JP2009067690A - Fluorescent contrast medium containing merocyanine dye - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fluorescent contrast medium having low toxicity and excellent water solubility, and exhibiting high clustering around a tumor. <P>SOLUTION: The fluorescent contrast medium contains a merocyanine dye [e.g. a compound represented by general formula (I) (wherein, Z<SB>1</SB>forms a nitrogen-containing heterocyclic ring which may be substituted with a sulfonic acid or the like; Z<SB>2</SB>forms an acidic nuclear which may be substituted with a sulfonic acid group or the like; R<SB>1</SB>is an alkyl group or the like which may be substituted with a sulfonic acid group; M<SB>1</SB>to M<SB>4</SB>are each independently a methine group or the like; m is 0 or 1; n is an integer of 0-4; CI is an ion for neutralizing charge; and y is the number of CIs required for neutralizing the charge)] as an effective ingredient. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

The present invention relates to a fluorescent contrast agent, and more particularly to a fluorescent contrast agent containing a merocyanine dye.

In treating a disease, it is crucial to detect morphological and functional changes caused by the disease in the body at an early stage of the disease. Especially when treating cancer, the location and size of the tumor is an important determinant in making an effective treatment plan. Methods already known for this purpose include biopsy by puncture or the like and imaging diagnostics such as X-ray imaging, MRI, ultrasound imaging and the like. Biopsy is effective for definitive diagnosis, but at the same time puts a heavy burden on the subject and is not suitable for tracking changes over time of the lesion. X-ray imaging and MRI necessarily expose the subject to radiation and electromagnetic waves. Furthermore, the conventional imaging diagnosis as described above requires complicated operations and a long time for measurement and diagnosis. The size of the device used for this purpose also makes it difficult to use these methods for operation.

One imaging diagnosis is fluorescence imaging (Non-patent Document 1). This method uses a substance that emits fluorescence when irradiated with excitation light having a specific wavelength as a contrast agent. For example, the body is irradiated with excitation light from the outside, and fluorescence emitted from the fluorescent contrast agent in the body is detected.
Such a fluorescent contrast agent is, for example, a porphyrin compound that accumulates in a tumor, and is used for photodynamic therapy (PDT) like hematoporphyrin. Other examples include photofurin and benzoporphyrin (see Non-Patent Document 1; Non-Patent Document 2; Patent Document 1 and the like). These compounds are originally used in PDT and have phototoxicity, which is the performance required by PDT. Therefore, they are not desirable as diagnostic agents.

On the other hand, retinal circulatory microangiography using known fluorescent dyes such as fluorescein, fluoresamine and riboflavin is known (Patent Document 2). These fluorescent dyes emit fluorescence in the visible light region of 400-600 nm. In this region, light transmission through the living tissue is very low, so that it is almost impossible to detect lesions in the inner part of the body.

Furthermore, utilization of cyanine compounds including indocyanine green (hereinafter abbreviated as ICG) used for measuring liver function and cardiac output as a fluorescent contrast agent has been reported (Non-patent document 3, Non-patent document 3). Reference 4). Cyanine compounds absorb in the near infrared region (700-1300 nm).

Near-infrared light is highly transmissive through living tissue and can pass through the skull of about 10 cm. Therefore, it is getting more and more attention in the field of clinical medicine. For example, an optical CT method using optical transmission of a medium has attracted attention as a new technology in the clinical field. This is because near-infrared light can penetrate the living body and can be used to monitor the oxygen concentration in the living body and its circulation.

