Fibroblast activated protein targeted diagnosis and treatment integrated molecular probe

Abstract

The invention relates to a fibroblast activated protein targeted diagnosis and treatment integrated molecular probe, and belongs to the technical field of nuclear medicine. The invention provides a fibroblast activating protein targeted diagnosis and treatment integrated molecular probe, a precursor compound of the molecular probe is composed of an FAP targeted group, a radioactive element complexed group DOTA and 4-(4-hydroxyphenyl) butyric acid, and PEG, glutamic acid and D-lysine are used as connecting arms. The precursor compound is complexed with < 68 > Ga or < 177 > Lu to obtain a high-purity PET (Polyethylene Terephthalate) imaging molecular probe < 68 > Ga-FAPI-JNU or a high-purity therapeutic molecular probe < 177 > Lu-FAPI-JNU. Researches show that < 68 > Ga-FAPI-JNU and < 177 > Lu-FAPI-JNU have better targeting property and higher target affinity, and have obvious tumor targeting uptake in tumor-bearing mice with positive expression of fibroblast activated protein.

Description

Technical Field

[0001] The invention relates to a fibroblast activation protein-targeted integrated diagnosis and treatment molecular probe, belonging to the technical field of nuclear medicine. Background Art

[0002] Fibroblast activation protein (FAP) is a type II transmembrane protein with both dipeptidase and endopeptidase activities. It is highly expressed on the surface of activated fibroblasts and lowly expressed on the surface of quiescent fibroblasts. FAP is highly expressed in many pathological conditions, such as various organ fibrosis, wound healing, atherosclerosis, autoimmune diseases, and cancer, and the expression of FAP may be associated with the progression of some diseases. FAP is transiently expressed during the embryonic period and is expressed in the cervix and endometrium, which may be associated with tissue remodeling. In the healthy tissues of normal adults, FAP is lowly expressed or even not expressed.

[0003] In extensive connective tissue hyperplasia tumors, cancer-associated fibroblasts occupy the main components of the tumor microenvironment. As one of the surface markers of cancer-associated fibroblasts, fibroblast activation protein plays an enzymatic and non-enzymatic role in promoting tumor tissue growth, invasion, metastasis, tumor angiogenesis and immunosuppression. Tumor tissue highly expresses fibroblast activation protein, while healthy tissue hardly expresses fibroblast activation protein, which makes fibroblast activation protein a potential target for cancer diagnosis and treatment. Fibroblast activation protein has a large extracellular domain and the catalytic site is also located outside the cell. These characteristics ensure that targeted fibroblast activation protein imaging has the characteristics of low background activity, high image contrast and low side effects.

[0004] At present, in terms of imaging, there have been many studies that "light up tumors" by developing targeted fibroblast activation protein imaging agents, and many developed tracers have entered the clinical trial stage. In terms of treatment, there have been many studies that inhibit cancer growth by targeting fibroblast activation protein molecules with radionuclides that have radiation damage to surrounding cancer cells, or by combining targeted fibroblast activation protein molecules with anti-tumor drugs, targeting fibroblast activation protein to break the matrix barrier and enhance the efficacy of other anti-tumor drugs. Fibroblast activation protein targeting molecules have been applied to many different solid tumors and designed diagnostics and combined therapies targeting tumor cells and tumor matrix components. Among them, quinoline-based radiolabeled small molecules FAPI and its derivatives are widely used in preclinical and clinical studies due to their rapid renal excretion, good targeting of fibroblast activation protein and rapid tumor internalization.

[0005] However, the existing quinoline-based radiolabeled small molecule FAPI and its derivatives still have certain defects. For example, FAPI-46 does not stay in the tumor for a long time, which affects its therapeutic effect in fibroblast activation protein-positive tumors. There are also studies that try to form polymers based on FAPI to improve the uptake of drugs in tumors and prolong the retention time of drugs in tumors. However, the effect of this structural improvement is not significant. For example, FAPI-46 dimer 68 Ga-DOTA-2P(FAP) 2 In the U87 tumor model, the average tumor uptake SUV at 1 hour was still only 0.42±0.03, and FAPI-46 tetramer 68 Ga-DOTA-4P(FAP) 4 In the U87 tumor model, the average tumor uptake SUV at 1 hour was still only 0.72±0.02. 68 The average tumor uptake SUV of Ga-FAPI-46 at 1 hour in the U87 tumor model is still only 0.16±0.01 (see the literature "Zhao, L.; Niu, B.; Fang, J.; Pang, Y.; Li, S.; Xie, C.; Sun, L.; Zhang, X.; Guo, Z.; Lin, Q.; et al. Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of (68) Ga-Labeled FAPI Dimer. J. Nucl. Med. 2022, 63(6), 862-868."). Therefore, in order to improve the detection rate of smaller lesions in clinical work, it is urgent to find radiolabeled molecular probes with higher tumor uptake and more suitable intratumoral retention time, which will also provide new options for the diagnosis and treatment of targeted solid tumors in the future. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a fibroblast activation protein-targeted integrated molecular probe for diagnosis and treatment, wherein the fibroblast activation protein-targeted integrated molecular probe for diagnosis and treatment comprises a precursor compound and a radionuclide; the molecular formula of the precursor compound is Y-Glu-[X-PEG 4 ] 2 -Lys-Z, wherein X is a fibroblast activation protein targeting group, Y is a chelating group DOTA, and Z is 4-(4-hydroxyphenyl)butanoic acid; the radionuclide is complexed to the chelating group DOTA of the precursor compound;

[0007] The structural formula of the precursor compound is as follows:

[0008]

[0009] In one embodiment of the present invention, the radionuclide comprises 68 Ga, 64 Cu, 177 Lu, 90 Y or 111 In.

[0010] In one embodiment of the present invention, the radionuclide is 68 Ga or 177 Lu.

[0011] In one embodiment of the present invention, the structural formula of the integrated diagnosis and treatment molecular probe is as follows:

[0012]

[0013] Alternatively, the structural formula of the integrated diagnosis and treatment molecular probe is as follows:

[0014]

[0015] The present invention also provides a kit for preparing the above-mentioned integrated diagnosis and treatment molecular probe, the components of the kit include a precursor compound and a radionuclide labeled compound; the molecular formula of the precursor compound is Y-Glu-[X-PEG 4 ] 2 -Lys-Z, wherein X is a fibroblast activation protein targeting group, Y is a chelating group DOTA, and Z is 4-(4-hydroxyphenyl)butyric acid; the radionuclide is complexed to the chelating group DOTA of the precursor compound;

[0016] The structural formula of the precursor compound is as follows:

[0017]

[0018] In one embodiment of the present invention, the radionuclide labeled compound comprises 68 GaCl 3 , 64 CuCl 2 , 177 LuCl 3 , 90 YC 3 or 111 InCl 3 .

[0019] In one embodiment of the present invention, the radionuclide labeled compound is68 GaCl 3 or 177 LuCl 3 .

[0020] In one embodiment of the present invention, the components of the kit further include an organic solvent and / or a buffer solution.

[0021] In one embodiment of the present invention, the organic solvent comprises dimethyl sulfoxide; and the buffer solution comprises acetic acid / sodium acetate buffer solution.

