Fibroblast activating protein targeted multivalent radioactive probe as well as preparation method and application thereof

Abstract

The invention relates to a fibroblast activating protein targeted multivalent radioactive probe as well as a preparation method and application thereof, and belongs to the technical field of radiopharmaceutical chemistry. The structural formula of the multivalent radioactive probe is shown in the formula I. The fibroblast activation protein targeted multivalent radioactive probe provided by the invention has high affinity and high specificity, also has a very high in-vivo focus uptake value and long residence time, and can be used for a fibroblast activation protein PET imaging agent and an internal irradiation therapeutic agent; therefore, the technical problems of poor targeting property and short residence time of the fibroblast activated protein probe in the prior art are solved.

Description

Technical Field

[0001] This invention belongs to the field of radiopharmaceutical chemistry, and more specifically, relates to a multivalent radioactive probe targeting fibroblast activation proteins, its preparation method, and its application, particularly relating to a... 68 Ga / 177 Lu-labeled inhibitory molecular probe targeting fibroblast activation protein (FAP) for diagnosis and treatment ( 68 Ga / 177 Lu-TJFAP1) and its preparation method and application. Background Technology

[0002] Fibroblast activating protein (FAP) is a type II transmembrane serine protease belonging to the dipeptidyl peptidase (DPP) family. FAP is expressed at extremely low levels in normal adult tissues, but is significantly upregulated in the tumor microenvironment (TME), particularly in cancer-associated fibroblasts (CAFs) of over 90% of epithelial malignancies (such as breast cancer, hepatocellular carcinoma, colorectal cancer, lung cancer, ovarian cancer, and pancreatic cancer). Furthermore, FAP is involved in various pathological processes, including chronic inflammation (such as rheumatoid arthritis and osteoarthritis) and cardiac remodeling after myocardial infarction. Due to its low background signal in normal tissues and high expression specificity in pathological tissues, FAP is considered an important biomarker and potential therapeutic target for tumors and fibrotic diseases. In recent years, positron emission tomography (PET) based on FAP has become a new pan-cancer molecular imaging diagnostic tool.

[0003] The emergence of small molecule FAP inhibitors (FAPIs) has further propelled the development of this field. Based on a quinoline structure, FAPIs can bind to FAPs with high affinity and are labeled with 68-Ga by chelating agents such as dodecanetetraacetic acid (DOTA) for use in PET imaging. 68 The Ga-FAPI-04 PET / CT can obtain specific and clear images in a variety of tumors, especially in 18 F-fluorodeoxyglucose ( 18 F-Fluorodeoxyglucose, 18F-FDG uptake showed a significant advantage in tumor types with low uptake (such as gastric cancer, pancreatic cancer, and liver cancer). Furthermore, FAPI-04 can also be used to label therapeutic radionuclides. 177 Lu is used for targeted radionuclide therapy, enabling integrated diagnosis and treatment.

[0004] Early treatment attempts for FAP primarily used the monoclonal antibody sibrotuzumab, in patients with colorectal cancer, specifically in free form. 131 Studies using I-labeled formulations have shown limited efficacy. Subsequently, various targeting strategies have been developed, including bispecific antibodies, antibody fragments, chimeric antigen receptor T cells (CAR-T), and antibody-drug conjugates (ADCs), some of which have entered Phase I clinical trials. Strategies based on small-molecule FAPIs can be used not only for imaging but also to carry therapeutic radionuclides, providing new avenues for precision tumor diagnosis and treatment. However, the relatively short retention time of FAPIs in tumors limits the effectiveness of their radionuclide therapy.

[0005] To improve tumor retention and enhance therapeutic efficacy, studies have reported on FAPI dimers (such as DOTA(SA.FAPi)2 and DOTAGA(SA.FAPi)2). By combining two FAP inhibitor monomers with a trifunctional chelator, not only is tumor retention prolonged, but therapeutic radionuclides can also be carried, achieving integration of diagnosis and treatment. Furthermore, a dual-target strategy for FAPIs combines FAP inhibitors with other tumor markers (such as integrin αvβ3), simultaneously targeting both the cancer cell fibrosis (CAF) and tumor angiogenesis, improving tumor uptake and prolonging tumor retention time. However, these molecules have complex structures and large molecular weights, potentially leading to pharmacokinetic instability, increased non-specific binding, and increased manufacturing challenges.

