is 0.3 or less.

Examples of the divalent linking group having a π value smaller than 0.3 include —O—, —NHCO—, and —NH.
SO ₂ —, —NHCOO—, —NHCONH—, —COO—, —CO—, —SO ₂ — and the like can be mentioned.

Examples of the substituted or unsubstituted methyl group or ethyl group having a π value of 0.3 or less include a methoxy group, 2-sulfoethyl group, 2-hydroxyethyl group, methylaminocarbonyl group, methoxycarbonyl group, acetyl group, and acetamide group. , Poropionylamino group, ureido group, methanesulfonylamino group, ethanesulfonylamino group, ethylaminocarbonyloxy group, methanesulfonyl group and the like. A preferred group as a substituent having a π value smaller than 0.3 is a sulfonic acid group.

  A particularly required property for using a cyanine compound as a fluorescent contrast agent in vivo is water solubility. In the near-infrared fluorescent contrast agent of the present invention, by introducing at least three sulfonic acid groups into the cyanine compound, a remarkable improvement effect is seen with respect to the water solubility of the compound. Since the cyanine compound is water-soluble, the preferred number of sulfonic acid groups is 4 or more.

Sulfonic acid group in the general formula (I) in the position of R _1, R _2, Z ₁ and / or Z _2, to the general formula (II) the position of Z ₁ and / or Z _2, the general formula (III ) Is preferably introduced at any position of R ₅ , R ₆ and R _{10 to} R ₁₇ . Further, the sulfonic acid group is preferably introduced into L _{4 of the} conjugated methine chain via a divalent group such as an alkylene group.

In general formula (I), general formula (II), and general formula (III), m represents an integer of 2 to 4, and n represents an integer of 0 to 2. In particular, n is preferably 1.

The cyanine compound according to the present invention is a compound represented by the general formula (I) to the general formula (III), and has at least three, preferably four or more sulfonic acid groups in the molecule. It is desirable to be. As the cyanine compound of the present invention, a lower alkyl having 3 to 5 carbon atoms represented by the general formula (III), wherein R ₅ and R ₆ are substituted with a nonionic water-solubilizing group and a sulfonic acid group. Particularly preferred is a sodium salt of a compound having 3 or more sulfonic acid groups in the molecule.

Specific examples of the compounds represented by the general formula (I) (including the compounds represented by the general formula (II) and the general formula (III)) used in the present invention are shown below. It is not limited.

The cyanine compound according to the present invention is FM Hamer in The Cyanine Dyes and Related Compounds, John Wiley and Sons, New York, 1964, Cytometry, 10 (1989) 3-10, Cytometry, 11 (1990) 418-430. , Cytometry, 12 (1990) 723-730, Bioconjugate Chem. 4 (1993) 105-111, Anal. Biochem. 217 (1994) 197-204, Tetrahedron 45 (1989) 4845-4866, European Patent Application Specification 05982020A1, According to the known methods for producing cyanine compounds described in European Patent Application 0580145A1, Japanese Published Patent Nos. 4-147131, 2003-48891, 2003-64063, 2003-261464, etc. Further, it can be synthesized from a commercially available cyanine compound by a known method. More specifically, it can be synthesized by a reaction between a dianyl compound and a heterocyclic quaternary salt.

  About the manufacturing method of the cyanine type compound represented by general formula (I) of this invention, for example, the compound (11) which is a member of the cyanine type compound represented by general formula (I) is carried out by the method shown in the following schemes. Synthesized. Other compounds can be synthesized in the same manner.

  Since the cyanine compound of the present invention is used in vivo, it is important that the cyanine compound is not finally accumulated in the body but is quickly discharged out of the body, and is required to be substantially water-soluble. As means for improving the water solubility of the cyanine compound, anionic carboxylic acid and sulfonic acid salts are preferable. The cyanine compound of the present invention is remarkably improved in water solubility by introducing three sulfonic acid groups. In order to obtain such excellent water solubility, the number of sulfonic acid groups is desirably 3 or more, preferably 4 or more. However, in order to facilitate the synthesis of the cyanine compound, the number of sulfonic acid groups is desirably 10 or less, preferably 8 or less.

  Any salt that is tolerated when administered in vivo should be one that forms a non-toxic salt with the compound of formula (I). Examples include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts such as magnesium salts and calcium salts; tryptophan, methionine, lysine, phenylalanine, leucine, isoleucine, valine, threonine, arginine. And amino acid salts such as salts thereof. The cyanine compound is particularly preferably a sodium salt having low toxicity in vivo.

Microcarrier In the fluorescent contrast agent according to the present invention and its use, the fluorescent dye or near-infrared fluorescent dye is encapsulated in a microcarrier, particularly a targeting microcarrier (for example, a liposome or other type of carrier particle described below). Preferably it is. This “encapsulation” refers to the state of being encapsulated in microcarriers and / or bound, trapped or embedded in its inner surface wall. In order to improve the resolution performance (contrast power) that can distinguish normal tissue from diseased tissue, in addition to developing suitable near-infrared fluorescent dyes themselves, microcarriers that selectively deliver existing fluorescent dyes to target sites It can be said that the use of is an effective method.

  Particles that can serve as a carrier for delivering the fluorescent dye to the target tissue, that is, specific examples of microcarriers collectively called microcarriers include microcapsules, microspheres, microparticles, nanoparticles, nanospheres, nanocapsules, etc. Is mentioned. The microcarrier in the present invention may be formed from any substance capable of encapsulating, capturing or embedding a fluorescent dye. Such substances include, for example, lipids or modified lipids (specifically, as part of liposomes or liposome-based particles), proteins (eg, protein coat forms such as viral protein coats or “capsids”) or lipids and proteins (For example, when a protein is embedded in a lipid vesicle and has a structure similar to a normal protein-embedded lipid bilayer in a cell). The microcarrier particles may be made of a synthetic material such as a synthetic polymer. Examples of synthetic materials / polymers used to encapsulate fluorescent dyes include cationic polymers such as polylysine-based complexes as described in Wagner et al., 1992, PNAS USA, 89: 7934-7938.

