Bimodal FAP targeting probe as well as preparation method and application thereof

Abstract

The invention provides a precursor compound capable of targeting FAP. The structure of the precursor compound is shown as a formula (I) or a formula (II). Based on the precursor compound, the invention further provides a bimodal FAP targeting probe which is a complex formed by the precursor compound as shown in the formula (I) or the formula (II) as a ligand and radionuclide. The radionuclide is a radionuclide capable of forming stable complexation with a DOTA group, preferably 68Ga. The invention also provides an application of the bimodal FAP targeting probe in preparation of a living body imaging agent. The bimodal FAP targeting probe disclosed by the invention can accurately describe the tumor edge, and has the potential of guiding intra-operative decision in future clinical application.

Description

Technical Field

[0001] This invention relates to the manufacture of biopharmaceuticals in the biopharmaceutical industry, and more particularly to a targeted cyclic polypeptide molecule, specifically a cyclic polypeptide molecule that can target fibroblast activation protein α, its radionuclide label, its preparation method, and its application in tumor resection. Background Technology

[0002] Fibroblast activating protein α (FAP) is a type II transmembrane serine protease selectively expressed on activated fibroblasts. Although FAP expression levels are extremely low in most normal tissues, its expression is significantly upregulated in cancer-associated fibroblasts (CAFs) of various solid tumors. Numerous studies have shown that FAP plays a crucial role in tumor progression through mechanisms such as promoting tumor growth, invasion, metastasis, and immune escape. In recent years, PET imaging based on radiolabeled FAP small molecule inhibitors (FAPIs) and peptide probes has shown great potential in the diagnosis and molecular characterization of various malignant tumors. In addition to its use in tumor diagnosis and staging, FAP-targeted PET imaging can also provide auxiliary information for clinical treatment. For example, it can clearly delineate tumor boundaries and accurately assess tumor volume, thus providing important information for precise radiotherapy planning and prognostic prediction.

[0003] Surgical resection remains the cornerstone of treatment for many solid tumors. Despite continuous advancements in preoperative imaging technology, intraoperative decision-making still largely relies on the surgeon's visual inspection and palpation, a situation that has room for improvement given the increasing prevalence of minimally invasive and function-preserving surgeries. Intraoperative frozen sections are widely considered the gold standard for assessing surgical margins, but their accuracy is significantly affected by the sampling location and the operator's experience. Intraoperative fluorescence imaging, as a visual and real-time feedback aid, has received considerable attention in recent years. This technology can be applied during surgery by incubating fluorescent probes locally in tissue or by intravenous injection, helping to distinguish tumors from normal tissues and aiding in the determination of surgical margin status.

[0004] Among existing fluorescence imaging techniques, near-infrared II (NIR-II) imaging has attracted considerable attention due to its superior tissue penetration depth, lower photon scattering, and higher signal-to-noise ratio (SBR) compared to conventional fluorescence imaging techniques. These optical advantages make NIR-II imaging particularly suitable for high-resolution intraoperative navigation. FAP-2286 and 3BP-3940 (also referred to as "FAP-3940") are two existing structurally similar cyclic peptides that specifically target FAP. Previous studies have shown that these cyclic peptides exhibit higher in vivo stability and longer tumor retention time compared to conventional FAPI analogs, including FAPI-04 and FAPI-46.

[0005] Therefore, it is necessary to develop a dual-modal probe targeting FAP based on the aforementioned cyclic peptide and apply it to tumor resection to improve intraoperative imaging support. Summary of the Invention

[0006] The first objective of this invention is to provide a precursor compound that can target FAP and has higher in vivo stability and longer tumor retention time in living tissue.

[0007] Another object of the present invention is to provide a bimodal FAP targeting probe based on the aforementioned precursor compound, which can function simultaneously in in vivo PET and fluorescence imaging, enabling more accurate delineation of tumor margins during tumor resection.

[0008] Another object of the present invention is to provide a method for preparing the dual-modal FAP targeting probe.

[0009] Another object of the present invention is to provide the application of the aforementioned dual-modal FAP targeting probe in the preparation of in vivo imaging agents.

