Fluorescent probe for detecting ovarian cancer and disseminated lesions thereof

Abstract

[Problem] To provide a fluorescent probe with which ovarian cancer and disseminated lesions originating from ovarian cancer can be detected. [Solution] A fluorescent probe for detecting ovarian cancer and disseminated lesions thereof comprises at least one compound represented by general formula (I) or salt thereof.　

Description

Fluorescent probe for detecting ovarian cancer and its disseminated lesions

[0001] This invention relates to a fluorescent probe for detecting ovarian cancer and disseminated lesions originating from ovarian cancer.

[0002] Ovarian malignancies are on the rise in Japan, with 13,049 cases reported in 2018. The number of deaths was 4,876, making it the leading cause of death among female reproductive organ malignancies (Non-Patent Literature 1). Over 90% of ovarian malignancies in Japan are epithelial ovarian cancers, which often have few noticeable symptoms in the early stages, with stage III and IV cases accounting for over 40%. The prognosis for stage III and IV ovarian cancer is poor, making improvement in treatment outcomes for advanced cases a crucial challenge in ovarian cancer treatment.

[0003] The degree of surgical completion is a particularly important prognostic factor among treatment factors, and in advanced cancer, the size of the residual tumor after surgery correlates with the prognosis. The principle is to perform debulking surgery to the greatest extent possible, aiming for complete removal of the lesion. The maximum residual tumor size is said to correlate with the prognosis, and when the maximum residual tumor size is reduced to less than 1 cm by primary debulking surgery (PDS), it is called optimal surgery, and when it is 1 cm or more, it is called suboptimal surgery. It is said that the prognosis improves when optimal surgery is performed (Non-Patent Literature 2-4). Furthermore, it has been shown that when complete surgery is performed, resulting in no macroscopic residual tumor, the prognosis is significantly improved compared to suboptimal surgery (Non-Patent Literature 5-7).

[0004] A fluorescent probe is a functional fluorescent substance that changes its molecular structure upon reaction with a specific molecule, resulting in strong fluorescence emission or a change in the color of the fluorescence. It has become an indispensable research tool in current biological research, enabling real-time observation of the dynamics of physiologically active substances. One of the inventors, Urano et al., have successfully developed a novel fluorescent probe that reacts with an enzyme specifically expressed in cancer cells, allowing the fluorescent substance to be taken up by cancer cells and identify them as strong fluorescence (Non-Patent Literature 8). Specifically, they focused on the fact that the activity of γ-glutamyl transpeptidase is enhanced specifically in tumor sites, and developed γ-glutamylhydroxymethylrhodamine green (gGlu-HMRG), a fluorescent probe activated by this enzyme. This method allows for rapid identification of tumor sites with a simple spraying operation and is expected to be a new technique for rapid diagnosis and imaging-guided surgery.

[0005] National Cancer Center Japan, Cancer Information Service, <URL: https: / / ganjoho.jp / reg_stat / atatistics / atat / summary.html> J Clin Oncol. 2002; 20: 1248-59 J Natl Cancer Inst. 2005; 97: 560-6 J Clin Oncol. 2005; 23: 8802-11 Gynecol Oncol. 2013; 130: 493-8 Cancer. 2009; 115: 1234-44 Gynecol Oncol. 2017; 145: 21-26 Sci Transl Med 2011; 3: 110-119

[0006] As mentioned earlier, residual lesions significantly impact the prognosis of ovarian cancer. However, identifying and completely resecting numerous, minute disseminated lesions is extremely difficult, and it is practically impossible to examine all resected specimens with rapid pathological diagnosis to confirm whether all cancer cells have been removed by macroscopic examination. Therefore, it is thought that if disseminated lesions can be identified by performing fluorescence imaging during surgery, it may be possible to completely resect them.

[0007] Therefore, an object of the present invention is to provide a fluorescent probe capable of detecting ovarian cancer and seeded lesions originating from ovarian cancer. Another object of the present invention is also to provide a method for detecting ovarian cancer and seeded lesions originating from ovarian cancer using the fluorescent probe.

[0008] The inventors applied a fluorescent probe library targeting various enzyme activities to ovarian cancer seeded specimens, searched for highly cancer-specific fluorescent probes, and as a result of studying aiming at realizing a new diagnostic technique, found that an HMRG derivative probe having a specific structure can detect ovarian cancer and its seeded lesions, and completed the present invention.

[0009] That is, the present invention provides [1] a fluorescent probe for detecting ovarian cancer and its seeded lesions, comprising one or more compounds represented by the following general formula (I) or salts thereof. (In the formula, R １ represents a hydrogen atom or 1 to 4 identical or different substituents bonded to a benzene ring; R ２ , R ３ *, R ４ , R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ８ , R ９ and R １０ each independently represents a hydrogen atom or an alkyl group; X is C １ -C ３[2] A fluorescent probe according to [1], comprising two or more compounds represented by general formula (I) or salts thereof, wherein (P2, P1) is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (aspartic acid residue, alanine residue), or (asparagine residue, lysine residue), where P1 is linked to the nitrogen in the adjacent formula by forming an amide bond, and P2 is linked to P1 by forming an amide bond. [3] R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ A fluorescent probe according to [1] or [2], wherein is a hydrogen atom and X is a methylene group. [4] A fluorescent probe for detecting ovarian cancer and its disseminated lesions, comprising at least one compound selected from the following group or a salt thereof. (In the formula, R １ R represents one to four identical or different substituents bonded to a hydrogen atom or a benzene ring; ２ , R ３ , R ４ , R ５ , R ６ , and R ７ Each of these independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ８ , R ９ and R １０ Each independently represents a hydrogen atom or an alkyl group; X is C １ -C ３[5] A composition for detecting ovarian cancer and its disseminated lesions, comprising a fluorescent probe according to any one of [1] to [4]. [6] A kit for detecting ovarian cancer and its disseminated lesions, comprising a fluorescent probe according to any one of [1] to [4]. [7] A diagnostic composition for ovarian cancer and its disseminated lesions, comprising a fluorescent probe according to any one of [1] to [4]. [8] The diagnostic composition for ovarian cancer and its disseminated lesions according to [7], used in cancer surgical treatment or cancer examination. [9] The diagnostic composition for ovarian cancer and its disseminated lesions according to [8], wherein the cancer surgical treatment is open surgery or laparoscopic surgery.

[10] A method for detecting ovarian cancer and its disseminated lesions, comprising the steps of: applying the fluorescent probe according to any one of [1] to [4] to tissue collected from the ovary, peritoneum, or omentum of a subject; irradiating the tissue after application with excitation light; and detecting fluorescence from the tissue.

[0010] The present invention provides a fluorescent probe capable of detecting ovarian cancer and disseminated lesions originating from ovarian cancer. Furthermore, by using the fluorescent probe of the present invention, a method for detecting ovarian cancer and disseminated lesions originating from ovarian cancer can be provided. By performing fluorescence imaging during surgery using the fluorescent probe of the present invention, real-time identification of disseminated lesions originating from ovarian cancer is possible.