  Given the positive significance of encapsulating, capturing or embedding fluorescent dyes, what is needed for microcarriers in the present invention is that the nature of the microcarrier particles is the lesion site being diagnosed, such as cancer cells or cancer. It is important to have an affinity for a target entity such as a tissue, in other words, to function as a “targeting microcarrier”. The “targeting microcarrier” as used herein means a microcarrier having directivity to a specific location / part in the body, specifically a specific organ, tissue, or cell. Such microcarriers are equipped with the “equipment” on the chemical structure necessary for this purpose. Therefore, the fluorescent dye to be delivered or the near-infrared fluorescent dye itself does not necessarily have affinity for cancer tissue or the like. It is preferable that the microcarrier encapsulating the fluorescent dye is selectively directed to such organs / tissues, and as a result, the delivered fluorescent dye is efficiently accumulated in the target cancer cells, cancer tissues, etc. . This means that various structural devices and devices are not necessarily required so that the fluorescent dye itself has targeting properties. Instead, the fluorescent dye may be selected in consideration of other characteristics such as fluorescence characteristics such as fluorescence quantum yield, stability, biotoxicity, and water solubility.

  Another significance of encapsulating fluorescent dyes or near-infrared fluorescent dyes in microcarriers is to improve the stability of these dyes in vivo, specifically to enhance plasma stability and blood retention. Can be pointed out. Since all the fluorescent dyes are organic low molecules, they are inevitably subject to degradation metabolism by drug metabolizing enzymes such as the liver, intestinal tract, and kidney as in the case of general low molecular weight drugs. In the case of water-soluble dyes, those that have not been taken into tissues or cells are rapidly excreted in the urine via the kidneys. Alternatively, it may bind to plasma proteins such as albumin and globulin in the blood. As a result, the disappearance of the fluorescent dye or near-infrared fluorescent dye from the blood, a decrease in the amount effectively present as a contrast medium, and the like are caused. Such a decrease in plasma stability or blood retention of the fluorescent dye or near-infrared fluorescent dye is inconvenient from the viewpoint of use as a contrast function. If a fluorescent dye or the like is previously enclosed in a microcarrier and administered, it can be protected from such effects as catabolism and can stably move to the target site in vivo.

  The targeting microcarrier used in the present invention is particularly preferably a liposome, particularly a functional liposome with an enhanced targeting function. Liposomes have a behavior similar to that of red blood cells, so they are not rapidly excreted via the kidneys, are relatively long in the bloodstream, and remain stable for a reasonable time for imaging purposes. Because.

Liposomes The fluorescent contrast agent of the present invention, particularly the near-infrared fluorescent contrast agent, is preferably a targeting agent in order to selectively and efficiently deliver the fluorescent dye to a target site such as a target organ or tissue. It is used in a form in which the dye is encapsulated in a liposome as a microcarrier. As used herein, “liposome” means all known liposomes and liposome-based vesicle particles. In the broadest sense, “liposome” means a vesicular structure based on any lipid. The liposome particles are microscopic level spherical particles, and usually a part of the solvent in which the particles are suspended is encapsulated inside a membrane composed of one or more lipid bilayers. In general, phospholipids and / or glycolipids are preferably used as components of the lipid bilayer membrane of liposomes. It may consist of one kind of lipid or a mixture of lipids having different physical properties, and modified lipids such as polyethylene glycol modified lipids can be used as described later.

  Liposomes and methods for their preparation are well known in the art and there are numerous references. Accordingly, the physical properties of suitable liposome particles into which the fluorescent dye is introduced can be manipulated and selected using methods known in the art in the literature.

  The appropriate amount of each type of lipid incorporated into the liposome is determined according to the use of the liposome. Each lipid constituting the liposome may be positively or negatively charged as a whole, or may be electrically neutral. Therefore, depending on the specific combination and amount of lipids used, the liposome particles themselves are totally positively charged (cations), negatively charged (anions) or electrically neutral.

  Methods of adjusting liposome density / weight are well known in the art and many examples and literature are known. For example, the liposome density can be increased by filling the core core of the liposome with a denser solution such as cesium chloride (or other heavy compound) or a concentrated salt solution. Preferably, the liposome density can be increased by impregnating the liposome with a polysaccharide such as blue dextran or dextran sulfate. The polysaccharide is incorporated into the core of the liposome and / or the lipid membrane.

  The fluorescent dye or near-infrared fluorescent dye can be introduced into the liposome particles by well-known standard methods. In this case, the fluorescent dye is usually encapsulated in the aqueous phase or central region inside the liposome and / or adhered to the internal surface of the liposome, such as by electrostatic force, or in a lipid membrane forming vesicles, Preferably, it is captured or embedded on the inner surface of the membrane. In the fluorescent contrast agent of the present invention, in particular, the near-infrared fluorescent contrast agent, it is a more preferable embodiment to encapsulate (near-infrared) fluorescent dye in “functionalized liposome” with enhanced targeting function as described above.

-Functionalized liposomes The contrast agent of the present invention preferably uses liposomes with excellent targeting properties to improve plasma stability and retention in blood, thereby realizing selective and efficient delivery of fluorescent dyes. ing. The term “functionalized liposome” as used herein refers to a special liposome proposed to meet such a demand, and will be described in detail below.

In order to produce EPR (Enhanced permeability and retention) effect, which is effective for obtaining excellent tumor visualization, retention is achieved by stabilizing the liposome structure and stabilizing the retention of the encapsulated substance. The contrast agent is required to have characteristics such as plasma stability and retention in blood while improving efficiency.

In the fluorescent contrast agent or near-infrared fluorescent contrast agent of the present invention, the particle diameter (also referred to as particle diameter, which is the particle diameter) of “functionalized liposome” encapsulating a fluorescent dye that is a contrast material and the bimolecules thereof. The targeting function can be realized by appropriately designing the membrane. For this purpose, both passive targeting and active targeting are considered. The former can control the in vivo behavior through adjustment of the particle size, lipid composition, charge, etc. of the liposome. Adjustment to align the liposome particle size in a narrow range is easily performed based on the method described later. The design of the surface of the liposome membrane can impart desired characteristics by changing the type, composition, and mixed substances of phospholipids.

