JPH09127115A - Diagnostic marker - Google Patents

Abstract

PROBLEM TO BE SOLVED: To specify a focus at an infrared endoscopic process or during a surgical operation by generating fluorescence having wavelength equal to or above 780nm under the irradiation with near or far infrared rays. SOLUTION: This diagnostic marker contains a detection system for an antibody or the like, and a fluorescent functional group bonded to the detection system and expressed by the formula. In the formula. R<1> and R<2> stand independently for hydrogen atom. an alkyl group or the like, R<3> for an alkyl group, an alkyl sulfonate group or the like, and X for an anion type whenever necessary. Furthermore. Y stands for an alkylene group having carbons between 1 and 10, or an alkylene group having carbons between 1 and 10 and containing one or more atoms selected from the group of oxygen atom, hydrogen atom and sulfur atom.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a diagnostic marker. More specifically, the present invention is produced by binding an antibody or the like that specifically recognizes cancer cells to a labeling compound that emits fluorescence when excited by irradiation of near-infrared rays or far-infrared rays, which causes less tissue damage. The present invention relates to a diagnostic marker applicable to a living body.

[0002]

2. Description of the Related Art In recent years, the spread of electronic endoscopes has facilitated endoscopic diagnosis, and it has become possible to reliably detect gastric cancer, colon cancer, etc. in the initial cancer state. However,
Regarding the diagnosis of micro-cancer, the diagnostic ability when using an electronic endoscope and a normal endoscope is almost the same. This means that a new diagnostic method utilizing the function of the electronic endoscope has not been established yet. Even microscopic lesions that are usually indistinguishable with an endoscope can be easily detected by imaging them using computer processing if they can be labeled with a labeled antibody that can be detected under an electronic endoscope. There is a possibility, but such a method has not yet been put to practical use.

In order to establish the above-mentioned diagnostic method using an electronic endoscope, it is necessary to directly stain a living tissue by an immunohistochemical staining method. The staining method for fixed specimens is an established technology. However, the staining method for non-immobilized specimens is not yet available to those skilled in the art. For example, although there has been a report on the immunostaining method for non-immobilized specimens (Shikoku Medical Journal, 29, 180, 1987), the immunostaining method for the resected fresh specimens in the relevant region or the living tissue itself using near infrared rays has not yet been reported. Not reported.

On the other hand, the above diagnostic method also requires a diagnostic marker (such as a labeled antibody) that can be detected under an electronic endoscope. A diagnostic marker in which an antibody is bound to a labeling compound that emits ultraviolet / visible light fluorescence when excited by ultraviolet rays is known, and is commonly used to detect cancer cells and cancer tissues present in explanted tissue. Has been done. However, the method using a fluorescent diagnostic marker that is excited by ultraviolet rays cannot be applied to a living body because ultraviolet rays cause damage to living tissues and DNA. Diagnostic markers that can be directly applied to the living body have not yet been known.

It is known that indocyanine green (ICG) exhibits specific absorption under infrared endoscope and emits fluorescence. An example of using indocyanine green with an infrared endoscope is known (Gastroenterological
Endoscopy, 34, pp.2287-2296, 1992; and Gastroint
estinal Endoscopy, 40, pp.621-2; 628, 1994), all of which are intravascularly administered ICG. Also,
In general, fluorescent dyes such as indocyanine green are highly hydrophobic, and are rapidly absorbed when administered alone in the intestinal tract, so hydrophilic sulfonyl groups such as in the nucleus or side chain are used. Attempts have been made to increase the water solubility by introducing the above, improve the measurement efficiency and avoid the problem of toxicity after absorption. However, a water-soluble labeling compound that emits fluorescence as high as that of indocyanine green has not yet been known.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a diagnostic marker useful for directly staining a living tissue by an immunohistochemical staining method. Another object of the present invention is to provide a diagnostic marker that emits fluorescence when irradiated with near infrared rays or far infrared rays and has less tissue damage and can be directly used in a living body. That is, it is an object of the present invention to provide a diagnostic marker that can be applied to a living body without fear of damage to living tissue or DNA due to ultraviolet excitation. It is also an object of the present invention to provide a diagnostic marker having such characteristics and excellent water solubility.

The present invention also provides a lesion at the time of early diagnosis or surgical operation of a malignant neoplasm or infectious disease in epithelial tissues such as esophageal cancer, gastric cancer, or colon cancer using an infrared endoscope or the like. It is an object to provide a diagnostic marker applicable to a living body, which is useful for identifying or diagnosing. It is another object of the present invention to provide a method of directly staining a living tissue by an immunohistochemical staining method using such a diagnostic marker.

[0008]

The present inventors have made diligent efforts to solve the above-mentioned problems, and by synthesizing various derivatives of indocyanine green, indocyanine which emits fluorescence by being excited by near infrared rays or far infrared rays. Succeeded in producing a green derivative. Further, the present inventors, by using the above indocyanine green derivative as a labeling compound and reacting with an anti-cancer antigen antibody or the like, it is possible to produce a diagnostic marker directly applicable to a living body, and Have found that such a diagnostic marker is useful for directly staining living tissue by immunohistochemical staining.

Further, as a result of intensive studies by the present inventors to provide a diagnostic marker having excellent water solubility, it is possible to form an intramolecular counter ion (Zwitter ion) among the above indocyanine green derivatives. For these compounds,
The formation of a counterion may lower the ionicity of the molecule and impair the water solubility, but when sodium iodide or the like is caused to act on the derivative, a counterion is not formed in the molecule,
It was found that the ionicity of the whole molecule is maintained and the water solubility is significantly increased. The present invention has been completed based on these findings.

That is, the present invention provides (a) a detection system and (b) the following formula (I) bound to the detection system:

Embedded image (In the formula, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, or a sulfonic acid group; R ³ represents an alkyl group, a sulfonic acid alkyl group, or an amino-substituted alkyl group; X ⁻ represents an anion species if necessary; Y is an alkylene group having 1 to 10 carbon atoms, or 1 to 10 carbon atoms containing at least one atom selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom. The present invention provides a diagnostic marker containing a fluorescent functional group represented by

According to another aspect of the present invention, (a) a detection system, and (b) the following formula (II) bound to the detection system:

Embedded image (In the formula, R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group, an alkoxy group, or a sulfonate group; R ⁶ represents an alkylene group; M ⁺ represents an alkali metal ion;
Q ⁻ represents a halogen ion, a perchlorate ion, or a thiocyanate ion; Y represents an alkylene group having 1 to 10 carbon atoms, or one or more atoms selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom. And a fluorescent functional group represented by C 1 to C 10 alkylene group).

