Iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe as well as preparation method and application thereof

Abstract

The invention relates to an iodine-131 labeled FAPI-TFMP-Y4 targeting molecular probe as well as a preparation method and application of the iodine-131 labeled FAPI-TFMP-Y4 targeting molecular probe. The probe combines the tumor targeting property of FAPI-46 and the characteristics that TFMP-Y4 small molecule polypeptide is safe and can stably stay in tumor cells for a long time, the novel molecular probe can obviously increase the uptake of the tumor cells and effectively prolong the residence time in the tumor cells, and the treatment effect on pancreatic cancer, non-small cell lung cancer and other tumors is improved. The invention also provides a preparation method of the probe. The preparation method comprises the following steps: synthesizing an ATE-FAPI-TFMP-Y4 precursor and carrying out radioiodine labeling. Experimental results show that the probe shows good tumor targeting, stability and anti-tumor activity in vitro and in vivo, and has low toxic and side effects on normal cells. The invention provides a new thought and method for treating pancreatic cancer and non-small cell lung cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and more specifically, to an iodine-131-labeled ATE-FAPI-TFMP-Y4 targeted molecular probe, its preparation method and application. Background Art

[0002] Pancreatic cancer is one of the most lethal cancers globally, with a very low 5-year survival rate. Developing new treatment methods is crucial for improving the survival rate and quality of life of patients. Fibroblast activation protein (FAP) is overexpressed in various cancers and is a promising target for tumor imaging and therapy. Radioactively labeled FAP inhibitors (FAPIs) have shown potential as cancer diagnostic and therapeutic agents. However, the rapid kinetics of FAPIs result in their too rapid clearance and too short retention time in tumors, limiting their application in the field of radionuclide therapy. Therefore, it is of great significance to develop new FAPI derivatives with tumor targeting and the ability to stably retain in tumor cells for a long time. In addition, non-small cell lung cancer (NSCLC), as one of the most common malignant tumors, has been maintaining a high incidence and mortality rate, and there is an urgent need to develop new treatment means. Summary of the Invention

[0003] The purpose of the present invention is to provide an iodine-131-labeled ATE-FAPI-TFMP-Y4 (hereinafter referred to as FAPI-Y4) targeted molecular probe, its preparation method and application, so as to solve the deficiencies of existing FAPI derivatives in tumor treatment, especially for the treatment of pancreatic cancer and non-small cell lung cancer.

[0004] The present invention modifies the Caerin1.1 polypeptide to obtain a new small molecule polypeptide TFMP-Y4, connects FAPI-46 with TFMP-Y4 through a linker, and 131 labels it with

[0005] I to obtain a novel molecular probe.

[0006] The probe of the present invention combines the tumor targeting property of FAPI-46 and the characteristics of the TFMP-Y4 small molecule polypeptide, which is safe and can stably remain in tumor cells for a long time. The novel molecular probe can significantly increase the uptake of tumor cells and effectively prolong the retention time in tumor cells, improving the therapeutic effect on tumors such as pancreatic cancer and non-small cell lung cancer. In addition, the present invention also provides the application of the probe in the treatment of pancreatic cancer and non-small cell lung cancer, and verifies its inhibitory effect on pancreatic cancer and non-small cell lung cancer cells and good biosafety through experiments.

[0007] The preparation method of the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe of the present invention comprises the following steps:

[0008] 1. Synthesize the ATE-FAPI-TFMP-Y4 precursor: Connect FAPI-46 and TFMP-Y4 through PEG2 and Lys, and introduce the chelator ATE at the Lys end to obtain the ATE-FAPI-TFMP-Y4 (abbreviated as FAPI-Y4) precursor.

[0009] 2. Radioactive iodine labeling: Adopt the radioactive iodine desmethyltin reaction to label 131 I onto the FAPI-Y4 precursor to obtain the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe (abbreviated as 131 I-FAPI-Y4).

[0010] Furthermore, the purity of the FAPI-Y4 precursor is greater than 98%.

[0011] The present invention also provides the application of the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe in the preparation of drugs for treating tumors, specifically the application of the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe in the preparation of drugs for intracavitary irradiation treatment of tumors.

[0012] Preferably, the tumors include but are not limited to pancreatic cancer and non-small cell lung cancer.

[0013] The present invention also provides a tumor treatment drug, which contains the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe.

[0014] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0015] The administration routes of the drug of the present invention include but are not limited to intratumoral injection, intravenous injection, etc., and the administration dose of the drug is determined by a doctor according to the specific conditions of the patient.

[0016] To verify and compare the therapeutic effects of FAPI-caerin1.1, FAPI-Y4, and FAPI-46 on pancreatic cancer, the present invention verified the ability of FAPI-caerin1.1, FAPI-Y4, and FAPI-46 to inhibit the proliferation of PANC-1, PANC-1-FAP, and HPDE6-C7 cells in vitro through CCK-8 experiments and plate clone experiments; indirectly labeled FAPI-caerin1.1, FAPI-Y4, and FAPI-46 through radioactive iodine desmethylstannylation reaction, detected the lipid-water partition coefficients of the three, and preliminarily evaluated and compared their stabilities at different temperatures and in different solutions; observed and compared 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4, and 131 whether I-FAPI-46 could be taken up by PANC-1, PANC-1-FAP, and HPDE6-C7 cells and stably retained in the cells; established a nude mouse tumor model of pancreatic cancer cells (PANC-1 cell line) in vivo, and further compared 131 I-FAPI-caerin1.1, 131 I-FAPI-Y4, 131 I-FAPI-46, FAPI-caerin1.1, FAPI-Y4, and FAPI-46 for the differences in anti-pancreatic cancer activity in vivo. And H&E staining was used to detect dead cells in tumor tissue sections and immunohistochemical staining was used to analyze the expression of FAP antibody in tumor cells.

