KR20200069613A - Targeting compound of cancer and use thereof - Google Patents

Abstract

The present invention relates to a compound for targeting cancer. More specifically, the present invention relates to a dual mode contrast agent containing the compound for targeting cancer, a composition for diagnosing cancer, and a method for providing information for monitoring cancer. When the compound for targeting cancer according to the present invention is treated in an organ or a tissue in vivo, the compound acts on the cancer tissues to simultaneously acquire radiographic images and fluorescence images, which can be provided as images for cancer diagnosis and stag setting, and surgical guide, respectively, and thus the compound can be used in cancer diagnosis and treatment using molecular imaging techniques.

Description

A compound for targeting cancer and its use

The present invention relates to a compound for cancer targets, and more particularly, to a dual mode contrast agent comprising the compound for cancer targets, a composition for diagnosis of cancer and a method for providing information for monitoring cancer.

Imaging using a dual mode contrast agent is a combination of two or more imaging methods, and is intended to overcome the limitations of conventional imaging techniques. In order to overcome this limitation, an imaging method using a contrast agent incorporating a radionuclide and a near-infrared fluorescent dye may be useful. Nuclear imaging has the advantage of excellent sensitivity and tissue penetration. However, imaging using nuclides has limitations in that acquisition time is long and space resolution is low. Another imaging method, optical imaging, has the advantage of being able to provide real-time and high-resolution images, but has the disadvantage of low tissue penetration. Therefore, dual-type imaging combining two or more imaging methods has many advantages over conventional imaging methods.

Meanwhile, folate is a nutrient essential for the division and growth of cancer cells and normal cells, and is absorbed into the cells through the folate receptor and metabolized. Folic acid receptors are attractive molecular targets that can be used for diagnosis and treatment because they are overexpressed in several types of cancer cells (renal cancer, ovarian cancer, pituitary tumor, colon cancer, etc.).

Accordingly, the present inventors have completed the present invention by developing a compound for a cancer target labeled with a radioactive isotope and a fluorescent material, in order to overcome the limitations of the conventional imaging technique as described above.

Accordingly, an object of the present invention is to provide a compound for cancer targets capable of specifically detecting cancer.

Another object of the present invention is to provide a dual mode contrast agent comprising the compound for cancer targets, a composition for diagnosing cancer, and a method for monitoring cancer using a compound for cancer targets.

In order to achieve the above object, the present invention provides a compound for a cancer target comprising a chelating ligand and folate, which is combined with a fluorescent material, a radioactive isotope, and represented by the amino acid sequence of SEQ ID NO: 2.

In addition, the present invention provides a dual mode contrast agent comprising the compound for cancer targets.

In addition, the present invention provides a composition for diagnosing cancer comprising the compound for cancer targeting.

In addition, the present invention provides a method for providing information for monitoring cancer, comprising the step of treating the cancer target compound with a biological sample.

When the compound for cancer target according to the present invention is treated in an organ or tissue in vivo, it is absorbed into cancer tissue, and thus radiographic images and fluorescence images can be simultaneously acquired. As it can be provided, it can be used for cancer diagnosis and treatment using molecular imaging techniques.

1 is a view showing the chemical structure of a compound for cancer target according to the present invention.
2 is a diagram showing the results of cancer cell affinity analysis of a radioactive isotope labeled cancer target compound according to the present invention.
3 is a view showing the results of analyzing the cellular uptake of the radioisotope labeled cancer target compound according to the present invention.
4 and 5 show the results of in vivo gamma camera imaging using the radioisotope labeled cancer target compound according to the present invention and the graphed results.
6 is a view showing the optical imaging results of the radioisotope labeled cancer target compound according to the present invention.
7 is a diagram showing the results of immunohistochemical staining of a tumor administered with a radioisotope labeled cancer target compound according to the present invention.
8 and 9 is a diagram showing the results of performing a tumor removal operation using a radioactive isotope labeled cancer target compound according to the present invention.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides a compound for cancer targets comprising a chelating ligand represented by the amino acid sequence of SEQ ID NO: 2 and folate, which is associated with a fluorescent substance, a radioactive isotope.

In the present invention, "cancer" is a malignant tumor and refers to a mass that is abnormally grown by overgrowth. Examples of the cancer include prostate cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, glioblastoma, head and neck cancer, kidney cancer, liver cancer, lung cancer, glioma, ovarian cancer, pancreatic cancer, stomach cancer, thyroid cancer, and uterine cancer.

