A CXCR4-targeting polypeptide and its application

Abstract

The present invention belongs to the field of medicine, and particularly relates to a CXCR4-targeting polypeptide and its application. The present invention first provides a CXCR4-targeting polypeptide, which is selected from any one of SEQ ID NO.1-10; then provides a molecular probe, which is composed of a fluorescent label or a radionuclide conjugated to the above-mentioned CXCR4-targeting polypeptide; finally, it also provides the use of any one of the above-mentioned CXCR4-targeting polypeptides and any one of the above-mentioned molecular probes in the preparation of reagents for cancer diagnosis or treatment. The CXCR4-targeting polypeptide provided by the present invention can specifically target CXCR4, conjugate with a fluorescent label or a radionuclide, and achieve high-sensitivity in vivo imaging of tumors; it can be used for tumor screening and early diagnosis, and can also realize the treatment monitoring of tumors. The polypeptide of the present invention has good biocompatibility and safety. The polypeptides of the present invention are all reported for the first time, and are easy to obtain and convenient to promote.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a CXCR4-targeting polypeptide and its application. Background Art

[0002] Malignant tumors have become one of the main causes of abnormal deaths of humans worldwide, posing a great threat to human health. Traditional tumor treatment methods mainly include surgery, chemotherapy, radiotherapy, etc. However, traditional treatment methods usually bring great toxic side effects to patients and increase the burden on patients.

[0003] Tumor targeted therapy is a method to inhibit tumor growth by interfering with molecular markers related to the occurrence, deterioration and metastasis of tumor cells at the cellular and molecular levels.

[0004] A treatment method that inhibits tumor progression by interfering with key molecules involved in the occurrence, development and spread of tumor cells. Compared with traditional treatment means, tumor targeted therapy has high selectivity for tumor cells, good curative effect and small side effects, and has good application prospects in tumor treatment. Currently, researchers are also developing various tumor targeted therapies. Among various targeted therapies, using targeted polypeptides for treatment is a relatively ideal means, which has the following advantages: 1) It has good penetrability and is easily taken up by tumor cells; 2) It has a fast plasma clearance rate, high selectivity, high affinity, and the binding time can be long or short. 3) It is easy to chemically synthesize and has low immunogenicity, which can avoid the deficiencies of monoclonal antibody therapy. Therefore, specific targeted polypeptides are ideal and attractive targets for medical research, clinical application and molecular imaging.

[0005] Chemokines are proteins with relatively low molecular weights (8,000 - 10,000) belonging to the cytokine superfamily, and are key regulatory factors for cell migration during development, homeostasis and immune surveillance. Chemokines are involved in various cancer development processes, such as angiogenesis, cancer cell proliferation, invasion and metastasis, and are key determinants of disease progression. Chemokine 12 (CXCL12), also known as stromal cell-derived factor-1 (SDF-1), belongs to the chemokine protein family.

[0006] CXCR4 (CXC motif chemokine receptor 4), also known as fusin or CD184, is a seven-transmembrane G protein-coupled receptor (GPCR), which is activated after the extracellular region binds to the ligand. CXCR4 is overexpressed in more than 20 types of human tumors, including ovarian cancer, prostate cancer, esophageal cancer, lung cancer, melanoma, neuroblastoma, renal cell carcinoma, etc.

[0007] CXCR4 is a specific receptor for CXCL12. After their binding, they are involved in embryonic growth and development processes such as embryonic hematopoiesis, organogenesis, and angiogenesis, as well as a series of physiological and pathological processes such as inflammation and tissue immune surveillance. Activation of the CXCL12 / CXCR4 signaling pathway can lead to a series of biological processes such as tumor proliferation, angiogenesis, invasion and migration, and tumor drug resistance.

[0008] The existing detection methods are mainly immunohistochemistry (IHC) detection. However, due to the temporal and spatial heterogeneity of tumors, IHC detection has fundamental limitations and cannot perform dynamic detection of tumors.

[0009] Relatively speaking, molecular imaging can perform personalized diagnosis and treatment of tumors. After the molecular probe enters the body, it binds to specific markers, and signal conversion is performed through specific imaging devices in vitro, such as positron emission tomography (PET-CT), single photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and fluorescence imaging (FL), etc. for acquisition and imaging.