[0017] The results showed that (1) both FAPI-caerin1.1 and FAPI-46 inhibited the in vitro proliferation of PANC-1, PANC-1-FAP, and HPDE6-C7 cells in a concentration-dependent manner, while FAPI-Y4 had no obvious inhibitory effect on the in vitro proliferation of pancreatic cancer cells and normal pancreatic duct epithelial cells. And there were significant differences in the inhibitory effects of FAPI-caerin1.1 and FAPI-46 on the proliferation of PANC-1, PANC-1-FAP, and HPDE6-C7 cells (P<0.05). (2) The plate clone formation experiment further verified that with the increase in the concentration of FAPI-caerin1.1, the clone proliferation ability of PANC-1 and PANC-1-FAP cells decreased. While there was no obvious change in the clonal proliferation of PANC-1 and PANC-1-FAP cells in the FAPI-Y4 group (P>0.05). (3) The radioactive labeled products 131 I-FAPI-caerin1.1 and 131 I-FAPI-Y4 were lipophilic, while 131I-FAPI-46 is water-soluble, with labeling rates of all three greater than 95%, and can maintain stability in different solutes and at different temperatures. (4) CCK-8 experiments showed that compared with Na 131 I, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 could all inhibit the proliferation of pancreatic cancer cells and normal pancreatic epithelial cells, and the difference was statistically significant (P < 0.05); 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 could all be taken up and stably retained by PANC-1, PANC-1-FAP, and HPDE6-C7 cells, and the uptake rate of 131 I-FAPI-Y4 was the highest among the three cells, and a plateau phase appeared after 24 h. (5) In vivo experiments showed that compared with the PBS group or the Na 131 I group, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 groups showed a significant reduction in tumor volume, and the difference was statistically significant (P < 0.05), and 131 the therapeutic effect of I-FAPI-Y4 was more significant. The results of H&E staining showed that compared with the PBS group or the Na 131 I control group, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 groups all showed varying degrees of cell necrosis and degeneration (P < 0.05).

[0018] The research of the present invention shows that 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 all have tumor targeting, can target and bind to pancreatic cancer cells, and have obvious cell killing effects; however, FAPI-caerin1.1 also has a killing effect on normal pancreatic epithelial cells, and its IC 50 is lower than that of tumor cells, while FAPI-Y4 does not have a killing effect on normal cells, and 131 I-FAPI-Y4 has a more significant toxic effect on pancreatic cancer. Therefore, 131 I-FAPI-Y4 is a potential drug for internal radiotherapy of pancreatic cancer.

[0019] To explore131 The value of the novel molecular probe I-FAPI-Y4 in the internal radiotherapy of non-small cell lung cancer, and compare it with 131 I-FAPI-46 and 131 I-TFMP-Y4. Through the uptake and internalization experiments of non-small cell lung cancer cells, the present invention evaluates the 131 in vitro kinetic characteristics of I-FAPI-Y4; through the cytotoxicity proliferation experiment, compare 131 I-FAPI-Y4, 131 I-FAPI-46, 131 I-TFMP-Y4, and the cytotoxic effects on A549-FAP cells (high FAP expression), A549 cells and Beas-2b cells; establish a tumor-bearing mouse model, perform intratumoral injection, and further verify the anti-tumor activity of the above drugs.

[0020] The results show that: ① The MTT experiment proves that FAPI-Y4 and TFMP-Y4 have no obvious toxic effects on A549-FAP cells, A549 cells and Beas-2b cells, while FAPI-46 has a proliferation inhibitory effect on the growth of all three cell lines, and it is concentration-dependent; ② 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 labeling rates are all above 95%, and they have good stability; 131 I-FAPI-Y4 shows weak water solubility, 131 I-FAPI-46 and 131 I-TFMP-Y4 show weak lipid solubility; ③ In the cell uptake and internalization experiments, 131 I-FAPI-Y4 has a high tumor cell uptake rate and a long retention time, 131 I-FAPI-46 is rapidly taken up and rapidly eluted, 131 I-TFMP-Y4 is slowly taken up; ④ 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 all have the effect of inhibiting the proliferation activity of tumor cells, among which 131 I-FAPI-Y4 is the strongest; ⑤ The in vivo experiment proves that 131 I-FAPI-Y4 has good anti-tumor proliferation activity.

[0021] The application of the iodine-131-labeled FAPI-Y4 targeting molecular probe of the present invention in the preparation and treatment of anti-tumor drugs, especially for the treatment of pancreatic cancer and non-small cell lung cancer. This probe can significantly inhibit the proliferation of tumor cells, and has low toxic and side effects on normal cells, with good drug safety and tumor targeting. Brief Description of the Drawings

[0022] Figure 1 It is a schematic structural diagram of FAPI-Y4.

[0023] Figure 2 It is a schematic structural diagram of FAPI-caerin1.1.