In one embodiment of the present invention, the cancer is preferably a cancer that expresses a folate receptor, for example, lung cancer, breast cancer, prostate cancer, colorectal cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, endometrial adenocarcinoma, It is preferable, but not limited to, one or more selected from the group consisting of nasopharyngeal cancer, pituitary cancer, and thyroid medulla cancer.

In the present invention, "folic acid receptor" is a receptor that functions to reduce folic acid derivatives by binding to folic acid, and mediates the delivery of folic acid into cells after binding to folic acid or folic acid derivatives. The folic acid receptor may be limitedly expressed in normal tissues, but is overexpressed in tumors, and thus is used as a target for selectively delivering drugs to tumor tissues.

In an embodiment of the present invention, the cancer target compound contains folic acid, and thus binds to a folic acid receptor overexpressed in tumor tissue. Radiographic and fluorescence images can be simultaneously acquired through a cancer target compound coupled to a folate receptor in tumor tissue.

In the present invention, "fluorescent material" is a material that develops in response to light of a specific wavelength, and does not limit its type. Fluorescent materials include molecules that emit light in an excited state, metal ions, complex compounds, organic dyes, conductors, semiconductors, non-conductors, and quantum dots.

Examples of the fluorescent material include fluorescent proteins such as enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP). have.

In addition, as examples of the fluorescent material, pyrene (Pyrene) or a derivative thereof, cyanine (Cyanine, Cy) series, Alexa Fluor (Alexa Fluor) series, bodypi (BODIPY) series, DY series, rhodamine (rhodamine) or Derivatives thereof, Fluorescein or derivatives thereof, coumarin or derivatives thereof, Acridine homodimer or derivatives thereof, Acridine Orange or derivatives thereof, 7-amino liquid Tinomycin D (7-aminoactinomycin D, 7-AAD) or derivatives thereof, Actinomycin D or derivatives thereof, ACMA, 9-amino-6-chloro-2-methoxyacridine or derivatives thereof , DAPI or derivatives thereof, Dihydroethidium or derivatives thereof, Ethidium bromide or derivatives thereof, Ethidium homodimer-1 (EthD-1) or derivatives thereof, Ethidium homo Dimer-2 (EthD-2) or derivatives thereof, Ethidium monoazide or derivatives thereof, Hexidium iodide or derivatives thereof, bisbenzimide (Hoechst 33258) or derivatives thereof , Hoechst 33342 or derivatives thereof, Hoechst 34580 or derivatives thereof, hydroxystilbamidine or derivatives thereof, LDS 751 (LDS 751) or derivatives thereof, propidium Propidium Iodide (PI) or derivatives thereof, Calcein or derivatives thereof, Oregon Green or derivatives thereof, Magnesium Green or its derivatives Conductor, Calcium Green or derivatives thereof, JOE or derivatives thereof, Tetramethylrhodamine or derivatives thereof, TRITC or derivatives thereof, Tamra (N,N,N',N'-tetrametyl-6-carboxyrhodamine , TAMRA) or derivatives thereof, Pyronin Y or derivatives thereof, Lysamine or derivatives thereof, 5-ROX (5-Carboxy-X-rhodamine) or derivatives thereof, Calcium Crimson Or derivatives thereof, Texas Red or derivatives thereof, Nile Red or derivatives thereof, Thiadicarbocyanine or derivatives thereof, dansylamide or derivatives thereof, cascade blue blue), DAPI (4',6-diamidino-2-phenylindole).

A quantum dot may be used as the fluorescent material. The quantum dot is a particle composed of nano-sized II-IV or III-V semiconductor particles, and a shell composed mainly of ZnS and the like having a core of about 2 to 10 nm. shell), even in the same material, the fluorescence wavelength varies depending on the size of the particles, so that fluorescence in various wavelength bands can be obtained. Group II-VI or III-V compounds forming the quantum dot are CdSe, CdSe/ZnS, CdTe/CdS, CdTe/CdTe, ZnSe/ZnS, ZnTe/ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP/ZnS and HgTe It may be selected from the group consisting of, may be a single core (core) or core (core) / shell (shell) form.

In a specific embodiment of the present invention, 5-ROX (5-Carboxy-X-rhodamine) was used as the fluorescent material.

