A CXCR4 targeting polypeptide and its preparation method and application

Abstract

The present invention discloses a CXCR4 targeting polypeptide and a preparation method and application thereof. The present invention designs and synthesizes a series of polypeptides targeting chemokine receptor 4 (CXCR4), and determines the affinity of the discovered polypeptides to the CXCR4 protein by SPR. The polypeptide of the present invention can specifically bind to a variety of tumor cells, preferably melanoma, lymphoma, breast cancer, lung cancer, colorectal cancer, glioma, prostate cancer, etc. The present invention also relates to a method for preparing the polypeptide; the use of the polypeptide directly or through ligand coupling fluorescent dyes or radionuclides to achieve the treatment or diagnosis of tumors.

Description

Technical Field

[0001] The present invention relates to a CXCR4 targeting polypeptide and a preparation method and application thereof, belonging to the field of polypeptides. Background Art

[0002] Malignant tumors have become one of the main causes of abnormal death in the world, posing a great threat to human health. Traditional tumor treatments mainly include surgery, chemotherapy, radiotherapy, etc., but traditional treatments usually bring great toxic side effects to patients and increase the burden on patients.

[0003] Tumor targeted therapy is a method of inhibiting tumors 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 specific molecules involved in the occurrence, development and spread of tumor cells. Compared with traditional treatments, tumor targeted therapy has high selectivity for tumor cells, good efficacy and few side effects, and has good prospects in treating tumors. Researchers are currently developing various tumor targeted therapies. Among the various targeted therapies, the use of targeted peptides to treat tumors is an ideal method with the following advantages: 1) It has good penetrability and is easily taken up by tumor cells; 2) It has fast plasma clearance, high selectivity and high affinity. 3) It is easy to chemically synthesize and has low immunogenicity, which can avoid the shortcomings of monoclonal antibody therapy. Therefore, specific targeted peptides are ideal and attractive targets for medical research, clinical applications and molecular imaging.

[0005] Chemokines are low molecular weight (8,000-10,000) proteins belonging to the cytokine superfamily and are key regulators of cell migration in development, homeostasis, and immune surveillance. Chemokines are involved in regulating a variety of biological processes such as angiogenesis, morphogenesis, autoimmunity, tumor growth, and metastasis, and they also guide the migration of various types of cells. Chemokine 12 (CXCL12), also known as stromal cell derived factor-1 (SDF-1), belongs to the chemokine protein family.

[0006] Chemokine receptor type 4 (CXCR4), also known as fusin or CD184, is a seven-transmembrane G protein-coupled receptor (GPCR) that is activated when the extracellular domain binds to the ligand. CXCR4 is overexpressed in more than 20 human tumor types, including ovarian cancer, prostate cancer, esophageal cancer, lung cancer, melanoma, neuroblastoma, renal cell carcinoma, etc.

[0007] CXCR4 (CXC motif chemokine receptor 4) is a specific receptor for CXCL12. After binding, the two participate in a series of physiological and pathological processes such as embryonic hematopoiesis, organogenesis, angiogenesis, embryonic growth and development, inflammation, tissue immune monitoring, etc. 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 resistance.

[0008] Currently, immunohistochemistry (IHC) testing is required for tumor diagnosis. However, due to the high temporal and spatial heterogeneity of tumor tissue and the uneven sampling of biopsy tissue, it is impossible to make an accurate pathological diagnosis of the tumor. Therefore, there is an urgent need for marker detection methods that can reflect accurate diagnosis in vivo. Summary of the invention

[0009] Purpose of the invention: The technical problem to be solved by the present invention is to provide a CXCR4 targeting polypeptide and a preparation method and application thereof.

[0010] Technical solution: To solve the above technical problems, the present invention provides a CXCR4 targeting polypeptide, the amino acid sequence of which is as follows:

[0011] Gly—Gly—X1—X2—X3—X4—X5—X6—X7—X8, or a pharmaceutically acceptable salt thereof,

[0012] in:

[0013] In the polypeptide, X1 is selected from one of Ala, Pro, DVal, DPro, HYP, OIC, and α-Me-Pro, X2 is selected from one of Tyr, DTyr, Ala, and N-Me-Tyr, X3 is selected from one of Arg, HAR, N2-Me-Arg, and Ala, X4 is selected from one of DVal and Ala, X5 is selected from one of Gly, Ala, β-Ala, and Sar, X6 is selected from one of Arg, Ala, D-2-Nal, HAR, N2-Me-Arg, and Pro, X7 is selected from one of 2-Nal, D-2-Nal, Ala, and Arg, and X8 is selected from one of Ala and Pro.

