Targeted FAP trimer compound, probe, and preparation method and application thereof

Abstract

The invention relates to a radionuclide labeled trimer probe 68Ga-TRAP-(FAPI) 3 targeting FAP and a preparation method thereof, Trap is used as a trivalent chelating platform, and three FAPI targeting units are covalently connected by clicking a chemical linking arm to form a trimer structure; the invention further relates to application of the 68Ga-TRAP-(FAPI) 3 probe in preparation of imaging agents or therapeutic drugs for diagnosing FAP positive diseases, experiments prove that the trimer probe is good in affinity and high in stability, and in cell experiments and animal experiments, the uptake rate of FAP positive cells is kept at a high level within 240 minutes; preclinical and preliminary clinical applications show that the tumor detection rate of the < 68 > Ga-TRAP-FAPI 3 in various cancer models is superior to that of < 18 > F-FDG, and compared with < 68 > Ga-FAPI-04, the < 68 > Ga-TRAP-FAPI 3 is clearer to display focuses (particularly tiny metastases), higher in uptake intensity and particularly outstanding in delayed imaging, and the < 68 > Ga-TRAP-FAPI 3 can be used for preparing a medicine for treating cancer. And a novel drug candidate with better performance is provided for precise diagnosis and staging of tumors and subsequent radionuclide targeted therapy.

Description

Technical Field

[0001] This invention belongs to the field of radiopharmaceutical technology, and particularly relates to a trimeric compound, probe, preparation method and application of FAP targeting. Background Technology

[0002] In the field of nuclear medicine for tumors, radiotherapeutic probes targeting fibroblast activating protein (FAP) have become a research hotspot in recent years. Numerous preclinical and clinical studies have confirmed that FAP is specifically highly expressed in cancer-associated fibroblasts of various epithelial tumors and is closely related to tumor proliferation, invasion, metastasis, and poor prognosis, making it a highly promising diagnostic and therapeutic target.

[0003] Currently, several FAP inhibitor (FAPI) derivatives have been developed and applied in clinical practice, such as FAPI-02, FAPI-04, and the subsequently optimized FAPI-46 probes. These probes are typically in monomeric form, and while they can effectively target FAP-positive tumors, they exhibit significant limitations in practical applications. The core issue lies in their suboptimal pharmacokinetic properties: these monomeric probes are rapidly cleared from the systemic circulation, exhibiting extremely short blood half-lives. For example, FAPI-02 and FAPI-04 are largely cleared within one hour of injection. This directly results in a very short retention time of the probe at the tumor site, with significantly reduced uptake in the tumor by the following day.

[0004] This rapid metabolism and clearance present a dual challenge. In diagnosis, the short tumor retention time limits the optimal time window for imaging techniques such as positron emission tomography (PET), making it difficult for probes to accumulate sufficiently in tumor lesions, especially small or low-metabolic metastases, thus affecting image contrast and lesion detection rates. In treatment, therapeutically labeled probes require a sufficiently long intratumoral retention time to release a radiation dose sufficient to kill tumor cells. The rapid "elution" of existing monomeric probes results in insufficient effective radiation dose absorbed by tumor tissue, often requiring increased dosage or frequency of administration to compensate. This undoubtedly increases the patient's systemic radiation exposure and potential toxic side effects, limiting therapeutic efficacy and safety. Although FAPI-46 has improved pharmacokinetics to some extent through optimization of the linker group, its retention time in blood and tumors is limited, failing to fundamentally solve the aforementioned bottlenecks. Therefore, developing a novel FAP-targeting probe capable of high uptake and long-lasting retention at the tumor site is an urgent clinical need for improving tumor diagnosis and treatment. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention proposes a trimeric compound targeting FAP, a probe, its preparation method, and its application.

[0006] The technical solution of this invention is as follows: A trimer compound targeting FAP has the following structure: ; Where R1 is O or N-ME.

[0007] The method for preparing the FAP-targeting trimer compound includes the following steps: S1. Compound 3 Trap is subjected to a first amidation reaction with compound 4 containing an alkyne group to obtain compound 5; S2. The compound 5 obtained in step S1 is reacted with compound 6 containing azide group and carboxylic acid under the catalysis of copper catalyst and reducing agent to obtain compound 7; S3. Compound 7 obtained in step S2 is subjected to a second amidation reaction with an FAP inhibitor to obtain a trimer compound targeting FAP.