[0010] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0011] In a first aspect, the present invention provides a precursor compound that can target FAP, the structure of which is shown in formula (I) or (II) below:

[0012]

[0013] The precursor compound structure described in this invention comprises a cyclic peptide molecule structure that can target FAP, a near-infrared II region fluorophore IRDye800cw structure, and a radionuclide chelating group DOTA, which are connected by a linker; the linker further comprises a maleimide structure; the cyclic peptide molecule structure that can target FAP is derived from the targeting peptides FAP-2286 and FAP-3940, respectively.

[0014] Based on the aforementioned precursor compounds, this invention further provides a dual-modal FAP targeting probe that can be used for PET and near-infrared II region fluorescence imaging. This probe is a complex formed by the precursor compound of formula (I) or (II) of this invention as a ligand and a radionuclide; the radionuclide is preferably a radionuclide capable of forming a stable complex with the DOTA group. 68 Ga.

[0015] The dual-modal FAP targeting probe structure described in this invention simultaneously contains a near-infrared II region fluorescent group and a radionuclide, thus enabling it to function in both PET and fluorescence imaging.

[0016] In a second aspect, the present invention also provides the use of the precursor compound described in the first aspect as a labeling precursor in the preparation of a PET and near-infrared II region fluorescent dual-modal FAP targeting probe, or the use of the precursor compound described in the first aspect as a fluorescent imaging agent in in vivo tumor imaging; and the use of the dual-modal FAP targeting probe described in the first aspect as an imaging agent in in vivo imaging of organisms for non-therapeutic or diagnostic purposes.

[0017] In this invention, we conjugate cyclic peptide structures from FAP-2286 and FAP-3940 with the NIR fluorophore IRDye800cw, and also combine them with the macrocyclic chelating agent DOTA. 68 The labeling of radionuclides such as Ga. This innovative approach has led to the development of bimodal FAP-targeting probes that can function simultaneously in PET and fluorescence imaging. Through systematic in vitro and in vivo characterization, we have demonstrated that these bimodal FAP-targeting probes of the present invention can accurately delineate tumor margins and have the potential to guide intraoperative decisions in future clinical applications. Attached Figure Description

[0018] Figure 1 This is a liquid chromatography-mass spectrometry (LC-MS) chromatogram of DOTA-IRDye800cw-FAP-2286 synthesized in Example 1.

[0019] Figure 2 The image shows a high-performance liquid chromatogram of DOTA-IRDye800cw-FAP-2286 synthesized in Example 1, illustrating the purity and quality control results of IRDye800cw-FAP-2286.

[0020] Figure 3 This is a liquid chromatography-mass spectrometry (LC-MS) chromatogram of DOTA-IRDye800cw-FAP-3940 synthesized in Example 2.

[0021] Figure 4 The image shows a high-performance liquid chromatogram of DOTA-IRDye800cw-FAP-3940 synthesized in Example 2, illustrating the purity and quality control results of IRDye800cw-FAP-3940.

[0022] Figure 5 This demonstrates the in vitro evaluation of IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2.

[0023] Figure 6 The in vivo PET imaging evaluation of IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2 is demonstrated.

[0024] Figure 7 The in vivo fluorescence imaging evaluation of IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2 is demonstrated.

[0025] Figure 8 These are the results of in vitro fluorescence imaging and quantitative analysis of the IRDye800cw labeled probe.

[0026] Figure 9 This demonstrates the results of in vitro fluorescence validation using the IRDye800cw-FAP-3940 from Example 2 in human lung cancer tissue. Parts A and B represent lung adenocarcinoma patients, respectively. 68 Ga-FAPI-46 PET / CT coronal and axial images, with arrows indicating tumor location. Parts C and D show macroscopic and fluorescence images of tumor tissue excised in vitro after incubation with IRDye800cw-FAP-3940, respectively. Parts E and F show the corresponding hematoxylin-eosin staining and FAP immunohistochemical (IHC) staining, used to confirm tumor localization and FAP expression. Detailed Implementation

[0027] The technical solution and technical effects of the present invention will be further illustrated below by listing embodiments and experimental examples.

[0028] All chemicals were purchased from commercial suppliers. 3BP-4089 was purchased from Nanchang Tanzhen Biotechnology Co., Ltd.

