Molecular probe targeting prostate specific membrane antigen and application

Abstract

The invention relates to a molecular probe for targeting a prostate specific membrane antigen and application, and belongs to the technical field of nuclear medicine. The invention provides a molecular probe for targeting a prostate specific membrane antigen, and the molecular probe successfully obtains a PSMA targeting molecular probe by introducing iodobenzene butyric acid and a PEG4 group on the basis of a PSMA-617 molecular structure on the basis of a double-ligand covalent coupling technology. The molecular probe has the advantages that the residence time of the molecular probe in a tumor area is obviously prolonged, the treatment effect is improved, the requirement for radioactive dose is reduced, and radiation damage to surrounding normal tissues is reduced; the specificity is excellent, a tumor area with high PSMA expression can be accurately targeted, and tumors with different PSMA expression levels can be effectively distinguished; the speed of reaching a target tumor is high, and an ideal imaging effect can be realized only in 20 minutes. In conclusion, the molecular probe has great advantages in diagnosis and treatment of PSMA high-expression tumors, and has high specificity and high efficiency.

Description

Technical Field

[0001] The present invention relates to a molecular probe targeting prostate-specific membrane antigen and its application, belonging to the technical field of nuclear medicine. Background Art

[0002] Prostate cancer (PCa) is a common malignant tumor in men. Early diagnosis is crucial for improving the treatment effect and the survival rate of patients. However, due to the lack of specific symptoms and the limitations of existing screening methods (such as PSA detection), the early diagnosis of prostate cancer remains challenging. Prostate-specific membrane antigen (PSMA) is a type II transmembrane protein with folate hydrolase activity. It is expressed at low levels in normal prostate and other tissues, while in prostate cancer, especially in the castration-resistant and advanced metastatic stages, its expression level increases significantly. The PSMA protein consists of an intracellular segment, a transmembrane segment, and an extracellular segment, among which the extracellular segment accounts for 95% of the total structure. Small molecule probes or antibody drugs can bind to it and deliver labeled radioisotopes or therapeutic drugs into cancer cells through the internalization mechanism. This property makes PSMA an important molecular target for the precise diagnosis and targeted treatment of prostate cancer (see the literature: Real-World Treatment Patterns and Overall Survival of Patients with Metastatic Castration-Resistant Prostate Cancer in the US Prior to PARP Inhibitors. Adv. Ther. 2021, 38(8), 4520–4540.).

[0003] Combined with the frontier concept of "diagnosis and treatment integration" in nuclear medicine, the research value of the PSMA target is further highlighted. This concept realizes the combination of diagnosis and treatment on the same molecular target, specifically manifested as using a molecular probe labeled with a diagnostic radionuclide for precise diagnosis and quantitative analysis, and then using the same probe labeled with a therapeutic radionuclide for targeted treatment, thereby organically integrating imaging diagnosis and internal radiation therapy to form a precise treatment mode. For example, 68 Ga]Ga-PSMA-617 can be applied to Positron Emission Computed Tomography (PET), which can accurately locate prostate cancer lesions and perform functional analysis; while 177The Lu-PSMA-617 probe shows significant anti-tumor effects through efficient targeted radiotherapy, especially in patients with advanced drug-resistant prostate cancer. This "integrated precision diagnosis and treatment" not only promotes the progress of nuclear medicine technology but also provides new theoretical bases and practical directions for personalized medicine. However, the therapeutic effect of PSMA-617 is limited by its short retention time at the tumor site, which requires the use of higher radioactive doses and increased dosing frequencies, thereby increasing the risk of radiation exposure to normal tissues and toxic side effects. At the same time, its non-specific uptake in the salivary glands and kidneys further limits the safety and therapeutic effect. Therefore, it is urgent to optimize the probe design, extend the tumor retention time, and reduce non-specific uptake to improve the diagnostic and therapeutic performance of PSMA-617 (see the literature: PSMA-targeted radiotheranostics in modern nuclear medicine: then, now, and what of the future?. Theranostics, 14(8), 3043–3079.). Summary of the Invention

[0004] To solve the above problems, the present invention provides a molecular probe targeting prostate-specific membrane antigen, and the molecular probe has the following structure:

[0005]

[0006] Wherein, R is a radioactive nuclide labeling group.