[0008] Furthermore, compound 4 in step S1 is propargylamine.

[0009] Furthermore, compound 6 in step S2 is 5-azidopentanoic acid.

[0010] Furthermore, the FAP inhibitor mentioned in step S3 is FAPI.

[0011] Furthermore, when R1 is O, FAPI is the compound with CAS number 2471983-20-5, and when R1 is N-ME, FAPI is the compound with CAS number 2883407-81-4.

[0012] Further, the molar ratio of compound 3 and compound 4 in step S1 is 1:(8-15).

[0013] Furthermore, the molar ratio of compound 5 and compound 6 in step S2 is 1:(8-15).

[0014] Further, the molar ratio of compound 7 and the FAP inhibitor in step S3 is 1:(2-6).

[0015] A radionuclide-labeled trimer probe for targeting FAP is obtained by labeling a radionuclide with the FAP-targeting trimer compound of claim 1, and its structure is shown below, wherein R represents a radionuclide labeling group. ; Where R1 is O or N-ME.

[0016] Furthermore, the nuclide labeling group is 18 F, 43 Sc、 44 Sc、 47 Sc、 55 Co、 58m Co、 61 Cu、 64 Cu、 67 Cu、 68 Ga、 86 Y、 89 Zr、 89 Sr、 90 Y、 99m Tc, 111 In、 123 I, 124 I, 125 I, 131 I, 153 Sm、 159 Gd, 161 Tb, 177 Lu、 186 Re、 188 Re、 203 Pb, 211 At、 212 Pb, 213 Bi、 223 Ra、 225 At least one of Ac.

[0017] Furthermore, the nuclide labeling group is 18 F, 68 Ga、 99m Tc, 161 Tb, 177 Lu and 211 At least one of At.

[0018] The application of the radionuclide-labeled FAP-targeting trimer probe in the preparation of imaging agents or therapeutic drugs for diagnosing FAP-positive diseases.

[0019] Furthermore, the FAP-positive diseases are selected from tumors, fibrotic diseases, arthritis, or atherosclerosis.

[0020] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to a radionuclide-labeled trimer probe for targeting FAP. 68Ga-TRAP-(FAPI)3 and its preparation method use Trap as a trivalent chelation platform to covalently connect three FAPI targeting units through click chemical linker arms to form a trimer structure. The raw materials are readily available, and the trimer compound can efficiently coordinate with diagnostic and therapeutic radionuclides, making it suitable for large-scale production. This solves the problems of existing FAPI monomer probes being cleared too quickly in vivo and having a short tumor retention time.

[0021] 2. The present invention also relates to the trimer probe. 68 The application of Ga-TRAP-(FAPI)3 in the preparation of imaging agents or therapeutic drugs for diagnosing FAP-positive diseases has been experimentally verified. The trimeric probe described in this invention exhibits good affinity and excellent stability both in vivo and in vitro, with an uptake rate of 2.31% ± 1.08% in FAP-positive cells at 240 minutes. In a tumor-bearing mouse model, tumor uptake remained at a high level 240 minutes after injection (SUVmax: 3.03 ± 0.66 %ID / g). Preclinical and preliminary clinical applications show that… 68 Ga-TRAP-(FAPI)3 showed superior tumor detection rates in various cancer models. 18 F-FDG, and compared to 68 Ga-FAPI-04 provides clearer visualization and higher uptake intensity for lesions (especially micrometastases), with particularly outstanding advantages in delayed imaging. It offers a superior new drug candidate for the accurate diagnosis, staging, and subsequent radionuclide targeted therapy of tumors. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.