  The adoption of active targeting that allows for higher levels of accumulation and selectivity of contrast agents should also be considered. As an example, introduction of a polyalkylene oxide polymer chain, particularly polyethylene glycol (PEG), onto the liposome surface is extremely beneficial because the induction process to the target site can be controlled. Liposomes suitable for the fluorescent contrast agent of the present invention are modified by adding polyalkylene oxide, particularly PEG, to their surface, resulting in a further increase in blood retention and phagocytosis of reticuloendothelial cells such as the liver. Liposomes that are difficult to be treated. Fluorescent contrast agents that have not reached cancerous tissues, diseased sites, etc., are not accumulated at normal sites, but are decomposed and excreted from the body before the side effects are manifested. This can be done by appropriately controlling the stability in relation to the extracorporeal discharge time when designing the liposome. When a water-soluble near-infrared fluorescent dye is used as the fluorescent dye, it is rapidly excreted in the urine via the kidney. Therefore, it is possible to prevent harmful effects caused by staying in the body and delayed side effects.

  The design and production method of the above-mentioned “functionalized liposome” used in the delivery system of the fluorescent contrast agent of the present invention will be described in detail below.

  Preferred neutral phospholipids in the liposome of the present invention include lecithin, lysolecithin and / or hydrogenated products and hydroxide derivatives obtained from soybeans, egg yolks and the like.

Other phospholipids derived from egg yolk, soy or other animals or plants, or semi-synthetic phosphatidylcholine, phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), phosphatidylethanolamine, sphingomyelin, synthetic And alkyl or alkenyl derivatives such as phosphatidic acid (PA) obtained by, preferably dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), dimyristol phosphatidylcholine (DMPC), dioleyl phosphatidylcholine (DOPC), di Palmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylserine (DSPS), distearoyl phosphatidylglycerol (DSPG), dipalmitoy Phosphatidylinositol (DPPI), distearoyl phosphatidylinositol (DSPI), dipalmitoyl phosphatidic acid (DPPA), distearoyl lysophosphatidic acid (DSPA), and the like hexadecylamine (HDA).

  These phospholipids are usually used alone, but may be used in combination of two or more. However, when two or more kinds of charged phospholipids are used, it is desirable to use them between negatively charged phospholipids or between positively charged phospholipids from the viewpoint of preventing liposome aggregation.

  Examples of glycolipids include glycerolipids such as digalactosyl diglyceride and galactosyl diglyceride sulfate, galactosylceramide, galactosylceramide sulfate, lactosylceramide, sphingoglycolipids such as ganglioside G7, ganglioside G6, and ganglioside G4.

In addition to the above lipids, other substances can be added as necessary as the membrane constituent of the liposome. Examples thereof include sterols such as cholesterol, dihydrocholesterol, cholesterol ester, phytosterol, sitosterol, stigmasterol, campesterol, cholestanol, lanosterol or 2,4-dihydrolanosterol which act as a membrane stabilizer. It has also been shown that sterol derivatives such as 1-O-sterol glucoside, 1-O-sterol maltoside or 1-O-sterol galactoside are effective in stabilizing liposomes (JP-A-5-245357).
The amount of sterols used is desirably 0.05 to 1.5 parts by weight, preferably 0.2 to 1 part by weight per 1 part by weight of phospholipid. If the amount is less than 0.05 parts by weight, the sterol stabilizing effect of improving the dispersibility of the mixed lipid is not exhibited, and if it is more than 1.5 parts by weight, the formation of liposomes is inhibited or unstable even if formed.

Cholesterol in the liposome membrane can also serve as an anchor for introducing polyalkylene oxide. Japanese Patent Application Laid-Open No. 09-3093 discloses a novel “functional substance” that can be efficiently immobilized by covalent bonding at the end of a polyoxyalkylene chain and can be used as a liposome-forming component. Cholesterol derivatives are disclosed.

  Examples of other compounds that can be added include dialkyl phosphates such as dicetyl phosphate, which are negatively charged substances, and examples of compounds that impart a positive charge include aliphatic amines such as stearylamine, and glycols such as ethylene glycol and propylene glycol. These substances have a liposome stabilizing effect.

In the present invention, preferably phospholipids or compounds having polyalkylene oxide (PAO) groups or similar groups are used as a component of the liposome membrane for the intended purpose of the fluorescent contrast agent. As a method for solving the problem of being trapped by reticuloendothelial cells and the instability of liposome itself such as disintegration and aggregation, a polyethylene glycol (PEG) chain that is a polymer chain on the surface of the liposome, that is, − (CH ₂ CH ₂ O) _n
Attempts have been made to introduce -H (for example, JP-A-1-249717, FEBS
letters, 268 , 235 (1990)).

By attaching a polyalkylene oxide chain (polyoxyalkylene chain) or PEG chain, which is an inert hydrophilic polymer, to the liposome surface, a new function can be imparted to the liposome. For example, PEGylated liposomes, so-called “stealth”
Liposomes (Lasic, 1995, CRC Press) can be expected to have an effect of being hardly recognized by the immune system. Alternatively, it has been clarified that liposomes have a tendency to be hydrophilic, thereby increasing blood stability and maintaining the concentration in blood over a long period of time (Biochim. Biophys. Acta., 1066 , 29-36 (1991)). ).

Utilizing these properties, organ specificity can be imparted to the fluorescent contrast medium. Specifically, since lipid components are likely to be stored in the liver, it is preferable not to use PEG or to use liposomes with a low PEG content when aiming at selective contrast of the liver. Further, when the particle size is increased to 200 nm or more, the possibility of rapid uptake by the phagocytosis of liver Kupffer cells increases, and it accumulates at the site of the liver. In imaging of liver cancer, since the cancer tissue has few Kupffer cells, the amount of contrast agent liposome taken up is relatively small, and the contrast becomes clear. The same applies to the spleen. On the other hand, in the case of contrast imaging of other organs, the introduction of PEG makes it possible to steal the liposome and make it difficult to collect in the liver and the like, and therefore the use of PEGylated liposome is recommended. Since the hydration layer is formed by the introduction of PEG, the liposome is stabilized and the retention in blood is also improved. By appropriately changing the length of the oxyethylene unit of PEG and the ratio of introduction, the function can be adjusted. As PEG, polyethylene glycol having 10 to 3500 oxyethylene units is preferred. In addition, when PEG is used, the amount used is 0.1 to 30% by mass with respect to the lipid constituting the liposome, preferably 1
It is good to contain about ~ 15% by mass.