[0007] In one embodiment of the present invention, the radioactive nuclide labeling group is 68 Ga, 64 Cu, 90 Y, 177 Lu, 161 Tb, 225 Ac or 111 In.

[0008] In one embodiment of the present invention, when the radioactive nuclide labeling group is 68 Ga, the molecular probe has the following structure:

[0009]

[0010] In one embodiment of the present invention, when the radioactive nuclide labeling group is 177 Lu, the molecular probe has the following structure:

[0011]

[0012] In one embodiment of the present invention, the labeling precursor of the molecular probe has the structure shown below:

[0013]

[0014] The present invention also provides a method for preparing the above-mentioned molecular probe, the method comprising: synthesizing the labeling precursor PSMA-DIM of the molecular probe; performing radionuclide labeling on the labeling precursor PSMA-DIM of the molecular probe to obtain the molecular probe;

[0015] The labeling precursor PSMA-DIM has the structure shown below:

[0016]

[0017] The present invention also provides the application of the above-mentioned molecular probe in the preparation of a prostate-specific membrane antigen imaging agent, a tumor imaging agent or an anti-tumor drug.

[0018] In one embodiment of the present invention, the tumor includes a prostate-specific membrane antigen-positive tumor; the prostate-specific membrane antigen-positive tumor includes prostate cancer, pancreatic cancer, breast cancer, lung cancer and / or gastric cancer.

[0019] The present invention also provides a prostate-specific membrane antigen imaging agent, and the components of the imaging agent include the above-mentioned molecular probe.

[0020] The present invention also provides a tumor imaging agent, and the components of the imaging agent include the above-mentioned molecular probe.

[0021] In one embodiment of the present invention, the tumor includes a prostate-specific membrane antigen-positive tumor; the prostate-specific membrane antigen-positive tumor includes prostate cancer, pancreatic cancer, breast cancer, lung cancer and / or gastric cancer.

[0022] The present invention also provides an anti-tumor drug, and the components of the anti-tumor drug include the above-mentioned molecular probe.

[0023] In one embodiment of the present invention, the tumor includes a prostate-specific membrane antigen-positive tumor; the prostate-specific membrane antigen-positive tumor includes prostate cancer, pancreatic cancer, breast cancer, lung cancer and / or gastric cancer.

[0024] The technical solution of the present invention has the following advantages:

[0025] The present invention provides a molecular probe targeting prostate-specific membrane antigen. This molecular probe is based on the dual-ligand covalent coupling technology. On the basis of the molecular structure of PSMA-617, by introducing iodophenbutyric acid and PEG4 groups, a PSMA-targeted molecular probe is successfully obtained. This molecular probe has the following advantages:

[0026] First, the structural modification of this molecular probe significantly prolongs its blood circulation time in vivo. Research shows that 68 the clearance half-life of [68Ga]Ga-PSMA-DIM reaches 99.55 min, which is 57.73 min longer than that of the control probe 68 [68Ga]Ga-PSMA-617 (41.82 min). The prolonged blood circulation time may help improve the uptake and retention of the probe in tumor tissues, thus providing potential possibilities for improving the clarity of tumor imaging and treatment effects;

[0027] Second, this molecular probe has the advantages of strong targeting specificity and high sensitivity. It can accurately target tumors with high PSMA expression, effectively distinguish tumors with different PSMA expression levels. Moreover, this molecular probe can quickly reach the target tumor, and an ideal imaging effect can be achieved in only 20 min in the PSMA-positive tumor model;

[0028] Third, the retention time of this molecular probe in the tumor region is significantly increased. Research shows that 3 h after injection, 68 [68Ga]Ga-PSMA-DIM still has good uptake values in the PSMA-positive tumor model (LNCaP: 3.85 ± 0.05% ID / mL, 22Rv1: 3.50 ± 0.20% ID / mL). Compared with the control probe 68 [68Ga]Ga-PSMA-617 (LNCaP: 2.29 ± 0.05% ID / mL, 22Rv1: 1.39 ± 0.05% ID / mL), it shows higher uptake rates and retention effects. Research shows that in the SPECT imaging of LNCaP mice, 177 [177Lu]Lu-PSMA-DIM still has good tumor uptake 24 h after injection. This improvement helps to enhance the therapeutic effect of the molecular probe, reduce the radioactive dose required for the molecular probe, and at the same time reduce the radiation damage of the molecular probe to surrounding normal tissues;