[0014] Wherein, the amino acid sequence of the CXCR4 targeting polypeptide is shown in any one of SEQ ID NO.1-27.

[0015] The present invention also provides a method for preparing the CXCR4 targeting polypeptide, the method comprising the following specific steps:

[0016] (1) The amino acid was coupled to Rink Amide MBHA resin, the Fmoc-protected amino acid was dissolved in DMF and activated using the HCTU / DIEA protocol of FastMoc chemistry; the N-terminal Fmoc was then removed using a 20% piperidine / DMF solution; the peptide was then treated with a solution of TFA / H2O / TIS / anisole at room temperature to remove the side chain protection and cleave the peptide from the resin; the system was then dried with nitrogen, washed twice with methyl tert-butyl ether, and centrifuged to obtain a precipitate;

[0017] (2) The amino acids are coupled to the Rink Amide MBHA resin one by one in sequence. During the coupling process, the HCTU and Fmoc-protected amino acids are dissolved in DMF containing DIPEA. The coupling time for each time is >1 h. Then, a deprotection process is performed: the Fmoc group is removed using a DMF solution containing piperidine. Then, the side chain protecting group is removed by reacting for 2-3 h under the action of a strong acid. The strong acid includes, by weight, 90-95% TFA, 2-5% H2O, 2-5% TIS, and 2%-5% EDT.

[0018] The present invention also provides a molecular probe, which is formed by coupling the CXCR4 targeting polypeptide with a fluorescent marker or a radioactive nuclide.

[0019] Wherein, the fluorescent marker includes one or more of IRDye800, Cy5, Cy7, ICG, rhodamine, and FITC.

[0020] The fluorescent marker can be labeled by click chemistry such as NHS, EDC, MAL, etc.

[0021] 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.

[0022] The radionuclide chelating agent includes HYNIC, DOTA, NOTA or DTPA and their derivatives.

[0023] Wherein, the ligand of the radionuclide includes tris(hydroxymethyl)methylglycine or sodium meta-sulfonate triphenylphosphine.

[0024] The present invention also provides the use of the CXCR4 targeting polypeptide or the molecular probe in preparing a reagent or a medicine for diagnosing a tumor characterized by overexpression of CXCR4.

[0025] The tumor characterized by CXCR4 overexpression includes one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, and pancreatic cancer.

[0026] Furthermore, the tumor is one or more of melanoma, breast cancer, cervical cancer, lung cancer, and prostate cancer.

[0027] The present invention also provides the use of the CXCR4 targeting polypeptide or the molecular probe in preparing fluorescent imaging or radioactive imaging reagents.

[0028] Wherein, the imaging agent is one or more of a radionuclide, a radionuclide marker, a magnetic resonance contrast agent or a molecular imaging agent.

[0029] The imaging preparation further comprises a pharmaceutically acceptable carrier, such as PLGA polymer, Dendrimer dendrimer, hydrogel, micelle, liposome or inorganic nanoparticles.

[0030] The present invention also protects the use of the imaging preparation described above in the preparation of a drug for the detection of CXCR4-positive tumor imaging diagnosis or surgical navigation precise resection.

[0031] The positive tumors are all tumors characterized by overexpression of CXCR4, such as one or more of melanoma, lymphoma, breast cancer, cervical cancer, lung cancer, colorectal cancer, glioma, prostate cancer, and pancreatic cancer.

[0032] The present invention designs a novel class of CXCR4 targeting polypeptides, which have high selectivity and affinity for CXCR4. By coupling with fluorescent dyes and using molecular imaging surgical navigation equipment, precise tumor resection can be achieved during surgery. Furthermore, the CXCR4 targeting polypeptides can also be coupled with radionuclides for radionuclide imaging to achieve the purpose of early diagnosis and treatment of tumors.

[0033] The targeted peptide method used in the present invention innovatively combines the diversity of peptide amino acid structure and the difference of peptide sequence, and can accurately reflect the dynamic expression changes of living CXCR4. In addition, due to the diversity of peptide sequences, it is not easy and difficult to think of synthesizing a peptide with excellent targeting ability.

