Oligopeptides targeting vegfr2 and uses thereof

Abstract

The application discloses an oligopeptide targeting VEGFR2 and application thereof, and belongs to the technical field of biological medicines. The oligopeptide targeting VEGFR2 is intercepted from ramucirumab, and the applicant finds that the intercepted oligopeptide is specifically targeted to VEGFR2, and a tracer prepared by using the oligopeptide has higher tumor uptake, has ideal distribution in the body, meets the imaging requirement of clinical nuclear medicine, and has the potential of diagnosing or treating diseases related to VEGFR2 expression.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically involving oligopeptides targeting VEGFR2 and their applications. Background Technology

[0002] Vascular endothelial growth factor receptor 2 (VEGFR2), also known as kinase insertion domain receptor (KDR) or fetal liver kinase-1 (Flk-1), is a transmembrane receptor tyrosine kinase that plays a crucial role in angiogenesis. Its basic structure includes an intracellular, transmembrane, and extracellular domains. The extracellular domain is primarily responsible for the specific binding of vascular endothelial growth factor (VEGF). VEGFR2 has biological functions such as promoting angiogenesis and regulating vascular permeability. As a core receptor for regulating angiogenesis, after VEGF binds to VEGFR2, it activates a series of downstream signaling pathways, promoting the proliferation, migration, and survival of vascular endothelial cells and guiding the formation of new blood vessels. Simultaneously, VEGFR2 activation also increases vascular permeability, allowing plasma proteins and other molecules to more easily leak out of blood vessels, providing nutrients and growth factors to tissues.

[0003] VEGFR2 is widely expressed on the surface of endothelial cells in tumor angiogenesis and plays a crucial role in tumor growth, invasion, and metastasis. During tumorigenesis and development, VEGF secreted by tumor cells activates VEGFR2, increasing tumor vascular permeability and facilitating tumor cell entry into the bloodstream and distant metastasis. Therefore, targeting VEGFR2 for tumor imaging and therapy is of great significance. For example, ramucirumab, approved by the FDA on April 21, 2014, targets VEGFR2 and inhibits ligand-stimulated VEGFR2 activation, thereby preventing the proliferation and migration of vascular endothelial cells.

[0004] Functional-anatomical fusion imaging is a technique that combines imaging information reflecting human physiological functions with imaging information displaying anatomical structures, aiming to provide more comprehensive and accurate information for medical diagnosis and research. SPECT / CT (Single Photon Emission Tomography / Computed Tomography) and PET / CT (Positron Emission Tomography / Computed Tomography) are two important functional-anatomical fusion imaging techniques in modern medical imaging, playing a significant role in tumor diagnosis, staging, efficacy evaluation, and follow-up. SPECT utilizes radiolabeled drugs as tracers. After being introduced into the human body, the tracers participate in specific physiological and biochemical processes and accumulate in corresponding organs or lesion sites. When the radionuclide decays, it emits gamma photons. The SPECT detector rotates around the body, acquiring these gamma photon signals from multiple angles. Through computer reconstruction algorithms, the acquired projection data is reconstructed into a tomographic image reflecting the distribution of the tracer within the body, thereby displaying the functional and metabolic information of organs or lesion sites. The latter uses a tracer labeled with a radioactive nuclide that emits positrons. Once inside the body, the tracer participates in metabolic processes, being absorbed in large quantities by metabolically active cells. When the positron emitter decays, it emits positrons, which annihilate with electrons in surrounding matter over a very short distance, producing a pair of gamma photons in opposite directions. PET detectors, using coincidence detection technology, simultaneously detect this pair of gamma photons, determining the location of the annihilation event. By collecting a large amount of annihilation event information, computer reconstruction algorithms generate functional images reflecting the tracer distribution, demonstrating the level of metabolic activity within the body. In nuclear medicine, this method, combining radioactive nuclides with biological carriers (such as antibodies, peptides, oligopeptides, or small molecule compounds), enables the aggregation of diagnostic or therapeutic nuclides at target sites, thereby achieving high-resolution imaging or specifically eliminating cancer cells.

[0005] In summary, radiolabeled VEGFR2-targeting tracers can achieve non-invasive imaging of solid tumors and kill tumor cells. However, current VEGFR2-targeting tracers generally have some problems, such as low tumor uptake and high liver uptake.

