Alphavbeta3 and PD-L1 double-target molecular probe and application thereof

Abstract

The invention relates to an alpha v beta 3 and PD-L1 double-target molecular probe and application thereof, and belongs to the technical field of nuclear medicine. The double-target molecular probe provided by the invention has the following advantages that the targeting property is excellent, alpha v beta 3 and PD-L1 in tumor tissues can be targeted at the same time, and compared with a single-target probe, the double-target molecular probe can more accurately position tumor or lesion areas and effectively reduce non-specific uptake possibly caused by the single-target probe; tumor heterogeneity can be overcome through a double-targeting strategy, missing detection or misdiagnosis caused by tumor marker expression difference of a single-target probe is avoided, and the tumor diagnosis efficiency is improved; tumor uptake and residence time is prolonged, and accumulation of the probe at a tumor part is ensured, so that the imaging effect is enhanced, and the diagnosis precision is improved; compared with a single-target probe, the double-target probe has the advantages that higher tumor uptake and clearer tumor positioning can be provided, and the accuracy and reliability of clinical diagnosis are further improved.

Description

Technical Field

[0001] The present invention relates to a dual-target molecular probe for α v β3 and PD-L1 and its application, belonging to the technical field of nuclear medicine. Background Art

[0002] Although single-target molecular probes have played an important role in clinical diagnosis and treatment, in some cases, there are higher requirements for imaging contrast and specificity of diseased tissues. Compared with single-target probes, dual-target molecular probes show better specific uptake in tumor tissues, and the specific interactions with two different targets make these dual-target probes exhibit stronger affinity and better sensitivity (see the literature: Dual-targeted molecular imaging of cancer. J. Nucl. Med. 2018; 59(3): 390-395.). Therefore, the application of dual-target molecular probes can improve the accuracy of tumor diagnosis.

[0003] Tumor angiogenesis is a key link in tumor progression, and integrin plays an important role in it, especially integrin α v β3, and the dysregulation of cell adhesion and signal transduction mediated by it is one of the pathogenesis of many diseases. Research shows that α v β3 is an important marker of tumor angiogenesis, with low expression in most normal cells and high expression in a variety of tumor cell lines (see the literature: A comparison of 18 F]AlF-and 68 Ga-labeled dual targeting heterodimer FAPI-RGD in malignant tumor: preclinical evaluation and pilot clinical PET / CT imaging. Eur. J. Nucl. Med. Mol. Imaging. 2024; 51(6): 1685-1697.). The high expression of α v β3 is closely related to the invasion and metastasis of tumors. Therefore, it has become an important target in tumor diagnosis and treatment. Accurately detecting the expression level of α v β3 has important clinical significance for the early diagnosis, treatment and prognosis evaluation of tumors.

[0004] Immunotherapy based on the PD-1 (Programmed cell death-1) / PD-L1 (Programmed cell death ligand-1) pathway is currently popular in the field of cancer immunotherapy. PD-L1 is a transmembrane protein mainly expressed on the surface of tumor cells, immune cells, and many other cell types. It regulates the body's immune response by inhibiting the proliferation of T cells and the production of Interferon-γ (IFN-γ). After PD-L1 binds to its receptor PD-1, it can promote tumor immune escape and inhibit the immune response. Therefore, accurately detecting the expression level of PD-L1 is more beneficial for precisely screening potential patients for immunotherapy, which can improve patient benefits (see the literature: High PD-1 / PD-L1 checkpoint interaction infers tumor selection and therapeutic sensitivity to anti-PD-1 / PD-L1 treatment. Cancer Res. 2020, 80(19): 4244-4257.).

[0005] In view of α v the key roles of β3 and PD-L1 in tumorigenesis and development, and α v β3 can regulate the expression of PD-L1 in the tumor microenvironment and is also one of the key components of the tumor immune escape mechanism (see the literature: α v β3-integrin regulates PD-L1 expression and is involved in cancer immune evasion. Proc. Natl. Acad. Sci. U.S.A. 2019, 116(40): 20141-20150.). Therefore, developing a dual-target molecular probe targeting α v β3 and PD-L1 molecules has relatively broad clinical application potential, and it is expected that the probe will show stronger affinity, thereby improving the sensitivity and accuracy of tumor diagnosis. At present, no dual-target molecular probes targeting α v β3 and PD-L1 have been reported. Summary of the Invention

[0006] To solve the above problems, the present invention provides a dual-target molecular probe targeting α v β3 and PD-L1, and the molecular probe has the following structure:

[0007]

[0008] Among them, R is a radionuclide-labeled group.

[0009] In one embodiment of the present invention, the radionuclide-labeled group is 68 Ga, 18 F]AlF, 64 Cu or 89 Zr.

[0010] In one embodiment of the present invention, when the radionuclide-labeled group is 68 Ga, the molecular probe has the following structure:

[0011]

[0012] In one embodiment of the present invention, when the radionuclide-labeled group is 18 F]AlF, the molecular probe has the following structure:

[0013]

[0014] In one embodiment of the present invention, the labeling precursor of the molecular probe has the following structure:

[0015]

[0016] The present invention also provides a method for preparing the above-mentioned molecular probe, the method comprising: dissolving the compound IMB-RGD and Boc-5-aminopentanoic NHS in an organic solvent to obtain a solution; adjusting the pH of the solution to alkaline with N,N-diisopropylethylamine, and then reacting under the protection of nitrogen to obtain a reaction solution A; concentrating, ether precipitating, centrifuging and drying the reaction solution A to obtain a reaction product; dissolving the reaction product in a mixed solution of trifluoroacetic acid and acetonitrile for reaction to obtain a reaction solution B; concentrating, ether precipitating, centrifuging and drying the reaction solution B to obtain the compound IMB-RGD-1;

