KR20190021739A - Targeting compound of cancer and use thereof - Google Patents

Abstract

The present invention relates to a compound for targeting cancer. More particularly, the present invention relates to a dual-mode contrast medium, a composition for diagnosing cancer, and a method for providing information for monitoring cancer containing the compound for targeting cancer. When the compound for targeting cancer according to the present invention is administered to organs or tissues in vivo, the compound can be taken into cancer tissues, thereby being able to simultaneously acquire a radiological image and a fluorescent image, and since the radiological image and the fluorescent image are provided as images for cancer diagnosis, clinical staging, and an operation guide, it is possible to be used for cancer diagnosis and treatment using molecular imaging techniques.

Description

Cancer targeting compounds and uses thereof < RTI ID = 0.0 >

More particularly, the present invention relates to a dual contrast agent, a composition for diagnosing cancer, and a method for providing information for monitoring cancer.

 Molecular imaging techniques have been used in the treatment and diagnosis of diseases as the localization and staging of disease progresses. For the diagnosis and treatment of various diseases, various molecular targets must be targeted. Accordingly, various imaging probes are being developed that can target a variety of molecular targets. In particular, radioactive isotope labeled peptides are emerging as valuable biological tools for tumor receptor imaging and radionuclide therapy.

On the other hand, tumor invasion involves the interaction of tumor cells and extracellular matrix (ECM). More specifically, tumor invasion occurs by cell migration through receptor-mediated recognition, attachment, and extracellular matrix degradation by chemotactic agents. At this time, some enzymes induce the degradation of elastin and induce the production of elastin-derived peptides (EDPs). The function of the elastin-derived peptide includes cell proliferation, cell migration, tumor progression, endothelial cell migration, and angiogenesis in normal cells and tumor cells. The function of the elastin-derived peptides is mediated primarily by elastin binding proteins, integrin av [beta] 3 and galactin-3 receptors. Thus, a substance that specifically binds to the elastin binding protein, integrin av [beta] 3 and the galactin-3 receptor can be used as a target molecule for molecular imaging of tumor cells.

However, despite the identification of substances specifically binding to elastin binding protein, integrin avß3 and galactin-3 receptors, there is no initial study of contrast agents using them.

It is difficult to overcome the limitations (sensitivity, resolution, transmissivity, etc.) inherent in molecular imaging techniques because most cancer-causing compounds can only be used in a single molecular imaging technique (Zanzonico P et al.

Accordingly, the present inventors have completed the present invention by developing a cancer-screening compound labeled with a radioisotope and a fluorescent substance in order to solve the problems of the prior art as described above.

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

It is another object of the present invention to provide a dual contrast agent, a composition for diagnosing cancer, and a method for providing information for cancer monitoring using a cancer screening compound.

In order to achieve the above object, the present invention provides a method for screening a cancer comprising a chelating ligand bound to a fluorescent substance, a radioactive isotope, an amino acid sequence of SEQ ID NO: 2, and a cancer target peptide represented by an amino acid sequence of SEQ ID NO: 3 ≪ / RTI >

The present invention also provides a dual contrast agent and a composition for diagnosing cancer, which comprises a compound to be administered as a cancer marker.

Also provided is a method for providing information for cancer monitoring comprising the step of treating the cancer-screening compound with a biological sample.

When the cancer screening compound according to the present invention is administered to an organ or tissue in vivo, the compound can be taken into cancer tissue to simultaneously acquire a radiological image and a fluorescence image, and these are provided as images for cancer diagnosis, And can be used for diagnosis and treatment of cancer using molecular imaging techniques.

Figure 1 shows the chemical structure of the cancer-screening compound and the scrambled peptide.
FIG. 2 shows the results of measurement of the cancer cell binding affinity of the cancer-screening compound and the ssklembide peptide.
FIG. 3 shows the results of measurement of cell uptake of cancer-scoring compounds and scrambled peptides.
Figures 4 and 5 show gamma camera imaging results and graphical results of radioactive isotope labeled cancer labeled compounds and scrambled peptides.
Figure 6 shows optical imaging results of radiolabeled cancer labeled compounds and scrambled peptides.
FIG. 7 shows the results of immunohistochemical staining of tumors to which the cancer targeting compound was administered.

