Molecular probe targeting EDB-FN as well as preparation method and application of molecular probe

Abstract

The invention provides a molecular probe targeting EDB-FN as well as a preparation method and application of the molecular probe. The structural formula of the molecular probe targeting EDB-FN is shown in the specification, according to the molecular probe targeting EDB-FN, the probe is based on a cyclic peptide probe with Lys-Glu side chain lactam bridge conformation fixed, the connection mode of a DOTA chelating agent is optimized, and the clinical transformation bottlenecks that an existing probe is poor in stability, low in affinity, insufficient in targeting specificity, poor in diagnosis and treatment integration compatibility and the like are solved. The probe can be used for early diagnosis and molecular imaging research of tumors, and provides a reliable basis for tumor malignancy degree evaluation and treatment effect monitoring.

Description

Technical Field

[0001] This invention relates to the field of radiopharmaceutical chemistry, and in particular to a molecular probe targeting EDB-FN, its preparation method, and its application. Background Technology

[0002] Invasion and metastasis of malignant tumors are the leading causes of cancer-related deaths. Significant heterogeneity exists among different tumor types and even within the same tumor, with vastly different invasive capabilities, ranging from indolent, slowly growing subtypes to highly aggressive metastatic ones. Therefore, this heterogeneity makes accurate assessment of tumor aggressiveness crucial for clinical decision-making, directly impacting treatment strategy selection and patient prognosis.

[0003] In clinical evaluation, molecular imaging methods are used in addition to tissue biopsy. These methods utilize molecular probes targeting specific tumor markers (such as PSMA in prostate cancer, HER2 in breast cancer, and SSTR in neuroendocrine tumors) for imaging detection, and have achieved some success in clinical applications. However, this method has limitations: First, tumor markers are often tumor-type specific; this makes existing molecular imaging methods often used for the diagnosis of specific tumor types, and difficult to use as an assessment tool for multiple tumor types. Second, the expression level of tumor markers is not always consistent with the degree of tumor malignancy. Taking prostate cancer as an example, the expression level of prostate-specific membrane antigen (PSMA) is not entirely consistent with the degree of tumor aggressiveness; its expression level cannot directly reflect the biological behavior of cancer. As the disease progresses to castration-resistant prostate cancer (CRPC), the expression of prostate-specific antigen (PSA) decreases, while PSMA expression, although upregulated in most advanced cases, still shows significant heterogeneity among different patients and lesions.

[0004] Therefore, there is an urgent need for a molecular probe applicable to a variety of tumors and capable of accurately reflecting their biological behavior and invasive potential. Among numerous targets, fibronectin (FN) is a relatively ideal pan-tumor target. Specifically, fibronectin (FN), as an important component of the tumor microenvironment, exhibits specific high expression in a variety of highly malignant tumors and is closely related to metastatic potential. In particular, its type III splice variant, Extradomain B (EDB-FN), is highly expressed at the invasive front of various malignant tumors, while its expression level is extremely low compared to normal tissues, making it an ideal pan-tumor target.

[0005] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a molecular probe targeting EDB-FN, its preparation method and application, aiming to solve the problem that the molecular probes in the prior art are difficult to be applied to multiple tumor types at the same time and are difficult to accurately reflect the true biological behavior and malignancy of tumors.

[0007] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a molecular probe for targeting EDB-FN, the structural formula of which is shown below: ; Where M is a radioactive metallic nuclide.

[0008] Optionally, the radioactive metal nuclide is 68 Ga or 177 Lu.

[0009] A second aspect of the present invention provides a method for preparing a molecular probe targeting EDB-FN, comprising the following steps: (1) Using glutamic acid as the first amino acid, aspartic acid, alanine, serine, threonine, arginine, valine, threonine and lysine are sequentially linked; then the carboxyl group of the glutamic acid is linked to the amino group of lysine to obtain a cyclic peptide; (2) The cyclic peptide in step (1) is coupled with Fmoc-Ahx-OH and then coupled with DOTA-tris(tert-butyl) ester N-hydroxysuccinimide ester to obtain a molecular probe precursor targeting EDB-FN; (3) React the molecular probe precursor targeting EDB-FN and the radioactive metal nuclide in solution in step (2) to obtain the molecular probe targeting EDB-FN.