[0034] The principle of the present invention is that several key amino acid residues in the CXCR4 targeting polypeptide are tightly bound to several amino acid residues in the binding region of the target CXCR4 by forming hydrogen bonds, salt bonds and van der Waals forces, so that the polypeptide is targeted to the binding region. And by connecting different chelating agents to the CXCR4 targeting polypeptide, different radionuclides such as 18F, 68 Ga, 64 Cu, or 99m Tc, 90 Y, or 111 In, 125 I, 131 I or 177 Lu. For tumors characterized by overexpression of CXCR4, CXCR4-targeted peptides linked to radionuclides are injected in vitro, and the radiation signals are detected by instruments such as positron emission tomography (PET-CT) and single photon emission computed tomography (SPECT), thereby performing tumor imaging diagnosis. If the linked radionuclide has a long half-life and the radiation dose is appropriate, radiotherapy can be performed on the tumor area.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention designs a series of targeting polypeptides, which has guiding significance for the development of CXCR4 receptor polypeptide antagonists, and also has a certain explanation for the study of the structure-activity relationship between CXCR4 and various amino acid residues; the present invention constructs a diagnostic radioactive probe that specifically targets CXCR4-positive tumors, which provides a new and effective tool for nuclear medicine to diagnose tumors, and also has a certain guiding significance for surgery and drug treatment of cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the synthesis diagram of peptide YQ-R-3;

[0037] Figure 2 is the mass spectrum of HYNIC-NHS;

[0038] Figure 3 for 99m Radiochemical purity of Tc-HYNIC-YQ-R-3;

[0039] Figure 4 For probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-3 in mice bearing melanoma cells B16;

[0040] Figure 5 For probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-4 in mice bearing melanoma cells B16;

[0041] Figure 6 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-11 in mice bearing melanoma cells B16;

[0042] Figure 7 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-12 in mice bearing melanoma cells B16;

[0043] Figure 8 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-13 in mice bearing human lung cancer cells H460;

[0044] Fig. 9 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-14 in mice bearing melanoma cells B16;

[0045] Fig.10 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-17 in mice bearing melanoma cells B16;

[0046] Fig.11 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-17 in mice bearing breast cancer cells 4T1;

[0047] Fig.12 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-21 in mice bearing melanoma cells B16;

[0048] Fig.13 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-21 in mice bearing human lung cancer cells H460;

[0049] Fig.14 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-22 in melanoma cell B16 tumor-bearing mice;

[0050] Fig.15 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-26 in mice bearing melanoma cells B16;

[0051] Fig.16 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-27 in mice bearing human lung cancer cells H460;

[0052] Fig.17 for 99mSPECT-CT imaging of Tc-HYNIC-YQ-R-29 in mice bearing melanoma cells B16;

[0053] Fig.18 for 99m The cellular uptake rate of Tc-HYNIC-YQ-R-11 in melanoma B16 cells and human colon cancer cells HCT116;

[0054] Fig.19 for 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-11 in mice bearing melanoma cells B16 and human colon cancer cells HCT116. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0056] The following examples involve N, N-diisopropylethylamine (DIPEA), piperidine, trifluoroacetic acid (TFA), dichloromethane (DCM), N, N-dimethylformamide (DMF), methanol, phenol, ninhydrin, anhydrous ether, resin, triisopropylsilane (TIS), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (HATU), 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU ), dimethyl sulfoxide (DMSO), 4-toluene hydroamine resin (MBHA resin), 1,2-ethanedithiol (EDT), triphenylphosphine trissulfonic acid sodium salt (TPPTS), 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxysuccinimide (NHS), 2-(N-morpholine)ethanesulfonic acid buffer (MES buffer), various Fmoc-protected amino acids, peptide synthesis tubes, shakers, vacuum water pumps and other instruments were obtained from commercial sources.

[0057] Example 1

[0058] This embodiment provides a method for synthesizing polypeptide YQ-R-3, the polypeptide sequence of which is Gly-Gly-Ala-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO. 1). Figure 1 As shown, YQ-R-3 was synthesized by Fmoc solid phase synthesis method, and its specific synthesis method is as follows:

[0059] 1. Weigh 400 mg of Rink Amide MBHA resin (manufacturer: Jiangsu Jitai Peptide Technology Co., Ltd., catalog number: JT230630-49101) and add it to the solid phase synthesis tube, then add 6 mL of DCM and shake on a shaker for 20 min to swell the resin;

[0060] 2. Then drain the reaction tube, shake and wash it twice with DMF and drain it, add 5mL of piperidine and DMF mixture (piperidine:DMF=2:8, v / v) solution, react for 5min, repeat twice, and drain it. Wash the synthesis tube with 5mL DMF five times and drain it, then add 1mmol of Fmoc-Ala, 0.4mL DIPEA, 400mg HCTU, 5mL DMF and place it on a shaker to react for 1 hour;

[0061] 3. Repeat step 2 and add Fmoc amino acids of equal molar mass from the carboxyl end to the amino end according to the amino acid sequence of the designed polypeptide, and keep the other components unchanged until all the amino acids of the polypeptide sequence are added;

[0062] 4. After removing the Fmoc protection of the last amino acid using piperidine / DMF (2 / 8, v / v) solution, wash the system twice with DMF, DCM, and MeOH respectively and drain, add 5 mL of mixed solution (cutting solution TFA 95%, H2O 2%, TIS 1%, EDT 2%) and treat at room temperature for 3 hours. Use nitrogen flow to quickly evaporate the cutting solution to a constant volume, add 5 mL of methyl tert-butyl ether, shake and centrifuge to obtain the precipitated peptide.