Hereinafter, the present invention will be described in detail.

The present invention provides a cancer targeting compound comprising a fluorescent substance, a chelating ligand bound to a radioactive isotope, an amino acid sequence of SEQ ID NO: 2, and a cancer target peptide represented by the amino acid sequence of SEQ ID NO: 3.

In the present invention, cancer refers to a malignant tumor, which 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, renal cancer, liver cancer, lung cancer, glioma, ovarian cancer, pancreatic cancer, gastric cancer, thyroid cancer and uterine cancer. In one embodiment of the present invention, cancer targeting compounds that target colon cancer as the cancer were prepared.

In the present invention, a fluorescent substance is a substance that is developed by reacting with light of a specific wavelength, and its kind is not limited. Examples of the fluorescent material include molecules that emit light in an excited state, metal ions, complex compounds, organic dyes, conductors, semiconductors, nonconductors, 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 have.

Examples of the fluorescent material include pyrene or derivatives thereof, cyanine (Cy) series, Alexa Fluor series, BODIPY series, DY series, rhodamine or Acridine orange or derivatives thereof, derivatives thereof, fluorescein or derivatives thereof, coumarin or derivatives thereof, acridine homodimer or derivatives thereof, acridine orange or derivatives thereof, The present invention relates to a method for producing 7-aminoactinomycin D, 7-AAD or a derivative thereof, Actinomycin D or a derivative thereof, ACMA (9-amino-6-chloro-2-methoxyacridine) Diethanolamine or its derivative, dihydroxyaniline or diethanolamine, diapyril or its derivative, dihydroxyidene or its derivative, ethidium bromide or its derivative, ethidium homodimer-1 or its derivative, Dimmer- (EthD-2) or a derivative thereof, Ethidium monoazide or a derivative thereof, Hexidium iodide or a derivative thereof, bisbenzimide (Hoechst 33258) or a derivative thereof, Hoechst 34580 or a derivative thereof, hydroxystilbamidine or a derivative thereof, LDS 751 or a derivative thereof, propidium iodide, Or a derivative thereof, propidium iodide (PI) or a derivative thereof, Calcein or a derivative thereof, Oregon Green or a derivative thereof, Magnesium Green or a derivative thereof, Calcium Green or a derivative thereof, JOE or a derivative thereof, tetramethylrhodamine or a derivative thereof, TRITC or a derivative thereof, N, N, N ', N'-tetrametyl-6-carboxyrhodamine, TAMRA Pyronin Y or a derivative thereof, lissamine or a derivative thereof, ROX or a derivative thereof, calcium cream or a derivative thereof, Texas Red or a derivative thereof, , Nile Red or a derivative thereof, thiadicarbocyanine or a derivative thereof, dansylamide or a derivative thereof, cascade blue, DAPI (4 ', 6-diamidino-2 -phenylindole. < / RTI >

Quantum dots can be used as the fluorescent material. The quantum dots are grains centered on nano-sized II-IV or III-V semiconductor grains, and have a core of about 2 to 10 nm in size and a shell mainly composed of ZnS shell). Even if the same material is used, fluorescence of various wavelength ranges can be obtained by changing the fluorescence wavelength according to the particle size. The II-VI or III-V compound constituting the quantum dot may be at least one selected from the group consisting of CdSe, CdSe / ZnS, CdTe / CdS, CdTe / CdTe, ZnSe / ZnS, ZnTe / ZnSe, PbSe, PbS InAs, InP, InGaP, InGaP / ZnS and HgTe And may be in the form of a single core or a core / shell.

In one embodiment of the present invention, TAMRA was used as the fluorescent material.