[0029] All statistical analyses were performed using SPSS 22.0 software (IBM, USA). Student's t test was used for mean comparisons, and a two-sided p-value <0.05 was considered statistically significant.

[0030] Example 1. Synthesis of DOTA-IRDye800cw-FAP-2286

[0031] The dual-modal FAP targeting probe DOTA-IRDye800cw-FAP-2286 was synthesized according to the following route:

[0032]

[0033]

[0034] The specific steps of the above synthetic route are as follows:

[0035] Synthesized compound 3:

[0036] Fmoc-Cys(Trt)-OH (compound 2) (1.2 eq) was dissolved in 10 mL of DMF, followed by the addition of DCC (1.2 eq) and HOSu (1.2 eq). The reaction was stirred at room temperature for 6 hours. 3BP-4089 (compound 1) (1 eq) and DIPEA (3 eq) were then added, and the reaction was continued at room temperature for 1 hour. The reaction was monitored by LC-MS, and the DMF was then evaporated. 10 mL of 20% piperidine / DMF solution was added, and the mixture was stirred at room temperature for 10 minutes. The product was washed twice with diethyl ether, and the solvent was removed by rotary evaporation. The crude product was purified by reverse-phase preparative HPLC to give compound 3 (yield: 45.3%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 32% to 52%; flow rate = 20 mL / min; λ = 220 nm.

[0037] Synthesized compound 5:

[0038] Compound 3 (1 eq) and DOTA-NHS (compound 4) (1 eq) were dissolved in 10 mL of DMF, followed by the addition of DIEA (3 eq). The reaction was stirred at room temperature for 2 hours, and the reaction was confirmed by LC-MS. The solvent was evaporated. 5 mL of TFA solution was added, and the reaction was stirred at room temperature for 2 hours. Then 50 mL of diethyl ether was added, and a solid precipitate was formed. The precipitate was collected by centrifugation and filtered dry. The crude product was purified by reverse-phase HPLC to obtain compound 5 (yield: 35.5%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 28% to 48%; flow rate = 20 mL / min; λ = 220 nm.

[0039] Synthesized compound 8:

[0040] IRDye 800cw Acid (compound 6) (1 eq) was dissolved in 5 mL of DMF, followed by the addition of HATU (1 eq) and DIEA (3 eq). Finally, N-(2-aminoethyl)maleimide (compound 7) (1 eq) was added, and the mixture was stirred at room temperature for 2 hours. The reaction was confirmed by LC-MS, and the solvent was evaporated. The crude product was purified by reverse-phase HPLC to obtain compound 8 (yield: 25.9%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 10% to 100%; flow rate = 20 mL / min; λ = 220 nm.

[0041] Synthesized compound DOTA-IRDye800cw-FAP-2286:

[0042] Compounds 5 (1 eq) and 8 (1 eq) were dissolved in 10 mL of MECN / H2O (1:1, V / V), followed by the addition of 10 mL of PBS buffer (pH = 7.2). The reaction mixture was stirred at room temperature for 1 hour, and the reaction was confirmed to be complete by LC-MS. The reaction mixture was directly purified by reverse-phase preparative HPLC to obtain DOTA-IRDye800cw-FAP-2286 (yield: 20.6%), and the precursor compound targeted by this FAP was designated as "P220588". The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–20 min, from 10% to 100%, flow rate = 3 mL / min; λ = 220 nm. The LC-MS (ESI+) result was 900.3 [M+3H] / 3. The chemical purity of IRDye800cw-FAP-2286 exceeded 95%, as determined by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). For the HPLC-MS analysis of DOTA-IRDye800cw-FAP-2286, see [link to HPLC analysis]. Figure 1 . Figure 2 The high-performance liquid chromatograms show the purity and quality control results of IRDye800cw-FAP-2286.