[0024] Figure 3 It is a schematic structural diagram of FAPI-46.

[0025] Figure 4 It shows the effects of P3 (control peptide), FAPI-46, FAPI-Y4, and FAPI-caerin1.1 at different concentrations on the proliferation of PANC-1, PANC-1-FAP, and HPDE6-C7 cells and the IC 50 value. Among them, A, B, and C are the survival rates of PANC-1, PANC-1-FAP, and HPDE6-C7 cells under the action of different concentrations of P3, FAPI-46, FAPI-Y4, and FAPI-caerin1.1; D, E, and F are the IC 50 values of P3, FAPI-46, FAPI-Y4, and FAPI-caerin1.1 acting on PANC-1, PANC-1-FAP, and HPDE6-C7 cells. (Ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0026] Figure 5 It is the γ-counting curve of iodine-131-labeled FAPI-Y4, FAPI-caerin1.1, and FAPI-46. Among them, A is the 131 γ-counting curve of 131 I-FAPI-Y4, B is the 131 γ-counting curve of

[0027] Figure 6 It is the radiochemical purity (RCP) of iodine-131-labeled FAPI-Y4, FAPI-caerin1.1, and FAPI-4 on the mixture with fetal bovine serum (FBS) or normal saline (NS) stored at room temperature (25°C), (37°C) for different times (0h, 24h). Among them, A is the 131 RCP of 131The radiochemical purity (RCP) of I-FAPI-46 stored in fetal bovine serum (FBS) at room temperature (25 °C, 37 °C) for different times (0 h, 24 h), where C is 131 The radiochemical purity (RCP) of I-FAPI-caerin1.1 stored in normal saline (NS) at room temperature (25 °C, 37 °C) for different times (0 h, 24 h), where D is 131 The radiochemical purity (RCP) of I-FAPI-caerin1.1 stored in fetal bovine serum (FBS) at room temperature (25 °C, 37 °C) for different times (0 h, 24 h), where E is 131 The radiochemical purity (RCP) of I-FAPI-Y4 stored in normal saline (NS) at room temperature (25 °C, 37 °C) for different times (0 h, 24 h), where F is 131 The radiochemical purity (RCP) of I-FAPI-Y4 stored in fetal bovine serum (FBS) at room temperature (25 °C, 37 °C) for different times (0 h, 24 h).

[0028] Figure 7 Results of cell uptake washing and elution experiments for FAPI-Y4, FAPI-caerin1.1, and FAPI-46. Among them, A, B, and E are the uptake rates of 131 I-FAPI-46, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and Na 131 I by PANC-1, PANC-1-FAP, and HPDE6-C7 cells at different time points (3 h, 6 h, 24 h, 48 h). C, D, and F are the elution kinetic characteristics of 131 I-FAPI-46, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and Na 131 I by the above three cell lines at different time points (3 h, 6 h, 24 h, 48 h) after 24 hours of drug binding.

[0029] Figure 8 For 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 in the cytotoxicity proliferation experiment results. Among them, A, B, and C represent the 131 I-FAPI-Y4, 131I-FAPI-caerin 1.1 and 131 I-FAPI-46 or Na 131 Comparison of survival rates under the action of I. (ns P>0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0030] Figure 9 For the tumor volume, tumor weight, and nude mouse weight of nude mice bearing tumors (human pancreatic cancer tumors) in 8 treatment groups. Among them, A is the changing trend of tumor volume in each treatment group at different time points; B is the comparison of tumor weights separated at the end of each group of experiments; C is the dynamic change of nude mouse weight in each group during the treatment process. (ns P>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0031] Figure 10 For the photos of human pancreatic cancer tumors separated in 8 treatment groups.

[0032] Figure 11 For the H&E staining results of tumor sections in 8 treatment groups.

[0033] Figure 12 For the statistical chart of the difference in the area of cell degeneration and necrosis in 8 treatment groups.

[0034] Figure 13 For the MTT experimental results of FAPI-Y4, FAPI-46, and TFMP-Y4. Among them, A, B, and C are the survival rates of A549-FAP cells, A549 cells, and Beas-2b cells under the action of different concentrations of P3, FAPI-Y4, FAPI-46, and TFMP-Y4 respectively; D, E, and F are the IC 50 values (NSP>0.05; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0035] Figure 14 For 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 labeling experimental results. Among them, A and B are the curve graphs drawn by measuring the γ counter values by paper chromatography after labeling 131 I-FAPI-Y4, 131 I-FAPI-46 with the formylation method; C is the curve graph drawn by measuring the γ counter values by paper chromatography after labeling 131 I-TFMP-Y4 with the chloramine-T method, and the labeling rate is greater than 95%. D represents131 I-FAPI-Y4, 131 I-FAPI-46 and 131 The labeling rate results of I-TFMP-Y4 had no statistical difference (NSP > 0.05).

[0036] Figure 15 For 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 The results of the labeling stability experiment of I-TFMP-Y4. Among them, A, C, and E are pure 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 The labeling rates of I-TFMP-Y4 samples at each time point; B, D, and F are 131 I-FAPI-Y4, 131 I-FAPI-46 or 131 The labeling rates of I-TFMP-Y4 samples mixed with NS or FBS samples at different time points at room temperature (25 °C) and 37 °C.