[0011] The fluorescent probe library used in this study is shown. The results of the primary screening (non-acetylated HMRG, T value) after 2 hours in the cancer-specific enzyme activity search using the peptidase probe library are shown. The results of the primary screening (non-acetylated HMRG, T / N) after 2 hours in the cancer-specific enzyme activity search using the peptidase probe library are shown. The results of the primary screening (non-acetylated HMRG, T value) after 3 hours in the cancer-specific enzyme activity search using the peptidase probe library are shown. The results of the primary screening (non-acetylated HMRG, T / N) after 3 hours in the cancer-specific enzyme activity search using the peptidase probe library are shown. The results of probe extraction in the cancer-specific enzyme activity search using the peptidase probe library are shown. The fluorescence increase values ​​30 minutes after dropping six types of fluorescent probes are shown. The ROC curves at 30 minutes for six types of fluorescent probes for ovarian cancer metastasis detection are shown. The fluorescence increase values ​​20 minutes after dropping six types of fluorescent probes are shown. The ROC curves at 20 minutes for six types of fluorescent probes for detecting ovarian cancer metastasis are shown. The fluorescence increase values ​​at 10 minutes after dropping the six types of fluorescent probes are shown. The ROC curves at 10 minutes for six types of fluorescent probes for detecting ovarian cancer metastasis are shown. An overview of the DEG assay method used in the examples is shown. The results of a DEG assay performed using lysate of ovarian cancer peritoneal dissemination HGSC #2278 as the sample and EK-HMRG as the fluorescent probe are shown. The results of a DEG assay performed using lysate of ovarian cancer peritoneal dissemination HGSC #2705 as the sample and KQ-HMRG as the fluorescent probe are shown. The fluorescence increase of 1 μM EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG in the presence or absence of puromycin and in the presence or absence of 5 ng PSA are shown. The sample and test conditions (upper part of Figure 12a) for Example 2(A), and fluorescence images 20 and 30 minutes after dropping the probe solution are shown. Fluorescence images before dropping the probe solution and 0, 1, 3, 5, and 10 minutes after dropping the probe solution for Example 2(A) are also shown. The time-dependent changes in fluorescence intensity for each probe (EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG) with and without puromycin are shown.The fluorescence intensity 30 minutes after administration of each probe (EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG) with and without the inhibitor is shown. The results of a two-dimensional DEG assay of 40 μg ovarian cancer peritoneal dissemination lysate using 10 μM NK-HMRG in DPBS(+) after isoelectric focusing (pH 3-7) and native PAGE are shown. The results of fluorescence imaging using tumor-normal serial samples from Example 3 (using the EK-HMRG probe) are shown. The results of fluorescence imaging using tumor-normal serial samples from Example 4 (using the NA-HMRG probe) are shown. The results of fluorescence imaging using tumor-normal serial samples from Example 4 (using the DA-HMRG probe) are shown. The results of fluorescence imaging using tumor-normal serial samples during surgical procedure in Example 5 (using the EK-HMRG probe) are shown.

[0012] 1. Definitions In this specification, “halogen atom” means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom.

[0013] In this specification, "alkyl" may be any aliphatic hydrocarbon group consisting of a linear, branched, cyclic, or combination thereof. The number of carbon atoms in the alkyl group is not particularly limited, but for example, it may have 1 to 20 carbon atoms (C １～２０ ), 3 to 15 carbon atoms (C ３～１５ ), 5 to 10 carbon atoms (C ５～１０ ) If the number of carbon atoms is specified, it means an "alkyl" having a number of carbon atoms within that range. For example, C １～８Alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neo-pentyl, n-hexyl, isohexyl, n-heptyl, n-octyl, and the like. In this specification, alkyl groups may have one or more substituents. Examples of such substituents include, but are not limited to, alkoxy groups, halogen atoms, amino groups, mono- or disubstituted amino groups, substituted silyl groups, or acyl groups. If an alkyl group has two or more substituents, they may be the same or different. The same applies to the alkyl portion of other substituents containing an alkyl portion (e.g., alkosyl groups, arylalkyl groups, etc.).

[0014] In this specification, when a functional group is defined as "may have substituents," the type of substituent, the position of substitution, and the number of substituents are not particularly limited, and if there are two or more substituents, they may be the same or different. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, hydroxyl groups, carboxyl groups, halogen atoms, sulfo groups, amino groups, alkoxycarbonyl groups, and oxo groups. These substituents may have further substituents. Examples of such substituents include, but are not limited to, alkyl halides and dialkylamino groups.

[0015] In this specification, "alkoxy group" refers to a structure in which the alkyl group is bonded to an oxygen atom, and examples include saturated alkoxy groups that are linear, branched, cyclic, or combinations thereof. For example, methoxy group, ethoxy group, n-propoxy group, isopropoxy group, cyclopropoxy group, n-butoxy group, isobutoxy group, s-butoxy group, t-butoxy group, cyclobutoxy group, cyclopropylmethoxy group, n-pentyloxy group, cyclopentyloxy group, cyclopropylethyloxy group, cyclobutylmethyloxy group, n-hexyloxy group, cyclohexyloxy group, cyclopropylpropyloxy group, cyclobutylethyloxy group, or cyclopentylmethyloxy group are examples of preferred groups.

[0016] In this specification, "alkylamino" and "arylamino" are defined as -NH ２ This refers to an amino group in which one or two of the hydrogen atoms of the group are substituted with the alkyl or aryl groups mentioned above. Examples include methylamino, dimethylamino, ethylamino, diethylamino, ethylmethylamino, and benzylamino. Similarly, "alkylthio" and "arylthio" refer to a group in which one of the hydrogen atoms of the -SH group is substituted with the alkyl or aryl groups mentioned above. Examples include methylthio, ethylthio, and benzylthio.

[0017] As used herein, "amide" includes both RNR'CO- (where R = alkyl, alkylaminocarbonyl-) and RCONR'- (where R = alkyl, alkylcarbonylamino-).

[0018] In this specification, the term “ring structure” means a heterocyclic or carbocyclic group formed by a combination of two substituents, such groups may be saturated, unsaturated, or aromatic. Thus, it includes the cycloalkyl, cycloalkenyl, aryl, and heteroaryl groups defined above. Examples include cycloalkyl, phenyl, naphthyl, morpholinyl, piperdinyl, imidazolyl, pyrrolidinyl, and pyridyl. In this specification, substituents may form ring structures with other substituents, and when such substituents are bonded together, those skilled in the art will understand that certain substitutions, such as bonding to hydrogen, are formed. Therefore, where it is stated that certain substituents together form a ring structure, those skilled in the art will understand that such ring structures can be formed by ordinary chemical reactions and are readily generated. Both such ring structures and the processes of their formation are within the realm of knowledge of those skilled in the art.

[0019] "Dissemination" refers to cancer cells detaching from their original site of origin within the body and spreading throughout the body via the flow of bodily fluids or metastasizing to other organs. "Peritoneal dissemination" refers to the state in which cancer cells leave the primary tumor and spread to the peritoneum, a state also known as "peritoneal metastasis." In cases of peritoneal dissemination, "carcinomatous peritonitis" and "carcinomatous ascites" may occur concurrently. "Carcinomatous ascites" refers to the leakage of blood components into the abdominal cavity due to peritoneal dissemination of cancer, and is also known as "malignant ascites" or "ascites."

