CD9-targeted radionuclide labeled polypeptide as well as preparation method and application thereof

Abstract

The invention relates to a radionuclide labeled polypeptide targeting CD9, a preparation method and application, and belongs to the technical field of radioactive medicine. A radionuclide 177Lu is used for labeling a polypeptide specifically targeting CD9 through a difunctional chelating agent DOTA, a maleimide group is used for modification, the radionuclide labeled polypeptide is prepared, the radionuclide binding rate of the radionuclide labeled polypeptide is larger than 95%, and the radionuclide labeled polypeptide has high biological safety and stability, can be efficiently combined with serum albumin, and can be used for preparing the serum albumin labeled polypeptide. Mouse in-vivo experiments prove that the compound specifically targets tumor parts, can be applied to preparation of diagnosis or imaging reagents for glioblastoma, can also remarkably inhibit growth of tumors in mice, and has a positive treatment effect.

Description

Technical Field

[0001] The present invention relates to the technical field of radioactive medicine, and particularly to a radionuclide-labeled polypeptide targeting CD9, a preparation method and an application thereof. Background Art

[0002] Glioma is an invasive primary brain tumor with a poor prognosis. At present, the effective treatment methods for it are limited. Except for surgical operations, therapies such as chemotherapy, radiotherapy, and immunotherapy are all negatively affected by the blood-brain barrier and the immune "cold" microenvironment. Among them, the treatment of glioblastoma (GBM) is particularly hindered, and the main reason is the tumor proliferation and recurrence caused by the cancer cell subsets of glioma stem cells (GSCs) or tumor-initiating cells.

[0003] CD9 is an integral membrane protein belonging to the tetraspanin family, with a molecular weight between 24-27 Kd. It forms a multimeric complex with other cell surface proteins to regulate different cell processes, including cell fusion, adhesion, and motility. In addition, CD9 is also involved in the processes of tumor growth and metastatic implantation, as well as the formation of immune synapses during antigen presentation. Recent studies have shown that CD9 is a biomarker for maintaining the characteristics of glioma stem cells (GSCs), and it has been demonstrated that CD9 promotes IL6-gp130-BMX-STAT3 signal transduction by blocking ubiquitin-dependent lysosomal degradation of gp130, so as to maintain GSC self-renewal and tumorigenic ability, and the high expression of CD9 has a strong positive correlation with the poor prognosis of glioma. More and more evidence indicates that CD9 is expected to become a new target for radionuclide imaging and treatment of glioblastoma, providing new ideas and guidance for non-invasive biological typing and treatment of glioblastoma.

[0004] Currently, there is a lack of in vivo targeting drugs for CD9. Therefore, how to design and provide a radionuclide-labeled polypeptide drug targeting CD9 and successfully apply it in the diagnosis and treatment of glioblastoma is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] To solve the above technical problems, the present invention uses the radionuclide 177 Lu to label a polypeptide specifically targeting CD9 through a bifunctional chelating agent DOTA. The amino acid sequence of the polypeptide is RSHRLRLH, and a radionuclide-labeled polypeptide is prepared and applied to the imaging and treatment of glioblastoma.

[0006] The first object of the present invention is to provide a polypeptide probe targeting CD9, which is composed of a polypeptide with an amino acid sequence shown in SEQ ID NO.1, a maleimide group, and a bifunctional chelator DOTA.

[0007] Further, the amino acid sequence of the polypeptide is RSHRLRLH (arginine - serine - histidine - arginine - leucine - arginine - leucine - histidine).

[0008]

[0009] Further, the structural formula of the polypeptide probe is shown as follows:

[0010] The second object of the present invention is to provide a preparation method of the above - mentioned polypeptide probe, the steps of which include carrying out a coupling reaction between the polypeptide and the bifunctional chelator, and purifying after the reaction to obtain the polypeptide probe.

[0011] The advantages of polypeptide - based targeted radionuclide drugs are mainly manifested as moderate molecular weight, high affinity, low immunogenicity, simple metabolic pathway, etc.

[0012] The third object of the present invention is to provide a radioisotope - labeled polypeptide, which is obtained by labeling the above - mentioned polypeptide probe with a radioisotope, and the radioisotope is 177 Lu.