[0023] Figure 1 This is Embodiment 3 of the present invention. 68 Ga-Trap-(FAPI)3and 68 Figure showing the hydrophilicity test results of the Ga-FAPI-04 probe; Figure 2 This is Embodiment Four of the present invention. 68 Figures showing the in vivo and in vitro stability test results of the Ga-Trap-(FAPI)3 probe; Figure 3 This is the fifth embodiment of the present invention. 68 Figure 1; Results of Ga-Trap-(FAPI)3 probe cell binding and uptake experiment; Figure 4 This is the sixth embodiment of the present invention. 68 Ga-Trap-(FAPI)3 probe affinity (IC) 50 ) Measurement results graph; Figure 5 This is the seventh embodiment of the present invention. 68 PET / CT imaging of tumor-bearing mice with Ga-Trap-(FAPI)3 probe; Figure 6 This is the eighth embodiment of the present invention. 68 Figure showing the results of a study on the biodistribution of the Ga-Trap-(FAPI)3 probe; Figure 7 This is the ninth embodiment of the present invention. 68 Figure 1; Acute toxicity test results of Ga-Trap-(FAPI)3 probe; Figure 8 This is the tenth embodiment of the present invention. 68 Human radiation dose assessment diagram of Ga-Trap-(FAPI)3 probe; Figure 9 This is the eleventh embodiment of the present invention. 68 Preliminary clinical application case of Ga-Trap-(FAPI)3 probe: PET imaging image; Figure 10 This is the eleventh embodiment of the present invention. 68 Comparison of Ga-Trap-(FAPI)3 probe head-to-head with control probe and immunohistochemical verification image. Detailed Implementation

[0024] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0025] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.

[0026] Unless otherwise specified, the abbreviations used in this invention shall have the following meanings: FAP: Fibroblast Activation Protein; FAPI: Fibroblast Activation Protein Inhibitor; PET: Positron Emission Tomography; CT: Computed Tomography; DMF: N,N-Dimethylformamide; HBTU: O-benzotriazol-tetramethyluronium hexafluorophosphate, O-(1H-Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; DIPEA: N,N-Diisopropylethylamine; LC-MS: Liquid Chromatography-Mass Spectrometry; THF: Tetrahydrofuran; VC: Ascorbic Acid; PBS: Phosphate Buffered Solution; FBS: Fetal Bovine Serum; HPLC: High Performance Liquid Chromatography; TFA: Trifluoroacetic acid; IC 50 Half-maximal inhibitory concentration (HMC). VOI: Volume of Interest; SUV: Standardized Uptake Value; ID / g: Percentage of injected dose per gram of tissue. ALT: Alanine Aminotransferase; AST: Aspartate Aminotransferase; UA: Uric Acid; CREA: Creatinine; FDG: Fluorodeoxyglucose.

[0027] Example 1: Synthesis of Precursor Compounds The precursor compound Trap-(FAPI)3 (compound 2) of this invention is prepared by the following steps, wherein compound 3 is the chelating agent Trap, CAS number: 1242003-07-1; compound 4 is propargylamine, CAS number: 2450-71-7; compound 6 is 5-azidopentanoic acid, CAS number: 79583-98-5; and compound 8 is FAPI, CAS number: 2471983-20-5. The structure of the precursor compound Trap-(FAPI)3 is shown below: S1. Compound 3 (1 equivalent) was dissolved in DMF, HBTU (18.7 equivalents) and DIPEA (28.8 equivalents) were added, and finally compound 4 (10 equivalents) was added. The mixture was stirred overnight at room temperature. After the reaction was completed, the reaction was confirmed to be complete by liquid chromatography-mass spectrometry (LC-MS). The solvent was removed by rotary evaporation, and compound 5 was purified by reversed-phase preparative liquid chromatography with a yield of 45.3%. The purification parameters were as follows: buffer A: 0.1% TFA / H2O, buffer B: ACN, Bondysil-C18 column (250×30mm); 0-7 min, 10% buffer B; 7-67 min, buffer B increased from 10% to 100%; flow rate 25 mL / min, retention time: 20 min.

[0028] S2. Compound 5 (1 equivalent) and compound 6 (10 equivalent) were dissolved in a mixed solution of THF and H2O (volume ratio 2:1), followed by the addition of CuSO4·5H2O (2 equivalent) and ascorbic acid (VC, 4 equivalent), and the reaction was carried out at room temperature for 4 hours. After the reaction was completed, the reaction was confirmed to be complete by LC-MS monitoring. The solvent was evaporated, and compound 7 was purified by reversed-phase preparative liquid chromatography with a yield of 53.6%. The purification process parameters were as follows: buffer A: 0.1% TFA / H2O, buffer B: ACN, Bondysil-C18 column (250×30mm); 0-7 min, 100% buffer A; 7-67 min, buffer B increased from 0% to 60%; flow rate 25 mL / min, retention time: 28 min.

