Targeted therapy for glioma 211 At-labeled polypeptide and preparation method thereof

Abstract

The invention discloses a method for targeted treatment of brain glioma 211 At-labeled polypeptide and its preparation method belong to the field of new biomedical technology applications. 211 At-labeled peptides, made from the following components: NPY1R-targeting peptide KINW, bifunctional chelator m‑MeATE, and α-nuclides 211 At, the primary amine on the peptide KINW targeting NPY1R is coupled to the succinimide of the chelator m‑MeATE through an amide bond, and the α-nuclides 211 At is chelated to the chelating agent m-MeATE. Compared with the prior art, the 211 At-labeled polypeptides have the characteristics of high preparation efficiency, high uptake rate of brain glioma cells, high in vitro stability, good therapeutic effect and low toxicity to normal tissues.

Description

Technical Field

[0001] The present invention belongs to the field of new biomedical technology applications, and specifically relates to a method for targeted treatment of brain gliomas. 211 At-labeled polypeptide and preparation method thereof. Background Art

[0002] Glioblastoma multiforme (GBM) is the highest grade astrocytic tumor, with a median survival of less than 15 months and a 5-year survival rate of only 5%. Due to the strong invasiveness of GBM, it is difficult to completely remove it surgically. Radiotherapy and chemotherapy are still the main treatment methods, but the therapeutic effect on GBM still shows limitations. In recent years, targeted alpha therapy (TAT) has played an increasingly important role in cancer treatment. Compared with β-nuclides, the energy transfer line density of α particles released by α-nuclides is 50-230keV / μm, which can effectively trigger DNA double-strand breaks in tumor cells. In addition, the average path length of α particles is only 50-100μm, which is equivalent to the length of 5-10 cell diameters. Therefore, TAT has a short killing distance, which effectively damages tumor cells while avoiding damage to normal organs and adjacent healthy tissues. Therefore, the development of new α-nuclides targeted drugs is of great significance to conquer cancer.

[0003] 211 The half-life of At is 7.21h. 211 At-labeled targeted drugs have been used in the treatment of tumors such as ovarian cancer, melanoma, thyroid cancer and glioma, and have potential for clinical application. Discovering GBM-specific expression targets and screening high-affinity ligands are prerequisites for the development of GBM targeted drugs. The Y1R and Y2R subtypes of neuropeptide Y (NPY) are upregulated in many types of human tumors, including breast cancer, adrenal tumors, renal cell carcinoma, ovarian cancer and glioma, and are potential targets for tumor diagnosis and treatment. Peptides have the advantages of easy structural modification, small molecular weight, rapid tumor uptake and rapid clearance from normal tissues. Currently, there are already methods for using 211 At 225 Ac and 213 Bi and other α-nuclides labeled substance P (natural peptide ligand of neurokinin 1) are used to prepare α-nuclides targeting drugs for glioma and applied to relevant research on clinical trials. Therefore, for the developed NPY1R targeting peptide, preparation 211 At-labeled α-nuclides targeted therapeutic drugs have potential clinical practical value in the treatment of brain gliomas. Summary of the invention

[0004] Based on the above background, the purpose of the present invention is to provide a method for treating glioma. 211 At-labeled polypeptide and preparation method thereof.

[0005] The targeted treatment of brain glioma provided by the present invention 211 At-labeled peptides, made from the following components: NPY1R-targeting peptide KINW, bifunctional chelator m-MeATE, and α-nuclides 211 At, the primary amine on the peptide KINW targeting NPY1R is coupled to the succinimide of the chelator m-MeATE through an amide bond, α-nuclides 211 At is chelated to the chelating agent m-MeATE;

[0006] The amino acid sequence of the NPY1R-targeting polypeptide KINW is as follows: KINWKFRLRY, lysine-isoleucine-asparagine-tryptophan-lysine-phenylalanine-arginine-leucine-arginine-tyrosine, Lys-Ile-Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINW.

[0007] The structural formula of the chelating agent m-MeATE is as follows:

[0008]

[0009] The targeted treatment of brain glioma 211 At-labeled polypeptide is expressed as: 211 At-m-MeATE-KINW, abbreviated as 211 At-ATE-KINW, where 211 At is the α-nuclides, m-MeATE is the chelating agent, and KINW is the peptide targeting NPY1R protein.

[0010] The present invention prepares an α-nuclide targeted therapeutic drug with a specific targeted therapeutic effect on human brain glioma cell transplanted tumors, and explores the 211 The results showed that At-labeled KINW has an in vivo targeted therapeutic effect on brain glioma cells U87-MG cells and transplanted tumors. 211 At-labeled KINW has a therapeutic effect on brain glioma and has potential application value in targeted treatment of brain glioma.