Cyanine compounds emit fluorescence in the near infrared region. The fluorescence in this region can penetrate biological tissues and has the potential as a fluorescent contrast agent. Various cyanine compounds have been recently developed and tried as fluorescent contrast agents (Patent Document 3, Patent Document 4, etc.). However, there is no contrast agent having sufficient water solubility and sufficient safety for a living body, together with the ability to distinguish normal tissue from affected tissue (selectivity with respect to an imaging target site). Indocyanine green (ICG) is known as an example of a diagnostic near-infrared fluorescent compound that has already been approved for human administration. This drug is mainly used for liver function tests and fundus angiography. However, in order to apply it to cancer diagnosis, there are problems such as low fluorescence intensity, low tumor site accumulation, and high toxicity. There was still a big gap in the conversion.
From the aspect of the compound, for example, Patent Document 5 describes a merocyanine dye as a dye for a hologram recording material, but there is no report as a fluorescent contrast agent.
Lipspn RL et al., J. Natl. Cancer Inst., 26, 1-11 (1961) Meng TS et al., SPIE, 1641, 90-98 (1992) Haglund MM et al., Neurosurgery, 35, 930 (1994) Li, X. et al., SPIE, 2389, 789-797 (1995) WO84 / 04665 U.S. Pat.No. 4,945,239 WO96 / 17628 WO97 / 13490 JP 2006-268030

An object of the present invention is to provide a fluorescent contrast agent having low toxicity, excellent water solubility, and high accumulation in a tumor.

The present inventors have found that merocyanine dyes have high accumulation in tumors, high water solubility, and low toxicity, and have completed the present invention based on this finding.
That is, the present invention provides a fluorescent contrast agent containing a merocyanine dye as an active ingredient.
According to a preferred aspect of the present invention, there is provided the fluorescent contrast agent, wherein the merocyanine dye is a compound represented by the following general formula (I).

In General Formula (I), Z ₁ is an atomic group that forms a nitrogen-containing heterocyclic ring that may have a substituent together with the nitrogen atom and carbon atom to which Z ₁ is bonded, and-(M ₁ -M ₂ ) _m-. Z ₂ represents an atomic group which forms an acidic nucleus which may have a substituent together with a carbonyl group to which Z ₂ is bonded and a carbon atom which is bonded to the carbonyl group, and R ₁ is a hydrogen atom, a substituent Or an unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group, and M _{1 to} M ₄ are each independently substituted or unsubstituted methine. Represents a group, M ₁ and M ₂ may combine to form a ring, M ₃ and M ₄ , M ₃ and M ₃ , or M ₄ and M ₄ may combine to form a ring. , M Represents 0 or 1, n represents an integer of 0 to 4, and when n is 2 or more, a plurality of M ₃ and M ₄ may be the same or different, and CI represents an ion that neutralizes charge. , Y represents the number of CIs required for charge neutralization.
According to a more preferred aspect of the present invention, there is provided the fluorescent contrast agent, wherein the merocyanine dye is a compound represented by the following general formula (II).

In general formula (II), R ₁ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group, n represents an integer of 0 to 4, Z ₂ represents an atomic group that forms an acidic nucleus which may have a substituent together with a carbonyl group to which Z ₂ is bonded and a carbon atom to which the carbonyl group is bonded; Represents —O—, —S—, —NR ₅ —, or —CR ₆ R ₇ —, wherein R _{5 to} R ₇ each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl. A group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group, wherein R ₃ and R ₄ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, Substituted or unsubstituted aryl group, substituted or unsubstituted Borocyclic group, halogen atom, nitro group, alkoxy group, substituted or unsubstituted aryloxy group, substituted or unsubstituted acylamino group, substituted or unsubstituted carbamoyl group, hydroxy group, substituted or unsubstituted alkylthio group, or Represents a cyano group, Z ₃ represents an atomic group which forms an aromatic ring which may have a substituent together with two carbon atoms to which Z ₃ is bonded, and when n is 2 or more, a plurality of R ₃ , R ₄ may be the same or different, CI represents an ion for neutralizing the charge, and y represents the number of CIs necessary for neutralizing the charge.

According to a further preferred aspect of the present invention, any one of the above fluorescent contrast agents in which the merocyanine dye has two or more sulfonic acid groups in the molecule; any one of the above fluorescent contrast agents in which CI is a sodium ion; Any one of the above-described fluorescent contrast agents having absorption and fluorescence in an external light region; any one of the above-described fluorescent contrast agents used for tumor imaging is provided.

Another aspect of the present invention is the use of the above merocyanine dye for the production of a fluorescent contrast agent; a fluorescence imaging method comprising the step of imaging after administering the above merocyanine dye to mammals including humans A method comprising tumor imaging, comprising the step of imaging after administering the merocyanine dye described above to a mammal, including a human.