[0010] Optionally, in step (2), after the cyclic peptide is coupled with Fmoc-Ahx-OH, the reaction further includes Fmoc deprotection reaction and cleavage reaction; the cleavage reaction uses a mixed solution of trifluoroacetic acid, triisopropylsilane and water.

[0011] Optionally, in step (2), after obtaining the molecular probe precursor targeting EDB-FN, the process also includes removing the protecting group.

[0012] A third aspect of the present invention provides the application of the above-described molecular probe targeting EDB-FN in the preparation of products for targeted screening, diagnosis, treatment, or prognostic assessment of diseases.

[0013] Optionally, the disease is one of abnormal EDB-FN expression.

[0014] Optionally, the product is a tumor imaging drug.

[0015] Optionally, the tumor is prostate cancer.

[0016] In a fourth aspect, the present invention provides a diagnostic reagent or kit comprising the above-described molecular probe targeting EDB-FN.

[0017] Optionally, the diagnostic reagent or kit is for diagnosing diseases with abnormal EDB-FN expression.

[0018] Optionally, the diagnostic reagent or kit is a tumor imaging drug; preferably, the tumor is prostate cancer.

[0019] Beneficial effects: (1) Cyclic peptide ring formation method: "i" is formed by using Lys–Glu side chain lactam bridges. The (i+5)” cyclic structure replaces the traditional disulfide bond cyclic structure, achieving conformational constraint, spatial stability and enhanced target recognition conformation of the cyclic peptide, solving the problems of low affinity, poor stability and insufficient specificity of existing peptide probes.

[0020] (2) DOTA conjugation method: The DOTA chelating agent is conjugated to the N-terminus of the cyclic peptide through the Ahx flexible linker, rather than the side chain or C-terminus, to avoid interference of the chelating agent with the target conformation of the cyclic peptide, so as to maintain high target affinity when achieving "therapeutic integration" and solve the problem of decreased target ability after the introduction of existing probe chelating agents.

[0021] (3) Target adaptability: Based on the binding characteristics of EDB-FN "superficial gap target", the specific binding of hydrogen bonds and electrostatic interactions is enhanced through cyclic peptide conformation design, thereby improving the uptake difference between tumor and non-tumor tissues and realizing risk stratification of tumors such as prostate cancer. Attached Figure Description

[0022] Figure 1 This is a synthetic route diagram of the DOTA-KTVRTSADE compound in Example 1 of the present invention.

[0023] Figure 2 This is the mass spectrum (MS) of the DOTA-KTVRTSADE compound in Example 1 of the present invention.

[0024] Figure 3 In Embodiment 1 of the present invention 68 Radioactive HPLC spectrum and stability HPLC spectrum of Ga-DOTA-KTVRTSADE.

[0025] Figure 4In Embodiment 1 of the present invention 177 Radioactive HPLC chromatogram and stability HPLC chromatogram of Lu-DOTA-KTVRTSADE.

[0026] Figure 5 The TGFβ-induced increase in the migration rate of 22Rv1 cells and 68 Figure showing increased cellular uptake of Ga-DOTA-KTVRTSADE.

[0027] Figure 6 for 68 Ga-DOTA-DTVRTSAD and 68 PET / CT images of Ga-DOTA-KTVRTSADE in PC3 (EDB-FN positive) tumor-bearing mice and 22Rv1 (EDB-FN negative) tumor-bearing mice.

[0028] Figure 7 for 68 Ga-DOTA-DTVRSTAD and 68 Tumor uptake SUVmax and time variation curves (TAC) of Ga-DOTA-KTVRTSADE in 22Rv1 and PC3 subcutaneous tumor models. Detailed Implementation

[0029] This invention provides a molecular probe targeting EDB-FN, its preparation method, and its application. To better understand this invention, the following examples further illustrate the invention, but these should not be construed as limiting the invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above-described invention are also considered to fall within the protection scope of this invention.