[0011] Another object of the present invention is to provide the above-mentioned targeted treatment of brain glioma 211 Method for preparing At-labeled polypeptide.

[0012] The targeted treatment of brain glioma provided by the present invention 211 The preparation method of At-labeled polypeptide comprises the following steps:

[0013] 1) Preparation of Chelating Agent-Peptide

[0014] Under alkaline conditions, the bifunctional chelator m-MeATE and the polypeptide KINW are coupled to obtain the chelator-polypeptide m-MeATE-KINW, abbreviated as ATE-KINW;

[0015] 2) Preparation of α-nuclides labeled chelating agent-peptide

[0016] Add α-nuclides to the chelating agent-peptide m-MeATE-KINW and methanol reaction solution 211 At and N-iodosuccinimide (NIS), incubate, and terminate the reaction to obtain 211 At-labeled chelator-peptide, a targeted therapy for brain glioma 211 At-labeled peptide 211 At-m-MeATE-KINW, abbreviated as 211 At-ATE-KINW.

[0017] In step 1) of the above method, the coupling reaction is carried out in dimethyl sulfoxide containing 1% (volume) triethylamine, and the temperature of the coupling reaction can be 37-40° C., specifically 37° C.;

[0018] The time can be 12-18h, specifically 12h;

[0019] The molar ratio of the polypeptide to the chelating agent is 1:4-1:6, specifically 1:6;

[0020] Specifically, the operation of step 1) of the above method is:

[0021] The polypeptide (KINWKFRLRY) is dissolved in dimethyl sulfoxide containing 1% (volume) triethylamine, the polypeptide molar concentration is 2 μmol, 4-6 times the molar amount of m-MeATE (specifically 12 μmol) is added for coupling reaction, the mixture is reacted in a 37°C water bath for 12 hours, and purified by reversed-phase high performance liquid chromatography, the initial gradient of the mobile phase is: an initial gradient of 60% mobile phase A (0.1% trifluoroacetic acid aqueous solution) and 40% mobile phase B (acetonitrile containing 0.1% trifluoroacetic acid); the ratio is uniformly changed to 20% mobile phase A and 80% mobile phase B in 0.1-20 minutes; the ratio is uniformly changed to 5% mobile phase A and 95% mobile phase B in 20-30 minutes; the mobile phase ratio is maintained for 10 minutes (30-40 minutes); the retention time difference of different compounds on a C18 reversed-phase column is used to determine and separate and purify the coupling compound.

[0022] In step 2), the α nuclide 211 The activity of At is about 180MBq;

[0023] The mass ratio of NIS to chelator-polypeptide can be 3:20.

[0024] The incubation temperature may be 37°C, and the incubation time may be 10-15 minutes, specifically 10 minutes;

[0025] The solution used to terminate the reaction is Na2S2O3 solution, and the mass ratio of Na2S2O3 to NIS in the Na2S2O3 solution is 20:3.

[0026] Specifically, the operation of step 2) of the above method is:

[0027] The freeze-dried ATE-KINW was dissolved in methanol (10 mg / mL), and 10 μL of ATE-KINW solution was mixed with 50 μL of 211 At (about 180 MBq) and 5 μL of N-iodosuccinimide (NIS) (dissolved in methanol containing 1% acetic acid, concentration of 3 mg / mL) were mixed and incubated at 37°C for 10 min; 5 μL of Na2S2O3 solution (dissolved in ultrapure water, concentration of 20 mg / mL) was added to terminate the labeling reaction; the product was purified by SephadexTM G-10 column, and 0.1 mol / L phosphate buffered saline (PBS, pH 7.6) was used as the elution solution; radioactive TLC (thin layer chromatography) was used for quality control, and acetone: water (v / v = 3:1) was used as the mobile phase ([R f ] 211 At-ATE-peptide = 0-0.1, [R f ] 211 At=0.9-1).

[0028] The above targeted therapy for glioma 211 The use of At-labeled polypeptides in the preparation of tumor treatment reagents also falls within the protection scope of the present invention.

[0029] In the application, the tumor includes a brain glioma, and specifically may be a glioblastoma multiforme.

[0030] According to the embodiment of the present invention, the selected human brain glioma cells are U87-MG cells, and the selected in vivo brain glioma transplanted tumor is a BABL / c subcutaneous transplanted tumor.

[0031] The present invention also provides a drug for treating brain glioma, wherein the drug contains the above-mentioned targeted treatment of brain glioma. 211 At-labeled peptide.