According to another aspect of the present invention, a detection system and a fluorescent functional group which emits fluorescence having a wavelength of 780 nm or more upon irradiation with excitation light having a wavelength of 600 to 800 nm, which is bound to the detection system. Diagnostic markers including and are provided. In addition to the above, according to a preferred embodiment of each of the above inventions, each of the above-mentioned diagnostic markers in which a fluorescent functional group is directly bound to the detection system; the fluorescent functional group is bound to the detection system via a linker or a protein. Each of the above diagnostic markers; the detection system is an antibody,
Each of the above-mentioned diagnostic markers selected from the group consisting of nucleic acids and amplification substances; each of the above-mentioned diagnostic markers using an anti-cancer antigen antibody as an antibody; used for identifying lesions during infrared endoscopy or surgery Provided are each of the above-mentioned diagnostic markers; and each of the above-mentioned diagnostic markers used for directly staining tissue of a living body by an immunohistochemical staining method; and a diagnostic agent containing each of the above-mentioned diagnostic markers as an active ingredient. To be done.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The fluorescent functional groups represented by the above general formulas (I) and (II) are bonded to a detection system via the carbonyl group of the --Y--CO-- group bonded to the mother nucleus. . The above general formula (I)
Wherein R ¹ and R ² are each independently a hydrogen atom, an alkyl group,
An alkoxy group or a sulfonic acid group (—SO ₃ H) is shown. R ¹ and R ² can each be substituted at any position on the phenyl group. The alkyl group has 1 to 6 carbon atoms.
A straight-chain or branched lower alkyl group, preferably a straight-chain or branched lower alkyl group having 1 to 4 carbon atoms, can be used. For example, methyl group, ethyl group, propyl group, isopropyl group, n-butyl group, sec-butyl group, tert
-Butyl group is preferred.

As the aryl group represented by R ¹ and R ² , a substituted or unsubstituted phenyl group, naphthyl group, pyridyl group or the like can be used. Alkoxy group has 1 carbon
To about 6, preferably a linear or branched lower alkoxy group having 1 to 4 carbon atoms, and more specifically, a methoxy group, an ethoxy group, a propoxy group, an isopropoxy group, an n-butoxy group. , Sec-butoxy group, tert-
It is preferable to use a butoxy group or the like. As the sulfonic acid group, in addition to the -SO ₃ H group in the free form, a base addition salt (sulfonate group) of a sulfonic acid group, for example, sodium salt, potassium salt and the like can be used. Of these, R ¹ and R ² are each independently a hydrogen atom, an alkyl group,
It is preferably an alkoxy group or a sulfonate group.

R ³ represents an alkyl group, an alkyl sulfonate group or an amino-substituted alkyl group. In these groups,
As the alkyl group, for example, those mentioned above can be used. The sulfonic acid group of the alkyl sulfonate group or the amino group of the amino-substituted alkyl group may be substituted at any position on the alkyl group, and, for example, one substituted at the terminal of the alkyl group can be used.

The sulfonic acid group and the amino group may form a base addition salt and an acid addition salt, respectively. For example,
Those in which sulfonic acid forms a sodium salt or potassium salt, those in which an amino group forms a salt such as ammonium halide, and those in which an amino group is quaternized are also preferable. Further, a substituted or unsubstituted amino group can be used as the amino group. Examples of the alkyl sulfonate group or the amino-substituted alkyl group include, for example, a methyl sulfonate group (-
CH ₂ SO ₃ H), an ethyl sulfonate group, an aminomethyl group, an aminoethyl group, a methylaminoethyl group, and salts thereof.

In the fluorescent functional group represented by the formula (I),
X ⁻ represents an anion species which may be optionally present, for example, halogen ion, acetate ion, perchlorate ion, carbonate ion and the like. X ^- The anion species represented by the, Y-CO- group cancel the positive charge on the nitrogen atom in the nucleus to be substituted; maintain neutral as a whole fluorescent functional group represented by the formula (I) Act to do. So, for example, the expression
When ^one of R ¹ , R ² , and R ^{3 in} the fluorescent functional group represented by (I) is an anionic group, the negative charge of the group is quaternary in the mother nucleus. X ⁻ may not be needed because it cancels the positive charge on the nitrogen atom to form an intramolecular Zwitter ion. On the other hand, for example, when either R ¹ or R ² is a sulfonic acid group and R ³ is an amino-substituted alkyl group, the charge is balanced between these groups,
X ^- may be required.

In the fluorescent functional group represented by the above general formula (II), R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group, an alkoxy group, or a sulfonate group. R ⁴ and
Each R ⁵ can be substituted at any position on the phenyl group. The alkyl and alkoxy groups can be used as described above, a sulfonate group (-SO ₃ ^- M
⁺ : M ⁺ represents an alkali metal ion and may be the same as or different from M ⁺ which is a counter ion of Q ⁻ ), for example, a sodium sulfonate group or a potassium sulfonate group can be used.

R ⁶ represents a linear or branched alkylene group, for example, having about 1 to 6 carbon atoms, preferably 2 to 2 carbon atoms.
A linear or branched lower alkylene group of 5, more preferably a trimethylene group, a tetramethylene group, or a pentamethylene group can be used. The —SO ₃ ^— group to be substituted on R ⁶ can be substituted at any position of the alkylene group, but it is preferable to use, for example, a group substituted at the terminal of the alkylene group. More specifically, R ⁶ —SO ₃ ^— is preferably a group represented by, for example, — (CH ₂ ) _k —SO ₃ ^— (k represents an integer of 2 to 4).

M ⁺ represents an alkali metal ion. As the alkali metal ion, sodium ion or potassium ion can be preferably used. Q ⁻ represents a halogen ion, a perchlorate ion, or a thiocyanate ion, and preferably, a chlorine ion, a bromine ion, an iodine ion, or the like can be used. Of these, iodine ion is particularly preferable. Without wishing to be bound by any particular theory, the above fluorescent functional groups are not represented by a positive charge (denoted by N ⁺ in the above formula) on the nitrogen atom that the Y-CO- group replaces.
R ³ -SO ₃ ^- and a negative charge derived from but, M ⁺ Q ^-
When in the alkali metal salt represented coexist, positive charge on the nitrogen atom (indicated by the formula N ⁺⁾ Q ^- between, and R ³
Ionic bonds are formed between -SO ₃ ^- and M ⁺ , respectively. As a result, the formation of counterions in the molecule is blocked, the ionicity of the entire molecule is maintained, and the water solubility is significantly increased.

In the above general formulas (I) and (II), Y is a linear or branched alkyl group having 1 to 10 carbon atoms, preferably a linear or branched alkylene group having 3 to 5 carbon atoms, More preferably, it represents a trimethylene group, a tetramethylene group or a pentamethylene group. Or, Y is 1 selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom.
A straight-chain or branched alkyl group having 1 to 10 carbon atoms containing the above atoms is shown. The group represented by -Y-CO-, for _{example, -CH 2 -CO-; - (CH} 2) 2 -CO-; - (CH 2) 3 -CO-; - (CH 2) 4 -CO
-;-(CH ₂ ) ₅ -CO-; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-;-(CH ₂ ) ₂ -CO-
NH- (CH ₂ ) ₅ -CO-;-(CH ₂ ) ₃ -CO-NH- (CH ₂ ) ₅ -CO-;-(CH ₂ ) ₄ -C
O-NH- (CH ₂ ) ₅ -CO-; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-NH- (CH ₂ ) ₂ -CO
-;-(CH ₂ ) ₄ -CO- (N, N'-piperadinyl)-(CH ₂ ) ₂ -CO (N, N'-p
iperadinyl is the 1-position of the piperazine - indicates that the (CH _{2) 2} -Z group is substituted - (CH _{2) 4} -CO- group is replaced, the 4-position. Hereinafter, the same applies. ); Or -CH ₂ -CO-NH-
_{(CH 2) 5 -CO- (N} , N'-piperadinyl) - (CH 2) 2 -CO- , etc. can be utilized.