In the present invention, a radioactive isotope is not limited to an isotope that is unstable and emits radiation and converts to a stable atomic nucleus. Examples of the radioisotope include Technetium-99m, Tc-99m, Gallium-67, Gallium-68, Copper-64, Copper-67, 18, bismuth-213, samarium-153, oxygen-15, cesium-18, rhenium-188, rubidium-82, ruthenium-177, manganese-177, molybdenum-99, fluorine- 137, selenium-75, sodium-24, strontium-85, strontium-89, strontium-90, americium-241, zinc-65, erbium-169, chlorine-36, iodine- -125, iodine-129, iodine-131, uranium-234, uranium-235, silver-110 m, iridium-192, ytterbium-169, ytterbium-177, yttrium- , Indium-111, germanium-68, xenon-133, nitrogen-13, iron-55, cadmium-109, calcium-47, californium-252, cobalt-57, cobalt-60, , Krypton-81, Krypton-85, Carbon-11, Carbon-14, Thallium-201, Thallium-204, Thorium-229, Thorium-230, Tritium, Palladium- 210, Promethium-147, Plutonium-238, Holmium-166 and Sulfur-35.

Technetium-99m (Tc-99m) was used as the radioisotope in one embodiment of the present invention.

In 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.

In the present invention, a peptide means a linear molecule formed by peptide bonds and amino acid residues bonded to each other. The peptide can be produced according to a chemical synthesis method known in the art, and can be prepared according to a solid phase synthesis technique, but is not limited thereto.

The cancer target peptide is preferably represented by the amino acid sequence of SEQ ID NO: 3.

The cancer targeting peptide (VAPG) is derived from an elastin derived peptide (EDP) and has a property of binding directly to a surface receptor (elastin binding protein, integrin avß3 and galactin-3 receptor). Due to these properties, it has been confirmed through the examples that cancer target peptide (VAPG) accumulates in cancer tissues.

The cancer targeting compound may further comprise a spacer to reduce fluorescence perturbation of the cancer target peptide. Specifically, the spacer is preferably represented by the amino acid sequence of SEQ ID NO: 1. Also, as a spacer, histidine may be inserted into the sequence of the peptide hydrazinonicotinamide (HYNIC) conjugate. By inserting the spacer as described above into the cancer targeting compound, it is possible to improve tumor-targeting performance in vitro and in vivo. It can also reduce disturbance of cancer targeting peptides and fluorescent materials of cancer targeting compounds.

The cancer-screening compound is preferably arranged in the form of the following structural formula (1).

[Structural formula 1]

Fluorescent-spacer-chelating ligand-target peptide

The cancer-screening compound is preferably represented by the following formula (1).

[Chemical Formula 1]

In another aspect, the present invention provides a dual contrast imaging agent comprising a cancer targeting compound.

In the present invention, the contrast agent is a substance to be injected into the tissue for enhancing the contrast of the image during the radiation examination, but the present invention is not limited thereto. When using conventional contrast agents, only radiation images such as magnetic resonance imaging (MRI) or computed tomography (CT) imaging could be acquired. However, when a dual contrast agent containing the compound of the present invention is used, a radiographic image and a fluorescence image can be simultaneously obtained.

The bi-system contrast agent comprising the cancer-targeting compound may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

Preferably, the dual contrast agent is administered parenterally to the individual. In the case of parenteral administration, intravenous infusion, intramuscular injection, intra-articular injection, intra-synovial injection, intrathecal injection, intrahepatic injection, intralesional injection, Intracranial injection or the like. Suitable doses of the dual contrast agents may be varied depending on factors such as formulation, mode of administration, age, weight, sex, pathology, food, time of administration, route of administration, excretion rate and responsiveness of the patient have.

A method of obtaining a radiological image and a fluorescence image using the dual contrast agent can be carried out according to a conventional method.

In another aspect, the present invention provides a cancer diagnostic composition comprising a cancer targeting compound.

In the present invention, the diagnosis is medically judged on the pathology, pathogenesis, morbidity, severity, disease state, and prognosis.