[0020] Ovarian cancer is mainly classified into four types based on the tissue in which the cancer originates: serous ovarian carcinoma (including high-grade serous carcinoma), mucinous ovarian carcinoma, endometrioid ovarian carcinoma, and clear cell ovarian carcinoma. Clear cell ovarian carcinoma is a type of ovarian cancer that is highly metastatic and malignant. In this specification, "ovarian cancer" includes one or more of these types.

[0021] In this specification, "disseminated lesions originating from ovarian cancer" and "disseminated lesions of ovarian cancer" refer to a condition in which cancer cells or tumors originating in the ovary spread beyond the ovary to the peritoneum, omentum, or other organs. Furthermore, "peritoneal dissemination originating from ovarian cancer," "peritoneal dissemination in ovarian cancer," and "peritoneal dissemination of ovarian cancer" refer to a condition in which cancer cells or tumors originating in the ovary spread beyond the ovary to the peritoneum.

[0022] 2. Fluorescent probe for detecting peritoneal dissemination of ovarian cancer One embodiment of the present invention is a fluorescent probe for detecting ovarian cancer and its disseminated lesions, comprising one or more compounds represented by the following general formula (I) or salts thereof (hereinafter also referred to as "the fluorescent probe of the present invention").

[0023] In the above general formula (I), R １ R represents one to four substituents bonded to a hydrogen atom or a benzene ring. Examples of substituents include, but are not limited to, alkyl groups, alkoxy groups, halogen atoms, amino groups, mono- or disubstituted amino groups, substituted silyl groups, or acyl groups. If there are two or more substituents on the benzene ring, they may be the same or different. １ A hydrogen atom is preferred as the atom.

[0024] R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ Each of these independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom. ２ and R ７ It is preferable that R is a hydrogen atom. ３ , R ４ , R ５ , R ６ It is also preferable that it be a hydrogen atom. ２ , R ３ , R ４ , R ５ , R ６ , and R ７ It is even more preferable that all of them are hydrogen atoms.

[0025] R ８ , R ９ and R １０ Each of these independently represents either a hydrogen atom or an alkyl group. ８ and R ９ If both represent alkyl groups, they may be the same or different. For example, R ８ and R ９ When both are hydrogen atoms, and R ８ is an alkyl group, and R ９ It is preferable that R is a hydrogen atom. ８ and R ９ It is even more preferable that both of them are hydrogen atoms. Also, R １０ It is preferable that it is a hydrogen atom.

[0026] X is C １ -C ３ This represents an alkylene group. The alkylene group may be either a linear alkylene group or a branched alkylene group. For example, a methylene group (-CH ２ -), ethylene group (-CH ２ -CH ２ -), propylene group (-CH ２ -CH ２ -CH ２ -) In addition, -CH(CH) is used as a branched alkylene group. ３ ) -, -CH ２ -CH(CH ３ )-,-CH(CH ２ CH ３ Other groups can also be used. Of these, a methylene group or an ethylene group is preferred, and a methylene group is more preferred.

[0027] In the fluorescent probe of the present invention, P1 and P2 of general formula (I) each independently represent an amino acid residue. Here, it is important that the combination of P2 and P1, (P2, P1), is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (aspartic acid residue, alanine residue), or (asparagine residue, lysine residue). Here, P1 is linked to the nitrogen in the adjacent formula by forming an amide bond, and P2 is linked to P1 by forming an amide bond. In the fluorescent probe of the present invention, the amino acid residue of P1 has a structure corresponding to the remaining substructure obtained by removing the hydroxyl group from the carboxyl group of the amino acid and the hydrogen atom from the amino group. The amino acid residue of P2 also has a structure corresponding to the remaining substructure obtained by removing the hydroxyl group from the carboxyl group of the amino acid. Furthermore, the N-terminus of the amino acid residue of P2 may be protected by a protecting group. Examples of protecting groups include acetyl, glutaryl, succinyl, tert-butoxycarbonyl, and benzyloxycarbonyl groups, but other substituents may also be used.

[0028] In this study, a screening test was conducted using a library of enzyme probes consisting of approximately 400 types of HMRG derivative probes. The results showed that fluorescent probes with the following combinations as (P2, P1): (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (asparagine residue, lysine residue), (lysine residue, glutamine residue), and (lysine residue, valine residue) exhibited high fluorescence elevation values ​​(T values) in tumors in lysates, and also showed a high ratio of fluorescence elevation value (T) in tumor tissue to fluorescence elevation value (N) in normal tissue.

[0029] Furthermore, the inventors conducted a secondary screening of fluorescent probes having these (P2, P1) combinations using live samples. As a result, they found that fluorescent probes having the combinations (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (aspartic acid residue, alanine residue), and (asparagine residue, lysine residue) as (EK-probe, NA-probe, DA-probe, and NK-probe, respectively) showed high fluorescence values ​​in tumor tissue and were promising probes for specifically detecting disseminated lesions of ovarian cancer. These fluorescent probes having these (P2, P1) combinations can preferably be applied to peritoneal dissemination of ovarian cancer among disseminated lesions of ovarian cancer. Hereinafter, these fluorescent probes can be applied to disseminated lesions originating from various types of ovarian cancer. One preferred aspect of the fluorescent probe of the present invention is that it is a fluorescent probe for detecting disseminated lesions originating from at least one type of ovarian cancer selected from the group consisting of ovarian serous carcinoma (high-grade serous carcinoma, etc.), ovarian mucinous carcinoma, ovarian endometrioid carcinoma, and ovarian clear cell carcinoma.

[0030] Furthermore, the inventors confirmed an increase in fluorescence values ​​in primary ovarian cancer lesions using fluorescent probes having this (P2, P1) combination, and found that these fluorescent probes can also be applied to ovarian cancer. In addition, fluorescent probes having this (P2, P1) combination can be applied to ovarian serous carcinoma, preferably ovarian serous carcinoma, and particularly preferably high-grade serous carcinoma (HGSC).

[0031] Furthermore, fluorescent probes (EK-probe, NA-probe, DA-probe) having combinations of (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), and (aspartic acid residue, alanine residue) as (P2, P1) showed high fluorescence values ​​in tumor tissue even at short intervals such as 10 minutes after probe application, and also showed a large AUC obtained from the ROC curve. Therefore, fluorescent probes having these combinations as (P2, P1) enable the identification of disseminated lesions in a short time after application to specimens, tissues, etc.

[0032] In addition, the present inventors have found that the target enzyme of fluorescent probes (EK-probe, NA-probe, DA-probe) having a combination of (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (aspartic acid residue, alanine residue) as (P2, P1) is PSA (Puromycin-sensitive aminopeptidase). On the other hand, the fluorescent probe (NK-probe) in which (P2, P1) is (asparagine residue, lysine residue) shows spots at the site of PSA enzyme in the DEG assay, serves as a substrate for the PSA enzyme in the lysate, but no inhibitory effect is observed with puromycin in tissue specimens, suggesting the involvement of other enzymes.