[0030] In the embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0031] The inventors discovered that molecular probes for EDB-FN include antibody fragments, ZD2 peptide (disulfide cyclic) (CTVRTSAD), and linear peptide (DTVRTSAD) ligands, but they have the following significant drawbacks: First, disulfide cyclic peptide probes (such as ZD2 peptide) are prone to breakage in the in vivo reducing environment, and the peptide chain is rapidly degraded by proteases after unfolding, resulting in rapid metabolism and short tumor retention time; Second, linear peptides (DTVRTSAD) are too flexible, making it difficult to form specific binding through a stable conformation for the "shallow cleft" binding pocket of EDB-FN, resulting in a high proportion of non-target conformations, large entropy loss, low receptor binding efficiency, and poor imaging contrast; Third, metal chelators (such as DOTA) are mostly linked by side chains or C-terminal grafts, which easily interfere with the binding conformation of the peptide chain and the target. The targeting ability is significantly reduced after the introduction of chelators, making it impossible to meet the "therapeutic integration" requirements of imaging and treatment.

[0032] Based on this, embodiments of the present invention provide a molecular probe targeting EDB-FN, the structural formula of which is shown below: ; Where M is a radioactive metallic nuclide.

[0033] The molecular probe provided by this invention is a cyclic peptide probe with a conformation fixed by the lactam bridge of the Lys–Glu side chain, and the connection mode of the DOTA chelating agent is optimized, which solves the clinical translation bottlenecks of existing probes such as poor stability, low affinity, insufficient targeting specificity, and poor compatibility with integrated diagnosis and treatment.

[0034] As can be seen from the structural formula, the molecular probe of the present invention comprises a cyclic peptide structure and a DOTA chelating agent. Specifically, in the cyclic peptide structure, a Lys–Glu side-chain lactam bridge is used to form an “i”. The (i+5) ring-forming mechanism allows the cyclic peptide backbone to form a pre-organized β-turn-like configuration, thereby significantly enhancing conformational constraint and steric stability: Firstly, this cyclic peptide structure strengthens conformational constraint: "i The (i+5)” lactam bridge and pre-organized β-turn-like configuration reduce the proportion of non-target conformations and decrease entropy loss, thereby increasing the binding rate (kon) of the cyclic peptide to EDB-FN and decreasing the dissociation constant (Kd), significantly enhancing binding affinity. In the animal experiments of Test Example 2 of this application, the tumor uptake rate (1.59±0.65 %ID / g) of the PC3 (EDB-FN positive) model was much higher than that of the traditional DTVRSTAD peptide (0.66±0.44 %ID / g), verifying the affinity enhancement effect. Secondly, the cyclic peptide structure enhances spatial stability: the lactam bridge stabilizes in the in vivo reducing environment, avoiding the risk of disulfide bond breakage; at the same time, it inhibits peptide chain unfolding, reduces the probability of protease degradation, prolongs the blood circulation half-life of the cyclic peptide, increases tumor retention time, and accelerates background tissue clearance. During imaging, the tumor / muscle ratio (T / M) and tumor / blood ratio (T / B) are significantly improved, resulting in PET... The imaging signal-to-noise ratio is improved, as shown in Table 1. Thirdly, the target recognition specificity is enhanced: the spatial orientation of the key motif TVRTSA is optimized to match the binding characteristics of EDB-FN "shallow cleft targets" (dependent on hydrogen bonding and electrostatic interactions). Compared with the non-specific binding of linear peptides, the selectivity of cyclic peptides for EDB-FN positive tumors (such as PC3) is significantly higher than that for negative tumors (such as 22Rv1). This can explain the difference in tumor uptake between PC3 and 22Rv1 in animal experiments, providing a basis for tumor risk stratification.