[0006] Multivalent binding strategies, by introducing multiple FAP inhibitor monomers onto a single molecule, can enhance binding affinity, significantly improve tumor targeting and retention time, and provide a new direction for the application of FAPI in precision oncology. Despite the numerous advantages of multivalent probes, their design and development still face several challenges. Achieving a balance between probe targeting affinity, in vivo stability, and tumor penetration, as well as metabolic clearance properties and synthetic feasibility, remain key issues that urgently need to be addressed in this field. Furthermore, the synthesis process of multivalent probes is generally more complex and costly; optimizing the synthesis steps and reducing synthesis costs are also crucial for the development of multivalent probes. Summary of the Invention

[0007] This invention provides a multivalent radioactive probe targeting fibroblast activation proteins, its preparation method, and its application. The structural formula of the multivalent radioactive probe is shown in Formula I. The multivalent radioactive probe targeting fibroblast activation proteins provided by this invention has a high tumor lesion uptake value and a long tumor lesion retention time, thereby solving the technical problems of poor targeting and short lesion retention time of existing fibroblast activation protein probes.

[0008] According to a first aspect of the present invention, a precursor for a multivalent radioactive probe targeting fibroblast activation proteins is provided, the structure of which is shown in Formula I: .

[0009] According to another aspect of the present invention, a multivalent radioactive probe targeting fibroblast activation proteins is provided, with a structure as shown in Formula II or Formula III: , .

[0010] According to another aspect of the present invention, a method for preparing the precursor of the aforementioned fibroblast activation protein-targeting multivalent radioactive probe is provided, comprising the following steps: (1) Diisopropylethylamine and 1-hydroxy-7-azabenzotriazolyl-oxo-tris(pyrrolidinyl)urea hexafluorophosphate were added to the solution of compound C1. After thorough mixing, the solution of compound C2 was added. After the reaction was completed, the solution was purified to obtain a white solid compound C3. Compound C3 was dissolved in trifluoroacetic acid for deprotection and then purified to obtain a white solid compound C4. (2) Add 1-hydroxy-7-azabenzotriazolyl-oxo-tris(pyrrolidinyl)urea hexafluorophosphate to the solution of compound C5, then add diisopropylethylamine and mix thoroughly to obtain a mixture of compound C5; then add the mixture of compound C5 dropwise to the solution of compound C4, while simultaneously adding diisopropylethylamine; after the reaction is complete, purify to obtain orange solid compound C6, which is the precursor of the multivalent radioactive probe targeting fibroblast activation protein shown in Formula I; the reaction route diagram is shown below: .

[0011] Preferably, the purification method is high performance liquid chromatography.

[0012] According to another aspect of the present invention, a method for preparing the aforementioned multivalent radioactive probe targeting fibroblast activation proteins is provided, comprising the following steps: The precursor of the multivalent radioactive probe targeting the fibroblast activation protein as described in claim 1, namely the compound shown in C6, is combined with... 68After mixing the GaCl3 solution, a buffer solution was added to adjust the pH of the system to acidic. The mixture was then heated, and after the reaction was complete, it was cooled. The product was then purified to obtain compound C7, which is the multivalent radioactive probe targeting fibroblast activation proteins as shown in Formula II. The reaction route is as follows: ; Alternatively, the precursor of the multivalent radioactive probe targeting fibroblast activation protein as described in claim 1 can be combined with... 177 The LuCl3 solution was mixed, and the pH of the system was adjusted to acidic by adding buffer solution. The mixture was then heated, and after the reaction was complete, it was cooled. The product was purified to obtain compound C8, which is the multivalent radioactive probe targeting fibroblast activation proteins as shown in Formula III. The reaction route is as follows: .

[0013] Preferably, the heating temperature for preparing the multivalent radioactive probe targeting fibroblast activation protein as shown in Formula II is 95-105°C, and the heating time is 10-20 min.

[0014] Preferably, the heating temperature for preparing the multivalent radioactive probe targeting fibroblast activation protein as shown in Formula III is 90-100℃, and the heating time is 10-20 min.

[0015] Preferably, the buffer solution is a hydrochloric acid-sodium acetate buffer solution.

[0016] According to another aspect of the present invention, the application of the fibroblast activation protein-targeting multivalent radioactive probe described in Formula II is provided, wherein the fibroblast activation protein-targeting multivalent radioactive probe is used to prepare tumor PET imaging reagents or radionuclide internal irradiation therapy reagents.

[0017] According to another aspect of the present invention, the application of the multivalent radioactive probe targeting fibroblast activation proteins is provided, characterized in that the multivalent radioactive probe targeting fibroblast activation proteins represented by Formula III is used in the preparation of radionuclide internal irradiation therapy reagents.