[0033] Specific examples of the compound of the general formula (I) include the following compounds. However, it is not limited thereto. In the above formula, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ Regarding the details of X, it is the same as those described in detail for the compound represented by the general formula (I) or a salt thereof. In addition, compounds in which the N-terminus of the above compounds is protected by a protecting group are also included in the scope of the compounds of the general formula (I). Examples of the protecting group include an acetyl group, a glutaryl group, a succinyl group, a tert-butoxycarbonyl group, a benzyloxycarbonyl group, etc., but other substituents may also be used.

[0034] In one embodiment of the fluorescent probe of the present invention, R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ are hydrogen atoms, and X is a methylene group, and includes a compound of the general formula (I).

[0035] Compounds represented by general formula (I) may exist as salts. Examples of such salts include base addition salts, acid addition salts, and amino acid salts. Examples of base addition salts include metal salts such as sodium salts, potassium salts, calcium salts, and magnesium salts, ammonium salts, or organic amine salts such as triethylamine salts, piperidine salts, and morpholine salts. Examples of acid addition salts include mineral salts such as hydrochloride salts, sulfate salts, and nitrate salts, carboxylate salts, methanesulfonate salts, p-toluenesulfonate salts, citrate salts, and oxalate salts. Examples of amino acid salts include glycine salts. However, the salts are not limited to these.

[0036] Compounds represented by general formula (I) may have one or more chiral carbons depending on the type of substituent, and may exist as stereoisomers such as optical isomers or diastereoisomers. Pure stereoisomers, any mixture of stereoisomers, racemates, etc., are all included within the scope of the present invention.

[0037] Compounds represented by general formula (I) or salts thereof may exist as hydrates or solvates, but all of these substances are included within the scope of the present invention. The type of solvent that forms the solvate is not particularly limited, but examples of solvents include ethanol, acetone, and isopropanol.

[0038] Compounds represented by general formula (I) can be easily produced by using, for example, a xanthene compound having amino groups at positions 3 and 6 and a 2-carboxyphenyl group or a 2-alkoxycarbonylphenyl group at position 9 as a raw material, converting the 2-carboxyphenyl group or 2-alkoxycarbonylphenyl group at position 9 to a hydroxyalkyl group, and then acylating the amino group at position 3. Examples of 3,6-diaminoxanthene compounds that can be used as raw materials include commercially available rhodamine 110 and rhodamine 123, but are not limited to these, and an appropriate xanthene compound can be selected depending on the structure of the target compound.

[0039] Another embodiment of the fluorescent probe of the present invention is a fluorescent probe comprising two or more compounds or salts thereof represented by general formula (I), wherein (P2, P1) is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), or (aspartic acid residue, alanine residue), and (P2, P1) is (asparagine residue, lysine residue), comprising one or more compounds or salts thereof represented by general formula (I), and a compound or salt thereof represented by general formula (I) where (P2, P1) is (asparagine residue, lysine residue) (hereinafter also referred to as "fluorescent probe 2 of the present invention").

[0040] In the fluorescent probe 2 of the present invention, the N-terminus of the amino acid residue P2 may also be protected with a protecting group. Examples of protecting groups include acetyl group, glutaryl group, succinyl group, tert-butoxycarbonyl group, and benzyloxycarbonyl group, but other substituents may also be used.

[0041] As described above, fluorescent probes (EK-probes, NA-probes, DA-probes) with combinations of (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), and (aspartic acid residue, alanine residue) as (P2, P1) exhibit high fluorescence values ​​in tumor tissue even in short periods such as 10 minutes after probe application, and also have the characteristic of a large AUC obtained from the ROC curve. On the other hand, fluorescent probes (NK-probes) where (P2, P1) is (asparagine residue, lysine residue) take longer to show high fluorescence values ​​in tumor tissue compared to the above-mentioned fluorescent probes. Furthermore, it has been confirmed that the target enzyme for EK-probes, NA-probes, and DA-probes is PSA, while for NK-probes, the involvement of enzymes other than PSA is considered. Therefore, by using the fluorescent probe 2 of the present invention, which includes a combination of one or more fluorescent probes selected from EK-probes, NA-probes, or DA-probes, and an NK-probe, disseminated lesions such as peritoneal dissemination targeting PSA can be identified in a short time using one or more fluorescent probes selected from EK-probes, NA-probes, or DA-probes, and disseminated lesions such as peritoneal dissemination targeting enzymes other than PSA can be identified using the NK-probe, which takes time to increase in fluorescence intensity. Thus, it becomes possible to identify disseminated lesions targeting various enzymes.

[0042] The fluorescent probe of the present invention and the fluorescent probe 2 of the present invention (hereinafter collectively referred to as "the fluorescent probe of the present invention") may be used as a composition by incorporating additives commonly used in the preparation of reagents as needed. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for use in a physiological environment, and the amounts of these additives can be appropriately selected by those skilled in the art. These compositions can be provided in appropriate forms such as powder mixtures, freeze-dried products, granules, tablets, and liquids.

[0043] The fluorescent probe of the present invention may be a compound represented by general formula (I) or a salt thereof, but may also be used as a composition by adding additives commonly used in reagent preparation as needed. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used as additives for using the reagent in a physiological environment, and the amounts of these additives can be appropriately selected by those skilled in the art. These compositions are generally provided in the form of a powder mixture, lyophilized product, granules, tablets, liquid, or other appropriate form, but can be dissolved in injection-grade distilled water or an appropriate buffer solution before use.

[0044] Furthermore, the fluorescent probe of the present invention can be used, for example, during surgery, during examination, and after surgery. In this specification, the term "surgery" encompasses any surgery, including endoscopic or laparoscopic surgery. The term "examination" encompasses examinations using endoscopes and procedures such as tissue excision and collection associated with examinations, as well as examinations performed on tissues separated and collected from living organisms. These terms must be interpreted in the broadest sense and should not be interpreted restrictively in any way.

[0045] Furthermore, in this specification, the term “cancer tissue” means any tissue containing cancer cells. The term “tissue” must be interpreted in the broadest sense, including part or all of an organ, and must not be interpreted restrictively in any way. Furthermore, in this specification, the term “diagnosis” must be interpreted in the broadest sense, including visual or microscopic confirmation of the presence of cancer tissue in any living site.

[0046] One aspect of the present invention is a composition for detecting ovarian cancer and its disseminated lesions, comprising the fluorescent probe of the present invention (hereinafter also referred to as "the detection composition of the present invention"). The detection composition of the present invention can be applied to peritoneal dissemination of ovarian cancer, among disseminated lesions of ovarian cancer. Furthermore, the detection composition of the present invention can be applied to ovarian serous carcinoma, among ovarian cancers, preferably high-grade serous carcinoma (HGSC).

[0047] Another aspect of the present invention is a diagnostic composition for ovarian cancer and its disseminated lesions, comprising the fluorescent probe of the present invention.