[0010] Based on the above considerations, the applicant has designed a novel class of CXCR4-targeted polypeptides, which have high selectivity and affinity for CXCR4. By conjugating with fluorescent dyes and using a molecular imaging surgical navigation device, precise resection of tumors can be achieved during surgery; further, the CXCR4-targeted polypeptides can also be used for radionuclide imaging by conjugating with radionuclides to achieve the purpose of early precise diagnosis and treatment of tumors. Summary of the Invention

[0011] To solve the problems existing in the prior art, the present invention provides a CXCR4-targeted polypeptide, a molecular probe and their applications.

[0012] The technical solution adopted by the present invention to solve its technical problems is:

[0013] In the first aspect, the present invention claims a CXCR4-targeted polypeptide, and the CXCR4-targeted polypeptide is selected from any one of the following amino acid sequences:

[0014] YQ-X-1: G-G-P-Y-R-R-G-R-G-P, as shown in SEQ ID NO.1;

[0015] YQ-X-2: G-G-P-A-F-X-S-R-G-P, as shown in SEQ ID NO.2;

[0016] YQ-X-3: G-G-G-H-R-R-G-R-G-P, as shown in SEQ ID NO.3;

[0017] YQ-X-4: P-Y-R-X-S-R-G-P, as shown in SEQ ID NO.4;

[0018] YQ-X-5: G-G-P-Y-R-R-P-R-G-P, as shown in SEQ ID NO.5;

[0019] YQ-X-6: G-G-P-Y-R-V-G-R-W-P, as shown in SEQ ID NO.6;

[0020] YQ-X-7: G-G-P-Y-R-R-G-R-X-P, as shown in SEQ ID NO.7;

[0021] YQ-X-8: G-G-P-Y-R-R-G-R-W-P, as shown in SEQ ID NO.8;

[0022] YQ-X-9: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO.9;

[0023] YQ-X-10: G-G-P-Y-R-V-G-R-X-P, as shown in SEQ ID NO.10.

[0024] Among them, in polypeptide YQ-X-2 and YQ-X-4, X is citrulline; in polypeptide YQ-X-7 and YQ-X-9, X is D-2-naphthylalanine; in YQ-X-10, X is L-2-naphthylalanine.

[0025] Among them, the involved amino acid residues can be L-type, D-type, or a mixture of L and D types.

[0026] Furthermore, the CXCR4-targeting polypeptide is selected from any one of the following amino acid sequences:

[0027] YQ-X-1: G-G-P-Y-R-R-G-R-G-P;

[0028] YQ-X-2: G-G-p-A-F-X1-S-R-G-p;

[0029] YQ-X-3: G-G-G-H-R-R-G-R-G-p;

[0030] YQ-X-4: P-Y-R-X1-S-R-G-p;

[0031] YQ-X-5: G-G-P-y-R-R-p-R-G-P;

[0032] YQ-X-6: G-G-P-y-R-v-G-R-W-P;

[0033] YQ-X-7: G-G-P-Y-r-R-G-R-X2-P;

[0034] YQ-X-8: G-G-P-Y-r-R-G-R-W-P;

[0035] YQ-X-9: G-G-P-y-R-v-G-R-X2-P;

[0036] YQ-X-10: G-G-P-y-R-v-G-R-X3-P;

[0037] In the above sequences, lowercase letters represent D-amino acids, X1 represents L-citrulline, X2 represents D-2-naphthylalanine, and X3 represents L-2-naphthylalanine.

[0038] In some specific embodiments, proline (Pro) can also be replaced by hydroxyproline (Hyp), and arginine (Arg) can be replaced by homo-arginine (homo-Arg) or citrulline.

[0039] In a second aspect, the present invention also protects a method for preparing the CXCR4-targeting polypeptide described above, and the CXCR4-targeting polypeptide is prepared by the Fmoc solid-phase polypeptide synthesis method.