[0037] Figure 16 For Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 The results of the cellular uptake experiment of I-TFMP-Y4. Among them, A and B represent the uptake rates of A549-FAP cells for Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 at different time points (1 / 6 h, 1 / 2 h, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h); C and D represent the uptake rates of A549 cells for Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4.

[0038] Figure 17 For Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46 and 131Cell internalization experiment results of I-TFMP-Y4. Among them, E, F, and G respectively represent the internalization and the ratio of binding to the cell membrane of A549-FAP cells to Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46, and 131 I-TFMP-Y4 at different time points (1 / 6 h, 1 / 2 h, 1 h, 2 h, 4 h, 6 h, 24 h, 48 h).

[0039] Figure 18 For 131 I-TFMP-Y4, 131 I-FAPI-46, and 131 I-FAPI-Y4 cytotoxicity proliferation experiment results. Among them, A represents the comparison of the survival rates of A549-FAP cells under the action of Na- 131 I, 131 I-FAPI-Y4, 131 I-FAPI-46, and 131 I-TFMP-Y4 at different radioactive concentrations (2000 KBq / mL, 4000 KBq / mL, 8000 KBq / mL, 16000 KBq / mL); B and C are respectively the comparison of the survival rates of A549 cells and Beas-2b cells; D is the comparison of the survival rates of A549-FAP cells, A549 cells, and Beas-2b cells under the action of 131 I-FAPI-Y4 at different radioactive concentrations; E and F are respectively 131 I-FAPI-46 and 131 I-TFMP-Y4 acting on the survival rates of the three types of cells. (NSP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0040] Figure 19 For 131 I-FAPI-Y4, 13 1I-FAPI-46, and 131 I-TFMP-Y4 in vivo treatment experiment results (non-small cell carcinoma). Among them, A represents the change in body weight of nude mice in each treatment group after starting treatment; B and C represent the change in tumor volume of nude mice in each treatment group after treatment; D represents the tumor weight of each group. (NSP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0041] Figure 20 For the photos of non-small cell carcinoma tumors isolated from 9 treatment groups.

[0042] Figure 21 The H&E staining results of tumor sections of A549-FAP tumor-bearing nude mice in 9 treatment groups. Among them, A is the PBS group, B is the FAPI-Y4 group, C is the FAPI-46 group, D is the TFMP-Y4 group, E is 131 Group I, F is 131 Group I-FAPI-Y4, G is 131 Group I-FAPI-46, H is 131 Group I-TFMP-Y4, I is 131 Group I-FAPI-Y4 / A549. Specific implementation manners

[0043] The present invention will be further described below in conjunction with specific embodiments. The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0044] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0045] Example 1. Internal irradiation therapy of a targeted FAPI molecular probe for human pancreatic cancer

[0046] Cell line and cell culture

[0047] Human pancreatic cancer cells (PANC-1 cells) and pancreatic duct epithelial cells (HPDE6-7 cells) were both provided by the Stem Cell Bank of the Chinese Academy of Sciences. PANC-1-FAP cells were transfected by Lipuvo Biotechnology Co., Ltd. The culture media for all three cell types were (by volume) 89% DMEM (GIBCO, USA), 10% heat-inactivated fetal bovine serum (FBS, Corning, USA), and 1% penicillin-streptomycin solution (GIBCO, USA). The cells were cultured in an incubator (Thermo, USA) at 37°C and 5% CO2.

[0048] Synthesis of FAPI-TFMP-Y4 precursor

[0049] FAPI-Y4, FAPI-caerin1.1, and FAPI-46 were synthesized by Jiangxi Tenzhen Biotechnology Co., Ltd. and determined by reverse-phase high-performance liquid chromatography to have a purity > 98%. FAPI-Y4, FAPI-caerin1.1, and FAPI-46 were dissolved in a mixed solution of DMSO and phosphate-buffered saline (PBS) (DMSO:PBS = 1:1) to different concentrations (5 mg / mL, 1 mg / mL, and 0.1 mg / mL) and used immediately after preparation.

[0050] Preparation of Iodine-131-Labeled FAPI-Y4, FAPI-caerin1.1 and FAPI-46

[0051] Indirect labeling of FAPI-Y4, FAPI-caerin1.1 and FAPI-46 was carried out by the radioactive iodine demethylstannylation reaction: After dissolving the three FAPI compounds in methanol, they were mixed with the Na 131 I solution activated by acetic acid and N-iodosuccinimide (NIS) and reacted for 30 minutes. Subsequently, the reaction was quenched by sodium ascorbate, and the 131 I-labeled products ( 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 I-FAPI-46) were obtained by centrifugal purification. High-efficiency binding of radioactive iodine was achieved by controlling the reagent ratio and reaction conditions during the labeling process. The CPM value was obtained by using a gamma counter (Zhongjia Optoelectronics Co., Ltd., China). The gamma counting curve was plotted with GraphPad Prism, and the area under the curve was calculated to obtain the labeling rate. (See Figure 5 )

[0052] CCK-8 Assay

[0053] The CCK-8 method was used to detect the proliferation inhibitory effects of FAPI-caerin1.1, FAPI-Y4, FAPI-46 and the control peptide P3 on PANC-1, PANC-1-FAP and HPDE6-C7 cells. Cells in the logarithmic growth phase were seeded in 96-well plates (5×10 3 cells / well). After 24 hours, six concentration gradient treatment groups (3 replicates per group) of 1 - 40 μg / mL were set up, and at the same time, a PBS zeroing group and a cell control group were established. After 24 hours of drug treatment, CCK-8 reagent was added, and after continuing to culture for 2 hours, the absorbance value at 450 nm was measured. The cell survival rate (IC 50 ) was calculated by the formula (experimental group OD - zeroing group OD) / (control group OD - zeroing group OD)×100%. (See Figure 4 )