[0017] Dissolving the compound IMB-RGD-1 and NOTA-NHS in an organic solvent to obtain a solution; adjusting the pH of the solution to alkaline with N,N-diisopropylethylamine, and then reacting under the protection of nitrogen to obtain a reaction solution; concentrating, ether precipitating, centrifuging, drying and purifying the reaction solution to obtain the labeling precursor NOTA-IMB-RGD of the molecular probe; performing radionuclide labeling on the labeling precursor NOTA-IMB-RGD of the molecular probe to obtain the molecular probe;

[0018] The compound IMB-RGD has the following structure:

[0019]

[0020] In one embodiment of the present invention, the preparation method of the compound IMB-RGD comprises: dissolving the compound IMB and FmocNH-PEG2-CH2COONHS in an organic solvent to obtain a solution; adjusting the pH of the solution to be alkaline with N,N-diisopropylethylamine, and then reacting under the protection of nitrogen to obtain a reaction solution A; concentrating, ether-precipitating, centrifuging, and drying the reaction solution A to obtain a reaction product; dissolving the reaction product in an organic solvent to obtain a mixed solution; adding piperidine to the mixed solution and reacting to obtain a reaction solution B; adjusting the pH of the reaction solution B to be neutral with an acidic solution, and then concentrating, ether-precipitating, centrifuging, and drying to obtain the compound IMB-1;

[0021] Dissolving the compound IMB-1 and Bis(2,5-dioxopyrrolidin-1-yl)succinate in an organic solvent to obtain a solution; adjusting the pH of the solution to be alkaline with N,N-diisopropylethylamine, and then reacting under the protection of nitrogen to obtain a reaction solution; concentrating, ether-precipitating, centrifuging, and drying the reaction solution to obtain the compound IMB-2;

[0022] Dissolving IMB-2 and c(RGDfk) in an organic solvent to obtain a solution; adjusting the pH of the solution to be alkaline with N,N-diisopropylethylamine, and then reacting under the protection of nitrogen to obtain a reaction solution A; concentrating, ether-precipitating, centrifuging, and drying the reaction solution A to obtain a reaction product; dissolving the reaction product in a mixed solution of trifluoroacetic acid, triisopropylsilane, and acetonitrile and reacting to obtain a reaction solution B; concentrating, ether-precipitating, centrifuging, drying, and purifying the reaction solution B to obtain the compound IMB-RGD;

[0023] The compound IMB has the following structure:

[0024]

[0025] The present invention also provides the application of the above molecular probe in the preparation of an integrin α v β3 and / or PD-L1 imaging agent.

[0026] The present invention also provides an integrin α v β3 and / or PD-L1 imaging agent, and the components of the imaging agent include the above molecular probe.

[0027] The present invention also provides the application of the above molecular probe in the preparation of a tumor imaging agent.

[0028] In one embodiment of the present invention, the tumor includes integrin α vβ3 and / or PD-L1 positive tumors; the integrin α v β3 and / or PD-L1 positive tumors include gliomas, malignant melanomas, colon cancers, lung cancers, and / or triple-negative breast cancers.

[0029] The present invention also provides a tumor imaging agent, and the composition of the imaging agent contains the above-mentioned molecular probe.

[0030] In one embodiment of the present invention, the tumor includes integrin α v β3 and / or PD-L1 positive tumors; the integrin α v β3 and / or PD-L1 positive tumors include gliomas, malignant melanomas, colon cancers, lung cancers, and / or triple-negative breast cancers.

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

[0032] The present invention provides a dual-target molecular probe for α v β3 and PD-L1, and this molecular probe has the following advantages:

[0033] First, this molecular probe has excellent targeting ability and can simultaneously target α v β3 and PD-L1 in tumor tissues. Compared with single-target probes, it can more accurately locate tumor or lesion areas and effectively reduce non-specific uptake that may be caused by single-target probes;

[0034] Second, this molecular probe can overcome tumor heterogeneity through a dual-targeting strategy, avoid missed detection or misdiagnosis caused by differences in tumor marker expression of single-target probes, and improve tumor diagnosis efficiency;

[0035] Third, the tumor uptake and retention time of this molecular probe are significantly improved, which can ensure the accumulation of the probe at the tumor site, thereby enhancing the imaging effect and improving the diagnostic accuracy;

[0036] Fourth, the imaging effect of this molecular probe is significantly improved. Compared with single-target probes, dual-target probes can provide higher tumor uptake and clearer tumor localization, further improving the accuracy and reliability of clinical diagnosis. Therefore, the molecular probe of the present invention can dynamically and real-time monitor the changes in the levels of α v β3 and PD-L1 in tumor cells through PET imaging technology, thereby significantly improving the sensitivity and accuracy of tumor diagnosis.

[0037] Furthermore, the radionuclide labeling group is 68 Ga and 18 F]AlF; both of these nuclides are radionuclides with short half-lives and release less radiation per unit time, further improving safety. Brief Description of the Drawings

[0038] Figure 1 : HPLC analysis chart of the compound NOTA-IMB-RGD.

[0039] Figure 2 : Electrospray mass spectrometry analysis chart of the compound NOTA-IMB-RGD.