[0040] In some specific embodiments, the preparation method includes:

[0041] (1) Couple amino acids to Rink Amide MBHA resin one by one according to the designed polypeptide sequence. During the reaction, DMF is used as the solvent to dissolve Fmoc-protected amino acids and the HCTU / DIEA scheme of FastMoc chemistry is used for activation. Each amino acid reacts for 1-2 hours; then, use a 20% piperidine / DMF solution to remove the Fmoc at the N-terminus, repeat twice, 5 minutes each time; then, use a solution composed of TFA / H2O / TIS / anisole (90 / 3 / 4 / 3, v / v / v / v) to treat the polypeptide at room temperature for 3 hours, while removing side-chain protection and cleaving the polypeptide from the resin; then, use nitrogen to blow dry the system, wash twice with methyl tert-butyl ether, and centrifuge to obtain a precipitate;

[0042] (2) The amino acids are sequentially coupled to the Rink Amide MBHA resin one by one according to the preset amino acid sequence. During the coupling process, HCTU and Fmoc-protected amino acids are dissolved in DMF containing 0.4 mol / L DIPEA, and the coupling time for each time is >1 h; then the deprotection process is carried out: the Fmoc group is removed using a DMF solution containing 20% piperidine, and the deprotection time for each time is 5 min, and it is repeated twice; then the side chain protecting group is removed by reacting for 2 - 3 h under the action of strong acid. By mass, the strong acid includes 90 - 95% TFA, 2 - 5% H2O, 2 - 5% TIS, and 2% - 5% EDT.

[0043] In a third aspect, the present invention also protects a molecular probe, which is a CXCR4-targeting polypeptide conjugated with a fluorescent label or a radionuclide as described above.

[0044] Further, the fluorescent label is one or more of fluorescent dyes such as IRDye800, Cy5, Cy7, ICG, rhodamine, FITC, etc.

[0045] Even further, the fluorescent label can be labeled by click chemistry methods such as NHS, EDC, MAL, etc.

[0046] Further, the radionuclide can be 18 F, 68 Ga, 64 Cu, or can also be 99m Tc, 90 Y, and can also be 111 In, 125 I, 131 I or 177 Lu.

[0047] Even further, the chelating agent for the radionuclide is HYNIC, DOTA, NOTA or DTPA and their derivatives, and the radionuclide ligand is tris(hydroxymethyl)methylglycine (Tricine) or sodium m-sulfonatotriphenylphosphine (TPPTS).

[0048] In a fourth aspect, the present invention also protects the use of the CXCR4-targeting peptide and the molecular probe as described above in the preparation of reagents for cancer diagnosis or treatment.

[0049] Further, the reagent is a fluorescence imaging or radioactive imaging reagent.

[0050] Even further, the imaging preparation is one or more of a radionuclide, a radionuclide-labeled substance, a magnetic resonance contrast agent or a molecular imaging preparation.

[0051] Furthermore, the reagent further includes its pharmaceutically acceptable carrier, such as PLGA polymer, Dendrimer, hydrogel, micelle, liposome or inorganic nanoparticles, etc.

[0052] Furthermore, the cancer is all tumors with overexpression of CXCR4, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.

[0053] Even further, the cancer is one or more of melanoma, breast cancer, cervical cancer, lung cancer, prostate cancer

[0054] The present invention also protects the use of the reagent described above in the preparation of a drug for imaging diagnosis of CXCR4-positive tumors or precise resection detection for surgical navigation.

[0055] The positive tumors are all tumors with overexpression of CXCR4, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, pancreatic cancer.

[0056] Beneficial effects

[0057] The CXCR4-targeting polypeptide provided by the present invention can specifically target CXCR4, and at the same time be conjugated with a fluorescent label or a radionuclide to achieve high-sensitivity in vivo imaging of tumors.

[0058] The CXCR4-targeting polypeptide of the present invention can be used for tumor screening and early diagnosis, and can also achieve tumor treatment monitoring.

[0059] The polypeptide of the present invention has good biocompatibility and safety.

[0060] The polypeptides of the present invention are all reported for the first time, and are convenient to obtain and easy to promote. Brief description of the drawings

[0061] Figure 1 Structure of polypeptide YQ-X-1.

[0062] Figure 2 Structure of MPA-YQ-X-1 in Example 2.