[0048] Another aspect of the present invention is a diagnostic composition for ovarian cancer and its disseminated lesions, comprising the fluorescent probe of the present invention, used in cancer surgical treatment or cancer screening. Here, cancer surgical treatment includes open surgery and laparoscopic surgery. These diagnostic compositions can be applied to peritoneal dissemination of ovarian cancer, among disseminated lesions of ovarian cancer. Furthermore, these diagnostic compositions can be applied to ovarian serous carcinoma, and particularly preferably high-grade serous carcinoma (HGSC), among ovarian cancers.

[0049] 3. Detection Method Using a Fluorescent Probe Another embodiment of the present invention is a method for detecting ovarian cancer and its disseminated lesions, comprising the steps of: applying the fluorescent probe of the present invention to tissue collected from the ovary, peritoneum, or omentum of a subject; irradiating the tissue with excitation light after application; and detecting fluorescence from the tissue (hereinafter also referred to as "the detection method of the present invention"). Here, the subject includes humans and non-human mammals (e.g., dogs, cats, etc.).

[0050] In step (a) above, the fluorescent probe can be applied to tissue collected from the subject's ovary, peritoneum, or omentum, for example, by using a lysate prepared from a cancerous or non-cancerous tissue sample and applying it to, for example, the wells of a 384-plate. However, this is not limited to this method.

[0051] Another embodiment of the present invention is a method for detecting ovarian cancer and its disseminated lesions, comprising the steps of (a) applying the fluorescent probe of the present invention to a clinical specimen of the ovary, peritoneum, or omentum, and (b) measuring the fluorescence image of the clinical specimen of the ovary, peritoneum, or omentum to which the fluorescent probe has been applied (hereinafter also referred to as "the detection method of the present invention").

[0052] To apply the fluorescent probe to the clinical specimen in step (a) above, this can be done, for example, by spraying a solution of the fluorescent probe locally or all over the clinical specimen.

[0053] The detection method of the present invention may further include observing a fluorescence response using fluorescence imaging means. The means for observing the fluorescence response may be a fluorometer with a wide measurement wavelength range, but the fluorescence response can also be visualized using fluorescence imaging means capable of displaying the fluorescence response as a two-dimensional image. By using fluorescence imaging means, the fluorescence response can be visualized in two dimensions, making it possible to instantly identify disseminated lesions. As the fluorescence imaging device, any device known in the art can be used. In some cases, it is also possible to detect the reaction between the sample to be measured and the fluorescence probe by changes in the ultraviolet-visible absorption spectrum (for example, changes in absorbance at a specific absorption wavelength). The detection method of the present invention can be applied to peritoneal dissemination of ovarian cancer, among disseminated lesions of ovarian cancer. Furthermore, the detection method of the present invention can be applied to ovarian serous carcinoma, and particularly preferably to high-grade serous carcinoma (HGSC), among ovarian cancers.

[0054] The method of using the fluorescent probe of the present invention is not particularly limited and can be used in the same way as conventionally known fluorescent probes. Typically, the compound of the present invention or a salt thereof is dissolved in an aqueous medium such as physiological saline or buffer solution, or in a mixture of an aqueous medium and a water-miscible organic solvent such as ethanol, acetone, ethylene glycol, dimethyl sulfoxide, or dimethylformamide, and this solution is added to a suitable buffer solution containing cells or tissues, and the fluorescence spectrum is measured. The fluorescent probe of the present invention may also be used in the form of a composition in combination with appropriate additives. For example, it can be combined with additives such as buffers, solubilizers, and pH adjusters. Furthermore, the concentration of the compound of the present invention in the fluorescent probe of the present invention can be appropriately determined according to the type of cells etc. to be measured and the measurement conditions.

[0055] Another embodiment of the present invention is a detection kit for ovarian cancer and its disseminated lesions, comprising the fluorescent probe of the present invention (hereinafter also referred to as "the detection kit of the present invention"). The detection kit of the present invention can be applied to peritoneal dissemination of ovarian cancer, among disseminated lesions of ovarian cancer. Furthermore, the detection kit of the present invention can be applied to ovarian serous carcinoma, and particularly preferably high-grade serous carcinoma (HGSC), among ovarian cancers.

[0056] In this kit, the fluorescent probe of the present invention is usually prepared as a solution, but it can also be provided as a composition in an appropriate form, such as a powder mixture, lyophilized product, granules, tablets, or liquid, and can be dissolved in injection-grade distilled water or an appropriate buffer solution before use.

[0057] Furthermore, the kit may contain other reagents as needed. For example, additives such as solubilizers, pH adjusters, buffers, and isotonic agents can be used, and the amounts of these additives can be appropriately selected by those skilled in the art.

[0058] The application concentration of the fluorescent probe of the present invention is not particularly limited, but for example, a solution with a concentration of about 0.1 to 10 μM can be applied.

[0059] 4. Application of the fluorescent probe of the present invention to surgical treatment of ovarian cancer Another embodiment of the present invention is a method for determining the presence of ovarian cancer and / or disseminated lesions in a subject and / or identifying the extent of ovarian cancer and / or disseminated lesions, comprising the steps of (a) applying a fluorescent probe containing one or more compounds represented by the following general formula (I) or salts thereof to a site in a subject where ovarian cancer and / or disseminated lesions are observed and / or a site suspected to have ovarian cancer and / or disseminated lesions, and (b) measuring the fluorescence image of the site where the fluorescent probe was applied (hereinafter also referred to as "the identification method of the present invention"). Here, the subjects include humans and non-human mammals (e.g., dogs, cats, etc.). The identification method of the present invention can be applied to peritoneal dissemination of ovarian cancer, among disseminated lesions of ovarian cancer. Furthermore, the identification method of the present invention can be applied to ovarian serous carcinoma, and more preferably to high-grade serous carcinoma (HGSC), among ovarian cancers.

[0060] In the above general formula (I), R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ , R １０ X, P1, and P2 are as described in detail above.

[0061] In one aspect of the identification method of the present invention, the fluorescent probe contains two or more compounds represented by general formula (I) or salts thereof, wherein the fluorescent probe contains one or more compounds represented by general formula (I) or salts thereof, in which (P2, P1) is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), or (aspartic acid residue, alanine residue), and a compound represented by general formula (I) or salts thereof, in which (P2, P1) is (asparagine residue, lysine residue). By using two or more fluorescent probes as described above in combination, peritoneal dissemination targeting PSA can be identified in a short time using one or more fluorescent probes selected from EK-probe, NA-probe, or DA-probe, and peritoneal dissemination targeting enzymes other than PSA can be identified using an NK-probe, which takes time for the fluorescence value to increase, thus making it possible to identify disseminated lesions targeting various enzymes.

[0062] Furthermore, in the identification method of the present invention, etc., in general formula (I), R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R８ , R ９ and R １０ Preferably, is a hydrogen atom and X is a methylene group.

[0063] The identification method of the present invention may further include visualizing a fluorescence image using a fluorescence imaging means. Details of the fluorescence imaging means are as described in the detection method and detection method of the present invention.

[0064] The identification method and other aspects of the present invention can be performed during surgical treatment of ovarian cancer. Here, surgical treatment of ovarian cancer includes open surgery and laparoscopic surgery.