[0040] Figure 3 : 68 Radioactive HPLC chart of the reaction solution before and after labeling with [

[0041] Figure 4 : 68 Stability HPLC analysis of [

[0042] Figure 5 : 68 Stability HPLC analysis of [

[0043] Figure 6 : 68 Cell uptake experiments of [

[0044] Figure 7 : 68 Cell uptake experiments of [

[0045] Figure 8 : 68 Results of pharmacokinetic analysis of [

[0046] Figure 9 : 68 microPET imaging results of [

[0047] Figure 10 : 68 Tumor and muscle uptake of [

[0048] Figure 11 :68 Biodistribution results of Ga]Ga-NOTA-IMB-RGD in U87, A375-hPD-L1, A375, B16-F10, MC38, and LLC tumor-bearing mice.

[0049] Figure 12 : 18 Radioactive HPLC chromatograms of the reaction solution before and after purification of F]AlF-NOTA-IMB-RGD.

[0050] Figure 13 : 18 HPLC analysis of the stability of F]AlF-NOTA-IMB-RGD incubated in PBS for 1, 2, and 4 hours.

[0051] Figure 14 : 18 HPLC analysis of the stability of F]AlF-NOTA-IMB-RGD incubated in FBS for 1, 2, and 4 hours.

[0052] Figure 15 : 18 Cell uptake experiments of F]AlF-NOTA-IMB-RGD in U87, A375, and A375-hPD-L1 cells.

[0053] Figure 16 : 18 Cell uptake experiments of F]AlF-NOTA-IMB-RGD in B16-F10, MC38, and LLC cells.

[0054] Figure 17 : 18 Results of the pharmacokinetic analysis of F]AlF-NOTA-IMB-RGD in mice.

[0055] Figure 18 : 18 MicroPET imaging results of F]AlF-NOTA-IMB-RGD in U87, A375-hPD-L1, A375, B16-F10, MC38, and LLC tumor-bearing mice.

[0056] Figure 19 : 18 Tumor and muscle uptake of F]AlF-NOTA-IMB-RGD in U87, A375-hPD-L1, A375, B16-F10, MC38, and LLC tumor-bearing mice.

[0057] Figure 20 : 18Biodistribution results of [¹⁸F]AlF-NOTA-IMB-RGD in U87, A375-hPD-L1, A375, B16-F10, MC38, and LLC tumor-bearing mice. Detailed implementation mode

[0058] The following examples are provided to better understand the present invention further. They are not limited to the described optimal implementation mode, and do not limit the content and protection scope of the present invention. Any product that is the same as 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.

[0059] For those not specifying specific experimental steps or conditions in the following examples, the operations or conditions of the conventional experimental steps described in the literature in this field can be followed. For reagents or instruments without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0060] Example 1: A dual-target molecular probe for α v β3 and PD-L1 68 [¹⁸Ga]Ga-NOTA-IMB-RGD

[0061] This example provides a dual-target molecular probe for α v β3 and PD-L1 68 [¹⁸Ga]Ga-NOTA-IMB-RGD. The dual-target molecular probe for α v β3 and PD-L1 68 [¹⁸Ga]Ga-NOTA-IMB-RGD has the following structure:

[0062]

[0063] Example 2: A method for preparing a dual-target molecular probe for α v β3 and PD-L1 68 [¹⁸Ga]Ga-NOTA-IMB-RGD

[0064] This example provides a method for preparing the dual-target molecular probe for α v β3 and PD-L1 described in Example 1 68 [¹⁸Ga]Ga-NOTA-IMB-RGD. The specific steps are as follows:

[0065] Step 1: Rinse the fritted funnel twice with dichloromethane, drain it, add 2-chlorotrityl chloride resin (loading amount: 1.106 mmol / g, 361.6 mg) to the drained fritted funnel, and add 10 mL of dichloromethane to soak and swell the 2-chlorotrityl chloride resin. After soaking and swelling for 10 min, drain it;

[0066] Step 2: Add Fmoc-L-leucine (132.5 mg, 0.375 mmol) to the sintered funnel obtained in Step 1, and dissolve it with 10 mL of ultradry DMF (N,N-dimethylformamide) to obtain a solution. After adding DIPEA (N,N-diisopropylethylamine) (329.92 μL, 2 mmol) to the solution to adjust the pH of the solution to 8, shake the solution at 25 °C for 3 h. After the shaking is completed, dry the solvent by suction. Add a mixed solution of 10 mL of DMF / CH3OH / DIPEA (DMF / CH3OH / DIPEA = 17:2:1, v / v / v) to the sintered funnel to wash the filter cake, shake (250 rpm) for 10 min and then filter by suction, and repeat the operation once to remove the unreacted amino acid. Add 10 mL of DMF (HPLC grade) to the sintered funnel to wash the filter cake, shake for 2 min and then filter by suction, and repeat the operation three times to purify the product. Add 10 mL of a DMF solution containing 20% (v / v) piperidine to the sintered funnel, shake for 10 min and then filter by suction, and repeat the operation three times to remove the Fmoc protecting group on the amino acid. Add 10 mL of DMF (HPLC grade) to the sintered funnel to wash the filter cake five times to wash away the excess piperidine. After the washing is completed, dry the solvent by suction, take a sample for Kaiser test, and the color of the reagent shows dark purple, indicating that the Fmoc group has been removed at this time, exposing the amino group, and the next amino acid can be linked;