[0013] The fourth object of the present invention is to provide a preparation method of the above - mentioned radioisotope - labeled polypeptide, the steps of which include mixing and reacting the polypeptide probe with the radioisotope to obtain the radioisotope - labeled polypeptide probe.

[0014] Further, the preparation method includes the following steps:

[0015] (1) Preparation of precursor: Dissolve DOTA - M - P in ultrapure water to prepare a 1 mM mother liquor;

[0016] (2) Reaction mixture: Take 1 μL of the precursor solution and mix it with 37 MBq 177 LuCl3, and add 50 μL of sodium acetate buffer solution (0.4 M) with pH 4.5;

[0017] (3) High - temperature chelation: Carry out a constant - temperature reaction in a metal bath at 95 °C for 30 minutes.

[0018] The fourth object of the present invention is to provide the application of the above - mentioned radioisotope - labeled polypeptide in the preparation of a diagnostic reagent for glioblastoma.

[0019] The fifth object of the present invention is to provide the application of the above - mentioned radioisotope - labeled polypeptide in the preparation of an imaging agent for glioblastoma.

[0020] The sixth object of the present invention is to provide the use of the above-mentioned radionuclide-labeled polypeptide in the preparation of a therapeutic drug for glioblastoma.

[0021] The seventh object of the present invention is to provide a therapeutic drug for glioblastoma, which comprises the above-mentioned radionuclide-labeled binding peptide and a pharmaceutically acceptable carrier.

[0022] Furthermore, the therapeutic drug for glioblastoma is administered by intravenous injection, and the dosage is 7.4 - 18.4 MBq.

[0023] Preferably, the dosage of the therapeutic drug for glioblastoma is 18.4 MBq.

[0024] Advantages of the present invention:

[0025] The present invention prepares a radionuclide-labeled polypeptide, the radionuclide binding rate of which is greater than 95%, and it has high biological safety and stability, can bind efficiently with serum albumin, and at the same time inhibits the growth of glioblastoma. The IC 50 value for human glioblastoma cells is 63.9 nM. Verified by in-vivo experiments on mice, it specifically targets tumor sites, can be applied to the preparation of diagnostic or imaging reagents for glioblastoma, and can also significantly inhibit the growth of tumors in mice, achieving a positive therapeutic effect, providing a brand-new tool and direction for the diagnosis, tracking and treatment of glioblastoma. Brief Description of the Drawings

[0026] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, where

[0027] Figure 1 is a schematic structural diagram of DOTA-M-P in Example 1 of the present invention;

[0028] Figure 2 is an MS mass spectrometry result diagram of DOTA-M-P in Example 1 of the present invention;

[0029] Figure 3 is the 1 H-NMR nuclear magnetic result diagram of DOTA-M-P in Example 1 of the present invention;

[0030] Figure 4 is the safety test result of DOTA-M-P in Example 2 of the present invention;

[0031] Figure 5 is in Example 3 of the present invention 177 TLC thin layer chromatography result of Lu-DOTA-M-P;

[0032] Figure 6 For Example 3 of the present invention 177 Radioactivity detection results of Lu-DOTA-M-P;

[0033] Figure 7 For Example 4 of the present invention 177 Binding rate detection results of Lu-DOTA-M-P with human serum albumin;

[0034] Figure 8 For Example 5 of the present invention 177 Inhibitory effect detection of Lu-DOTA-M-P on CD9-transfected human brain astrocytoma cells;

[0035] Figure 9 For Example 6 of the present invention 177 Verification of in vitro targeting effect of Lu-DOTA-M-P on CD9-transfected human brain astrocytoma cells;

[0036] Figure 10 For Example 7 of the present invention 177 Growth inhibition experimental results of Lu-DOTA-M-P on CD9-transfected human brain astrocytoma cells;

[0037] Figure 11 For Example 8 of the present invention 177 Determination results of the distribution of Lu-DOTA-M in mice;

[0038] Figure 12 For Example 9 of the present invention 177 Determination results of the plasma half-life of Lu-DOTA-M in mice;