The cancer diagnostic composition may further comprise a pharmaceutically acceptable carrier in addition to the above-described effective ingredients for administration. Pharmaceutically acceptable carriers are those conventionally used in the formulation and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition for diagnosing cancer including the cancer-screening compound may be used as a cancer diagnostic kit.

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

The biological sample is preferably a variety of in vivo organs or tissues.

The information providing method for cancer monitoring can provide objective basic information necessary for monitoring the position, prognosis and treatment process of cancer in vivo or in vitro, Judgments or observations are excluded.

The sample treated with the cancer targeting compound can simultaneously acquire the radiological image and the fluorescence image by performing gamma camera imaging and optical imaging according to the conventional method. The acquired radiation image and fluorescence image can be used to monitor the cancer location, prognosis, and treatment process.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. It is to be understood that these examples and experimental examples are provided only for illustrating the present invention and that the scope of the present invention should not be construed as being limited by these examples and experimental examples, will be.

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

1-1 Synthesis and Characterization of Cancer Applications

The novel cancer targeting compounds according to the present invention are prepared to include fluorescent materials, spacers, chelating ligands and cancer target peptides. More specifically, in order to increase the water solubility of the cancer-targeting compound, the fluorescent material was TAMRA. The spacer is represented by SEQ ID NO: 1 (GHEG) and is a site capable of enhancing tumor-targeting performance in vitro and in vivo. The chelating ligand was used to chelate the radioactive isotope. More specifically, the amino acid of SEQ ID NO: 2 (ECG) was selected to exhibit a very stable chelation with Tc-99m used in this Example. Further, the cancer target peptide is represented by SEQ ID NO: 3 (VAPG), and the cancer target composition is accumulated in the cancer tissue due to the peptide.

The cancer targeting compound (TAMRA-GHEG-ECG-VAPG) designed as described above was synthesized in Peptron, Inc. (Daejeon, Korea) so that the purity exceeded 96%. Specifically, the cancer-screening compound was synthesized using Fmoc solid-phase peptide synthesis (Fomc-SPPS) with ASP48S (Peptron, Inc.).

In order to purify the synthesized cancer-labeled compounds, TAMRA-succinimidyl ester and diisopropylethylamine were treated with the peptide binding resin. The synthesized cancer labeled compounds were purified by reverse phase high performance liquid chromatography (RP-HPLC) with a Vydac Everest column (C28, 220 x 22 mm, 10 μm). The RP-HPLC used 0.1% aqueous solution of Trifluoroacetic acid (TFA) containing 0 to 80% acetonitrile for concentration gradient formation.

Liquid chromatography / mass spectrometry (LC / MS) was used to determine the molecular weight of the purified cancer labeled compounds.

The control group, the scrambled peptide (TAMRA-GHEG-ECG-PGVA) and the target peptide (VAPG) were also synthesized and purified as described above.

Synthesis and characterization results of cancer targeting compounds, scrambled peptides and target peptides are shown in Fig.

As shown in FIG. 1, it was confirmed that the synthesized cancer-inhibiting compound had a formula of C ₆₅ H ₈₂ N ₁₅ O ₂₀ S ₁ and a molecular weight of 1423.84. Also, it can be confirmed that the molecular weight of the Schrambd peptide is C ₆₅ H ₈₂ N ₁₅ O ₂₀ S ₁ and the molecular weight is 1423.84.

1-2 Radioactive isotopic labels for cancer-resistant compounds

The cancer labeled compound (TAMRA-GHEG-ECG-VAPG) prepared in Example 1-1 was labeled with radioactive isotope Tc-99m.

Specifically, 300 μl of cancer-labeled 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 solution was adjusted with 1 ml of Tc-99m and Tc-99m pertechnetate (approximately 1,110 MBq) and tin chloride (SnCl ₂ ) (1 mg / ml in nitrogen-purged 0.01 M HCl) Respectively. The calibrated solution was heated for 15 minutes and then cooled.