[0006] Therefore, there is a need for a tracer that can specifically bind to the tumor vascular marker VEGFR2 and has low uptake by non-target tissues to indicate tumor location and assist in clinical diagnosis. Summary of the Invention

[0007] 1. Purpose of the invention

[0008] The purpose of this application is to provide an oligopeptide targeting VEGFR2 and its application. This oligopeptide specifically binds to VEGFR2. A tracer (imaging compound) is prepared using this oligopeptide, comprising the VEGFR2-targeting oligopeptide and an imaging agent. The VEGFR2-targeting oligopeptide specifically binds to VEGFR2, while the imaging agent is used for tracking. This tracer has higher tumor uptake and ideal distribution in vivo, meeting the imaging needs of clinical nuclear medicine.

[0009] 2. Technical Solution

[0010] To achieve the aforementioned objectives, the technical solution adopted in this application is as follows:

[0011] In a first aspect, this application provides an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof, wherein the VEGFR2-targeting oligopeptide is selected from any of the following oligopeptides:

[0012] (1) Oligopeptide P1, amino acid sequence: ISSSSY (SEQ ID NO.1);

[0013] (2) Oligopeptide P2, amino acid sequence: RVTDAF (SEQ ID NO.2);

[0014] (3) Oligopeptide P3, amino acid sequence: GIDNWL (SEQ ID NO.3);

[0015] (4) Oligopeptide P4, amino acid sequence: QAKAFPP (SEQ ID NO.4);

[0016] As a further explanation of this application, flow cytometry results show that any one of the oligopeptides P1-P4 can specifically bind to human umbilical vein endothelial cells (HUVECs) that highly express VEGFR, especially oligopeptide P4, which has a higher affinity.

[0017] Preferably, the oligopeptide targeting VEGFR2 is selected from oligopeptide P1.

[0018] Preferably, the oligopeptide targeting VEGFR2 is selected from oligopeptide P2.

[0019] Preferably, the oligopeptide targeting VEGFR2 is selected from oligopeptide P3.

[0020] Preferably, the oligopeptide targeting VEGFR2 is selected from oligopeptide P4.

[0021] Secondly, this application also provides a chelate precursor oligopeptide targeting VEGFR2, the chelate precursor oligopeptide comprising the above-mentioned VEGFR2-targeting oligopeptide or its pharmaceutically acceptable salt, and a molecular structural unit with the ability to chelate metal ions linked thereto.

[0022] Preferably, the molecular structural unit is selected from any one of HYNIC (hydrazine nicotinamide), DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), NOTA (2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid), DTPA (diethylenetriaminepentaacetic acid), or a derivative thereof.

[0023] Preferably, the molecular structural units are selected from derivatives of HYNIC.

[0024] Preferably, the structure of the HYNIC derivative is as shown in formula (1).

[0025] In equation (1), P represents an oligopeptide targeting VEGFR2.

[0026] Preferably, the aforementioned molecular structural unit is linked to the N-terminus of the peptide chain of the aforementioned VEGFR2-targeting oligopeptide or a pharmaceutically acceptable salt thereof.

[0027] Thirdly, this application provides an imaging compound targeting VEGFR2, the imaging compound comprising the aforementioned VEGFR2-targeting oligopeptide or a pharmaceutically acceptable salt thereof, and an imaging agent bound thereto, the imaging agent being selected from fluorescent groups or radionuclides.

[0028] Preferably, the imaging agent is selected from fluorescent groups, which are selected from any one or more of ICG (indocyanine green), IRDye800, Cy7 (cyanine dye 7), Cy5.5 (cyanine dye 5.5), Cy5 (cyanine dye 5), Cy3 (cyanine dye 3), FITC (fluorescein isothiocyanate) and rhodamine.

[0029] Preferably, the imaging agent is selected from fluorescent groups, and the fluorescent group is selected from FITC.

[0030] Preferably, the imaging agent is selected from radionuclides.

[0031] Preferably, the radionuclides mentioned above are selected from... 99m Tc, 18 F, 64 Cu、 67 Ga、 90 Y、 111 In、 177 Lu or 125 I.

[0032] Preferably, the radionuclides mentioned above are selected from... 99m Tc.

[0033] Preferably, the aforementioned radionuclide binds to an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof via a chelating group.

[0034] Preferably, the chelating group is selected from HYNIC (hydrazine nicotinamide), HYNIC / EDDA (hydrazine nicotinamide / 1,2-ethylenediamine-diacetic acid), HYNIC / TPPTS / Tricine (hydrazine nicotinamide / sodium triphenylphosphine tri-methylsulfonate / trihydroxymethylglycine), HYNIC / (Tricine)2 (hydrazine nicotinamide / trihydroxymethylglycine) or derivatives thereof.