[0022] The present invention also provides a method for preparing the above-mentioned integrated diagnosis and treatment molecular probe, the method comprising: using the above-mentioned kit to prepare the above-mentioned integrated diagnosis and treatment molecular probe.

[0023] In one embodiment of the present invention, the method includes: dissolving a precursor compound in an organic solvent to obtain a dissolving solution; mixing the dissolving solution and a buffer solution, and adjusting the pH to 3.5-4.0 with the buffer solution to obtain a mixed solution; reacting the mixed solution at 80-100°C for 10-20 minutes to obtain a reaction solution containing the above-mentioned integrated diagnostic and therapeutic molecular probe.

[0024] The present invention also provides the use of the above-mentioned integrated diagnosis and treatment molecular probe or the above-mentioned kit in the preparation of fibroblast activation protein imaging agents, tumor imaging agents or anti-tumor drugs.

[0025] In one embodiment of the present invention, the tumor includes a fibroblast activation protein-positive tumor; the fibroblast activation protein-positive tumor includes a solid tumor; the solid tumor includes a glioma, lung cancer and / or breast cancer.

[0026] The present invention also provides a fibroblast activation protein imaging agent, the components of which include the above-mentioned integrated diagnosis and treatment molecular probe.

[0027] The present invention also provides a tumor imaging agent, the components of which include the above-mentioned integrated diagnosis and treatment molecular probe.

[0028] In one embodiment of the present invention, the tumor includes a fibroblast activation protein-positive tumor; the fibroblast activation protein-positive tumor includes a solid tumor; the solid tumor includes a glioma, lung cancer and / or breast cancer.

[0029] The present invention also provides an anti-tumor drug, the components of which include the above-mentioned integrated diagnosis and treatment molecular probe.

[0030] In one embodiment of the present invention, the tumor includes a fibroblast activation protein-positive tumor; the fibroblast activation protein-positive tumor includes a solid tumor; the solid tumor includes a glioma, lung cancer and / or breast cancer.

[0031] The technical solution of the present invention has the following advantages:

[0032] The present invention provides a fibroblast activation protein-targeted integrated diagnosis and treatment molecular probe, wherein the precursor compound of the molecular probe is composed of a FAP targeting group, a group DOTA for complexing radioactive elements, and 4-(4-hydroxyphenyl)butyric acid, and PEG, glutamic acid, and D-lysine are used as connecting arms. 68 Ga or 177 High-purity PET imaging molecular probe obtained after Lu 68 Ga-FAPI-JNU or high-purity therapeutic molecular probe 177 Lu-FAPI-JNU. Studies have shown that PET imaging molecular probes 68 Ga-FAPI-JNU and therapeutic molecular probes 177 Lu-FAPI-JNU has good in vitro stability, good water solubility, can be metabolized by the kidneys and cleared quickly from non-target tissues in vivo. It has better targeting and higher target affinity. In tumor-bearing mice with positive expression of fibroblast activation protein, the tumor-targeted uptake is obvious, and the uptake in important organs such as muscle, bone, heart, lung, liver, etc. is low. Among them, animal experiments have shown that PET imaging molecular probes 68 The uptake of Ga-FAPI-JNU in fibroblast activation protein-positive tumors was 68 Ga-FAPI-46 is 8 times higher, has higher tumor imaging contrast, better target and non-target ratio (T / NT ratio can reach 6.75±1.70), stronger resolution, can clearly distinguish the lesion site, improves the accuracy of early diagnosis and imaging of fibroblast activation protein-positive tumors, and promotes the diagnostic application of FAPI molecular probes in fibroblast activation protein-positive tumors. 177 Lu-FAPI-JNU can inhibit the tumor growth of FAP-positive tumor-bearing mice to a certain extent, promoting the therapeutic application of FAPI-type molecular probes in FAP-positive tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 : Characterization results of precursor FAPI-JNU.

[0034] Figure 2 :Standard FAPI-JNU and prepared PET molecular probe68 HPLC quality control chart of Ga-FAPI-JNU.

[0035] Figure 3 : Affinity determination results of standard substance FAPI-JNU and fibroblast activation protein.

[0036] Figure 4 : 68 The in vitro stability results of Ga-FAPI-JNU (i.e. 68 Radio-HPLC results of Ga-FAPI-JNU in PBS buffer and fetal bovine serum at 2 h).

[0037] Figure 5 : 68 Biocompatibility experimental results of Ga-FAPI-JNU in living ICR mice. Figure 5 In, A: 68 Effect of Ga-FAPI-JNU intervention on body weight of ICR mice; B: 68 HE staining results of various organs of ICR mice treated with Ga-FAPI-JNU.

[0038] Figure 6 : 68 PET / CT images of Ga-FAPI-JNU in U87 tumor-bearing mouse model. Figure 6 Middle, A: U87 tumor-bearing mouse model injection 68 Imaging images of Ga-FAPI-JNU at 2.5 minutes, 27.5 minutes, 60 minutes, 120 minutes and 180 minutes, and imaging images of Ga-FAPI-JNU at 2.5 minutes, 27.5 minutes, 60 minutes, 120 minutes and 180 minutes after precursor blocking; B: Trend change chart of quantitative results of tumor uptake in U87 tumor-bearing mouse model before and after blocking.

[0039] Figure 7 : 68 Drug biodistribution profile of Ga-FAPI-JNU in U87 tumor-bearing mouse model (in vivo).

[0040] Figure 8 : 68 Ga-FAPI-JNU and 68 Comparison of imaging of Ga-FAPI-46 in U87 tumor-bearing mouse model.

[0041] Fig. 9 : 68 Ga-FAPI-JNU and 68 The uptake results of Ga-FAPI-46 in tumor tissue of U87 tumor-bearing mouse model. Fig. 9 In, A: 68Ga-FAPI-JNU and 68 Comparison of the average tumor uptake SUV of Ga-FAPI-46 in the U87 tumor-bearing mouse model at 1 hour (1.33±0.17vs 0.16±0.03P<0.001); B: 68 Ga-FAPI-JNU and 68 Comparison of tumor / muscle uptake SUV values ​​of Ga-FAPI-46 in the U87 tumor-bearing mouse model at 1 hour (8.52±1.93vs 4.67±1.10P<0.05).

[0042] Fig.10 : 68 Results of autoradiography, immunohistochemistry and HE staining of Ga-FAPI-JNU in human tumor specimens.

[0043] Fig.11 :Standard FAPI-JNU and 177 HPLC quality control chart of Lu-FAPI-JNU.

[0044] Fig.12 : 177 Figure 3 shows the in vitro stability results of Lu-FAPI-JNU. Fig.12 In, A: 177 Radio-HPLC results of Lu-FAPI-JNU in PBS buffer at 2h and 72h; B: 177 Radio-HPLC results of Lu-FAPI-JNU in fetal bovine serum at 2h and 72h.

[0045] Fig.13 : 177 Drug biodistribution profile of Lu-FAPI-JNU in U87 tumor-bearing mouse model (in vitro).

[0046] Fig.14 : 177 SPECT images of Lu-FAPI-JNU in U87 tumor-bearing mouse model.