[0018] In summary, compared with the prior art, the above-described technical solutions conceived by this invention mainly possess the following technical advantages: (1) The probe in this invention has excellent tumor uptake value in FAP-positive tumors in vivo (U87MG cells to 68The Ga-TJFAP1 uptake value is above 6270.80 CPM, reaching 10758.9 CPM at 4 hours, and its long tumor lesion retention time (over 4 hours) improves the efficiency of diagnosis and treatment. The probe in this invention has better affinity for FAP, with higher uptake in tumors with high FAP expression than in tumors with low FAP expression, enabling quantitative analysis of intratumoral FAP expression levels. 177 The Lu-TJFAP1 radiopharmaceutical, with its high affinity, high tumor uptake value, and long tumor retention time, can efficiently accumulate and deliver the radiotherapy nuclide Lu-177 in tumors that overexpress the FAP protein, achieving highly efficient internal irradiation.

[0019] (2) In this invention 68 The Ga-TJFAP1 molecular probe can monitor the expression level and changes of FAP in primary and metastatic lesions in real time, dynamically, and systemically at the molecular level using PET imaging, thereby evaluating the efficacy of FAP immunotherapy in cancer patients. It can be used as a FAP / PET imaging agent. In this invention... 177 The Lu-TJFAP1 radiopharmaceutical, with its high affinity, high tumor uptake, and long tumor retention time, acts as a radiotherapy agent in tumors overexpressing the FAP protein. This probe molecule holds promise as a therapeutic molecular probe for FAP-targeting in precision oncology.

[0020] (3) The probe in this invention has good stability and maintains a single peak for up to 4 hours when incubated in PBS and FBS (fetal bovine serum). Attached Figure Description

[0021] Figure 1 The synthesis equation for the labeled precursor Dota-TJFAP1(C6).

[0022] Figure 2 This is the mass spectrum of compound Dota-TJFAP1(C6).

[0023] Figure 3 for 68 Ga / 177 The marking route of the Lu-TJFAP1 radioactive probe.

[0024] Figure 4 Radioactive probe 68 Ga / 177 Radioactive HPLC chromatogram of Lu-TJFAP1 labeling.

[0025] Figure 5 HPLC analysis was performed to assess the stability of the radioactive probe after incubation in PBS (pH 7.4) and FBS systems for 0, 1, 2, and 4 hours.

[0026] Figure 6 Radioactive probe 68 Results of in vitro cellular uptake studies of Ga-TJFAP1.

[0027] Figure 7 Radioactive probe 68 Results of Ga-TJFAP1 cell blockade experiment.

[0028] Figure 8 for 68 PET / CT fusion imaging of Ga-TJFAP1 labeled probes in U87MG subcutaneous tumor mice, quantitative analysis of SUVmax of tumor tissues at different time points and tumor / background ratio (TBR): (A) PET / CT fusion imaging of different 68-Ga labeled probes in U87MG subcutaneous tumor mice at 1, 2 and 4 hours; (B) Quantitative analysis of SUVmax of probes in tumor tissues at different time points; (C) Quantitative comparison of tumor / background ratio (TBR) in each group and at each time point.

[0029] Figure 9 for 68 Organ metabolism curves (heart, liver, kidney, muscle) of Ga-TJFAP1 in U87MG subcutaneous tumor mice over 2 hours and biodistribution over 4 hours.

[0030] Figure 10 Radioactive probe 68 Experimental and quantitative analysis of Ga-TJFAP1 autoradiography.

[0031] Figure 11 Radioactive probe 68 Figure 1: Results of in vivo competitive binding study of Ga-TJFAP1: (A) PET / CT imaging showing changes in tumor radioactivity distribution in U87MG subcutaneous tumor mice after injection of 68Ga-TJFAP1 alone or co-injected with different molar amounts of TJFAP1, C4, FAPI-46, and FAPI-04 precursors; (B) Quantitative analysis of SUVmax of each probe in tumor tissue in the 10-molar-level blocking group; (C) Comparison of SUVmax of each probe in tumor tissue in the 40-molar-level blocking group; (D) Quantitative analysis of SUVmax of each probe in the 100-molar-level blocking group; (E) Quantitative analysis of the biodistribution of 68Ga-TJFAP1 in major organs and tumors of mice at 4 hours.

[0032] Figure 12SPECT / CT biodistribution and tumor treatment results of the radioactive probe 177Lu-TJFAP1 in tumor-bearing mice: (A) Schematic diagram of experimental procedure; (B) SPECT imaging of 177Lu-TJFAP1 at various time points; (C) Tumor volume change curve after treatment; (D) Mouse survival curve analysis; (E) Statistical analysis of mouse weight change during treatment; (F, G) Quantitative analysis of probe biodistribution in tumors and major organs at different time points.

[0033] Figure 13 HE staining image of tumor section for pathological analysis.

