Novel fluorescent compound for marking tumor tissue

Abstract

The subject matter of the present invention is novel fluorescent compounds useful for marking tumor tissue, methods for their preparation, and their use as monitoring or diagnostic tools or tools for assisting cancer surgery.

Description

[0001] This application is a divisional application of the invention patent application filed on May 12, 2021, with application number 202180035401.9 and invention title "Novel Fluorescent Compounds for Labeling Tumor Tissue".

[0002] This invention relates to novel fluorescent compounds that can be used to label tumor tissues, their preparation methods, and their applications as monitoring, diagnostic tools, or as adjuncts to cancer surgery. Technical Field

[0003] Labeling tumor tissue with fluorescent compounds has generated considerable interest in the field of medical imaging because, among other things, it allows for the localization of tumors. Background Technology

[0004] Fluorescent compounds have been used in medicine for over 50 years as biomarkers for monitoring and / or diagnosis in non-invasive imaging techniques.

[0005] Technical issues The emergence of new fluorescence imaging techniques in surgical services, requiring improved sensitivity and accuracy, has led to research into new fluorescent molecules that offer increasingly better performance.

[0006] In the case of diseases such as cancer, there is a particular need for the preferential distribution of fluorescent molecules in tumor tissue relative to healthy tissue, as well as sufficiently long fluorescence persistence, to obtain improved labeling specificity and provide assistance for surgical interventions, such as delineating tumor areas to be removed.

[0007] Some existing fluorescent biomarkers have limited fluorescence persistence, necessitating surgery on the patient shortly after biomarker injection and not allowing for satisfactory delineation of tumor tissue. In other cases, insufficient accumulation of the biomarker in the tissue leads to poor labeling and thus detection problems. The localization of the lesion or tumor, and its subsequent removal, for example, by surgery, is incomplete.

[0008] Another problem with existing fluorescent markers, particularly indocyanine green (ICG), is the requirement for the presence of neovascularization in the tumor to obtain labeling of the tumor tissue, where ICG is one of the few dyes used to label tumors during surgery. Furthermore, like other dyes in the prior art, ICG is only visible in tumor tissue for a maximum of 24 hours after injection. This short duration does not adequately eliminate circulating dye outside the tumor tissue, resulting in poor visualization due to the low signal-to-noise ratio.

[0009] Another major drawback of existing compounds is that they cannot be used directly. In fact, in order to be used, they need to be conjugated with other targeting molecules, such as antibodies, proteins, tumor tissue-specific molecules, folic acid, or steroids.

[0010] This invention allows us to overcome the aforementioned problems of the prior art by providing fluorescent molecules that exhibit preferential distribution and sufficient persistence in tumor tissue relative to healthy tissue, said fluorescent molecules for use in imaging techniques for monitoring, diagnosis, and / or as surgical aids. A key advantage of these new molecules is that, due to their specific affinity for tumor tissue, they can be used alone and directly without prior conjugation. Furthermore, compared to prior art molecules, they retain in tumor tissue for a much longer time, up to several days, allowing for more thorough elimination of these fluorescent molecules circulating outside the tumor tissue, and thus improved visualization due to a better signal-to-noise ratio. Invention Overview This invention relates to compounds of formula (I). [Chemical Formula 1] (I) in n1 and n2 are both integers between 0 and 15. R1, R2, R3, R4, R5, and R6 are each independently selected from H, OH, SH, and NH. 2、 SO3R 10 and XR 11 -Y, R 10 、R' 10 Independently, it can be H, Na, or K. X, X', X'' are independently O, S, or NH. R 11 、R' 11 、R'' 11 Independently selected from C1-C 15 Alkyl, aryl, heteroaryl, (C1-C) 15 alkyl)aryl, (C1-C 15 Alkyl) heteroaryl, aryl (C1-C) 15 Alkyl groups and heteroaryl groups (C1-C50) 15 alkyl); Y, Y', and Y'' are independently selected from H, halogen, and COOR'. 10 or amide; R7 and R8 are each independently selected from H, OH, SH, and NH. 2、 C 1- C 15 Alkyl and X'-R' 11 -Y'; R9 is selected from H, OH, SH, NH2, and X''-R''. 11 -Y'', The compound contains at least one XR group.11 -Y、X'-R' 11 -Y' or X''-R'' 11 -Y'', where Y, Y' and / or Y'' are COOR' 10 .