[0035] In the DOTA chelator, the N-terminus of the cyclic peptide is coupled via an Ahx flexible linker, avoiding interference from the bulky structure of DOTA with the binding conformation of the cyclic peptide and EDB-FN. Furthermore, the N-terminal grafting does not disrupt the spatial orientation of the key recognition motif of the cyclic peptide, thus achieving… 68 Ga-labeled PET imaging and 177 Lu-labeled targeted radiotherapy achieves "therapeutic integration" while maintaining high targeting affinity.

[0036] In summary, the molecular probe provided by this invention has excellent chemical and metabolic stability, can resist in vivo reducing environment and protease hydrolysis, prolongs the blood circulation half-life and tumor retention time, and combined with rapid background clearance characteristics, significantly improves the imaging signal-to-noise ratio and detection sensitivity. In some embodiments, M is 68 Ga or 177 Lu.

[0037] In this embodiment, the label 68 Ga can be used in PET tumor imaging; labeling 177 When Lu is used in targeted therapy, it can achieve precise delivery of radiation dose, enhance anti-tumor effects and reduce systemic toxicity, providing a reliable tool for non-invasive assessment and precision diagnosis and treatment of pan-tumor malignancy.

[0038] This invention provides a method for preparing a molecular probe targeting EDB-FN, comprising the following steps: (1) Using glutamic acid as the first amino acid, aspartic acid, alanine, serine, threonine, arginine, valine, threonine and lysine are sequentially linked; then the carboxyl group of the glutamic acid is linked to the amino group of lysine to obtain a cyclic peptide; (2) The cyclic peptide in step (1) is coupled with Fmoc-Ahx-OH and then coupled with DOTA-tris(tert-butyl) ester N-hydroxysuccinimide ester to obtain a molecular probe precursor targeting EDB-FN; (3) React the molecular probe precursor targeting EDB-FN and the radioactive metal nuclide in solution in step (2) to obtain the molecular probe targeting EDB-FN.

[0039] This invention achieves a cyclic peptide structure by forming a lactam bridge between lysine and glutamic acid; by introducing Ahx as a flexible linker and chelating it with DOTA, it can achieve labeling of various radioactive metal nuclides (such as...). 68 Ga、 177 Lu).

[0040] In some embodiments, in step (2), after the cyclic peptide is coupled with Fmoc-Ahx-OH, the reaction further includes Fmoc deprotection and cleavage reaction; the cleavage reaction uses a mixed solution of trifluoroacetic acid, triisopropylsilane and water.

[0041] Fmoc deprotection effectively exposes the free amino group at the N-terminus of the cyclic peptide, ensuring that it can undergo a coupling reaction with DOTA-tris(tert-butyl) ester N-hydroxysuccinimide ester.

[0042] Using a mixed solution of trifluoroacetic acid, triisopropylsilane, and water as the cleavage system improved the integrity and purity of the target cyclic peptide.

[0043] In some embodiments, after obtaining the molecular probe precursor targeting EDB-FN in step (2), the process further includes the removal of protecting groups.

[0044] This invention provides the application of the above-mentioned molecular probe targeting EDB-FN in the preparation of products for targeted screening, diagnosis, treatment or prognostic assessment of diseases.

[0045] In some embodiments, the disease is a disease characterized by abnormal expression of EDB-FN.

[0046] In some embodiments, the product is a tumor imaging drug.

[0047] In some embodiments, the tumor is prostate cancer.

[0048] This invention provides a diagnostic reagent or kit, including the aforementioned molecular probe targeting EDB-FN.

[0049] In some embodiments, the diagnostic reagent or kit is for diagnosing diseases with abnormal EDB-FN expression.

[0050] In some embodiments, the diagnostic reagent or kit is a tumor imaging drug; preferably, the tumor is prostate cancer.

[0051] The present invention will be further described below through specific embodiments.