[0034] Figure 14 Immunohistochemical analysis and quantitative statistical charts: (A) Ki-67 immunohistochemical analysis and positive rate statistics; (B) γ-H2AX protein expression analysis; (C) α-SMA protein expression analysis; (D) FAP protein expression analysis and quantitative statistics of each group's proportion. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0036] The structure of the multivalent radioactive probe targeting the fibroblast activation protein FAP of this invention is as follows:

[0037] The precursor structure of the multivalent radioactive probe targeting fibroblast activation proteins in this invention is as follows:

[0038] Example 1: A FAP-targeted radiotherapeutic molecular probe 68 Ga / 177 Lu-TJFAP1 Step 1: The precursor of the multivalent radioactive probe of this invention is prepared through the following steps (synthesis equation as follows). Figure 1 ): To a 10.0 mL solution of compound C1 (680 mg, 743 μmol, 1.00 equivalent) in N,N-dimethylformamide (DMF), diisopropylethylamine (DIEA) (192 mg, 1.49 mmol, 259 μL, 2.00 equivalent) and 1-hydroxy-7-azabenzotriazolyl-oxo-tris(pyrrolidinyl)urea hexafluorophosphate (HATU) (254 mg, 669 μmol, 0.90 equivalent) were added. The reaction mixture was stirred at 25°C for 0.5 h. Subsequently, a 2.00 mL solution of compound C2 (677 mg, 624 μmol, 0.84 equivalent) in DMF was added to the mixture, and the mixture was stirred at 25°C for 1 h (pH 8–9). The reaction product was directly purified by HPLC (A: 0.1% TFA in H2O, B: 0.075% TFA in ACN) to give a white solid compound C3 (540 mg, 272.65 μmol, yield 36.6%). Compound C3 (540 mg, 272 μmol, 1.00 equivalent) was dissolved in TFA (8.44 g, 66.6 mmol, 5.50 mL, 90% purity, 244 equivalent) and stirred at 25 °C for 1.5 h. The mixture was purified by high performance liquid chromatography (A: 0.1% TFA in H2O, B: 0.075% TFA in ACN) to give a white solid compound C4 (385 mg, 206 μmol, 75.6% yield, 97.8% purity, TFA).

[0039] HATU (125 mg, 329 μmol, 1.85 equivalents) was added to a DMF (7.00 mL) solution of compound C5 (245 mg, 329 μmol, 1.85 equivalents), followed by DIEA (161 mg, 1.25 mmol, 217 μL, 7.00 equivalents), and the mixture was stirred at 25 °C for 0.5 h. Then, the mixture of compound C5 was added dropwise in three portions to a DMF (5.00 mL) solution of compound C4 (333 mg, 178 μmol, 1.00 equivalents, TFA), simultaneously with DIEA (46.1 mg, 356 μmol, 62.1 μL, 2.00 equivalents). The reaction mixture was stirred at 25 °C for 5 h. The reaction mixture was purified by HPLC (A: 0.1% TFA in H2O, B: 0.075% TFA in ACN) to give an orange solid compound C6 (377 mg, 143 μmol, yield 80.4%, purity 97.1%, TFA).

[0040] Confirmation of compound Dota-TJFAP1(C6): Mass spectrometry verification: Theoretical molecular weight C114H166F2N24O27S3 [(M / 2)+H]+ 1219.6, measured molecular weight 1219.9 ( Figure 2 ).

[0041] The precursor preparation route is as follows:

[0042] Step 2: Labeling of the 68-Ga / 177-Lu radioactive probe: (Synthesis equation as follows) Figure 3 The C6 (labeled precursor) obtained in step one was mixed with 2 mL of... 68 After mixing with GaCl3 solution (296 MBq), 1 mL of hydrochloric acid-sodium acetate buffer (1M) was added to adjust the pH of the system to 3.9-4.0. The total reaction volume was 3 mL. After thorough mixing, the mixture was reacted at 100 °C for 15 minutes. After the reaction, it was cooled to room temperature, and the product was purified using Oasis HLB Plus Light filter cartridge (186005125, Waters) and C18Sep-Pak filter cartridge (WAT020515, Waters), with 0.5 mL ethanol as the eluent. Quality control was performed using radioactive high-performance liquid chromatography (HPLC). The radiochemical purity (RCP) of the radioactive molecular probe was calculated by dividing the peak area of ​​the radioproduct by the total peak area. Alternatively, it could be mixed with 177-Lu solution in a hydrochloric acid-sodium acetate system (HCl-NaAc, pH = 5). After thorough mixing, the mixture was reacted at 95 °C for 15 minutes. After the reaction, it was cooled, and the product was purified using HPLC. Quality control was performed using HPLC. The roadmap is as follows:

[0043] Step 3: Radioactive probe 68 Ga / 177 Lu-TJFAP1 radiochemical purity test: Radioactive HPLC chromatogram of the labeled reaction solution ( Figure 4 The graph shows that 68 Ga-TJFAP1 has a radiochemical purity greater than 99% and 177 The radiochemical purity of Lu-TJFAP1 is greater than 97%.