[0032] The invention provides a method for treating glioma by targeting 211 At-labeled polypeptide (specifically 211Compared with the existing technology, At–m-MeATE-KINW) has the following beneficial effects:

[0033] The present invention prepares 211 At-labeled polypeptides have the characteristics of high preparation efficiency, high uptake rate of brain glioma cells, high in vitro stability, good therapeutic effect and low toxicity to normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the structural formula of polypeptide KINW in Example 1 of the present invention.

[0035] Figure 2 The HPLC and MS identification results of the polypeptide KINW prepared in Example 1 of the present invention are shown.

[0036] Figure 3 is the structural formula of ATE-KINW prepared in Example 1 of the present invention.

[0037] Figure 4 HPLC and MS identification results of ATE-KINW prepared in Example 1 of the present invention.

[0038] Figure 5 Prepared in Example 1 of the present invention 211 TLC test results of At-ATE-KINW, where A is 211 At-ATE-KINW marking rate, B is 211 At-ATE-KINW purification elution curve, C is 211 Radiochemical purity of At-ATE-KINW after purification.

[0039] Figure 6 Prepared in Example 1 of the present invention 211 The structural formula of At-ATE-KINW.

[0040] Figure 7 In Example 2 of the present invention 211 In vitro stability experimental results of At-ATE-KINW.

[0041] Figure 8 The U87-MG cell pair in Example 3 of the present invention 211 Uptake results of At-ATE-KINW. **p﹤0.01, ***p﹤0.001.

[0042] Fig. 9 In Example 4 of the present invention 211 Uptake and distribution of At-ATE-KINW in various tissues and organs of normal mice.

[0043] Fig.10 In Example 5 of the present invention211 Effect of At-ATE-KINW on the survival rate of U87-MG cells. **p﹤0.01, ***p﹤0.001.

[0044] Fig.11 In Example 5 of the present invention 211 Effect of At-ATE-KINW on the apoptosis rate of U87-MG cells. *p﹤0.05, **p﹤0.01.

[0045] Fig.12 In Example 5 of the present invention 211 Effect of At-ATE-KINW on the survival of U87-MG cell clones. ***p﹤0.001.

[0046] Fig.13 In Example 5 of the present invention 211 Effect of At-ATE-KINW on the invasion ability of U87-MG cells. ***p﹤0.001.

[0047] Fig.14 In Example 5 of the present invention 211 Effect of At-ATE-KINW on the migration ability of U87-MG cells. **p﹤0.01, ***p﹤0.001.

[0048] Fig.15 In Example 6 of the present invention 211 The results of the effect of At-ATE-KINW on the body weight of nude mice bearing subcutaneous U87-MG cell transplanted tumors.

[0049] Fig.16 In Example 6 of the present invention 211 The inhibitory effect of At-ATE-KINW on subcutaneous transplanted tumors of U87-MG cells in nude mice.

[0050] Fig.17 In Example 6 of the present invention 211 Effect of At-ATE-KINW on prolonging the survival rate of nude mice bearing subcutaneous U87-MG cell transplanted tumors.

[0051] Fig.18 In Example 6 of the present invention 211 Toxicological effects of At-ATE-KINW on subcutaneous transplanted tumors of U87-MG cells and different organs of tumor-bearing nude mice. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0053] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0054] Example 1. Synthesis of KINW polypeptide

[0055] 1) Artificially synthesized peptide KINW

[0056] The structural formula of polypeptide KINW is as follows Figure 1As shown, the synthesis sequence is from C-terminus to N-terminus. ① The resin is swollen, 2-Chlorotrityl Chloride Resin is placed in a reaction tube, DCM (dichloromethane) (15 mL / g) is added, and the mixture is shaken for 30 minutes; ② Synthesis is from C-terminus to N-terminus, the first amino acid is connected, the solvent is filtered out through a sand core, 3 times molar excess of Fmoc-Tyr(tBu)-OH is added, DMF (dimethylformamide) is added to dissolve, and then 10 times molar excess of DIEA (diisopropylethylamine) is added, and the mixture is shaken for 60 minutes. Block with methanol; ③ Deprotection, remove DMF, add 20% piperidine DMF solution (15mL / g), react for 5min, remove and add 20% piperidine DMF solution (15mL / g), react for 15min; ④ Detection, remove the piperidine solution, take more than a dozen resins, wash with ethanol three times, add detection reagent for detection, heat at 105℃~110℃ for 5min, turn dark blue for positive reaction; ⑤ Wash, DMF (10mL / g) twice, DCM (10mL / g) twice, DMF (10mL / g) twice; ⑥ Condensation, protect amino acid three times excess, HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate) three times excess, all dissolved with as little DMF as possible, add to the reaction tube, immediately add DIEA ten times excess, react for 30min; ⑦ Detection, take more than a dozen resins, wash with ethanol three times, add detection reagent for detection, heat at 105℃~110℃ for 5min, colorless for negative reaction. ⑧ Washing, DMF (10mL / g) twice, DCM (10mL / g) twice, DMF (10mL / g) twice; ⑨ Repeat steps ② to ⑧, connecting the amino acids in the sequence from right to left; ⑩ Draining, washing steps: DMF (10mL / g) twice, methanol (10mL / g) twice, DMF (10mL / g) twice, DCM (10mL / g) twice, draining for 10 minutes. 11) Cut the peptide from the resin, prepare a cutting solution (10mL / g) TFA (trifluoroacetic acid) 95%; water 1%; EDT (ninhydrin) 2%; TIS (triisopropylsilane) 2%, cutting time: 120min; blow dry and wash, blow the cleavage solution as dry as possible with nitrogen, wash with ether six times, and then evaporate at room temperature; analyze and purify, purify the crude product with high performance liquid chromatography; freeze dry, collect the target peptide solution and put it into a freeze dryer for concentration, and freeze dry it into powder. The HPLC and MS identification results of KINW are shown in Figure 2 .