  A known technique can be used for PEGylation of the liposome. An anchor to which PEG is bound (for example, cholesterol, phospholipid, etc.) may be mixed with a phospholipid that is a membrane component to prepare a liposome, and activated PEG may be bound to the anchor. Alternatively, PEG with some modification at the PEG tip can be bound to phospholipid and included as a liposome constituent to prepare liposomes. As an example of a phospholipid derivative of polyethylene glycol, PEG-PE (N- (ω-methoxypoly- (oxyethylene) oxycarbonyl-DSPE) (DSPE is 1,2-distearoyl-3-sn-phosphatidylethanolamine) is Can be mentioned.

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

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

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

  When n is 2 or more, the types of oxyalkylene groups may be the same or different. In the latter case, it may be added in a random manner or a block shape. In the case of imparting hydrophilicity to the polyalkylene oxide chain, AO is preferably an ethylene oxide added alone, and in this case, n is preferably 10 or more. When different types of alkylene oxides are added, it is desirable that ethylene oxide is added in an amount of 20 mol% or more, preferably 50 mol% or more. When imparting lipophilicity to the polyalkylene oxide chain, the number of added moles other than ethylene oxide is increased.

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

  In addition to the effects of introducing polyalkylene oxide chains, liposomes with a polyalkylene oxide chain immobilized at the tip of a "functional substance" can be used without being disturbed by the polyalkylene oxide chain. Functions such as specific organ directivity, cancer tissue directivity, etc. are sufficiently exerted as a “recognition element”. Specifically, it may be designed to contain a protein originally present in the target cancer cell membrane.

A phospholipid or compound having a polyalkylene oxide group can be used alone or in combination of two or more. The content is 0.001 to 50 mol%, preferably 0.01 to 30 mol%, more preferably 0.1 to 10 mol%, based on the total amount of the liposome membrane-forming components.

  A known technique can be used to introduce the polyalkylene oxide chain into the liposome. As a preferred production method, it is preferable to prepare liposomes in advance by including a phospholipid polyalkylene oxide (PEO) derivative in the raw material phospholipids. For this purpose, modified phospholipids such as polyethylene oxide (PEO) derivatives such as phosphatidylethanolamine, such as distearoylphosphatidylethanolamine polyethylene oxide (DSPE-PEO) have been proposed (Japanese Patent Laid-Open No. 7-165770). . Furthermore, JP 2002-37883 A discloses high-purity polyalkylene oxide-modified phospholipids for producing water-soluble polymer-modified liposomes with increased blood retention. It is described that when a polyalkylene oxide-modified phospholipid having a low monoacyl body content is used in preparing a liposome, the temporal stability of the liposome dispersion is good.

-Methods for preparing liposomes Various methods have been proposed so far for preparing liposomes. When the production method is different, the shape and properties of the final liposome are also often significantly different (Japanese Patent Laid-Open No. 6-80560). Therefore, the production method is appropriately selected according to the desired form and characteristics of the liposome. In general, liposomes are almost completely dissolved in lipid components such as phospholipids, sterols, and lecithins, and are first dissolved and mixed in a container with organic solvents such as chloroform, dichloromethane, ethyl ether, carbon tetrachloride, ethyl acetate, dioxane, and THF. It is prepared by. Such preparations of liposomes always contain an organic solvent. Such residual organic solvents, particularly chlorinated organic solvents, are difficult to remove completely, and there are concerns about adverse effects on living bodies.

In order to prepare liposomes used in the present invention, particularly “functionalized liposomes”, it is desirable to use a liposome preparation method using supercritical or subcritical carbon dioxide that can avoid the above-mentioned problems. Carbon dioxide has a critical temperature of 31.1 ° C and a critical pressure of 75.3 kg / cm ^2, and is relatively easy to handle. It is preferable for reasons such as Furthermore, the liposome produced by this method has various preferable characteristics and advantages for encapsulating the fluorescent dye of the fluorescent contrast agent as described later.

In the case of producing liposomes using supercritical or subcritical carbon dioxide, it is necessary to dissolve, disperse or mix the lipid membrane component in carbon dioxide in a supercritical state (including a subcritical state). At that time, when one or more alcohols such as lower alcohols, glycols, and glycol ethers are used as a solubilizing agent, the solubility of the lipid membrane component is further improved. For example, alcohols may be used as a co-solvent in a proportion of 0.1 to 10% by mass, preferably 1 to 8% by mass of supercritical carbon dioxide. Of these, ethanol is preferred from the viewpoint of safety and ease of removal. Suitable pressure of carbon dioxide in the supercritical state used in the production method of the present invention (including semi-critical state), 50~500kg / cm ^2, preferably 100~400 kg / cm ^2. The temperature of the carbon dioxide gas in a suitable critical state is 31 to 200 ° C, preferably 33 to 100 ° C, and more preferably 35 to 80 ° C.

The preferred method for preparing the liposome used in the fluorescent contrast agent of the present invention is specifically performed as follows. In the presence of the liposome membrane component, liquid carbon dioxide is added to the supercritical or subcritical state under the above-described suitable pressure and temperature. The above phospholipid as a membrane lipid component, preferably at least one compound selected from a cationic substance, a polyalkylene oxide-modified phospholipid, a compound having a polyalkylene oxide group, a compound having a polyethylene glycol group, a sterol, and a glycol Mix together and dissolve. Alternatively, liquid carbon dioxide is added to a pressure vessel to which these compounds have been added in advance, and then the temperature and pressure are adjusted to dissolve in a supercritical state. In that case, the said dissolution aid can be used together as needed. Subsequently, an aqueous solution containing a fluorescent dye and a formulation aid described below (for example, a water-soluble amine buffer and a chelating agent) is continuously added to form an aqueous phase / carbon dioxide emulsion. In this emulsion system, it is presumed that the lipid component is in a micelle form and is separated and concentrated. Further, the aqueous solution is continuously added until the carbon dioxide phase and the aqueous phase are separated. As the aqueous phase increases, the phase transition of the system occurs, resulting in a two-phase system of water / carbon dioxide emulsion + carbon dioxide / water emulsion. When the system is depressurized and carbon dioxide is discharged, an aqueous dispersion in which liposomes encapsulating a fluorescent dye are dispersed is generated. In this case, a formulation aid (for example, a water-soluble amine buffer and a chelating agent) is also added to the aqueous phase other than the inside of the liposome membrane (that is, the aqueous medium in which the liposome is dispersed) in addition to the fluorescent dye. include. Since the aqueous solution is also encapsulated inside the liposome membrane, the fluorescent dye and the formulation aid exist in the aqueous phase inside the liposome in addition to the aqueous medium outside the liposome, and are in a so-called “encapsulation” state. Further, the liposome is passed through a filtration membrane having a pore size of 0.1 to 0.4 μm. Next, the fluorescent contrast agent of the present invention is prepared through a preparation process such as sterilization and packaging as required.