[0043] Example 2. Synthesis of DOTA-IRDye800cw-FAP-3940

[0044] The dual-modal FAP targeting probe DOTA-IRDye800cw-FAP-3940 was synthesized according to the following route:

[0045]

[0046]

[0047] The specific steps of the above synthetic route are as follows:

[0048] Synthesized compound 11:

[0049] First, Fmoc-Cys(Trt)-OH was grafted onto CTC resin under alkaline conditions. Subsequently, Fmoc-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, and Fmoc-Cys(Trt)-OH were coupled sequentially. All amino acid couplings were performed in DMF using DIC (3 eq) and HOBt (3 eq) as coupling agents. The Fmoc protecting group was removed with 20% Pip / DMF solution. n-Butylamine was coupled to the resin-bound peptide using DSC (6 eq) to form a urea bond. Finally, the crude peptide was cleaved from the resin at room temperature with TFA solution for 2 hours. The crude peptide was purified by HPLC using a C18 preparative column to obtain pure product compound 11 (yield: 23.4%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. B gradient conditions: 0-60 minutes, from 20% to 40%; flow rate = 20 ml / min; λ = 220 nm.

[0050] Synthesized compound 13:

[0051] Compound 11 (1 eq) was dissolved in 15 mL of ACN, followed by the addition of 15 mL of 0.2 M PBS buffer and 1,3,5-tris(bromomethyl)benzene (compound 12) (1.2 eq). The reaction was carried out at room temperature for 1 hour under N2 protection. Then, 2-aminoethanethiol (mercaptoethylamine) was added, and the reaction continued for 30 minutes. The reaction progress was monitored by LC-MS. The solvent was then evaporated, and the crude product was purified by reverse-phase preparative HPLC to give compound 13 (yield: 35.4%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 22% to 42%; flow rate = 20 mL / min; λ = 220 nm.

[0052] Synthesized compound 15:

[0053] Fmoc-Cys(Trt)-OH (compound 2) (1.2 eq) was dissolved in 10 mL DMF, followed by the addition of DCC (1.2 eq) and HOSu (1.2 eq). The mixture was stirred at room temperature for 6 hours. Compound 13 (1 eq) and DIPEA (3 eq) were added, and the reaction was carried out at room temperature for 1 hour. The reaction progress was monitored by LC-MS. DMF was removed, and the residue was treated with 10 mL of 20% piperidine / DMF solution at room temperature for 10 minutes. The resulting mixture was washed twice with diethyl ether, and the solvent was evaporated under reduced pressure. The crude product was purified by reverse-phase HPLC to obtain compound 15 (yield: 40.3%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 28% to 48%; flow rate = 20 mL / min; λ = 220 nm. The LC-MS (ESI+) result was 716.02.

[0054] Synthesized compound 17:

[0055] Compound 15 (1 eq) and DOTA-NHS (compound 4) (1 eq) were dissolved in 10 mL of DMF, and then DIEA (3 eq) was added. The reaction mixture was stirred at room temperature for 2 hours, and the reaction progress was monitored by LC-MS. The solvent was then removed. The residue was treated with 5 mL of TFA solution and stirred at room temperature for 2 hours. Then 50 mL of diethyl ether was added to precipitate the product, giving a large amount of solid. The mixture was centrifuged and the solid was dried. Finally, the crude product was purified by reverse-phase HPLC to obtain compound 17 (yield: 46.2%). The HPLC conditions were as follows: water with 0.1% TFA was used as mobile phase A; acetonitrile was used as mobile phase B. Gradient conditions B: 0–60 min, from 25% to 45%; flow rate = 20 mL / min; λ = 220 nm. LC-MS (ESI+) showed a value of 788.46.

[0056] Synthesize DOTA-IRDye800cw-FAP-3940:

[0057] Compound 17 (1 eq) and compound 8 (1 eq) synthesized in Example 1 were dissolved in 10 mL of a 1:1 (v / v) formonitrile / water mixture, followed by the addition of 10 mL of phosphate buffered saline (PBS, pH 7.2). The reaction mixture was stirred at room temperature for 1 hour, and the reaction was confirmed to be complete by LC-MS. The reaction mixture was directly purified by reversed-phase preparative HPLC to give DOTA-IRDye800cw-FAP-3940, the FAP-targeted precursor compound named "P230721" (yield: 15.8%). The HPLC conditions were as follows: water containing 0.1% TFA as mobile phase A; acetonitrile as mobile phase B. Gradient conditions B: 0–20 min, from 10% to 100%, flow rate = 3 mL / min; λ = 220 nm. LC-MS (ESI+) showed a concentration of 900.53 [M+3H] / 3. The chemical purity of IRDye800cw-FAP-3940 exceeded 95%, as determined by mass spectrometry (MS) and high-performance liquid chromatography (HPLC). For the HPLC-MS analysis of DOTA-IRDye800cw-FAP-3940, see [link to HPLC analysis]. Figure 3 . Figure 4 The high-performance liquid chromatograms show the purity and quality control results of IRDye800cw-FAP-3940.