[0035] Preferably, the chelating group is selected from HYNIC / EDDA, ​​and the structure of the imaging compound targeting VEGFR2 is shown in formula (2):

[0036] Equation (2); where P represents any one of the oligopeptides P1-P4.

[0037] Preferably, the chelating group is selected from HYNIC / TPPTS / Tricine, and the structure of the imaging compound targeting VEGFR2 is shown in formula (3):

[0038] Equation (3); where P represents any one of the oligopeptides P1-P4.

[0039] Preferably, the chelating group is selected from HYNIC / (Tricine)2, and the structure of the imaging compound targeting VEGFR2 is shown in formula (4):

[0040] Equation (4); where P represents any one of the oligopeptides P1-P4.

[0041] Preferably, the chelating group is linked to the N-terminus of the peptide chain of the above-mentioned VEGFR2-targeting oligopeptide or its pharmaceutically acceptable salt.

[0042] Fourthly, this application provides the use of the above-mentioned VEGFR2-targeting oligopeptides or their pharmaceutically acceptable salts, the above-mentioned VEGFR2-targeting chelate precursor oligopeptides, or the above-mentioned VEGFR2-targeting imaging compounds in the preparation of diagnostic tumor tracers and / or drugs for the prevention and / or treatment of tumors; as a further explanation of this application, such as radionuclide-labeled tracers, non-invasive imaging can be achieved; at the same time, the radiation emitted by the radionuclide can kill tumor cells, that is, it can simultaneously have diagnostic and therapeutic functions.

[0043] Preferably, the tumor is a tumor that highly expresses VEGFR2.

[0044] Preferably, the tumors are selected from any one or more of the following: breast cancer, renal cell carcinoma, liver cancer, colorectal cancer, non-small cell lung cancer, ovarian cancer, glioblastoma, pancreatic cancer, gastric cancer, and thyroid cancer, and all of the above tumors highly express VEGFR2.

[0045] Fifthly, this application also provides a tumor tracer targeting VEGFR2, including the aforementioned imaging compound targeting VEGFR2.

[0046] Sixthly, this application also provides a kit for detecting vascular cells or VEGFR2 protein on the surface of solid tumors, the kit comprising the aforementioned tumor tracer targeting VEGFR2.

[0047] In a seventh aspect, this application also provides a pharmaceutical composition for the prevention or treatment of tumors targeting VEGFR2, the pharmaceutical composition comprising the above-mentioned VEGFR2-targeting oligopeptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier thereof.

[0048] Preferably, the pharmaceutical composition further includes a tumor-killing agent that binds to an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof.

[0049] Preferably, the above-mentioned reagents for killing tumor cells are selected from any one or more of cyclophosphamide, cisplatin, methotrexate, 5-fluorouracil, camptothecin, doxorubicin, vincristine, docetaxel, capecitabine, colchicine, mitomycin, teniposide, and doxorubicin.

[0050] Preferably, the above-mentioned agent for killing tumor cells is selected from radionuclides that bind to an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof via a chelating group.

[0051] Preferably, the radionuclides mentioned above are selected from... 99m Tc, 18 F, 64 Cu、 67 Ga、 90 Y、 111 In、 177 Lu or 125 I.

[0052] Preferably, the radionuclides mentioned above are selected from... 99m Tc.

[0053] Preferably, the chelating group is selected from HYNIC (hydrazine nicotinamide), HYNIC / EDDA (hydrazine nicotinamide / 1,2-ethylenediamine-diacetic acid), HYNIC / TPPTS / Tricine (hydrazine nicotinamide / sodium triphenylphosphine tri-methylsulfonate / trihydroxymethylglycine), HYNIC / (Tricine)2 (hydrazine nicotinamide / trihydroxymethylglycine) or derivatives thereof.

[0054] Preferably, the chelating group is linked to the N-terminus of the peptide chain of the above-mentioned VEGFR2-targeting oligopeptide or its pharmaceutically acceptable salt.

[0055] Eighthly, this application also provides a method for diagnosing tumors, including administering the above-mentioned tumor tracer targeting VEGFR2 or imaging compound targeting VEGFR2 to a subject.