[0065] By using the fluorescent probe of the present invention and performing the identification method of the present invention during surgical treatment of ovarian cancer, it becomes possible to clearly distinguish peritoneal dissemination from surrounding non-cancerous tissue as a fluorescent region in the peritoneum or omentum of the patient. Furthermore, by performing the identification method of the present invention, it becomes possible to rapidly identify cancerous tissue based on the viability of peritoneal dissemination in real time. As a result, it is possible to reduce the need for pathological examination of tissue suspected of containing cancer (peritoneal dissemination) during surgery.

[0066] The present invention will be described in more detail below with reference to examples, but the present invention is not limited thereto.

[0067] 1. Cancer-Specific Enzyme Activity Exploration Using a Peptidase Probe Library: The Department of Pharmaceutical and Metabolic Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, created approximately 380 fluorescent probes targeting aminopeptidases and dipeptidyl peptidases by introducing one or two amino acids into hydroxymethylrhodamine green (HMRG) as the fluorescent nucleus. Screening was conducted using human biopsy samples to find peptidase probes specific to peritoneal dissemination of ovarian cancer from this library.

[0068] (1) Methods Lysate screening was performed on biopsy specimens of ovarian cancer with peritoneal dissemination using a library of 381 non-acetylated HMRG fluorescent probes. Five cases from the same patient were used, including both tumored and normal peritoneal tissue. The fluorescent probe library used in this study is shown in Figure 1. A list of biopsy specimens of ovarian cancer with peritoneal dissemination is shown in Table 1.

[0069]

[0070] The lysate screening protocol is as follows: [Protocol] - Dispense 5 μl of 0.1 mg / ml lysate into a 384 black plate using an 8-channel pipette. - Dispense 5 μl of probe diluent (1.8 μM) into a 384 black plate using a 384 dispenser. - Measure fluorescence values ​​at 0h, 1h, 2h, and 3h using En Vision. - Perform screening three times for each lysate and analyze the average fluorescence intensity.

[0071] [Probe Extraction Criteria] Probes that showed a tumor fluorescence elevation value (T value) of 0.05 (Conversion Rate ≥ 5%) in the lysate, and whose tumor tissue fluorescence elevation value (T) / normal tissue fluorescence elevation value (N) ratio was in the top 10%, were color-coded in the table. Furthermore, probes with a T / N ratio of 2.5 or higher and a Conversion Rate of ≥ 10% were extracted, and probes that met the criteria in 3 or more out of 5 cases were selected.

[0072] The Conversion Rate (an indicator of the proportion of the added fluorescent probe that was converted to its hydrolysis product, HMRG) was calculated using the following formula (1).

[0073]

[0074] The results of the initial screening (non-acetylated HMRG) after 2 hours are shown in Figures 2-1 and 2-2. The T values ​​were color-coded as follows: 0.05 (5%), 0.3 (30%) (marked in gray), and 0.5 (50%) (marked in black) (Figure 2-1), and the top 10% of T / N ratios were extracted (Figure 2-2).

[0075] Furthermore, the results of the initial screening (non-acetylated HMRG) after 3 hours are shown in Figures 3-1 and 3-2. The T values ​​were color-coded as follows: 0.05 (5%), 0.3 (30%) (marked in gray), and 0.5 (50%) (marked in black) (Figure 3-1), and the top 10% of T / N ratios were extracted (Figure 3-2).

[0076] Figure 4 shows the results of extracting probes that met the criteria in 3 or more cases out of 5 cases, with a T / N ratio of 2.5 or higher and a Conversion Rate of 10% or higher.

[0077] [Example 1] (A) Fluorescent probe drop experiment using frozen and live specimens A drop experiment was performed as a secondary screening using six types of fluorescent probes extracted from the HMRG probe library. (1) Method Tissue biopsies were taken from the tumor and non-tumor portions of frozen and live specimens of human peritoneum or human omentum, and HMRG peptidase probes were dropped onto them, and the change in fluorescence intensity over time was measured. [Probe] EK-HMRG, KQ-HMRG, KV-HMRG, DA-HMRG, NA-HMRG, NK-HMRG [Equipment used] Maestro In Vivo Imaging System Exu (Filter set: Blue (Ex.435-480nm / Em.490nm LP), Acquisition Setting; 500 to 720 nm in 10nm steps)

[0078] [Protocol] ↓ Divide the excised specimen into approximately 2-3 mm pieces each of tumor tissue and normal tissue, and place them on an 8-well microslide (ibidi, #ib80826). ↓ Add 200 μL of 50 μM probe solution to each dish. ↓ Obtain fluorescence images using Maestro before addition and at 0, 1, 3, 5, 10, 20, and 30 minutes after addition. ↓ Calculate the average signal of the ROI (Region of Interest) using the 540 nm image.

[0079]

[0080] Figures 5-1 and 5-2 show the fluorescence increase values ​​30 minutes after dropping six types of fluorescent probes, and the ROC curves at 30 minutes for six types of fluorescent probes used to detect ovarian cancer metastasis, respectively. Figures 6-1 and 6-2 show the fluorescence increase values ​​20 minutes after dropping six types of fluorescent probes, and the ROC curves at 20 minutes for six types of fluorescent probes used to detect ovarian cancer metastasis, respectively. Figures 7-1 and 7-2 show the fluorescence increase values ​​10 minutes after dropping six types of fluorescent probes, and the ROC curves at 10 minutes for six types of fluorescent probes used to detect ovarian cancer metastasis, respectively.

[0081] Figure 5-1 shows that EK-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG showed high fluorescence values ​​in tumor tissue 30 minutes after probe application, suggesting they are promising probes. Furthermore, Figure 7-1 shows that EK-HMRG, NA-HMRG, and DA-HMRG still showed high fluorescence values ​​in tumor tissue 10 minutes after probe application, and the large AUC obtained from the ROC curve suggests that a high effect can be obtained in a short time after probe addition.

[0082] (B) Selection of target enzymes and LC-MS / MS analysis using the Diced electrophoresis gel (DEG) assay The DEG assay is a method developed by Toru Komatsu et al. of the Faculty of Pharmaceutical Sciences, University of Tokyo. It involves fractionating enzyme extracts such as cell lysates by non-denaturing two-dimensional electrophoresis, then subdividing the gel and dispensing it into multi-well plates, where an enzyme assay using a fluorescent probe is performed (Komatsu T, Hanaoka K, Adibekian A, Yoshioka K, Terai T, Ueno T, Kawaguchi M, Cravatt BF, Nagano T. Diced electrophoresis gel assay for screening enzymes with specified activities. Journal of the American Chemical Society. 2013; 135: 6002-6005). Figure 8 shows an overview of the DEG assay method used in the example, referring to Fig. 1(d) in the aforementioned paper by Komatsu et al.