[0063] Figure 3 Flow cytometry affinity test chart of probe MPA-YQ-X-9 on B16 cells.

[0064] Figure 4 Fluorescent imaging of probe MPA-YQ-X-1 on A549 tumor-bearing mice.

[0065] Figure 5 Structure of HYNIC-YQ-X-1.

[0066] Figure 6 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-1 in mice bearing B16 melanoma cells.

[0067] Figure 7 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-1 in mice bearing A549 lung cancer cells.

[0068] Figure 8 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-2 in mice bearing A549 lung cancer cells.

[0069] Figure 9 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-3 in mice bearing B16 melanoma cells.

[0070] Figure 10 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-4 in mice bearing B16 melanoma cells.

[0071] Figure 11 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-5 in mice bearing PC3 prostate cancer cells.

[0072] Figure 12 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in mice bearing B16 melanoma cells.

[0073] Figure 13 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in mice bearing Hela cervical cancer cells.

[0074] Figure 14 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in mice bearing B16 melanoma cells.

[0075] Figure 15 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in mice bearing Hela cervical cancer cells.

[0076] Figure 16 99mSPECT-CT imaging of Tc-HYNIC-YQ-X-8 in mice bearing B16 melanoma cells.

[0077] Figure 17 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in mice bearing B16 melanoma cells.

[0078] Figure 18 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in mice bearing MCF7 breast cancer cells.

[0079] Figure 19 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in mice bearing Hela cervical cancer cells.

[0080] Figure 20 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-10 in mice bearing B16 melanoma cells. Detailed implementation mode

[0081] The preparation of the compounds in the present invention described in the following examples is only for illustrative purposes and does not limit the scope of the present invention. Reagents or instrument and equipment not specified by the manufacturer are regarded as conventional products that can be purchased through the market.

[0082] N,N-Diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N,N-dimethylformamide (DMF), methanol, phenol, ninhydrin, anhydrous ether, resin, triisopropylsilane (TIS), 2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), 6-chlorobenzotriazol-1-yl-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), dimethyl sulfoxide (DMSO), various Fmoc-protected amino acids, peptide synthesis tubes, shakers, vacuum water pumps and other instruments involved in the following examples are all obtained through commercial channels.

[0083] Example 1

[0084] This example provides a preparation method of polypeptide YQ-X-n (n = 1 - 10). The polypeptide is synthesized by the FMOC solid-phase synthesis method, and the specific synthesis method is as follows:

[0085] 1. Weigh 400 mg of Rink Amide MBHA resin and add it to a solid-phase synthesis tube. Then add 6 mL of DCM (dichloromethane) and shake it on a shaker for 20 min to swell the resin. 2. Then drain the reaction tube, wash it twice with DMF by shaking and then drain it. Add 5 mL of piperidine / DMF (2 / 8, v / v) solution and react for 5 min, repeat twice and then drain. Wash the synthesis tube five times with 5 mL of DMF and drain it. Then add 1 mmol of FMOC amino acid, 0.4 mL of DIEA, 400 mg of HCTU, and 5 mL of DMF and place it on a shaker to react for 1 hour. 3. Repeat the operation in step 2. Each time, according to the amino acid sequence of the designed polypeptide, add FMOC amino acids in turn from the carboxyl terminus to the amino terminus, and the other components remain unchanged until all the amino acids of the polypeptide sequence are added. 4. After removing the FMOC protection of the last amino acid with piperidine / DMF (2 / 8, v / v) solution, wash the system twice with DMF, DCM, and MeOH in turn and then drain. Add 5 mL (cutting solution: 90 - 95% TFA, 2 - 5% H2O2, 2 - 5% TIS, 2 - 5% EDT) and treat it at room temperature for 3 hours. Use a nitrogen stream to quickly evaporate the volume of the cutting solution until it remains unchanged. Add 5 mL of methyl tert-butyl ether, shake it and then centrifuge to obtain the precipitated peptide.