Liposome preparation methods using supercritical or subcritical carbon dioxide have been shown to have higher liposome production rates, encapsulated substance encapsulation rates, and encapsulated substance retention rates in liposomes than conventional methods (Japanese Patent Application Laid-Open No. 2003-260260). 2003-119120). It can also be applied on an industrial scale,
This method capable of efficiently encapsulating a nonionic and water-soluble substance in a liposome without substantially using an organic solvent is a useful method for producing the fluorescent contrast agent of the present invention.

In the fluorescent contrast agent of the present invention, as described later, fluorescent dyes are encapsulated in liposomes at an appropriate weight ratio with respect to the liposome membrane lipid weight from the viewpoint of retention stability. Furthermore, the retention stability of the fluorescent dye in the liposome can also be achieved by the fact that the liposome usually has a central particle diameter of 50 to 300 nm and is substantially a single film. Single-membrane liposomes are liposomes substantially composed of a unilamellar vesicle in which a phospholipid bilayer is formed as one layer. Here, “substantially” means that a liposome is composed of a phospholipid bilayer in which a replica is generally recognized as one layer in a transmission electron microscope (TEM) observation by a freeze fracture replica method. That means. That is, for the observed traces of particles left on the carbon film, the one without a step was determined as a single film, and the one with two or more steps was determined as a “multilayer film”. In order to increase the ratio of unilamellar liposomes, an operation of irradiating ultrasonic waves may be further added. Single-membrane liposomes also have the advantage that the dose of liposomes, in other words, the dose of lipids does not increase compared to MLV.

Single membrane liposomes, particularly large unilamellar veislcles (LUV), have the advantage of providing a large encapsulation capacity compared to multilamellar liposomes. Liposomes used in the contrast agent of the present invention have an LUV having a particle size of 200 to 1000 nm and a particle size of 50 nm.
It is located in the middle of SUV (Small unilamellar vesicles), which are smaller unilamellar liposomes. For this reason, the holding volume is also larger than that of SUV, and the encapsulation rate of fluorescent dyes, particularly water-soluble fluorescent dyes, is remarkably excellent as described later. Further, unlike MLV and LUV, they are not taken up by reticuloendothelial cells and rapidly disappear from the bloodstream.

In general, liposomes can be prepared in various sizes from small vesicles of 50 to 150 nm (in many cases, a single membrane) to large vesicles of several μm (in many cases, a multilayer membrane). The above size range is appropriately selected by a compromise between liposome loading efficiency (increasing with increasing size) and liposome stability (optimal size range, decreasing with increasing size beyond 80-200 nm). Is done. In fact, the size of liposome particles and their distribution are closely related to the high blood retention, targeting and delivery efficiency that the fluorescent contrast agent of the present invention aims at. The particle size can be measured by freezing the dispersion containing the liposome encapsulating the fluorescent dye, then depositing carbon on the crushed interface, and observing the carbon with an electron microscope (freeze crushing TEM method). Here, the “center particle size” refers to the particle size having the highest appearance frequency in the particle distribution. The adjustment of the particle size can be carried out according to the formulation or process conditions. For example, when the above supercritical pressure is increased, the liposome particle size formed is reduced. In order to make the particle size distribution of the liposome to be produced in a narrower range, the liposome dispersion liquid may be filtered by a method of extruding from a filter such as a polycarbonate membrane. In this case, by passing through an extruder equipped with a filter having a pore size of 0.1 to 0.4 μm as a filtration membrane, it is possible to efficiently prepare liposomes having an optimum size that is 100 to 300 nm or less as the central particle size of one membrane. . Extrusion filtration methods and selection of appropriate pore sizes are based on known techniques and literature. For example, Biochim. Biophys. Acta 557, 9 (1979). By incorporating such an “extrusion” operation, in addition to the above sizing, it is possible to adjust the concentration of the fluorescent dye existing outside the liposome, exchange the liposome dispersion, and remove undesirable substances. There is also an advantage.

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

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

  The contrast agent leaking from the hole in the blood vessel wall does not return to the blood vessel again because the lymphatic vessel is not sufficiently developed around the cancer cell, and remains in the place for a long time. Since the EPR effect is a passive transport using the bloodstream, the retention in the blood must be improved as a requirement for its effective expression. That is, it is required that the contrast agent particles (liposome particles containing the fluorescent dye) stay in the blood for a long time and pass through the blood vessels near the cancer cells many times. Since the fluorescent contrast agent of the present invention is not particularly large particles, it is difficult to be captured by reticuloendothelial cells. Liposomes behave like erythrocytes, and are not rapidly excreted via the kidneys. If they are stealthed, they are phagocytosed by reticuloendothelial cells. But stays relatively long in the bloodstream. The EPR effect inevitably increases the transferability of the contrast compound to the target organ or tissue, and achieves selective concentration and accumulation of the contrast agent in the cancer tissue. Increasing the tumor cell / normal cell accumulation ratio of contrast material enhances the contrast performance of the fluorescent contrast agent. Such improvement in tumor visibility makes it possible to even discover micrometastatic cancers that have been difficult to detect.

Fluorescent contrast agent The fluorescent contrast agent of the present invention contains a fluorescent dye, preferably a near-infrared fluorescent dye, encapsulated in a microcarrier, preferably a targeting microcarrier. As the near-infrared fluorescent dye, the above cyanine compound, and as the targeting microcarrier, a liposome is particularly preferable. In particular, since the cyanine compound has low toxicity and excellent water solubility, and emits near-infrared fluorescence that can be transmitted through living tissue, a contrast agent containing the compound as a contrast medium is used for tumors and / or blood vessels. Allows non-invasive imaging.