[0047] Fig.15 : 177 The results of the Lu-FAPI-JNU treatment experiment on the U87 tumor-bearing mouse model. Fig.15 In, A: 177 Effect of Lu-FAPI-JNU intervention on tumor volume in U87 tumor-bearing mouse model; B: 177 Effect of Lu-FAPI-JNU intervention on tumor weight in U87 tumor-bearing mouse model; C: 177 Tumor photos of U87 tumor-bearing mouse model treated with Lu-FAPI-JNU.

[0048] Fig.16 : After 177 The HE staining results of each organ in the U87 tumor-bearing mouse model intervened by Lu-FAPI-JNU. Specific implementation manners

[0049] The following embodiments are provided to better further understand the present invention, which are not limited to the described best implementation manner, and do not constitute a limitation to the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with those of other existing technologies falls within the protection scope of the present invention.

[0050] For those not specifying specific experimental steps or conditions in the following embodiments, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments not specifying the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0051] Example 1: A fibroblast activation protein-targeted molecular probe 68 Ga-FAPI-JNU

[0052] This example provides a fibroblast activation protein-targeted molecular probe 68 Ga-FAPI-JNU, and the fibroblast activation protein-targeted molecular probe 68 Ga-FAPI-JNU has the following structure:

[0053]

[0054] Example 2: A method for preparing a fibroblast activation protein-targeted molecular probe 68 Method for Ga-FAPI-JNU

[0055] This example provides a method for preparing the fibroblast activation protein-targeted molecular probe 68 Ga-FAPI-JNU described in Example 1, and the specific steps are as follows:

[0056] 1. Preparation of the precursor compound FAPI-JNU

[0057] Step 1: Prepare compound 1 according to the literature “Loktev, A.; Lindner, T.; Burger, EM; Altmann, A.; Giesel, F.; Kratochwil, C.; Debus, J.; Marme, F.; Jager, D.; Mier, W.; et al. Development of Fibroblast Activation Protein-Targeted Radiotracers with Improved Tumor Retention. J. Nucl. Med. 2019, 60 (10), 1421-1429.”; Take compound 1 (499.57 mg, 1 eq, 1 mmol) and compound 2 (Fmoc-NH-PEG 4 -CH 2 COOH, 434.3 μL, 1.1 eq, 1.1 mmol) was dissolved in N,N-dimethylformamide (DMF, 3 mL) to obtain a solution; 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 456.30 mg, 1.2 eq, 1.2 mmol) and N,N-diisopropylethylamine (DIPEA, 695.42 μL, 4 eq, 4 mmol) were added to the solution and mixed well, and then stirred (150 rpm) at room temperature (25°C) for 16 h to obtain a reaction product A; a DMF solution (1 mL) containing 20% ​​(v / v) piperidine was added to the reaction product A, and then stirred (150 rpm) at room temperature (25°C) for 3 h to remove the Fmoc protecting group to obtain a reaction product B; the reaction product B was purified by semi-preparative HPLC to obtain a light yellow solid compound 3;

[0058] Among them, compound 1 has the following structure:

[0059]

[0060] Compound 2 has the following structure:

[0061]

[0062] Compound 3 has the following structure:

[0063]

[0064] Step 2: Boc-Lys-OMe (312.40 mg, 1.2 eq, 1.2 mmol) was dissolved in N,N-dimethylformamide (DMF, 20 mL) to obtain a solution; 4-(4-hydroxyphenyl)butyric acid (180.2 mg, 1 eq, 1 mmol), 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC, 310.5 mg, 2 eq, 2 mmol), 1-hydroxybenzotriazole (HOAT, 272.2 mg, 2 eq, 2 mmol) and N,N-diethylethylamine (DIPEA, 153 μL, 2.75 eq, 1.1 mmol) were added to the solution and mixed well, and then stirred at room temperature (25°C) for 15 min. 0rpm) for 16 hours to obtain a reaction product A; the reaction product was first extracted with ethyl acetate and saturated sodium bicarbonate aqueous solution to remove DMF, and then purified by silica gel column chromatography (the eluent used for silica gel column chromatography is a mixture of chloroform and methanol, and the volume ratio of chloroform to methanol in the mixture is 5:1), and then the solvent was removed by vacuum rotary evaporation to obtain a brown oily compound before deprotection; trifluoroacetic acid (TFA, 4 mL) was added dropwise to the brown oily compound, and then stirred (150rpm) for reaction for 3 hours at room temperature (25°C) to remove the Boc protecting group, to obtain a reaction product B; the reaction product B was first subjected to vacuum rotary evaporation to remove the solvent, and then precipitated with anhydrous ether to obtain a brown solid powder compound A1;

[0065] Boc-Lys-OMe has the structure shown below:

[0066]

[0067] Compound A1 has the structure shown below:

[0068]

[0069] Step 3: Compound A1 (157.98 mg, 1 eq, 0.490 mmol) and Fmoc-L-glutamic acid-γ-methyl ester (Fmoc-Glu(OMe)-OH, 229.33 mg, 1.1 eq, 0.539 mmol) were dissolved in N,N-dimethylformamide (DMF, 5 mL) to obtain a solution; 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 223.58 mg, 1.2 eq, 0.588 mmol) and N,N-diisopropylethylamine (DIPEA, 340.75 μL, 4 eq, 1.96 mmol) were added to the solution and mixed well, and then stirred (150 rpm) at room temperature (25°C) for 3 h to obtain a reaction product; the reaction product was subjected to reduced pressure rotary evaporation to remove the solvent to obtain a brown oily compound A2;

[0070] Fmoc-Glu(OMe)-OH has the following structure:

[0071]

[0072] Compound A2 has the structure shown below:

[0073]

[0074] Step 4: After adding HCl solution (HCl solution concentration is 6N, 2 mL) to compound A2, stirring (150 rpm) for reaction at room temperature (25°C) for 3 h to remove the OMe protecting group to obtain a reaction product; the reaction product is first extracted with ethyl acetate and saturated sodium bicarbonate aqueous solution to remove the HCl solution, then precipitated with anhydrous ether, and then the solvent is removed by rotary evaporation under reduced pressure to obtain a rotary evaporation product; after adding DMF solution (1 mL) containing 20% ​​(v / v) piperidine dropwise to the rotary evaporation product, stirring (150 rpm) for reaction at room temperature (25°C) for 1 h to remove the Fmoc protecting group to obtain a reaction solution; the reaction solution is purified by semi-preparative HPLC to obtain a white solid powder compound A3;

[0075] Compound A3 has the structure shown below:

[0076]

[0077] Step 5: Compound A3 (96.50 mg, 1 eq, 0.221 mmol) and DOTA-tri-tert-butyl ester-active ester (DOTA-(COOt-Bu)3-NHS, 177.63 mg, 1.2 eq, 0.265 mmol) were dissolved in N,N-dimethylformamide (DMF, 5 mL) to obtain a solution; N,N-diisopropylethylamine (DIPEA, 307.37 μL, 8 eq, 1.768 mmol) was added to the solution and mixed, and then stirred (150 rpm) at room temperature (25°C) under nitrogen protection for 3 h to obtain a reaction product; the reaction product was first subjected to reduced pressure rotary evaporation to remove the solvent, and then purified by semi-preparative HPLC to obtain an off-white solid powder compound 4;