[0044] Example 2: 68 In vitro stability of Ga-TJFAP1 ~7.4 MBq, 100 μL 68The Ga-TJFAP1 radioactive probe was added to 100 μL of a mixture of 0.01 M PBS buffer and FBS (fetal bovine serum) and incubated at 37 °C. Samples were taken at 1, 2, 3, and 4 hours for detection. Figure 5 ).

[0045] Depend on Figure 5 It was found that the FAP-targeted radioactive molecular probe 68Ga-TJFAP1 maintained a single peak for 4 hours after incubation at 37℃ in 0.01 M PBS buffer and FBS system, indicating that 68Ga-TJFAP1 has good stability in both systems.

[0046] Figure 5 The results showed that the probe exhibited good radiochemical stability in both PBS and FBS systems.

[0047] Example 3: 68 In vitro cell uptake and cell blockade assays of Ga-TJFAP1 In cell uptake assays, human glioblastoma cell line (U87MG) and mouse hepatocyte carcinoma cell line were cultured in DMEM medium supplemented with 1% penicillin-drug antibiotics and 10% fetal bovine serum. U87MG cells, which highly expressed FAP, and Hepa1-6 cells, which expressed low FAP, were seeded into 6-well plates containing 10% FBS and cultured for 48 hours to achieve a cell density of approximately 80%. Before the experiment, the medium was replaced with FBS-free DMEM. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 68 Ga-TJFAP1 or 68 Ga-C4 or 68 Ga-FAPI-46 or 68 Ga-FAPI-04 was incubated with U87MG and Hepa1-6 for 1 h, 2 h, 3 h, and 4 h, respectively, and counts were performed using a γ-counter. Figure 6 ).

[0048] The blocking group received 10, 40, and 100 times the molar amounts of unlabeled TJFAP1, C4, FAPI-46 variants, and FAPI-04. Cells in each group were incubated at 37 °C for 1, 2, 3, and 4 hours, after which the reaction was terminated, the culture medium was discarded, and the cells were washed twice with 1 mL PBS. Then, 200 μL of NaOH solution was added to digest the cells. After all time points, the NaOH solution in each well was transferred to a gamma counter tube for measurement (1% of the volume of the added diluent was used as the marker). Figure 7 ).

[0049] Figure 6 and Figure 7 The results showed that U87MG was effective against FAP-targeted radioactive molecular probes.68 Ga-TJFAP1 uptake was higher in Hepa1-6 cells and the U87MG blockade group at every time point; although the uptake of Ga-TJFAP1 in Hepa1-6 cells decreased with increasing incubation time. 68 Ga-TJFAP1 uptake increased continuously, reaching a peak of 5659.50 CPM, but U87MG cells showed resistance to... 68 The uptake value of Ga-TJFAP1 remained above 6270.80 CPM, reaching 10758.9 CPM at 4 hours, and was significantly better than... 68 Ga-C4 (5583.23 CPM) and 68 Ga-FAPI-04 (4426.5 CPM) showed a significant difference.

[0050] In the TJFAP1 blocking experiment, U87MG cells were incubated with 10-fold and 40-fold unlabeled TJFAP1 precursor. Figure 7 In this context, A represents the unblocked U87MG. 68 Ga-TJFAP1 cell uptake; Figure 7 B in the figure shows the effect at 10 times the molar amount. 68 Ga-TJFAP1 uptake inhibition is limited, while a 40-molar dose achieves significant blockade, with a blocking effect comparable to that of 100-molar doses of C4 and FAPI-46. 68 The blocking effect of Ga-TJFAP1 is quite good. Figure 7 C in Figure 7 D and Figure 7 (E in the text). These results indicate that the FAP-highly expressed cell line U87MG is effective against FAP-targeting radioactive molecular probes. 68 Ga-TJFAP1 uptake was significantly higher than that in the FAP-low-expressing cell line Hepa1-6, and this uptake could be blocked by FAP-targeting non-radioactive molecular probes, i.e., FAP-targeting radioactive molecular probes. 68 Ga-TJFAP1 exhibits good in vitro affinity for the target FAP.