[0063] The precipitated peptide was diluted with water and purified using standard preparative HPLC techniques. The chromatographic filler used for purification was a 10 μm reverse phase C18 column. Under 220 nm UV detection, the mobile phase system was a 0.1% TFA aqueous solution and an acetonitrile solution containing 0.1% TFA for gradient elution. After mass spectrometry analysis to further determine the final product, the collected eluate was placed in a freeze dryer for concentration and freeze-dried into a white powder to obtain the polypeptide YQ-R-3.

[0064] Example 2

[0065] The sequence of polypeptide YQ-R-4 is Gly-Gly-Pro-Ala-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.2)

[0066] The synthesis and preparation were carried out as in Example 1, except that the third Fmoc-Ala was replaced by an Fmoc-Pro of equal molar mass, and the fourth Fmoc-D-Tyr(tBu) was replaced by an Fmoc-Ala of equal molar mass, and other raw materials and proportions remained unchanged.

[0067] The polypeptide YQ-R-5~29 was synthesized according to the method of Example 1.

[0068] Among them, the sequence of polypeptide YQ-R-5 is Gly-Gly-Pro-DTyr-Ala-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.3)

[0069] The sequence of polypeptide YQ-R-6 is Gly-Gly-Pro-DTyr-Arg-Ala-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.4)

[0070] The sequence of polypeptide YQ-R-7 is Gly-Gly-Pro-DTyr-Arg-DVal-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.5)

[0071] The sequence of polypeptide YQ-R-8 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Ala-D-2-Nal-Pro (SEQ ID NO.6)

[0072] The sequence of polypeptide YQ-R-9 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Arg-Ala-Pro (SEQ ID NO.7)

[0073] The sequence of polypeptide YQ-R-10 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Ala (SEQ ID NO.8)

[0074] The sequence of polypeptide YQ-R-11 is Gly-Gly-Pro-Tyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.9)

[0075] The sequence of polypeptide YQ-R-12 is Gly-Gly-DVal-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.10)

[0076] The sequence of polypeptide YQ-R-13 is Gly-Gly-Hyp-DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.11)

[0077] The sequence of polypeptide YQ-R-14 is Gly-Gly-OIC--DTyr-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.12)

[0078] The sequence of polypeptide YQ-R-15 is Gly-Gly-Pro-DTyr-HAR-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.13)

[0079] The sequence of polypeptide YQ-R-16 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-HAR-D-2-Nal-Pro (SEQ ID NO.14)

[0080] The sequence of polypeptide YQ-R-17 is Gly-Gly-Pro-DTyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.15)

[0081] The sequence of polypeptide YQ-R-18 is Gly-Gly-Pro-DTyr-N2-ME-Arg-DVal-Gly-Arg-D-2-Nal-Pro (SEQ ID NO.16)

[0082] The sequence of polypeptide YQ-R-19 is Gly-Gly-Pro-DTyr-Arg-DVal-Gly-N2-ME-Arg-D-2-Nal-Pro (SEQ ID NO.17)

[0083] The sequence of polypeptide YQ-R-20 is Gly-Gly-Pro-DTyr-Arg-DVal-β-Ala-D-2-Nal-Arg-Pro (SEQ ID NO.18)

[0084] The sequence of polypeptide YQ-R-21 is Gly-Gly-Pro-N-ME-Tyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.19)

[0085] The sequence of polypeptide YQ-R-22 is Gly-Gly-Pro-DTyr-Arg-DVal-β-Ala-Arg-2-Nal-Pro (SEQ ID NO. 20)

[0086] The sequence of polypeptide YQ-R-23 is Gly-Gly-Pro-DTyr-Arg-DVal-Sar-Arg-D-2-Nal-Pro (SEQ ID NO.21)

[0087] The sequence of polypeptide YQ-R-24 is Gly-Gly-Pro-DTyr-Arg-DVal-β-Ala-HAR-D-2-Nal-Pro (SEQ ID NO.22)

[0088] The sequence of polypeptide YQ-R-25 is Gly-Gly-Pro-Tyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.23)

[0089] The sequence of polypeptide YQ-R-26 is Gly-Gly-DPro-Tyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.24)

[0090] The sequence of polypeptide YQ-R-27 is Gly-Gly-DPro-DTyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.25)

[0091] The sequence of polypeptide YQ-R-28 is Gly-Gly-α-Me-Pro-DTyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.26)

[0092] The sequence of polypeptide YQ-R-29 is Gly-Gly-α-Me-Pro-Tyr-Arg-DVal-β-Ala-Arg-D-2-Nal-Pro (SEQ ID NO.27)

[0093] The mass spectrometry characterization of peptide YQ-Rn (n=3~29) is shown in Table 1.