[0067] Step 3: On the basis of Step 2, replace Fmoc-L-leucine (132.5 mg, 0.375 mmol) successively with N-fluorenylmethoxycarbonyl-L-alanine (117 mg, 0.375 mmol), N-(fluorenylmethoxycarbonyl)-L-serine tert-butyl ester (144 mg, 0.375 mmol), Fmoc-L-isoleucine (132.5 mg, 0.375 mmol), Fmoc-N-trityl-L-asparagine (224 mg, 0.375 mmol), Fmoc-L-proline (126.5 mg, 0.375 mmol), N-fluorenylmethoxycarbonyl-L-alanine (117 mg, 0.375 mmol), Fmoc-L-leucine (132.5 mg, 0.375 mmol), N-(fluorenylmethoxycarbonyl)-L-serine tert-butyl ester (144 mg, 0.375 mmol), Fmoc-S-trityl-L-cysteine (219.6 mg, 0.375 mmol), N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine (149 mg, 0.375 mmol), Fmoc-L-leucine (132.5 mg, 0.375 mmol), tert-butoxycarbonyl-fluorenylmethoxycarbonyl-lysine (175.7 mg, 0.375 mmol). While replacing, additionally add HBTU (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) (379 mg, 1 mmol), and repeat the operation of Step 2 to obtain the polypeptide chain Ac-KLTCSLAPNIISAL-OH;

[0068] The polypeptide chain Ac-KLTCSLAPNIISAL-OH has the structure shown below:

[0069]

[0070] Step 4: Add 10 mL of a CH2Cl2 solution containing 1% (v / v) TFA (trifluoroacetic acid) to the fritted funnel obtained in Step 3 to obtain a mixed solution; shake the mixed solution at 25 °C for 10 min. After shaking, filter out the filtrate with the compound IMB. Repeat this operation until the 2-chlorotrityl chloride resin turns wine red and does not fade; remove the solvent from the collected filtrate using a rotary evaporator, precipitate with cold diethyl ether (4 °C), transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant; take the precipitate and dry it to obtain the compound IMB (197 mg, yield 28%);

[0071] The compound IMB has the structure shown below:

[0072]

[0073] Step 5: Dissolve compound IMB (190 mg, 0.0865 mmol) and FmocNH-PEG2-CH2COONHS (50.08 mg, 0.1038 mmol) in 200 μL of DMF to obtain a solution; add DIPEA (50 μL, 0.28 mmol) to the solution to adjust the pH of the solution to 8, and then stir at 25 °C and 150 rpm for 3 h under the protection of nitrogen to obtain reaction solution A; spin-dry reaction solution A with an oil pump, precipitate with cold ether (4 °C), ultrasonicate at 25 °C for 30 s, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation); take the precipitate and dry it to obtain the reaction product; dissolve the reaction product in 160 μL of DMF to obtain a mixture; add 40 μL of piperidine to the mixture, and then stir at 25 °C and 150 rpm for 20 min to obtain reaction solution B; adjust the pH of reaction solution B to neutral (pH = 7) with 4 M hydrochloric acid in an ice bath, remove piperidine and water with a rotary evaporator, precipitate with cold ether (4 °C), ultrasonicate at 25 °C for 30 s, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation); take the precipitate and dry it to obtain compound IMB-1 (115 mg, yield 57%);

[0074] Compound IMB-1 has the structure shown below:

[0075]

[0076] Step 6: Dissolve compound IMB-1 (112 mg, 0.0479 mmol) and Bis(2,5-dioxopyrrolidin-1-yl)succinate (18 mg, 0.0575 mmol) in 200 μL of DMF to obtain a solution; add DIPEA (50 μL, 0.28 mmol) to the solution to adjust the pH of the solution to 8, and then stir at 25 °C and 150 rpm for 3 h under the protection of nitrogen to obtain the reaction solution; spin-dry the reaction solution with an oil pump, precipitate with cold ether (4 °C), ultrasonicate at 25 °C for 30 s, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation); take the precipitate and dry it to obtain compound IMB-2 (85 mg, yield 70%);

[0077] Compound IMB-2 has the structure shown below:

[0078]

[0079] Step 7: Dissolve compound IMB-2 (76 mg, 0.0300 mmol) and c(RGDfK) (15 mg, 0.0250 mmol) in 200 μL of DMF to obtain a solution. After adding DIPEA (25 μL, 0.14 mmol) to adjust the pH of the solution to 8, stir the solution at 25 °C and 150 rpm for 3 h under the protection of nitrogen to obtain reaction solution A. Spin-dry reaction solution A with an oil pump, precipitate with cold ether (4 °C), ultrasonicate for 30 s at 25 °C, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation). Take the precipitate and dry it to obtain the reaction product. Dissolve the reaction product in a mixed liquid of 2 mL of TFA, 80 μL of acetonitrile, and 80 μL of Tips (triisopropylsilane), and stir at 25 °C and 150 rpm for 2 h to obtain reaction solution B. Remove the organic solvent from reaction solution B with a rotary evaporator, precipitate with cold ether (4 °C), transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant. Take the precipitate and dry it to obtain compound IMB-RGD (40 mg, yield 58%);

[0080] The compound IMB-RGD has the structure shown below:

[0081]

[0082] Step 8: Dissolve compound IMB-RGD (35 mg, 0.01270 mmol) and Boc-5-aminopentanoic NHS (4.8 mg, 0.0153 mmol) in 200 μL of DMF to obtain a solution. After adding DIPEA (25 μL, 0.14 mmol) to adjust the pH of the solution to 8, stir the solution at 25 °C and 150 rpm for 3 h under the protection of nitrogen to obtain reaction solution A. Spin-dry reaction solution A with an oil pump, precipitate with cold ether (4 °C), ultrasonicate for 30 s at 25 °C, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation). Take the precipitate and dry it to obtain the reaction product. Dissolve the reaction product in a mixed liquid of 2 mL of TFA and 80 μL of acetonitrile, and stir at 25 °C and 150 rpm for 2 h to obtain reaction solution B. Remove the organic solvent from reaction solution B with a rotary evaporator, precipitate with cold ether (4 °C), transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant. Take the precipitate and dry it to obtain compound IMB-RGD-1 (16 mg, yield 55%);