In the present invention, the "radioactive isotope" is an isotope that is unstable in the atomic nucleus, which emits radiation and converts it into a stable atomic nucleus, and does not limit its type. Examples of the radioactive isotopes include: Technetium-99m (Tc-99m), Gallium-67, Gallium-68, Copper-64, Copper-67, Gold-198, Lead-210, Nickel-63, Dysprosium-165, rhenium-186, rhenium-188, rubidium-82, ruthenium-177, manganese-177, molybdenum-99, fluorine-18, bismuth-213, samarium-153, oxygen-15, cesium- 137, selenium-75, sodium-24, strontium-85, strontium-89, strontium-90, americium-241, zinc-65, erbium-169, chlorine-36, iodine-123, iodine-124, iodine -125, Iodine-129, Iodine-131, Uranium-234, Uranium-235, Silver-110m, Iridium-192, Ytterbium-169, Ytterbium-177, Yttrium-90, Phosphorus-32, Phosphorus-33 , Indium-111, Germanium-68, Xenon-133, Nitrogen-13, Iron-55, Cadmium-109, Calcium-47, California-252, Cobalt-57, Cobalt-60, Quarium-244, Chromium-51 , Krypton-81, Krypton-85, Carbon-11, Carbon-14, Thallium-201, Thallium-204, Thoria Tungsten, Thorium-229, Thorium-230, Tritium, Palladium-103, Potassium-42, Polonium-210, There are therapeutic radioactive isotopes such as promethium-147, plutonium-238, holmium-166 and sulfur-35.

In an embodiment of the present invention, as the radioactive isotope, Technium-99m (Technetium-99m, Tc-99m) was used.

In an embodiment of the present invention, the chelating ligand is preferably represented by the amino acid sequence of SEQ ID NO: 2. The chelating ligand exhibits strong and stable chelation, especially with the radioactive isotope Tc-99m.

The cancer target compound may further include a spacer for reducing fluorescence disturbance of the cancer target peptide. Specifically, the spacer is preferably represented by the amino acid sequence of SEQ ID NO: 1. In addition, histidine may be used as a spacer in which histidine is inserted in the sequence of a conjugate of peptide hydrazinonicotinamide (HYNIC). By inserting a spacer as described above into the compound for cancer targeting, it is possible to improve tumor-targeting performance in vitro and in vivo. In addition, it is possible to reduce the disturbance between the cancer target peptide and the fluorescent substance of the cancer target compound.

In an embodiment of the present invention, the compound for cancer target is preferably arranged in the form of Structural Formula 1 below.

[Structural Formula 1]

Folic acid-spacer-chelating ligand-fluorescent material

In an embodiment of the present invention, the compound for cancer target is preferably represented by the following formula (1).

According to another aspect of the present invention, the present invention provides a dual modality contrast agent comprising the compound for cancer targeting.

In the present invention, the contrast medium is a material injected into tissues to increase the contrast of an image during radiographic examination, but is not limited thereto. When using conventional contrast agents, only radiographic images, such as magnetic resonance imaging (MRI) or computed tomography (CT) imaging, could be obtained. However, when using the dual-contrast contrast agent containing the compound for cancer target according to the present invention, it is possible to simultaneously acquire a radiographic image and a fluorescent image.

The dual mode contrast agent comprising the compound for cancer targeting may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto.

It is preferred that the dual mode contrast agent is administered to a subject parenterally. For parenteral administration, intravenous infusion, intramuscular infusion, intra-articular infusion, intra-synovial infusion, intramedullary infusion, intrarahepatic infusion, intralesional infusion, or It can be administered by intracranial injection. Suitable dosages of the dual mode contrast medium can be variously prescribed by factors such as the formulation method, the mode of administration, the patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. have.

A method of obtaining a radiographic image and a fluorescence image using the dual-contrast contrast agent may be performed according to a conventional method.

According to another aspect of the present invention, the present invention provides a composition for diagnosing cancer comprising the compound for targeting cancer.

In the present invention, "diagnosis" is not limited to medical, disease, etiology, pathology, severity, condition of the disease, and prognosis.

The composition for diagnosing cancer may further include a pharmaceutically acceptable carrier in addition to the active ingredients described above for administration. Pharmaceutically acceptable carriers are commonly used in formulations, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly Vinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but is not limited thereto.

The composition for diagnosing cancer comprising the compound for cancer target may be used as a cancer diagnostic kit.

According to another aspect of the present invention, the present invention provides a method for providing information for monitoring cancer, comprising the step of treating the cancer target compound with a biological sample.

In the present invention, "biological sample" is preferably a variety of organs or tissues in vivo.

It is preferable that the information providing method for cancer monitoring according to the present invention provides information such as location, prognosis and treatment process for cancer in vivo or in vitro.