[0056] Ninthly, this application also provides a method for treating tumors, including administering the above-described pharmaceutical composition for the prevention or treatment of tumors targeting VEGFR2 to a subject.

[0057] 3. Beneficial effects

[0058] Compared with the prior art, the advantages of this application are as follows:

[0059] (1) The oligopeptides P1-P4 provided in this application are derived from ramucirumab. Through the applicant's research, it was found that any one of the oligopeptides can specifically bind to human umbilical vein endothelial cells that highly express VEGFR, indicating that it can specifically recognize the expression of VEGFR2 in tumor tissue. In particular, oligopeptide P4 has a higher affinity.

[0060] (2) The oligopeptides P1-P4 provided in this application are oligopeptides composed of 6-7 amino acids. Compared with monoclonal antibodies, they are smaller in size and have a lower molecular weight, enabling them to quickly penetrate tissues and clear background signals in the blood. When used as carriers to conjugate imaging agents to prepare tracers or to conjugate with agents that kill tumor cells to prepare drugs, they have high tumor affinity, higher tumor uptake, ideal in vivo distribution, reduced radiation exposure to normal tissues, and improved signal-to-noise ratio of target lesions, meeting the imaging needs of clinical nuclear medicine. They can be used for tumor screening and early diagnosis, and can also be used for real-time non-invasive in situ monitoring of early malignant tumors and treatment. At the same time, the applicant's research found that, with the same ramucirumab, as the number of amino acids decreased to 4 (P5), the tumor uptake of the tracer in vivo decreased sharply, i.e., the affinity decreased; while as the number increased (P6), the metabolic characteristics of the tracer in vivo changed, mainly being taken up in the liver. Further optimization of the number of peptides and the hydrophilic-lipophilic properties of oligopeptides is needed.

[0061] (3) The imaging compound targeting VEGFR2 provided in this application has good biocompatibility, can be used safely in vivo and is metabolized rapidly, reducing the risk of toxicity and adverse reactions, and is more suitable for clinical application in nuclear medicine. Attached Figure Description

[0062] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0063] Figure 1 This is a mass spectrometry (MS) characterization of oligopeptide P1.

[0064] Figure 2 This is a mass spectrometry (MS) characterization of oligopeptide P2.

[0065] Figure 3 This is a mass spectrometry (MS) characterization of oligopeptide P3.

[0066] Figure 4 This is a mass spectrometry (MS) characterization of oligopeptide P4.

[0067] Figure 5 This is the mass spectrometry (MS) characterization of HYNIC-P1.

[0068] Figure 6 This is the mass spectrometry (MS) characterization of HYNIC-P2.

[0069] Figure 7 This is the mass spectrometry (MS) characterization of HYNIC-P3.

[0070] Figure 8 This is the mass spectrometry (MS) characterization of HYNIC-P4.

[0071] Figure 9 The results show the screening results for oligopeptide affinity.

[0072] Figure 10 for 99m SPECT imaging results of Tc-HYNIC-P1 hepatocellular carcinoma HepG2 mice.

[0073] Figure 11 for 99m SPECT imaging results of Tc-HYNIC-P2 hepatocellular carcinoma HepG2 mice.

[0074] Figure 12 for 99mSPECT imaging results of Tc-HYNIC-P3 hepatocellular carcinoma HepG2 mice.

[0075] Figure 13 for 99m SPECT imaging results of Tc-HYNIC-P4 hepatocellular carcinoma HepG2 mice.

[0076] Figure 14 for 99m SPECT imaging results of Tc-HYNIC-P4 mice with colorectal cancer HCT116, where P represents a side view.

[0077] Figure 15 for 99m SPECT imaging results of Tc-HYNIC-P4 lung cancer A549 mice, where P represents a side view.

[0078] Figure 16 for 99m SPECT imaging results of Tc-HYNIC-P4 breast cancer MDA-MB-231 mice, where P represents a side view.

[0079] Figure 17 for 99m SPECT imaging results of Tc-HYNIC-P5 hepatocellular carcinoma HepG2 mice.

[0080] Figure 18 for 99m SPECT imaging results of Tc-HYNIC-P6 hepatocellular carcinoma HepG2 mice. Detailed Implementation

[0081] The present application will be further described below with reference to specific embodiments.

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0083] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0084] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0085] As used herein, the term “at least one of…” is intended to be synonymous with “one or more of…”. For example, “at least one of A, B, and C” explicitly includes only A, only B, only C, and combinations thereof.