[0083] First, using lysates from samples that showed increased fluorescence in tumors in a biopsy experiment, the gels were subjected to two-dimensional electrophoresis using isoelectric focusing (IEF) and native polyacrylamide gel electrophoresis (PAGE). The gels were placed on a 384 plate, pressed down from above with a lid, and cut into cubes. Then, the plates were centrifuged at 3,000 rpm at 4°C for 5 minutes, and 70 μL of fluorescent probe was added to each well. The fluorescence intensity at the time of addition was measured using EnVision. After incubation at 37°C for approximately 19 hours, the fluorescence intensity was measured again using EnVision. The experiment was repeated with four gels, and the fractions of gels that showed high fluorescence elevation were collected in 1.5 mL collection tubes and stored at -80°C. Then, Pharma Foods Co., Ltd. was commissioned to analyze the proteins in the fractions using LC-MS / MS to select candidate target enzymes for the probe.

[0084] Figure 9 shows the results of a DEG assay using lysate of ovarian cancer peritoneal dissemination HGSC #2278 as the sample and EK-HMRG as the fluorescent probe. Figure 9 shows the results of a two-dimensional DEG assay of 30 μg ovarian cancer peritoneal dissemination lysate using 10 μM EK-HMRG in DPBS(+) after isoelectric focusing (pH 3-7) and native PAGE. The fluorescence increase rate after incubation at 37°C for 1 hour was plotted.

[0085] Figure 10 shows the results of a DEG assay using lysate of ovarian cancer peritoneal dissemination HGSC #2705 as the sample and KQ-HMRG as the fluorescent probe. Figure 10 shows the results of a two-dimensional DEG assay of 60 μg ovarian cancer peritoneal dissemination lysate using 10 μM EK-HMRG in DPBS(+) after isoelectric focusing (pH 3-7) and native PAGE. The fluorescence increase rate after incubation at 37°C for 1 hour was plotted.

[0086] Based on the above, LC / MS-MS analysis was performed on the gels of lysate #2278 and EK-HMRG, and the gels of lysate #2705 and KQ-HMRG. The enzymes detected in the analysis are listed below.

[0087] Sample: Lysate of ovarian cancer peritoneal dissemination HGSC #2278. Probe: EK-HMRG. Enzymes with aminopeptidase / protease activity were color-coded.

[0088]

[0089] Sample: Lysate probe of ovarian cancer peritoneal dissemination HGSC #2705: KQ-HMRG enzyme with aminopeptidase / protease activity was colored.

[0090]

[0091] Both EK-HMRG and KQ-HMRG are thought to target PSA (Puromycin-sensitive aminopeptide) / NPEPPS, and the plan is to confirm this through purified enzyme and inhibitor experiments.

[0092] (C) Reaction of purified enzyme and fluorescent probe: The enzyme reaction was carried out by adding 5 ng of PSA to a 1 μM probe solution. In addition, the inhibitory effect was confirmed by adding 30 μM of puromycin, a PSA inhibitor.

[0093] [Protocol] ↓ Adjust the final probe concentration to 1 μM. ↓ Set the final inhibitor concentrations to 30 μM and 0 M. ↓ Add 10 μL of probe and inhibitor to 384 plate wells (using 4 wells). ↓ Adjust PSA to 0.25 ng / μL and add 10 μL to each well. ↓ Measure every minute for 120 minutes.

[0094] The results are shown in Figure 11. Figure 11 shows the increased fluorescence of 1 μM EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG in the presence or absence of puromycin and with or without 5 ng PSA. All assays were performed at 37°C in 20 μL total volume phosphate-buffered saline (pH 7.4) with 0.6% v / v DMSO as the cosolvent. PSA enzyme activity was confirmed with five different probes at excitation / emission wavelengths of 485 / 535 nm.

[0095] [Example 2] (A) Fluorescent probe drop experiment using tissue samples The effect of the target enzyme (PSA) inhibitor Puromycin on the reaction of EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, NK-HMRG and tissue samples from ovarian cancer peritoneal dissemination was measured using the following protocol.

[0096] [Protocol] ↓ Divide the tumor tissue into pieces approximately 2-3 mm in size and place them on an 8-well microslide (μ-Slide 8well; Ibidi). ↓ Add 200 μL of 50 μM probe solution to each dish. Add 500 μM puromycin. ↓ Acquire fluorescence images using Maestro before addition and at 0, 1, 3, 5, 10, 20, and 30 minutes after addition. ↓ Calculate the average signal of the ROI (Region of Interest) using the 540 nm image.

[0097] Figures 12a and 12b show the sample and test conditions (upper part of Figure 12a), as well as fluorescence images before dropping the probe solution and at 0, 1, 3, 5, 10, 20, and 30 minutes after dropping. Figure 13 shows the time course of fluorescence intensity for each probe (EK-HMRG, KQ-HMRG, NA-HMRG, DA-HMRG, and NK-HMRG) with and without puromycin. Figure 14 shows the fluorescence intensity 30 minutes after dropping for each probe, with and without the inhibitor.

[0098] In samples without the inhibitor, an increase in fluorescence values ​​over time was observed. However, in samples with the inhibitor, the fluorescence of EK-HMRG, KQ-HMRG, NA-HMRG, and DA-HMR was inhibited. Furthermore, NK-HMRG was not inhibited compared to the other probes, suggesting that another target enzyme may be involved.

[0099] (B) Selection of target enzymes using Diced electrophoresis gel (DEG) assay A DEG assay was performed to select target enzymes for NK-HMRG. The DEG assay was performed in the same manner as in Example 1.

[0100] Sample: Lysate from ovarian cancer peritoneal dissemination HGSC #2916. Probe: NK-HMRG

[0101] Figure 15 shows the results of a two-dimensional DEG assay of 40 μg ovarian cancer peritoneal dissemination lysate using 10 μM NK-HMRG in DPBS(+) after isoelectric focusing (pH 3-7) and native PAGE.

[0102] Since a spot (lower arrow in the figure) was also observed near PSA, it was considered that NK-HMRG also acts as a substrate for PSA. Spots were also observed in other areas indicated by arrows (upper arrow in the figure). Because the magnitude of activity on the gel and the magnitude of contribution of activity in the tissue do not necessarily correlate (as they reflect differences in localization, lysate adjustment, and changes in activity within the gel), the gel in the area indicated by the upper arrow was subjected to PMF analysis to evaluate the enzyme's contribution. The spot on the far left may show activity derived from proteins that were partially aggregated during loading, and was therefore not considered a significant signal.

[0103] (C) DEGassay LC-MS / MS results of NK-HMRG and peritoneal dissemination lysate. The gel showing spots (indicated by the arrows above) from the DEGassay experiment was submitted for LC-MS / MS analysis.

[0104] As a result of LC / MS-MS analysis, Cathepsin B and 26S Protein were considered to be the target enzymes (the list of proteins detected by LC / MS-MS analysis is omitted). Therefore, we decided to conduct in vitro experiments using purified enzymes and inhibitors.

[0105] Next, a reaction test was performed between purified Cathepsin B enzyme and a fluorescent probe (NK-HMRG). The results showed no reaction between Cathepsin B and NK-HMRG, confirming that NK-HMRG is not a substrate of Cathepsin B (details of the results are omitted). Similarly, a reaction test was performed between purified Human 20S Proteasome enzyme and a fluorescent probe (NK-HMRG). The results showed no reaction between Human 20S Proteasome and NK-HMRG, confirming that NK-HMRG is not a substrate of Human 20S Proteasome (details of the results are omitted).