According to the present invention, a fluorescent contrast agent having low toxicity, excellent water solubility, and high accumulation in a tumor is provided. Fluorescent imaging that allows specific imaging of tumors and / or blood vessels using the fluorescent contrast agent is possible.

In this specification, when a group is “substituted or unsubstituted” or “may have a substituent”, it means that the group may have one or more substituents. However, unless otherwise specified, the number of substituents to be bonded, the substitution position, and the type are not particularly limited. When it has two or more substituents, they may be the same or different.
Examples of the substituent include a halogen atom (in the present specification, “halogen atom” may be any of fluorine, chlorine, bromine, or iodine), an alkyl group (in the present specification, “alkyl group”). May be linear, branched, cyclic, or combinations thereof, and the cyclic alkyl group includes a polycyclic alkyl group such as a bicycloalkyl group, etc. The alkyl part of other substituents including the alkyl part This also applies to alkenyl groups (including cycloalkenyl groups and bicycloalkenyl groups), alkynyl groups, aryl groups, heterocyclic groups, cyano groups, hydroxyl groups, nitro groups, carboxyl groups, alkoxy groups, aryloxy groups, Silyloxy group, heterocyclic oxy group, acyloxy group, carbamoyloxy group, alkoxycarbonyloxy Group, aryloxycarbonyloxy group, amino group (including anilino group), acylamino group, aminocarbonylamino group, alkoxycarbonylamino group, aryloxycarbonylamino group, sulfamoylamino group, alkyl and arylsulfonylamino group, mercapto Group, alkylthio group, arylthio group, heterocyclic thio group, sulfamoyl group, alkyl and arylsulfinyl group, alkyl and arylsulfonyl group, acyl group, aryloxycarbonyl group, alkoxycarbonyl group, carbamoyl group, aryl and heterocyclic azo group, Examples include imide group, phosphino group, phosphinyl group, phosphinyloxy group, phosphinylamino group, silyl group, and sulfonic acid group. The above substituents may be substituted with the above substituents.
The sulfonic acid group may mean a sulfonate (—SO ₃ ⁻ ) when the sulfonic acid group forms a salt.

Merocyanine dye is a general term for dyes in which both ends of a conjugated methine chain are an oxygen atom and a nitrogen atom. Specifically, as merocyanine dyes, JP-A-2006-268030, F.I. M.M. Harmer, Heterocyclic Compounds-Cyanine Dies and Related Compounds, John & Wiley & Sons, New York, London, 1964, pages 511 to 611, etc. Any compound can be used.
As the merocyanine dye, a compound represented by the following general formula (I) is particularly preferable.

In General Formula (I), Z ₁ is an atomic group that forms a nitrogen-containing heterocyclic ring that may have a substituent together with the nitrogen atom and carbon atom to which Z ₁ is bonded, and-(M ₁ -M ₂ ) _m-. Represents.
The nitrogen-containing heterocyclic ring is preferably a 5-membered or 6-membered nitrogen-containing heterocyclic ring or a ring in which a 5-membered or 6-membered nitrogen-containing heterocyclic ring and an aromatic ring are condensed.
Examples of the nitrogen-containing heterocycle include indoline ring, benzoindoline ring, benzoimidazoline ring, naphthimidazoline ring, oxazoline ring, benzoxazoline ring, naphthoxazoline ring, thiazoline ring, benzothiazoline ring, naphthothiazoline ring, selenazoline ring, and Examples thereof include a benzoselenazoline ring and a ring in which these rings and other rings are condensed. Examples of other rings include a benzene ring, a benzofuran ring, a pyridine ring, a pyrrole ring, an indole ring, and a thiophene ring.
As the nitrogen-containing heterocycle, an indoline ring, a benzoindoline ring, a benzoxazoline ring, and a benzothiazoline ring are preferable.