[0058] Example 3.

[0059] Commercially available targeting peptides FAP-2286 and FAP-3940, IRDye800cw-FAP-2286 synthesized in Example 1, and IRDye800cw-FAP-3940 synthesized in Example 2 were used as precursor compounds for radiolabeling. Eluting was performed using a gallium-68 generator (ITM, Germany) to obtain... 68 Ga was used for the radiolabeling of the aforementioned precursor compounds. 20 nmol of each precursor was dissolved in 1 mL of sodium acetate buffer (0.25 M, pH 8.2-8.3), and then mixed with 4 mL of... 68 The reaction was carried out in Ga solution (1.0 GBq dissolved in 0.6 M HCl) at 100 °C for 15 min. The four products were purified using a C18 Sep-Pak column (WAT020515; Waters, USA) with ethanol (0.5 mL) as the eluent. The four products were incubated in phosphate-buffered saline (PBS) and fetal bovine serum (FBS) at 25 °C for 3 h, followed by analysis by thin-layer chromatography (TLC) to determine their precipitates. 68 Ga-IRDye800cw-FAP-2286 and 68 Stability of Ga-IRDye800cw-FAP-3940.

[0060] Four precursors 68Ga radiolabeling yields 68 Ga-FAP-2286, 68 Ga-FAP-3940 68 Ga-IRDye800cw-FAP-2286 and 68 Four products, Ga-IRDye800cw-FAP-3940, were obtained. The radiochemical purity of all four products exceeded 95%, and their molar activity was 37.0 ± 0.6 GBq / μmol. Thin-layer chromatography (TLC) results showed that... 68 Ga-IRDye800cw-FAP-2286 and 68 Ga-IRDye800cw-FAP-3940 showed good stability in PBS and FBS for up to 3 hours, with no obvious dechelation observed.

[0061] Experimental Example 1. In vitro properties of IRDye800cw-FAP-2286 and IRDye800cw-FAP-3940

[0062] The HK293T-FAP cell line was established by stably transfecting HK293T cells with human FAP. In in vitro studies, cells were seeded in 24-well plates and cultured to 80% confluency in serum-containing standard medium. The medium was then replaced with serum-free medium for further experiments. In cell uptake assays, 10 nmol of a non-radioactive precursor (FAP) was added or not added. nat After Ga-FAP-2286), the products prepared in Example 3 were used with 3 μCi / mL. 68 Ga-FAP-2286, 68 Ga-FAP-3940 68 Ga-IRDye800cw-FAP-2286 or 68 Cells were treated with Ga-IRDye800cw-FAP-3940 and incubated for 60 minutes. For the FAP radioligand binding assay, HK293T-FAP cells were treated with different concentrations of unlabeled FAP-2286, FAP-3940, IRDye800cw-FAP-2286, or IRDye800cw-FAP-3940 (3.70 × 10⁻⁶). -7 Up to 10 -13 M, n=3) and 68 Incubation was performed with Ga-FAP-2286 (as a radioligand). After 60 minutes of incubation, the cells were washed three times with PBS to remove free tracer, and radioactivity was measured. Cells were lysed with 0.5 mL of 1 M NaOH, and radioactivity was measured using a gamma counter (Wizard 2480; PerkinElmer Inc., USA).

[0063] In the cellular uptake and blockade studies, cell binding assays were performed in HEK293T-FAP cells using the four products prepared in Example 3. This was achieved by using an excess of unlabeled... nat The binding specificity of Ga-FAP-2286 was confirmed by blocking experiments. The binding affinity of IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2 to FAP was evaluated by determining the half-maximum inhibitory concentration (MCMC).