[0029] Fourth, the synthesis process of the precursor of this molecular probe is simple. All synthesis steps are carried out in a polypeptide tube, effectively improving the reaction efficiency and product purity, reducing the risk of contamination. Moreover, the use of this closed system simplifies the cleaning and separation process, is suitable for large-scale production, and has good cost-effectiveness.

[0030] Therefore, the molecular probe of the present invention has significant advantages in the diagnosis and treatment of tumors with high PSMA expression, combines high specificity, high efficiency and good cost-effectiveness, and has broad clinical application prospects.

[0031] Furthermore, the radionuclide labeling group is 68 68Ga and 177 177Lu;68 Ga is a radioactive nuclide with a short half-life, releasing a relatively low amount of radioactivity per unit time, which can improve safety; while 177 Lu has a relatively long half-life and is suitable for use in therapeutic radiopharmaceuticals, providing a relatively persistent radiation effect. At the same time, its radioactive release is also relatively controllable to ensure safety. Description of the Drawings

[0032] Figure 1 : High-performance liquid chromatography analysis chart of the compound PSMA-DIM.

[0033] Figure 2 : Electrospray mass spectrometry chart of the compound PSMA-DIM.

[0034] Figure 3 : 68 High-performance liquid chromatography analysis chart of the reaction solution before and after labeling with [Ga]Ga-PSMA-DIM.

[0035] Figure 4 : 68 Stability HPLC analysis of [Ga]Ga-PSMA-DIM incubated in PBS (A), mouse serum (B), and human serum (C) for 0.5, 1, and 2 h.

[0036] Figure 5 : 68 Saturation binding curve of [Ga]Ga-PSMA-DIM to LNCaP cells with high PSMA expression.

[0037] Figure 6 : 68 Results of the study on cell uptake (A) and cell internalization (B) of [Ga]Ga-PSMA-DIM.

[0038] Figure 7 : 68 In vivo stability of [Ga]Ga-PSMA-DIM in normal mice.

[0039] Figure 8 : 68 Results of the pharmacokinetic analysis of [Ga]Ga-PSMA-DIM in mice.

[0040] Figure 9 : 68 Results of the pharmacokinetic analysis of [Ga]Ga-PSMA-617 in mice.

[0041] Figure 10 : 68 Results of the microPET dynamic imaging of [Ga]Ga-PSMA-DIM in tumor-bearing mice of LNCaP, 22Rv1, and PC-3.

[0042] Figure 11 : 68 Tumor and muscle uptake curves (A, B, C) and tumor-to-muscle ratio (D) of [Ga]Ga-PSMA-DIM in LNCaP, 22Rv1, and PC-3 tumor-bearing mice.

[0043] Figure 12 : 68 MicroPET static imaging results (A), tumor and muscle uptake curves (B), and tumor-to-muscle ratio (C) of [Ga]Ga-PSMA-DIM in LNCaP and 22Rv1 tumor-bearing mice.

[0044] Figure 13 : 68 MicroPET static imaging results (A), tumor and muscle uptake curves (B), and tumor-to-muscle ratio (C) of [Ga]Ga-PSMA-617 in LNCaP and 22Rv1 tumor-bearing mice.

[0045] Figure 14 : 68 Biodistribution experimental results of [Ga]Ga-PSMA-DIM in LNCaP, 22Rv1, and PC-3 tumor-bearing mice.

[0046] Figure 15 : 177 High-performance liquid chromatography analysis chart of the reaction solution before and after labeling with [Lu]Lu-PSMA-DIM.

[0047] Figure 16 : 177 Stability high-performance liquid chromatography analysis of [Lu]Lu-PSMA-DIM incubated in PBS (A) and mouse serum (B) for 4, 8, 12, 24, 48 h.