The sample treated with the compound for cancer target can simultaneously acquire a radiographic image and a fluorescent image by performing gamma camera imaging and optical imaging according to a conventional method. The acquired radiographic and fluorescence images can provide objective basic information necessary for monitoring cancer location, prognosis and treatment process, and excludes clinical judgment or opinions of doctors.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1. Synthesis and radioactive isotope labeling of cancer target compounds

1-1. Synthesis and characterization of compounds for cancer targets

The novel cancer target compound according to the present invention was prepared to include a fluorescent substance, a spacer, a chelating ligand, and folic acid. More specifically, in order to increase the water solubility of the compound for cancer target, 5-ROX (5-Carboxy-X-rhodamine) was used as the fluorescent material. The spacer is represented by SEQ ID NO: 1 (GHEG), and is a site capable of improving tumor-targeting performance in vitro and in vivo. The chelating ligand was used to chelate radioactive isotopes, and more specifically, amino acids of SEQ ID NO: 2 (ECG) were selected to exhibit very stable chelation with Tc-99m used in this example. In addition, the folic acid is represented by the following Chemical Formula 2, and functions to allow the composition for cancer targets to accumulate in cancer tissue by binding to a folic acid receptor overexpressed in cancer cells.

The cancer target compound (Folate-Gly-His-Glu-Gly-Glu-Cys-Gly-Lys(-5-Carboxy-X-rhodamine)-NH ₂ , Folate-ECG-ROX) designed as above is 96% pure. Was synthesized by Anygen, Inc. (Gwangju, Korea). Specifically, the compound for cancer target was synthesized using Fmoc solid-phase peptide synthesis (Fomc-SPPS). The peptide-bound resin was treated with folic acid hydrate in DMSO and 2M HOBt/2M DIC and incubated for 4 hours. After incubation, 5-ROX in DMF and 2M DIEA was added to the peptide-binding resin and incubated for 24 hours. The synthesized compound for cancer targets is purified through reverse phase high-performance liquid chromatography (RP-HPLC) equipped with a Shimadzu C18 analytical column (C18, 5 μm, 100 mm 2 column, 4.6 × 250 mm). Did. For the RP-HPLC, a 0.1% aqueous solution of trifluoroacetic acid (TFA) containing 0 to 70% acetonitrile was used to form a concentration gradient.

In addition, the molecular weight of the purified compound for cancer targets was confirmed using a mass spectrometer (AXIMA-CFR, MALDI-TOF Mass Spectrometer).

The structural formula of the synthesized compound for cancer targets is shown in FIG. 1.

As shown in Figure 1, the synthesized cancer target compound has a chemical formula of C ₈₃ H ₉₅ N ₂₁ O ₂₁ S ₁ and a molecular weight of 1754.83.

1-2. Radioactive isotope labeling of compounds for cancer targets

The cancer target compound (Folate-ECG-ROX) prepared in Example 1-1 was labeled with a radioactive isotope Tc-99m.

Specifically, 300 μl of a cancer target compound (0.005 mg/ml in nitrogen purged water) and 300 μl of sodium tartrate (100 mg/ml in nitrogen purged water) were mixed in a microcentrifuge tube.

The prepared compound-containing tartrate salt solution was corrected with 1 ml of Tc-99m pertechnetate (about 1,110 MBq) and tin chloride (SnCl ₂ ) (1 mg/ml in nitrogen-purged 0.01 M HCl) Did. The calibrated solution was heated at 95° C. for 15 minutes and then cooled at room temperature. Characterization of the compound for labeled cancer targets was performed by Gilson 321 HPLC pump (Gilson, Inc., Middleton, WI, USA), Bioscan FC-1000 radioactive detector (Bioscan, Inc., Washington DC, USA), Trilution LC software (Gilson , Inc.) and YMC-Triart C18 column (4.6×100 nm; YMC, Kyoto, Japan). In addition, the HPLC used solvents A and B, and more specifically, solvent A was 0.1% TFA in water, and solvent B was used 0.1% TFA in acetonitrile. The linear gradient of 0-35% solvent B for 35 minutes eluted at a flow rate of 1 mL/min. An ultraviolet detector (230 nm) and a gamma radiodetector were used for monitoring compounds for cancer targets.

As a result of radioisotope labeling and purification, a compound for cancer target (Folate-ECG-ROX) labeled with radioisotope was obtained. In addition, when performing radio-RP-HPLC, the retention time was 22.4 minutes. In addition, the radioisotope-labeled compound for cancer targets was prepared with a yield of 97% or more.