[0039] Figure 13 For Example 10 of the present invention 177 Verification results of the targeting of Lu-DOTA-M in mice;

[0040] Figure 14 For Example 10 of the present invention 177 Determination of the retention of Lu-DOTA-M at the tumor site in mice;

[0041] Figure 15 For Example 11 of the present invention 177 Tumor growth curve in mice after injection of Lu-DOTA-M;

[0042] Figure 16 For Example 11 of the present invention 177 Actual picture of the tumor in mice after injection of Lu-DOTA-M;

[0043] Figure 17 The structural formula of A-P in the comparative example of the present invention;

[0044] Figure 18 In the comparative example of the present invention 125 The labeling rate result of I-A-P;

[0045] Figure 19 In the comparative example of the present invention 125 The MS mass spectrum result diagram of I-A-P;

[0046] Figure 20 In the comparative example of the present invention 125 of I-A-P in the present invention 1 The 1H-NMR nuclear magnetic result diagram;

[0047] Figure 21 In the comparative example of the present invention 125 The in vitro stability test result of I-A-P;

[0048] Figure 22 In the comparative example of the present invention 125 The inhibitory effect test of I-A-P. Detailed implementation manners

[0049] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the specific embodiments cited are not intended to limit the present invention.

[0050] Example 1: Preparation of DOTA-M-P

[0051] The structural formula of DOTA-M-P is as Figure 1 shown, where P is a polypeptide with the amino acid sequence RSHRLRLH (shown in SEQ ID NO.1), M is a maleimide group (Mal), and DOTA is a bifunctional chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid.

[0052] The specific synthesis steps of DOTA-M-P are as follows:

[0053] (1) Resin swelling: Put dichloride resin (2-Chlorotrityl Chloride Resin) into a solid-phase reaction tube, add anhydrous dichloromethane (DCM), and the ratio of anhydrous dichloromethane to resin is (15-20):1 (mL:g), and shake for 20-30 min.

[0054] (2) Linkage of the first amino acid to the dichloro resin. Blow off the DCM, dissolve 3-fold N-Fmoc-N'-trityl-L-histidine and 3.6-fold N,N-diisopropylethylamine (DIPEA) with anhydrous N,N-dimethylformamide (DMF) at 15 - 20 mL / g resin, and add it to the solid-phase reaction tube.

[0055] (3) Capping. Dissolve 10-fold methanol and DIPEA with DMF at 15 - 20 mL / g resin, add it to the solid-phase reaction tube, and react for 10 min.

[0056] (4) Removal of the Fmoc protecting group. Blow off the reaction solution in step (3), wash the resin three times with DMF, add piperidine / DMF (20% / 80%, V / V) solution with an addition amount of 12 - 18 mL / g resin, react for 10 min, and repeat three times.

[0057] (5) Detection. Take a little resin, add 50 μL of ninhydrin solution, heat at 105 - 110 °C for 3 - 5 min. A dark blue color change indicates a positive reaction, and then wash the resin with DMF;

[0058] (6) Condensation. Add 2-fold raw material containing an exposed carboxyl group, 2.4-fold O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and 2.4-fold DIPEA dissolved in DMF, detect with ninhydrin after reacting for 4 h, and wash the resin three times with DMF.

[0059] (7) Repeat steps (4) - (6) to sequentially link each component. The remaining components are N-Fmoc-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, N-Fmoc-L-leucine, N-Fmoc-L-serine tert-butyl ester, trans-4-[(9H-fluoren-9-ylmethoxycarbonylamino)-methyl]-cyclohexanecarboxylic acid, N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl-N'-Fmoc-L-lysine, DOTA-tris(t-Bu) ester (2-(4,7,10-tris(2-(t-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid).

[0060] (8) Removal of the Dde protecting group. Add hydrazine hydrate / DMF (2% / 98%, V / V) solution with an addition amount of 12 - 18 mL / g resin, react for 5 min, and repeat three times. Add 2-fold 6-maleimidohexanoic acid, 2.4-fold HBTU, and 2.4-fold DIPEA dissolved in DMF, and detect with ninhydrin after reacting for 4 h.

[0061] (9) Wash the resin, and wash it successively with DMF, methanol, petroleum ether, and DCM.