[0086] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0087]

Material

[0088] The amino acids, Rink Amide resin, and synthetic reagents used in this application were purchased from Bid Pharmaceutical (Shanghai) Co., Ltd. and Shanghai McLean Biotechnology Co., Ltd., respectively. HUVECs were existing cell lines in the laboratory, and the DMEM culture medium used was purchased from Jiangsu Kaiji Biotechnology Co., Ltd. For details, please refer to Table 1.

[0089] Table 1 Reagent List

[0090]

[0091] As used in this application, unless otherwise specified, a radioactive nuclide, also known as an "unstable nuclide," refers to an unstable atomic nucleus that is radioactive and can spontaneously emit radiation (such as alpha rays, beta rays, gamma rays, etc.) and decay into a stable nuclide.

[0092] As used in this application, unless otherwise specified, "targeted" means specific binding, such as "targeting VEGFR2" means being able to specifically bind to VEGFR2.

[0093] As used in this application, unless otherwise specified, "oligopeptide" refers to a peptide containing 2 to 20 amino acid residues.

[0094] As used in this application, unless otherwise specified, "chelate precursor oligopeptide" refers to an oligopeptide that binds a molecular structural unit (such as DOTA, NOTA, EDTA, etc.) capable of chelating metal ions. This molecular structural unit capable of chelating metal ions can be attached to the oligopeptide chain by chemical modification or coupling. "Chelate precursor oligopeptide" provides the basic structure for the chelation of metal ions.

[0095] As used in this application, unless otherwise specified, oligopeptide (P or Pn) refers to any one of oligopeptide P1, oligopeptide P2, oligopeptide P3, and oligopeptide P4, and those skilled in the art can accurately understand the meaning of P in context. Furthermore, unless otherwise specified, P-1, P1, and oligopeptide P1 have the same meaning and all refer to oligopeptide P1.

[0096] As used herein, unless otherwise specified, Fmoc refers to 9-fluorenylmethoxycarbonyl, a commonly used amino protecting group. During peptide synthesis, the amino group of amino acids needs to be protected to prevent it from participating in reactions at inappropriate times. 9-fluorenylmethoxycarbonyl can form a stable carbamate bond with the amino group of amino acids, thus protecting the amino group. In subsequent synthetic steps, this protecting group can be selectively removed under mild conditions, restoring the amino group's reactivity and allowing it to participate in peptide bond formation. For example, Fmoc-glycine refers to fluorenylmethoxycarbonyl-glycine.

[0097] As used herein, unless otherwise specified, the Fmoc-SPPS method (9-Fluorenylmethyloxycarbonyl-Solid-Phase Peptide Synthesis) is a widely used method in the field of peptide synthesis. This application does not limit the method of preparing oligopeptides; they can also be obtained through recombinant expression using synthetic biology methods. As an example, this application provides the Fmoc-SPPS method for preparing the oligopeptides described in this application.

[0098] As used in this application, unless otherwise specified, 1.0 eq means "1.0 equivalent".

[0099] As used in this application, unless otherwise specified, MBq is the unit of radioactivity, or megabecquerel. Radioactivity refers to the number of atoms of a radioactive element or isotope decaying per second. One becquerel (Bq) is defined as the number of nuclear decays of a radionuclide that occurs once per second.

[0100] Example 1

[0101] This embodiment provides an oligopeptide targeting VEGFR2 and its preparation.

[0102] In this embodiment, four oligopeptides (P) targeting VEGFR2 are provided:

[0103] Oligopeptide P1: ISSSSY (SEQ ID NO.1);

[0104] Oligopeptide P2: RVTDAF (SEQ ID NO.2);

[0105] Oligopeptide P3: GIDNWL (SEQ ID NO.3);

[0106] Oligopeptide P4: QAKAFPP (SEQ ID NO. 4).

[0107] In this embodiment, the oligopeptide was prepared by the conventional Fmoc-SPPS method, specifically including:

[0108] (1) Resin activation: Take 300 mg of Rink Aimde resin (0.1 mmol equivalent) into a peptide synthesis tube, add 5 mL of dichloromethane (DCM), shake in a shaker for 20 min, and then dry.