[0094] Table 1 Mass spectrometry characterization of peptide YQ-Rn (n=3~29)

[0095]

[0096] Example 3

[0097] This example provides a method for determining the affinity of polypeptide YQ-Rn (n=3-29) with human CXCR4 protein (manufacturer: DIMA Biotechnology (Wuhan) Co., Ltd., catalog number: PME100834), mainly through surface ion resonance technology (SPR technology), and selects the listed drug AMD3100 (manufacturer: Shanghai Bide Pharmaceutical Technology Co., Ltd., catalog number: BD208340) as a comparison. The specific operation steps are as follows:

[0098] 1. Rinse the chip surface with pure water. After drying, soak the chip in 2.18 mg / mL (10 mmol / L) 11-mercaptoundecanoic acid MUA (prepared in pure ethanol) for overnight treatment. Rinse with ethanol and pure water respectively on the next day.

[0099] 2. Use covalent binding method to fix CXCR4 protein to the gold film on the chip surface. The specific operation is as follows: (1) Set the injection speed to 10 μL / min. After the baseline is stable, start injecting 150 μL of 0.4 mol / L EDC solution and 150 μL of 0.1 mol / L NHS solution (both prepared in MES buffer, the concentration and pH of MES buffer are 0.1 mol / L, pH = 5.5) for 10 min to activate the carboxyl groups on the gold film surface. (2) Inject 150 μL of 10 μg / mL CXCR4 protein solution (prepared in sodium acetate solution, the concentration and pH of sodium acetate solution are 0.01 mol / L, pH = 4.8) and let it flow through the chip surface to achieve its covalent binding on the gold film surface. (3) After the binding of CXCR4 protein is stable, pass PBS to rinse the gold film surface. (4) Pass 10 mg / mL BSA solution (prepared in sodium acetate solution, the concentration and pH of sodium acetate solution are 0.01 mol / L, pH = 4.8) pH = 4.8) to block nonspecific sites.

[0100] 3. After the CXCR4 protein was fixed on the chip surface, PBS buffer was used as the working solution to flow through the system, and the blank channel was used as the control channel to eliminate nonspecific binding as much as possible. The injection speed was set to 20 μL / min and the temperature was maintained at 25°C. After the baseline was stable, different concentrations of peptide YQ-R-3~29 solutions (prepared in PBS buffer, the concentration of PBS buffer was 0.01M, pH was 7.4, Table 2) or small molecule solutions (AMD3100, prepared in PBS buffer) were injected to monitor the binding process in real time. After that, PBS buffer was passed to clean the chip surface. GraphPad 9.0 software was used for data processing, and the KD value results are shown in Table 3.

[0101] Table 2

[0102]

[0103] Table 3 KD values ​​determined by SPR

[0104]

[0105] The results are shown in Table 3. The binding abilities of peptides YQ-R-3~29 to the target (CXCR4 protein) vary, but most of them are better than the marketed drug AMD3100.

[0106] Example 4 Preparation of prodrug HYNIC-YQ-Rn (n=3~29)

[0107] 1. Synthesis of HYNIC-NHS:

[0108] Synthesis route:

[0109]

[0110] 1.79 g of 6-chloronicotinic acid (compound 1, 11.36 mmol, 1eq) was added to a 100 mL round-bottom flask, and 6.2 mL of 80% hydrazine hydrate (124.98 mmol, 11 eq) and 11 mL of deionized water were added. The mixture was stirred and heated to 100°C for 6 h. The solvent was removed by vacuum distillation, and the pH was adjusted to 5.5 with hydrochloric acid. The mixture was then concentrated by vacuum distillation. After standing overnight, a precipitate appeared. The precipitate was filtered and dried to obtain 6-hydrazinonicotinic acid, which was then weighed for the next step of reaction.