[0083] The compound IMB-RGD-1 has the structure shown below:

[0084]

[0085] Step 9: Dissolve compound IMB-RGD-1 (15 mg, 0.0063 mmol) and NOTA-NHS (10 mg, 0.025 mmol) in 200 μL of DMF to obtain a solution; add DIPEA (10 μL, 0.056 mmol) to the solution to adjust the pH of the solution to 8, and then stir the solution at 25 °C and 150 rpm for 3 h under the protection of nitrogen to obtain a reaction solution; spin-dry the reaction solution with an oil pump, precipitate with cold ether (4 °C), sonicate at 25 °C for 30 s, transfer to a 50 mL centrifuge tube, and centrifuge to remove the supernatant (place at -20 °C for 10 min before centrifugation); take the precipitate and dry it, and then purify the reaction solution using semi-preparative HPLC to obtain the labeling precursor NOTA-IMB-RGD of the α v β3 and PD-L1 targeted molecular probe;

[0086] The labeling precursor NOTA-IMB-RGD has the following structure:

[0087]

[0088] Step 10: Use 1.0 mL of 0.05 M HCl to elute 68 Ge / 68 Ga generator (ITG) to obtain an eluate; mix the eluate with 0.25 M NaOAc buffer to adjust the pH value to 4.0 to obtain a mixture; directly transfer the mixture to a 5 mL centrifuge tube containing 20 μg of the labeling precursor NOTA-IMB of the PD-L1 targeted molecular probe, mix well, and incubate the mixture in an oil bath at 37 °C for 15 min to obtain the molecular probe 68 Ga]Ga-NOTA-IMB-RGD; analyze the product by radio-HPLC. 68 Perform ESI-MS analysis on the labeling precursor NOTA-IMB-RGD using an electrospray ionization source, and perform HPLC detection on the labeling precursor NOTA-IMB-RGD using Waterse2695. The analysis and detection results are shown in

[0089] . Use a POMONova radioactive detector to perform radioactive HPLC detection on the reaction solutions before and after the labeling of Figures 1 - 2 Ga]Ga-NOTA-IMB-RGD. The detection results are shown in 68 Figure 3 . Calculate the radiochemical purity (RCP) of 68 Ga]Ga-NOTA-IMB-RGD by the peak area of the radioactive product / total peak area. The calculation result is: the radiochemical purity of the labeled product is higher than 95%.

[0090] ​In the above radio-high performance liquid chromatography, 68 The retention time of [¹⁷⁷Lu]Lu-NOTA-IMB-RGD was 17.7 min, which was similar to the peak time of the labeled precursor NOTA-IMB-RGD (17.8 min). After attenuation correction, the final radiochemical yield (RCY) of [¹⁷⁷Lu]NOTA-IMB was calculated by radioactive HPLC detection. The calculated result was >95%. The molar activity (Am) = product radioactivity / M (molar mass of the precursor) was used to calculate 68 the molar activity of [¹⁷⁷Lu]Lu-NOTA-IMB-RGD, and the calculated result was 17.82 ± 2.26 GBq / μmol. 68 Ga]Ga-NOTA-IMB-RGD's molar activity was calculated to be 17.82 ± 2.26 GBq / μmol.

[0091] Example 3: A dual-target molecular probe for α v β3 and PD-L1 18 [¹⁸F]AlF-NOTA-IMB-RGD

[0092] This example provides a dual-target molecular probe for α v β3 and PD-L1 18 [¹⁸F]AlF-NOTA-IMB-RGD. The dual-target molecular probe for α v β3 and PD-L1 18 [¹⁸F]AlF-NOTA-IMB-RGD has the following structure:

[0093]

[0094] Example 4: A method for preparing a dual-target molecular probe for α v β3 and PD-L1 18 [¹⁸F]AlF-NOTA-IMB-RGD

[0095] This example provides a method for preparing the α v β3 and PD-L1-targeted molecular probe 18 [¹⁸F]AlF-NOTA-IMB-RGD described in Example 1. The specific steps are as follows:

[0096] Mix aluminum chloride (6 μL, 2 mM), glacial acetic acid (10 μL, 2 mM) and acetonitrile (544 μL, 2 mM) to obtain a mixed solution; add 60 μg of the labeled precursor NOTA-IMB-RGD of the PD-L1-targeted molecular probe prepared in Example 2 to the mixed solution to obtain a mixture; place the mixture in 100 μL of target water (by bombarding a silver cyclotron target with 30 MeV protons, which is rich in 98% of l8In the target water produced by [O] water, it was heated at 100 °C for 10 min to obtain a reaction solution; after the reaction solution was cooled to 25 °C, it was diluted with 30 mL of deionized water to obtain a dilution solution; after the dilution solution was purified by a C18 column, it was first washed with 30 mL of water to remove impurities, and then eluted with 0.3 mL of ethanol containing 10 mM HCl to obtain a molecular probe 18 F]AlF-NOTA-IMB-RGD.