[0109] (2) Deprotection: Take 10 mL of 20% (v / v) morpholine / DMF solution, add it to the polypeptide synthesis tube, shake for 10 min, dry, wash twice with DMF, and repeat twice;

[0110] (3) Extend:

[0111] i) Take Fmoc-L-amino acids (0.5 mmol), 310 mg HCTU (0.75 mmol), and 270 μL DIPEA (1.5 mmol), dissolve them in 10 mL DMF, add them to the peptide synthesis tube, shake for 1.5 h; dry, and wash twice with DMF;

[0112] ii) Take 10 mL of 20% (v / v) morpholine / DMF solution (v / v), add it to the polypeptide synthesis tube, shake for 10 min, dry, wash twice with DMF, and repeat twice.

[0113] iii) Repeat steps i) and ii) until the last amino acid has reacted completely;

[0114] (4) Cutting: Take 10 mL of TFA:TIPS:H2O (95:2.5:2.5) cutting solution, add it to the peptide synthesis tube, and shake for 120 min;

[0115] (5) Purification: Filter the cutting solution into a centrifuge tube and blow TFA to a volume of less than 1 mL using an air pump; add 10 times the volume of methyl tert-butyl ether, mix thoroughly, centrifuge at 4000 rpm for 10 min, and discard the supernatant; add methyl tert-butyl ether again, mix well, centrifuge, and discard the supernatant; wash twice with ethyl acetate using the same method; dry the polypeptide under vacuum for 1 h to obtain a white powdery crude oligopeptide.

[0116] The crude product was dissolved in 2 mL of DMSO and purified by preparative C18 reversed-phase high-performance liquid chromatography (Agilent Preparative Liquid Chromatography (1260II); column (410910-102); eluent: 0 min (20% A: acetonitrile; 80% B: 0.1% trifluoroacetic acid water), 35 min (80% A: acetonitrile; 20% B: 0.1% trifluoroacetic acid water)). The collected solution was freeze-dried to obtain a white solid, which was the pure oligopeptide (P). The molecular weight was confirmed by mass spectrometry (Agilent 1260II-6125B).

[0117] The results are as follows Figures 1-4 As shown, all the measured mass spectrometry data are [M+H] for P1-P4. + The peak indicates that the oligopeptide P1-P4 was successfully synthesized.

[0118] Example 2

[0119] This embodiment uses flow cytometry to verify the binding of oligopeptides to VEGFR.

[0120] In this embodiment, the binding of fluorescently labeled oligopeptides to the HUVECs cell line was determined by flow cytometry. Oligopeptides with stronger affinity exhibited higher average fluorescence intensity; that is, higher average fluorescence intensity indicated stronger affinity.

[0121] The specific method is as follows:

[0122] 1) Oligopeptides (Pn) were fluorescently labeled with FITC. Taking P-1 as an example, P-1 (15 mg, 20.6 μM, 1.0 eq) was dissolved in 1 mL of DMSO, followed by the addition of FITC (13.6 mg, 30.9 μM, 1.5 eq) and DIPEA (6.64 mg, 51.5 μM, 2.5 eq). After reacting at room temperature in the dark for 1 h, the mixture was purified by preparative liquid chromatography, and the target fraction was lyophilized to obtain FITC-P-1 (8 mg, pale yellow powder). The target molecular weight was confirmed by mass spectrometry. The labeling of other peptides followed the same method as the synthesis of FITC-P-1.

[0123] 2) Human umbilical vein endothelial cells (HUVECs) with high VEGFR expression were used at a concentration of 1.0 × 10⁻⁶. 5 Cells were seeded in 24-well plates and cultured at 37°C with 5% CO2 for 24 h.

[0124] 3) Remove the culture medium and incubate with DMEM medium containing 20 μM FITC-Pn drug for 2 h.

[0125] 4) After 2 hours, digest the cells, wash them twice with PBS, and then analyze the average fluorescence intensity of each sample by flow cytometry.

[0126] Results analysis:

[0127] The fluorescence intensity of each sample is as follows Figure 9 As shown, compared with the pure dye FITC and Control groups (without added dye), FITC-P-1 to FITC-P-4 have good cell binding ability in HUVECs cells, that is, FITC-P-1 to FITC-P-4 specifically bind to HUVECs cells with high VEGFR expression, among which FITC-P-4 has relatively higher affinity.

[0128] Example 3

[0129] This embodiment provides a chelate precursor oligopeptide targeting VEGFR2 and its preparation.

[0130] VEGFR2-targeting chelate precursor oligopeptides include an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof, and a molecular structural unit linked thereto with the ability to chelate metal ions.