An aqueous fluorescent dye solution to be encapsulated in a targeting microcarrier such as a liposome can be prepared by dissolving the fluorescent dye in a solvent such as distilled water for injection, physiological saline or Ringer's solution. In the aqueous solution, various formulation aids based on the formulation technology may be dissolved as other components. As a “formulation aid”, it is added together with a fluorescent dye at the time of formulation, and various substances are used based on conventional contrast agent production techniques. Specifically, various physiologically acceptable buffers, chelating agents, osmotic pressure regulators, stabilizers, viscosity modifiers, antioxidants (for example, α-tocopherol) as necessary, and further necessary If there is a preservative such as methyl paraoxybenzoate. As the buffer, phosphate buffer, citrate buffer, amine buffer, carbonate buffer, and the like are used. Preferably, both a water-soluble amine buffer and a chelating agent are included. As chelating agents, EDTA, EDTANa ₂ -Ca (edetate disodium calcium), E
Examples thereof include DTANa ₂ and hexametaphosphoric acid. Preferably, a EDTANa ₂ -Ca. The preferred pH range of the solution is 6.5 to 8.5, more preferably 6.8 to 7.8 at room temperature. A preferred buffer solution for the water-soluble near-infrared fluorescent dye is a buffer solution having a negative temperature coefficient as described in US Pat. No. 4,278,654. The amine-based buffer has properties that satisfy such requirements, and tris (trometamol) is particularly preferable. This type of buffer has a low pH at the autoclave temperature, which increases the stability of the fluorescent contrast agent in the autoclave, while returning to a physiologically acceptable pH at room temperature.

  The contrast agent of the present invention is encapsulated in the liposome in the form of an isotonic solution or suspension with respect to the osmotic pressure in the body so that the liposome is stably maintained in the body after administration. To obtain an isotonic solution or suspension, the contrast agent is dissolved or suspended in the medium at a concentration that provides the isotonic solution. Other non-toxic water-soluble substances such as sodium chloride such as sodium chloride, saccharides such as mannitol, glucose, sucrose, sorbitol may be added to the aqueous medium so that an isotonic solution is formed. The contrast agent of the present invention may contain a fluorescent dye and a formulation aid in both the aqueous phase inside the liposome membrane and the aqueous medium in which the liposome is dispersed. There is no significant osmotic pressure difference between the inside and outside of the membrane, thereby maintaining the structural stability of the liposomes.

  When the fluorescent dye is encapsulated in a microcarrier called liposome as in the present invention, the lipid dose must be considered in addition to the delivery efficiency and retention stability of the fluorescent dye. As the amount of lipid increases, the viscosity of the contrast agent increases. The viscosity of the fluorescent contrast agent solution produced by the method of the present invention is 6 cPa or less, preferably 0.9 to 3 cPa at 37 ° C.

Regarding the amount of fluorescent dye encapsulated in the liposome, the fluorescent dye in the aqueous solution encapsulated in the liposome is 1-10, preferably 3-8, more preferably 5-8, relative to the liposome membrane lipid weight. It is desirable to contain by weight ratio. If the weight ratio of the fluorescent dye encapsulated in the aqueous phase in the liposome is less than 1, it is necessary to inject a relatively large amount of lipid, resulting in poor delivery efficiency of the fluorescent dye. Since the viscosity of the fluorescent contrast agent of the present invention depends on the amount of lipid in the liposome, the superiority of the single membrane liposome with excellent retention volume and encapsulation efficiency is clear. On the other hand, when the encapsulated weight ratio of the fluorescent dye to the liposome membrane lipid weight exceeds 10, the liposome becomes structurally unstable, and the diffusion and leakage of the fluorescent dye outside the liposome membrane is injected during storage or in vivo. Inevitable even after. In addition, even if 100% encapsulation is achieved immediately after the liposome suspension drug is produced and isolated, the encapsulated components decrease as soon as possible based on destabilization due to the osmotic pressure effect. (Japanese National Publication No. 9-505821).

From the above, an example of a preferred embodiment of the fluorescent contrast agent of the present invention having particularly good targeting properties and tumor visualization is a polyalkylene oxide chain or a polyethylene glycol chain on the lipid membrane surface of the liposome encapsulating the cyanine compound. A near-infrared fluorescent contrast agent characterized by comprising the liposome introduced and having a central particle size of 50 to 200 nm.

The fluorescent contrast agent of the present invention is produced by means such as injection, instillation, spraying or application into blood vessels (veins, arteries), oral, intraperitoneal, subcutaneous, intradermal, intravesical, intratracheal (branch), etc. Can be administered into the body. The dose of the fluorescent contrast agent of the present invention is not particularly limited as long as it can detect the site to be finally diagnosed, and the total amount of fluorescent dye in the liposome, or the amount outside the liposome, according to the conventional fluorescent dye contrast agent The total amount of the fluorescent dyes may be the same as the conventional dosage. For example, it can be appropriately increased or decreased depending on the type of cyanine compound emitting near-infrared fluorescence to be used, the age and body size of the subject to be administered, and the target organ, but usually the cyanine compound is 0.1 to 100 mg / kg ( Body weight), preferably 0.5 to 20 mg / kg (body weight). The near-infrared fluorescent dye content is usually 0.1 to 500 mg / mL, preferably 1 to 300 mg / mL in the assumed dosage of 10 to 300 mL of the preparation solution.

  The fluorescent contrast agent of the present invention can also be suitably used as a contrast agent for animals, and its administration form, administration route, dosage and the like are appropriately selected depending on the body weight and state of the subject animal.

  The fluorescence contrast method according to the present invention is characterized by using the above fluorescent contrast agent. The measurement method is performed using a method known to those skilled in the art, and the optimum conditions such as the excitation wavelength and the fluorescence wavelength for detection are determined according to the fluorescent dye compound to be administered in order to obtain the highest resolution. It is determined appropriately according to the type, subject to be administered and the like. The time required to start the measurement using the fluorescence contrast method of the present invention after the fluorescent contrast agent of the present invention is administered to the measurement target also varies depending on the type of the fluorescent contrast agent to be administered, the subject to be administered, etc. For example, when administering for the purpose of tumor or cancer imaging, it is preferable to select an elapsed time of about 10 minutes to 6 hours after administration. If the elapsed time is too short, fluorescence is scattered throughout and it is difficult to distinguish between the target site and other sites, and if it is too long, the contrast medium is excreted outside the body. For the purpose of angiography, it is preferable to measure at an elapsed time of 1 hour after administration.