[0062] (10) Cut the resin. Use a DCM solution of 1% TFA to cut the resin, 15 - 20 mL / g resin each time, with a cutting time of 10 min each time, and collect the cutting solution.

[0063] (11) Remove the protecting group. Add 5 mL of a solution of TFA / H2O (95% / 5%, V / V) to the solution after concentration under reduced pressure, and stir at room temperature for 4 h.

[0064] (12) Add 20 mL of DCM and concentrate under reduced pressure. Drop the concentrated liquid into 10 times its volume of ether to precipitate the deprotected crude peptide, and dry it under vacuum.

[0065] (13) Purification and preparation. Take 10 mg of the crude peptide, add 1 mL of HPLC-grade methanol and 1 mL of water dropwise to dissolve it; take 20 μL of the sample and analyze it on an HPLC analyzer to determine the elution time corresponding to the target peak; use a C18 reversed-phase chromatography preparation system to collect the target peak solution, and set the HPLC parameters: Wavelength: 220 nm; Flow Rate: 9 mL / min; Inj.Vol: 20 mL ColumnTemp: 25 °C; Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in methanol. Collect the target peak solution and lyophilize it to obtain a white powdery substance, namely the DOTA-M-PDOTA-M-P structure, which is identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) (the detection results are as Figure 2 shown) and nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) (the detection results are as Figure 3 shown).

[0066] Example 2: Safety analysis of DOTA-M-P

[0067] (1) Take human umbilical vein endothelial cells (HUVEC) in the logarithmic growth phase, digest them with 0.25% trypsin and adjust to 6×10 4 / mL, seed in 96-well plates, 100 μL per well, with a cell concentration of 6×10 3 cells per well, and incubate overnight in a 37 °C, 5% CO2 incubator;

[0068] (2) Change the medium for each group of cells, add 100 μL of the culture medium at the specified concentration, and the dose concentrations of DOTA-M-P are 0, 2, 4, 8, 10, 20, 40, 80 μM;

[0069] (3) After 24 h, aspirate the culture medium, add 100 μL containing 10 μL of CCK8 solution (10%), and continue to incubate in the incubator for 1 h;

[0070] (4) After 1 h, measure the absorbance at 450 nm in each well using a microplate reader. Then calculate the relative cell viability based on the measured OD value. Cell viability = (drug-treated group - blank) / (control group - blank) × 100%. Figure 4 The results of the biosafety of DOTA-M-P incubated in HUVEC cells for 24 h are shown. It can be seen that DOTA-M-P has high biosafety and does not affect the survival of HUVEC cells after 24 h of incubation.

[0071] Example 3: Radionuclide labeling and labeling stability of DOTA-M-P

[0072] (1) Accurately weigh a certain mass of DOTA-M-P into a sample vial and add an appropriate amount of ultrapure water to prepare a 1 mM solution.

[0073] (2) Pipette 1 μL of the precursor solution and mix it with 37 MBq 177 LuCl3, and then add 50 μL of sodium acetate (NaOAc) solution (0.4 M, pH = 4.5) and mix well to obtain the reaction solution.

[0074] (3) Place the above reaction tube in a 95 °C metal bath and react for 30 min.

[0075] (4) After the reaction solution is cooled to room temperature, centrifuge it briefly and analyze the quality control by radio-thin layer chromatography (radio-TLC). The stationary phase is fast silica gel paper and the developing agent is 1% EDTA solution. The results are as Figure 5 shown, and the labeling rate is higher than 95% and no purification is required.

[0076] (5) In the in vitro radiostability study, 177 Lu-DOTA-M-P is incubated with PBS or mouse serum at 37 °C for 48 h. Collect a small amount of the mixture at different time points and measure the radioactivity using a radio-TLC scanner. The results are as Figure 6 shown, indicating that 177 Lu-DOTA-M-P has good stability in vivo and in vitro.

[0077] Example 4: Human serum albumin binding experiment

[0078] According to the method provided in Example 3, use 125 I to label the polypeptide (amino acid sequence as shown in SEQ ID NO.1) to obtain 125 I-P as a control.