[0131] In this embodiment, the oligopeptide targeting VEGFR2 is selected from P1-P4, a derivative of the molecular structural unit HYNIC with the ability to chelate metal ions, and its structural formula is as follows:

[0132] .

[0133] Its preparation methods include:

[0134] The oligopeptide (2 μM) and HYINC-NHS (3 μM) were dissolved in 300 μL DMSO, and 1 μL DIPEA was added. The reaction was carried out at 37°C for 5 h. After the reaction, the product was purified by preparative C18 reversed-phase high-performance liquid chromatography (same as in Example 1). The collected solution was freeze-dried to obtain a yellow solid, which was the pure chelate precursor oligopeptide (HYNIC-P). The molecular weight was confirmed by mass spectrometry.

[0135] Table 2 Mass Spectrometry Characterization

[0136]

[0137] The results are as follows Figures 5-8 As shown in Table 2, all measured mass spectrometry data are for HYINC-P1 to HYINC-P4 [M+H]. + The peak indicates that the synthesis of HYINC-P1~HYINC-P4 was successful.

[0138] Example 4

[0139] This embodiment provides imaging compounds targeting VEGFR2 and their preparation.

[0140] The imaging compound includes an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof, and an imaging agent bound thereto; the imaging agent is selected from radionuclides and binds to the oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof via a chelating group.

[0141] In this embodiment, the oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof is selected from P1-P4, and the imaging agent is selected from radionuclides. 99m Tc binds to oligopeptides targeting VEGFR2 via chelating groups HYNIC / TPPTS / Tricine.

[0142] The general structural formula of imaging compounds is:

[0143] .

[0144] Preparation method (VEGFR2-targeting chelate precursor oligopeptide) 99m The Tc tagging process includes the following steps:

[0145] The chelate precursor oligopeptide (HYINC-P) was dissolved in DMSO to prepare a 1 nmol / μL solution;

[0146] Add 10 μg TPPTS, 13 μg Tricine, and 2 nmol of chelate precursor oligopeptide to a vial, and dilute to 200 μL with physiological saline to prepare a reaction solution. Heat the reaction solution in a 100℃ metal bath for 20 min to obtain the imaging compound targeting VEGFR2. 99m Tc-HYNIC-P).

[0147] Example 5

[0148] This embodiment provides SPECT (single-photon emission computed tomography) verification of the imaging compound targeting VEGFR2 prepared in Example 4 in tumor-bearing mice.

[0149] The imaging compound (probe) targeting VEGFR2 prepared in Example 4 was used. 99m Tc-HYNIC-P was prepared as a physiological saline solution and injected via tail vein (11 MBq) into the tail veins of nude mice bearing HepG-2 hepatocellular carcinoma, HCT116 colorectal cancer, A549 non-small cell lung cancer, and MDA-MB-231 breast cancer. SPECT signal acquisition was performed 2 h after administration.

[0150] The results are as follows Figure 10-16 As shown, the probe 99m Tc-HYNIC-P1~99mTc-HYNIC-P4 exhibited good tumor-targeting ability in HepG-2 tumor-bearing nude mice with hepatocellular carcinoma, with significant uptake at the tumor site. 99m Tc-HYNIC-P1 ( Figure 10 )and 99m Tc-HYNIC-P2 ( Figure 11 There was a relatively obvious kidney signal, and the probe... 99m Tc-HYNIC-P3 ( Figure 12 Partial intestinal uptake, probe 99m Tc-HYNIC-P4 ( Figure 13 The probe showed significant tumor uptake and bladder signal, indicating that the probe... 99m Tc-HYNIC-P1~99mTc-HYNIC-P4 shows promising diagnostic potential in liver cancer and has the potential for further development.

[0151] Further research revealed probes 99m Tc-HYNIC-P4 exhibited significant tumor uptake and intestinal uptake in breast cancer MDA-MB-231 and colorectal cancer HCT116 tumor-bearing mice, while it showed good tumor uptake and metabolic characteristics in non-small cell lung cancer A549 tumor-bearing mice. It has the potential to be further developed and applied to clinical tumor diagnosis, improve the technology for early diagnosis, early screening and early detection of tumor patients, provide a basis for patient treatment, and thus improve their effective survival.

[0152] Comparative Example

[0153] This comparative example provides a comparison of SPECT imaging in tumor-bearing mice of imaging compounds prepared from oligopeptides truncated or extended from the oligopeptides screened in this application.