[0057] 2) ATE-KINW preparation

[0058] Dissolve KINW in dimethyl sulfoxide containing 1% (volume) triethylamine, take 2 μmol of KINW solution and 6 times the molar amount of m-MeATE (12 μmol) dissolved in methanol for coupling reaction, and react the mixture in a 37°C water bath for 12 hours. Purify by reversed-phase high performance liquid chromatography, the initial gradient of the mobile phase is: the initial gradient is 60% mobile phase A (0.1% trifluoroacetic acid aqueous solution) and 40% mobile phase B (acetonitrile containing 0.1% trifluoroacetic acid); the ratio is uniformly changed to 20% mobile phase A and 80% mobile phase B in 0.1-20 minutes; the ratio is uniformly changed to 5% mobile phase A and 95% mobile phase B in 20-30 minutes; and the mobile phase ratio is maintained for 10 minutes (30-40 minutes). Using the difference in retention time of different compounds on the C18 reverse phase column, the coupling compound is determined and separated and purified. The structural formula of ATE-KINW is shown in Figure 3 (m-MeATE is linked to the N-end of KINWKFRLRY, such as Figure 3 A; or the side chain of lysine K, such as Figure 3 B, C), the HPLC and MS identification diagrams after purification are shown in Figure 4 In the coupling reaction, the primary amine on the peptide and the succinimide of ATE form a stable compound through an amide bond. Under the set elution conditions of the C18 column, the retention time of the peptide is about 13.2 minutes. The retention time of ATE is about 29 minutes. The other high peaks are all ATE-peptide conjugates, because there are three primary amines that can be amidated in the peptide structure, so a variety of conjugates will be formed.

[0059] 3) 211 At Mark ATE-KINW

[0060] The freeze-dried ATE-KINW was dissolved in methanol (10 mg / mL), and 10 μL of ATE-KINW solution was mixed with 50 μL of 211 At (about 180 MBq) and 5 μL of N-iodosuccinimide (NIS) (NIS dissolved in methanol containing 1% acetic acid at a concentration of 3 mg / mL) were mixed and incubated at 37°C for 10 min. 5 μL of Na2S2O3 solution (Na2S2O3 dissolved in ultrapure water at a concentration of 20 mg / mL) was added to terminate the labeling reaction. The product was purified by SephadexTM G-10 column, and 0.1 M PBS was used as the elution solution. Radioactive TLC was used for quality control, and acetone: water (v / v = 3:1) was used as the mobile phase ([R f ] 211 At-ATE-peptide = 0-0.1, [R f ] 211 At = 0.9-1, the labeling rate is about 70% (such as Figure 5The labeled mixture was purified using a Sep G10 gel column, and an elution curve was obtained (as shown in FIG. Figure 5 B), the purified target product is known according to the elution curve 211 At-ATE-KINW appeared in the third tube. The product of the third tube was subjected to TLC analysis (acetone: water = 3:1), and the radiochemical purity reached about 95% (such as Figure 5 C). Figure 6 For the preparation 211 The structural formula of At-ATE-KINW.

[0061] Embodiment 2, 211 In vitro stability study of At-ATE-KINW

[0062] Take the purified labeled drug 211 At-ATE-KINW was placed in PBS and fetal bovine serum (FBS) at 37°C, and samples were taken at 1, 3, 6, 12, and 24 hours, respectively, and the radiochemical purity at different time points was analyzed by TLC. The TLC condition was acetone: water = 3:1. Figure 7 As shown, the purified labeled drug 211 At-ATE-KINW was placed in PBS and FBS, and the results showed that it could maintain good stability in the first 6 hours, and its radiochemical purity was >80%. The radiochemical purity in PBS was >85% at 24h, and the radiochemical purity in serum was >70% at 24h; thus, it can be seen that there is a difference in the stability of the labeled drug placed in PBS and FBS, and the stability in PBS is better than that in FBS.