[0079] (1) Dissolve 2 mg of human serum albumin HSA in 400 μL of physiological saline.

[0080] (2) Add the same radioactive count of177 Lu-DOTA-M-P and 125 I-P were co-incubated at 37 °C and 800 rpm for 1 h and 4 h.

[0081] (3) The samples were transferred to a 30 kDa ultrafiltration tube for separation and purification, centrifuged at 14,000 rpm for 15 minutes to obtain a concentrated solution, and the radioactivity of the concentrated solution and the filtrate was measured separately using a gamma counter, denoted as A and B. The binding rate to HSA was defined as A / (A + B) × 100%, and the binding rate results are as Figure 7 shown. It can be seen that 177 the binding rate of Lu-DOTA-M-P to human serum albumin is significantly higher than 125 that of I-P.

[0082] Example 5: Inhibitory experiment on human brain astrocytoma cells

[0083] (1) U87 cells (human brain astrocytoma cells) transfected with CD9 in the logarithmic growth phase were digested with 0.25% trypsin, seeded in a 24-well plate, and 10 6 cells were inoculated in each well. Incubated overnight in a 37 °C, 5% CO2 incubator.

[0084] (2) The next day, after the cells were completely adhered, the medium was replaced with medium containing 74 kBq 177 Lu-DOTA-M-P and different concentrations of DOTA-M-P (0.01, 0.1, 1, 10, 100, and 1000 nM), and 3 replicates were set for each group.

[0085] (3) The medium containing the radiolabeled drug ( 177 Lu-DOTA-M-P) was incubated with the cells for 4 h.

[0086] (4) The supernatant was removed, the cells were washed twice with PBS, lysed with 0.5 mL NaOH (0.5 M) at the bottom of the well, and washed twice with PBS and collected in a radioimmunoassay tube.

[0087] (5) The radioactivity accumulated in the cells was measured using a gamma counter, and the data was fitted using a non-linear regression algorithm (GraphPad Software) to calculate the 50% inhibitory concentration (IC 50 value), and the results are as Figure 8 shown. The IC 50 value was 63.9 nM.

[0088] Example 6: 177 Verification of the in vitro targeting of Lu-DOTA-M-P

[0089] (1) Take the logarithmically growing U87 cells transfected with CD9, U87 cells and Raji cells, digest the cells with 0.25% trypsin, seed them in 24-well plates, and inoculate 10 6 cells per well. Incubate overnight in a 37 °C, 5% CO2 incubator.

[0090] (2) After the cells adhered completely the next day, replace the medium with the medium supplemented with 74 kBq 177 Lu-DOTA-M-P and an excess of DOTA-M-P (100 μM). Set up 3 replicate wells for each group.

[0091] (3) Incubate the medium supplemented with the radiolabeled drug with the cells for 4 h.

[0092] (4) Aspirate the medium, wash the cells twice with cold PBS, collect the liquid in a radioimmunoassay tube, and measure its radioactivity using a gamma counter, which is designated as A.

[0093] (5) Lyse the cells at the bottom of the wells with 0.5 mL NaOH (0.5 M) and wash twice with PBS, then collect all in a radioimmunoassay tube. Measure its radioactivity using a gamma counter, which is designated as B. The cell uptake rate is recorded as T = B / (A + B) × 100%. The results are as Figure 9 shown. After the addition of an excess of cold probe DOTA-M-P, an inhibitory effect on the uptake of U87-CD9 and U87 cells was produced, while there was no similar phenomenon in Raji cells that do not express CD9, indicating the specific binding of the probe to the CD9 protein....

[0094] Example 7: 177 Experiment on the growth inhibitory effect of Lu-DOTA-M-P on CD9-transfected U87 cells

[0095] (1) Take the logarithmically growing CD9-transfected U87 cells, digest with 0.25% trypsin and adjust the cell concentration to 6×10 4 / mL, seed in 96-well plates, 100 μL per well, and the cell concentration is 6×10 3 cells per well. Incubate overnight in a 37 °C, 5% CO2 incubator.