[0154] In this embodiment, the following is provided:

[0155] Oligopeptide P5:RVTD (SEQ ID NO.5) is an oligopeptide consisting of 4 amino acid segments truncated from oligopeptide P2;

[0156] Oligopeptide P6: QAKAFPPSSY (SEQ ID NO.6) is an oligopeptide that extends the length of oligopeptide P4 by 3 amino acids.

[0157] The imaging compound obtained is: 99m Tc-HYNIC-P5 and 99m Tc-HYNIC-P6.

[0158] Referring to Example 5, the animal experiment results are as follows: Figure 17 and 18 As shown, the probe 99m After a reduction in Tc-HYNIC-P5 amino acid levels, there is virtually no tumor uptake, but abundant signaling is observed in the kidneys and bladder; probe 99m Tc-HYNIC-P6 showed increased liver uptake with increasing amino acid content, while tumor uptake was weaker. These results indicate that an appropriate amino acid content can regulate tumor uptake and in vivo metabolism of the probe.

Claims

1. An oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof, characterized in that, The oligopeptide targeting VEGFR2 is oligopeptide P4, with the amino acid sequence QAKAFPP.

2. A chelate precursor oligopeptide targeting VEGFR2, characterized in that, The chelate precursor oligopeptide comprises the VEGFR2-targeting oligopeptide of claim 1 or a pharmaceutically acceptable salt thereof, and a molecular structural unit connected thereto with the ability to chelate metal ions. The molecular structural units are selected from HYNIC, DOTA, NOA, DTPA, or compounds with structural units as shown in formula (1): Equation (1).

3. An imaging compound targeting VEGFR2, characterized in that, The imaging compound targeting VEGFR2 comprises the VEGFR2-targeting oligopeptide of claim 1 or a pharmaceutically acceptable salt thereof, and an imaging agent bound thereto; the imaging agent is selected from fluorescent groups or radionuclides.

4. The imaging compound targeting VEGFR2 according to claim 3, characterized in that, The imaging agent is selected from fluorescent groups; the fluorescent group is selected from any one or more of ICG, IRDye800, Cy7, Cy5.5, Cy5, Cy3, FITC and rhodamine.

5. The imaging compound targeting VEGFR2 according to claim 4, characterized in that, The fluorescent group is selected from FITC.

6. The imaging compound targeting VEGFR2 according to claim 3, characterized in that, The imaging agent is selected from radionuclides; the radionuclides are selected from... 99m Tc, 18 F, 64 Cu、 67 Ga、 90 Y、 111 In、 177 Lu or 125 I.

7. The imaging compound targeting VEGFR2 according to claim 6, characterized in that, The radionuclides are selected from 99m Tc.

8. The imaging compound targeting VEGFR2 according to claim 6 or 7, characterized in that, The radionuclide binds to an oligopeptide targeting VEGFR2 or a pharmaceutically acceptable salt thereof via a chelating group.

9. The imaging compound targeting VEGFR2 according to claim 8, characterized in that, The chelating group is selected from HYNIC, HYNIC / EDDA, ​​HYNIC / TPPTS / Tricine, and HYNIC / (Tricine)2.

10. The imaging compound targeting VEGFR2 according to claim 9, characterized in that, The chelating group is selected from HYNIC / TPPTS / Tricine.

11. The use of the VEGFR2-targeting oligopeptide of claim 1 or a pharmaceutically acceptable salt thereof, the VEGFR2-targeting chelate precursor oligopeptide of claim 2, or the VEGFR2-targeting imaging compound of any one of claims 3-10 in the preparation of a diagnostic tumor tracer; wherein the tumor is a tumor that highly expresses VEGFR2; and the tumor is selected from any one or more of breast cancer, liver cancer, colorectal cancer, and non-small cell lung cancer.

12. A tumor tracer targeting VEGFR2, characterized in that, The tumor tracer comprises the imaging compound targeting VEGFR2 as described in any one of claims 3-10; the tumor is a tumor that highly expresses VEGFR2; the tumor is selected from any one or more of breast cancer, liver cancer, colorectal cancer, and non-small cell lung cancer.

13. A kit for detecting vascular cells or VEGFR2 protein on the surface of solid tumors, characterized in that, The kit includes the VEGFR2-targeting tumor tracer of claim 12; the solid tumor is a tumor that highly expresses VEGFR2; the solid tumor is selected from any one or more of breast cancer, liver cancer, colorectal cancer, and non-small cell lung cancer.

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