[0111] 0.55 g of 6-hydrazinonicotinic acid (compound 2, 3.59 mmol, 1 eq) and 0.54 g of dimethylaminobenzaldehyde (compound 3, 3.59 mmol, 1 eq) were dissolved in 8 mL of DMF and stirred at room temperature for 3 h to obtain compound 4; 0.62 g of NHS (5.39 mmol, 1.5 eq) and 1.8 g of EDCI (9.39 mmol, 2.5 eq) were added, and 15 mL of DMF was added to continue stirring for 18 h. The DMF was concentrated by vacuum distillation and the system was added to 50 mL of deionized water to obtain a yellow precipitate, which was filtered and washed with methanol three times. After drying, it was heated to reflux in ethyl acetate, filtered while hot, and dried to obtain the product HYNIC-NHS and weighed. The product was confirmed by mass spectrometry. The mass spectrum is shown in FIG. Figure 2 HYNIC-NHS: calculate[M+H+]: 382.15, found[M+H+]:382.22.

[0112] 2. Preparation of prodrug HYNIC-YQ-Rn (n=3~29), the specific steps are as follows:

[0113] 1 mg of purified peptide YQ-Rn (n=3~29) and 1 mg of HYNIC-NHS were dissolved in 0.1 mL DMSO, and 1 μL of DIPEA was added to react at 50°C for 1.5 h. After the reaction was completed, separation and purification were performed by preparative high performance liquid phase (Agilent ZORBAXSB-C18, 5 μm) to obtain the product. Taking HYNIC-YQ-R-3 as an example, its structure is shown below:

[0114]

[0115] HYNIC-YQ-R-3: Calculated value: [1 / 2M+H +]: 697.86, Actual value: 697.98. The purity of HYNIC-YQ-R-3 is 98%.

[0116] The separation and purification steps are as follows: use preparative HPLC gradient elution for 35 minutes at a flow rate of 1 mL / min, where mobile phase A is ultrapure water (containing 0.1% TFA) and mobile phase B is acetonitrile (containing 0.1% TFA). The elution gradient is set as: 85% A and 15% B to 60% A and 40% B at 0-20 minutes, 50% A and 50% B at 25 minutes, and 10% A and 90% B at 25-35 minutes. HYNIC-YQ-R-4~29 was also prepared according to the above method. The chemical purity of all products is greater than 95%.

[0117] Example 5 99m Preparation of Tc-HYNIC-YQ-Rn (n=3~29)

[0118] A TPPTS solution with a concentration of 100.0 mg / mL, a Tricine (trimethylglycine) with a concentration of 130.0 mg / mL, and a succinic acid-sodium succinate buffer solution with a concentration of 102.4 mg / mL (wherein the mass ratio of succinic acid to sodium succinate is 77.0:25.4) were prepared respectively, and 10.0 μL of TPPTS solution, 10.0 μL of Tricine solution, 10.0 μL of succinic acid-sodium succinate buffer solution and 10.0 μL (1.0 mg / mL) of the prodrug described in Example 4 were mixed in a vial, and then 10 mCi Na 99m TcO4 (Senko (Nanjing) Pharmaceutical Technology Co., Ltd.) was heated in a 100 °C metal bath for 20 minutes and cooled to room temperature after the reaction to obtain peptide radiopharmaceuticals. 99m Tc-HYNIC-YQ-Rn (n=3~29), the product was analyzed and identified by Agilent ZORBAX SB-Aq analytical column. The HPLC method used was an Agilent 1220 Infinity II series HPLC system equipped with an online radioactive detector (Flow-RAM) and an Agilent ZORBAX SB-Aq analytical column (4.6×250 mm, 5 μm). The gradient elution was 45 minutes, the flow rate was 1 mL / min, and the mobile phase A was ultrapure water (0.01% TFA) and B was acetonitrile (0.01% TFA). The elution gradient was set as: 95%A and 5%B at 0-5 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. 99mThe radiochemical purity of Tc-HYNIC-YQ-Rn (n=3~29) is not less than 95%. 99m Take Tc-HYNIC-YQ-R-3 as an example, the radiochemical purity is Figure 3 shown.

[0119] Example 6 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-3 in tumor-bearing mice

[0120] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-3 was prepared into physiological saline solution and 500 μCi was injected into the tail vein of at least three nude mice bearing melanoma B16 cells (Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.). SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results after administration are shown in Figure 2. Figure 4 As shown, the probe 99m Tc-HYNIC-YQ-R-3 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0121] Example 7 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-4 in tumor-bearing mice

[0122] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-4 was prepared into physiological saline solution and 500 μCi was injected into the tail vein of at least three nude mice bearing melanoma B16 cells. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results after administration are shown in Figure 2. Figure 5 As shown, the probe 99m Tc-HYNIC-YQ-R-4 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0123] Example 8 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-11 in tumor-bearing mice