[0063] Embodiment 3, 211 Cellular uptake experiments of At-ATE-KINW

[0064] U87-MG cells were expressed at 1.0×10 6 / well density were respectively plated in 6-well plates and incubated overnight to allow them to adhere completely to the wall. 211 At-ATE-KINW and 211 At solution was added to a 6-well plate containing 1.0 mL of culture medium and incubated at different time points (1, 3, 6, 12, and 24 h). The culture medium was collected into a supernatant tube, and each well was washed twice with PBS. The washing solution was also transferred to the supernatant tube. Subsequently, the cells in the well plate were lysed with 1 M NaOH, and the cell lysate was collected. The supernatant and cell lysate were counted using a calibrator. Three replicate wells were set up. The supernatant and cell lysate were counted using a γ counter to calculate the number of cells. 211 At-ATE-KINW and 211The uptake rate of At. Uptake rate (%) = lysate count / (lysate count + supernatant count) × 100%. The results are shown in Figure 8 As shown, 211 At-ATE-KINW can rapidly bind to U87-MG cells (10.54±1.15%; 1h), and the binding rate is significantly higher than 211 At (1.63 ± 0.4%; 1h). The binding rate showed an upward trend within 12h, reaching 15.23 ± 2.31% at 3h, 23.31 ± 3.45% at 6h, and 33.6 ± 2.34% at 12h. The binding rate at 24h did not increase significantly, indicating that the cells were sensitive to 211 The uptake of At-ATE-KINW tends to be stable. 211 The binding rate of At to U87-MG cells did not increase significantly over time. 211 At-ATE-KINW has a good affinity for brain glioma cells U87-MG cells.

[0065] Embodiment 4, 211 Distribution of At-ATE-KINW in normal mice

[0066] Twenty four-week-old female Kunming mice were randomly divided into five groups and each mouse was injected with 211 At-ATE-KINW (0.1 mL, 180 kBq) was administered to the animals at 1, 3, 6, 12, and 24 h, and the heart, liver, spleen, lung, kidney, stomach, intestine, muscle, thyroid gland, tumor, blood, and brain were collected and weighed. The radioactivity count of each organ was measured using a γ counter, and the radioactivity of each organ was calculated. 211 The uptake percentage of At-ATE-KINW. The radioactive uptake percentage (%ID / g) = [tissue count / (injected drug count × tissue mass)] × 100%. The results show that ( Fig. 9 ) 211At-ATE-KINW is rapidly metabolized in vivo, mainly through the liver and spleen. The liver has a higher uptake (1h, 9.64±1.29%ID / g; 3h, 6.67±0.57%ID / g; 6h, 1.86±0.68%ID / g; 12h, 0.64±1.65%ID / g; 24h, 2.11±0.035%ID / g). The stomach also has radioactive uptake (1h, 3.07±0.78%ID / g; 3h, 2.41±0.79%ID / g). / g; 6h, 1.16±0.24%ID / g; 12h, 0.4±0.046%ID / g; 24h, 0.11±0.05%ID / g), while the thyroid gland had a certain amount of radioactive uptake (1h, 1.2±0.22%ID / g; 3h, 1.05±0.230%ID / g; 6h, 0.7±0.22%ID / g; 12h, 0.3±0.024%ID / g; 24h, 0.08±0.05%ID / g), and the uptake in other organs was relatively small.

[0067] Embodiment 5, 211 Cellular functional detection of At-ATE-peptide

[0068] 1) Cell viability assay

[0069] The cell viability experiment was detected by CCK-8 method, and the control group, ATE-KINW group, and free 211 At and 211 At-ATE-KINW group, where 211 At and 211 The equivalent activity of the At-ATE-KINW group was 37KBq / mL, and the concentration of the ATE-KINW group was 2μg / mL. The drug to be tested was diluted to the specified activity and concentration with culture medium in advance, and the cell status was observed. When the cell confluence was 70%, the drug was added, and the culture was continued for 24 hours. The cells were digested and counted. Each group was set up with 5 replicate wells, and 3000 cells were inoculated in each well. After 48 hours of culture, 10μL CCK-8 solution was added to each well and incubated for 4 hours. The 96-well plate was placed in an enzyme reader and the absorbance was measured at a wavelength of 450nm. The cell survival rate formula is [A(sample well)–A(blank)] / [A(PBS)–A(blank)]×100%. The results showed that U87-MG cells were treated with drugs for 24 hours, compared with free 211 At contrast, 211 At-ATE-KINW can significantly inhibit the viability of U87-MG cells, indicating that the targeting effect of the peptide increases the proliferation of glioma cells. 211 The uptake efficiency of At enhances the toxic effect of labeled drugs on brain glioma cells ( Fig.10 ).