The nitrogen-containing heterocycle may have a substituent. Preferred examples of the substituent for the nitrogen-containing heterocyclic ring include a sulfonic acid group and an alkyl group (for example, a methyl group, an ethyl group, and a propyl group) that may be substituted with a sulfonic acid group.
As the indoline ring, a 3,3-dialkylindoline ring, particularly a 3,3-dimethylindoline ring is preferable, and as the benzoindoline ring, a 3,3-dialkylbenzoindoline ring, particularly a 3,3-dimethylbenzoindoline ring is preferable. . When the nitrogen-containing heterocyclic ring is a ring in which a 5-membered or 6-membered nitrogen-containing heterocyclic ring and an aromatic ring are condensed and has a sulfonic acid group as a substituent, the sulfonic acid group is directly bonded to the aromatic ring. It is preferable.

In the general formula (I), Z ₂ represents an atomic group that forms an acidic nucleus which may have a substituent together with a carbonyl group to which Z ₂ is bonded and a carbon atom to which the carbonyl group is bonded. An acidic nucleus is an electron-accepting cyclic unit containing ketomethylene or active methylene, for example, “The Theory of the Photographic Process” edited by James, 4th edition. McMillan Publishers, 1977, 198-200. Specifically as an acidic nucleus, U.S. Pat. Nos. 3,567,719, 3,575,869, 3,804,634, 3,837,862, 4,002,480, No. 4,925,777, JP-A-3-167546, US Pat. No. 5,994,051, US Pat. No. 5,747,236, and the like. The acidic nucleus in the general formula (I) may be any nucleus that meets this definition.
The acidic nucleus is preferably a 5-membered or 6-membered heterocycle or a ring condensed with two or more 5-membered or 6-membered heterocycles.

Specific examples of the acidic nucleus include 2-pyrazolone-5-one, pyrazolidine-3,5-dione, imidazolin-5-one, hydantoin, 2 or 4-thiohydantoin, and 2-iminooxazolidine-4-one. 2-oxazolin-5-one, 2-thiooxazoline-2,4-dione, isorhodanine, rhodanine, indan-1,3-dione, thiophen-3-one, thiophen-3-one-1,1-dioxide, Indoline-2-one, indoline-3-one, 2-oxoindazolium, 5,7-dioxo-6,7-dihydrothiazolo [3,2-a] pyrimidine, 3,4-dihydroisoquinoline-4- ON, 1,3-dioxane-4,6-dione, barbituric acid, 2-thiobarbituric acid, coumarin-2,4-dione, indazoline 2-one, pyrido [1,2-a] pyrimidine-1,3-dione, pyrazolo [1,5-b] quinazolone pyrazolopyridone, and 3-dicyanomethylidene Ride-3- phenylpropionitrile and the like. Of these, more preferably, pyrazolidine-3,5-dione, rhodanine, indan-1,3-dione, thiophen-3-one, thiophen-3-one-1,1-dioxide, 1,3-dioxane- Examples include 4,6-dione, barbituric acid, and 2-thiobarbituric acid.

The acidic nucleus may be substituted. Of the aforementioned substituents, the acidic nucleus preferably includes a sulfonic acid group, an alkyl group, a sulfonic acid group-substituted alkyl group, an aryl group, and a sulfonic acid group-substituted aryl group. In this case, the alkyl group is preferably a linear alkyl group having 1 to 6 carbon atoms. As the aryl group, a phenyl group is preferable. In the sulfonic acid group substituent, the number of sulfonic acid groups to be substituted is preferably 1 or 2, and more preferably 1. In the sulfonic acid group-substituted alkyl group, the sulfonic acid group is preferably substituted with a terminal carbon.