[0052] Example 1 This embodiment provides a method for synthesizing DOTA-KTVRTSADE, and the specific steps are as follows: like Figure 1 As shown, the linear peptide sequence (KTVRTSADE) was synthesized using the Fmoc / tBu solid-phase peptide synthesis method. First, Rink amide resin (0.45 mmol, 789 mg, loading 0.57 mmol / g) was swollen in N,N-dimethylformamide (DMF), followed by two Fmoc deprotection reactions (20 min each, for a total of two reactions) under nitrogen protection using a 20% piperidine / DMF solution. The amino acid coupling reactions used HBTU (511.5 mg, 1.35 mmol, 3 mmol) as the coupling reagent and DIPEA (469.5 μL, 2.7 mmol) as the activating reagent. The following amino acids were sequentially linked in a DMF system: Fmoc-Glu(Phipr)-OH (657 mg, 1.35 mmol), Fmoc-Asp(OtBu)-OH (555 mg, 1.35 mmol), Fmoc-Ala-OH (420 mg, 1.35 mmol), Fmoc-Ser(tBu)-OH (517.5 mg, 1.35 mmol), Fmoc-Thr(Trt)-OH (535.5 mg, 1.35 mmol), Fmoc-Arg(Pbf)-OH (874.5 mg, 1.35 mmol), and Fmoc-Val-OH (457.5 mg, 1.35 mmol). The linear protective peptides were assembled through repeated coupling and deprotection cycles. The formulations included Fmoc-Thr(Trt)-OH (535.5 mg, 1.35 mmol) and Fmoc-Lys(Dde)-OH (718.5 mg, 1.35 mmol).

[0053] Selective deprotection of the side chains was then performed: first, the glutamic acid was treated three times (15 minutes each) with 1% trifluoroacetic acid / DMF (v / v) solution to remove the Phir protecting group, and then treated twice (20 minutes each) with 2% hydrazine monohydrate / DMF solution to remove the Dde protecting group of lysine, thereby exposing the carboxyl group of the glutamic acid side chain and the amino group of the lysine side chain. Cyclocyclization of the carboxyl and amino groups of the side chains was carried out in DMF for 3 hours using HATU (516 mg, 1.35 mmol), Oxyma Pure (204 mg, 1.44 mmol), and DIPEA (469.5 μL, 2.7 mmol). Mass spectrometry was used to confirm whether the cyclization was complete. Fmoc-Ahx-OH was coupled in a DMF system using HBTU (511.5 mg, 1.35 mmol, 3 equivalents) as the coupling agent and DIPEA (469.5 μL, 2.7 mmol) as the activating agent. After coupling, the resin was washed with DMF (15 mL, 3 × 2 min), and then the N-terminal Fmoc was deprotected with 20% piperidine / DMF (v / v, 2 × 10 min). The cleavage reaction was carried out for 2 hours using a TFA:TIS:H2O (95:2.5:2.5, v / v, total volume 7.5 mL) mixture. The crude peptide was purified by precipitation with pre-cooled diethyl ether (45 mL × 3 times). The peptide was coupled with DOTA-tris(tBu)ester N-hydroxysuccinimide ester (DOTA-trs(tBu)ester NHS, 0.53 g, 0.8 mmol) and N,N-diisopropylethylamine (DIPEA, 0.72 g, 2.4 mmol) in N,N-dimethylformamide (DMF, 15 mL). After the reaction, the mixture was extracted with EtOAc / H2O, dried, and rotary evaporated to obtain the crude product. Finally, the product was deprotected with TFA, purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and lyophilized. The molecular weight of DOTA-KTVRTSADE was verified by MALDI-TOF mass spectrometry: C 62 H 107 N 19 O 23 The calculated value is [M+2H]. 2+ 744.89, measured value 744.15, such as Figure 2 As shown.

[0054] Example 2 This embodiment provides 68 Ga-DOTA-KTVRTSADE and 177 The marking method for Lu-DOTA-KTVRTSADE is as follows: (1) 68 Ga tagging method 68Ga-DOTA-KTVRTSADE labeling method: Dissolve 30 μg of the labeling precursor DOTA-KTVRTSADE in 1 mL of 0.25 N sodium acetate buffer, rinse the germanium-gallium generator with 0.1 N hydrochloric acid solution, and then... 68 Add 2 mL of Ga]GaCl3 to a mixture of the precursor and sodium acetate, mix thoroughly, and react at 95℃ for 10 min. Cool to room temperature. Determine the labeling rate using high-performance liquid chromatography (radio-HPLC) with a radiochemical detector, yielding a product with a radiochemical purity greater than 95%. 68 Ga-DOTA-KTVRTSADE, such as Figure 3 As shown.