[0096] (2) After the cells adhered overnight, change the medium for each group, add 100 μL of the specified dose of culture medium, and add different dose concentrations of 177 Lu-DOTA-M-P (0, 0.5, 1, 2, 4, 8 and 16 uCi, 100 μL / well) to the 96-well plates.

[0097] (3) After incubating for 24 h, aspirate the cell medium, add fresh medium (90 μL / well), and mix with CCK-8 (5 mg / mL, 10 μL / well)

[0098] (4) After incubation for 4 h, the absorbance of CCK-8 at 450 nm was recorded using a microplate reader, and the cell viability was calculated based on the ratio of the absorbance of the experimental wells to that of the cell control wells. Cell viability = (drug-treated group - blank) / (control group - blank) × 100%. The results are as Figure 10 shown. It can be seen that as 177 the concentration of Lu-DOTA-M-P increases, the growth of CD9-transfected U87 cells is significantly inhibited.

[0099] Example 8: 177 Biodistribution determination of Lu-DOTA-M

[0100] Each U87-CD9 tumor-bearing athymic nude mouse was injected with 150 μCi 177 Lu-DOTA-M-P via the tail vein. At 1, 2, 4, 8, 24, and 48 h after injection, the tumors and each important organ were dissected and separated. The weight of each organ was measured, and the %ID / g of each organ was calculated as %ID / g = (radioactivity count of the organ) / (weight of the organ × source count) × 100%. Three mice were used at each time point. The results are as Figure 11 shown. The radioactive uptake at the tumor site increased with time and reached a relatively high level at 4 h and 8 h. At the same time, the probe was cleared by the kidney, avoiding long-term radioactive accumulation in non-target organs and causing damage.

[0101] Example 9: 177 Plasma half-life determination of Lu-DOTA-M

[0102] Mice were injected with 177 Lu-DOTA-M-P and 125 I-P via the tail vein. Blood samples were collected from the retinal vein at 1, 5, 15, 30 min, 1, 2, 4, 8, and 24 h for gamma counter measurement. The radioactive counts of the organs were calculated from the mouse blood to obtain ID% / g. Three mice were used at each time point, and the average values at each time point were used to fit the plasma half-life parameters using a two-compartment model. The results are as Figure 12 shown. 177 The plasma half-life of Lu-DOTA-M-P in mice was higher than that of 125 I-P, indicating a shorter drug elimination time.

[0103] Example 10: 177 Verification of in vivo targeting and specificity of Lu-DOTA-M

[0104] (1) U87 cells transfected with CD9 in the logarithmic growth phase were digested with trypsin and resuspended, and then inoculated into the right forelimb of 5-week-old nude mice to form tumors. The cell injection amount for each mouse was 4 × 10 6 .

[0105] (2) Wait for the tumor to grow to 400 cm 3 or so, and then inject 7.4 MBq 177 of Lu-DOTA-M-P into the tail veins of different mice. (In the BLCOK group, 100 μL of physiological saline containing 500 μg of DOTA-M-P was injected through the tail vein in advance, and static imaging was performed for 10 minutes by U-SPECT+ / CT (MILabs) at 1, 2, 4, 8, 24, and 48 h after administration (after pre-anesthetizing with a 3% volume fraction of isoflurane-oxygen mixed gas, placing on the PET / CT scanning bed, and then maintaining anesthesia with a 1.5% volume fraction of isoflurane-oxygen mixed gas). The imaging results are as Figure 13 shown, and it can be seen that 177 Lu-DOTA-M-P has good in vivo targeting and retention. The quantitative result of ID% / g uptake at the tumor site is as Figure 14 shown. Compared with the control group, 177 the retention of Lu-DOTA-M-P at the tumor site has been significantly improved.

[0106] Example 11: 177 Targeted radionuclide therapy with Lu-DOTA-M-P and evaluation of efficacy

[0107] (1) Take U87 cells transfected with CD9 in the logarithmic growth phase, resuspend them after trypsin digestion to 4×10 7 / mL, and inoculate tumors on the right forelimbs of nude mice aged 4-5 weeks. The injection volume for each mouse is 50 μL, and the cell quantity is 4×10 6 .