In general formula (I), R ₁ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. As the substituent, a sulfonic acid group is preferable. As the alkyl group, an alkyl group having 1 to 20 carbon atoms is preferable, and an alkyl group having 1 to 6 carbon atoms is more preferable. Examples of the substituted or unsubstituted alkyl group include methyl, ethyl, n-propyl, isopropyl, n-butyl, n-pentyl, benzyl, 3-sulfopropyl, 4-sulfobutyl, carboxymethyl, 5-carboxypentyl and the like. It is done. As the alkenyl group, an alkenyl group having 1 to 20 carbon atoms is preferable, and an alkenyl group having 1 to 6 carbon atoms is more preferable. Examples of the substituted or unsubstituted alkenyl group include vinyl, allyl, 2-butenyl, 1,3-butadienyl, and the like. As the cycloalkyl group, a cycloalkyl group having 3 to 20 carbon atoms is preferable, and a cycloalkyl group having 3 to 6 carbon atoms is more preferable. Examples of the substituted or unsubstituted cycloalkyl group include cyclopentyl and cyclohexyl. As the aryl group, an aryl group having 6 to 20 carbon atoms is preferable, and an aryl group having 6 to 10 carbon atoms is more preferable. Examples of the substituted or unsubstituted aryl group include phenyl, 2-chlorophenyl, 4-methoxyphenyl, 3-methylphenyl, 1-naphthyl, 2-nitrophenyl and the like. As the heterocyclic group, a heterocyclic group having 1 to 20 carbon atoms is preferable, and a heterocyclic group having 1 to 10 carbon atoms is more preferable. Examples of the substituted or unsubstituted heterocyclic group include pyridyl, thienyl, furyl, thiazolyl, imidazolyl, pyrazolyl, pyrrolidino, piperidino, morpholino and the like.

R ₁ is most preferably a substituted or unsubstituted alkyl group, and among them, a sulfonic acid group-substituted linear alkyl group having 1 to 6 carbon atoms is preferable. The number of sulfonic acid groups to be substituted is preferably 1 or 2, and more preferably 1. In the sulfonic acid group-substituted linear alkyl group having 1 to 6 carbon atoms, the sulfonic acid group is preferably substituted with a terminal carbon.

In the general formula (I), M _{1 to} M ₄ each independently represents a substituted or unsubstituted methine group. Among the above-mentioned substituents, the substituent of the methine group is preferably an alkyl group having 1 to 20 carbon atoms (for example, methyl, ethyl, i-propyl) and the like. M ₁ and M ₂ may combine to form a ring, and M ₃ and M ₄ , M ₃ and M ₃ , or M ₄ and M ₄ may combine to form a ring. Examples of the ring formed include a benzene ring, a cyclohexane ring, and a cyclopentane ring. M _{1 to} M ₄ are each independently more preferably an unsubstituted methine group, an ethyl group-substituted methine group, or a methyl group-substituted methine group, and more preferably an unsubstituted methine group.

In general formula (I), m Represents 0 or 1, preferably 0.
In general formula (I), n Represents an integer of 0 to 4, preferably an integer of 2 to 4. When n is 2 or more, the plurality of M ₃ and M ₄ may be the same or different.

CI represents an ion for neutralizing the charge, and y represents a number necessary for neutralizing the charge.
CI may be any as long as it forms a non-toxic salt with the compound of general formula (I). Examples of CI include alkali metal ions such as sodium ion and potassium ion; alkaline earth metal ions such as magnesium ion and calcium ion; ammonium ion, triethylammonium ion, tributylammonium ion, pyridinium ion and the like Examples include organic ammonium ions; amino acid ions such as lysine ions and arginine ions. Particularly preferred is sodium ion which has low toxicity in vivo.

As the merocyanine dye, a compound having a sulfonic acid group is preferable. When a merocyanine dye is used as a fluorescent contrast agent in a living body, it is preferable that water solubility is high, but a sulfonic acid group improves water solubility. Preferably, by using a merocyanine dye having two or more sulfonic acid groups, a more excellent water-soluble contrast agent can be obtained. From the viewpoint of ease of synthesis, a merocyanine dye having 6 or less, preferably 4 or less sulfonic acid groups is preferred.

When the compound represented by the general formula (I) is used as the merocyanine dye, the sulfonic acid group is preferably present as a part of any one or more of R ¹ , Z ¹ , and Z ² . Specifically, the sulfonic acid group is preferably present as a substituent such as a substituted alkyl group represented by R ¹ , instead of or in addition to the ring substituent formed by Z ¹ and / or Z ² or It is preferably present as part of the substituent.
Furthermore, the compound represented by the general formula (I) is preferably a compound represented by the following general formula (II).

一般式（II）中、Ｒ₁、ｎ、Ｚ₂、ＣＩ、ｙは一般式（Ｉ）における定義と同様である。

In general formula (II), R ₁ , n, Z ₂ , CI, and y are the same as defined in general formula (I).