Further, the carbonyl group of the —Y—CO— group is further selected from the group consisting of a linear or branched alkylene group having 1 to 10 carbon atoms; an oxygen atom, a nitrogen atom, and a sulfur atom.
It may be bonded to the detection system via a linear or branched alkylene group having 1 to 10 carbon atoms containing the above atoms; or a —NH—NH— group.

Among the diagnostic markers of the present invention, it is preferable to use a labeling compound of the following formula (III) for producing a diagnostic marker having a fluorescent functional group represented by the formula (I). . Therefore, according to one aspect of the present invention, there is provided a method for producing a diagnostic marker having a fluorescent functional group represented by the formula (I), which comprises the following formula (III):

Embedded image (In the formula, R ¹ , R ² , R ³ , n, and X ⁻ are as described above, and Z is the following formula (IV):

Embedded image Represents a group selected from the group consisting of: W and Y are each independently an alkyl group having 1 to 10 carbon atoms, or 1 selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.
A method is provided which comprises the step of reacting a labeling compound represented by the above-mentioned alkyl group having 1 to 10 carbon atoms containing an atom) with a detection system.

Further, for producing the diagnostic marker of the present invention having a fluorescent functional group represented by the formula (II), it is preferable to use the labeling compound of the following formula (V). Therefore, according to another aspect of the present invention, there is provided a method for producing a diagnostic marker having a fluorescent functional group represented by the formula (II), which comprises the following formula (V):
:

Embedded image ^{(Wherein, R 4, R 5, R} 6, n, Y, M +, Q -, and W are as defined above, Z is the following formula (VI):

Embedded image Represents a group selected from the group consisting of: W and Y are each independently an alkyl group having 1 to 10 carbon atoms, or 1 selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.
A method is provided which comprises the step of reacting a labeling compound represented by the above-mentioned alkyl group having 1 to 10 carbon atoms containing an atom) with a detection system.

In the compounds of the above general formulas (III) and (V), the group represented by Y-CO-Z is, for example, -CH ₂ -CO-Z;
-(CH ₂ ) ₂ -CO-Z;-(CH ₂ ) ₃ -CO-Z;-(CH ₂ ) ₄ -CO-Z;-(CH ₂ ) ₅
-CO-Z; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-Z;-(CH ₂ ) ₂ -CO-NH- (CH ₂ )
₅ -CO-Z;-(CH ₂ ) ₃ -CO-NH- (CH ₂ ) ₅ -CO-Z;-(CH ₂ ) ₄ -CO-NH-
(CH ₂ ) ₅ -CO-Z; -CH ₂ -CO-NH- (CH ₂ ) ₅ -CO-NH- (CH ₂ ) ₂ -CO-Z;-
(CH ₂ ) ₄ -CO- (N, N'-piperadinyl)-(CH ₂ ) ₂ -Z; or -CH ₂ -C
_{O-NH- (CH 2) 5} -CO- (N, N'-piperadinyl) - (CH 2) or the like can be used ₂ -Z.

In the group represented by Z, W is as described above.
The group corresponding to Y in the examples of Y-CO-Z can be preferably used. In addition, M ⁺ of the counter ion of the sulfonic acid group of the N-sulfosuccinimidyloxy group and the labeling compound of the formula (V)
Although it may be the same as or different from M ⁺ of the Q ⁻ counterion, it is preferable that both are sodium ions. Compounds represented by among formula of the labeled compound (V) is, for example, M ⁺ X in the compound of formula (V) ^- after producing the counterion type absence of alkali metal salt represented by compounds It can be easily produced by dissolving the compound in a solvent such as dimethyl sulfoxide or dimethylformamide at a high concentration and adding an alkali metal salt to the solution.

Of the above-mentioned labeling compounds used for the production of the diagnostic marker of the present invention, the production examples of preferred compounds are specifically described in Examples, but those skilled in the art will refer to these Examples. Thus, it will be easily understood that the desired labeling compound can be easily produced by appropriately modifying / altering the starting materials, reagents, reaction conditions and the like.

An N-succinimidyloxy group as Z and
When N-sulfosuccinimidyloxy groups are used, these groups form a reactive active ester together with the carbonyl group to which Z is bound, and therefore, the amino groups included in the detection system
(H ₂ NR) is replaced with Z in the active ester to form -Y-CO-N
Form HR. When a 2- (N-maleimido) -alkyl group is used as Z, the thiol group (H
SR) reacts to form -Y-CO-alkyl-SR. Further, Z in the case of -NH-NH ₂ has, reducing sugar terminus of the aldehyde group (OHC-R) react -Y-CO-NH included in the detection system
-NH-CO-R is formed.

Therefore, by reacting the labeling compound with a detection system, the labeling compound represented by the formula (III) or (V) is bound to the mother nucleus and the mother nucleus corresponding to the linker moiety -Y.
The -CO- moiety is stored in the diagnostic marker of the present invention as a fluorescent functional group. However, the diagnostic marker of the present invention having such a chemical structure is not limited to the one produced by the above method, and the one produced by any method is included in the scope of the present invention. Needless to say.

Further, the fluorescent functional groups represented by the above formulas (I) and (II), the labeling compounds represented by the formulas (III) and (V), and the compounds shown in the schemes of the examples. In the above, for convenience, the positive charge on the nitrogen atom (indicated by N ⁺ in the above formula) was fixed on one nitrogen atom in the mother nucleus, but the positive charge on the nitrogen atom was changed to another positive charge via the conjugated double bond. It is easily understood by those skilled in the art that it can be transferred to one nitrogen atom. Therefore, it goes without saying that all such tautomers based on conjugation are included in the scope of the present invention.

The detection system to be bound to the above-mentioned fluorescent functional group is an antibody that recognizes various antigens such as an antibody highly specific to cancer, a nucleic acid that can be used as a probe, or an amplification system such as biotin-avidin. The amplification system substance etc. which are used can be used. As the antibody, for example, an anti-cancer antigen antibody that specifically binds to cancer cells or cancer tissues, preferably early cancer cells or early cancer tissues can be used. For example, CEA, AFP, CA19-9, NSE, DU-PAN-2, CA
50, SPan-1, CA72-4, CA125, HCG, p53, STN (Sialyl T
n-antigen), c-erbB-2 protein-specific antitumor antibody for the stomach, and antitumor specifically reacting with tumor antigens such as esophageal cancer, colon cancer, rectal cancer, skin cancer, and uterine cancer. Antibodies can be used. Alternatively, an anti-pathogen protein antibody may be used. As the nucleic acid, a nucleic acid that can be used as a probe for a specific gene or a pathogen gene can be used.

When the above-mentioned antibody or nucleic acid is used as a detection system, the above-mentioned detection system contained in the diagnostic marker of the present invention specifically binds to a cancer antigen, a cancer gene or the like. The lesion is immunostained with the diagnostic marker of the present invention. Then, by irradiating near-infrared rays or far-infrared rays with an infrared laser or the like, the lesions emitting fluorescence can be confirmed.