[0070] 2) U87-MG cell apoptosis experiment

[0071] The control group, ATE-KINW group, and free 211 At and 211 At-ATE-KINW group, where 211 At and 211 The equivalent activity of the At-ATE-KINW group was 37KBq / mL, and the concentration of the ATE-KINW group was 2μg / mL. The drug to be tested was diluted to the specified activity and concentration with culture medium in advance, and the cell state was observed. When the cell confluence was 70%, the drug was added, and the culture was continued for 24 hours. The cells were washed with PBS three times, digested and collected with 0.25% trypsin without EDTA, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and the cells were washed with PBS twice to remove the residual culture medium. The apoptotic cells were detected using the AnnexinV / PI double staining kit, 100μL 1×Binding Buffer was added to resuspend the cells, 5μL Annexin V-FITC and 5μL PI were added respectively, and the cells were gently mixed. The cells were protected from light and reacted at room temperature for 10min. 400μL Binding Buffer was added and the cells were tested on the machine. The results showed that 211 The apoptosis rate of At-ATE-KINW group was greater than 211 At group and ATE-KINW group, indicating that the targeting effect of the peptide enables brain glioma cells to take up 211 At has a higher efficiency, resulting in a stronger ability to induce apoptosis in brain glioma cells ( Fig.11 ).

[0072] 3) U87-MG cell cloning experiment

[0073] The control group, ATE-KINW group, and free 211 At and 211 At-ATE-KINW group, where 211 At and 211The equivalent activity of the At-ATE-KINW group was 37KBq / mL, and the concentration of the ATE-KINW group was 2μg / mL. The drug to be tested was diluted to the specified activity and concentration with the culture medium in advance, and the cell state was observed. When the confluence was 70%, the drug was added. After continuing to culture for 24 hours, the cells were washed with PBS three times, digested and collected with 0.25% trypsin, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and the cells were washed with PBS twice to wash away the residual serum; the cells were resuspended in serum-free DMEM culture medium, counted with a cell counting plate, and inoculated in a 35mmΦ culture dish at a cell density of 1300 cells. Gently rotate to evenly disperse the cells. The culture dish was placed in a 37℃, 5% CO2 incubator and cultured for 3 weeks. When clones appeared, the supernatant was discarded, and the cells were washed twice with PBS and fixed with 75% ice-hexane for 15min. The fixative was discarded, and an appropriate amount of 0.5% crystal violet was added for staining for 30min. Washed with PBS and counted. Calculation formula: clone formation rate = number of clones / number of inoculated cells × 100%. The results showed that after drug treatment, U87-MG cells showed a significant difference compared with the control group and ATE-KINW group. 211 At and 211 The cell clone survival of the At-ATE-KINW group was significantly reduced, and 211 At Group ﹥ 211 At-ATE-KINW Group( Fig.12 ), indicating that due to the targeted binding of KINW, cells can take up more 211 At, therefore, 211 The cells in the At-ATE-KINW group had the least survival, and almost no clones were observed.

[0074] 4) U87-MG cell invasion assay

[0075] The control group, ATE-KINW group, and free 211 At and 211 At-ATE-KINW group, where 211 At and 211The equivalent activity of the At-ATE-KINW group was 37KBq / mL, and the concentration of the ATE-KINW group was 2μg / mL. The drug to be tested was diluted to the specified activity and concentration with culture medium in advance, and the cell status was observed. When the cell confluence was 70%, the drug was added, and the cells were cultured for 24 hours. The cells were washed with PBS for 3 times, digested and collected with 0.25% trypsin, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and the cells were washed twice with PBS to wash away the residual serum; the cells were resuspended in serum-free DMEM medium and counted on a cell counting plate. 800 μL of DMEM culture medium (containing double antibodies) containing 10% FBS was pre-added to a 24-well plate, and a Transwell chamber was placed. One hour later, 200 μL of cell suspension containing 30,000 cells of each group was inoculated into the upper chamber of the Transwell, and cultured in a 37°C, 5% CO2 incubator for 24 hours; the Transwell was removed, the chamber was carefully washed with PBS, and the cells were fixed with 70% ice ethanol solution for 1 hour; they were stained with 0.5% crystal violet stain, placed at room temperature for 20 minutes, and after washing with PBS, the non-migrated cells on one side of the upper chamber were wiped clean with a clean cotton ball; the Transwell chamber was placed under a microscope for observation and photography, 400 times magnification, and 3 fields of view were taken for each group. The results showed that after drug treatment, U87-MG cells were compared with the control group and ATE-KINW group. 211 At and 211 The cell invasion ability of At-ATE-KINW group was significantly weakened. 211 At Group ﹥ 211 At-ATE-KINW Group( Fig.13 ), indicating that due to the targeted binding of KINW, cells can take up more 211 At, therefore, 211 The cell invasion ability of the At-ATE-KINW group was the weakest.