[0086] Dilute the precipitated peptide with water and complete the purification of the peptide using high-performance liquid chromatography technology. The chromatographic packing material for purification is a 10 - μm reversed-phase C18 column, and gradient elution is carried out using a mobile phase system of 0.1% TFA / water (V / V) and 0.1% TFA / acetonitrile (v / v) under UV detection at 220 nm. Further determine the final product through mass spectrometry analysis. Put the collected eluate into a freeze dryer for concentration and freeze-dry it into a white powder.

[0087] YQ-X-n (n = 1 - 10) was confirmed by mass spectrometry. Taking YQ-X-1 as an example, its structure is as Figure 1 shown, and its [M + H] + cal.: 1071.59, [M + H] + obs.: 1071.67; For YQ-X-2, its [M + H] + cal.: 1001.53, [M + H] + obs.: 1001.64; For YQ-X-3, its [M + H] + cal.: 1005.56, [M + H] + obs.: 1005.79; For YQ-X-4, its [M + H] + cal.: 988.54, [M + H] + obs.: 988.98; For YQ-X-5, its [M + H] +Calcd.: 1111.62, [M+H] + Obsd.: 1111.98; YQ-X-6, [M+H] + Calcd.: 1143.62, [M+H] + Obsd.: 1143.88; YQ-X-7, [M+H] + Calcd.: 1211.65, [M+H] + Obsd.: 1211.98; YQ-X-8, [M+H] + Calcd.: 1200.65, [M+H] + Obsd.: 1200.86; YQ-X-9, [M+H] + Calcd.: 1154.62, [M+H] + Obsd.: 1154.90; YQ-X-10, [M+H] + Calcd.: 1154.62, [M+H] + Obsd.: 1154.32.

[0088] Example 2

[0089] This example provides a preparation method of YQ-X-n (n = 1-10) conjugated with MPA, where MPA is a near-infrared fluorescent dye from the invention patent (CN101440282) applied by this research group. The specific synthesis steps are as follows:

[0090] Dissolve 2.0 mg of the YQ-X-n (n = 1-10) polypeptide obtained in Example 1, 3.0 mg of HATU, and 3.0 mg of the fluorescent dye MPA (the structure is shown in the figure below) in 200 μL of DMSO solution, then add 1.0 μL of DIPEA, and react in a 50 °C metal bath for 2 h. Then, separation and purification are carried out by high-performance preparative liquid chromatography. The preparative liquid chromatography conditions are as follows: an Agilent 1220 Infinity II series HPLC system equipped with an Agilent ZORBAX SB-C18 semi-preparative column (9.4×250 mm, 5 μm) is used, gradient elution for 60 minutes, flow rate 2 mL / min, where mobile phase A is 0.01% TFA / ultrapure water (v / v), and B is 0.01% TFA / acetonitrile (v / v). The elution gradient is set as follows: 90% A and 10% B at 0-5 minutes, 70% A and 30% B at 20-25 minutes, 65% A and 35% B at 30 minutes, 50% A and 50% B at 40 minutes, and 10% A and 90% B at 50-60 minutes. Finally, the product MPA-YQ-X-n (n = 1-10) is obtained. Taking MPA-YQ-X-1 as an example, its structure is as Figure 2 shown.

[0091] Example 3

[0092] In this example, the affinity of MPA-YQ-X-9 for the CXCR4-highly expressed cell line B16 was detected by flow cytometry. The specific procedure is as follows:

[0093] Seed B16 cells in a six-well plate at a cell density of 1×10 6 After culturing for 24 h, add 1 mL of fresh medium containing 10 μM / L of MPA-YQ-X-9, 1 mL of fresh medium containing 10 μM / L of MPA-YQ-X-9 and 100 μM / L of AMD3100, 1 mL of fresh medium containing 10 μM / L of MPA-YQ-X-9 and 200 μM / L of AMD3100, 1 mL of fresh medium containing 10 μM / L of MPA, and 1 mL of fresh blank medium into each well in sequence, and incubate at 37 °C for 2 h. Then wash the cells in each well 3 times with PBS (pH 7.4) to remove unbound drugs. Then digest the cells with trypsin, centrifuge at 1000 rpm for 3 min, and then resuspend them in 300 μL of PBS and store them in a flow tube in the dark. Load and analyze the samples with a flow cytometer equipped with a near-infrared FL 4 channel by itself. Finally, use FlowJo 7.0 software and GraphPad Prism 8.0 software to analyze the original data and express it as the mean fluorescence intensity (MFI).