[0051] Example 4: Radioactive Probe 68 Ga-TJFAP1 in PET / CT imaging experiments and quantitative differences in tumor-bearing mice PET / CT imaging of tumor-bearing mice: 5 x 10⁵ cells were injected subcutaneously into the right shoulder of female 5-week-old Balbc / nude mice (purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.) 6 U87MG cells (5×10) 6 A tumor-bearing mouse model was constructed by suspending cells in 100 μL phosphate-buffered saline (PBS) and administering 0.2 mL (3.7 MBq) via tail vein injection. 68Ga-TJFAP1 underwent static PET / CT scans at 1, 2, and 4 hours post-injection. To quantify radiopharmaceutical uptake in vivo, the Standard Uptake Value (SUV) was used to evaluate drug uptake, and quantitative analysis of SUVmax and the tumor / background ratio was performed. SUV = lesion radioactivity concentration (kBq / ml) / injected dose (MBq, calculated decay) / body weight (kg). A higher value indicates a higher concentration of radioactive probes at that site.

[0052] like Figure 8 As shown in A, 68 Ga-TJFAP1 clearly visualized the tumor outline at 1, 2, and 4 hours post-injection, observed significant radioactive accumulation at the tumor site, and maintained a high level of tumor uptake signal throughout the imaging cycle, significantly higher than the other three probes. 68 Ga-C4, 68 Ga-FAPI-46, 68 Ga-FAPI-04).

[0053] Figure 8 Quantitative analysis of B in 8 and C in 8 showed that 68 Ga-TJFAP1 maintained high SUVmax values ​​at 1, 2, and 4-hour time points, with an average SUVmax of 5.12 at 4 hours. Regarding the tumor / background ratio, 68 Ga-TJFAP1 exhibits extremely high TBR, which increases over time, with an average TBR of 56.07 at 4 hours.

[0054] Example 5: Radioactive Probe 68 Biodistribution of Ga-TJFAP1 in tumor-bearing mice U87MG subcutaneous tumor mice were injected with 3.7 MBq. 68 Ga-TJFAP1 was administered and euthanized at 4 hours. Blood, tumor, muscle, and major organs were weighed and measured using a gamma counter. Data were normalized to 1% of the total count as a percentage of the injected dose per gram (%ID / g).

[0055] Figure 9 This indicates that within a timeframe of 0 to 120 minutes, 68 The SUV values ​​of Ga-TJFAP1 in the heart, liver, kidneys, and muscles remained relatively stable, indicating that... 68 Ga-TJFAP1 was not significantly metabolized or cleared during this time period. Compared with other organs, after 4 hours... 68The uptake of Ga-TJFAP1 in tumor tissues was significantly higher than in other tissues (such as blood, heart, liver, lungs, spleen, kidneys, stomach, small intestine, large intestine, muscles, and bones), reaching nearly 20% ID / g, followed by the kidneys (7.8% ID / g), indicating that it is mainly metabolized by the kidneys.

[0056] The above results indicate that FAP-targeted radioactive molecular probes 68 Ga-TJFAP1 exhibits good stability, high tumor specificity, and prolonged tumor retention in vivo, enabling specific imaging of FAP-overexpressing models.

[0057] Example 6: Radioactive Molecular Probe 68 Autoradiography experiment of Ga-TJFAP1 U87MG mice were euthanized, and subcutaneous tumor tissue was harvested and prepared into 25 μm thick frozen sections. Before the experiment, the sections were fixed with 4% paraformaldehyde for 15 minutes and then washed three times with PBS. The unblocked tumor tissue was then... 68 Ga-TJFAP1, along with unlabeled cold TJFAP1 solutions containing 10 and 40 molar amounts respectively, and mixtures of unlabeled cold C4, FAPI-46, and FAPI-04 containing 10, 40, and 100 molar amounts respectively, were added to tissue sections and incubated at room temperature for 1 hour. After incubation, the radioactive liquid on the surface of the sections was aspirated, and the sections were washed three times with PBS in a shaker. Subsequently, the sections were covered with a phosphorus screen for 1 hour, and autoradiography was performed.

[0058] In further autoradiographic normalization experiments, 68-Ga-labeled TJFAP1, C4, and FAPI-46 were incubated in tumor tissue, and different radioactivities were set as internal references for signal normalization.

[0059] Figure 10 A and Figure 10 B in the figure indicates that in the TJFAP1 blocking experiment, i.e., co-incubation of U87MG cells with 10-fold and 40-fold unlabeled TJFAP1 precursors, the results showed that the 10-fold molar amount had a significant effect on TJFAP1 inhibition. 68 Ga-TJFAP1 uptake inhibition is limited (high-intensity autoradiographic signal), while a 40-molar dose achieves significant blocking, with blocking effects comparable to 100-molar doses of C4 and FAPI-46. 68 The blocking results of Ga-TJFAP1 were comparable (significantly reduced autoradiographic signal in tissue).