In another embodiment of the diagnostic marker of the present invention, a fluorescent functional group is introduced into an amplification system substance such as avidin or biotin which is a detection system. For example, by applying a conventionally used biotin-labeled anti-cancer antigen antibody or the like to a living body, labeling can be performed by binding the anti-cancer antigen antibody and the biotin-labeled antibody. After performing such labeling, by binding the diagnostic marker of the present invention containing avidin as a detection system to the biotin-labeled antibody, the labeling with the biotin-labeled antibody can be amplified by the diagnostic marker of the present invention. It is possible to detect the initial cancer by infrared laser. As the amplification substance, any substance may be used as long as it can amplify the label as described above.

Furthermore, the above-mentioned antibody or nucleic acid bound to the above-mentioned amplification system substance may be used as a detection system for the diagnostic marker of the present invention. For example, an antibody obtained by binding an avidin to an anti-cancer antigen antibody can be used as a detection system for the diagnostic marker of the present invention. Alternatively, a secondary antibody can be used as the detection system of the diagnostic marker of the present invention. The above-described detection system is an example, and the detection system that can be used for the diagnostic marker of the present invention is not limited to the above. Any substance may be used as the detection system as long as it has a property of substantially specifically binding to the target cell or target tissue for the purpose of inspection / diagnosis.

The diagnostic marker of the present invention is not limited to one in which the fluorescent functional group as described above is bound to the detection system directly or via a linker. For example, a protein such as albumin has a fluorescent functional group. The fluorescent functional group and the detection system may be bound to each other via a protein such as albumin by introducing the detection system. One or more, preferably about 10 or more, of the above-mentioned fluorescent functional groups can be introduced into a protein such as albumin, and a detection system such as an antibody can be easily introduced. The diagnostic marker described above is a preferred embodiment of the present invention.

Furthermore, the diagnostic marker according to another embodiment of the present invention is a fluorescence which emits fluorescence having a wavelength of 780 nm or more, preferably 780 to 840 nm or more when irradiated with excitation light having a wavelength of 600 to 800 nm. It is composed of a detection system to which a sexual functional group is bound. As such a fluorescent functional group, in addition to those derived from the above formula (I) or (II), if it emits fluorescence having a wavelength of 780 nm or more by irradiating with excitation light having a wavelength of 600 to 800 nm. However, any one may be used.
A compound for introducing such a fluorescent functional group can be appropriately selected by those skilled in the art depending on the purpose of use of the diagnostic marker, the type of excitation light used, and the like.

In order to bind such a fluorescent functional group to a detection system, for example, a near-infrared absorbing agent such as cyanine, phthalocyanine, dithiol nickel complex, naphthoquinone / anthraquinone, indophenol or azo is used. Compounds can be used. When these compounds are bound to the detection system directly or via a linker, the absorption wavelength of the compound may shift to the long wavelength side.
The wavelength of the excitation light of the compound used to introduce the fluorescent functional group is 60
It can be below 0 nm. Further, in order to introduce a fluorescent functional group into the detection system, it is preferable that the compound used has a group represented by Z in the general formula (III) or (V). If no such group exists,
The fluorescent functional group can be coupled to the detection system via a linker available to those of skill in the art.

The diagnostic agent provided by the present invention is characterized by containing the above-mentioned diagnostic marker as an active ingredient. INDUSTRIAL APPLICABILITY The diagnostic agent of the present invention is useful because it can be excited by near-infrared rays or far-infrared rays and does not cause damage to living tissues or DNA during diagnosis. Further, the diagnostic agent of the present invention is characterized by being extremely excellent in water solubility. In the diagnostic agent of the present invention, a diagnostic agent including a diagnostic marker that emits fluorescence having a wavelength longer than about 820 nm when irradiated with excitation light of 780 nm is particularly preferable. The diagnostic agent of the present invention may contain one or more diagnostic markers.

The diagnostic agent of the present invention comprises, for example, the above-mentioned diagnostic marker in an aqueous medium such as various buffers, preferably,
Form of solution dissolved in buffer solution such as physiological saline or phosphate buffer, or particulate powder or frozen that can be dissolved by adding buffer solution such as saline or phosphate buffer at the time of diagnosis / test Although it is provided as a solid agent in the form of dry powder, the form of the diagnostic agent is not limited to the above, and can be appropriately selected by those skilled in the art according to the purpose of use.

In order to produce the diagnostic agent of the present invention, pharmacologically and pharmaceutically acceptable additives such as excipients, disintegrants or disintegration aids, binders, lubricants and coatings are used. Agents, dyes, diluents, bases, solubilizers or solubilizers, isotonic agents, pH adjusters, stabilizers, propellants, adhesives and the like can be used. For example, glucose, lactose,
Excipients such as D-mannitol, starch, or crystalline cellulose; disintegrants or disintegration aids such as carboxymethylcellulose, starch, or carboxymethylcellulose calcium; petrolatum, liquid paraffin, polyethylene glycol, gelatin, kaolin, glycerin, purified water,
Or bases such as hard fat; isotonic agents such as glucose, sodium chloride, D-mannitol, glycerin; pH adjusting agents such as inorganic acids, organic acids, inorganic bases or organic bases; vitamin A, vitamin E, coenzyme Q For example, a pharmaceutical additive such as a drug that can contribute to stabilization may be added.

Regarding the method of using the diagnostic agent containing the diagnostic marker of the present invention, for example, the examination method using an infrared endoscope will be described. The above-mentioned diagnosis is performed endoscopically on a tissue suspected of having a lesion. After spraying or applying a drug (concentration of about 0.1 to 1,000 mg / ml) to stain the lesion area and washing appropriately to remove excess diagnostic agent from the tissue, near infrared or far infrared, more specifically For example,
By irradiating with laser excitation light having a wavelength of 800 nm, preferably about 780 nm, it is possible to detect a lesion site emitting fluorescence. The diagnostic marker of the present invention is characterized in that it can be directly applied to a living body, but the mode of use of the diagnostic marker of the present invention is not limited to that applied to a living body, and immobilization such as paraffin-immobilized specimen It goes without saying that it can be applied to a sample.

A means such as an infrared endoscope or an infrared microscope can be used for detecting fluorescence. For example, a filter having a transmission characteristic, specifically, a filter having an excitation light blocking characteristic and a fluorescence are used. One detection filter or a combination of two or more detection filters can be used. When performing the endoscopic examination by applying the diagnostic marker of the present invention to a living body, the magnification of the endoscope is 10 to 1,000.
It is possible to use a graded one, and it is preferable to use, for example, an infrared endoscope having a microscope-level magnification. Further, it is preferable that the endoscope is provided with a spraying or applying means for the diagnostic agent of the present invention and a cleaning means.

When the diagnostic marker of the present invention is applied to a tissue or specimen excised in vitro, an infrared microscope can be used for detecting fluorescence. In addition, after observing the preparation under normal light and confirming the stained area, it is also possible to shoot with an infrared film under an infrared light source in a dark room or record it on a video film and perform image analysis with a computer. .

[0044]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples.

Example 1 Preparation of Labeling Compound According to the following scheme, a labeling compound of the formula (III) used for preparing the diagnostic marker of the present invention was prepared. The compound numbers in the following production examples correspond to the compound numbers in the scheme.