[0054] Labeling Rate Determination Experiment

[0055] Thin layer chromatography was used, with No. 1 Whatman filter paper as the stationary phase and acetone as the mobile phase. The experiment was repeated three times to determine 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131Labeling efficiency of I-FAPI-46. The specific steps are as follows: cut the filter paper and mark the origin, spot the sample and let it dry, cut the developed and dried filter paper into sections and put them into the counting tube, measure the radioactivity intensity with a γ radioimmunoassay counter, draw a curve after attenuation correction and calculate the labeling rate according to the formula, and determine the migration position of the component according to the Rf value. (See Figure 5 )

[0056] Stability determination

[0057] By measuring 131 the changes in the radiochemical purity (RCP) of I-labeled FAPI-Y4, FAPI-caerin1.1, and I-FAPI-46 in fetal bovine serum and physiological saline to evaluate their in vitro stability. After mixing the labeled substances with serum or physiological saline respectively, store them at 25 °C room temperature and 37 °C, and use paper chromatography to measure the RCP values at 0 h and 24 h. Independently repeat the experiment three times to analyze the stability differences of the labeled substances in different temperatures and media. (See Figure 6 )

[0058] Determination of lipid-water partition coefficient

[0059] Add 500 μL of n-octanol (Macklin, China), 500 μL of NS, and 50 μL of 131 I-FAPI-TFMP-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 into three 1.5 mL Ep tubes respectively, seal and shake for 2 min, then centrifuge at 4000 rpm / min for 5 min to obtain an equilibrium state between n-octanol and NS. Take 50 μL of lipid phase and aqueous phase samples respectively, and measure the γ counts of each tube. Repeat this operation 3 times. The lipid-water partition coefficient (log P) is calculated as follows: logP = log[(γ count in lipid phase - background γ count) / (γ count in aqueous phase - background γ count)].

[0060] Cell uptake assay

[0061] In this example, 4-6-week-old SPF-grade BALB / C female nude mice were selected as experimental animals. The experimental animals were housed in the SPF-grade experimental animal center of the First Affiliated Hospital of Guangdong Pharmaceutical University (Experimental Animal Use License No.: SYXK (Guangdong) 2017-0124), and were raised and managed in a strict sterile barrier system, provided with sterilized feed and drinking water. All animal experiment operations strictly complied with the experimental protocol approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Guangdong Pharmaceutical University. At the end of the experiment, in accordance with the relevant regulations of the "Implementation Rules for the Administration of Medical Experimental Animals" (Order No. 55 of the Ministry of Health of the People's Republic of China), the experimental animals were euthanized by cervical dislocation to ensure compliance with animal welfare ethics requirements.

[0062] PANC-1, PANC-1-FAP, and HPDE6-C7 cells in the logarithmic growth phase with good morphology were selected. After preparing single-cell suspensions and adjusting the density, they were inoculated into 24-well plates and incubated for 24 hours. After the cells adhered to the wall, the medium was discarded and the cells were washed. Serum-free medium was added, and the cells were divided into 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, 131 I-FAPI-46, and Na 131 I. There were three replicate wells and one positive control well in each group, and the drug concentration in each group was 259 KBq (7 μCi) / 2 μL / well. Samples were taken at 3 h, 6 h, 24 h, and 48 h after adding the drug. After the incubation ended, the cells were washed, digested, and the washing solution was collected. The radioactivity count was measured using a γ radioimmunoassay counter. The cell binding rate was calculated by the formula: cell binding rate = (average γ count of three replicate wells in each group / γ count of the corresponding positive control well in each group) × 100%. This was used to evaluate 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131 I-FAPI-46 labeled product binding characteristics with PANC-1, PANC-1-FAP, and HPDE6-C7 cells. (See Figure 7 )

[0063] Cell elution experiment

[0064] PANC-1, PANC-1-FAP, and HPDE6-C7 cells were inoculated into 8 24-well cell culture plates at 5×10 4 cells (500 μL) / well and cultured for 24 h. There were three replicate experimental wells and one positive control well for each drug. After culturing, the supernatant was discarded, and 0.5 mL of serum-free medium and 2 μL of Na 131 I, 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1, and 131I-FAPI-46 solution (7 μCi / well). After incubating in the incubator for 24 hours, discard the supernatant from all experimental wells, wash each well twice with PBS, and add 0.5 mL of serum-free medium to each well. Incubate the plate in the incubator for 3, 6, 24, and 48 h, and then take it out. Discard the supernatant of the experimental group, and wash the experimental wells twice with PBS (the supernatant of each positive control well is collected in the corresponding test tube). After adding 200 μL of trypsin to each well for digestion, wash the experimental wells three times with PBS, and collect the samples in the corresponding test tubes. Measure the γ count of each tube, and calculate the drug retention rate using GraphPad Prism. (See Figure 7 )