After the fluorescent contrast agent of the present invention is administered into the body of the measurement object, the measurement object is irradiated with excitation light by an excitation light source, and fluorescence from the fluorescence contrast agent (for example, cyanine compound) generated by the excitation light is detected by a fluorescence detector. Detect with. The excitation wavelength varies depending on the fluorescent dye used, and is not particularly limited as long as the dye emits fluorescence efficiently. For near-infrared fluorescent dyes, it is excited by near-infrared light with excellent biological permeability, usually 600 to 1000 nm, more preferably 700 to 850 nm, and the fluorescence is detected with a highly sensitive fluorescence detector. It is desirable to detect. In this case, as the fluorescence excitation light source, various laser light sources such as an ion laser, a dye laser, a semiconductor laser, or a normal excitation light source such as a halogen light source or a xenon light source may be used. Various optical filters can be used to obtain. Similarly, when detecting fluorescence, the fluorescence detection sensitivity can be increased through various optical filters that select only fluorescence from the fluorescent contrast agent.

  The near-infrared contrast agent of the present invention has a property that when it exceeds a certain concentration, it accumulates in the tumor tissue, and when it falls below a certain concentration, it tends to be discharged out of the body. It can be used as a fluorescent contrast agent that makes it possible to selectively and specifically image a tumor tissue using its characteristics. Further, the compound of the present invention is difficult to diffuse out of the blood vessel wall once injected into the blood vessel, and has a high property of remaining in the blood vessel, and can also be used as an angiographic agent.

[Example]
In order to describe the present invention in more detail, examples and experimental examples are shown below, but the present invention is not limited to these examples.

Preparation of near-infrared fluorescent contrast agent solution The following four types of fluorescent dyes for near-infrared contrast are prepared. Each dye is adjusted to a concentration of 0.5 mg / ml, and 50 mg phosphate buffer (PBS, pH 7.4) was added to make 100 ml. The obtained near-infrared contrast agent solutions were designated as Nos. 1 to 4.

Near-infrared contrast agent solution No. 1: Indocyanine green Near-infrared contrast agent solution No. 2: No. 29 dye used in the examples of JP-A-2000-95758 Near-infrared contrast agent solution No. 3 : No. 7 dye used in Examples of JP-A-2003-261464 Near-infrared contrast agent solution No. 4: Exemplary compound (11) represented by the above general formula (I)
The phosphate buffer (PBS) used had the following composition (finalized to 1 liter (L) with water for injection).

NaCl: 8 g, Na ₂ HPO ₄ · 12H ₂ O: 2.9 g, KCl: 0.2 g, KH ₂ PO ₄ : 0.2 g
86 mg of dipalmitoylphosphatidylcholine (DPPC) and 38.4 mg of cholesterol were dissolved in 0.7 ml of ethanol, charged into a special stainless steel pressure vessel heated to 60 ° C., and capped.
Thereafter, 13 g of liquefied carbon dioxide was injected while stirring the inside of the pressure vessel with a magnetic stirrer. The pressure inside the container, which was 50 kg / cm ² , was reduced to 120 kg / cm ² by reducing the volume of the pressure container, and carbon dioxide was brought into a supercritical state. Thereafter, 10 ml of the near-infrared contrast agent solution No. 1 was injected into the pressure vessel at a flow rate of 0.1 ml / min using an HPLC liquid feed pump. After completion of the injection, the carbon dioxide in the pressure vessel was gradually withdrawn over about 20 minutes to normal pressure, and then the solution inside was taken out.

  The obtained solution was put into a dialysis membrane, dialyzed 3 times with PBS, 1 L, and then concentrated using an ultrafiltration membrane to obtain a near infrared contrast agent solution No. 5 that was made into a liposome. A part of this contrast agent solution No. 5 was taken out, ethanol was added to destroy the liposome structure, and spectral absorption was measured. As a result, it was found that indocyanine green was contained at 0.6 mg / ml.

86 mg of hydrogenated soybean phospholipid (COATSOME NC-21, manufactured by NOF Corporation), 38.4 mg of cholesterol, and 19.2 mg of PEGylated lipid (SUNBRIGHT DSPE-020CN, manufactured by NOF Corporation) were dissolved in 0.7 ml of ethanol at 75 ° C. The sample was placed in a special pressure vessel made of stainless steel heated to 1 and covered.
Thereafter, 13 g of liquefied carbon dioxide was injected while stirring the inside of the pressure vessel with a magnetic stirrer. The pressure inside the container, which was 50 kg / cm ² , was reduced to 120 kg / cm ² by reducing the volume of the pressure container, and carbon dioxide was brought into a supercritical state. Thereafter, 10 ml of the near-infrared contrast agent solution No. 1 was injected into the pressure vessel at a flow rate of 0.1 ml / min using an HPLC liquid feed pump. After completion of the injection, the carbon dioxide in the pressure vessel was gradually withdrawn over about 20 minutes to normal pressure, and then the solution inside was taken out.

  The obtained solution was put in a dialysis membrane and dialyzed 3 times with 1 L of PBS, and then concentrated using an ultrafiltration membrane to obtain a near infrared contrast agent solution No. 6 that was made into a liposome. A part of this contrast medium solution No. 6 was taken out, ethanol was added to destroy the liposome structure, and the spectral absorption was measured. As a result, it was found that 0.8 mg / ml indocyanine green was contained.

  In the same manner as in the near-infrared contrast agent solution No. 6, the near-infrared contrast agent solutions No. 2 to 4 were made into liposomes to obtain near-infrared contrast agent solutions No. 7 to 9, respectively.

Preparation of Breast Cancer Carcinogenesis Model Mouse A carcinogen, 7,12-dimethylbenz [a] anthracene (DMBA) was administered to an aging-promoting mouse, a so-called SAM strain, a SAMP6 / Ta mouse, in order to develop breast cancer. The method for carcinogenesis in mice was performed according to the description in JP-A-2003-033125. DMBA was administered 6 times at a dose of 0.5 mg / mouse / week to 20 SAMP6 / Ta mice each. As a feed, a high protein, high calorie CA-1 solid (manufactured by Claire Japan) was given. After the 6th administration of the carcinogen, the period from the 1st administration to the 20th week was taken as a drug holiday. Breast cancer and lung metastases of breast cancer were searched histopathologically.

In this way, DMBA was administered 6 times at weekly intervals, and then the drug was withdrawn from the start of administration until the 20th week, and breast cancer was produced using a mouse (75% breast cancer incidence). A carcinogenic model mouse was created. Tumor tissue fragments (2 mm × 2 mm square pieces) of the above breast cancer mice were transplanted subcutaneously into the breast of the left breast of BALB / c nude mice (5 weeks old, supplied by Clare Japan). Ten days later, the mouse when the tumor grew to about 5 mm was subjected to the following contrast test.