X represents —O—, —S—, —NR ₅ —, or —CR ₆ R ₇ —, and R _{5 to} R ₇ each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted group. An alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group is represented. X is preferably —O—, —S—, or —CR ₆ R ₇ —.

R ₃ and R ₄ are each independently a hydrogen atom, alkyl group, alkenyl group, aryl group, heterocyclic group, halogen atom, nitro group, alkoxy group, aryloxy group, acylamino group, carbamoyl group, hydroxy group, alkylthio group, Or represents a cyano group. R ₃ and R ₄ preferably represent a hydrogen atom or an alkyl group, most preferably a hydrogen atom, a methyl group, or an ethyl group.

Z ₃ represents an atomic group that forms a substituted or unsubstituted aromatic ring together with two carbon atoms to which Z ₃ is bonded. The formed aromatic ring is preferably a ring having a benzene ring, and is preferably an aromatic ring having 4 to 10 carbon atoms. Examples of the aromatic ring formed include a benzene ring, naphthalene ring, benzofuran ring, quinoline ring, indole ring, and benzothiophene ring. Although the condensation position of these rings with a 5-membered ring containing X is not particularly limited, it is preferable that the benzene ring is condensed with a 5-membered ring containing X.
Preferred examples of the substituent of the aromatic ring include a sulfonic acid group, a sulfonic acid group-substituted alkyl group (more preferably a sulfonic acid group-substituted linear alkyl group having 1 to 6 carbon atoms), and more preferably a sulfonic acid group. .

n When R is 2 or more, the plurality of R ₃ and R ₄ may be the same or different, and n When R is 2 or more, the plurality of R ₃ and R ₄ may be the same or different.
Specific examples of the compound represented by formula (I) are shown below, but the present invention is not limited thereto.

The compounds represented by the general formula (I) are described in Journal of American Chemical Society, 1951, 73, 5326 (J. Am. Chem. Soc, 73, 5326 (1951)) and US Pat. No. 2,177,402. It can be easily synthesized by the method described in 1.

The fluorescent contrast agent of the present invention is not particularly limited as long as it contains a merocyanine dye, preferably a merocyanine dye represented by the formula [I] or [II]. The merocyanine dye may be contained in the fluorescent contrast agent alone or in combination of two or more. The merocyanine dye contained in the fluorescent contrast agent of the present invention preferably has a molar extinction coefficient of 50,000 or more.

The fluorescent contrast agent of the present invention exhibits absorption and fluorescence at 400-1300 nm, and preferably exhibits absorption and fluorescence in the near infrared region of 700-1300 nm, particularly preferably about 700-900 nm.
The fluorescent contrast agent of the present invention can be preferably used as a near-infrared fluorescent contrast agent.

The fluorescent contrast agent of the present invention may contain the above compound in a state suspended or dissolved in a solvent such as distilled water for injection, physiological saline, Ringer's solution and the like. You may add the pharmacologically acceptable additive to the fluorescent contrast agent of this invention as needed. Examples of the additive include carriers, excipients, pharmacologically acceptable electrolytes, buffers, detergents, and substances for adjusting osmotic pressure to improve stability and solubility (eg, cyclodextrin, liposomes). And various additives generally used in related fields can be used. The fluorescent contrast agent of the present invention is preferably produced through sterilization treatment for medical use.
The fluorescent contrast agent of the present invention can be administered to a living body by injection, injection or application, intravascular (vein, artery), oral, intraperitoneal, transdermal, subcutaneous, intravesical or intrabronchial administration. Preferably, the fluorescent contrast agent of the present invention is administered into a blood vessel in the form of a solution, emulsion or suspension.

The dose of the fluorescent contrast agent of the present invention is not particularly limited as long as the site to be finally diagnosed can be detected by the dose, and the type, age, weight, and target organ to be administered are used. It is adjusted appropriately by such as. Typically, the dosage is 0.1-100 mg / kg body weight, preferably 0.5-20 mg / kg body weight as the amount of the compound.
The fluorescent contrast agent of the present invention can be appropriately used for various animals other than humans. The form, route and dose of administration are appropriately determined according to the weight and condition of the subject animal.