[0094] The results are as Figure 3 shown, indicating that the polypeptide MPA-YQ-X-9 has strong affinity for the CXCR4-highly expressed cell line and the CXCR4 inhibitor AMD3100 has a blocking effect on it, and the polypeptide probe can specifically target CXCR4.

[0095] Example 4

[0096] In this example, the probe MPA-YQ-X-1 prepared in Example 2 was used for in vivo fluorescence imaging in tumor-bearing mice. The specific procedure is as follows:

[0097] Subcutaneously inoculate A549 cells into female mice at 6-8 weeks old. When the tumors are formed, they are used for imaging experiments. Inject 15 μg of the probe MPA-YQ-X-1 into the tail vein of one group of mice, and inject 15 μg of pure dye MPA into the tail vein of another group of mice. Perform fluorescence detection on a small animal in vivo imaging system at 1 h, 2 h, 4 h, 6 h, 9 h, 12 h, and 24 h after administration. The results are as Figure 4As shown, the probe MPA-YQ-X-1 showed strong fluorescence signals in tumors within 1 h. Compared with the pure dye, the fluorescence intensity of tumors in mice injected with the probe was stronger, demonstrating that the probe of the present invention has CXCR4 targeting and can achieve high-sensitivity in vivo imaging of tumors. It can also be seen from the distribution in major organs that the probe has good biocompatibility and safety.

[0098] Example 5

[0099] This example provides the preparation of HYNIC-YQ-X-n (n = 1 - 10)

[0100] (1) Synthesis of bifunctional chelating agent HYNIC-NHS

[0101] Weigh 5.0 g of 6-chloronicotinic acid, then add 18 mL of 80% hydrazine hydrate, dissolve it in water, and heat under reflux for 6 h. Then concentrate the system by rotary evaporation under reduced pressure, adjust the pH to 5.5 with concentrated hydrochloric acid, precipitate the solid, filter it by suction and dry it to obtain a yellow solid, which is 2-hydrazinopyridine-4-carboxylic acid.

[0102] Weigh 4.0 g of 2-hydrazinopyridine-4-carboxylic acid and 3.8 g of p-dimethylaminobenzoic acid, dissolve them in 25 mL of DMF, and stir at room temperature for 3 h. Then add 4.5 g of NHS, 9.0 g of EDCI, and 15 mL of DMF, stir overnight at room temperature, precipitate the yellow solid with water, filter it by suction, wash it with methanol, dry it by suction, boil it in ethyl acetate, filter it while it is hot, and dry it by suction to obtain a bright yellow solid, which is HYNIC-NHS.

[0103] (2) Synthesis of HYNIC-YQ-X-n (n = 1 - 10)

[0104] Dissolve 2 mg of purified polypeptide YQ-X-n (X = 1 - 10) and 2 mg of HYNIC-NHS in 0.2 mL of DMSO, add 1 μL of DIPEA, and react at 50 °C for 3 h. After the reaction is completed, separate and purify by preparative liquid phase. Finally, obtain the product. Taking HYNIC-YQ-X-1 as an example, its structure is as Figure 5 shown.

[0105] Example 6

[0106] This example provides 99m the preparation of 99mTc-HYNIC-YQ-X-n (n = 1 - 10)