[0078] DOTA-(COOt-Bu)3-NHS has the following structure:

[0079]

[0080] Compound 4 has the following structure:

[0081]

[0082] Step 6: Compound 3 (200 mg, 3 eq, 0.273 mmol) and compound 4 (90.3 mg, 1 eq, 0.091 mmol) were dissolved in N,N-dimethylformamide (DMF, 5 mL) to obtain a solution; 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU, 836.53 mg, 2.2 eq, 0.200 mmol) and N,N-diisopropylethylamine (DIPEA, 79. 1 μL, 5 eq, 0.455 mmol) was mixed, and then stirred (150 rpm) in an 80°C oil bath under nitrogen protection for 4 h to obtain a reaction product A; the reaction product A was transferred to an ice bath, and a trifluoroacetic acid solution (a TFA aqueous solution with a volume percentage concentration of 95%, 133 μL) was added to the reaction product A, and then stirred (150 rpm) for another 2 h at room temperature (25°C) under nitrogen protection to remove the OtBu protecting group, to obtain a reaction product B; saturated NaHCO was added to the reaction product B 3 After adjusting the pH value of the reaction product B to 6, the solution was purified by semi-preparative HPLC to obtain a brown-red precursor compound FAPI-JUN; the precursor compound FAPI-JUN was lyophilized to obtain a precursor FAPI-JNU lyophilized powder;

[0083] The precursor compound FAPI-JUN has the following structure:

[0084]

[0085] The precursor compound FAPI-JUN was subjected to mass spectrometry analysis. The mass spectrometry analysis results of the compound FAPI-JUN are as follows: Figure 1 As shown, the theoretical predicted value of the molecular weight (m / z) of the compound FAPI-JNU is 2253.53, while the ion peak value appearing in the mass spectrum analysis diagram is 1127.45, which is consistent with [(M+2H) / 2] + That is, m / z=1127.76 is consistent.

[0086] 2. Molecular probes 68 Preparation of Ga-FAPI-JNU

[0087] 40 μg of precursor FAPI-JNU lyophilized powder was taken and dissolved in a reaction bottle with 20 μL of dimethyl sulfoxide to obtain a solution; 0.2 mL of acetic acid / sodium acetate buffer solution (pH=7.2, concentration 0.15 M, purchased from Wuxi Nuoyu Pharmaceutical Technology Co., Ltd.) was added to the reaction bottle to obtain a mixed solution; 68 GaCl 3 The 0.1M HCl solution (purchased from Merck & Co., Ltd.) was used to elute from a germanium gallium generator (purchased from Etchi Radiology and Medical Technology Co., Ltd.) to obtain 68GaCl 3 Solution; take 1mL 68 GaCl 3 The solution was added to the reaction bottle and mixed with the mixed solution to obtain a reaction solution; acetic acid / sodium acetate buffer solution (pH=7.2, concentration 0.15M, purchased from Wuxi Nuoyu Pharmaceutical Technology Co., Ltd.) was added to the reaction bottle to adjust the pH value of the reaction solution to 4.0, and the reaction bottle was placed in a 95° C. heater to react for 15 minutes to obtain a reaction product;

[0088] Step 2: Activate the Sep-Pak C18 column with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection; after the reaction product is cooled to room temperature (25°C), extract the reaction product with a 10 mL syringe, pass the reaction product through a C18 solid phase extraction column, and rinse the C18 solid phase extraction column with 20 mL of sterile water for injection; after the rinsing is completed, rinse the C18 solid phase extraction column with 1 mL of 70% (v / v) ethanol solution to obtain the target product 68 Ga-FAPI-JNU; draw out the final product with a syringe 68 Ga-FAPI-JNU was filtered through a 0.22 μm sterile filter membrane to obtain a sterile 68 Ga-FAPI-JNU.

[0089] Using FAPI-JNU as the standard, the standard and 68 Ga-FAPI-JNU was used for quality control. HPLC conditions were as follows: using a YMC C-18 reverse phase column; column temperature: 37°C; UV at 245nm; flow rate of 1mL / min; mobile phase A was an aqueous phase containing 0.02% (v / v) trifluoroacetic acid, and phase B was an acetonitrile phase containing 0.04% (v / v) trifluoroacetic acid. The gradient elution method is shown in Table 1.

[0090] Standard products and 68 The HPLC quality control results of Ga-FAPI-JNU are as follows Figure 2 The retention time of the standard is 11.07min, and after being labeled with radionuclides, 68 The retention time of Ga-FAPI-JNU was 11.27 min. The radioactive chemical purity of the Ga-FAPI-JNU was 95% by integrating the radioactive spectrum area of ​​the PET imaging molecular probe.

[0091] Table 1 Gradient elution method

[0092] time Flow rate Mobile phase A LiquidityB 0min 1mL / min 95％ 5％ 3min 1mL / min 95％ 5％ 20min 1mL / min 10％ 90％ 30min 1mL / min 95％ 5％

[0093] Experimental Example 1: Binding affinity experiment of precursor compound FAPI-JUN and target protein FAP

[0094] This experimental example provides a binding affinity experiment between the precursor compound FAPI-JUN prepared in Example 2 and the target protein FAP. The experimental process is as follows:

[0095] We commissioned Science Compass to conduct the measurement using surface plasmon resonance technology. The specific steps are as follows:

[0096] Step 1: Coupling of fibroblast activation protein (SA chip)

[0097] Biotinylated fibroblast activation protein (FAP, purchased from Biopsies Biotech Co., Ltd.) was prepared into a solution with a concentration of 5 μg / mL using HBS-N buffer and then loaded onto the SA chip for coupling. The coupling conditions were 5 μL / min, 4200 s, and the coupling amount was about 5000 RU.

[0098] Step 2: Dilution of small molecule FAPI-JNU

[0099] The small molecule FAPI-JNU was diluted into HBS-EP buffer to obtain test samples with concentrations of 100 nM, 50 nM, 25 nM, 12.5 nM, and 6.25 nM, respectively;

[0100] Step 3: Affinity test (single cycle kinetics)

[0101] The running buffer of the affinity test was HBS-EP buffer solution. The single-cycle kinetic test method was used to load the small molecule FAPI-JNU solution that was gradient diluted in the previous step onto the fibroblast activation protein chip in order from low to high concentrations. The loading conditions were 30 μL / min, 120 s, and the dissociation conditions were 30 μL / min, 600 s. The test curve was recorded.

[0102] Step 4: Data Analysis

[0103] A 1:1 binding model was used to fit the raw data to obtain kinetic data and affinity constants.

[0104] Figure 3 is the affinity constant of the precursor compound FAPI-JNU and fibroblast activation protein, and the KD value is 90.98 pM. This result suggests that the precursor compound FAPI-JNU has a picomolar affinity with fibroblast activation protein.

[0105] Experimental Example 2: Molecular Probe 68 In vitro stability experiment of Ga-FAPI-JNU

[0106] This experimental example provides the molecular probe prepared in Example 2 68 In vitro stability test of Ga-FAPI-JNU, the experimental process is as follows:

[0107] Experiment 1: Take 200μCi 68 Ga-FAPI-JNU was mixed with 200 μL PBS buffer (pH=7.4, concentration 1×, purchased from Pronocell Life Science Co., Ltd.) by shaking and incubated at 37°C for 2 h to obtain an incubation solution. The incubation solution was sampled and analyzed by Radio-HPLC. 68 Stability of Ga-FAPI-JNU in PBS buffer.