[0108] (2) When the tumor grows to a major axis exceeding 6 mm, divide the tumor-bearing mice into 3 groups (5 mice in each group), and inject through the tail vein respectively: physiological saline group (150 μL), 177 Lu-DOTA-M-P group (150 μL, containing 9.25 MBq 177 of Lu-DOTA-M-P), 177 Lu-DOTA-M-P group (150 μL, containing 18.5 MBq 177 of Lu-DOTA-M-P).

[0109] (3) Measure the tumor size of each group of mice using a vernier caliper every three days, and the volume calculation = (tumor length) × (tumor width) 2 / 2. Stop the experiment when the tumor volume of the control group exceeds 1500 mm 3 . The tumor growth curve is as Figure 15 shown. When the MBq is 18.5, the growth rate of tumors in mice significantly slows down. The physical picture of the tumor is as Figure 16 shown. For 18.5 MBq of 177The inhibitory effect of the Lu-DOTA-M-P treatment group was significant, and overall, it presented a dose-dependent tumor growth inhibition result.

[0110] Comparative example

[0111] Synthesize A-P (where A is 4-(p-iodophenyl)butyric acid and P is a polypeptide, and the amino acid sequence of the polypeptide is as shown in SEQ ID NO.1), and the structural formula is as Figure 17 shown. Use 125 I to label A-P to obtain 125 I-A-P, and the labeling rate is as Figure 18 shown. Perform matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) (the detection results are as 125 Figure 19 shown) and nuclear magnetic resonance hydrogen spectrum ( 1 1H-NMR) identification (the detection results are as Figure 20 shown) on the structure of 125

[0112] The specific synthesis steps of A-P are as follows:

[0113] (1) Resin swelling: Put 2-Chlorotrityl Chloride Resin into a solid-phase reaction tube, add anhydrous dichloromethane (DCM), and the ratio of anhydrous dichloromethane to resin is (15 - 20):1 (mL:g), and shake for 20 - 30 min.

[0114] (2) Connection of the first amino acid to the dichloride resin. Blow off the DCM, dissolve 3 times of N-fluorenylmethoxycarbonyl-N'-triphenylmethyl-L-histidine and 3.6 times of N,N-diisopropylethylamine (DIPEA) with 15 - 20 mL / g resin of anhydrous N,N-dimethylformamide (DMF), and add it to the solid-phase reaction tube.

[0115] (3) Capping. Dissolve 10 times of methanol and DIPEA with 15 - 20 mL / g resin of DMF, and add it to the solid-phase reaction tube, and react for 10 min.

[0116] (4) Removal of the Fmoc protecting group. Blow off the reaction solution in step (3), wash the resin three times with DMF, add piperidine / DMF (20% / 80%, V / V) solution, and the addition amount is 12 - 18 mL / g resin, and react for 10 min, and repeat three times.

[0117] (5) Detection. That is, take a little resin, add 50 μL of ninhydrin solution, heat it at 105 - 110 °C for 3 - 5 min, and a dark blue color change is a positive reaction, and wash the resin with DMF;

[0118] (6) Condensation: Add 2-fold of the raw material containing exposed carboxyl groups, 2.4-fold of benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (HBTU), and 2.4-fold of N,N-diisopropylethylamine (DIPEA) dissolved in DMF. After reacting for 4 h, detect with ninhydrin and rinse the resin three times with DMF.

[0119] (7) Repeat steps (4)-(6) to sequentially connect each component. For the remaining components, use N-[(9H-fluoren-9-yl)methoxycarbonyl]-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine, N-[(9H-fluoren-9-yl)methoxycarbonyl]-L-leucine, N-[(9H-fluoren-9-yl)methoxycarbonyl]-L-serine tert-butyl ester, trans-4-[(9H-fluoren-9-ylmethoxycarbonylamino)methyl]cyclohexanecarboxylic acid, Nα-[(9H-fluoren-9-yl)methoxycarbonyl]-Nε-tert-butoxycarbonyl-L-lysine.

[0120] (8) Remove the Dde protecting group: Add a hydrazine hydrate / DMF (2% / 98%, V / V) solution in an amount of 12 - 18 mL / g of resin, react for 5 min, and repeat three times. Add 2-fold of 4-(4-iodophenyl)butyric acid, 2.4-fold of HBTU, and 2.4-fold of DIPEA dissolved in DMF. After reacting for 4 h, detect with ninhydrin.