[0048] Figure 17 : 177 Results of cell uptake (A) and cell internalization (B) studies of [Lu]Lu-PSMA-DIM.

[0049] Figure 18 : 177 SPECT imaging results (A) and tumor uptake values (B) of [Lu]Lu-PSMA-DIM in LNCaP tumor-bearing mice.

[0050] Figure 19 : 177 Biodistribution of [Lu]Lu-PSMA-DIM in LNCaP tumor-bearing mice. Detailed implementation

[0051] The following embodiments are provided to better further understand the present invention. They are not limited to the best mode described, and do not limit 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 prior arts falls within the protection scope of the present invention.

[0052] 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 without indicating the manufacturer, they are all conventional reagent products that can be obtained through commercial purchase.

[0053] Example 1: A PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM

[0054] This example provides a PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM. The PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM has the following structure:

[0055]

[0056] Example 2: A method for preparing a PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM

[0057] This example provides a preparation method of the PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM in Example 1. The specific steps are as follows:

[0058] Step 1: Rinse the solid-phase peptide synthesis tube twice with 10 mL of ultradry dichloromethane, evacuate and dry. Then add 2-chlorotrityl chloride resin (loading amount: 1.147 mmol / g, 1 g) to the solid-phase peptide synthesis tube, and add 10 mL of ultradry dichloromethane to soak and swell the 2-chlorotrityl chloride resin. Place it on a new type of wrist-type oscillator and oscillate (285 rpm) for 10 min, then evacuate and dry;

[0059] Step 2: Add Fmoc-Glu(otBu)-OH (425.27 mg, 1 eq, 1 mmol) and 10 mL of ultradry DMF (N,N-dimethylformamide) to the solid-phase peptide synthesis tube obtained in Step 1 to obtain a reaction system; add DIEA (N,N-diisopropylethylamine) (348.36 μL, 2 eq, 2 mmol) to the reaction system to adjust the pH of the reaction system to 9, then place the solid-phase peptide synthesis tube on a new wrist-type oscillator and oscillate for 3 h. After the oscillation is completed, drain the solvent;

[0060] Step 3: First, add 10 mL of a mixed solution of DMF / CH3OH / DIPEA (DMF / CH3OH / DIPEA = 17:2:1, v / v / v) to the drained solid-phase peptide synthesis tube to wash the resin. After oscillating for 10 min, perform suction filtration, and repeat the operation 3 times to remove excess unreacted amino acids; then wash the resin with 10 mL of DMF (HPLC analytical grade). After manually oscillating for 2 min, perform suction filtration, and repeat the operation 3 times;

[0061] Step 4: After the washing is completed, first take a sample for the Kaiser test. The color of the Kaiser reagent shows transparency, indicating that the amino group of the amino acid is protected at this time and not exposed; then, add 10 mL of a DMF solution containing 20% (v / v) piperidine to the solid-phase peptide synthesis tube. After oscillating for 10 min, perform suction filtration, and repeat the operation three times to remove the Fmoc protecting group on the amino acid; then wash with 10 mL of DMF (HPLC analytical grade) to wash away the excess piperidine;

[0062] Step 5: After the washing is completed, drain the solvent, take a sample and perform the Kaiser test again. The color of the Kaiser reagent shows dark purple, indicating that the Fmoc group of the amino acid has been removed at this time, exposing the amino group, and the next amino acid can be linked;

[0063] Step 6: On the basis of Step 5, sequentially replace Fmoc-Glu(otBu)-OH in Step 2 with N,N'-carbonyldiimidazole (162.15 mg, 1 eq, 1 mmol), Fmoc-Lys-OtBu (424.54 mg, 1 eq, 1 mmol), and repeat the operations of Steps 2 to 5;

[0064] Step 7: On the basis of Step 6, sequentially replace Fmoc-Glu(otBu)-OH in Step 2 with Fmoc-2-Nal-OH (437.49 mg, 1 eq, 1 mmol), Fmoc-4-amino-(1-carboxymethyl)piperidine (380.44 mg, 1 eq, 1 mmol), Fmoc-NH-PEG4-CH2COOH (473.52 mg, 1 eq, 1 mmol), Fmoc-Glu (369.37 mg, 1 eq, 1 mmol), Fmoc-Lys(Dde)-OH (532.63 mg, 1 eq, 1 mmol). Each time of replacement, additionally add HBTU (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) (454.80 mg, 1.2 eq, 1.2 mmol), and repeat the operations of Step 2 to Step 5;