[0108] Experiment 2: Take 200μCi 68 Ga-FAPI-JNU was mixed with 200 μL fetal bovine serum by shaking and evenly incubated at 37°C for 2 h to obtain an incubation solution; 40 μL of the incubation solution was mixed with 50 μL of acetonitrile and shaken for 5 seconds at room temperature (25°C) to precipitate the protein to obtain a treated incubation solution; the treated incubation solution was centrifuged at 12000 r / min for 5 min; after the centrifugation, the supernatant was taken and analyzed by Radio-HPLC 68 Stability of Ga-FAPI-JNU in fetal bovine serum.

[0109] Figure 4 for 68 Radio-HPLC results of Ga-FAPI-JNU in PBS buffer and fetal bovine serum at 2h. Figure 4 It can be seen that after 2 hours of incubation, 68 The main peak of the radioactivity of Ga-FAPI-JNU in the PBS buffer and fetal bovine serum systems did not change significantly, and no other 68 Ga 3+ Plasma noise peaks, visible, 68 Ga-FAPI-JNU has good stability in vitro.

[0110] Experimental Example 3: Molecular Probe 68 Biocompatibility experiment of Ga-FAPI-JNU

[0111] This experimental example provides the molecular probe prepared in Example 2 68 The biocompatibility experiment of Ga-FAPI-JNU, the experimental process is as follows:

[0112] ICR mice (purchased from Vital River) were randomly divided into two groups, the experimental group (n=5) and the control group (n=4). The mice in the experimental group were injected with 189.0±14.1μCi / mouse of the drug through the tail vein. 68Ga-FAPI-JNU (solvent is 200 μL saline), and the control group mice were injected with the same volume of saline by tail vein once. After the injection, the weight of the mice was monitored daily, and the mental state, diet and drinking water of the mice were observed. The monitoring lasted for 14 days. After the monitoring, the important organs (heart, liver, spleen, lung and kidney) of the mice were taken for HE staining to observe whether there were any differences in the microscopic level of the important organs between the two groups.

[0113] Figure 5 A in the figure represents the weight changes of the experimental group and the control group during the monitoring period. Figure 5 B in the figure shows HE staining of important organs. Figure 4 As shown in A, injection 68 There was no significant difference in the weight gain trend between the Ga-FAPI-JNU group and the saline injection group. 68 The weight of mice in the Ga-FAPI-JNU group did not show a significant downward trend, and during the monitoring period, there were no abnormalities in the diet, water, skin, and stool of mice in the experimental group. Figure 4 As shown in B, no significant differences were observed in the microscopic aspects of the important organs of the experimental group and the control group. 68 There was no obvious damage to the important organs of the mice in the Ga-FAPI-JNU group at the microscopic level. 68 Ga-FAPI-JNU has good biocompatibility.

[0114] Experimental Example 4: Molecular Probe 68 Micro-PET / CT imaging experiment of Ga-FAPI-JNU in tumor-bearing mice

[0115] This experimental example provides the molecular probe prepared in Example 2 68 Micro-PET / CT imaging experiment of Ga-FAPI-JNU in tumor-bearing mice. The experimental process is as follows:

[0116] 1. Animal model establishment:

[0117] U87 cells (purchased from Pronocell Life Science Co., Ltd., U87 cells are fibroblast activation protein-positive tumor cells) were cultured at 5×10 6 The inoculum size was 100 mL and inoculated into MEM medium (purchased from Pronocell Life Science Co., Ltd.) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (purchased from Meilun Biotechnology Co., Ltd.) and incubated at 37°C and 5% (v / v) CO 2 The cells were cultured in a sterile cell culture incubator; when the cell density reached 90%, the cells were collected and counted; after the counting was completed, the cells were resuspended to a concentration of 1×10 using 0.25% trypsin (purchased from Meilun Biotechnology Co., Ltd.). 6Nude mice (purchased from Vital River) were inoculated with the bacterial suspension at a volume of 100 μL / mouse into the subcutaneous area under the right forelimb of the nude mice to obtain tumor-bearing mice; after the inoculation, the tumor volume grew to 150-400 mm 3 The tumor-bearing mice can then be used for experiments.

[0118] 2. Micro-PET / CT imaging of tumor-bearing mice

[0119] Each tumor-bearing mouse was weighed before administration. After weighing, the tumor-bearing mice were injected with 100±20μCi / mouse via the tail vein. 68 Ga-FAPI-JNU (solvent is 100 μL saline); after the injection, the 68 The tumor-bearing mice were subjected to static Micro-PET / CT scans at 2.5 minutes, 27.5 minutes, 60 minutes, 120 minutes and 180 minutes after the administration of Ga-FAPI-JNU to observe the distribution of the test substance in the tumor-bearing mice at different time points after administration. 68 FAPI-JNU, a precursor not labeled with radionuclides, was injected into the tail vein 1 hour before Ga-FAPI-JNU for target blocking as a control.

[0120] Each tumor-bearing mouse was weighed before administration. After weighing, the tumor-bearing mice were injected with 100±20μCi / mouse via the tail vein. 68 Ga-FAPI-JNU (solvent is 100 μL saline); after the injection, the 68 The tumor-bearing mice were subjected to static Micro-PET / CT scanning at 1 hour after administration of Ga-FAPI-JNU to observe the distribution of the test substance in the tumor-bearing mice after administration. The next day after the scan, the same batch of tumor-bearing mice were injected with 100±20μCi / mouse of Ga-FAPI-JNU by tail vein. 68 Ga-FAPI-46 was used as a control ( 68Ga-FAPI-46, see the literature "Zhao, L.; Niu, B.; Fang, J.; Pang, Y.; Li, S.; Xie, C.; Sun, L.; Zhang, X.; Guo, Z.; Lin, Q.; et al. Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of (68) Ga-Labeled FAPI Dimer. J. Nucl. Med. 2022, 63 (6), 862-868."). The scanning process is: first pre-anesthetize the tumor-bearing mice with isoflurane anesthetic, fix the tumor-bearing mice on the scanning bed, record the position of the tumor-bearing mice on the bed, and then start Micro-PET / CT scanning, while recording the injection dose, injection time, syringe residual dose, residual dose measurement time, data formation folder and other scanning information. After the scan is completed, the original data is reconstructed. After reconstruction, the image and data are processed using the image processing software PMOD, and the brain, heart, liver, kidney, bone (bone joint), muscle and other organs are outlined as regions of interest. After the outline is completed, the radioactivity value of the region of interest per unit volume is obtained, and the %ID / g value of each organ is calculated.