[0064] The results showed that the sample prepared in Example 3 68 Ga-FAP-2286, 68 Ga-FAP-3940 68 Ga-IRDye800cw-FAP-2286 and 68 Ga-IRDye800cw-FAP-3940 can be effectively taken up by FAP-positive HEK293T-FAP cells (see [link]). Figure 5 (Part a of the text). Excessive Nat Ga-FAP-2286 significantly blocked the binding of these four radiotracers to HEK293T-FAP cells, indicating that this binding is specific to FAP. We used HEK293T-FAP cells and 68 Competitive binding assays were performed using Ga-FAP-2286 (as a radioligand) to determine the FAP binding affinity of FAP-2286, FAP-3940, IRDye800cw-FAP-2286 prepared in Example 1, and IRDye800cw-FAP-3940 prepared in Example 2. All four radiotracers obtained after radiolabeling these four compounds as precursors according to the method in Example 3 exhibited high binding affinity for FAP (see [link to example]). Figure 5 (Part b of the text). The competitive binding test results show that FAP-3940 has a slightly higher affinity than FAP-2286 (IC). 50 (2.4±0.2 nM vs. 11.5±2.3 nM), correspondingly, the affinity of IRDye800cw-FAP-3940 prepared in Example 2 was also slightly higher than that of IRDye800cw-FAP-2286 prepared in Example 1 (IC). 50 ,11.2±1.2nM vs.23.7±2.6nM).

[0065] Experimental Example 2. Confocal Microscopy Imaging

[0066] Confocal fluorescence images were obtained of HEK293T-FAP cells after incubation with IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2, respectively. Binding specificity was verified by competition with excess FAP-2286.

[0067] Immunofluorescence staining: Tumor cells were seeded in confocal culture dishes and fixed with 4% paraformaldehyde for 10 minutes. After washing three times with PBS, they were blocked with 10% goat serum for 30 minutes to reduce nonspecific binding. FAP-2286 was used as a competitive agent in the blocking group during incubation. With or without a competitive agent, cells were incubated for 1 hour with a 3.3 nmol / mL diluted fluorescent probe (IRDye800cw-FAP-2286 or IRDye800cw-FAP-3940). After washing three times with PBS, the cell nuclei were stained with DAPI (D4080; Everbright, USA, China). Fluorescence images were acquired using a laser confocal microscope (Evident FV4000, Japanese supplier) at an excitation wavelength of 774 nm and an emission wavelength of 789 nm.

[0068] We used confocal microscopy to observe the specific binding of IRDye800cw-FAP-2286 (prepared in Example 1) and IRDye800cw-FAP-3940 (prepared in Example 2) to HEK293T-FAP cells (results are shown in [link to results]). Figure 5 (Part c in the text). From Figure 5 As shown in section C, after 60 minutes of incubation, we detected strong fluorescence signals of IRDye800cw-FAP-2286 prepared in Example 1 and IRDye800cw-FAP-3940 prepared in Example 2 in HEK293T-FAP cells. However, in the FAP-2286 overdose blocking group, we observed almost no fluorescence signal of IRDye800cw.

[0069] Experimental Example 3. Small Animal PET Imaging

[0070] Obtain the results of injecting HEK293T-FAP tumor-bearing mice as described in Example 3 68 Ga-FAP-2286, 68 Ga-FAP-3940 68 Ga-IRDye800cw-FAP-2286 and 68 Representative static PET images after Ga-IRDye800cw-FAP-3940. And for... 68 Ga-IRDye800cw-FAP-2286 and 68Quantitative analysis of PET signals in tumor tissue using Ga-IRDye800cw-FAP-3940.