[0065] Cytotoxicity proliferation experiment

[0066] Evaluate using the CCK-8 method 131 I-FAPI-Y4, 131 I-FAPI-caerin1.1 and 131 The cytotoxic effects of I-FAPI-46 on PANC-1, PANC-1-FAP, and HPDE6-C7 cells. Select three types of cells in the logarithmic growth phase with good morphology. After processing and adjusting the density according to the standard procedure, inoculate them into a 96-well plate and incubate for 24 hours until 80% confluence. Subsequently, add different concentrations of the three probe solutions, with three replicates in each group and three drug-free control groups. After adding the drugs for 24 hours, add the CCK-8 solution and continue to incubate for 2 hours. Measure the OD value at a wavelength of 450 nm using a full-wavelength microplate reader. Calculate the cell survival rate of each group according to the formula: Cell survival rate = (Average OD value of each experimental group - Average OD value of the zero-adjustment group) / (Average OD value of the control group - Average OD value of the zero-adjustment group) × 100%, and compare the effects of different probes on the cell survival rate. (See Figure 8 )

[0067] Establishment of nude mouse model

[0068] Establish a PANC-1 cell xenograft nude mouse model through the following steps: First, select the PANC-1 cell line in the logarithmic growth phase with good morphology. After treatment, adjust the cell density to 1×10 7 cells / mL with pre-cooled sterile PBS at 4°C and store for later use. Then disinfect the right axillary area of the nude mouse with a 75% ethanol cotton ball and slowly inject 200 μL of the cell suspension (containing 5×10 6 cells) at a 45° angle. After inoculation, a transparent vesicle appears at the injection site. After about one week, the vesicle is absorbed and a rice-grain-like neoplasm is formed. Then regularly observe the status of the nude mouse, and measure the tumor volume with an electronic vernier caliper every 48 hours. When the tumor volume approaches 9 - 10 mm 3When the time came, 32 nude mice with similar tumor volumes were selected for subsequent experiments by calculating the tumor volume according to the formula volume = long diameter × short diameter × height diameter × π / 6.

[0069] In vivo treatment experiment

[0070] Starting 72 hours before the experiment, nude mice were pretreated with 0.1% potassium iodide solution to block the thyroid gland and reduce non-specific uptake. The nude mice with established pancreatic cancer models were randomly divided into 8 groups (4 mice in each group), namely FAPI-46 group, FAPI-Caerin1.1 group, FAPI-Y4 group, PBS group, Na 131 I group, 131 I-FAPI-caerin1.1 group, 131I-FAPI-Y4 group and 131I-FAPI-46 group. Intratumoral injection was performed every 3 days for a total of 4 times. The injection regimens for each group were as follows: the PBS group was injected with pure PBS; the FAPI-Caerin1.1, FAPI-46, and FAPI-Y4 groups were injected with a mixed solution of PBS and methanol (volume ratio 1:1) containing 30 μg of polypeptide; Na 131 I group was injected with 7.4×10 6 Bq Na 131 I solution; 131 I-FAPI-caerin1.1, 131 I-FAPI-46, 131 I-FAPI-Y4 groups were injected with the labeled product containing 30 μg of polypeptide and 7.4×10 6 Bq Na 131 I, and the injection volume was 50 μL for all. On the 7th day after the last administration, the nude mice were euthanized according to animal ethics regulations, the tumor tissues were isolated and weighed, and GraphPad Prism software was used to analyze the body weight, tumor size and weight of the nude mice to evaluate the therapeutic effects of the probes in each group.

[0071] To observe the changes in pancreatic cancer cells after treatment, H&E staining was performed. The tumor tissues were fixed with 4% paraformaldehyde, embedded in paraffin, sectioned (4-6 μm), dried, and then stained with hematoxylin and eosin (H&E), and observed and photographed under a microscope. (See Figure 9 、 Figure 10 、 Figure 11 、 Figure 12 )

[0072] Experimental results

[0073] The results showed that FAPI-caerin1.1 and FAPI-46 significantly inhibited the proliferation of pancreatic cancer cells (PANC-1, PANC-1-FAP) and normal pancreatic duct epithelial cells (HPDE6-C7) in a concentration-dependent manner (IC 50Range: 9.65 - 29.82 μg / mL, P < 0.05). Among them, FAPI-caerin1.1 showed the strongest inhibitory effect on HPDE6-C7, while FAPI-Y4 had no significant inhibitory effect, suggesting its good safety; the radiolabeled products 131 I-FAPI-caerin1.1 (liposoluble), 131 I-FAPI-Y4 (liposoluble) and 131 I-FAPI-46 (water-soluble) had labeling rates > 95%, good stability, and 131 I-FAPI-Y4 had the highest uptake rate and stable retention in cells. In in vivo experiments, 131 The three I-labeled compounds all significantly inhibited tumor growth (P < 0.05), especially 131 I-FAPI-Y4 had the most significant effect, accompanied by obvious tumor cell necrosis.

[0074] Example 2. Internal irradiation therapy of targeted FAPI molecular probes for non-small cell lung cancer

[0075] Experimental materials

[0076] Human non-small cell lung cancer cells (A549) and human normal lung epithelial cells (Beas-2b) were both purchased from the Stem Cell Bank of the Chinese Academy of Sciences; A549-FAP cells stably transfected with FAP were transfected from the above A549 cells by Lipuvo Biotechnology Co., Ltd.