[0011] The present invention also relates to a method for preparing a compound of formula (I) according to the invention, and a method for labeling tumor tissue with one of the compounds according to the invention or a compound prepared according to the method of the invention. Invention Details This invention primarily relates to compounds of formula (I). [Chemical Formula 2] (I) in n1 and n2 are both integers between 0 and 15. R1, R2, R3, R4, R5, and R6 are each independently selected from H, OH, SH, and NH. 2、 SO3R 10 and XR 11 -Y, R 10 、R' 10 Independently, it can be H, Na, or K. X, X', X'' are independently O, S, or NH. R 11 、R' 11 、R'' 11 Independently selected from C1-C 15 Alkyl, aryl, heteroaryl, (C1-C) 15 alkyl)aryl, (C1-C 15 Alkyl) heteroaryl, aryl (C1-C) 15 Alkyl groups and heteroaryl groups (C1-C50) 15 alkyl); Y, Y', and Y'' are independently selected from H, halogen, and COOR'. 10 or amide; R7 and R8 are each independently selected from H, OH, SH, and NH. 2、 C 1- C 15 Alkyl and X'-R' 11 -Y'; R9 is selected from H, OH, SH, NH2, and X''-R''. 11 -Y'', The compound contains at least one XR group. 11 -Y、X'-R' 11 -Y' or X''-R''11 -Y'', where Y, Y' and / or Y'' are COOR' 10 .

[0013] In the sense of this invention, "C1-C" 15 "Alkyl" refers to a cyclic, straight-chain or branched hydrocarbon chain containing 1-15 carbon atoms, preferably 2-6 carbon atoms, even more preferably 4-6 carbon atoms, and especially 5 carbon atoms, and may be, in particular, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, isopentyl, neopentyl, 2-pentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, heptyl, octyl, nonyl, decyl, dodecyl or palmityl chains.

[0014] In the context of this invention, "aryl" refers to an aromatic group containing one or more optionally substituted aromatic rings.

[0015] In the context of this invention, "heteroaryl" refers to an aromatic group containing one or more optionally substituted aromatic rings and comprising at least one heteroatom other than carbon and hydrogen.

[0016] In the context of this invention, "aryl group" refers to an aryl group substituted with one or more alkyl groups; the alkyl group may be C1-C6. 15 Alkyl groups, preferably containing 1-15 carbon atoms.

[0017] In the context of this invention, "heteroaryl" refers to a heteroaryl group substituted with one or more alkyl groups; the alkyl group may be C1-C2. 15 Alkyl groups, preferably containing 1-15 carbon atoms.

[0018] According to one implementation scheme, in the above formula, n1 or n2 is independently equal to 1, 2, 3, 4 or 5, or even more preferably 3 or 4.

[0019] According to another specific implementation, in the above formula, n1=n2 and is preferably equal to 1, 2, 3, 4 or 5, even more preferably 3 or 4.

[0020] According to another specific embodiment, the molecule is symmetrical. In this case, it contains a single group X''-R'' 11 -Y'', where Y'' is the COOR carried by R9. 10 and / or two groups XR 11 -Y, where Y is COOR' 10 One is carried by one of R1, R2, R3 or R7, preferably one of R1, R2 or R3, and the other is carried by one of R4, R5, R6 or R8, preferably one of R4, R5 or R6.

[0021] According to the specific implementation plan, in the above formula, R 10 and / or R' 10 They can be the same. Similarly, X, X', and / or X'' can be the same, Y, Y', and / or Y'' can be the same, and R... 11 、R' 11 and / or R'' 11 They can be the same.

[0022] The compounds according to the invention may be particularly selected from compounds of the following general formula: Where X'' can be O, S, or NH, corresponding to the following formula: Compounds of formula (I) according to the invention may be particularly selected from those of formula (I) in which R1, R2, R3, R4, R5 and R6 are not simultaneously H, excluding compounds according to formula (II): (II).