[0065] Step 8: On the basis of Step 7, add DOTA-tri(t-butyl ester) (572.73 mg, 1.5 eq, 1.5 mmol) and DIEA (1393.42 μL, 8 eq, 8 mmol) into the solid-phase peptide synthesis tube, and fully oscillate and react at 25 °C for 3 h;

[0066] Step 9: On the basis of Step 8, first add the DMF solution containing 2% (v / v) hydrazine hydrate into the solid-phase peptide synthesis tube, oscillate for 10 min and then perform suction filtration, and repeat the operation three times to remove the Dde protecting group on the amino acid; then add 4-(p-iodophenyl)butyric acid (290.1 mg, 1 eq, 1 mmol) and HBTU (454.80 mg, 1.2 eq, 1.2 mmol), and repeat the operations of Step 2 to Step 3;

[0067] Step 10: Dry the solid-phase peptide synthesis tube obtained in Step 9, add 10 mL of CH2Cl2 solution containing 1% (v / v) TFA (trifluoroacetic acid) to cleave the resin; oscillate at 25 °C for 10 min. After the oscillation is completed, filter out the filtrate, and repeat the operation 3 times until the 2-chlorotrityl chloride resin shows a wine red color and does not fade; then use the mixed solution of DCM / TFA (DCM / TFA = 1:1, v / v) to remove the tBu protecting group on the amino acid;

[0068] Step 11: Remove the organic solvent from the reaction solution obtained in Step 10 using a rotary evaporator. After precipitation with cold ethyl ether (4 °C), transfer it to a 50 mL centrifuge tube. After centrifugation and discarding the supernatant, place the compound precipitate in a vacuum drying oven and dry it for 30 min. Then, purify the compound using semi-preparative HPLC (the purification conditions are shown in Table 1; the process of semi-preparative HPLC purification is as follows: select the mobile phase gradient in Table 1 and purify the sample dissolved in DMF through a C18 reverse chromatographic column) to obtain the PSMA-targeted molecular probe precursor PSMA-DIM;

[0069] Table 1 Purification conditions for semi-preparative HPLC

[0070]

[0071] Step 12: Elute 68 Ge / 68 Ga ions from a 68 Ga generator (ITG) using 1.4 mL of 0.05 M HCl. Take out 100 μL and add it to a 0.25 M NaOAc buffer solution to mix and adjust the pH value to 4.0 to obtain a mixture. Transfer the mixture directly to a 5 mL EP tube containing 4 μg of the PSMA-targeted molecular probe precursor PSMA-DIM. After mixing, incubate the mixture in an oil bath at 95 °C for 15 min to obtain the molecular probe 68 Ga]Ga-PSMA-DIM; Analyze the product by radio-HPLC.

[0072] Perform ESI-MS analysis on the precursor PSMA-DIM using an electrospray ionization source, and perform HPLC detection on the precursor PSMA-DIM using Waters1525. The analysis and detection results are shown in Figures 1 - 2 .. Use a Gabi Nova radioactive detector to perform radioactive HPLC detection on the reaction solution after 68 Ga]Ga-PSMA-DIM labeling. The detection results are shown in Figure 3 .. Calculate the radiochemical purity (RCP) of 68 Ga]Ga-PSMA-DIM by the peak area of the radioactive product / total peak area. The calculation result is that the radiochemical purity of the labeled product is higher than 99%.

[0073] In the above radio-high performance liquid chromatography, 68 The retention time of 68The final radiochemical yield (RCY) of [[Ga]]Ga-PSMA-DIM, the calculated result > 98%, was calculated by molar activity (Am) = product radioactivity / M (molar mass of the precursor). 68 The molar activity of [[Ga]]Ga-PSMA-DIM, the calculated result was 24.77 GBq / μmol.