[0077] Female BALB / C nude mice aged 4 - 6 weeks, SPF grade, were selected. All experimental nude mice used were purchased from the Guangdong Provincial Medical Experimental Animal Center (License number: SCYK(Guangdong)2018 - 0002) and were raised in the SPF-grade experimental animal center of the First Affiliated Hospital of Guangdong Pharmaceutical University (License number: SYXK(Guangdong)2017 - 0124). Breeding conditions: SPF-grade environment, constant temperature (25 °C), constant humidity (40 - 60%), 12-hour light / dark cycle. After the experiment, the mice were euthanized by cervical dislocation in accordance with the "Implementing Rules for the Administration of Medical Experimental Animals" (Order No. 55) of the Ministry of Health of the People's Republic of China.

[0078] MTT experiments of FAPI-Y4, FAPI-46, and TFMP-Y4

[0079] The MTT method was used to detect the proliferation inhibitory effects of FAPI-Y4, FAPI-46, and TFMP-Y4 on A549-FAP, A549, and Beas-2b cells. In the experiment, cells were seeded in 96-well plates (drugs were administered when the density was approximately 80%), and a blank group (PBS), a negative control group, and three drug treatment groups (each with 3 replicates) were set up. The drug concentration gradient was 1 - 40 μg / mL. After 24 hours of treatment, MTT solution was added and incubated for 4 hours. After dissolving the formazan crystals, the OD value at a wavelength of 570 nm was measured. The cell survival rate was calculated according to the formula (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%. The dose-effect curve was plotted using the experimental data obtained from the MTT experiment, and the IC 50 value of FAPI-Y4, FAPI-46, and TFMP-Y4 was calculated using GraphPad Prism 9.0 software.

[0080] (See Figure 13 )

[0081] 131 I-FAPI-Y4, 131 I-FAPI-46, and 131 Preparation of I-TFMP-Y4

[0082] Utilizing the structural characteristics of tyrosine (Tyr) in the TFMP-Y4 molecule, its efficient 131 I labeling was achieved using the chloramine-T method: A 1 mg / mL TFMP-Y4 solution was mixed with chloramine-T (1 mg / mL) and Na 131 I (37 MBq), and vortex reaction was carried out at room temperature for 1 - 2 minutes to complete the radioiodine labeling. This method realizes stable labeling through the covalent binding of active iodine (I+) with tyrosine residues.

[0083] Aiming at the problem of low labeling efficiency of FAPI-Y4 and FAPI-46 by the chloramine-T method, an improvement was made by combining the formylation method with 131 I chelator ATE: After the Na 131 I solution was activated by N-iodosuccinimide (NIS) and acetic acid, it was reacted with FAPI-Y4 and FAPI-46 solutions for 30 minutes respectively, and then sodium ascorbate was added to quench, finally achieving stable radioiodine labeling. This method optimized the iodination reaction conditions by introducing a chelator and improved the labeling efficiency. The dose-effect curve was plotted using the experimental data obtained from the MTT experiment, and the IC 50 value of FAPI-Y4, FAPI-46, and TFMP-Y4 was calculated. (See Figure 14 ) Determination of the lipophilic-hydrophilic partition coefficient and determination of labeling stability

[0084] Similar to Example 1. (SeeFigure 14 , Figure 15 )

[0085] Cell experiments

[0086] Cell uptake experiment: Take the cell suspension of A549-FAP cells or A549 cells with a concentration of 1×10 5 cells / mL, evenly inoculate it into a 24-well plate, 500 μL / well, and incubate it in an incubator for 24 h. Add 0.5 mL of DMEM medium to each well, and add 131 I-FAPI-Y4, 131 I-FAPI-46, 131 I-TFMP-Y4 solution respectively. After incubating for different times, measure the cell binding rate with a γ counter. (See Figure 16 )

[0087] Cell internalization experiment: Determine the internalization rate and membrane binding rate of 131 I, 131 I-TFMP-Y4, 131 I-FAPI-46 and 131 I-FAPI-Y4 in A549-FAP and A549 cells. In the experiment, the cells were respectively exposed to four drugs with equal radioactive doses (259 KBq / well). After treatment at 8 time points from 1 / 6 to 48 hours, the membrane-bound part was separated by washing with pre-cooled PBS (eluted with Gly-HCl buffer), and then the internalized part was obtained by cell lysis, and the radioactive counts were measured respectively. The internalization rate calculation formula is: CPM of the internalized part / (CPM of the internalized + membrane-bound part) × 100%, to compare the dynamic differences in the uptake of different radioactive drugs in cells. (See Figure 17 )

[0088] Cell cytotoxicity proliferation experiment: Take the cell suspension with a concentration of 5×10 4 cells / mL and inoculate it into a 96-well plate, add 100 μL to each well, and incubate it in an incubator. Add 131 I-FAPI-TFMP-Y4, 131 I-FAPI-46, 131 I-TFMP-Y4 solution with different concentrations. After incubating for 24 h, add MTT solution and incubate for 4 h under light-proof conditions. Measure the absorbance value (OD value) at a wavelength of 570 nm for each well with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the cell survival rate. In vivo treatment experiment: Establish A549-FAP cell and A549 cell xenograft nude mouse models. Randomly divide the nude mice into multiple groups and inject 131 I-FAPI-Y4, 131 I-FAPI-46, 131 I-TFMP-Y4 and other drugs, and observe the changes in tumor volume and weight. (SeeFigure 18 )

[0089] Establishment of tumor-bearing nude mouse model: A tumor-bearing model was established by subcutaneously injecting 1×10 6 suspensions of A549-FAP or A549 cells into the right axilla of female BALB / c nude mice aged 4-6 weeks. The tumor volume was monitored daily (calculated according to the formula π×length×width×height / 6). When the tumor diameter reached 6-8 mm, the model was determined to be successfully established and used for subsequent experimental studies.