[0121] 3. Experimental results

[0122] Figure 6 A is the injection of U87 tumor-bearing mice 68 From the imaging images at 2.5 minutes, 27.5 minutes, 60 minutes, 120 minutes, and 180 minutes after Ga-FAPI-JNU, it can be observed that at 2.5 minutes, except for the tumor and metabolic organs kidney and bladder, the uptake of other tissues and organs was low, and a clearer tumor outline could be observed. By the imaging images at the 3rd hour, an even clearer tumor outline could be seen. After precursor blocking, it could be observed that the tumor uptake was significantly reduced, and no obvious uptake was observed in other tissues and organs. Figure 6 B in the figure shows the U87 tumor-bearing mice injected without blocking and after blocking 68 The quantitative results of tumor uptake after Ga-FAPI-JNU showed that the tumor uptake was (8.44±1.53%ID / g) after 3 hours without precursor blocking, while the tumor tissue uptake value after precursor blocking was (0.99±0.06%ID / g) (P<0.01), confirming that the tumor 68 The uptake of Ga-FAPI-JNU was specific.

[0123] Figure 7 Injection of U87 tumor-bearing mice 68 The drug distribution in the body at 1 hour and 3 hours after Ga-FAPI-JNU can be observed. 68Ga-FAPI-JNU is mainly enriched in tumor sites, while the uptake of non-target tissues and organs is at a low level. The uptake in tumor sites is significantly higher than that in muscles, bones, lungs and other organs or tissues (T / NT ratio can reach 6.75±1.70). In addition to tumor uptake, 68 Ga-FAPI-JNU is mainly distributed in the metabolic organs kidney and bladder.

[0124] Figure 8 Injection of U87 tumor-bearing mice 68 Ga-FAPI-JNU& 68 Comparison of the images 1 hour after Ga-FAPI-46, we can see the 68 Ga-FAPI-JNU comparison 68 Ga-FAPI-46 has a more obvious radioactive concentration.

[0125] Fig. 9 A is the injection of U87 tumor-bearing mice 68 Ga-FAPI-JNU& 68 Ga-FAPI-46 PET / CT images with the tumor as the region of interest to outline the difference in uptake of the two tracers in the same tumor model. Fig. 9 B in the equation is the target-to-body ratio (tumor muscle uptake ratio) of the two tracers. 68 The tumor uptake of Ga-FAPI-JNU at 1 hour was (SUV mean ,1.33±0.17), and 68 The tumor uptake of Ga-FAPI-46 was only (SUV mean ,0.16±0.03), tumor uptake 68 Ga-FAPI-JNU comparison 68 Ga-FAPI-46 is 8 times higher. 68 The tumor-to-muscle ratio of Ga-FAPI-JNU was (8.52±1.93) at 1 hour. 68 The tumor-muscle ratio of Ga-FAPI-46 was (4.67±1.10) at 1 hour. 68 Compared with Ga-FAPI-46, 68 Ga-FAPI-JNU has higher tumor uptake and accumulation and better target-to-target ratio.

[0126] In addition, the literature reported in "Zhao, L.; Niu, B.; Fang, J.; Pang, Y.; Li, S.; Xie, C.; Sun, L.; Zhang, X.; Guo, Z.; Lin, Q.; et al. Synthesis, Preclinical Evaluation, and a Pilot Clinical PET Imaging Study of (68) Ga-Labeled FAPI Dimer. J. Nucl. Med. 2022, 63(6), 862-868." 68 Ga-DOTA-2P(FAPI) 2 1-hour tumor uptake of SUV in the U87 tumor model mean is 0.42±0.03, 68 Ga-DOTA-4P(FAPI) 4 Tumor uptake of 1-hour SUV mean was 0.72±0.02, compared with these two probes. 68 Ga-FAPI-JNU had higher tumor uptake.

[0127] Experimental Example 5: Molecular Probe 68 Experimental study on the binding effect of Ga-FAPI-JNU and human tumor tissue

[0128] This experimental example provides the molecular probe prepared in Example 2 68 The experimental process of the combination effect of Ga-FAPI-JNU and human tumor tissue is as follows:

[0129] Step 1: After baking the serial sections of tumor tissue of human tumor tissue (provided by the Department of Pathology, Affiliated Hospital of Jiangnan University) in a 65°C oven for 2 hours, the sections were immersed in xylene for 3 times, each time for 10 minutes, and new xylene was replaced each time, and then the sections were immersed in ethanol for dehydration for 3 times, each time for 3 minutes, to obtain processed tissue sections;

[0130] Step 2: 68 Ga-FAPI-JNU was diluted with Tris-HCl buffer (purchased from Meilun Biotechnology Co., Ltd.) containing 1% (w / v, g / 100mL) bovine serum albumin (BSA, purchased from Feijing Biotechnology Co., Ltd.) to a concentration of 16 μCi / mL; the diluent was added dropwise to the treated tissue slices at a rate of 0.3 mL / slice, and incubated at room temperature (25°C) for 35 min to obtain bound tissue slices;

[0131] Step 3: First rinse the bound tissue slice with pure water for 30 seconds, then drop a Tris-HCl buffer solution containing 1% (w / v, g / 100mL) bovine serum albumin to the bound tissue slice, incubate at room temperature (25°C) for 1 minute, and then rinse the bound tissue slice with pure water for 30 seconds; repeat the above process 3 times to obtain a blocked tissue slice;

[0132] Step 4: After cleaning, wipe off the liquid around the sealed tissue slices, dry the sealed tissue slices with a hair dryer, and send them to the Jiangsu Institute of Atomic Medicine for exposure.

[0133] Unbound 68 Serial tumor tissue sections of Ga-FAPI-JNU were immunohistochemically stained with anti-fibroblast activation protein antibody (purchased from Abcam, ab207178) to verify the expression of fibroblast activation protein. Serial sections of tumor tissue were HE stained to verify the cell types that mainly expressed fibroblast activation protein in tumor tissue.

[0134] Fig.10 for 68 The results of Ga-FAPI-JNU autoradiography in human tumor tissues and the results of fibroblast activation protein immunohistochemical staining and HE staining. Fig.10 visible, 68 Ga-FAPI-JNU binds to tumor tissue in vitro, and the binding area is roughly consistent with the expression area of ​​fibroblast activation protein in the tissue. This result once again confirms that 68 The uptake of Ga-FAPI-JNU was correlated with fibroblast activation protein expression, and this result was also 68 Provides a basis for human body imaging of Ga-FAPI-JNU.

[0135] Example 3: A molecular probe targeting fibroblast activation protein 177 Lu-FAPI-JNU

[0136] This embodiment provides a molecular probe targeting fibroblast activation protein 177 Lu-FAPI-JNU, a molecular probe targeting the fibroblast activation protein 177 Lu-FAPI-JNU has the following structure:

[0137]

[0138] Example 4: Preparation of a molecular probe targeting fibroblast activation protein 177 Lu-FAPI-JNU method

[0139] This example provides a molecular probe targeting fibroblast activation protein as described in Example 3. 177 The preparation method of Lu-FAPI-JNU, the specific steps are as follows:

[0140] 1. Preparation of the precursor compound FAPI-JNU

[0141] The precursor compound FAPI-JNU was prepared according to the method of Example 2.