Additionally, the stability of compounds for cancer targets labeled with radioactive isotopes was analyzed. Specifically, the radioactive isotope-labeled compound for cancer target (0.1 ml) was incubated with saline (1 ml) at room temperature (25 °C) and with human serum (1 ml) at 37 °C, respectively. Take out 30 minutes, 1, 3 and 24 hours after incubation, respectively, and take a saline solution (Tc-99m Folate-ECG-ROX and Rf = 0.9-1.0 of free pertechnitium acid; Rf = 0.0-0.1 of colloid) and acetone ( Stability was analyzed using Instant Thin Layer Chromatography-Silica Gel (ITLC-SG) using Rf = 0.9-1.0 of free pertechnitic acid; Tc-99m Folate-ECG-ROX and Rf = 0.0-0.1 of colloid).

As a result of analyzing the stability of the compounds for cancer targets labeled with radioactive isotopes, the undamaged proportions of samples cultured in saline were 97.1 ± 0.4, 96.5 ± 0.5, 94.8 ± 0.8 at 30 minutes, 1, 3 and 24 hours, respectively. And 92.7±1.5%. The undamaged proportions of samples cultured in human serum were 96.2 ± 0.8, 96.2 ± 0.7, 93.7 ± 1.2 and 91.6 ± 1.7% at 30 minutes, 1, 3 and 24 hours, respectively. The above results indicate that the radioactive isotope-labeled compound for cancer targets according to the present invention is stable in saline and human serum.

Experimental Example 1.Measurement of cancer cell binding affinity of radioisotope-labeled compounds for cancer targets

The cancer cell binding affinity of the radioisotope labeled cancer target compound (Folate-ECG-ROX) prepared in Example 1-2 was measured using breast cancer cell line KB cells and fibrosarcoma cell line HT-1080 cells. . The KB cells are tumor cells that express folate receptors, and HT-1080 cells are tumor cells that do not express folate receptors. The two cell lines were cultured in RPMI 1640 medium at 5% CO ₂ and a temperature of 37°C. The RPMI 1640 medium contains 10% fetal bovine serum (FBS), 300 mg/l L-glutamine, 25 mM HEPES (4-(2-hydroxyethyl)-1-iperazineethanesulfonic acid) and 25 mM sodium hydrogen carbonate (NaHCO ₃ ). Includes.

The binding affinity of the radioisotope-labeled cancer targeting compound to each of the KB cell line and the HT-1080 cell line was performed by a saturated binding experiment method according to Wu, Chunying, et al. (2007). Specifically, cells with a concentration of 1 X 10 ⁶ cells/ml were plated at a uniform cell density in a 24-well plate and then cultured overnight. The cultured cells were washed twice each for 5 minutes with ice-cold binding buffer containing 25 mM HEPES and 1% bovine serum albumin. Thereafter, the washed cells were treated with radioisotope-labeled compounds for cancer targets and radioisotope-labeled scrambled peptides (0 to 500 μM), respectively, and incubated at 37° C. for 1 hour. Then, the cells were washed 3 times with cold binding buffer and then lysed in 200 μl of lysis buffer. Cell-associated radioactivity of lysed cells was measured using a 1480 Wizard 3 gamma counter (PerkinElmer Life and Analytical Sciences, Wallingford, CT, US). The measurement result was determined using a nonlinear regression model of Graphpad Prism (version Graph3, GraphPad Software, La Jolla, CA, USA) software to determine the binding constant (K _d ) and the maximum number of binding sites (B _max ). The results of measuring the cancer cell binding affinity are shown in FIG. 2.

As shown in FIG. 2, the binding constant (K _d ) of the radioisotope-labeled compound for cancer targets against the breast cancer cell line KB cell is 6.9 ± 0.9 nM (maximum number of binding sites (B _max ) = 243.9 ± 5.9, FIG. 2A). In contrast, the binding constant (K _d ) of the radioisotope labeled cancer target compound for the fibrosarcoma cell line HT-1080 cells was 4340 ± 3352 nM (B _max = 1621 ± 1142, Figure 2B) (p< 0.005). The above results indicate that the radioisotope labeled cancer target compound has a very high binding affinity to cancer cells expressing folate receptors.

Experimental Example 2. Cellular uptake analysis of radioisotope labeled cancer target compounds

To measure the cellular uptake of the radioisotope labeled cancer target compound (Folate-ECG-ROX) prepared in Example 1-2, a confocal microscopy (Confocal microscopy) was used.

First, the breast cancer cell line KB cells (1 X 10 ⁵ cells/well) were cultured on a cover slip slide (temperature 37° C., 24 hours). After removing the medium of KB cells, the cells were cultured by replacing with 500 μl of serum-free medium containing a compound for cancer target labeled with a radioactive isotope having a concentration of 200 μM (37° C., 1 hour). The cultured KB cells were washed three times with phosphate-buffered saline (PBS) to remove unbound peptide. The cover slip was placed on a slide containing a fluorescent binding medium (Fluorescent mounting medium, Dako, Glostrup, Denmark). A confocal laser scanning microscope was used with a 100X Oil immersion lens FV1200 confocal microscope (Olympus, Pittsburgh, PA, USA). In the same manner as described above, the cell uptake of the radioisotope labeled cancer target compound was analyzed using the fibrosarcoma cell line HT-1080. The cell uptake measurement results are shown in FIG. 3.