[0090] In vivo treatment experiment: The A549-FAP tumor-bearing nude mice were randomly divided into 8 groups (including PBS control group, non-radioactive drug group, Na 131 I group and 131 I-labeled drug group) and A549 tumor-bearing 131 I-FAPI-Y4 group. The treatment was carried out by intratumoral injection 3 times every other day (PBS group, non-radioactive drug 30 μg or 20 μg, radioactive drug 200 μCi). 0.1% potassium iodide was given continuously for 3 days before treatment to inhibit thyroid iodine uptake. The body weight and tumor volume of the nude mice were monitored daily. On the 7th day after the last treatment or when the tumor volume > 1000 mm 3 , the mice were sacrificed to remove the tumors and weigh them to evaluate the inhibitory effect of 131 I-labeled FAPI-Y4, FAPI-46 and TFMP-Y4 on tumor growth. After treatment, H&E staining was performed to understand the degeneration and necrosis of tumor cells. (See Figure 19 , Figure 20 , Figure 21 )

[0091] Experimental results

[0092] ① MTT experiment proved that FAPI-Y4 and TFMP-Y4 had no obvious toxic effects on A549-FAP cells, A549 cells and Beas-2b cells, and had good safety. While FAPI-46 had a proliferation inhibitory effect on the growth of the three cell lines, and showed a concentration-dependent manner; ② 131 I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 labeling rates were all above 95%, and had good stability; 131 I-FAPI-Y4 showed weak water solubility, 131 I-FAPI-46 and 131 I-TFMP-Y4 showed weak lipid solubility; ③ In the cell uptake and internalization experiments, 131 I-FAPI-Y4 had a high tumor cell uptake rate and a long retention time, 131 I-FAPI-46 was rapidly taken up and rapidly eluted, 131 I-TFMP-Y4 was slowly taken up; ④ 131I-FAPI-Y4, 131 I-FAPI-46 and 131 I-TFMP-Y4 all have the effect of inhibiting the proliferation activity of tumor cells, among which 131 I-FAPI-Y4 is the strongest; ⑤ In vivo experiments have proved that 131 I-FAPI-Y4 has good anti-tumor proliferation activity.

[0093] The iodine-131 labeled FAPI-Y4 tumor-targeted molecular probe of the present invention has good tumor targeting, stability and anti-tumor activity, and at the same time has low toxicity and side effects on normal cells. It is a potential drug for internal radiotherapy of pancreatic cancer and non-small cell lung cancer. The successful development of this probe provides new ideas and methods for the treatment of related tumors.

Claims

1. An ATE-FAPI-TFMP-Y4 targeted molecular probe, characterized in that, The amino acid sequence of TFMP-Y4 in the probe is YGLFGVLGSAKHVLPHVVPVIAEHL-NH2. TFMP-Y4 is linked to FAPI-46 through a linker, and a chelator ATE is introduced at the Lys terminus.

2. The ATE-FAPI-TFMP-Y4 targeting molecular probe according to claim 1, wherein It is characterized in that The linker is PEG2 and a Lys group.

3. An iodine-131 labeled ATE-FAPI-TFMP-Y4 targeting molecular probe, characterized in that, The amino acid sequence of TFMP-Y4 in the probe is YGLFGVLGSAKHVLPHVVPVIAEHL-NH2. TFMP-Y4 is linked to FAPI-46 through the linker PEG2 and a Lys group, and a chelator ATE is introduced at the Lys terminus. A radioactive label iodine-131 is bound to its amino acid group.

4. The preparation method of an iodine-131 labeled ATE-FAPI-TFMP-Y4 targeted molecular probe according to claim 3 comprises the following steps: a. Synthesis of FAPI-Y4 precursor: Connect FAPI-46 and TFMP-Y4 through PEG2 and Lys groups, and introduce the chelating agent ATE at the Lys end to obtain the FAPI-Y4 precursor; b. Radioiodine labeling: Use the radioiodine desmethyltin reaction to label 131 I onto the FAPI-Y4 precursor to obtain the iodine-131 labeled ATE-FAPI-TFMP-Y4 targeted molecular probe.

5. The preparation method according to claim 4, characterized in that, The purity of the FAPI-Y4 precursor is greater than 98%.

6. Use of the iodine-131-labeled ATE-FAPI-TFMP-Y4 targeting molecular probe according to claim 3 in the preparation of a drug for treating tumors.

7. The application according to claim 6, wherein The tumors are pancreatic cancer and non-small cell lung cancer.

8. A tumor treatment drug, characterized in that, Containing the iodine-131-labeled ATE-FAPI-TFMP-Y4 targeting molecular probe according to claim 3.

9. The medicament according to claim 8, characterized in that, It also includes pharmaceutically acceptable excipients.

10. The drug according to claim 8, characterized in that, The administration routes of the drug include but are not limited to intratumoral injection, intravenous injection, etc. The dosage of the drug is determined by a doctor according to the specific conditions of the patient.

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