[0121] (9) Rinse the resin: Wash the resin successively with DMF, methanol, petroleum ether, and dichloromethane (DCM).

[0122] (10) Cleave the resin: Use a 1% trifluoroacetic acid (TFA) solution in DCM to cleave the resin, 15 - 20 mL / g of resin each time, with a cleavage time of 10 min each time, and collect the cleavage solution.

[0123] (11) Deprotect: Add 5 mL of a TFA / H2O (95% / 5%, V / V) solution to the solution after concentration under reduced pressure, and stir at room temperature for 4 h.

[0124] (12) Add 20 mL of DCM and concentrate under reduced pressure. Drop the concentrated liquid into 10-fold volume of diethyl ether to precipitate the deprotected crude peptide, and dry it under vacuum.

[0125] (13) Purification and preparation: Take 10 mg of crude peptide, add dropwise 1 mL of HPLC-grade methanol and 1 mL of water to dissolve it; take 20 μL of the sample and analyze it on an HPLC analyzer to determine the elution time corresponding to the target peak; use a C18 reverse-phase chromatography preparation system to collect the target peak solution, and set the HPLC parameters: Wavelength: 220 nm; Flow Rate: 9 mL / min; Inj.Vol: 20 mL ColumnTemp: 25 °C; Buffer A: 0.1% TFA in water Buffer B: 0.1% TFA in methanol. Collect the target peak solution and lyophilize it to obtain a white powdery substance, namely A-P.

[0126] According to the method provided in Example 3, 125 the in vitro stability of I-A-P was detected, and the detection results are as Figure 21 shown. It can be seen that 125 I-A-P has certain problems in in vitro stability. According to the method provided in Example 5, 125 the inhibitory ability of I-A-P was detected, and the detection results are as Figure 22 shown, and the 125 IC 50 value of I-A-P was 244.6 nM, which is much higher than the Figure 8 IC 177 value of Lu-DOTA-M-P, which is 63.9 nM in 50 Example 5 (corresponding attachment Figure 8 ). This comparative example verified that even based on the same polypeptide sequence, different synthesis methods and different labeling methods will affect its stability and inhibitory ability.

[0127] Obviously, the above examples are only illustrations given for clarity and are not limitations on the implementation methods. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. A polypeptide probe targeting CD9, characterized in that: The polypeptide probe consists of a polypeptide with an amino acid sequence as shown in SEQ ID NO.1, a maleimide group, and a bifunctional chelator DOTA.

2. The polypeptide probe according to claim 1, wherein The structural formula of the polypeptide probe is as follows:

3. The preparation method of the polypeptide probe according to claim 1 or 2, characterized in that: The step includes carrying out a coupling reaction between the polypeptide and the bifunctional chelator, and purifying after the reaction to obtain the polypeptide probe.

4. A radioisotope-labeled polypeptide, characterized in that: The radioactive nuclide-labeled polypeptide probe is obtained by labeling the polypeptide probe according to Claim 1 or 2 with a radioactive nuclide, and the radioactive nuclide is 177 Lu.

5. The preparation method of the radionuclide-labeled polypeptide according to claim 4, characterized in that: The step includes mixing and reacting the polypeptide probe with the radionuclide to obtain the radionuclide-labeled polypeptide probe.

6. Use of the radionuclide-labeled polypeptide according to claim 4 in the preparation of a diagnostic reagent for glioblastoma.

7. Use of the radionuclide-labeled polypeptide according to claim 4 in the preparation of an imaging agent for glioblastoma.

8. Use of the radionuclide-labeled polypeptide according to claim 4 in the preparation of a therapeutic drug for glioblastoma.

9. A drug for treating glioma, characterized in that: The therapeutic drug for glioblastoma contains the radionuclide-labeled polypeptide according to claim 4 and a pharmaceutically acceptable carrier.

10. The glioma treatment drug according to claim 9, wherein: The therapeutic drug for glioblastoma is administered by intravenous injection, and the dosage is 7.4 - 18.4 MBq.

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