As shown in FIG. 3, strong fluorescence was observed in the cell membrane and cytoplasm of the KB cells cultured together with the radioisotope-labeled cancer target compound. On the other hand, it was confirmed that HT-1080 cells cultured with a radioisotope-labeled cancer target compound were very weak in fluorescence. The above results indicate that the radioisotope-labeled cancer target compound has a very high cell uptake rate in cancer cells expressing folate receptors.

Experimental Example 3. In vivo gamma camera of radioisotope labeled cancer target compound in tumor bearing mouse model Imaging (Gamma camera imaging) and competitive effect analysis

The mouse used was a female thymic nude mouse (BALB/c nu/nu), 6 weeks of age, 16 to 18 g. The mice were bred in cages and provided with tap water and food. The diet of common rodents is high in folic acid. Therefore, the mice used in this experiment were fed with folic acid-free diet (TD.95247, Harlan Laboratories, Indianapolis, IN, USA) for 3 weeks in order to reduce the serum folic acid concentration to the physiological range.

The mice were inoculated subcutaneously with 1×10 ⁷ KB (FR-positive) cells and HT-1080 (FR-negative, as an internal reference) cells contained in 0.1 ml PBS, respectively, on the right and left sides of the rear chest. After inoculation, the mice were kept for about 14 days to prepare tumor-bearing mice. The vertical dimension of the tumor formed in the chest of the prepared tumor bearing mouse was measured to be 400 to 550 mm ³ .

Using the tumor-bearing mouse, in vivo gamma camera imaging of the radioisotope-labeled cancer target compound prepared in Example 1-2 was performed. More specifically, after injecting 55.5 MBq of a radioactive isotope labeled cancer target compound into a tail vein in a tumor-bearing mouse, the tumor-bearing mouse is in vivo using a gamma camera (Vertex; ADAC Laboratories, Milpitas, CA, USA). Imaging was performed. Gamma camera images were acquired 1, 2 and 3 hours after injection, and acquisition times were 120 seconds. In the obtained gamma camera image, regions of interest (ROIs, 15 X 15 pixels) were drawn on the tumor region of the chest wall. In addition, the region of interest for normal muscle uptake measurement was extracted from the left arm muscle. The average number of cancer cells per pixel in the determined region of interest was measured, and the target (tumor)-non-target (normal muscle) absorption ratio was calculated.

In addition, it was evaluated whether the radioisotope-labeled cancer target compound prepared in Example 1-2 can specifically bind to a folic acid receptor and compete with free folic acid. Specifically, the radioisotope-labeled cancer target compound and free folic acid (4 mM, a concentration corresponding to about 20 times the radioisotope-labeled cancer target compound) prepared in Examples 1-2 of tumor-bearing mice were prepared. The tail vein was injected and gamma camera imaging was performed 1, 2 and 3 hours after injection.

The results of analyzing in vivo gamma camera imaging and competitive effects are shown in FIGS. 4 and 5.

As shown in Figures 4 and 5, the strongest activity in the kidney was observed, which means that the radioisotope labeled cancer target compound is excreted through the kidney. In addition, in tumor-bearing mice, it was confirmed that the radioisotope-labeled cancer target compound accumulated in folate receptor-positive KB cells (FIG. 4A, arrow). The target (tumor)-non-target (normal muscle) uptake ratio of radioisotope-labeled compounds for cancer targets tended to increase with time (3.4 ± 0.4, 4.4 ± 0.7 and 6.6 at 1, 2 and 3 hours, respectively). ± 0.8). In contrast, it was confirmed that the radioisotope-labeled cancer target compound does not accumulate in folate receptor-negative HT-1080 cells (Fig. 4, arrow head), and the target (tumor)-non-target (muscle) uptake ratio is It was significantly lower than KB cells (p<0.05) (FIG. 5 ).

In addition, as a result of evaluating the competitive effect of the free folic acid and the radioisotope-labeled compound for cancer targets, the absorption of KB cells was significantly decreased (tumor-normal muscle uptake ratio was 2.6±0.3 at 1, 2 and 3 hours, respectively). 2.9 ± 0.5 and 2.8 ± 0.3, Figure 4B, arrows). The results indicate that injecting a radioisotope labeled cancer target compound and excess free folic acid together inhibits the uptake of isotopes in the cancer target compound in KB cells (FIG. 5 ).