[0023] The compounds of formula (I) according to the present invention may preferably be selected from compounds of the following general formulas: [Chemical Formula 3] [Chemical Formula 4] R1, R2, R3, R4, R5, R6, R7, R8 and R9 are defined as above.

[0024] According to one specific embodiment, the compounds according to the present invention may be selected from compounds of the following formula. [Chemical Formula 5] [Chemical Formula 6] [Chemical Formula 7] R1, R2, R3, R4, R5, R6, R7, R8 and R9 are defined as above.

[0025] According to a preferred embodiment, the compound according to the present invention may be selected from the following compounds:

[01] [Chemical Formula 8]

[02]

[03]

[04] [Chemical Formula 9]

[05]

[06]

[07] [Chemical Formula 10]

[08]

[09]

[10] [Chemical Formula 11]

[11]

[12]

[13] [Chemical Formula 12]

[14]

[15]

[16] [Chemical Formula 13]

[17]

[18]

[19] [Chemical Formula 14]

[20] [twenty one]

[22] [Chemical Formula 15] [twenty three] [twenty four] [Chemical Formula 16] Secondly, the present invention relates to a method for preparing a compound of formula (I) according to the present invention, comprising the following reaction steps: [Chemical Formula 17] and [Chemical Formula 18] The reaction is preferably carried out by heating under reflux in a mixture of acetic acid and acetic anhydride in the presence of sodium acetate.

[0026] Furthermore, the present invention relates to a method for labeling tumor tissue with one of the compounds prepared according to the invention or by the method according to the invention.

[0027] In the context of this invention, "tumor tissue" refers to tissue composed of tumor cells, which are abnormally proliferating cells, and supporting tissue, which is also called tumor matrix or stromal tissue, and is composed of cells and extracellular material where tumor vascularization is located.

[0028] The fluorescent compounds according to the invention possess a specific characteristic of being captured in tumor tissue after they have diffused into the body, while they are eliminated from healthy tissue. This specific characteristic allows for the direct use of these fluorescent compounds without prior conjugation to another labeled molecule, thus making their use simpler, faster, and more effective than compounds of the prior art. This elimination from healthy tissue is observed to increase over time. Typically, their elimination from healthy tissue is complete 24-72 hours after application of these compounds, preferably 36-60 hours, more preferably 48 hours. However, they remain captured in tumor tissue. This property provides a clear distinction between tumor tissue and healthy tissue, thus these compounds can be used for monitoring, diagnostic applications, and / or as an adjunct to surgery in cases of cancer. This distinction lasts for 6-48 hours, preferably 12-36 hours, allowing for targeted procedures in diagnosis or surgery.

[0029] Therefore, the compounds according to the invention can be particularly useful in cancer cases, such as hormone-dependent cancers like breast cancer or digestive system cancers like pancreatic cancer. Indeed, in pancreatic cancer, tumors are particularly difficult to completely remove surgically because they are not easily delineated. Due to the distinction between tumor tissue and healthy tissue, the use of the compounds according to the invention allows for better visualization of the tumor outline, and thus more effective surgical removal of the tumor.

[0030] The present invention also relates to the use of one of the compounds according to the invention or one of the compounds prepared according to the method according to the invention in a method for labeling tumor tissue.

[0031] This method of labeling tissue requires administration of the compound via intravenous or intra-arterial route, or in another blood vessel, particularly a lymphatic vessel, or by local injection or local application, preferably via intravenous route.

[0032] The present invention also relates to compositions comprising a compound according to the invention or a compound prepared by the method according to the invention and at least one pharmaceutically acceptable adjuvant.

[0033] The present invention also relates to a method for labeling and / or detecting tumor tissue, and / or for surgical treatment of tumors, comprising one of the compounds according to the invention or a compound prepared according to the method according to the invention, or a composition comprising one of the compounds according to the invention or a compound prepared according to the method according to the invention.