[0060] Figure 10 C and Figure 10 The D in the text indicates 68The Ga-TJFAP1 group consistently exhibited the highest autoradiographic signal intensity (37 kBq) at the same activity, suggesting... 68 Ga-TJFAP1 competitively binds to TJFAP1, C4, and FAPI-46 variants at the FAP binding site, further confirming the probe's specific recognition ability for FAP.

[0061] Example 7: Radioactive Molecular Probe 68 In vivo competitive binding study of Ga-TJFAP1 U87MG tumor-bearing nude mice were divided into four groups to evaluate the specific competitive binding characteristics of the radioactive molecular probe in vivo. 68 Ga-TJFAP1 was co-administered via tail vein with 10 or 40 times the molar amount of unlabeled TJFAP1, or with 10, 40, and 100 times the molar amount of C4, FAPI-46, and FAPI-04. Static PET scans were performed 1 and 2 hours post-injection, and the SUVmax values ​​of the tumor region were quantitatively analyzed. Four hours after injection, mice were euthanized, and blood, tumor, muscle, and major organ samples were collected, weighed, and analyzed using a gamma counter. Results were expressed as percentage of injected dose per gram of tissue (%ID / g) and normalized to total injected activity.

[0062] Figure 11 The A PET / CT imaging results showed that, when given alone 68 After using Ga-TJFAP1 or in combination with 10-fold or 40-fold C4 and FAPI-46 precursors, the tumor site maintained a high intensity of radioactive signal, and there was no significant difference in signal intensity between the blocking groups and the unblocked group. However, when the molar amount of TJFAP1 precursor increased to 40-fold, or C4 and FAPI-46 reached 100-fold respectively, the radioactive signal in the tumor area was significantly weakened, and SUVmax decreased significantly, approaching the background level (see 11-B-11-D).

[0063] Figure 11 B in the data shows that only the TJFAP1 blockade group showed a partial decrease in SUVmax. The 40-fold molar blockade group ( Figure 11 In the C group, TJFAP1 precursor significantly inhibited tumor signaling, up to 100-fold blocking group ( Figure 11 The blocking effects of D), C4, and FAPI-46 in the precursor are only comparable to those of 40 times the TJFAP1 precursor, indicating that they can only compete with TJFAP1 for the same binding site at extremely high molar amounts.

[0064] Figure 11 The results showed that in the non-blocking and low-magnification precursor blockade groups, tumor tissue... 68Ga-TJFAP1 uptake (%ID / g) was high. In the TJFAP1 blockade and high molar amounts of other precursor blockade groups (100-fold), tumor uptake was significantly reduced, approaching that of background tissue.

[0065] Example 8: Radioactive Probe 177 SPECT / CT, biodistribution, and tumor treatment outcomes of Lu-TJFAP1 in tumor-bearing mice All animal experiments were conducted in strict accordance with the "Guidelines for the Management and Use of Laboratory Animals" issued by the Ministry of Health of China. U87MG glioblastoma cells (1×10⁻⁶) were used. 6 The right shoulder was subcutaneously inoculated into 6-week-old BALB / c nude mice (SPF Biotechnology Co., Ltd., China). When the tumor volume reached approximately 50 mm... 3 Each mouse received a single tail vein injection of 27.75 MBq of radioactive molecular probes. Tumor growth was monitored using calipers, and tumor volume was calculated using the formula (length diameter × width). 2 ) / 2. Throughout the experiment, mouse weight and survival data were continuously recorded.

[0066] Following imaging and treatment, tumor tissue from U87MG xenograft subcutaneous tumors was collected for histopathological evaluation. Analysis included hematoxylin and eosin (H&E) staining and Masson's trichrome staining to assess tumor morphology and fibrosis. Immunohistochemistry (IHC) and TUNEL (DAB) assays were used to evaluate the expression of FAP, type I collagen, Ki-67, γ-H2AX, α-SMA, and CD11b in the tumor microenvironment.

[0067] Imaging is performed according to the procedure. Figure 12 After A), the SPECT imaging results ( Figure 12 B) shows that 177 Lu-TJFAP1 radioactive signals remained significant in the tumor region even after 120 hours, demonstrating good tumor retention ability. Quantitative analysis of biodistribution (…) Figure 12 F in Figure 12 The result of G in the imaging is consistent with the imaging results. 177 Lu-TJFAP1 reached 8.5% ID / g in tumor tissue after 4 hours and 5.3% ID / g after 24 hours. In major non-target organs, it mainly accumulated in the kidneys, indicating that it is primarily metabolized by the kidneys.