[0046]

Embedded image

[0047]

Embedded image

Ethane iodide (2.84 ml, 35.5 mmol) was added to 100 ml of compound 1 (5 g, 23.9 mmol, obtained from Daiichi Pure Chemicals Co., Ltd.) / Acetonitrile, and the mixture was refluxed for 5 hours. The reaction mixture was concentrated under reduced pressure and the residue was diluted with 200 ml of ether.
After crystallization, the obtained crystals were filtered, washed with ether, and dried under reduced pressure to obtain Compound 2 as a light reddish brown powder. Yield 6.83 g (78.2% yield).

Compound 1 (5 g, 23.9 mmol) / 100 ml of DMF
6-Bromocaproic acid ethyl ester (6.32 ml, 35.5 m
mol) was added and the mixture was heated at 80 ° C. for 16 hours. The reaction mixture was concentrated under reduced pressure, 200 ml of ether was added to the residue for crystallization, and the obtained crystals were filtered, washed with ether, and dried under reduced pressure. 30 ml of a 1 M sodium hydroxide aqueous solution / methanol = 1/1 mixed solution was added, and the mixture was stirred at room temperature for 2 hours. Methanol was distilled off under reduced pressure, the aqueous solution was neutralized with a 4 M aqueous hydrochloric acid solution, and then washed three times with chloroform. The aqueous solution was concentrated under reduced pressure to obtain a red solid of Compound 3. yield
5.75 g (74.4% yield).

Compound 2 (5 g, 13.7 mmol) and glutaconaldehyde dianyl hydrochloride (3.90 g, 13.7 mmol) were suspended in 50 ml of acetic anhydride and heated at 100 ° C. for 1.5 hours. The red solution was poured into 300 ml of water, and the resulting black-red solid was collected by filtration.
After washing with water and drying under reduced pressure, a black-red powder of compound 4 was obtained. Yield 6.02 g (101% yield).

Compound 4 (220 mg, 0.505 mmol) and compound 3 (164 mg, 0.507 mmol) were dissolved in 3 ml of pyridine,
The mixture was stirred at 50 ° C for 1 hour. The reaction mixture was concentrated under reduced pressure, and the obtained residue was purified by a silica gel column (solvent: 1 to 20% methanol / chloroform) to obtain a black green solid of Compound 5. Yield 49 mg (15.5% yield).

Compound 5 (40 mg, 64 μmol) in 1 ml of DMF
N-Hydroxysuccinic anhydride (8.8mg, 76μmol) and N, N '
-Dicyclohexylcarbodiimide (DCC, 26.4 mg, 0.1
(28 mmol) was added and the mixture was reacted overnight at 4 ° C., 10 ml of ether was added to the reaction solution, and the resulting residue was washed with ether three times. Dissolve this solid in 400 μl of chloroform,
After adding 200 μl of 0.1 M hydrochloric acid aqueous solution and stirring, the chloroform phase was separated and washed three times with water. The chloroform solution was concentrated under reduced pressure to give compound 6 [labeling compound of formula (III)]
To obtain a black-green solid. Yield 38 mg (78.5% yield). MS
(FAB) m / e = 720 (M ⁺ ); Fluorescence spectrum: λ _ex = 769 nm,
λ _em = 820 nm (10% DMSO / water)

Compound 5 (20 mg, 32 μmol) in 1 ml DMF
Sodium N-hydroxysuccinate sulfonate (8.
3 mg, 38 μmol) and N, N′-dicyclohexylcarbodiimide (13.2 mg, 64 μmol) were added, and the mixture was reacted overnight at 4 ° C. and then filtered. 10 ml of ether was added to the filtrate, and the resulting residue was washed with ether three times. This solid is DMF 400
After dissolving in μl, 200 μl of 0.1 M hydrochloric acid aqueous solution was added and stirred, and then the chloroform phase was separated and washed 3 times with water. The chloroform solution was concentrated under reduced pressure to obtain a black green solid of compound 7 [labeling compound of formula (III)]. Yield 17 mg (Yield
66.4%). MS (FAB) m / e = 822 (M + Na); fluorescence spectrum
: λ _ex = 769 nm, λ _em = 820 nm (10% DMSO / water)

Compound 5 (20 mg, 32 μmol) / DMF 0.5 ml
N- (2- (N'-maleimido) ethyl) piperazine dihydrochloride (PEM, 4.4 mg, 38 μmol) and triethylamine (TEA, 2
0 μl, 0.146 mmol) and DCC (10.2 mg, 49 μmol) were added and the mixture was reacted overnight at 4 ° C., then the reaction solution was mixed with ether 10
ml was added and the resulting residue was washed with additional ether 3 times.
This solid was dissolved in 400 μl of chloroform, 200 μl of 0.1 M hydrochloric acid aqueous solution was added and stirred, and then the chloroform phase was separated and washed with water three times. The chloroform solution was concentrated under reduced pressure to obtain a black green solid of compound 8 [compound for labeling of formula (III)]. Yield 20 mg (70.5% yield). MS (FAB) m / e =
814 (M ⁺ ); Fluorescence spectrum: λ _ex = 773 nm, λ _em = 821 nm
(10% DMSO / water)

Example 2: Production of compound for labeling Hydrazine monohydrochloride (9.0 mg, 0.131 mmol) was added to 0.5 ml of compound 6 (20 mg, 26.4 μmol) / DMF, and the mixture was reacted overnight at room temperature. After filtering the reaction solution and concentrating the filtrate under reduced pressure,
The residue was dissolved in 0.5 ml of chloroform, and the chloroform solution was washed 3 times with water. The chloroform phase was concentrated under reduced pressure to obtain Compound 9 as a black-green solid. Yield 16 mg (Yield 9
0.0%). MS (FAB) m / e = 637 (M ⁺ ); Fluorescence spectrum:
λ _ex = 771 nm, λ _em = 820 nm (10% DMSO / water)

Compound 10 (5.0 g, 15.1 mmol) and glutaconaldehyde dianyl hydrochloride (4.40 g, 15.1 mmol) were suspended in a mixed solution of 20 ml of acetic anhydride and 300 ml of acetic acid and heated at reflux temperature for 5 hours. . The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the obtained residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of compound 11. Yield 5.33
g (yield 65.2%).

Compound 11 (167 mg, 0.308 mmol) and compound 3 (100 mg, 0.309 mmol) were dissolved in 3 ml of pyridine,
The mixture was stirred at 50 ° C for 1 hour. The reaction mixture is concentrated under reduced pressure,
The obtained residue was dissolved in 10 ml of water to adjust the pH of the solution to 3, and then purified with a Sephadex LH20 column (solvent: water) to obtain Compound 12 as a black-green solid. Yield 51 mg
(Yield 23.1%).