[0097] Using a POMO Nova radioactive detector to perform radioactive HPLC detection on the reaction solution before and after the 18 F]AlF-NOTA-IMB-RGD labeling, and the detection results are shown in Figure 12 . Calculate the 18 RCP of F]AlF-NOTA-IMB-RGD by the radioactive product peak area / total peak area, and the RCP of the labeled product > 98%.

[0098] In the above radio high performance liquid chromatography, 18 the retention time of F]AlF-NOTA-IMB-RGD is 18 min, which is close to the peak time of the labeling precursor NOTA-IMB (17.8 min). After attenuation correction, calculate the 18 final RCY of F]AlF-NOTA-IMB-RGD by radio-HPLC detection, and the calculation result is > 95%. Calculate the 18 molar activity of F]AlF-NOTA-IMB-RGD by molar activity (Am) = product radioactivity / M (molar mass of the precursor), and the calculation result is 18.39 ± 1.85 GBq / μmol.

[0099] Experimental Example 1: α v In vitro stability experiment of the αβ3 and PD-L1 dual-target molecular probe

[0100] This experimental example provides an in vitro stability experiment of the α v β3 and PD-L1 dual-target molecular probe, and the specific process is as follows:

[0101] Experiment 1: The α v β3 and PD-L1 dual-target molecular probe 68 Ga]Ga-NOTA-IMB-RGD prepared in Example 2 and the α v β3 and PD-L1 dual-target molecular probe 18F]AlF-NOTA-IMB-RGD was mixed with PBS buffer (pH 7.4, 0.01 M) respectively to obtain a mixed solution; the mixed solution was incubated at 37 °C for 1, 2, and 4 h; after the incubation, the incubation solution was taken for radio-HPLC analysis using a POMO Nova radioactive detector, and the analysis results are shown in Figure 4 and Figure 13 .

[0102] Experiment 2: The α v β3 and PD-L1 dual-target molecular probe 68 Ga]Ga-NOTA-IMB-RGD and the α v β3 and PD-L1 dual-target molecular probe 18 F]AlF-NOTA-IMB-RGD prepared in Example 4 were mixed with FBS (Biological Industries, Israel) respectively to obtain a mixed solution; the mixed solution was incubated at 37 °C for 1, 2, and 4 h; after the incubation, acetonitrile was added for precipitation, and the serum and protein were separated by high-speed centrifugation at 12000 g for 5 min. The supernatant was taken for radio-HPLC analysis using a POMO Nova radioactive detector, and the analysis results are shown in Figure 5 and Figure 14 .

[0103] It can be seen from Figures 4 - 5 that the proportion of the main peak in the HPLC chromatogram of the α v β3 and PD-L1 dual-target molecular probe 68 Ga]Ga-NOTA-IMB-RGD incubated in FBS and PBS at 37 °C for 4 h is greater than 85%, indicating good stability. It can be seen from Figures 13 - 14 that the proportion of the main peak in the HPLC chromatogram of the α v β3 and PD-L1 dual-target molecular probe 18 F]AlF-NOTA-IMB-RGD incubated in FBS and PBS at 37 °C for 4 h is greater than 95%, indicating good stability.

[0104] Experimental Example 2: Lipid-water partition coefficient experiment of the α v β3 and PD-L1 dual-target molecular probe

[0105] This experimental example provides a lipid-water partition coefficient experiment of the α v β3 and PD-L1 dual-target molecular probe, and the specific process is as follows:

[0106] Take three centrifuge tubes, add 1 mL of deionized water and 1 mL of n-octanol to each centrifuge tube, and then add the molecular probe prepared in Example 2 68Ga]Ga-NOTA-IMB-RGD (25 μCi) was used to obtain a mixed solution. After the mixed solution was shaken for 1 min, it was centrifuged at 4000 g for 5 min at high speed to break the emulsion and separate the two phases. 500 μL of the n-octanol phase and the aqueous phase were taken into a radioimmunoassay tube, and then the radioactivity of the n-octanol phase and the aqueous phase was detected using a γ counter and Log P was calculated (Log P = LogC o / C w ), where C o represents the radioactive dose of 68 Ga]Ga-NOTA-IMB-RGD in the n-octanol phase, and C w represents the radioactive dose of 68 Ga]Ga-NOTA-IMB-RGD in the aqueous phase. The average value of three groups of data was taken as the value of the lipid-water partition coefficient, and the result was expressed as the mean ± standard deviation. The same method was used to detect the lipid-water partition coefficient of the α v β3 and PD-L1 dual-target molecular probe 18 F]AlF-NOTA-IMB-RGD prepared in Example 4.

[0107] The experimentally measured lipid-water partition coefficients of the α v β3 and PD-L1 dual-target molecular probe 68 Ga]Ga-NOTA-IMB-RGD and 18 F]AlF-NOTA-IMB-RGD were -2.09 ± 0.05 and -1.33 ± 0.02 respectively, which indicates that 68 Ga]Ga-NOTA-IMB-RGD and 18 F]AlF-NOTA-IMB-RGD are both water-soluble and have the advantage of being excreted from the kidneys and reducing liver metabolism in terms of metabolism.