[0142] 2. Molecular probes 177 Preparation of Lu-FAPI-JNU

[0143] Step 1: Take 40 μg of precursor FAPI-JNU lyophilized powder and dissolve it in a reaction bottle with 20 μL of dimethyl sulfoxide to obtain a solution; add 0.03 mL of acetic acid / sodium acetate buffer solution (pH = 7.2, concentration 0.15 M) to the reaction bottle to obtain a mixed solution; take 370 MBq 177 LuCl 3 The solution (purchased from Chengdu Xinke Pharmaceutical Co., Ltd.) was added to the reaction bottle and mixed with the mixed solution to obtain a reaction solution; after adding acetic acid / sodium acetate buffer solution to the reaction bottle to adjust the pH value of the reaction solution to 4.0, the reaction bottle was placed in a 90° C. heater to react for 20 minutes to obtain a reaction product;

[0144] Step 2: Activate the Sep-Pak C18 column with 10 mL of anhydrous ethanol and 10 mL of sterile water for injection; after the reaction product is cooled to room temperature (25°C), extract the reaction product with a 10 mL syringe, pass the reaction product through a C18 solid phase extraction column, and rinse the C18 solid phase extraction column with 20 mL of sterile water for injection; after the rinsing is completed, rinse the C18 solid phase extraction column with 0.5 mL of 70% (v / v) ethanol solution to obtain the target product 177 Lu-FAPI-JNU; draw out the final product with a syringe 177 Lu-FAPI-JNU was filtered through a 0.22 μm sterile filter membrane to obtain a sterile 177 Lu-FAPI-JNU.

[0145] Using FAPI-JNU as a standard, the standard and 177 Lu-FAPI-JNU for quality control. Standards and 177 The HPLC quality control results of Lu-FAPI-JNU are as follows Fig.11 shown. 177 The retention time of Lu-FAPI-JNU was 11.43 min. The radioactive spectrum of the therapeutic molecular probe was integrated by area, and its radiochemical purity was found to be 97%.

[0146] Experimental Example 6: Molecular Probe 177 In vitro stability experiment of Lu-FAPI-JNU

[0147] This experimental example provides the molecular probe prepared in Example 4 177 In vitro stability test of Lu-FAPI-JNU, the experimental process is as follows:

[0148] Experiment 1: Take 200μCi 177 Lu-FAPI-JNU was mixed with 200 μL PBS buffer (pH=7.4, concentration 1×, purchased from Pronocell Life Science Co., Ltd.) by shaking and incubated at 37°C for 2 h or 72 h to obtain an incubation solution; the incubation solution was sampled and analyzed by Radio-HPLC. 177 Stability of Lu-FAPI-JNU in PBS buffer.

[0149] Experiment 2: Take 200μCi 177 Lu-FAPI-JNU was mixed with 200 μL fetal bovine serum by shaking and evenly incubated at 37°C for 2 h or 72 h to obtain an incubation solution; 30 μL of the incubation solution was mixed with 40 μL of acetonitrile and shaken for 5 s at room temperature (25°C) to precipitate the protein to obtain a treated incubation solution; the treated incubation solution was centrifuged at 12000 r / min for 5 min; after the centrifugation, the supernatant was taken and analyzed by Radio-HPLC 177 Stability of Lu-FAPI-JNU in fetal bovine serum.

[0150] Fig.12 for 177 Radio-HPLC results of Lu-FAPI-JNU in PBS buffer and fetal bovine serum at 2h and 72h. Fig.12 It can be seen that 177 After Lu-FAPI-JNU was incubated in PBS buffer and fetal bovine serum, the Radio-HPLC results still showed only one main radioactive peak. 177 Lu-FAPI-JNU has no obvious fragmentation and radionuclides 177 Lu ion shedding situation, it can be seen that 177 Lu-FAPI-JNU has good stability in vitro.

[0151] Experimental Example 7: Molecular Probe 177 Biodistribution experiment of Lu-FAPI-JNU

[0152] This experimental example provides the molecular probe prepared in Example 4 177 The biodistribution experiment of Lu-FAPI-JNU, the experimental process is as follows:

[0153] 1. Animal model establishment:

[0154] U87 cells (purchased from Pronocell Life Science Co., Ltd.) were cultured at 5×10 6 The inoculum size was 100 mL and inoculated into MEM medium (purchased from Pronocell Life Science Co., Ltd.) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (purchased from Meilun Biotechnology Co., Ltd.) and incubated at 37°C and 5% (v / v) CO 2 The cells were cultured in a sterile cell culture incubator; when the cell density reached 90%, the cells were collected and counted; after the counting was completed, the cells were resuspended to a concentration of 1×10 using 0.25% trypsin (purchased from Meilun Biotechnology Co., Ltd.). 6 Nude mice (purchased from Vital River) were inoculated with the bacterial suspension at a volume of 100 μL / mouse into the subcutaneous area under the axilla of the right forelimb of the nude mice. After the inoculation, the tumor volume grew to 100-200 mm. 3 The tumor-bearing mice can then be used for experiments.

[0155] 2. Biodistribution experiment in tumor-bearing mice

[0156] Tumor-bearing mice (purchased from Vitallife) were randomly divided into three groups (n=3). Each group of mice was injected with 30 μCi of 177 Lu-FAPI-JNU (solvent is 100 μL saline). After the injection, the mice were killed at 6 hours and 24 hours, and the blood, heart, liver, spleen, lung, kidney, stomach, intestine, pancreas, bone, muscle and tumor were weighed and counted using a gamma counter (γ-counter), and the results were finally calculated as %ID / g.

[0157] 3. Experimental results

[0158] Fig.13 U87 tumor-bearing mice at different time points 177 The in vitro distribution results of Lu-FAPI-JNU. Fig.13 It is known that injection 177 Within 24 hours after Lu-FAPI-JNU, the tumor was the main uptake tissue, while the radioactivity uptake in other non-target tissues was low. 177 Lu-FAPI-JNU has good tumor targeting and good biodistribution.

[0159] Experimental Example 8: Molecular Probe 177 SPECT imaging experiment of Lu-FAPI-JNU in tumor-bearing mice

[0160] This experimental example provides the molecular probe prepared in Example 4 177SPECT imaging experiment of Lu-FAPI-JNU in tumor-bearing mice. The experimental process is as follows:

[0161] 1. Animal model establishment:

[0162] U87 cells (purchased from Pronocell Life Science Co., Ltd.) were cultured at 5×10 6 The inoculum size was 100 mL and inoculated into MEM medium (purchased from Pronocell Life Science Co., Ltd.) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (purchased from Meilun Biotechnology Co., Ltd.) and incubated at 37°C and 5% (v / v) CO 2 The cells were cultured in a sterile cell culture incubator; when the cell density reached 90%, the cells were collected and counted; after the counting was completed, 0.25% trypsin (purchased from Meilun Biotechnology Co., Ltd.) was used to resuspend the cells to a concentration of 1×10 6 Nude mice (purchased from Vital River) were inoculated with the bacterial suspension at a volume of 100 μL / mouse into the subcutaneous area under the right forelimb of the nude mice to obtain tumor-bearing mice; after the inoculation, the tumor volume grew to 500-700 mm 3 The tumor-bearing mice can then be used for experiments.