Experimental Example 4. In vitro using a tumor-bearing mouse model ( ex vivo ) Optical Imaging (Optical imaging) and postmortem research

After performing Experimental Examples 2 and 3, tumors and organs were excised from tumor bearing mice. Resected tumors and organs were optically imaged in vitro using a fluorescence imaging system (FOBI-10BR, Neoscience, Suwon, Korea). The emission band of the fluorescent material ROX used in the present invention is 520 to 675 nm. When performing optical imaging, the exposure time was 2.5 seconds per image, and the acquired images were analyzed using dedicated software.

In addition, for post-mortem studies, tumors were divided into two parts after in vitro optical imaging. Each piece was used randomly for biodistribution studies or confocal microscopy analysis. Pieces for the confocal microscopy analysis were prepared by immediately freezing in an optimal cutting temperature embedding compound. The in vitro optical imaging results are shown in FIG. 6.

As shown in Figure 6, the radioisotope-labeled compound for cancer targets exhibited fluorescence activity in the kidney, a healthy organ, which means that the compound is excreted through the kidney. In addition, the fluorescence activity of the fluorescent substance ROX was detected high in the KB tumor (arrow), and the fluorescence activity was weakly detected in the HT-1080 tumor (arrow head).

Experimental Example 5. Biodistribution of radioisotope labeled cancer target compounds using tumor bearing mouse models

The organ of Experimental Example 4 was used to confirm the biodistribution of the radioisotope-labeled cancer target compound. Specifically, the organ of Experimental Example 4 is the tumor bearing mouse sacrificed 3 hours after the administration of the radioisotope-labeled cancer target compound. The organs of Experimental Example 4 were each stored in a previously measured gamma counter tube. Radioactivity of each organ was measured by a gamma counter (1480 Wizard 3, PerkinElmer Life and Analytical Sciences). The counts per minute were decay correction, and the corrected results were expressed as the percentage of dose per gram of wet tissue (% ID/g). Table 1 shows the biodistribution values (% ID/g) of the cancer target compound.

long time Dose per weight of tissue (%ID/g) (SD) After 1 hour 3 hours later Lung, Lu 2.75 (1.28) 1.06 (0.08) Heart (Hart) 1.57 (0.43) 0.45 (0.16) Blood 6.21 (2.74) 2.44 (0.17) Liver (Lv) 2.89 (1.35) 2.02 (0.82) Stomach, St 2.27 (0.89) 2.11 (1.12) Colon (Colon, Co) 1.25 (0.47) 0.35 (0.02) Kidney (Kdney) 20.03 (6.54) 10.52 (2.63) Muscle (Muscle, Mu) 1.68 (1.61) 0.46 (0.10) KB tumor (folate receptor positive) 2.50 (0.80) 4.08 (1.16) HT-1080 tumor (folate receptor negative) 0.94 (0.93) 0.78 (0.25)

As shown in Table 1, when the radioisotope labeled cancer target compound is administered, the biodistribution value of the kidney is highest. Due to the decrease in the activity of normal tissues, it was confirmed that the biodistribution value decreased 3 hours after administration of the compound. On the other hand, the biodistribution value increased in the hydrochloric acid receptor positive KB tumor.

Experimental Example 6. Immunohistochemistry staining of tumors administered with a radioisotope labeled cancer target compound.

The frozen tumors of Experimental Example 4 (KB tumors and HT-1080 tumors) were used to immunohistochemically stain tumors to which the radioisotope labeled cancer target compound was administered. Specifically, the frozen tumor of Experimental Example 4 is that of a tumor-bearing mouse sacrificed 3 hours after administration of a radioisotope-labeled cancer target compound. Frozen tumor tissue was cut to a thickness of 10 μm to prepare sections. After the prepared sections were completely dried, they were fixed to slides using 4% formaldehyde (in PBS). The slides were incubated for 10 min at -20 °C with cold 100% methanol. Then, the sections fixed to the slides were blocked with 5% goat serum for 30 minutes at room temperature. Blocked sections were incubated with primary antibody (mouse anti-FR antibody, sc-5155219, 1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 hour at room temperature. After incubation, it was washed with PBS and incubated with a secondary antibody (Alexa Fluor® 488-conjugated goat anti-Armenian hamster secondary antibody, 1:100; Jackson Immuno Research Inc., West Grove, PA, USA). Secondary antibody and cultured slides were covered with Prolong® Gold Antifade Reagent containing DAPI (4',6-diamidino-2-phenylindole, Invitrogen, Calsbad, CA, USA) to perform confocal laser scanning. The confocal laser scanning was performed with an FV1200 confocal microscope (Olympus) equipped with a 60x oil immersion lens. All acquired images were taken at the same exposure time, and the brightness and contrast adjustments were applied identically. The results of immunohistochemistry staining of tumors administered with a radioisotope labeled cancer target compound are shown in FIG. 7.