[0074] Example 3: A PSMA-targeted molecular probe 177 [[Lu]]Lu-PSMA-DIM

[0075] This example provides a PSMA-targeted molecular probe 177 [[Lu]]Lu-PSMA-DIM, the PSMA-targeted molecular probe 177 [[Lu]]Lu-PSMA-DIM has the following structure:

[0076]

[0077] Example 4: A method for preparing a PSMA-targeted molecular probe 177 The method for preparing [[Lu]]Lu-PSMA-DIM

[0078] This example provides the method for preparing the PSMA-targeted molecular probe described in Example 1 177 The method for preparing [[Lu]]Lu-PSMA-DIM, the specific steps are as follows:

[0079] Add the probe precursor PSMA-DIM (3 μg) to a vial containing 177 [[Lu]] (1 mCi / 100 μL sodium acetate solution), react at 90 °C for 20 min; after the reaction is completed, take a sample for radiochemical yield analysis, and the product is denoted as 177 [[Lu]]Lu-PSMA-DIM. Use a single-photon radioactive detector to perform radioactive HPLC detection on the reaction solution before and after the labeling of 177 [[Lu]]Lu-PSMA-DIM, and the detection results are shown in Figure 15 . Calculate the radiochemical purity (RCP) of [[Lu]]Lu-PSMA-DIM by the peak area of the radioactive product / total peak area, and the calculated result is: the radiochemical purity of the labeled product is higher than 99%. 177 In the above radio-high performance liquid chromatography,

[0080] the retention time of [[Lu]]Lu-PSMA-DIM was 15.1 min, which was close to the elution time of the labeling precursor PSMA-DIM (14.7 min). After attenuation correction, calculate by radioactive HPLC detection 177 [[Lu]]Lu-PSMA-DIM 177The final radiochemical yield (RCY) of Lu]Lu-PSMA-DIM, calculated to be >98%, was calculated by molar activity (Am) = product radioactivity / M (molar mass of the precursor). 177 The molar activity of Lu]Lu-PSMA-DIM was calculated to be 33.02 GBq / μmol.

[0081] Experimental Example 1: In vitro stability experiment of PSMA-targeted molecular probe

[0082] This experimental example provides an in vitro stability experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0083] Experiment 1: The PSMA-targeted molecular probe prepared in Example 2 68 Ga]Ga-PSMA-DIM and the PSMA-targeted molecular probe prepared in Example 4 177 Lu]Lu-PSMA-DIM were respectively mixed with PBS buffer (pH = 7.4, 0.01 M) at a volume ratio of 1:9 to obtain a mixed solution; the mixed solution was incubated at 37 °C for 0.5, 1, 2 h or 4, 8, 12, 24, 48 h respectively; after the incubation ended, the incubated solution was taken and analyzed by radioactive HPLC using an electron / single photon radioactive detector. The analysis results are shown in Figure 4 A in Figure 16 and A in

[0084] Experiment 2: The PSMA-targeted molecular probe prepared in Example 2 68 Ga]Ga-PSMA-DIM and the PSMA-targeted molecular probe prepared in Example 4 177 Lu]Lu-PSMA-DIM were respectively mixed with mouse serum (purchased from Nanjing Senbeijia Biotechnology Co., Ltd.) at a volume ratio of 1:9 to obtain a mixed solution; the mixed solution was incubated at 37 °C for 0.5, 1, 2 h or 4, 8, 12, 24, 48 h respectively; after the incubation ended, 20 μL of the incubated solution was taken, an equal volume of acetonitrile was added, and the mixture was centrifuged at 12000 g for 5 min to separate the serum from the protein. The supernatant was aspirated and analyzed by radioactive HPLC using a Gabi Nova radioactive detector. The analysis results are shown in Figure 4 B in Figure 16 and B in

[0085] Experiment 3: The PSMA-targeted molecular probe prepared in Example 2 68Ga]Ga-PSMA-DIM was mixed with human serum at a volume ratio of 1:9 to obtain a mixed solution; the mixed solution was incubated at 37 °C for 0.5, 1, and 2 h respectively; after the incubation, 20 μL of the incubation solution was taken, an equal volume of acetonitrile was added, and the mixture was centrifuged at 12,000 g for 5 min to separate the serum from the protein. The supernatant was aspirated and analyzed by radioactive HPLC using a Gabi Nova radioactive detector. The analysis results are shown in Figure 4 C in