[0124] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99mTc-HYNIC-YQ-R-11 was prepared into physiological saline solution and 500 μCi was injected into the tail vein of at least three nude mice bearing melanoma B16 cells. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results after administration are shown in Figure 2. Figure 6 As shown, the probe 99m Tc-HYNIC-YQ-R-11 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0125] Example 9 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-12 in tumor-bearing mice

[0126] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-12 was prepared into physiological saline solution and 500 μCi was injected into the tail vein of mice bearing melanoma B16 cells (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.), at least three mice of each type. SPECT signal acquisition was performed 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results are shown in Figure 7 As shown, the probe 99m Tc-HYNIC-YQ-R-12 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0127] Example 10 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-13 in tumor-bearing mice

[0128] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-13 was prepared into physiological saline solution and injected into the tail vein of at least three mice bearing human lung cancer cells H460 (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.) at 500 μCi. SPECT signal acquisition was performed 0.5 h, 1 h, and 2 h after administration. The imaging results are shown in Figure 8 As shown, the probe 99m Tc-HYNIC-YQ-R-13 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0129] Example 11 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-14 in tumor-bearing mice

[0130] The 18.5 MBq / 200 μL probe prepared in Example 5 was99m Tc-HYNIC-YQ-R-14 was prepared into physiological saline solution and injected into the tail vein of mice bearing melanoma cells B16 (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.) at 500 μCi, at least three mice of each type. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 3 h after administration. The imaging results are shown in Figure 2. Fig. 9 As shown, the probe 99m Tc-HYNIC-YQ-R-14 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0131] Example 12 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-17 in tumor-bearing mice

[0132] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-17 was prepared into physiological saline solution, and 500 μCi was injected into the tail vein of nude mice bearing melanoma cells B16 and breast cancer cells 4T1 (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.), at least three of each type. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The imaging results of nude mice bearing melanoma cells B16 are shown in Figure 2. Fig.10 As shown, the imaging results of nude mice injected with breast cancer cells 4T1 are as follows Fig.11 As shown, the probe 99m Tc-HYNIC-YQ-R-17 showed obvious uptake at the tumor site, indicating that the probe has good targeting ability.

[0133] Example 13 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-21 in tumor-bearing mice

[0134] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-21 was prepared into physiological saline solution, and 500 μCi was injected into the tail vein of nude mice bearing melanoma cells B16 and nude mice bearing human lung cancer cells H460 (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.), at least three of each type. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, 3 h, and 4 h after administration. The results of the imaging of nude mice bearing melanoma cells B16 are shown in the figure. Fig.12 As shown, the imaging results of nude mice bearing human lung cancer cells H460 are shown in Fig.13 As shown, the probe 99mTc-HYNIC-YQ-R-21 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0135] Example 14 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-22 in tumor-bearing mice

[0136] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-22 was prepared into physiological saline solution and injected into the tail vein of nude mice bearing melanoma cells B16 at 500 μCi for at least three times. SPECT signal acquisition was performed at 0.5 h, 1 h, 2 h, and 3 h after administration. The imaging results are shown in Figure 2. Fig.14 As shown, the probe 99m Tc-HYNIC-YQ-R-22 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0137] Example 15 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-26 in tumor-bearing mice

[0138] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-26 was prepared into physiological saline solution and injected into the tail vein of at least three nude mice bearing melanoma cells B16 through 500 μCi. SPECT signal acquisition was performed 0.5 h and 2 h after administration. The imaging results are shown in Figure 2. Fig.15 As shown, the probe 99m Tc-HYNIC-YQ-R-26 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0139] Example 16 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-27 in tumor-bearing mice

[0140] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-27 was prepared into physiological saline solution and injected into the tail vein of nude mice bearing human lung cancer cells H460 at 500 μCi for at least three times. SPECT signal acquisition was performed 2 h after administration. The imaging results are shown in Figure 2. Fig.16 As shown, the probe 99m Tc-HYNIC-YQ-R-27 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0141] Example 17 Radioactive Probe 99m SPECT-CT imaging of Tc-HYNIC-YQ-R-29 in tumor-bearing mice

[0142] The 18.5 MBq / 200 μL probe prepared in Example 5 was 99m Tc-HYNIC-YQ-R-29 was prepared into physiological saline solution and injected into the tail vein of at least three nude mice bearing melanoma cells B16 through the tail vein. SPECT signal acquisition was performed 1h, 2h, and 4h after administration. The imaging results are shown in Fig.17 As shown, the probe 99m Tc-HYNIC-YQ-R-29 was obviously taken up in the tumor site, indicating that the probe had good targeting ability.