[0076] 5) U87-MG cell migration assay

[0077] The control group, ATE-KINW group, and free 211 At and 211 At-ATE-KINW group, where 211 At and 211The equivalent activity of the At-ATE-KINW group was 37KBq / mL, and the concentration of the ATE-KINW group was 2μg / mL. The drug to be tested was diluted to the specified activity and concentration with culture medium in advance, and the cell state was observed. When the confluence was 70%, the drug was added. After continuing to culture for 24 hours, the cells were washed with PBS for 3 times, digested and collected with 0.25% trypsin, centrifuged at 1000rpm / min for 5min, the supernatant was removed, and the cells were washed with PBS twice to wash away the residual serum; the cells were resuspended in serum-free DMEM medium and counted on a cell counting plate; 800μL of 10% FBS DMEM culture medium (including double antibody) was placed in the Transwell chamber. After 1 hour, 200 μL of cell suspension containing 30,000 cells of each group was inoculated into the upper chamber of the Transwell, and cultured in a 37°C, 5% CO2 incubator for 24 hours; the Transwell was removed, the chamber was carefully washed with PBS, and the cells were fixed with 70% ice ethanol solution for 1 hour; they were stained with 0.5% crystal violet stain, placed at room temperature for 20 minutes, and after washing with PBS, the non-migrated cells on one side of the upper chamber were wiped clean with a clean cotton ball; the Transwell chamber was placed under a microscope for observation and photography, 400 times magnification, and 3 fields of view were taken for each group. The results showed that after drug treatment, U87-MG cells were compared with the control group and ATE-KINW group. 211 At and 211 The cell migration ability of At-ATE-KINW group was significantly weakened. 211 At Group ﹥ 211 At-ATE-KINW Group( Fig.14 ), indicating that due to the targeted binding of KINW, cells can take up more 211 At, therefore, 211 The cell migration ability of the At-ATE-KINW group was the weakest.

[0078] Embodiment 6, 211 In vivo therapeutic experiments of At-ATE-KINW

[0079] 1) Establishment of U87-MG cell subcutaneous tumor animal model

[0080] U87-MG cells in the logarithmic growth phase were injected subcutaneously in the left axilla of 4-week-old female nude mice. 6 When tumors reached 5 to 8 mm in diameter (1 to 2 weeks after inoculation), mice were used for in vivo experiments.

[0081] 2) In vivo treatment

[0082] Set up controls, 211 Intratumoral injection and tail vein injection groups, 211In the At-ATE-KINW intratumoral and tail vein injection groups, when the tumor size was about 100 mm 3 At 14:00 pm, 9 tumor-bearing mice in each group were injected with about 100 μL of 740 kBq of dapoxetine mixed with saline via the tail vein and intratumoral injection. 211 At and 211 At-ATE-KINW. The control group was injected with 100 μL of normal saline via the tail vein. The body weight and tumor size of the remaining mice were measured every two days. The tumor volume was calculated as follows: tumor volume (mm 3 )=(length×width 2 ) / 2. The treatment endpoint was when the tumor diameter reached 15mm, the mouse died, the tumor tissue ruptured, and the weight loss was ≥20%. After the treatment experiment observation, autopsy was performed within 24 hours. During the autopsy, some tissue samples of kidney, liver, stomach, etc. were fixed with 4% paraformaldehyde, embedded in paraffin, and subjected to immunohistochemical examination.

[0083] from Fig.15 The changes in the weight of tumor-bearing mice showed that the weight of the tumor-bearing mice in the free nuclide injection group did not decrease significantly after injection, but increased slightly over time. Fig.16 The tumor growth curves of tumor-bearing mice in each group showed that there was no significant difference in the average tumor size between the groups at the beginning of the experiment, and the tumor volume ranged from ∼100 mm 3 ±20%, with the extension of survival time, tail vein and intratumoral injection can be observed 211 The tumor growth of At-ATE-KINW-bearing mice was inhibited. 211 The tumor volume of the At-ATE-KINW group increased slowly within 16 days after administration. 211 Although the tumor growth in the At group was also inhibited within 12 days after administration, the inhibitory effect was inferior to that in the 211 At-ATE-KINW. At-ATE-KINW. 211 The control group of At-ATE-KINW tumor-bearing mice (including mice that received the same volume of saline and tail vein injection) 211 The fastest tumor growth was observed in At tumor-bearing mice, with no inhibitory effect. The tumor-bearing mice reached the sacrifice standard about 12 days after injection.