Purification of radioactive isotope labeled compounds was performed by radio-reverse phase high performance liquid chromatography (RP-HPLC). The radio-RP-HPLC used 0.1% Trifluoroacetic acid (TFA) aqueous solution containing 0 to 80% acetonitrile as a mobile phase for the formation of a concentration gradient, And eluted at a rate of 2 ml / min. An ultraviolet detector (220 nm) and a gamma radiodetector were used to monitor the eluted radioisotope labeled cancer labeled compounds.

Also, the control group, scrambled peptide (TAMRA-GHEG-ECG-PGVA), was labeled and purified with radioisotope as described above.

 (Tc-99m TAMRA-GHEG-ECG-VAPG) and a scrambled peptide (Tc-99m TAMRA-GHEG-ECG-PGVA) labeled with radioactive isotopes were obtained as a result of radioisotope labeling and purification. The retention time was 7.2 minutes for radiolabeled labeled compounds and 7.0 minutes for radiolabeled scrambled peptides in radio-RP-HPLC. In addition, the radioisotope labeled cancer labeled compounds were prepared with a yield of 96% or more and showed high stability in saline and serum.

Experimental Example  1. Tumor cell binding affinity of radioactive isotope labeled compounds

(Tc-99m TAMRA-GHEG-ECG-VAPG) and the radioisotope labeled scrambled peptide (Tc-99m TAMRA-GHEG-ECG-PGVA ), SW620, a colon cancer cell line, was used for cancer cell binding affinity measurement. The SW620 cell line was cultured in RPMI 1640 medium at 5% CO ₂ and 37 ° C. The RPMI 1640 medium contained 10% fetal bovine serum (FBS), 300 mg / l L-glutamine, 25 mM HEPES (4- hydroxyethyl) -1-iperazineethanesulfonic acid and 25 mM sodium hydrogencarbonate (NaHCO ₃ ) do.

Binding affinities of radioactive isotope labeled compounds and radioisotope labeled scrambled peptides to the SW620 cell line were determined by the saturation binding assay according to Wu, Chunying, et al. (2007).

Specifically, SW620 cells at a concentration of 1 × 10 ⁶ cells / ml were plated on a plate at a uniform cell density and then cultured overnight. The cultured SW620 cells were washed twice each with ice-cold binding buffer containing 25 mM HEPES and 1% bovine serum albumin for 5 minutes each. Subsequently, the washed SW620 cells were treated with a radioisotope labeled compound and a radioactive isotope labeled scrambled peptide, respectively (0 to 500 [mu] M), and cultured at 37 DEG C for 1 hour. SW620 cells were washed three times with cold binding buffer and then dissolved in 200 μl of lysis buffer. The cell-associated radioactivity of the dissolved SW620 cells was measured using a 1480 Wizard 3 gamma counter (PerkinElmer Life and Analytical Sciences, Wallingford, CT, US). The binding constant (K _d ) and the maximum number of binding sites (B _max ) were determined using a nonlinear regression model of GraphPad prism (Graphpad Prism, version 5.03, GraphPad Software, La Jolla, CA, USA) software.

The cancer cell binding affinity measurement result is shown in Fig.

As shown in FIG. 2, it was confirmed that the binding constant (K _d ) of the labeled compounds to be labeled was 16.8 ± 3.6 nM (maximum binding site number (B _max ) = 94.1 ± 4.6, FIG. 2A). In contrast, the binding constant (K _d ) of labeled scrambled peptides was 2364 ± 1071 nM (B _max = 2792 ± 1080, FIG. 2B), about 140 times the labeled cancer labeled compound (p <0.005). These results indicate that the cancer targeting compound containing the target peptide (VAPG) has a very high binding affinity for the SW620 cell line. It also suggests that the presence of the target peptide (VAPG) affects the binding affinity for the SW620 cell line.

Experimental Example  2. Cellular uptake and competitive effects of cancer-targeting compounds

Confocal microscopy was used to measure the cellular uptake of cancer labeled compounds (TAMRA-GHEG-ECG-VAPG) and scrambled peptide (TAMRA-GHEG-ECG-PGVA).