Compound 12 (30 mg, 41.8 μmol) / 50 v / v
% THF aqueous solution (1 ml), sodium N-hydroxysuccinate sulfonate (16.8 mg, 77.4 μmol) and N, N'-dicyclohexylcarbodiimide (25.8 mg, 0.125 mmol) were added, and the mixture was reacted overnight at 4 ° C and then filtered. The filtrate at 20 ° C.
It concentrated under reduced pressure below. Ethyl acetate (10 ml) was added to the residue, and the generated crystals were washed with ether three times. The crystals were dissolved in 200 μl of water and purified by a Sephadex LH20 column (solvent: water) to obtain a black-green solid of Compound 13 [labeling compound of formula (III)]. Yield 34 mg (Yield 88.8
%). MS (FAB) m / e = 892 (M -); fluorescence spectrum: lambda _ex =
771 nm, λ _em = 822 nm (water)

Example 3 Preparation of Labeling Compound According to the following scheme, a labeling compound of the formula (V) used for preparing the diagnostic marker of the present invention was prepared. The compound numbers in the following production examples correspond to the compound numbers in the scheme.

[0060]

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Compound 14 (5.0 g, 14.5 mmol) and glutaconaldehyde dianyl hydrochloride (4.23 g, 14.5 mmol) were suspended in a mixed solution of 20 ml of acetic anhydride and 300 ml of acetic acid and heated at reflux temperature for 5 hours. . The red solution was concentrated under reduced pressure, 200 ml of a mixed solution of ethyl acetate and water was added to the obtained residue to suspend it, and a black red solid was collected by filtration. After washing with water, it was dried under reduced pressure to obtain a black-red powder of compound 15. Yield 5.20
g (yield 66.1%).

Compound 15 (300 mg, 0.553 mmol) and compound 3 (179 mg, 0.553 mmol) were dissolved in 5 ml of pyridine,
The mixture was stirred at 50 ° C for 1 hour. The reaction mixture is concentrated under reduced pressure,
The obtained residue was dissolved in 10 ml of water to adjust the pH of the solution to 3, and then purified by a Sephadex LH20 column (solvent: methanol) to obtain a black green solid of Compound 16. yield
105 mg (25.9% yield).

Compound 16 (106 mg, 0.145 mmol) / 50 v /
To 3 ml of v% THF aqueous solution, sodium N-hydroxysuccinate sulfonate (65.2 mg, 0.287 mmol) and N, N'-dicyclohexylcarbodiimide (129 mg, 0.596 mmol) were added.
Was added and reacted overnight at 4 ° C, then filtered, and the filtrate was filtered.
The mixture was concentrated under reduced pressure at a temperature of not higher than ° C. 10 ml of ethyl acetate was added to the residue, and the resulting crystals were washed with ether 3 times to give compound 17
To obtain a black-green solid. Yield 110 mg (yield 80.7%). MS
(FAB) m / e = 906 (M -); fluorescence spectra: λ _ex = 768 n
m, λ _em = 807 nm (water)

The above compound 17 (100 mg, 0.11 mmol) was dissolved in 2 ml of methanol, and 1 g of sodium iodide (6.7
(5 mmol) methanol solution in 5 ml was added. The methanol solution was concentrated to 1/2 under reduced pressure, cooled at 4 ° C. overnight, and the precipitated crystals were collected by filtration. The green crystals obtained were dried in a vacuum desiccator to obtain Compound 18 (a labeling compound of the formula (V)) (85
mg, yield 72%). The water solubility data and infrared absorption spectrum data (IR) of sodium iodide were measured before salt formation (counterion type) and after salt formation. Further, the iodine content was determined by the Schöniger method.

Infrared absorption spectra of Compound 18 in which sodium iodide was incorporated as a salt and counterion type indocyanine green N-hexanoic acid sulfosuccinimide ester (Compound 17) used as raw materials were changed, 2360 observed in counterion compound 17 and
Absorption at 1740 cm ⁻¹ was abolished in compound 18 (see FIG. 1: (a) shows compound 18, (b) shows compound 17, that is, before salt formation). From these results, it is clear that the crystal structure is changed between the case where an intramolecular counter ion is formed and the case where a double salt of sodium iodide is formed.

Example 4: Preparation of labeling compound Indocyanine green-N-butanoic acid sulfosuccinimide ester (Compound 19) was prepared according to the method of Example 3. This compound 19 (150 mg, 0.17 mmol) was added to methanol 3
This was dissolved in ml, and a solution of 1.5 g (10 mmol) of sodium iodide in 8 ml of methanol was added. Methanol was concentrated to 1/2 under reduced pressure, cooled at 4 ° C. overnight, and the precipitated crystals were collected by filtration.
The obtained green crystals were dried with a vacuum desiccator to give the compound
20 (labeling compound of formula (V)) was obtained (117 mg, yield 66
%). Water solubility data and infrared absorption spectrum data (IR) before and after sodium iodide salt formation were measured. Further, the iodine content was determined by the Schöniger method.

Example 5: Preparation of labeling compound According to the method of Example 3, indocyanine green-N-hexanoic acid sulfosuccinimide ester (Compound 21) was prepared. This compound 21 (100 mg, 0.11 mmol) was added to methanol.
Dissolve in 2 ml and add 0.3 g of sodium perchlorate (2.5 mmo
A solution of l) in 20 ml of methanol was added. 1/4 of methanol
After concentration under reduced pressure and cooling at 4 ° C. overnight, the precipitated crystals were collected by filtration. The obtained green crystals were dried with a vacuum desiccator to obtain 23 mg of compound 22 [labeling compound of formula (V)] (yield 21%). The water solubility data and IR before and after salt formation of sodium perchlorate were measured.

[0068]

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[0069]

[Table 1] ──────────────────────── Labeling compound Counterion type Double salt forming type ────────────── ────────── Example 3 Compound 17 Compound 18 Water solubility: 1 mg / 10 ml or more 1 mg / 0.8 ml IR (cm ^-1 ): 2360, 1740 Absorption disappeared I content: -12.3% Example 4 Compound 19 Compound 20 Water solubility: 1 mg / 10 ml or more 1 mg / 0.9 ml IR (cm ^-1 ): 2370, 1790 Absorption disappeared I content: −13.5% Example 5 Compound 21 Compound 22 Water solubility: 1 mg / 10 ml or more 1 mg / 5 ml IR (cm ^-1 ): 2370, 1790 Absorption disappeared ────────────────────────

Example 6: Preparation of labeling compound Compound 13 was treated according to the method of Example 2 with a solution of sodium iodide in methanol to give compound 23. Comparing the infrared absorption spectrum of the compound 13 used as a raw material with the infrared absorption spectrum of the labeling compound 23, the absorptions of 2360 and 1740 cm ^-1 observed in the compound 13 were observed in the compound 23.
Was not observed in.

Example 7: Production of diagnostic marker (a) Purification of antibody Anti-EMA (Epitherial Membrane Antigen) antibody containing culture supernatant (Dako, M613) was used as an antibody using MabTrap GII (Pharmacia). The EMA monoclonal antibody was purified as follows. 4 ml of binding buffer to 4 ml of culture supernatant
Was added and applied to a MabTrap GII column previously equilibrated with binding buffer. After washing with 5 ml of binding buffer, elution buffer
3 ml was added and the elution buffer was fractionated every 0.5 ml. Using a spectrophotometer, collect fractions with an absorbance at 280 nm of 0.05 or more as a purified IgG fraction, and exchange the buffer with 0.1 M phosphate buffer (pH 7.5) using a PD-10 column (Pharmacia). Then, the degree of purification was confirmed by SDS-PAGE (manufactured by Daiichi Pure Chemicals Co., Ltd.). 725 μg of purified anti-EMA monoclonal antibody was obtained from 4 ml of the culture supernatant.