[0108] Experimental Example 3: Cellular uptake experiment of the α v β3 and PD-L1 dual-target molecular probe

[0109] This experimental example provides a cellular uptake experiment of the α v β3 and PD-L1 dual-target molecular probe, and the specific process is as follows:

[0110] 1 × 10 6 A375-hPD-L1 cells (α v β3 and PD-L1 positive cells, provided by Suzhou Zhinuclear Biopharmaceuticals Co., Ltd.), U87 cells (α v β3 and PD-L1 positive cells, purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and A375 cells (PD-L1 negative cells, α vβ3-positive cells, provided by Suzhou Zhinuclear Biopharmaceutical Co., Ltd.) were respectively placed in radioimmunoassay tubes and co-incubated with 68 Ga]Ga-NOTA-IMB-RGD (1 μCi / well). During co-incubation, A375-hPD-L1, U87 and A375 cells were cultured in serum-free DMEM medium (purchased from BI) in a 37 °C water bath for 30 min, 60 min, 90 min, 120 min, 240 min, and 4 parallel groups were set up. After co-incubation, 500 μL of cold (4 °C) PBS buffer (pH = 7.4, 0.01 M) was added to each tube, and centrifuged at 4000 r / min for 5 min. After centrifugation, the above operation was repeated once, and the CPM value of the sample was detected by a gamma counter. After the collected cells were lysed with RIPA lysis buffer, the cell protein was quantified using a BCA protein quantification kit. The cell uptake results were expressed as the ratio of CPM in each milligram of cell protein to the CPM of the total dose (%AD / mg), and the detection results are shown in Figure 6 . In addition, B16-F10 cells (α v β3 and PD-L1 positive cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai), MC38 cells (α v β3 and PD-L1 positive cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai) and LLC cells (α v β3 and PD-L1 negative cells, purchased from the Cell Bank of the Chinese Academy of Sciences, Shanghai) were placed in radioimmunoassay tubes and co-incubated with 68 Ga]Ga-NOTA-IMB-RGD (1 μCi / well). The experimental method was the same as that for A375-hPD-L1, U87 and A375 cells, and the detection results are shown in Figure 7 . Using the same method to detect the α v β3 and PD-L1 dual-target molecular probe 18 F]AlF-NOTA-IMB-RGD prepared in Example 4, the detection results are shown in Figures 15 - 16 .

[0111] As Figure 6 shown, the uptake of the tracer by A375-hPD-L1 and U87 cells gradually increased, and the maximum uptake amounts were 30.27 ± 0.74% AD / mg at 90 min and 27.50 ± 1.63% AD / mg at 120 min, respectively. Compared with the uptake of the tracer by A375-hPD-L1 and U87 cells, the uptake of the tracer by A375 cells within 2 h was significantly different. As Figure 7It was shown that the uptake of the tracer by MC38 and B16-F10 cells gradually increased, and the maximum uptakes were 37.71 ± 1.27% ID / mg at 90 min and 51.34 ± 1.11% ID / mg at 90 min, respectively. Compared with the uptake of the tracer by MC38 and B16-F10 cells, there were significant differences in the uptake of the tracer by LLC cells within 2 h. This indicated that 68 the uptake of [68Ga]Ga-NOTA-IMB-RGD in double-positive cells was higher than that in single-positive cells and double-negative cells, and it was specific for α v β3 and PD-L1 at the cellular level. As Figure 15 shown, the uptake of the tracer by U87 cells gradually increased, and the maximum uptake at 4 h was 26.06 ± 0.71% ID / mg. The uptake of the tracer by A375-hPD-L1 cells remained above 25.33% ID / mg within 4 h. Compared with the uptake of the tracer by U87 and A375-hPD-L1 cells, the uptake of the tracer by A375 cells was lower within 4 h, with significant differences. As Figure 16 shown, the uptake of the tracer by MC38 and B16-F10 cells gradually increased, and the maximum uptakes at 4 h were 77.75 ± 4.57% ID / mg and 77.04 ± 6.26% ID / mg, respectively. Compared with the uptake of the tracer by MC38 and B16-F10 cells, the uptake of the tracer by LLC cells was lower within 4 h, with significant differences. This indicated that 18 the uptake of [18F]AlF-NOTA-IMB-RGD in double-positive cells was higher than that in single-positive cells and double-negative cells, and it was specific for α v β3 and PD-L1.

[0112] Experimental Example 4: Pharmacokinetic analysis experiment of the α v β3 and PD-L1 dual-target molecular probe in mice

[0113] This experimental example provided a pharmacokinetic analysis experiment of the α v β3 and PD-L1 dual-target molecular probe. The specific process was as follows:

[0114] Female ICR mice (5 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) were intravenously injected with 200 μCi of the molecular probe prepared in Example 2 dissolved in 100 μL of physiological saline through the tail vein 68Ga]Ga-NOTA-IMB-RGD; After the probe injection, blood was taken from the tail vein of mice at different time points (1, 3, 5, 7, 10, 15, 20, 30, 45, 60, 90, 120 min). Equal volume of solution was taken as reference, and the CPM value was measured using a gamma counter. The obtained results were subjected to data fitting analysis in DAS 2.1 software; In the blood concentration analysis, with time (min) as the abscissa and dose absorption ratio (ID% / g) as the ordinate, a clearance curve with linear correlation (r 2 >0.95) was plotted, and the pharmacokinetic parameters were calculated, as shown in Figure 8 . Using the same method, the α v β3 and PD-L1 dual-target molecular probe 18 F]AlF-NOTA-IMB-RGD prepared in Example 4 was detected, and the detection results are shown in Figure 17 .

[0115] As Figure 8 shown, 68 Ga]Ga-NOTA-IMB-RGD showed linear kinetics in the metabolic process in healthy mice, conforming to the two-compartment model. The distribution half-life (t 1 / 2α ) was on average 1.41 min, and the elimination half-life (t 1 / 2β ) was on average 63.46 min. As Figure 17 shown, 18 the distribution half-life (t 1 / 2α ) of 1 / 2β F]AlF-NOTA-IMB-RGD was on average 1.14 min, and the elimination half-life (t 1 / 2β ) was on average 61.64 min. This indicates that these two probes can be rapidly removed from the blood.