[0163] 2. SPECT imaging of tumor-bearing mice

[0164] Each tumor-bearing mouse was weighed before administration. After weighing, the tumor-bearing mice were injected with 1 mCi of 177 Lu-FAPI-JNU / only 177 Lu-FAPI-JNU (solvent is 100 μL saline); after the injection, the tail vein was injected 177 Static SPECT / CT imaging of tumor-bearing mice was performed using a Micro SPECT / CT system {Mars (MSSC 101), PINGSENG Healthcare (Kunshan) Inc.} at 9, 12, and 26 hours after Lu-FAPI-JNU was applied. After scanning, the original SPECT data were reconstructed in the whole body area, and then the CT, SPECT images, and fused images were reconstructed using Avatar software (PINGSENG Healthcare (Kunshan) Inc.).

[0165] 3. Experimental results

[0166] Fig.14 U87 tumor-bearing mice were injected 177 SPECT image after Lu-FAPI-JNU. Fig.14 It is known that tumors are 177Lu-FAPI-JNU is mainly distributed in the kidney. 177 The main excretion organs of Lu-FAPI-JNU, other organs 177 Lu-FAPI-JNU has a lower distribution, which shows that 177 Lu-FAPI-JNU has good tumor targeting and biodistribution.

[0167] Experimental Example 9: Molecular Probe 177 Tumor therapy experiment of Lu-FAPI-JNU in tumor-bearing mice

[0168] This experimental example provides the molecular probe prepared in Example 4 177 The tumor treatment experiment of Lu-FAPI-JNU in tumor-bearing mice, the experimental process is as follows:

[0169] 1. Animal model establishment:

[0170] U87 cells (purchased from Pronocell Life Science Co., Ltd.) were cultured at 5×10 6 The inoculum size was 100 mL and inoculated into MEM medium (purchased from Pronocell Life Science Co., Ltd.) containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin (purchased from Meilun Biotechnology Co., Ltd.) and incubated at 37°C and 5% (v / v) CO 2 The cells were cultured in a sterile cell culture incubator; when the cell density reached 90%, the cells were collected and counted; after the counting was completed, 0.25% trypsin (purchased from Meilun Biotechnology Co., Ltd.) was used to resuspend the cells to a concentration of 1×10 6 Nude mice (purchased from Vital River) were inoculated with the cell suspension at a volume of 100 μL / mouse into the subcutaneous area under the right forelimb of the nude mice to obtain tumor-bearing mice; after the inoculation, the tumor volume grew to 70-100 mm 3 The tumor-bearing mice can then be used for experiments.

[0171] 2. Tumor treatment in tumor-bearing mice

[0172] The tumor-bearing mice were randomly divided into four groups, namely, a low-dose experimental group (n=5), a medium-dose experimental group (n=6), a high-dose experimental group (n=6) and a control group (n=6). The tumor-bearing mice in the high-dose experimental group were injected with 46 MBq / mouse via tail vein. 177 Lu-FAPI-JNU (solvent: 100 μL saline), medium-dose experimental group tumor-bearing mice were injected with 23 MBq / mouse via tail vein 177 Lu-FAPI-JNU (solvent: 100 μL saline), low-dose experimental group tumor-bearing mice were injected with 17 MBq / mouse via tail vein 177Lu-FAPI-JNU (solvent is 100 μL saline), the control group tumor-bearing mice were injected with the same volume of saline once through the tail vein. After the injection, the weight and tumor size of the tumor-bearing mice were monitored daily. 3 After the monitoring, the important organs of tumor-bearing mice were taken for HE staining.

[0173] Fig.15 A in the figure is the tumor growth curve of mice in each experimental group. Fig.15 B is the weight trend chart of mice in each experimental group. Fig.15 C in the figure is a visual picture of the tumors in each group of mice after the monitoring was completed. Fig.15 HE staining of important organs of mice in each group. Figure 15-16 It is known that injection 177 The tumor growth rates of mice in the three dose groups of Lu-FAPI-JNU were slower than those in the group injected with normal saline, and the inhibitory effect was dose-dependent. The larger the dose, the more obvious the inhibitory effect on tumor growth. During the treatment experiment, the body weight of mice in the two experimental groups injected with doses of 46MBq and 23MBq fluctuated slightly after injection, but the decrease did not exceed 0.5%, which was not statistically significant. After the monitoring was completed, the differences in tumor volume between the groups could be observed through direct visualization of the tumor dissection. Moreover, no obvious damage was observed at the microscopic level in the important organs of the three groups of experimental mice. In summary, 177 Lu-FAPI-JNU has a certain inhibitory effect on tumor growth, has potential tumor treatment effects, and the toxic side effects caused by radiation damage are controllable.

[0174] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A molecular probe for diagnosis and treatment targeting fibroblast activation protein, characterized in that: The fibroblast activation protein-targeted integrated molecular probe for diagnosis and treatment comprises a precursor compound and a radionuclide; the molecular formula of the precursor compound is Y-Glu-[X-PEG4]2-Lys-Z, wherein X is a fibroblast activation protein targeting group, Y is a chelating group DOTA, and Z is 4-(4-hydroxyphenyl)butyric acid; the radionuclide is complexed to the chelating group DOTA of the precursor compound; The structural formula of the precursor compound is as follows:

2. The molecular probe according to claim 1, wherein The radionuclides include 68 Ga, 64 Cu, 177 Lu, 90 Y or 111 In.

3. A kit for preparing the integrated diagnosis and treatment molecular probe according to claim 1 or 2, characterized in that: The components of the kit include a precursor compound and a radionuclide labeled compound; the molecular formula of the precursor compound is Y-Glu-[X-PEG4]2-Lys-Z, wherein X is a fibroblast activation protein targeting group, Y is a chelating group DOTA, and Z is 4-(4-hydroxyphenyl)butyric acid; the radionuclide is complexed to the chelating group DOTA of the precursor compound; The structural formula of the precursor compound is as follows:

4. The kit according to claim 3, characterized in that The radionuclide labeled compound comprises 68 GaCl3, 64 CuCl2, 177 LuCl3, 90 YCl3 or 111 InCl3.

5. A method for preparing the integrated diagnosis and treatment molecular probe according to claim 1 or 2, characterized in that: The method comprises: using the kit described in claim 3 or 4 to prepare the integrated diagnosis and treatment molecular probe described in claim 1 or 2.

6. The method according to claim 5, characterized in that The method comprises: dissolving a precursor compound in an organic solvent to obtain a dissolving solution; mixing the dissolving solution with a buffer solution, and adjusting the pH to 3.5 to 4.0 with the buffer solution to obtain a mixed solution; reacting the mixed solution at 80 to 100° C. for 10 to 20 minutes to obtain a reaction solution containing the integrated diagnosis and treatment molecular probe according to claim 1 or 2.

7. Use of the integrated diagnosis and treatment molecular probe according to claim 1 or 2 or the kit according to claim 3 or 4 in the preparation of fibroblast activation protein imaging agents, tumor imaging agents or anti-tumor drugs.

8. A fibroblast activation protein imaging agent, characterized in that: The components of the fibroblast activation protein imaging agent include the integrated diagnosis and treatment molecular probe according to claim 1 or 2.

9. A tumor imaging agent, characterized in that: The components of the tumor imaging agent include the integrated diagnosis and treatment molecular probe according to claim 1 or 2.

10. An anti-tumor drug, characterized in that: The components of the anti-tumor drug include the integrated diagnosis and treatment molecular probe according to claim 1 or 2.

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