SW620 cells (1 × 10 ⁵ cells / well) were cultured on a cover slip slide (temperature: 37 ° C., 24 hours). After the medium of SW620 cells was removed, 500 μl of the serum-free medium containing 200 μM of the cancer-screening compound and the scrambled peptide (at 37 ° C. for 1 hour) was replaced with 500 μl of the serum-free medium. The cultured SW620 cells were washed three times with phosphate-buffered saline (PBS) to remove unbound peptides. The cover slip was placed on a slide containing a fluorescence mounting medium (Dako, Glostrup, Denmark). The confocal laser scanning microscope was a 100X oil immersion lens FV1200 confocal microscope (Olympus, Pittsburgh, PA, USA). The results of the cell uptake measurement are shown in FIG.

As shown in Fig. 3, strong fluorescence activity was confirmed in SW620 cells cultured with the cancer marking compound (Fig. 3A). In contrast, SW620 cells cultured with the smebrand peptide showed weak fluorescence activity (Fig. 3B). In addition, the TAMRA-DAPI activity ratio of the cancer-screening compound was 0.66 ± 0.34, which was significantly higher than that of the TAMRA-DAPI activity of the scrambled peptide (0.04 ± 0.01) (p <0.005).

Experimental Example  3. In vivo gamma camera of radioisotope labeled cancer labeled compounds in a tumor-bearing mouse model Imaging (Gamma camera imaging) and target Of peptide (VAPG)  Confirm Competitive Effect

Six-week-old female thymic nude mice (BALB / c nu / nu) with 16 to 18 g of mice were used. SW620 cells (1 × 10 ⁷ cells / 0.1 ml) suspended in the mice were subcutaneously inoculated on the left side of the chest of the back. After inoculation, the mice were maintained for about 14 days to prepare tumor-bearing mice. The vertical dimension of the tumor formed on the left side of the chest of the tumor bearing mice was measured as 400 to 550 mm ³ .

Using the tumor-bearing mouse, the competitive effect of the target peptide (VAPG) contained in the in vivo gamma camera imaging and cancer-screening compound of the radiolabeled cancer labeled compounds prepared in Example 1-2 was confirmed Respectively.

More specifically, in vivo imaging of tumor bearing mice was performed using a gamma camera (Vertex; ADAC Laboratories, Milpitas, Calif., USA) after intravenous injection of 55.5 MBq radioisotope labeled cancer labeled compounds into tumor bearing mice Respectively. The gamma camera images were acquired 1, 2, and 3 hours after the injection, and the acquisition times were 120 seconds. In the acquired gamma camera images, regions of interest (ROIs, 15 X 15 pixels) were painted on the tumor site of the chest wall. In addition, the region of interest for normal muscle uptake measurements was extracted from the left arm muscles. 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 rate was calculated.

In the same manner as described above, intravenous injection of a radioisotope labeled scrambled peptide intravenously injected mouse, radioactive isotope labeled cancer labeled compound (0.5 mM) and excess target peptide (10 mM) Gamma camera imaging of a tumor bearing mouse was performed.

Gamma camera imaging of radioisotope labeled cancer labeled compounds and radioisotope labeled scrambled peptides and the competitive effect of the target peptide (VAPG) are shown in Table 1, Figures 4 and 5.

Target (tumor) - non-target (normal muscle) absorption rate Labeled
Cancer labeled compound Labeled
Schrambled peptide Labeled
Cancer labeled compound
+ Excess of target peptide 1 hours 3.0 ± 0.5 1.6 ± 0.3 1.7 ± 0.6 2 hours 4.8 ± 0.4 1.8 ± 0.5 2.1 ± 0.6 3 hours 6.6 ± 0.6 2.1 ± 0.8 2.3 ± 0.7

As shown in Table 1, the target (tumor) - non-target (normal muscle) uptake rate of the radioisotope labeled cancer labeled compounds in tumor bearing mice increased with time, and the control group, radioisotope labeled scram Peptide &lt; / RTI &gt;

Also, as a result of confirming the competitive effect of the target peptide, it can be seen that the ratio of target (tumor) to non-target (normal muscle) absorption of the cancer targeting compound decreased when the excessive amount of the target peptide and the cancer targeting compound were injected together.