[0068] Results of in vivo radiotherapy studies show 177 Lu-TJFAP1 group can significantly inhibit tumor growth ( Figure 12 (C) and extending the lifespan ( Figure 12 (D in the text), and there were no significant abnormalities in weight change ( Figure 12 The E in the formula shows good treatment tolerability and anti-tumor effect.

[0069] Pathological analysis ( Figure 13 Further explanation 177 After Lu-TJFAP1 treatment, the extent of tumor necrosis significantly increased, and the proportion of Ki-67 positivity significantly decreased. Figure 14 (A in the middle). 177 The expression of FAP in tumors decreased significantly in the Lu-TJFAP1 treatment group. Figure 14 (B in the text) indicates that the target molecule can effectively intervene in FAP-mediated tumor stroma remodeling, thereby promoting the improvement of the tumor microenvironment's structure and function. γ-H2AX staining results ( Figure 14 C) in the middle confirms 177 The Lu-TJFAP1 group showed the strongest DNA damage effect, suggesting that radiotherapy is effective. α-SMA ( Figure 14 D) in 177 The Lu-TJFAP1 treatment group showed significant downregulation, indicating a reduction in tumor stroma activation and effective suppression of the fibroblast phenotype.

[0070] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A precursor of a fibroblast activation protein targeted multivalent radioprobes, characterized in that, The structure is shown as formula I: 。 2. A fibroblast activation protein targeted multivalent radioprobes, characterized in that, The structure is shown as formula II or formula III: 、 。 3. The method for preparing the precursor of the multivalent radioactive probe targeting fibroblast activation proteins as described in claim 1, characterized in that, The method comprises the following steps: (1) adding diisopropylethylamine and 1-hydroxy-7-azabenzotriazolyl-oxo-tris(pyrrolidinyl) uronium hexafluorophosphate to a solution of compound C1, mixing thoroughly, then adding a solution of compound C2, purifying after the reaction is completed to obtain white solid compound C3; deprotecting compound C3 by dissolving in trifluoroacetic acid, and purifying to obtain white solid compound C4; (2) adding 1-hydroxy-7-azabenzotriazolyl-oxo-tris(pyrrolidinyl) uronium hexafluorophosphate to a solution of compound C5, then adding diisopropylethylamine, mixing thoroughly to obtain a mixture of compound C5; then adding the mixture of compound C5 dropwise to a solution of compound C4, and simultaneously adding diisopropylethylamine; purifying after the reaction is completed to obtain orange solid compound C6, which is a precursor of the fibroblast activation protein-targeted multivalent radioactive probe shown as formula I; the reaction scheme is shown as follows: 。 4. The method for preparing the precursor of the multivalent radioactive probe targeting fibroblast activation proteins as described in claim 3, characterized in that, The purification method is high performance liquid chromatography.

5. The method for preparing a multivalent radioactive probe targeting fibroblast activation proteins as described in claim 2, characterized in that, The method comprises the following steps: The precursor of the fibroblast activation protein targeting multivalent radioprobes of claim 1, i.e. the compound shown as C6, is mixed with 68 After mixing with GaCl3 solution, the pH of the system is adjusted to be acidic by adding buffer, then heating, cooling after the reaction is completed, using for purification of the product, to obtain compound C7, i.e. the fibroblast activation protein targeting multivalent radioprobes of formula II; the reaction scheme is as follows: ； or precursors of multivalent radioprobes targeting the fibroblast activation protein of claim 1 are administered to a patient in need of such treatment 177 The compound C8, i.e. the multivalent radioprobes targeting the fibroblast activation protein of formula III, is obtained by mixing LuCl3 solution, adjusting the pH of the system to be acidic by adding buffer, then heating, cooling after the reaction is completed, and purifying the product. The reaction scheme is as follows: 。 6. The production method according to claim 5, characterized by, The heating temperature for preparing the fibroblast activation protein-targeted multivalent radioactive probe shown as formula II is 95-105 DEG C, and the heating time is 10-20 min.

7. The production method according to claim 5, characterized by, The heating temperature for preparing the fibroblast activation protein-targeted multivalent radioactive probe shown as formula III is 90-100 DEG C, and the heating time is 10-20 min.

8. The production method according to claim 5, wherein The buffer is hydrochloric acid-sodium acetate buffer.

9. Use of a fibroblast activation protein targeted multivalent radioprobes according to claim 2, wherein, The fibroblast activation protein-targeted multivalent radioactive probe shown as formula II is used for preparing a tumor PET imaging reagent or a radionuclide internal irradiation therapy reagent.

10. Use of a fibroblast activation protein targeted multivalent radioprobes according to claim 2, wherein, The fibroblast activation protein-targeted multivalent radioactive probe shown as formula III is used for preparing a radionuclide internal irradiation therapy reagent.

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