[0029] S3. Compound 7 (1 equivalent) was dissolved in DMF, HBTU (3 equivalents) and DIPEA (10 equivalents) were added, and finally compound 8 (4 equivalents) was added. The reaction was carried out at room temperature for 2 hours. After the reaction was completed, the reaction was confirmed to be complete by LC-MS monitoring. The solvent was removed by rotary evaporation, and the target precursor compound Trap-(FAPI)3 (compound 2) was purified by reversed-phase preparative liquid chromatography with a yield of 23.7%. The purification process parameters were as follows: Buffer A: 0.1% TFA / H2O, Buffer B: ACN, Bondysil-C18 column (250×30mm); 0-7 min, 20% Buffer B; 7-67 min, Buffer B increased from 20% to 40%; flow rate 25 mL / min, retention time: 28 min.

[0030] The preparation method described in this invention has a simple reaction route, is easy to operate, uses inexpensive and readily available raw materials, and has a low production cost, making it suitable for industrial-scale production.

[0031] Example 2 68 Synthesis and Quality Control of Ga-Trap-(FAPI)3 Probes Add 1.1 ml of a 0.15 mol / L sodium acetate aqueous solution containing 20 μg Trap-(FAPI)3 to 4 mL of a solution dissolved in 0.05 mol / L hydrochloric acid. 68 A GaCl3 solution (activity 0.74 GBq) was mixed and subjected to radiolabeling at 105 °C for 5 min. After the reaction, the reaction solution was purified using a Sep-Pak C18 solid-phase extraction column, eluting with 0.7 ml of 70% ethanol, then diluted with 4.3 ml of physiological saline, and finally sterile filtered to obtain the final product. 68 The Ga-Trap-(FAPI)3 probe (compound 1) has the following structural formula.

[0032] 68Ga-Trap-(FAPI)3 Quality Control: Referring to the Guidelines and General Rules for Quality Control of Positron-emitting Radiopharmaceuticals in the 2020 edition of the Chinese Pharmacopoeia, the following quality inspection items and methods are formulated: (1) Appearance inspection: visual inspection is adopted, and the color and clarity of the sample are visually inspected through lead glass. (2) pH value inspection: precision pH test paper is used. A small amount of sample is spotted on the precision pH test paper, and compared with the standard colorimetric card after 30s. (3) Radiochemical purity determination and product identification: HPLC method was used, with B-FC-3200 γ-ray detector and ultraviolet detector. The ultraviolet detection wavelength was 254 nm. The analytical column was a C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase A was acetonitrile, and the mobile phase B was 0.1% TFA water. The gradient method was used: from 0 to 5 min, the mobile phase A was maintained at 5%; from 5 to 10 min, the mobile phase A was increased to 95%; from 10 to 15 min, the mobile phase A was maintained at 95%; from 15 to 20 min, the mobile phase A was decreased to 5%; and from 20 to 25 min, the mobile phase A was maintained at 5%. The flow rate was 1 mL / min. The ratio of the radiochromatographic peak area of ​​the sample was measured, and the radiochemical purity of the sample was calculated. (4) Nuclide identification: Half-life method was used. (5) Sterility detection: Bacterial culture method was used. (6) Bacterial endotoxin: Limulus amebocyte lysate (LAL) reagent method was used. The results are shown in Table 1.

[0033] Table 1 68 Quality control results of Ga-Trap-(FAPI)3 probe Example 3 68 Hydrophilicity test of Ga-Trap-(FAPI)3 probe By measurement 68 The lipophilicity of the Ga-Trap-(FAPI)3 probe was assessed by evaluating its partition coefficient in isooctanol and phosphate-buffered saline (PBS, pH 7.4). A simplified procedure was performed as follows: 500 μL of isooctanol and 490 μL of PBS were mixed with 10 μL of... 68 Ga-Trap-(FAPI)3 (37 kBq) was mixed in a microcentrifuge tube, vortexed vigorously for 5 minutes, and then centrifuged at 2000 rpm for 3 minutes. After the two phases were clearly separated, 100 μL of the organic phase and the aqueous phase were taken separately, and their radioactivity was measured using a gamma counter. The logarithm of the partition coefficient (Log D 7.4) was calculated as the ratio of the radioactivity in the organic phase to the radioactivity in the aqueous phase; the experiment was repeated in triplicate.