[0116] Experimental Example 5: Mouse PET imaging experiment of α v β3 and PD-L1 dual-target molecular probe

[0117] This experimental example provides a mouse PET imaging experiment of a PD-L1-targeted molecular probe. The specific process is as follows:

[0118] U87, A375, A375-hPD-L1, and LLC cells were respectively implanted subcutaneously into the right anterior axilla of female BALB / C mice (5 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.) at a dose of 5×10 6 cells; MC38 and LLC cells were respectively implanted subcutaneously into the right anterior axilla of female C57BL / 6 mice (5 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.) at a dose of 5×10 6 cells. The tumor diameter was monitored every other day. When the tumor volume reached 200.0±25.0 mm3 (Tumor volume calculation formula: 1 / 2 × long diameter × short diameter 2 ), anesthetize the mice with oxygen containing 2vt% isoflurane at a flow rate of 2 L / min; after fixing the limbs and tail of the mice, dissolve 150 μCi of the molecular probe prepared in Example 2 68 18 Figure 9 and Figure 18 in 100 μL of physiological saline and inject them into the tail vein respectively; 30 min after the injection of the probe, perform a 10-min static PET scan, and the PET imaging results are shown in Figure 10 and Figure 19 ; after the scan, use the region of interest (ROI) technique in the ASIPRO software to outline and analyze the distribution of the probe in the tumor site and other organ tissues, and the analysis results are shown in

[0119] As Figures 9 - 10 shown, 68 68 v 68 Figures 18 - 19 shown, 18 18 v 18

[0120] ​​​​​​​​​​Experimental Example 6: α v Biodistribution experiment of the dual-target molecular probe of β3 and PD-L1

[0121] Refer to the method in Experimental Example 6 to construct a tumor-bearing mouse animal model. Inject the probes 68 Ga]Ga-NOTA-IMB-RGD and 18 F]AlF-NOTA-IMB-RGD (200 μCi) into the tumor-bearing mice respectively. Sacrifice the tumor-bearing mice 60 minutes later, dissect them, collect the tumors and other normal tissue organs (heart, liver, spleen, lung, kidney, large intestine, small intestine, stomach, muscle, brain and bone), and weigh each tissue. Use a gamma counter to record the decay-corrected radioactivity. The distribution of the probes in the tumor and normal tissues is expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0122] As Figure 11 and Figure 20 shown, 68 Ga]Ga-NOTA-IMB-RGD and 18 F]AlF-NOTA-IMB-RGD (20 μCi) had obvious radioactivity at the tumor sites of U87, A375-hPD-L1, B16-F10 and MC38 mice, moderate radioactivity at the tumor site of A375 mice, and almost no radioactivity at the tumor site of LLC mice. It was further confirmed that the probe was specific for α v β3 and PD-L1 and was a promising radioactive tracer for tumor diagnosis.

[0123] Obviously, the above embodiments are only examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill 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 enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims

1. An alpha v The dual-target molecular probe of β3 and PD-L1 is characterized in that: The molecular probe has the following structure: Wherein, R is a radionuclide labeling group.

2. The molecular probe according to claim 1, wherein The radionuclide labeling group is 68 Ga, [ 18 F]AlF, 64 Cu or 89 Zr.

3. The molecular probe according to claim 2, characterized in that When the radionuclide labeling group is 68 When Ga, the molecular probe has the following structure:

4. The molecular probe according to claim 2, characterized in that When the radionuclide labeling group is [ 18 F]AlF, the molecular probe has the following structure:

5. The molecular probe according to any one of claims 1 to 4, characterized in that The labeling precursor of the molecular probe has the following structure:

6. A method for preparing the molecular probe according to any one of claims 1 to 5, characterized in that: The method comprises: dissolving the compound IMB-RGD and Boc-5-aminopentanoic NHS in an organic solvent to obtain a solution; adjusting the pH of the solution to alkaline using N,N-diisopropylethylamine, and reacting under the protection of nitrogen to obtain a reaction solution A; concentrating the reaction solution A, precipitating with ether, centrifuging, and drying to obtain a reaction product; dissolving the reaction product in a mixed solution of trifluoroacetic acid and acetonitrile to react to obtain a reaction solution B; concentrating the reaction solution B, precipitating with ether, centrifuging, and drying to obtain a compound IMB-RGD-1; Dissolving the compound IMB-RGD-1 and NOTA-NHS in an organic solvent to obtain a solution; adjusting the pH of the solution to alkaline using N,N-diisopropylethylamine, and reacting under the protection of nitrogen to obtain a reaction solution; concentrating the reaction solution, ether precipitating, centrifuging, drying, and purifying the reaction solution to obtain a labeled precursor of a molecular probe, NOTA-IMB-RGD; and radionuclide labeling the labeled precursor of the molecular probe, NOTA-IMB-RGD, to obtain a molecular probe; The compound IMB-RGD has the structure shown below:

7. The molecular probe according to any one of claims 1 to 5 for preparing integrin α v Application of β3 and / or PD-L1 imaging agents.

8. An integrin alpha v β3 and / or PD-L1 imaging agent, characterized in that The imaging agent comprises the molecular probe according to any one of claims 1 to 5.

9. Use of the molecular probe according to any one of claims 1 to 5 in the preparation of a tumor imaging agent.

10. A tumor imaging agent, characterized in that: The imaging agent comprises the molecular probe according to any one of claims 1 to 5.

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