[0071] All animal experiments were approved by the Animal Protection and Use Committee of Xiamen University. Six-week-old BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). 5 × 10 6 One HK293T-FAP cell (100 μL PBS) was subcutaneously injected into the right shoulder of each mouse. When the tumor volume reached approximately 500 mm², the cells were inoculated. 3 Imaging was performed at that time. Tumor-bearing mice (n=3 per group) were intravenously injected with 7.4 MBq. 68 Ga-FAP-2286, 68 Ga-FAP-3940 68 Ga-IRDye800cw-FAP-2286 or 68 Ga-IRDye800cw-FAP-3940. PET scans were performed 1 hour and 3 hours post-injection using an Inveon small animal PET scanner (Siemens, USA). In the blocking study, 15 nmol of the non-radioactive precursor was... nat Ga-FAP-2286 and 68 Ga-IRDye800cw-FAP-2286 or 68 Ga-IRDye800cw-FAP-3940 was injected simultaneously, and a static PET scan was performed 1 hour later. The PET images were iteratively reconstructed using the 3DOpMAP 256 protocol (Siemens Healthineers Solutions) and converted to the percentage of injected dose per gram of tissue (%ID / g) by delineation of the region of interest.

[0072] like Figure 6 The results showed that four types 68 Ga-labeled radiotracers accumulated significantly in tumors within 1 hour after injection, with a slight increasing trend after 3 hours (see results). Figure 6 (AC portion). Prepared in Example 3 68 Ga-IRDye800cw-FAP-3940 and 68 Tumor uptake of Ga-IRDye800cw-FAP-2286 was comparable (1 hour, 7.5 ± 0.7% ID / g vs. 5.9 ± 0.8% ID / g, p = 0.051; 3 hours, 7.8 ± 0.8% ID / g vs. 6.2 ± 0.9% ID / g, p = 0.091). Notably, the addition of the IRDye800cw group significantly increased the uptake of both probes in the kidneys, while no such increase was observed in other organs.

[0073] We used excessive amountsnat Blocking studies were conducted on Ga-FAP-2286 to evaluate the preparation of Example 3. 68 Ga-IRDye800cw-FAP-3940 and 68 Ga-IRDye800cw-FAP-2286 exhibits specificity for FAP receptors in vivo. One hour after injection, when... nat Ga-FAP-2286 is respectively compared with the above two types of the present invention. 68 When Ga-labeled fluorescent probes were administered in combination, we observed that tumor uptake of the two fluorescent probes described above was significantly inhibited. 68 Ga-IRDye800cw-FAP-2286, 0.9±0.1% ID / g, with a blocking rate of 84.7%; 68 Ga-IRDye800cw-FAP-3940, 0.9±0.03% ID / g, with an inhibition rate of 88.8%.

[0074] Experimental Example 4. Fluorescence Imaging

[0075] Representative fluorescence images at different time points were obtained after intravenous injection of IRDye800cw-FAP-2286 (synthesized in Example 1) and IRDye800cw-FAP-3940 (synthesized in Example 2) into HEK293T-FAP tumor-bearing mice. Quantitative analysis of tumor-related fluorescence signals of IRDye800cw-FAP-2286 (synthesized in Example 1) and IRDye800cw-FAP-3940 (synthesized in Example 2) was also performed.

[0076] Each group of mice (n=3) was intravenously injected with 18.5 nmol of either IRDye800cw-FAP-2286 synthesized in Example 1 or IRDye800cw-FAP-3940 synthesized in Example 2. The Lumina III in vivo imaging system (PerkinElmer, USA) was used to perform fluorescence imaging at 0.5, 1, and 3 hours post-injection. To evaluate the specificity of the fluorescent probe, a blocking experiment was performed: 226.4 nmol of unlabeled FAP-2286 was injected simultaneously, followed by imaging 1 hour post-injection.

[0077] To perform in vitro fluorescence imaging of three-dimensional tissues, mice were sacrificed one hour after injection of the fluorescent probe (with or without an inhibitor), and major organs were removed and used. The system performs fluorescence imaging.

[0078] from Figure 7The fluorescence images show that both fluorescent probes rapidly entered the tumor within 0.5 hours, their levels gradually increased within 1 hour, and remained relatively stable within 1 to 3 hours. Quantitative fluorescence intensity analysis showed that at 1 hour and 3 hours post-injection, the tumor fluorescence intensity of IRDye800cw-FAP-3940 synthesized in Example 2 was significantly higher than that of IRDye800cw-FAP-2286 synthesized in Example 1 (see [link to example]). Figure 7 (ab part). When we used excessive amounts of FAP-2286 as a competitive agent, tumor uptake of both probes was significantly reduced.