(B) Binding of Antibody to Labeling Compound ICG-OSu (compound 6 above) in DMF solution 0.1 ml in 0.5 ml of 100 mM phosphate buffer containing 500 μg of the above purified antibody.
25 ml (0.8 mg / ml) was added, and the mixture was left standing at 30 ° C for 30 minutes. This reaction mixture was previously equilibrated with the same reaction buffer PD-10.
It was applied to a column and unreacted ICG-OSu was separated. Further PD
-10 column was used to exchange the protein fractionation buffer with 0.1 M phosphate buffer-0.05% sodium azide (pH 7.4), and the labeling compound (compound 6) was bound with anti-EMA antibody of the present invention. Diagnostic marker 74 protein μg / ml was obtained. Then, 0.1% BSA was added for storage. The UV / visible absorption spectrum is shown in FIG.

The measurement conditions of the above spectrum are as follows. Measuring instrument: Hitachi U-3200 spectrophotometer; Measuring solvent: 100 mM phosphate buffer (pH 7.4), 0.05% sodium azide; Scan speed: 300.0 nm / min. Then, 0.1% BSA was added for storage. The binding number of the antibody to the labeling compound obtained from the concentration of the antibody obtained with the BCA protein assay reagent (manufactured by Pierce) and the molecular extinction coefficient of the labeling compound in DMSO is about 12 mol.
It was a mol antibody.

Example 8: Production of diagnostic marker Compound 18 (1 mg) was dissolved in 0.94 ml of water to give a 1 mM solution. 1 mg of human IgG was dissolved in 1 ml of 50 mM sodium carbonate buffer having a pH of 8.5, 0.2 ml of the above solution containing compound 18 was added to this solution, and the mixture was reacted at 30 ° C. for 1 hour. The reaction solution was applied to a Sephadex G-25 column to separate the diagnostic marker of the present invention. As a separation buffer, 50 mM phosphate buffer (pH 7.4) was used. Unreacted compound 18 remained on top of the Sephadex gel and was well separated from the diagnostic marker. The obtained diagnostic marker was freeze-dried and stored frozen at -20 ° C. Fig. 3 shows the result of measuring the fluorescence spectrum by taking 1 mg of the diagnostic marker and dissolving it in 100 ml of 50 mM phosphate buffer (pH 7.4).

Example 9: Preparation of diagnostic marker 0.5 ml of 100 mM phosphate buffer containing 500 μg of the purified antibody obtained in Example 7 was added to 0.125 ml (0.8 m) of DMF solution of compound 18.
(g / ml) was added and the mixture was allowed to stand at 30 ° C for 30 minutes. This reaction mixture was applied to a PD-10 column which had been equilibrated with the same reaction buffer in advance, and unreacted compound 18 was separated. Further, the buffer solution for protein fraction was added to 0.1 M phosphate buffer-0.05 using PD-10 column.
% 18 sodium azide (pH 7.4), Compound 18 is anti-EM
Diagnostic marker of the present invention bound to A antibody 74 protein μg /
ml was obtained. Then, 0.1% BSA was added for storage.

Example 10: Binding of labeling compound and protein (reference example) 10 mg (0.151 μmol) of BSA (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 3.0 ml of HEPES buffer to prepare a BSA solution. 2.0 mg (2.64 μmol) of the labeling compound (compound 6) was dissolved in 150 μl of DMSO, mixed with the above BSA solution, and left overnight in the refrigerator. Then, gel filtration was carried out using a PD-10 column in the same manner as above to obtain a labeled protein in which a fluorescent functional group derived from the labeling compound was introduced into BSA. During the gel filtration after the reaction, the labeling compound did not remain in the green solution, so all of the labeling compound reacted with BSA, and one fluorescent functional group per BSA molecule. It is considered that about 17.5 molecules were introduced.

Example 11: Preparation of diagnostic marker (a) 100 mM phosphate buffer containing BSA (10 mg) (pH)
7.5) 0.5 ml of DMF solution of compound 6 in 5 ml (4.6 mg / ml)
Was added and the mixture was allowed to stand at 30 ° C for 30 minutes. This reaction mixture was previously equilibrated with the same reaction buffer Sephadex G2
The mixture was attached to 5 columns, and the conjugate of the labeling compound and the protein (labeled BSA) was separated from the unreacted compound 6. Add 20% buffer to the fractions containing the conjugate using a Sephadex® G25 column.
The phosphate buffer was replaced with 0.15 M NaCl (pH 7.0) to obtain about 8 mg of fluorescent functional group-introduced BSA.

(B) Anti-CEA antibody 1.0 mg / ml (0.1 M citric acid / 0.15 M NaCl, pH 4.0) and pepsin concentration 0.004 mg / ml
Pepsin was added to the mixture so that the mixture was reacted at 37 ° C. for 2 hours. The reaction solution was 20 mM phosphate buffer, 0.15 M NaCl, 1
It was added to TSKgel G3000SW pre-equilibrated with mM EDTA to separate F (ab ') ₂ from undigested IgG and digested fragments (about 500 μg). Add 2-aminoethanethiol hydrochloride to the above F (ab ') ₂ fraction to a final concentration of 10 mM.
The reaction was carried out at 90 ° C for 90 minutes. The reaction solution was applied to a Sephadex G25 column to remove 2-aminoethanethiol to obtain a solution containing Fab '.

(C) Labeled BSA (2 mg, 20 mM phosphate buffer
50 μl sulfo-SMCS in -0.15 M NaCl, pH 7.0; PBS)
Solution (sulfo-EMCS, Dojindo Laboratories, 1 mg / ml,
PBS) was added dropwise with stirring, and the mixture was reacted at room temperature for 20 minutes. This reaction solution was pre-equilibrated with PBS to Sephadex.
Labeled BS activated with sulfo-SMCS on a G25 column
A was separated. This solution was added to the Fab 'solution of (b) above, and the mixture was reacted at room temperature for 2 hours with stirring. After the reaction was completed, the reaction mixture was separated and purified by gel filtration using TSKgel G3000SW to obtain BSA in which a fluorescent functional group and Fab 'were introduced.

Example 12: Immunohistochemical Staining Using Diagnostic Marker Using the diagnostic marker of the above Example 6 as the diagnostic marker of the present invention, paraffin section of esophageal mucosa confirmed to have anti-EMA antibody staining ( Approximately 10 × 10 mm, thickness 2.5 μm) or by applying a diagnostic marker to the excised raw section of esophageal cancer, ABC method (avidin-biotin complex method)
Then, the degree of staining was confirmed by observing under a microscope and under an ordinary endoscope. In addition, the preparation colored as described above was observed under an infrared endoscope to identify a stained site, and a comparison was made with the stained site identified by a normal endoscopic observation. Furthermore, the concentration of the diagnostic marker was diluted by multiples and observed with a normal endoscope and an infrared endoscope, and the discriminating ability between them was examined.