In addition, as shown in Figs. 4 and 5, it was confirmed that the mice showed the strongest activity in the kidney of tumor-bearing mice. This means that radiolabeled cancer labeled compounds are excreted primarily through the kidneys. In the tumor-bearing mouse, it is confirmed that the radioisotope-labeled cancer-labeled compound is accumulated in the tumor (SW620) in a large amount (FIGS. 4A and 5), and the radioisotope labeled scrambled peptide is significantly accumulated (Fig. 4B, Fig. 5) (p < 0.05).

In addition, as a result of confirming the competitive effect of the target peptide, it was confirmed that the cell uptake of cancer targeting compounds can be blocked by injecting an excessive amount of the target peptide and the cancer targeting compound (Fig. 5) (p < 0.05).

Experimental Example  4. In vitro &lt; RTI ID = 0.0 &gt; ex vivo ) Optical Imaging (Optical imaging) and postmortem study

After performing Experimental Examples 2 and 3, tumor (SW620) and organs were excised from tumor-bearing mice. Resected tumors and organs were imaged in vitro using an optical camera for TAMRA (FOBI-10BR, Neoscience, Suwon, Korea). The peak absorbance of the TAMRA is 565 nm and the peak emission is 580 nm. The exposure time for optical imaging was 2.5 seconds per image, and the acquired images were analyzed using dedicated software.

For post-hoc studies, tumor SW620 was divided into two sections after in vitro optical imaging. Each piece was randomly used for biodistribution studies or confocal microscopy analysis. The slices for confocal microscopy analysis were prepared by immediate freezing at the optimal cutting temperature embedding compound. The in vitro optical imaging results are shown in Fig.

As shown in FIG. 6, the fluorescence activity of TAMRA was high in the healthy organs (Lv, Liver) and kidney (Kd, Kidney). The fluorescence activity of the tumor-labeled compounds labeled with tumor SW620 was significantly higher than the labeled scrambled peptide.

Experimental Example  5. Biodistribution of cancer-screening compounds using tumor-bearing mouse models Biodistribution )

The organs of Experimental Example 4 were used to confirm the biodistribution of cancer target compounds. Specifically, the organ of Experimental Example 4 was a tumor-bearing mouse sacrificed 3 hours after administration. The organ of Experimental Example 4 was contained in a gamma counter tube previously measured. The radioactivity of each organ was measured with a gamma counter (1480 Wizard 3, PerkinElmer Life and Analytical Sciences). The counter per minute was decay corrected and the corrected result was expressed as a percentage of the administered dose per gram of tissue (% ID / g).

Table 2 shows the biodistribution values (% ID / g) of the cancer-screening compounds.

long time Dose per weight of tissue (% ID / g) (SD) Radioisotope Labeled cancer marking compound
(Tc-99m TAMRA-GHEG-ECG-VAPG) Radioisotope labeled scrambled peptide
(Tc-99m TAMRA-GHEG-ECG-PGVA) Lung (Lu) 1.12 (0.15) 0.95 (0.06) Heart (Heart, Hr) 0.31 (0.04) 0.32 (0.08) Blood 1.65 (0.11) 1.59 (0.33) Liver (Lv) 1.45 (0.10) 1.49 (0.36) Stomach, St, 0.53 (0.05) 0.45 (0.20) Colon (Co) 0.48 (0.04) 0.54 (0.34) Spleen, Sp 0.77 (0.10) 0.64 (0.21) Kidney, Kd 8.60 (2.44) 10.59 (2.57) Muscle (Mu) 0.22 (0.04) 0.19 (0.04) Tumor (Tomor, Tu) 1.37 (0.15) 0.34 (0.07)

As shown in Table 2, the kidney bivariate value was the highest when the radiolabeled cancer labeled compound and the radioisotope labeled scrambled peptide were administered. Other organs showed similar biodistribution values for radiolabeled cancer labeled compounds and radioisotope labeled scrambled peptides. However, the biodistribution value of the tumor (SW620) is about four times higher than that of the radiolabeled scrambled peptide labeled compound for radioactive isotope labeling.