[0034] The present invention also relates to a method for detecting tumor tissue, comprising the steps of labeling the tumor tissue with one of the compounds prepared according to the invention or by the method according to the invention, and the steps of detecting by medical fluorescence imaging or fluorescence spectroscopy. Attached Figure Description

[0035] Figure 1 [ Figure 1 The median and standard deviation of the tumor / abdominal intensity ratio as a function of time following injection of compound 2 (CJ215) and ICG are shown.

[0036] Figure 2 [ Figure 2 The results of ex vivo imaging of pancreatic tumors after injection of two compounds according to the present invention and a prior art fluorescent agent (ICG) are shown. Detailed Implementation

[0037] Example 1 [Chemical Formula 19] Compound (1) A mixture of 4-[(5-carboxypentyl)oxy]-6-sulfono-1-(4-sulfobutyl)-2,3,3-trimethyl-benzo(e)indole (inner salt and disodium salt) (9 g; 15 mmol), 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (1.30 g; 7.50 mmol), and sodium acetate (3 g; 36.6 mmol) was heated under reflux for 10 min in a 60 / 30 mixture of acetic acid and acetic anhydride. The reaction mixture was cooled to room temperature, and the precipitate was separated by filtration and washed with diethyl ether to give 4.33 g (yield: 43.9%) of a green solid. The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0038] Example 2 [Chemical Formula 20] Compound (2) Sodium methoxide (440 mg; 7.6 mmol) was added to a solution of compound (1) (1 g; 0.76 mmol) in 500 mL of methanol. The reaction mixture was heated under reflux for 16 hours, concentrated under vacuum, and then filtered. The resulting residue was washed with cold methanol and acetone and dried under vacuum to give 450 mg of green solid (yield: 45%). The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0039] Example 3 [Chemical Formula 21] Compound (3) MeSNa (106 mg; 1.5 mmol) was added to compound (1) (400 mg; 0.30 mmol) in 20 mL of a 50 / 50 mixture of methanol / NMP (N-methyl-2-pyrrolidone). The reaction mixture was heated under reflux for 4 hours, and then diethyl ether (20 mL) was added to the mixture. The precipitate was filtered and washed with the same solvent to give 254 mg of crude product (yield: 61%; sulfur odor). The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0040] Example 4 [Chemical Formula 22] Compound (4) A mixture of 6-sulfo-1-(4-sulfobutyl)-2,3,3-trimethylbenzo(e)indole (internal salt and DCHA salt) (2 g; 4.7 mmol), 2-chloro-1-formyl-3-(hydroxymethylene)-1-cyclohexene (0.40 g; 2.35 mmol), and sodium acetate (0.9 g; 11 mmol) was heated under reflux for 15 min in a 50 / 20 mixture of acetic acid and acetic anhydride. The precipitate was separated by filtration, washed with ethanol and acetone, and dried under vacuum to give 1.6 g of brick-red powder (yield: 63.8%). The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0041] Example 5 [Chemical Formula 23] Compound (5) 8 mL of 1M methanol (KOH), 16 mL of DMSO, and compound (4) (500 mg; 0.25 mmol) were added to 3-(4-hydroxyphenyl)propionic acid (660 mg; 4 mmol). The reaction mixture was stirred at room temperature for 8 hours, followed by the dropwise addition of 150 mL of ethyl acetate. The precipitate was separated by filtration, washed with ethanol and acetone, and dried under vacuum to give 260 mg (yield: 45%) of green powder. The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0042] Example 6 [Chemical Formula 24] Compound (6) 4-Aminohydrocinnamic acid (816 mg; 4.9 mmol), 25 mL DMSO, and triethylamine (500 mg; 4.9 mmol) were added to compound (4) (520 g; 0.49 mmol). The reaction mixture was stirred at room temperature for 8 hours, followed by the dropwise addition of 200 mL acetone. The precipitate was separated by filtration, washed with acetone, and dried under vacuum to give 430 mg (yield: 74%) of red powder. The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0–25% / water).

[0043] Example 7 [Chemical Formula 25] Compound (7) 1 mL of 1M methanol (KOH), 16 mL of DMSO, and compound (4) (500 mg; 0.25 mmol) were added to 4-mercaptocinnamic acid (91 mg; 0.5 mmol). The reaction mixture was stirred at room temperature for 30 minutes, followed by the addition of 50 mL of ethyl acetate. The precipitate was separated by filtration, washed with ethanol and acetone, and dried under vacuum to give 310 mg (yield: 54%) of green powder. The crude product was purified by rapid column chromatography (reversed-phase silica gel C18, acetonitrile 0-25% / water).