5 times and 10 times dilutions of the diagnostic marker were used for staining, the color development under the microscope was good, and compared with the anti-EMA antibody (50 times dilution) used as a control. The same level of dyeability was confirmed. Normally, when observing the preparation by endoscopy, the diagnostic marker diluted 10-fold was consistent with the esophageal mucosa in the same manner as the control.
Deposition of B (Diaminobenzidine) was observed.

[0082]

EFFECTS OF THE INVENTION By using the diagnostic marker of the present invention, for example, malignant neoplasia of epithelial tissue such as esophageal cancer, gastric cancer, or colon cancer is quasi-incorporated using an infrared endoscope or the like. Diagnosis and identification / diagnosis of lesions during surgery can be performed easily and accurately. The test or diagnosis using the diagnostic marker of the present invention is useful as a biological immunohistological staining method because there is no fear of damage to living tissue or DNA due to ultraviolet excitation and direct testing or diagnosis can be performed in the living position. . Further, since the labeling compound contained in the diagnostic marker of the present invention has the characteristic of being extremely water-soluble, the diagnostic marker of the present invention is not absorbed from the gastrointestinal tract and can be tested very safely. It is useful because it enables diagnosis.

[Brief description of the drawings]

FIG. 1 is a diagram showing an infrared absorption spectrum of a labeling compound (Compound 18). In the figure, (a) is an infrared absorption spectrum of Compound 18 that forms a salt by the addition of sodium iodide, and (b) is the indocyanine green-N-hexanoic acid sulfosuccinimide ester (Compound 17) used as a raw material. It is an infrared absorption spectrum.

FIG. 2 is a diagnostic marker of the present invention obtained by binding Compound 6 as a labeling compound and an anti-EMA antibody.
It is a figure which shows the ultraviolet-visible absorption spectrum of (74 microgram / ml).

FIG. 3 is a diagram showing a fluorescence spectrum of the diagnostic marker of the present invention in which the labeling compound, Compound 18, is bound to human IgG. In the figure, the solid line shows the emission spectrum and the broken line shows the excitation spectrum.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display location // A61K 39/44 A61K 39/44 C07D 209/60 C07D 209/60 403/14 207 403/14 207 C07H 21/00 C07H 21/00 (72) Inventor Kazuhiro Takesako 56-10 Koga, Mashiki-cho, Kamimashiki-gun, Kumamoto Prefecture (72) Inventor Hiroshi Takeuchi 1-13-44, Nishisatsuma, Kamagaya-shi, Chiba

Claims (21)

[Claims] 1. A detection system comprising: (a) a detection system; and (b) the following formula attached to the detection system: (In the formula, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, or a sulfonic acid group; R ³ represents an alkyl group, a sulfonic acid alkyl group, or an amino-substituted alkyl group; X ⁻ represents an anion species if necessary; Y is an alkylene group having 1 to 10 carbon atoms, or 1 to 10 carbon atoms containing at least one atom selected from the group consisting of oxygen atom, nitrogen atom, and sulfur atom. And a fluorescent functional group represented by 2. (a) a detection system, and (b) the following formula attached to the detection system: (In the formula, R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group, an alkoxy group, or a sulfonate group; R ⁶ represents an alkylene group; M ⁺ represents an alkali metal ion;
Q ⁻ represents a halogen ion, a perchlorate ion, or a thiocyanate ion; Y represents an alkylene group having 1 to 10 carbon atoms, or one or more atoms selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom. And a fluorescent functional group represented by C1 to C10). 3. The diagnostic marker according to claim 1, wherein the fluorescent functional group is directly bound to the detection system. 4. The diagnostic marker according to claim 1, wherein the fluorescent functional group is bound to the detection system via a linker or a protein. 5. The detection system according to claim 1, which is selected from the group consisting of an antibody, a nucleic acid, and an amplification system substance.
The diagnostic marker according to the item. 6. The diagnostic marker according to claim 5, wherein an anti-cancer antigen antibody, an anti-bacterial antibody, or an anti-virus antibody is used as the antibody. 7. By irradiation of near infrared rays or far infrared rays
The diagnostic marker according to any one of claims 1 to 6, which emits fluorescence having a wavelength of 780 nm or more. 8. The diagnostic marker according to claim 1, which is used for infrared endoscopy or for identifying a lesion during surgery. 9. The diagnostic marker according to any one of claims 1 to 8, which is used to directly stain a tissue of a living body by an immunohistochemical staining method. 10. A diagnostic agent comprising the diagnostic marker according to any one of claims 1 to 9 as an active ingredient. 11. The following formula: (In the formula, R ¹ and R ² each independently represent a hydrogen atom, an alkyl group, an aryl group, an alkoxy group, or a sulfonic acid group; R ³ represents an alkyl group, a sulfonic acid alkyl group, or an amino-substituted alkyl group; X ⁻ represents an anion species as necessary; Z represents the following formula: W and Y are each independently an alkylene group having 1 to 10 carbon atoms, or 1 selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.
The method for producing a diagnostic marker according to claim 1, further comprising a step of reacting a labeled compound represented by the above-mentioned alkylene group having 1 to 10 carbon atoms and containing a atom) with a detection system. 12. The following formula: (In the formula, R ⁴ and R ⁵ each independently represent a hydrogen atom, an alkyl group, an alkoxy group, or a sulfonate group; R ⁶ represents an alkylene group; M ⁺ represents an alkali metal ion;
Q ^- represents a halogen ion, a perchlorate ion, or a thiocyanate ion; Z is the following formula: W and Y are each independently an alkylene group having 1 to 10 carbon atoms, or 1 selected from the group consisting of an oxygen atom, a nitrogen atom, and a sulfur atom.
The method for producing a diagnostic marker according to claim 2, which comprises a step of reacting a labeled compound represented by the above-mentioned alkylene group having 1 to 10 carbon atoms and containing an atom) with a detection system. 13. A detection system and 600 to 80 bound to the detection system
A diagnostic marker containing a fluorescent functional group that emits fluorescence having a wavelength of 780 nm or more when irradiated with excitation light having a wavelength of 0 nm. 14. A fluorescent substance having a fluorescent functional group derived from a near-infrared absorbing compound selected from the group consisting of a cyanine type, a phthalocyanine type, a dithiol nickel complex type, a naphthoquinone / anthraquinone type, an indophenol type, and an azo type. 14. The diagnostic marker according to claim 13, which is a functional group. 15. The diagnostic marker according to claim 13 or 14, wherein the fluorescent functional group is directly bound to the detection system. 16. The diagnostic marker according to claim 13 or 14, wherein the fluorescent functional group is bound to the detection system via a linker or a protein. 17. The diagnostic marker according to any one of claims 13 to 16, wherein the detection system is selected from the group consisting of antibodies, nucleic acids, and amplification system substances. 18. The diagnostic marker according to claim 17, wherein an anti-cancer antigen antibody is used as the antibody. 19. The diagnostic marker according to any one of claims 13 to 17, which is used for identification of a lesion during infrared endoscopy or surgery. 20. The diagnostic marker according to any one of claims 13 to 19, which is used for directly staining tissue of a living body by an immunohistochemical staining method. 21. A diagnostic agent containing the diagnostic marker according to any one of claims 13 to 20 as an active ingredient.