[0106] In DEGassay, NK-HMRG showed spots at the PSA enzyme (puromycin amino peptide) site, and in lysate, it acts as a substrate for the PSA enzyme. However, in tissue samples, no inhibitory effect was observed on puromycin, suggesting the involvement of other enzymes.

[0107] [Example 3] Fluorescence imaging using tumor-normal continuous specimens (1) Fluorescence imaging was performed using an EK-HMRG probe with continuous peritoneal dissemination specimens of tumor and normal.

[0108] [Protocol] ↓ Place the excised specimen on a dish and drop 50 μM EK-HMRG probe solution onto the specimen until it is immersed. ↓ Acquire fluorescence images using Maestro before dropping and at 0, 1, 3, 5, 10, 20, and 30 minutes after dropping. ↓ Calculate the average signal of the ROI (Region of Interest) using the 540 nm image.

[0109] The results are shown in Figure 16. As shown in Figure 16, an increase in fluorescence was observed in the tumor area after 10 minutes, indicating successful visualization. Furthermore, the presence of the tumor was confirmed by histopathological examination of the visualized area.

[0110] [Example 4] Fluorescence imaging using tumor-normal continuous specimens (2) Fluorescence imaging was performed using tumor-normal continuous peritoneal dissemination specimens with NA-HMRG probe or DA-HMRG probe as the fluorescent probe.

[0111] [Protocol] ↓ Place the excised specimen on a dish and drop 50 μM NA-HMRG probe solution and DA-HMRG probe solution onto the specimen until it is submerged. ↓ Acquire fluorescence images using Maestro before drop and at 0, 1, 3, 5, 10, 20, and 30 minutes after drop. ↓ Calculate the average signal of ROI (Region of Interest) using the 540 nm image.

[0112] The results obtained using the NA-HMRG probe are shown in Figure 17, and the results obtained using the DA-HMRG probe are shown in Figure 18. As shown in Figures 17 and 18, in both cases using the NA-HMRG probe and the DA-HMRG probe, an increase in fluorescence was observed in the tumor area from 10 minutes later, and visualization was successful. Furthermore, the presence of the tumor was confirmed by histopathological examination of the visualized area.

[0113] [Example 5] Fluorescence imaging immediately after surgical excision using tumor-normal continuous specimens (3) Fluorescence imaging was performed using tumor-normal continuous peritoneal dissemination specimens.

[0114] [Protocol] ↓Immediately after specimen removal, 3 mL of 50 μM EK-HMRG probe solution is dropped in the operating room. ↓Fluorescence images are acquired 5, 10, 15, and 20 minutes after drop using a portable fluorescence observation device, Discovery.

[0115] The results are shown in Figure 19. Figure 19a shows a photograph of the specimen removed during surgery, and the left image in Figure 19b shows a magnified photograph of the peritoneal dissemination specimen, showing the area of ​​the peritoneum enclosed by the dotted line in Figure 19a. The areas numbered 1 to 3 indicate tumor portions. As shown in Figure 19, an increase in fluorescence was observed in the tumor portion after 10 minutes, indicating successful visualization. Furthermore, the presence of the tumor was confirmed by histopathological examination of the visualized area (right image in Figure 19b). Also, as shown in Figure 19c, no increase in fluorescence was observed in areas other than the tumor even immediately after the specimen was removed during surgery. Therefore, by using the fluorescent probe of the present invention, it is possible to identify the areas with ovarian cancer and its disseminated lesions even during surgery, and to determine the boundary between these areas and areas without them, thereby enabling complete resection of ovarian cancer and its disseminated lesions.

[0116] Furthermore, in the treatment of ovarian cancer, anticancer drugs are often administered to patients before surgery, and in Examples 3 to 5, fluorescence imaging tests were performed using samples from patients who had received anticancer drugs. Although the use of anticancer drugs can alter a patient's metabolism, it was confirmed that the fluorescent probe of the present invention can be used to identify the sites of ovarian cancer and its disseminated lesions.

Claims

1. A fluorescent probe for detecting ovarian cancer and its metastatic lesions, comprising one or more compounds represented by the following general formula (I) or salts thereof. (In the formula, R １ represents a hydrogen atom or 1 to 4 identical or different substituents bonded to a benzene ring; R ２ , R ３ , R ４ , R ５ , R ６ , and R ７ each independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ８ , R ９ and R １０ each independently represents a hydrogen atom or an alkyl group; X represents a C １ -C ３ alkylene group; P1 and P2 each independently represent an amino acid residue, and the combination (P2, P1) of P2 and P1 is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), (aspartic acid residue, alanine residue) or (asparagine residue, lysine residue), where P1 forms an amide bond with the adjacent nitrogen in the formula to be linked, and P2 forms an amide bond with P1 to be linked.) 2. A fluorescent probe according to claim 1, comprising two or more compounds represented by general formula (I) or salts thereof, wherein (P2, P1) is selected from (glutamic acid residue, lysine residue), (asparagine residue, alanine residue), or (aspartic acid residue, alanine residue), and a compound represented by general formula (I) or salt thereof, wherein (P2, P1) is (asparagine residue, lysine residue).

3. R １ , R ２ , R ３ , R ４ , R ５ , R ６ , R ７ , R ８ , R ９ and R １０ The fluorescent probe according to claim 1, wherein is a hydrogen atom and X is a methylene group.

4. A fluorescent probe for detecting ovarian cancer and its disseminated lesions, comprising at least one compound or salt thereof selected from the following group. (In the formula, R １ R represents one to four identical or different substituents bonded to a hydrogen atom or a benzene ring; ２ , R ３ , R ４ , R ５ , R ６ , and R ７ Each of these independently represents a hydrogen atom, a hydroxyl group, an alkyl group, or a halogen atom; R ８ , R ９ and R １０ Each independently represents a hydrogen atom or an alkyl group; X is C １ -C ３ (Represents an alkylene group.) 5. A composition for detecting ovarian cancer and its disseminated lesions, comprising the fluorescent probe described in any one of claims 1 to 4.

6. A kit for detecting ovarian cancer and its disseminated lesions, comprising a fluorescent probe according to any one of claims 1 to 4.

7. A diagnostic composition for ovarian cancer and its disseminated lesions, comprising a fluorescent probe according to any one of claims 1 to 4.

8. The diagnostic composition for ovarian cancer and its disseminated lesions according to claim 7, used in cancer surgical treatment or cancer examination.

9. The diagnostic composition for ovarian cancer and its disseminated lesions according to claim 8, wherein the cancer surgical treatment is open surgery or endoscopic surgery.

10. A method for detecting ovarian cancer and its disseminated lesions, comprising the steps of: applying a fluorescent probe according to any one of claims 1 to 4 to tissue collected from the ovary, peritoneum, or omentum of a subject; irradiating the tissue after application with excitation light; and detecting fluorescence from the tissue.

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