[0044] Example 8: The compound according to the present invention and the prior art fluorescent agent (ICG) for in vivo formation of breast tumors Comparison of images ICG (or indocyanine green) is a prior art fluorescent agent that has been approved in humans for assessing cardiac and liver function, as well as for retinal diseases in ophthalmology. It is also being evaluated in numerous clinical trials worldwide for guiding surgery during tumor resections or for mapping ganglia draining tumors using near-infrared imaging.

[0045] ICG was compared with compound (2) according to the invention, the synthesis of which is described in Example 2 above; the compound is referred to as CJ215 in this study.

[0046] The study included a total of 30 mice, distributed across three groups. Each mouse underwent tumor transplantation by injecting 50,000 cells (4T1-Dendra2 / 20 μl) into each of the two contralateral mammary glands.

[0047] Biomarkers (compound 2, referred to as CJ215 in this study, and ICG) were injected on day 9 after tumor transplantation (to limit the occurrence of necrosis in the tumor).

[0048] The changes in fluorescence signal intensity recorded for each biomarker over time were evaluated using microscopic images. The ability of the two biomarkers to produce tumor-specific signals was quantitatively assessed by calculating the ratio of tumor-associated specific signals to non-specific signals in surrounding tissues.

[0049] Imaging protocols were performed on all mice at 2h, 24h, 48h, 4, and 6 days post-injection. All images at each acquisition time point were acquired using an IVIS spectral imager (Perkin Elmer) with the following parameters:

[25] For the detection of GFP form of Dendra2 (tumor detection):

[26] - Excited at 465nm

[27] -520-580nm emission

[28] For the detection of biomarkers:

[29] - Excited at 745nm -800-840nm emission Quantitative measurements were performed on the undeconvolutioned raw image. For two fluorophores, the acquisition time was parameterized in automatic mode. In this mode, the system determined the acquisition time required to reach a specified target value (6000 counts) within an allowed time (fixed at 2 minutes).

[0050] Figure 1 The median and standard deviation of the tumor / abdominal intensity ratio as a function of time following CJ215 and ICG injections are reported.

[0051] The measurement of the tumor / abdomen intensity ratio shown in this figure allows us to demonstrate that: - Compared to ICG, compound 2 (CJ215) exhibited a significantly higher intensity, independent of time post-injection (from 1.5 times at 2 h to more than 3 times at D+6), which translates into the ability to identify tumor-specific signals earlier and more specifically with compound 2 (CJ215). These results also suggest the possibility that increasing the time between compound 2 (CJ215) injection and imaging can significantly improve the specificity of the signal to the tumor. For compound 2 (CJ215), the signal-to-noise ratio continued to increase until 6 days post-injection, the last day of examination considered in this protocol. During this phase, ICG was no longer observed in the tumor (starting from 48 hours). Therefore, the high stability of the intratumoral signal of compound 2 (CJ215) compared to surrounding tissue (its elimination products) provides an improved ability to identify tumors and thus contributes to a significant improvement in the fine delineation of tumor margins, which remains problematic when using ICG.

[0052] Example 9: In vitro use of two compounds according to the present invention and a prior art fluorescent agent (ICG) in pancreatic tumors. Comparison in imaging.

[0053] An orthotopic pancreatic cancer model was developed in mice. Tumor cells were subcutaneously expanded in SCID mice, and the resulting fragments were then surgically transplanted into the pancreas of irradiated BALB / c nude mice.

[0054] Tumor development was monitored in vivo by MRI (4.7T, PharmaScan, Bruker Biospin) at three time points, D14, 28, and 36. Animals were exposed to weak fluorescence to minimize autofluorescence. Fluorescence imaging was performed using a charge-coupled device (CCD) camera (PhotonRT, BiospaceLab) with excitation at 700 nm and emission through a 770 nm filter.