[0143] Example 18 Cellular uptake comparison experiment

[0144] This example compares 99m Cellular uptake rate of Tc-HYNIC-YQ-R-11 in CXCR4 high-expressing cells B16F10 and CXCR4 low-expressing cells HCT116.

[0145] HCT116 cells and B16F10 cells were seeded in two 12-well plates (2*10 5 1.5 mL of culture medium (DMEM medium containing 10% fetal bovine serum) was added to each well of HC116 cells, and 1.5 mL of culture medium (1640 medium containing 10% fetal bovine serum) was added to each well of B16F10 cells. The cells were incubated at 37°C for 24 h. The culture plates were removed and replaced with the corresponding fresh culture medium (1 mL per well). 37 KBq of 99m Tc-HYNIC-YQ-R-11 was added to each well and incubated at 37°C for 1h and 2h, respectively. After the incubation time, the supernatant was aspirated, and the cells were washed twice with PBS. The PBS was aspirated and merged into the supernatant. Then, it was digested with trypsin, aspirated and counted as the precipitate, and the residual cells were washed with PBS and aspirated and merged into the precipitate. The supernatant and cell gamma counts (CPM) were measured using a WIZARD 2470 gamma counter, and the cell uptake rate was calculated. The formula is as follows:

[0146]

[0147] The results are as follows Fig.18 As shown, it was found that the uptake rate was positively correlated with the expression of CXCR4 in cells. The expression of the radionuclide probe in B16F10 cells was significantly higher than that in HC116 cells, which proved the binding ability and specificity of the probe to cells with high expression of CXCR4.

[0148] Embodiment 19 99m Tc-HYNIC-YQ-R-11 imaging in tumor-bearing mice

[0149] To evaluate 99m The imaging ability and metabolic distribution of Tc-HYNIC-YQ-R-11 probe in vivo were investigated using B16F10 melanoma model mice and HCT116 tumor-bearing mice (purchased from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd.). Each mouse was injected with 18 MBq of 99m Tc-HYNIC-YQ-R-11 in saline solution. SPECT / CT scans were performed 30 min after injection. After each scan, the data was automatically reconstructed by the software to obtain specific SPECT / CT images. The mice were then scanned at different time points to observe changes in their distribution in vivo. Fig.19 As shown, 99m Tc-HYNIC-YQ-R-11 in B16F10 ( Fig.19 The radionuclide probe showed good imaging effects in B16F10 tumor-bearing mice from 30 min to 5 h 30 min after injection, and all imaging showed good effects, especially at 30 min and 1h30 min. The probe was mainly metabolized by the kidneys and excreted through the bladder, and the uptake in other organs was very low. By processing the images of B16F10 tumor-bearing mice at 1h30min in the software, the highest TBR was calculated to be 14.2±2.2. In HCT116 ( Fig.19 The in vivo imaging results of mice with xenografted subcutaneous tumors were used as a control, and the tumor uptake was very low and almost invisible, which proved that the specificity of the radionuclide probe was significant. 99m The tumor imaging experiment of Tc-HYNIC-YQ-R-11 demonstrated the repeatability of the imaging capability of the radionuclide probe, such as Figure 6 shown.

Claims

1. A CXCR4 targeting polypeptide or a pharmaceutically acceptable salt thereof, characterized in that: The amino acid sequence of the CXCR4 targeting polypeptide is shown in any one of SEQ ID NO.5, SEQ ID NO.15, SEQ ID NO.20, and SEQ ID NOs.23 to 27.

2. A molecular probe, characterized in that The molecular probe is formed by coupling the CXCR4 targeting polypeptide of claim 1 with a fluorescent marker or a radionuclide.

3. The molecular probe according to claim 2, characterized in that The fluorescent markers include one or more of IRDye800, Cy5, Cy7, ICG, rhodamine, and FITC.

4. The molecular probe according to claim 2, characterized in that The radionuclide is selected from 18 F, 68 Ga, 64 Cu, 99m Tc, 90 Y, 111 In, 125 I, 131 I or 177 Lu.

5. The molecular probe according to claim 2, characterized in that The radionuclide chelators include HYNIC, DOTA, NOTA or DTPA.

6. The molecular probe according to claim 2, characterized in that The ligand of the radionuclide includes tris(hydroxymethyl)methylglycine or sodium meta-sulfonate triphenylphosphine.

7. Use of the CXCR4 targeting polypeptide according to claim 1 or the molecular probe according to any one of claims 2 to 6 in the preparation of a reagent for diagnosing a tumor characterized by overexpression of CXCR4.

8. Use of the CXCR4 targeting polypeptide according to claim 1 or the molecular probe according to any one of claims 2 to 6 in the preparation of fluorescent imaging or radioactive imaging agents.

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