[0084] From the survival curve, we can see that Fig.17 ), tail vein injection of free 211 The median survival time of At and control groups was similar, 16 days and 18 days, respectively. 211 The median survival time of the At-ATE-KINW group was prolonged to 26 days. 211The median survival of the At group was 24 days. 211 The median survival time of the At-ATE-KINW group was 30 days, which was significantly different from that of all other groups. 211 At-ATE-KINW can prolong the survival time of tumor-bearing mice.

[0085] Pathological examination of stomach, liver, kidney and tumor tissue specimens ( Fig.18 ), found that injection of free 211 In the At group (whether in the caudal vein or in the tumor), mucosal necrosis and inflammatory cell infiltration occurred. 211 The results of gastric tissue samples in the At-ATE-KINW group were normal in the mucosal layer, submucosal layer, muscular layer and serosa layer, with no obvious pathological damage. All kidney samples showed slight dilation of glomerular capillaries and slight increase in red blood cells. According to the distribution of the drug in the body, the liver has a high uptake. Pathological studies have shown that: injection 211 The liver samples of the At-ATE-KINW group showed pathological changes such as slight increase in inflammatory cells and infiltration of inflammatory cells in the liver sinusoids. 211 In the At-ATE-KINW group, large areas of tumor cell necrosis and a large number of inflammatory cell infiltration occurred. 211 At-ATE-KINW can induce necrosis of brain glioma cells and inhibit cell proliferation. 211 At-ATE-KINW has little toxic effect on normal tissues.

[0086] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A targeted therapy for brain glioma 211 At-labeled peptides, made from the following components: NPY1R-targeting peptide KINW, bifunctional chelator m-MeATE, and α-nuclides 211 At, where The primary amine on the peptide KINW targeting NPY1R was coupled to the succinimide of the chelator m-MeATE via an amide bond, and the α-nuclides 211 At is chelated to the chelating agent m-MeATE; The amino acid sequence of the polypeptide KINW targeting NPY1R is as follows: KINWKFRLRY, Lysine-Isoleucine-Asparagine-Tryptophan-Lysine-Phenylalanine-Arg-Leucine-Arg-Tyr, Lys-Ile- Asn-Trp-Lys-Phe-Arg-Leu-Arg-Tyr, abbreviated as KINW; The structural formula of the chelating agent m-MeATE is as follows: The targeted treatment of brain glioma 211 At-labeled polypeptide is expressed as: 211 At-m-MeATE-KINW, abbreviated as 211 At-ATE-KINW, where 211 At is the α-nuclides, m-MeATE is the chelating agent, and KINW is the peptide targeting NPY1R protein.

2. Preparation of the targeted treatment of brain glioma according to claim 1 211 The method for At-labeling a polypeptide comprises the following steps: 1) Preparation of Chelating Agent-Peptide Under alkaline conditions, the bifunctional chelator m-MeATE and the polypeptide KINW are coupled to obtain the chelator-polypeptide m-MeATE-KINW, abbreviated as ATE-KINW; 2) Preparation of α-nuclides labeled chelating agent-peptide Add α-nuclides to the chelating agent-peptide m-MeATE-KINW and methanol reaction solution 211 At and N-iodosuccinimide (NIS), incubate, and terminate the reaction to obtain 211 At-labeled chelator-peptide, a targeted therapy for brain glioma 211 At-labeled peptide 211 At-m-MeATE-KINW, abbreviated as 211 At-ATE-KINW.

3. The method according to claim 2, characterized in that In step 1), the coupling reaction is carried out in dimethyl sulfoxide containing 1% triethylamine; The temperature of the coupling reaction is 37-40°C; Time is 12-18 hours; The molar ratio of polypeptide to chelating agent is 1:4-1:

6.

4. The method according to claim 2, characterized in that In step 2), the α nuclide 211 The activity of At is 180MBq; The mass ratio of NIS to chelating agent-polypeptide is 3:

20. The incubation temperature is 37°C and the incubation time is 10-15 min.

5. The targeted treatment of brain glioma according to claim 1 211 Application of At-labeled polypeptide in the preparation of tumor therapeutic reagent; the tumor is brain glioma.

6. A drug for treating brain glioma, the drug containing the targeted treatment of brain glioma according to claim 1 211 At-labeled peptide.

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