Fluorescence Contrast Test and Stability Test Near-infrared contrast agent solutions No. 1 to 9 were administered from the tail vein to the above-mentioned breast cancer carcinogenesis model mice at a dose of 1.0 mg / kg. After administration of the test contrast agent solution, the mouse was anesthetized with diethyl ether at regular intervals, and a fluorescent image of the whole mouse was taken.

In the fluorescence contrast test, a titanium / sapphire laser was used as a fluorescence excitation light source. Using a ring-type light guide (Sumita Optical Glass Co., Ltd.) so that the dispersion of irradiation was within 2%, the test mouse was uniformly irradiated with laser light. The irradiation power was adjusted to be about 36 μW / cm ² near the skin surface of the mouse. Fluorescence was excited at the maximum excitation wavelength of each compound, and fluorescence emission from the mouse was detected and contrasted by using a CCD camera (C4880, Hamamatsu Photonics) to cut the reflection of incident light with a short wavelength cut filter. The cut filter was selected to match the excitation wavelength (800-900 nm) of the compound. The irradiation time was adjusted according to the fluorescence intensity of each compound.

  Twelve minutes after administration of the near-infrared contrast agent solution, the mouse was anesthetized with diethyl ether, and a fluorescent image of the whole mouse was imaged. The sensor used was a photodiode (wavelength range 220-730 nm) of a fluorometer (JASCO FP-750).

The relative fluorescence sensitivity was expressed as the tumor part fluorescence intensity of each near infrared contrast agent when the near infrared contrast agent solution 2 was administered and the fluorescence intensity of the tumor part 12 minutes later was taken as 100. The fluorescence intensity was further measured 1 hour, 6 hours, 12 hours and 24 hours after administration.

In the stability test of the near-infrared fluorescent contrast agent solution, 1 ml of a sample was left in the dark at a storage temperature of 40 ° C. for 2 weeks to confirm the presence or absence of precipitation or precipitation. A level at which there is no precipitation or precipitates, ◎, a slight haze, but a level that disappears by stirring, a level that disappears by stirring, a level that does not disappear by stirring, and a level that precipitates It evaluated as x.
The results are shown in Table 1.

Claims (13)

  A fluorescent contrast agent comprising a fluorescent dye encapsulated in a microcarrier.   2. The fluorescent contrast agent according to claim 1, wherein the fluorescent dye is a near-infrared fluorescent dye.   2. The fluorescent contrast agent according to claim 1, wherein the microcarrier is a liposome.   The fluorescent contrast agent according to claim 3, wherein the liposome is a liposome produced by a supercritical carbon dioxide method. 4. The liposome is characterized in that a polyalkylene oxide chain or a polyethylene glycol chain is introduced on the lipid membrane surface and has a central particle size of 50 to 200 nm.
Or the fluorescent contrast agent according to 4.   The fluorescent contrast agent according to claim 2, wherein the near-infrared fluorescent dye is a cyanine-based near-infrared fluorescent dye.   The fluorescent contrast agent according to claim 6, wherein the cyanine near-infrared fluorescent dye is indocyanine green or a derivative thereof. The fluorescent contrast agent according to claim 6, wherein the cyanine near-infrared fluorescent dye is a compound represented by the following general formula (I):

[Wherein, R represents a hydrogen atom, a lower alkyl group or an aromatic group,
R ₁ and R ₂ may be the same or different and each represents an aliphatic group substituted with a water-solubilizing group;
R ₃ and R ₄ may be the same or different and each represents a lower alkyl group or an aromatic group, or R ₃ and R ₄ may combine to form a carbocycle, and n is 1 Alternatively, when it is 2, L ₆ and R ₃ or R ₄ may combine to form a carbocycle, and when n is 0, L ₄ and R ₃ or R ₄ are A nonmetallic atom group that may be bonded to form a carbocyclic ring.
L _{1 to} L ₆ each represent the same or different methine group.
Z ₁ and Z ₂ may be the same or different and each represents a group of nonmetallic atoms necessary to form a 5-membered or 6-membered fused ring by bonding to a hetero 5-membered ring.
X represents a counter ion necessary for neutralizing the charge of the molecule, and p represents the number of X necessary for neutralizing the charge of the whole molecule.
m represents an integer of 2 to 4, and n represents an integer of 0 to 2. ].   The fluorescent contrast agent according to claim 8, wherein the water-solubilizing group in the general formula (I) is a sulfonic acid group. The fluorescent contrast agent according to claim 8, wherein the cyanine near-infrared fluorescent dye is represented by the following general formula (II) and has at least two water-solubilizing groups in the molecule:

[Wherein, J ₁ and J ₂ may be the same or different and each represents an alkylene group having 1 to 5 carbon atoms, and R, R ₃ , R ₄ , L _{1 to} L ₆ , Z ₁ , Z ₂ , m, n, p and X are the same as defined in general formula (I). ].   The fluorescent contrast agent according to any one of claims 8 to 10, wherein the number of sulfonic acid groups in the molecule of the cyanine-based near-infrared fluorescent dye is at least four. The fluorescent contrast agent according to claim 10, wherein the cyanine near-infrared fluorescent dye is represented by the following general formula (III):

[Wherein R ₅ and R ₆ each represent a sulfoalkyl group having 3 to 5 carbon atoms substituted with a water-solubilizing group, and R, R ₃ , R ₄ , L _{1 to} L ₇ , m , N, p and X are the same as defined in the general formula (I), and R _{10 to} R ₁₇ may be the same or different and each represents a hydrogen atom or a substituent having a π value of less than 0.3. The π value is represented by the following formula:
π = logP (PhX) −logP (PhH)
(In the formula, P means the partition coefficient of the compound with respect to octanol / water, logP (PhX) indicates the logP value of benzene (PhX) having the substituent X, and logP (PhH) indicates the logP value of benzene (PhH). Is shown.)
It is represented by ].   Fluorescence including a step of introducing the fluorescent contrast agent according to any one of claims 2 to 12 into a living body, a step of irradiating the living body with excitation light, and a step of detecting near-infrared fluorescence from the fluorescent contrast agent Contrast method.