The fluorescent contrast agent of the present invention accumulates significantly in tumor tissue. By utilizing this characteristic, tumor tissue can be specifically imaged using the fluorescent contrast agent of the present invention. Furthermore, the fluorescent contrast agent of the present invention can remain in the blood vessel for a long time and is expected to function well as an angiographic contrast agent.

Fluorescence imaging of tumors and / or blood vessels is possible by using the fluorescent contrast agent of the present invention. Fluorescence imaging may be performed according to known methods, and each parameter such as excitation wavelength and fluorescence wavelength to be detected can be optimized for imaging and evaluation depending on the type of fluorescent contrast agent to be administered and the administration target. What is necessary is just to determine suitably. The time taken from the administration of the fluorescent contrast agent of the present invention to the measurement target to the start of the fluorescence imaging measurement varies depending on the type of fluorescent contrast agent used and the administration target. For example, when the fluorescent contrast agent of the present invention is administered to a living body for the purpose of tumor imaging, the disappearance time is considered to be about 2-120 hours after administration. If the disappearance time is too short, the fluorescence may be too strong to clearly separate the target site from other sites. If the length is too long, the contrast medium may be excreted from the body. When angiography is required, it is preferable to detect the fluorescent contrast agent of the present invention immediately after administration or within about 30 minutes.

The fluorescence imaging method using the fluorescent contrast agent of the present invention typically includes the following steps.
That is, the fluorescent contrast agent of the present invention is administered to a detection target, and the detection target is exposed to excitation light from an excitation light source. Next, the fluorescence generated by the excitation light from the fluorescent contrast agent is detected by a fluorescence detector.
The excitation wavelength varies depending on the fluorescent contrast agent used, and is not limited as long as the compound effectively emits fluorescence. Preferably, near-infrared light having excellent biological permeability is used.

The wavelength of the fluorescence to be detected also depends on the contrast agent used. Generally speaking, near infrared fluorescence in the wavelength region of 400-1000 nm, preferably 750-950 nm is detected using excitation light having a wavelength of 300-1000 nm, preferably 700-900 nm. In this case, the excitation light source may be a normal excitation light source such as various lasers (for example, an ion laser, a dye laser, and a semiconductor laser), a halogen light source, a xenon light source, or the like. If necessary, various optical filters can be used to obtain the optimum excitation wavelength. Similarly, fluorescence can be detected by capturing only fluorescence from the near-infrared fluorescent contrast agent using various optical filters.

The detected fluorescence is data-processed as fluorescence information and used to generate a recordable fluorescence image. The fluorescence image is generated by irradiating a wide area including the target tissue, detecting fluorescence with a CCD camera, and processing the obtained fluorescence information. Alternatively, an optical CT apparatus, an endoscope, or a fundus camera may be used.
By the fluorescence imaging method using the fluorescent contrast agent of the present invention, systemic diseases, tumors, blood vessels and the like can be visualized without damaging the living body.

The present invention will be described in more detail with reference to examples, but the present invention is not limited to the examples shown below. The compound numbers in the following examples and experimental examples correspond to the compound numbers described by the structural formulas.

Experimental example 1
The n-butanol / water partition coefficient (log Po / w) was measured for compounds (1) to (7). Compounds (1) to (7) are disclosed in Journal of American Chemical Society, 1951, 73, 5326 (J. Am. Chem. Soc, 73, 5326 (1951)) and US Pat. No. 2,177,402. Synthesized with reference to the description.
As control compounds, cyanine dyes NK-1967 (Nippon Sensitive Dye Research Co., Ltd.) and ICG (Tokyo Chemical Industry) were used. The results are shown in Table 1.