[0055] (2) 177 Lu tagging method 177 Lu-DOTA-KTVRTSADE labeling method: Dissolve 50 μg of the labeling precursor DOTA-KTVRTSADE in 30 μL of 3N pH 5.2 sodium acetate buffer, and add [ 177 Lu]LuCl3 solution was mixed into the precursor and sodium acetate mixture, and the mixture was thoroughly mixed. The mixture was reacted at 95°C for 30 minutes. After cooling to room temperature, the labeling rate was determined using high-performance liquid chromatography (radio-HPLC) with a radiodetector. Figure 4 As shown.

[0056] (3) Determination of radioactivity purity Both labeled products were analyzed using high-performance liquid chromatography (radio-HPLC) with a radiodetector. The first mobile phase was a 0.1% trifluoroacetic acid solution in acetic acid; the second mobile phase was a 0.1% trifluoroacetic acid aqueous solution. Gradient elution conditions were: 0-2 min, 100%-0% second mobile phase; 2-4 min, 100%-0% second mobile phase; 4-9 min, 100% first mobile phase; 9-10 min, 0%-100% second mobile phase; 10-15 min, 100% second mobile phase; the flow rate was 1 mL / min.

[0057] Comparative Example 1: This comparison provides 68 The preparation method of Ga-DOTA-DTVRSTAD is as follows: A linear peptide sequence (DTVRSTAD) was synthesized using the Fmoc / tBu solid-phase peptide synthesis method. The obtained linear peptide was then coupled with DOTA-tris(tert-butyl) ester N-hydroxysuccinimide ester and N,N-diisopropylethylamine in N,N-dimethylformamide to obtain DOTA-DTVRSTAD.

[0058] Dissolve 30 μg of the labeled precursor DOTA-DTVRSTAD in 1 mL of 0.25 N sodium acetate buffer, rinse the germanium-gallium generator with 0.1 N hydrochloric acid solution, and then […]. 68 Add 2 mL of Ga]GaCl3 to a mixture of the precursor and sodium acetate, mix thoroughly, and react at 95℃ for 10 min. Cool to room temperature. Determine the labeling rate using high-performance liquid chromatography (radio-HPLC) with a radiochemical detector, yielding a product with a radiochemical purity greater than 95%. 68 Ga-DOTA-DTVRSTAD.

[0059] Test Example 1: This test case is based on the results obtained in Example 2. 68 Ga-DOTA-KTVRTSADE was tested, specifically... 68 The cellular uptake experiment of Ga-DOTA-KTVRTSADE in 22Rv1 and 22Rv1-TGFβ cells was conducted as follows: (1) 22Rv1 cells were continuously cultured in 1640 complete medium containing 20 ng / mL TGFβ until the sixth generation to obtain 22Rv1-TGFβ cells.

[0060] (2) Take 22Rv1 and 22Rv1-TGFβ cells in the logarithmic growth phase, and use approximately 2 × 10⁻⁶ cells to extract the 22Rv1 and 22Rv1-TGFβ cells. 5 Cells / well, seeded in 12-well plates, after labeling. 68 The Ga-DOTA-KTVRTSADE solution was diluted with 1640 solution, and then approximately 0.5 μCi was added to each well plate. 68 Mix Ga-DOTA-KTVRTSADE with 1 mL of 1640 solution (blank culture).

[0061] (3) After incubating in a CO2 incubator (37℃) for 30, 60 and 120 min, rinse with PBS.