[0034] Hydrophilicity test results are as follows Figure 1 As shown, the results demonstrate that the product prepared in this invention... 68 Ga-Trap-(FAPI)3 exhibits high hydrophilicity, with a logD7.4 value of -2.71 ± 0.19, compared to... 68The Ga-FAPI-04 (-3.962±0.16) value indicates that the probe prepared in this invention has high hydrophilicity.

[0035] Example 4 68 Stability experiment of Ga-Trap-(FAPI)3 probe To assess in vitro stability, 100 μL of freshly prepared [material / material] was [used / distributed]. 68 Ga-Trap-(FAPI)3 solution was mixed with 900 μL PBS or fetal bovine serum (FBS) and incubated at 37°C. Radiochemical purity was analyzed by HPLC at 1, 2, and 3 hours after incubation. To assess in vivo stability, 11.1 MBq of the solution was injected into healthy mice via the tail vein. 68 Ga-Trap-(FAPI)3 was administered, and urine samples were collected 1, 2, and 3 hours after injection. The metabolic stability was analyzed by radioactive HPLC.

[0036] Stability test results are as follows Figure 2 As shown, 68 Ga-Trap-(FAPI)3 exhibited excellent in vitro stability after 3 hours of incubation in PBS and FBS, with >95% of the radioactive compound remaining intact as determined by HPLC. In vivo metabolic studies showed... 68 Ga-Trap-(FAPI)3 maintains high stability after circulation in vivo, demonstrating its excellent stability both in vivo and in vitro.

[0037] Example 5 68 Cell binding assay of Ga-Trap-(FAPI)3 probe For evaluation 68 The FAP-specific binding ability of the Ga-Trap-(FAPI)3 probe was investigated using the human glioblastoma cell line U87MG (high FAP expression) and the human non-small cell lung cancer cell line A549 (low FAP expression) for cell uptake experiments. Cells were routinely cultured in DMEM medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO2. During the experiment, cells were seeded into 24-well plates and incubated overnight with 500 μL of complete medium. After 24 hours, the medium was replaced with 500 μL of serum-free DMEM, and 100 μL (37 kBq) of [a specific substance / method] was added. 68 Ga-Trap-(FAPI)3 or 68 Ga-FAPI-04 was used as a control. Cells were incubated at 37°C and 5% CO2 for 10, 30, 60, 120, or 240 minutes, respectively. To verify binding specificity, an inhibition experimental group was set up, i.e., when Ga-FAPI-04 was added... 68Before using Ga-Trap-(FAPI)3, cells were pre-incubated with 10 μg of unlabeled FAPI-04.

[0038] The experimental results confirmed that 68 Ga-Trap-(FAPI)3 exhibits efficient FAP-specific binding, such as Figure 3 As shown, in FAP-positive U87MG cells, the accumulation of the trimer tracer of this invention was significantly higher than that of the control monomeric probe. 68 Ga-FAPI-04. At 240 minutes, 68 The uptake rate of Ga-Trap-(FAPI)3 reached 2.31% ± 0.42%, while 68 The uptake rate of Ga-FAPI-04 was only 0.07% ± 0.03%, which is attributed to the enhanced binding capacity and stability of the trimer structure.

[0039] The specificity of the probe binding to U87MG cells was confirmed by the following two key findings: (1) when pre-incubation with an excess of unlabeled FAPI-04 was used to block the binding, 68 Ga-Trap-(FAPI)3 uptake was significantly inhibited at 60 minutes (from 2.57% ± 0.41% to 0.27% ± 0.15%); (2) in FAP-negative A549 cells, 68 The uptake rate of Ga-Trap-(FAPI)3 at 60 minutes was extremely low, only 0.18% ± 0.08%, in stark contrast to the high uptake in U87MG cells (2.57% ± 0.41%); these data together indicate that... 68 Cellular uptake of Ga-Trap-(FAPI)3 is specifically mediated by the FAP protein, and its binding affinity and cell retention properties are superior to existing monomeric probes.