As shown in FIG. 7, strong fluorescence was detected in the KB tumor tissue, and the detected fluorescence originated from ROX and anti-FR antibodies. On the other hand, fluorescence due to ROX and anti-FR antibodies was weakly detected in HT-1080 tumor tissue.

Experimental Example 7. In vivo optics using compounds for radioactive isotope labeled cancer targets Imaging ( In vivo optical imaging) and tumor removal surgery using the same

It was evaluated whether in vivo optical imaging with a radioisotope labeled cancer target compound can guide tumors in tumor bearing mice during surgery. Specifically, mice with peritoneal carcinomatosis were prepared by intraperitoneal injection of KB cells 1 x 10 ⁷ (in 0.1 ml PBS) for 10 days after intraperitoneal injection of mice. Prepared peritoneal cancer-bearing mice were anesthetized immediately prior to surgery (intraperitoneal injection, ketamine [60 mg/kg]/xylazine [5 mg/kg]) and ROX under a fluorescence imaging system (FOBI-10BR, Neoscience, Suwon, Korea). Fluorescence was detected (peak absorption wavelength=575 nm, peak emission wavelength=602 nm) and surgery was performed. After surgery, optical imaging of mice and resected tumors was performed. The surgical procedure and results of tumor removal using in vivo optical imaging are shown in FIGS. 8 and 9.

8 and 9, it was confirmed that the tumor metastasized in the abdominal cavity through fluorescence imaging of mice bearing peritoneal cancer (FIG. 8A, arrow). In addition, it was confirmed that the tumor was removed through in vivo optical imaging (FIG. 9 ). However, it was found that the peritoneal cancer located on the surface was removed, but the small tumor was not removed (FIG. 8B ). As a result of performing optical imaging after surgery, it was confirmed that the excised tumor showed stronger fluorescence than normal muscles (FIG. 8C).

Overall, the present inventors have confirmed that the radioisotope labeled cancer target compound has a high absorption rate and biodistribution value when administered to cancer tissue. This means that when radioactive isotope-labeled compounds for cancer targets are administered, radiographic images and fluorescence images can be obtained together. .

As described above, since a specific part of the present invention has been described in detail, it is obvious to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. something to do. Therefore, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Wonkwang University Center for Industry-Academy Cooperation <120> Targeting compound of cancer and use thereof <130> 1-111P <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> spacer <400> 1 Gly His Glu Gly One <210> 2 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> chelating ligand <400> 2 Glu Cys Gly One

Claims (10)

Fluorescent,
A chelating ligand represented by the amino acid sequence of SEQ ID NO: 2, which is associated with a radioactive isotope and
A compound for targeting cancer, comprising folate.
According to claim 1,
The fluorescent material is a light-emitting molecule, a fluorescent protein, a metal ion, a complex, an organic dye, a conductor, a semiconductor, a nonconductor, a quantum dot or a quantum ray, a compound for cancer targeting.
According to claim 1,
The cancer is for cancer targets, at least one selected from the group consisting of lung cancer, breast cancer, prostate cancer, colorectal cancer, brain cancer, ovarian cancer, cervical cancer, endometrial cancer, endometrial carcinoma, nasopharyngeal cancer, pituitary cancer and thyroid medulla cancer compound.
According to claim 1,
The cancer target compound further comprises a spacer for reducing fluorescence disturbance of the target peptide, a cancer target compound.
The method of claim 4,
The spacer is represented by the amino acid sequence of SEQ ID NO: 1, a cancer target compound.
The method of claim 4,
The cancer target compound is to be arranged in the form of structural formula 1, a cancer target compound.
[Structural Formula 1]
Folic acid-spacer-chelating ligand-fluorescent material
The method of claim 4,
The compound for cancer targets is represented by Formula 1, a compound for cancer targets.
[Formula 1]

A dual modality contrast agent comprising a compound for a cancer target according to any one of claims 1 to 7.
A composition for diagnosing cancer comprising the compound for cancer target according to any one of claims 1 to 7.
A method for providing information for cancer monitoring, comprising the step of treating a compound for a cancer target according to any one of claims 1 to 7 in a biological sample.

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