[0062] (4) Pyrolyze with NaOH solution for 5 min, collect the lysate, and measure the radioactivity count; Experimental results: such as Figure 5 As shown, Figure 5 The TGFβ-induced increase in the migration rate of 22Rv1 cells and 68 The figure shows the results of increased uptake in Ga-DOTA-KTVRTSADE cells. As can be seen from the figure, the migration rate of 22Rv1 cells induced by TGF-β significantly increased to 21% after 96 h, while the untreated group only reached 5%. The difference between the two groups was statistically significant, indicating a significant enhancement in their migration and invasion abilities. Simultaneously, 22Rv1-TGFβ and 22Rv1 cells showed increased uptake. 68The uptake of Ga-DOTA-KTVRTSADE was 4.06 ± 1.76 %ID / 10. 6 cells and 1.11 ± 0.24 %ID / 10 6 The results showed that TGF-β induction significantly enhanced the invasion and metastasis ability of 22Rv1 cells, and there were significant differences between the two groups. 68 The uptake level of Ga-DOTA-KTVRTSADE is positively correlated with the invasiveness of tumor cells.

[0063] Test Example 2: This test case is based on the results obtained in Example 2. 68 Ga-DOTA-KTVRTSADE was tested, specifically... 68 The following are the specific steps for micro PET / CT imaging of Ga-DOTA-KTVRTSADE in PC3 and 22Rv1 subcutaneous xenograft mouse models: (1) Establishment of subcutaneous animal model: Human prostate cancer PC3 and 22Rv1 cells in the logarithmic growth phase were digested and prepared into PBS suspension. The cell suspension (approximately 1×10⁻⁶ cells) was then used to establish the subcutaneous animal model. 6 (cell) was injected subcutaneously into the right shoulder of male SCID mice that were 4-5 weeks old, and observed for 30-60 days.

[0064] (2) 68 After diluting the Ga-DOTA-KTVRTSADE solution with water, approximately 200 μCi was taken with a syringe. 68 Ga-DOTA-KTVRTSADE solution was injected via the tail vein, followed by micro PET / CT dynamic imaging for 1 hour.

[0065] (3) PET / CT imaging results show 68 Ga-DOTA-KTVRTSADE showed high specific uptake in the PC3 prostate cancer model.

[0066] (4) Observation by continuous PET / CT dynamic imaging within 1 hour after drug injection 68 Ga-DOTA-DTVRSTAD and 68 Tumor uptake and time-varying curves (TAC) of Ga-DOTA-KTVRTSADE in 22Rv1 and PC3 subcutaneous tumor models. In addition, the ROI of tumor, cardiac blood pool, liver, muscle and kidney were delineated, and the %ID / mL of each site was calculated. Then the ratios of tumor / cardiac blood pool, tumor / liver, tumor / muscle and tumor / kidney were calculated. The results are shown in Table 1.

[0067] Table 1. In PC3 (EDB-FN positive) tumor-bearing mice 68Ga-DOTA-KTVRTSADE and 68 Tumor-organ ratio 60 minutes after Ga-DOTA-DTVRSTAD injection

[0068] Experimental results are as follows Figure 6 and Figure 7 As shown, the experimental results indicate that, compared to 68 Ga-DOTA-DTVRSTAD, 68 Ga-DOTA-KTVRTSADE exhibited higher tumor uptake and tumor retention capabilities in the PC3 subcutaneous tumor model. At the 60-minute timepoint, 68 Ga-DOTA-KTVRTSADE and 68 The tumor uptake of Ga-DOTA-DTVRSTAD was 1.59±0.65 %ID / g and 0.66±0.44 %ID / g, respectively. In the 22Rv1 subcutaneous tumor model, 68 Ga-DOTA-KTVRTSADE and 68 There was no significant difference in tumor uptake of Ga-DOTA-DTVRSTAD. Tumor pathological immunohistochemical results showed that EDB-FN expression was positive in PC3 tumors, while no EDB-FN expression was found in 22Rv1 tumors.