[0040] Example 6 68 Affinity determination of Ga-Trap-(FAPI)3 probe To determine the affinity of the probe for FAP, a competitive binding experiment was conducted in this embodiment. 100 μL (37 kBq) of... 68 Ga-Trap-(FAPI)3 with a series of concentration gradients (from 10 to 10) -4 Up to 10 -13 Unlabeled Trap-(FAPI)3, a competitor of M), was co-incubated in U87MG cells for 60 minutes. After incubation, the culture medium was aspirated, and the cells were washed twice with pre-cooled PBS to remove unbound radioactive probes. Subsequently, the cells were lysed with 0.5 M NaOH, and the cell lysates were collected and their radioactivity was measured using a gamma counter.

[0041] Competition inhibition curves were plotted by measuring cellular radioactive uptake in the presence of different concentrations of competing agents, such as... Figure 4 As shown; calculations show that, 68 The half-maximum inhibitory concentration (IC50) of Ga-Trap-(FAPI)3 50 The value is 2.18 nM. This value is consistent with the reported values ​​for monomeric probes in the literature. 68 The affinity of Ga-FAPI-04 is at the same level, which indicates that the present invention significantly prolongs the binding time of the probe to the cell through multivalent design, while fully preserving the high intrinsic binding affinity of the FAPI molecule itself to the FAP target.

[0042] Example 7 68 PET / CT imaging of tumor models using Ga-Trap-(FAPI)3 probe All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of the Third Military Medical University (AMUWEC20257015). This involved using 100 μL of a solution containing 5 × 10⁶... 6 A cell suspension of U87MG (FAP positive) or A549 cells (FAP negative) was injected into the right shoulder region of male Balb / c nude mice (6 weeks old, ~22g) to establish a tumor-bearing mouse model; 150μL (9.25MBq) was injected intravenously into the tumor-bearing mice. 68 Ga-Trap-(FAPI)3 was administered, and whole-body in vivo PET scans were performed at 30, 60, 120, and 240 minutes post-injection. The imaging acquisition time was 600 seconds. As a control, 68 The Ga-FAPI-04 scan followed the same experimental protocol.

[0043] PET imaging results as follows Figure 5 It is clearly shown that in the U87MG tumor model, 68 Ga-Trap-(FAPI)3 exhibits superior in vivo performance. This probe achieves highly specific tumor uptake within 30 minutes of injection and continues to accumulate within the tumor over time. In contrast, 68 Ga-FAPI-04 exhibited only moderate initial uptake at 30 minutes, followed by rapid clearance from the tumor. Crucially, 68 Tumor uptake of Ga-Trap-(FAPI)3 was significantly competitively inhibited by co-injected unlabeled FAPI-04, demonstrating its high FAP specificity in binding. Regarding the normalized maximum uptake (SUVmax) within malignant lesions, 68 Ga-Trap-(FAPI)3 significantly outperforms other imaging techniques at extended imaging times (240 minutes). 68Ga-FAPI-04 (3.03 ± 0.66 %ID / g vs. 0.41 ± 0.04 %ID / g). In FAP-negative A549 tumors, 68 Ga-Trap-(FAPI)3 showed only minimal uptake. Immunohistochemical analysis of the resected tumor further confirmed that tracer accumulation in PET images was highly correlated with FAP expression levels within the tumor microenvironment (TME). These data collectively validated... 68 Ga-Trap-(FAPI)3 has the ability to specifically target FAP in vivo and can be used as an effective tool for non-invasive assessment of FAP expression.

[0044] Example 8 68 Biodistribution of Ga-Trap-(FAPI)3 probes Biodistribution studies were conducted in U87MG tumor-bearing nude mice to quantitatively analyze the distribution of the probe in vivo. Each mouse was injected via tail vein with 100 μL containing 7.4 MBq. 68 A solution of Ga-Trap-(FAPI)3 was prepared. Mice were anesthetized and sacrificed at specified time points (30, 60, 120, and 240 minutes) after injection, and major organs and tissues (such as heart, liver, spleen, lung, kidney, tumor, muscle, etc.) were subsequently harvested. The harvested samples were weighed and their radioactivity was measured using a gamma counter, with results expressed as a percentage of the injected dose per gram of tissue (%ID / g).