[0055] In vivo imaging was performed at 2h, 48h, and 164h to obtain in vitro fluorescence images. Thirty-nine days after tumor fragment implantation, fluorescent compounds 2 (CJ215) and CJ319 (the structures of which are detailed below) according to the present invention were administered intravenously at 2 mg / kg, with an average tumor volume of approximately 70 mm². 3 Indocyanine green (ICG), a dye widely used in tumor surgical imaging, was used as a control.

[0056] [Chemical Formula 26] (CJ319) Figure 2 The ex vivo fluorescence imaging described herein showed that 2 hours post-injection, the two fluorescent compounds according to the invention were present in nearly equal amounts in the pancreas and tumor. However, 48 hours post-injection, a distinctly preferential distribution was observed in the tumor, with the fluorescence signals produced by both compounds being approximately 4 times higher in the tumor than in the surrounding pancreatic tissue. This effect persisted for 6 days post-injection, although the signal decreased over time. In contrast, indocyanine green did not show any specific accumulation in either the pancreas or the tumor.

[0057] These results demonstrate the superiority of the compounds of the present invention over prior art fluorescent agents in terms of their specific distribution levels in tumor tissues.

Claims

1. Compounds of formula (I) [Chemical Formula 27] (I) in n1 and n2 are both integers between 0 and 15. R1, R2, R3, R4, R5, and R6 are each independently selected from H, OH, SH, and NH. 2、 SO3R 10 and XR 11 -Y, R 10 and R' 10 Independently, it can be H, Na, or K. X, X', and X'' are independently O, S, or NH. R 11 、R' 11 and R'' 11 Independently selected from C1-C 15 Alkyl, aryl, heteroaryl, (C1-C) 15 alkyl)aryl, (C1-C 15 Alkyl) heteroaryl, aryl (C1-C) 15 Alkyl groups and heteroaryl groups (C1-C50) 15 alkyl); Y, Y', and Y'' are independently selected from H, halogen, and COOR'. 10 or amide; R7 and R8 are each independently selected from H, OH, SH, and NH. 2、 C 1- C 15 Alkyl and X'-R' 11 -Y'; R9 is selected from H, OH, SH, NH2, and X''-R''. 11 -Y'', The compound contains at least one XR group. 11 -Y、X'-R' 11 -Y' or X''-R'' 11 -Y'', where Y, Y' and / or Y'' are COOR' 10 .

2. The compound according to claim 1, wherein R1, R2, R3, R4, R5 and R6 are not simultaneously H.

3. The compound according to claim 1 or 2, wherein the compound is selected from compounds of the following formula: [Chemical Formula 28] [Chemical Formula 29] R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as defined in claim 1.

4. The compound according to any one of the preceding claims, wherein the compound is selected from compounds of the following formula. [Chemical Formula 30] [Chemical Formula 31] [Chemical Formula 32] R1, R2, R3, R4, R5, R6, R7, R8 and R9 are as defined in claim 1.

5. The compound according to any one of the preceding claims, wherein the compound is selected from compounds of the following formula. [Chemical Formula 33] [Chemical Formula 34] [Chemical Formula 35] [Chemical Formula 36] [Chemical Formula 37] [Chemical Formula 38] [Chemical Formula 39] [Chemical Formula 40] [Chemical Formula 41] 。 6. A method for preparing the compound according to any one of claims 1-5, comprising the following reaction steps: [Chemical Formula 42] and [Chemical Formula 43] 。 7. A method for labeling tumor tissue with one of the compounds prepared according to any one of claims 1-5 or according to claim 6.

8. Use of one of the compounds according to any one of claims 1-5 or prepared according to claim 6 in a method for labeling tumor tissue.

9. A compound according to any one of claims 1-5 or prepared according to claim 6, or a composition comprising a compound according to any one of claims 1-5 or prepared according to claim 6, for use in methods of labeling and / or detecting tumor tissue, and / or for use in surgical treatment of tumors.

10. A method for detecting tumor tissue, comprising the steps of labeling the tumor tissue with one of the compounds described in any one of claims 1-5 or prepared according to claim 6, and the step of detecting it by medical fluorescence imaging or fluorescence spectroscopy.

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