[0079] To further evaluate the fluorescence distribution of IRDye800cw-FAP-2286 synthesized in Example 1 and IRDye800cw-FAP-3940 synthesized in Example 2, we performed ex vivo fluorescence imaging on tumors and major organs one hour after injection of IRDye800cw-FAP-2286 and IRDye800cw-FAP-3940. The results are as follows: Figure 8 As shown in Figure a, part a indicates that the probe mainly accumulates in tumor tissue and the kidney, with very little fluorescence retention in other normal organs. The IRDye800cw-FAP-3940 synthesized in Example 2 exhibited a fluorescence intensity as high as (4.5 ± 0.2) × 10⁻⁶ in HEK293T-FAP tumors. 9 p / s / cm 2 / sr, significantly higher than the fluorescence intensity of IRDye800cw-FAP-2286 prepared in Example 1 ([3.4±0.3]×10). 9 p / s / cm 2 / sr, p=0.008, see Figure 8 (Part b). Furthermore, excessive FAP-2286 significantly inhibited tumor uptake of both fluorescent probes (IRDye800cw-FAP-2286, [3.4±0.3]×10⁻⁶). 9 vs. [0.4±0.2]×10 9 p / s / cm 2 / sr, p<0.001; IRDye800cw-FAP-3940, [4.5±0.2]×10 9 vs. [0.4±0.1]×10 9 p / s / cm 2 / sr, p<0.001).

[0080] Experimental Example 5. In vitro validation using human lung cancer tissue.

[0081] This pilot clinical study has been approved by the Clinical Research Ethics Committee of the First Affiliated Hospital of Xiamen University, and written informed consent has been obtained from all participants. In four patients with early-stage lung adenocarcinoma, existing probes were used preoperatively. 68 PET imaging with Ga-FAPI-46 was performed for tumor staging and assessment of FAP expression. Immediately after surgical resection, tumor tissue was immersed in FBS-free DMEM medium and transported to the laboratory on ice. The tissue was gently washed three times with cold PBS (4°C) and then incubated with IRDye800cw-FAP-3940 (7.4 μM, dissolved in FBS-free DMEM medium) synthesized in Example 2 at 25°C for 20 minutes. After incubation, the tissue was washed three more times with cold PBS and then... Imaging was performed using the Lumina III fluorescence imaging system. Subsequently, the tissue was embedded and cut into two consecutive 4 μm thick sections, one of which was stained with hematoxylin and eosin (H&E), and the other was stained with FAP immunohistochemistry (IHC).

[0082] Given that the imaging performance of IRDye800cw-FAP-3940 synthesized in Example 2 was superior to that of IRDye800cw-FAP-2286 synthesized in Example 1 in preclinical experiments, we further performed fluorescence imaging in four lung cancer tissues. These tissues were diagnosed as lung cancer and... 68 Human lung cancer tissue confirmed as FAP-positive by Ga-FAPI-46 PET / CT was incubated with IRDye800cw-FAP-3940 synthesized in Example 2 for in vitro evaluation. After incubation, the tissue was subjected to fluorescence imaging using an IVIS fluorescence system. The results showed that the fluorescence intensity of the tumor tissue was significantly higher than that of the surrounding normal tissue. Further H&E staining and immunohistochemical staining confirmed that the fluorescence boundary precisely corresponded to the tumor and surrounding normal tissue (see results). Figure 9 (AF part in the text).

Claims

1. A precursor compound that can target FAP, having the structure shown in formula (I) or (II) below:

2. A dual-modal FAP targeting probe, characterized in that: It is a complex formed by the precursor compound of formula (I) or (II) as claimed in claim 1 as a ligand and a radionuclide; the radionuclide is a radionuclide that can form a stable complex with the DOTA group.

3. The dual-modal FAP targeting probe as described in claim 2, characterized in that: The radioactive nuclide mentioned is 68 Ga.

4. The application of the precursor compound according to claim 1 in the preparation of a dual-modal FAP targeting probe for PET and near-infrared II fluorescence.

5. The use of the precursor compound according to claim 1 in the preparation of a fluorescent imaging agent.

6. The application of the dual-modal FAP targeting probe of claim 2 as an imaging agent in in vivo imaging of organisms for non-therapeutic or diagnostic purposes.

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