[0045] Results of biological distribution data research, such as Figure 6 As shown, 30 minutes after injection, 68 Ga-Trap-(FAPI)3 exhibited significant uptake in both the kidneys and tumors (at which point the tumor-to-kidney ratio was 0.43), confirming that this hydrophilic radiotracer is primarily cleared by the kidneys. As time progressed to 4 hours post-injection, the tumor-to-kidney ratio increased to 2.33 due to the rapid clearance of the probe from normal tissues and a significantly prolonged retention time in the tumor. Simultaneously, the tumor-to-muscle and tumor-to-liver ratios reached 13.14 and 3.26, respectively. These data demonstrate the excellent tumor targeting and high target-to-background ratio of the probe of this invention.

[0046] Example 9 68 Toxicity test of Ga-Trap-(FAPI)3 probe Acute toxicity was assessed using male KM mice. The experimental group mice received a single tail vein injection of 100 μL of 37 MBq. 68 Ga-Trap-(FAPI)3 was administered to control mice, who were injected with an equal volume of 10% ethanol saline. Venous blood samples were collected 60 minutes after injection to assess key biochemical indicators of liver and kidney function.

[0047] The results of the analysis of key hematological parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acid (UA), urea, and serum creatinine (CREA), are as follows: Figure 7 As shown, compared with the control group, no significant changes were observed in any of the indicators in the experimental group mice injected with a high dose of tracer (1 mCi / mouse). This indicates that... 68 Ga-Trap-(FAPI)3 did not induce any acute adverse reactions at the stated dosage, demonstrating good biocompatibility.

[0048] Example 10 68 Radiation dose assessment of Ga-Trap-(FAPI)3 probe Using PMOD software, four participants were... 68 PET images of healthy volunteers injected with Ga-TRAP-(FAPI)3 at five time points were analyzed. Figure 8 a). Delineate the volumes of interest (VOIs) of major organs on CT images and map them onto registered PET images to quantify the normalized uptake values ​​(SUVs) of each organ. Figure 8 (b) Time-activity curves were generated by standardizing VOI data to reference organ weights. Bladder contents activity was estimated using the trapezoidal method, and the time-activity data were fitted with a biexponential method using OLIDA / EXM software to calculate the absorbed dose and effective dose for each organ in the adult model.

[0049] Dosimetric estimation results (see Table 2) show that the organs receiving the highest effective doses were the thyroid gland (5.09E-03 ± 1.58E-03 mSv / MBq), liver (2.10E-03 ± 2.24E-04 mSv / MBq), and stomach wall (2.10E-03 ± 2.31E-04 mSv / MBq). 68 The overall effective dose of Ga-TRAP-(FAPI)3 was 2.15E-02 ± 3.13E-03 mSv / MBq, which is consistent with... 68 It is comparable to Ga-FAPI-04 and falls within the clinically acceptable safety range.

[0050] Table 2. Average uptake of radiotracers in target organs Example 11 68 Preliminary clinical application of Ga-Trap-(FAPI)3 probe All clinical studies involving human subjects were approved by the Clinical Research Ethics Committee of Daping Hospital, Army Medical University, and registered with ClinicalTrials. Nine patients (including one with esophageal cancer, one with gastric cancer, one with ovarian cancer, one with pancreatic cancer, two with lung cancer, two with colon cancer, and two with liver cancer) were studied using a Siemens Biograph 64 scanner. 18 F-FDG, 68 Ga-Trap-(FAPI)3and 68 PET / CT contrast imaging with Ga-FAPI-04. (The sentence appears to be incomplete and requires further context.) 18 Prior to F-FDG (5.5 MBq / kg), patients had fasted for ≥6 hours and had a confirmed pre-scan blood glucose level ≤11.1 mmol / L. No special preparation was required for patients using 68Ga-labeled tracers (111–185 MBq). Static images were acquired 1 hour after injection for all tracers. 68 Ga-Trap-(FAPI)3and 68 The Ga-FAPI-04 additionally acquired 4 hours of images. The clinical application case demonstrates the superiority of the probe.