Experimental Example  6. Immunohistochemical staining of tumor treated with cancer-inducing compound ( Immunohistochemistry  staining)

The frozen tumor of Experimental Example 4 (SW620) was used to immunohistochemically stain the tumor to which the cancer target compound was administered. Specifically, the frozen tumor of Experimental Example 4 is a tumor-bearing mouse sacrificed 3 hours after administration. Frozen tumor tissues were cut to a thickness of 10 mu m to prepare slices. The prepared sections were thoroughly dried and fixed on slides using 4% formaldehyde (in PBS). The slides were incubated with cold 100% methanol at 20 DEG C for 10 minutes. The slice fixed on the slide was then blocked with 5% goat serum at room temperature for 30 minutes. Blocked sections were treated with primary antibody (American hamster anti-integrin β3 antibody, 1: 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and incubated at room temperature for 1 hour. After incubation, the cells were 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 antibodies and incubated slides were confocal laser scanned with a Prolong® Gold Antifade Reagent containing DAPI (4 ', 6-diamidino-2-phenylindole, Invitrogen, Calsbad, CA, USA). The confocal laser scanning was performed with an FV1200 confocal microscope (Olympus) equipped with a 60x oil immersion lens. All acquired images were photographed at the same exposure time, and the brightness and contrast adjustments were the same. Immunohistochemistry staining results of the tumors to which the cancer targeting compound was administered are shown in FIG.

As shown in Fig. 7, strong fluorescence was detected in tumor cells treated with radiolabeled cancer marking compound. The detected fluorescence appears to be strongly correlated with the fluorescence activity of β ₃ , and low fluorescence was detected in tumor cells treated with radioisotope labeled scrambled peptides.

Taken together, the present inventors confirmed that the absorption rate and the biodistribution value of the radioisotope labeled cancer-screening compound when administered to cancer tissues were high. This means that a radiological image and a fluorescence image can be obtained together with the application of the compound labeled with a radioisotope, and thus the cancer-screening compound of the present invention can be used in various fields in the field of cancer diagnosis.

Having described specific portions of the present invention in detail, those skilled in the art will appreciate that these specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual 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 compounds of cancer and use thereof <130> 1.97P <160> 3 <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 <210> 3 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> cancer targeting ligand <400> 3 Val Ala Pro Gly   One

Claims (10)

Fluorescent material,
A chelating ligand bound to a radioactive isotope and represented by the amino acid sequence of SEQ ID NO: 2, and
A cancer-targeting compound comprising a cancer-targeting peptide represented by the amino acid sequence of SEQ ID NO: 3.
The method according to claim 1,
Wherein the fluorescent material is a luminescent molecule, a fluorescent protein, a metal ion, a complex compound, an organic dye, a conductor, a semiconductor, an insulator, a quantum dot or a proton.
The method according to claim 1,
The cancer is selected from the group consisting of 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, gastric cancer, thyroid cancer and uterine cancer &Lt; / RTI &gt;
The method according to claim 1,
Wherein said cancer targeting compound further comprises a spacer to reduce fluorescence perturbation of the target peptide.
5. The method of claim 4,
Wherein the spacer is represented by the amino acid sequence of SEQ ID NO: 1.
5. The method of claim 4,
Wherein the cancer-screening compound is arranged in the form of a structural formula (1).
[Structural formula 1]
Fluorescent-spacer-chelating ligand-target peptide
5. The method of claim 4,
The cancer-screening compound is a compound represented by the formula (1)
[Chemical Formula 1]

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

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