[0107] Prepare TPPTS solution with a concentration of 100.0 mg / mL, Tricine (trimethylglycine) solution with a concentration of 130.0 mg / mL, and succinic acid-sodium succinate buffer solution with a concentration of 102.4 mg / mL (where 77.0 mg of succinic acid and 25.4 mg of sodium succinate). Take 10.0 μL of TPPTS solution, 10.0 μL of Tricine solution, and 10.0 μL of succinic acid-sodium succinate buffer solution respectively, and mix them with 10.0 μL (1.0 mg / mL) of HYNIC-YQ-X-n (n = 1 - 10) described in Example 5 in a vial. Then add 10 mCi Na 99m TcO4 and heat in a metal bath at 100 °C for 20 minutes. After the reaction is completed, cool to room temperature to obtain the peptide radiopharmaceutical 99m Tc-HYNIC-YQ-X-n (n = 1 - 10). The product is analyzed and identified by an Agilent ZORBAX SB-Aq analytical column. The HPLC method used is an Agilent 1220Infinity II series HPLC system equipped with a radioactive on-line detector (Flow-RAM) and an Agilent ZORBAX SB-Aq analytical column (4.6×250 mm, 5 μm). Gradient elution is carried out for 45 minutes at a flow rate of 1 mL / min. The mobile phase A is ultrapure water (0.01% TFA), and B is acetonitrile (0.01% TFA). The elution gradient is set as follows: 95% A and 5% B from 0 to 5 minutes, 70% A and 30% B at 15 minutes, 65% A and 35% B at 20 minutes, 45% A and 55% B at 25 minutes, and 5% A and 95% B at 45 minutes. 99m The purity of

[0108] Example 7

[0109] Radioactive probe 99m SPECT-CT imaging of

[0110] Tc-HYNIC-YQ-X-1 in tumor-bearing mice 99m Prepare the probe Figure 6 prepared in Example 6 Figure 7 Tc-HYNIC-YQ-X-1 into a physiological saline solution and inject 500 μCi through the tail vein into two types of tumor-bearing nude mice (B16, A549) via the tail vein, with at least three mice of each type. And collect SPECT signals at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as 99m shown. The probe

[0111] Example 8

[0112] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-2 in tumor-bearing mice

[0113] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-2 was formulated into a physiological saline solution and injected via the tail vein at a dose of 500 μCi into the tail veins of nude mice bearing lung cancer cells A549, with at least three mice. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 8 shown, the probe 99m Tc-HYNIC-YQ-X-2 had obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0114] Example 9

[0115] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-3 in tumor-bearing mice

[0116] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-3 was formulated into a physiological saline solution and injected via the tail vein at a dose of 500 μCi into the tail veins of nude mice bearing melanoma cells B16, with at least three mice. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 9 shown, the probe 99m Tc-HYNIC-YQ-X-3 had obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0117] Example 10

[0118] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-4 in tumor-bearing mice

[0119] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-4 was formulated into a physiological saline solution and injected via the tail vein at a dose of 500 μCi into the tail veins of nude mice bearing melanoma cells B16, with at least three mice. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 10 shown, the probe 99m Tc-HYNIC-YQ-X-4 had obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0120] Example 11

[0121] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-5 in tumor-bearing mice

[0122] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-5 was formulated into a saline solution and injected via the tail vein at 500 μCi. It was injected into the tail veins of nude mice bearing human prostate cancer cells PC3, with at least three mice. SPECT signals were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 11 As shown 99m Tc-HYNIC-YQ-X-5 had obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0123] Example 12

[0124] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-6 in tumor-bearing mice

[0125] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-6 was formulated into a saline solution and injected via the tail vein at 500 μCi. It was injected into the tail veins of nude mice bearing melanoma cells B16 and human cervical cancer cells Hela, with at least three mice for each type. SPECT signals were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 12 As with Figure 13 As shown 99m Tc-HYNIC-YQ-X-6 had obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0126] Example 13

[0127] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-7 in tumor-bearing mice

[0128] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-7 was formulated into a saline solution and injected via the tail vein at 500 μCi. It was injected into the tail veins of nude mice bearing melanoma cells B16 and human cervical cancer cells Hela, with at least three mice for each type. SPECT signals were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 14 As with 15 As shown 99mTc-HYNIC-YQ-X-7 showed obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0129] Example 14

[0130] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-8 in tumor-bearing mice

[0131] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-8 was formulated into a physiological saline solution and injected via the tail vein at a dose of 500 μCi into the tail veins of nude mice bearing melanoma cells B16, with at least three mice. SPECT signals were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 16 shown. The probe 99m Tc-HYNIC-YQ-X-8 showed obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0132] Example 15

[0133] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-9 in tumor-bearing mice

[0134] The probe prepared in Example 6 99m Tc-HYNIC-YQ-X-9 was formulated into a physiological saline solution and injected via the tail vein at a dose of 500 μCi into the tail veins of nude mice bearing melanoma cells B16, human breast cancer cells MCF-7, and human cervical cancer cells Hela, with at least three mice for each type. SPECT signals were collected at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as follows Figure 17 , Figure 18 and Figure 19 shown. The probe 99m Tc-HYNIC-YQ-X-9 showed obvious uptake at the tumor site, indicating that the probe had good targeting ability.