[0086] It can be seen from Figure 4 that the PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM incubated in mouse serum, human serum, and PBS at 37 °C for 0.5 - 2 h had a main peak ratio greater than 95% in the HPLC chromatogram, indicating good stability. It can be seen from Figure 16 that the PSMA-targeted molecular probe 177 Lu]Lu-PSMA-DIM incubated in mouse serum and PBS at 37 °C for 4 - 48 h had a main peak ratio greater than 95% in the HPLC chromatogram, indicating good stability.

[0087] Experimental Example 2: Lipid-water partition coefficient experiment of PSMA-targeted molecular probe

[0088] This experimental example provides a lipid-water partition coefficient experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0089] Take an EP tube, add deionized water (1 mL) and n-octanol (1 mL), and then add the molecular probe 68 Ga]Ga-PSMA-DIM (0.74 MBq) prepared in Example 2 to the mixed solution. Vortex and mix for 5 min to completely disperse the probe in the mixed solution. After centrifuging at 4000 r / min for 5 min to separate the two phases, 500 μL was taken from each of the n-octanol phase and the water phase, and the radioactive CPM value was measured using a γ counter, and log P (logP = log C O / C W ) was calculated. After each test, 500 μL of n-octanol and deionized water were added to the EP tube respectively to restore the volumes of the n-octanol phase and the water phase to 1 mL. Shake well, centrifuge and separate the layers, and resample to measure log P. Repeat the above experiment multiple times until the logP values of the three groups are close to each other. The results are expressed as the mean ± standard deviation of the three groups of data (mean ± SD, n = 3). The lipid-water partition coefficient of the PSMA-targeted molecular probe 177 Lu]Lu-PSMA-DIM prepared in Example 4 was detected using the same method.

[0090] The experimentally measured PSMA-targeted molecular probe 68Ga]Ga-PSMA-DIM and 177 The partition coefficients of 68 Ga]Ga-PSMA-DIM and 177 Lu]Lu-PSMA-DIM are -1.32 ± 0.003 and -1.56 ± 0.032 respectively, indicating that

[0091] Experimental Example 3: Binding Affinity Experiment of PSMA-Targeted Molecular Probe

[0092] This experimental example provides a binding affinity experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0093] 2×10 5 LNCaP cells (purchased from the Cell Bank of Shanghai Institute of Chinese Academy of Sciences) were seeded in polylysine-coated 24-well plates and cultured overnight (16 h) at 37°C. After the culture, the old medium was aspirated, and 300 μL of 1640 medium containing the molecular probe 68 Ga]Ga-PSMA-DIM prepared in Example 2 at different concentrations (0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, and 100 nM) was added to each well and co-incubated at 37°C, denoted as PSMA summary and experiment; in the non-specific binding experiment, 100-fold blocking non-radioactive probe precursor PSMA-DIM was added to each well and co-incubated with the above gradient concentrations of 68 Ga]Ga-PSMA-DIM (a total of 300 μL of 1640 medium) and LNCaP cells at 37°C for 1 h. After the co-incubation, the cells were washed twice with cold PBS buffer (pH = 7.4, 0.01 M), and then the radioactivity of the cells was detected using a γ counter. The specific binding curve was obtained by fitting the total binding value minus the non-specific binding value, and the dissociation constant (K d ) value was obtained through analysis, which can indicate the affinity of the probe for PSMA. The analysis results are shown in Figure 5 .