[0051] All scans covered the area from the top of the skull to the mid-femur, with the patient in a supine position. High-dose CT (120 kVp, 130 mA) was acquired for attenuation correction, followed by 3D PET acquisition (1.5 minutes / bed). Image reconstruction was performed using the TrueD algorithm on a Siemens MMWP workstation, fusing the attenuated PET and CT data to generate 3 mm thick axial, sagittal, and coronal fused images.

[0052] like Figure 9 As shown in Figure a, a patient's caudate lobe lesion was treated with an injection. 68 The SUVmax values ​​after 1 hour and 4 hours following Ga-Trap-(FAPI)3 were 7.44 and 10.34, respectively, indicating that its intake continued to increase over time rather than decrease.

[0053] like Figure 9 As shown in b, another patient's pelvic lymph node lesions appeared 1 hour after injection. 68 The Ga-Trap-(FAPI)3's SUVmax (8.30) is higher than 68 Ga-FAPI-04 (6.21).

[0054] In a head-to-head comparison of a patient with liver cancer, the tumor detection rate using FAPI-based probes was superior to that of other methods. 18 F-FDG ( 68 Ga-FAPI-04: 40 / 55; 68Ga-Trap-(FAPI)3: 55 / 55; 18 F-FDG: 36 / 55). Although both FAPI probes can visualize all tumors, 68 Ga-Trap-(FAPI)3 showed higher uptake intensity in the vast majority of lesions. Figure 10 a). Immunohistochemical staining of the tumor tissue after surgery confirmed the positive characteristics of FAP, which was highly consistent with the PET imaging results. Figure 10 b).

[0055] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A trimer compound targeting FAP, characterized in that, Its structure is as follows: ； Where R1 is O or N-ME.

2. The method for preparing the FAP-targeting trimer compound according to claim 1, characterized in that, Includes the following steps: S1. Compound 3 Trap is subjected to a first amidation reaction with compound 4 containing an alkyne group to obtain compound 5; S2. The compound 5 obtained in step S1 is reacted with compound 6 containing azide group and carboxylic acid under the catalysis of copper catalyst and reducing agent to obtain compound 7; S3. Compound 7 obtained in step S2 is subjected to a second amidation reaction with an FAP inhibitor to obtain a trimer compound targeting FAP.

3. The preparation method according to claim 2, characterized in that, Compound 4 mentioned in step S1 is propargylamine.

4. The preparation method according to claim 2, characterized in that, Compound 6 in step S2 is 5-azidopentanoic acid.

5. The preparation method according to claim 2, characterized in that, The FAP inhibitor mentioned in step S3 is FAPI.

6. A radionuclide-labeled trimer probe targeting FAP, characterized in that, It is obtained by labeling a radionuclide with the FAP-targeting trimer compound of claim 1, and its structure is shown below, where R represents the radionuclide labeling group; ； Where R1 is O or N-ME.

7. The radionuclide-labeled trimer probe targeting FAP according to claim 6, characterized in that, The nuclide labeling group is 18 F, 43 Sc、 44 Sc、 47 Sc、 55 Co、 58m Co、 61 Cu、 64 Cu、 67 Cu、 68 Ga、 86 Y、 89 Zr、 89 Sr、 90 Y、 99m Tc, 111 In、 123 I, 124 I, 125 I, 131 I, 153 Sm、 159 Gd, 161 Tb, 177 Lu、 186 Re、 188 Re、 203 Pb, 211 At、 212 Pb, 213 Bi、 223 Ra、 225 At least one of Ac.

8. The trimer probe according to claim 6, characterized in that, The nuclide labeling group is 18 F, 68 Ga、 99m Tc, 161 Tb, 177 Lu and 211 At least one of At.

9. The use of the trimer probe according to any one of claims 6-8 in the preparation of an imaging agent or therapeutic agent for diagnosing FAP-positive diseases.

10. The application according to claim 9, characterized in that, The FAP-positive diseases are selected from tumors, fibrotic diseases, arthritis, or atherosclerosis.

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