[0135] Example 16

[0136] Radioactive probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-X-10 in tumor-bearing mice

[0137] The probe prepared in Example 6 99mTc-HYNIC-YQ-X-10 was formulated into a physiological saline solution and 500 μCi was injected via the tail vein into the tail veins of at least three nude mice bearing melanoma cells B16. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are as Figure 20 shown, and the probe 99m Tc-HYNIC-YQ-X-10 had obvious uptake at the tumor site, indicating that the probe had good targeting properties.

[0138] The protection scope of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, variations and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims are used as the protection scope.

[0139] Sequence Listing

[0140] Name Polypeptide Sequence YQ-X-1 GGPYRRGRGP YQ-X-2 <![CDATA[GGpAFX1SRGp]]> YQ-X-3 GGGHRRGRGp YQ-X-4 <![CDATA[PYRX1SRGp]]> YQ-X-5 GGPyRRpRGP YQ-X-6 GGPyRvGRWP YQ-X-7 <![CDATA[GGPYrRGRX2P]]> YQ-X-8 GGPYrRGRWP YQ-X-9 <![CDATA[GGPyRvGRX2P]]> YQ-X-10 <![CDATA[GGPyRvGRX3P]]>

[0141] Among them, lowercase letters in the above sequences represent D-type amino acids, X1 represents L-citrulline, X2 represents D-2-naphthylalanine, and X3 represents L-2-naphthylalanine.

Claims

1. A CXCR4-targeting polypeptide, wherein the CXCR4-targeting polypeptide has the following amino acid sequence: YQ-X-1: G-G-P-Y-R-R-G-R-G-P, as shown in SEQ ID NO.

1.

2. A method for preparing the CXCR4-targeting polypeptide according to claim 1, wherein the CXCR4-targeting polypeptide is prepared by the Fmoc solid-phase polypeptide synthesis method.

3. A molecular probe, which is composed of a fluorescent label or a radionuclide conjugated to the CXCR4-targeting polypeptide according to claim 1.

4. The molecular probe according to claim 3, characterized in that, The fluorescent label is one or more of IRDye800, Cy5, Cy7, ICG, rhodamine, and FITC.

5. The molecular probe according to claim 4, characterized in that, The fluorescent label is labeled by NHS, EDC, or MAL.

6. The molecular probe according to claim 3, wherein The radionuclide is selected from 18 F, 68 Ga, 64 Cu, 99m Tc, 90 Y, 111 In, 125 I, 131 I or 177 Lu.

7. The molecular probe according to claim 6, characterized in that, The chelator of the radionuclide is HYNIC, DOTA, NOTA, DTPA, or their derivatives, and the radionuclide ligand is tris(hydroxymethyl)methylglycine or sodium 3-(trimethylammonio)phenylsulfonate.

8. Use of the CXCR4-targeting polypeptide according to claim 1 or the molecular probe according to any one of claims 3-7 in the preparation of a cancer diagnostic reagent for CXCR4 overexpression; The cancer is one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioblastoma, prostate cancer, and pancreatic cancer.

9. The use according to claim 8, characterized in that, The reagent further comprises a pharmaceutically acceptable carrier thereof.

10. The use according to claim 9, characterized in that, The carrier is a PLGA polymer, a Dendrimer, a hydrogel, a micelle, a liposome, or an inorganic nanoparticle.

11. The use according to claim 8, wherein, The reagent is a fluorescence imaging or a radioactive imaging reagent.

12. The use according to claim 11, characterized in that, The imaging reagent is one or more of a radionuclide, a radionuclide-labeled substance, a magnetic resonance contrast agent, or a molecular imaging agent.

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