Experimental Example 2: Evaluation of tumor selectivity Evaluation of selectivity to tumor cells was performed by using a ratio of the discharge rate between cells as an index when the pigment was taken up and discharged by tumor cells and normal cells. The tumor cells were evaluated using KB cells (Dainippon Sumitomo Pharma) and normal cells using CHL cells (Dainippon Sumitomo Pharma). 8.0 × 10 ³ cells were seeded in each well of 12 wells and cultured at 37 ° C. in a CO 2 incubator for 2 days. Each dye was dissolved in physiological saline and added to the medium to a concentration of 0.5 g / ml. The cells were further cultured for 24 hours in a CO ₂ incubator at 37 ° C. to incorporate the dye into the cells. Thereafter, the cells were washed three times with physiological saline, and a medium was added. Immediately after the addition of the medium and 8 hours after the addition of the medium, the amount of pigment in the cells when cultured at 37 ° C. in a CO ₂ incubator was quantified to compare the discharge rate from the cells. For quantification of intracellular dyes, the medium was taken out, washed 3 times with physiological saline, 0.5 ml of 0.1% SDS solution was added, and the cells were lysed by treatment at 37 ° C. for 30 minutes. ml was added as a sample. The sample was quantified by HPLC analysis. When the amount of intracellular dye immediately after the addition of the medium was taken as 100, the amount of dye discharged from the cell after 8 hours was defined as the relative discharge rate. Tumor selectivity was compared between the dyes as (tumor selectivity): (exclusion rate from normal cells) / (exclusion rate from tumor cells). The results are shown in Table 2.

From the above results, it can be seen that the merocyanine dye represented by the general formula (I) is excellent in tumor selectivity as compared with the comparative dye, and can be effectively used as a fluorescent contrast agent for tumor diagnosis.

Claims (7)

A fluorescent contrast agent containing a merocyanine dye as an active ingredient. The fluorescent contrast agent according to claim 1, wherein the merocyanine dye is a compound represented by the following general formula (I).

(In the general formula (I), Z ₁ is an atom that forms a nitrogen-containing heterocycle optionally having a substituent together with the nitrogen atom and carbon atom to which Z ₁ is bonded, and-(M ₁ -M ₂ ) _m- ). Z ₂ represents a group of atoms forming an acidic nucleus which may have a substituent together with a carbonyl group to which Z ₂ is bonded and a carbon atom to which the carbonyl group is bonded; R ₁ is a hydrogen atom; Represents a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group, and M _{1 to} M ₄ are each independently substituted or unsubstituted. Represents a methine group, M ₁ and M ₂ may combine to form a ring, M ₃ and M ₄ , M ₃ and M ₃ , or M ₄ and M ₄ combine to form a ring. Well, m Represents 0 or 1, n represents an integer of 0 to 4, and when n is 2 or more, a plurality of M ₃ and M ₄ may be the same or different, and CI represents an ion that neutralizes charge. , Y represents the number of CIs required for charge neutralization. ) The fluorescent contrast agent according to claim 1 or 2, wherein the merocyanine dye is a compound represented by the following general formula (II).

(In the general formula (II), R ₁ represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group. , N represents an integer of 0 to 4, Z ₂ represents an atomic group that forms an acidic nucleus which may have a substituent together with a carbonyl group to which Z ₂ is bonded and a carbon atom bonded to the carbonyl group, X represents —O—, —S—, —NR ₅ —, or —CR ₆ R ₇ —, and R _{5 to} R ₇ each independently represents a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted group. An alkenyl group, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocyclic group, wherein R ₃ and R ₄ are each independently a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group; Substituted or unsubstituted aryl group, substituted or unsubstituted Terocyclic group, halogen atom, nitro group, alkoxy group, substituted or unsubstituted aryloxy group, substituted or unsubstituted acylamino group, substituted or unsubstituted carbamoyl group, hydroxy group, substituted or unsubstituted alkylthio group, or Represents a cyano group, Z ₃ represents an atomic group which forms an aromatic ring which may have a substituent together with two carbon atoms to which Z ₃ is bonded, and when n is 2 or more, a plurality of R ₃ , R ₄ may be the same or different, CI represents an ion that neutralizes the charge, and y represents the number of CIs necessary for charge neutralization.) The fluorescent contrast agent according to claim 2 or 3, wherein CI is sodium ion. The fluorescent contrast agent according to any one of claims 1 to 4, wherein the merocyanine dye has two or more sulfonic acid groups in a molecule. The fluorescence contrast agent as described in any one of Claims 1-5 which has absorption and fluorescence in a near-infrared-light area | region. The fluorescent contrast agent according to any one of claims 1 to 6, which is used for tumor imaging.