[0069] The data in Table 1 shows that... 68 In the PC3 tumor-bearing mouse model, the ratios of Ga-DOTA-KTVRTSADE to the heart / blood pool, liver, muscle, and kidney were 3.32, 3.93, 10.39, and 0.89, respectively, all higher than those of other tumor-bearing mice. 68 The ratio of Ga-DOTA-DTVRSTA in the corresponding tissues. The results show that, compared with... 68 Compared with Ga-DOTA-DTVRSTA, 68 Ga-DOTA-KTVRTSADE exhibits stronger tumor targeting ability and higher tumor-specific uptake, with a higher ratio of targeted to non-targeted tissues, demonstrating its potential as an excellent diagnostic probe.

[0070] In summary, the molecular probe of this invention achieves multiple beneficial effects through its overall structural design: On the one hand, the lactam-bridged cyclic peptide structure endows the probe with high affinity, high stability, and high specificity, exhibiting higher tumor uptake levels and longer in vivo retention time in highly invasive EDB-FN positive tumors (such as PC3), with significantly improved tumor / muscle ratio (T / M) and tumor / blood ratio (T / B), thereby enhancing the contrast and diagnostic sensitivity of PET imaging; on the other hand, by introducing Ahx-DOTA coupling at the N-terminus, the probe of this invention achieves high chelation efficiency and therapeutic compatibility, maintaining stable radiochemical purity above 95%, and reducing demetallization rate, thus... 68 Ga imaging and 177 Lu treatment can be performed using the same molecular probe, increasing the local dose concentration to the tumor while helping to reduce systemic toxicity to normal tissues. Furthermore, the overall structural design of the molecular probe in this invention enables it not only to specifically identify EDB-FN positive lesions but also to distinguish between tumor subtypes with different invasiveness and / or different EDB-FN expression levels. This provides the capability for pan-tumor risk stratification assessment, offering more precise imaging evidence for the development of personalized clinical treatment strategies.

[0071] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A molecular probe targeting EDB-FN, characterized in that, The structural formula of the molecular probe targeting EDB-FN is shown below: ； Where M is a radioactive metallic nuclide.

2. The molecular probe targeting EDB-FN according to claim 1, characterized in that, The radioactive metal nuclide is 68 Ga or 177 Lu.

3. A method for preparing a molecular probe targeting EDB-FN as described in claim 1 or 2, characterized in that, Includes the following steps: (1) Using glutamic acid as the first amino acid, aspartic acid, alanine, serine, threonine, arginine, valine, threonine and lysine are sequentially linked; then the carboxyl group of the glutamic acid is linked to the amino group of lysine to obtain a cyclic peptide; (2) The cyclic peptide in step (1) is coupled with Fmoc-Ahx-OH and then coupled with DOTA-tris(tert-butyl) ester N-hydroxysuccinimide ester to obtain a molecular probe precursor targeting EDB-FN; (3) React the molecular probe precursor targeting EDB-FN and the radioactive metal nuclide in solution in step (2) to obtain the molecular probe targeting EDB-FN.

4. The method for preparing the molecular probe targeting EDB-FN according to claim 3, characterized in that, In step (2), after the cyclic peptide is coupled with Fmoc-Ahx-OH, the reaction further includes Fmoc deprotection reaction and cleavage reaction; the cleavage reaction uses a mixed solution of trifluoroacetic acid, triisopropylsilane and water.

5. The method for preparing the molecular probe targeting EDB-FN according to claim 3, characterized in that, In step (2), after obtaining the molecular probe precursor targeting EDB-FN, the process also includes the removal of protecting groups.

6. The use of the molecular probe targeting EDB-FN as described in claim 1 or 2 in the preparation of products for targeted screening, diagnosis, treatment or prognostic assessment of diseases.

7. The application according to claim 6, characterized in that, The disease in question is caused by abnormal expression of EDB-FN.

8. The application according to claim 6, characterized in that, The product in question is a tumor imaging drug.

9. The application according to claim 8, characterized in that, The tumor is prostate cancer.

10. A diagnostic reagent or kit, characterized in that, Including the molecular probe targeting EDB-FN as described in claim 1 or 2.

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