[0094] As Figure 5 shown, 68 the dissociation constant (K d ) of 68 Ga]Ga-PSMA-DIM is approximately 37.09 nM, indicating that

[0095] Experimental Example 4: Cellular Uptake Experiment of PSMA-Targeted Molecular Probe

[0096] This experimental example provides a cell uptake experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0097] Seed 2×10 5 LNCaP, 22Rv1, and PC-3 cells (all prostate cancer cell lines, purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) in a 24-well plate coated with polylysine and culture overnight (16 h) at 37 °C. After the culture, aspirate the old medium, and add 400 μL of RPMI 1640 medium containing the molecular probe 68 Ga]Ga-PSMA-DIM prepared in Example 2 (0.5 μCi / 400 μL) to each well, and incubate at 37 °C for 15 min, 30 min, 1 h, and 2 h. After the incubation, discard the medium and add 500 μL of cold (4 °C) PBS buffer (pH = 7.4, 0.01 M) to wash, then add 500 μL of lysis buffer (NaOH, 1 M) to lyse the cells. Collect the lysate and measure the CPM value of the sample using a gamma counter. The cell uptake %AD result is expressed as the ratio of the CPM in the cells to the CPM of the total dose. The detection results are shown in Figure 6 A in. Use the same method to incubate for 1, 2, 4, 6, 8, 12, 24, 48, 72 h to detect the cell uptake of the PSMA-targeted molecular probe 177 Lu]Lu-PSMA-DIM prepared in Example 4. The detection results are shown in Figure 17 A in.

[0098] As Figure 6 shown in A in, the uptake of 68 Ga]Ga-PSMA-DIM by LNCaP cells increased with the prolongation of the culture time, from 0.97 ± 0.04% AD at 15 min to 1.72 ± 0.06% AD at 120 min. The uptake rate of 22Rv1 cells was significantly lower than that of LNCaP cells, and the maximum uptake at 120 min was 0.75 ± 0.04% AD. The maximum uptake of PSMA-negative cells PC-3 was only 0.47 ± 0.01% AD within 2 h, indicating that 68 Ga]Ga-PSMA-DIM has good targeting specificity and sensitivity to PSMA at the cellular level. As Figure 17 shown in A in, the uptake of 177 Lu]Lu-PSMA-DIM by LNCaP cells increased with the prolongation of the culture time, from 0.81 ± 0.06% AD at 1 h to the maximum uptake of 1.72 ± 0.05% AD at 24 h. The uptake rate of 22Rv1 cells was significantly lower than that of LNCaP cells, and the maximum uptake at 48 h was 0.79 ± 0.08% AD. The maximum uptake of PSMA-negative cells PC-3 was only 0.37 ± 0.06% AD within 48 h, indicating that177 Lu]Lu-PSMA-DIM can specifically target PSMA-positive cells at the cellular level.

[0099] Experimental Example 5: Cellular Internalization Experiment of PSMA-Targeted Molecular Probe

[0100] Seed 2×10 5 LNCaP, 22Rv1, and PC-3 cells (purchased from the Cell Bank of the Shanghai Institute of Chinese Academy of Sciences) in a 24-well plate coated with polylysine and culture overnight (16 h) at 37 °C. After the culture, aspirate the old medium, and add 400 μL of RPMI 1640 medium containing the molecular probe 68 Ga]Ga-PSMA-DIM prepared in Example 2 (0.5 μCi / 400 μL) to each well, and incubate at 37 °C for 15 min, 30 min, 1 h, and 2 h. After incubation, collect the supernatant into a radioimmunoassay tube, then wash twice with 500 μL of cold (4 °C) PBS buffer and collect into the radioimmunoassay tube, which is denoted as extracellular; treat each well with 1 mL of Gly-HCl buffer (pH = 3) for 5 min, collect the supernatant, and wash twice with 500 μL of cold PBS buffer, and collect all for γ counting, which is denoted as membrane-bound; finally, lyse with 500 μL of NaOH (1 M) for 5 min, collect all the lysates into the radioimmunoassay tube, and rinse twice with PBS, and collect all for γ counting, which is denoted as internal binding. Calculate the internalization rate % = internal / (internal + membrane-bound) × 100%. The detection results are shown in Figure 6 B in. Incubate for 1, 2, 4, 6, 8, 12, 24, 48, and 72 h using the same method to detect the cellular uptake of the PSMA-targeted molecular probe 177 Lu]Lu-PSMA-DIM prepared in Example 4, and the detection results are shown in Figure 17 B in.

[0101] As Figure 6 shown in B in, after 15 min of culture, the internalization rates of LNCaP and 22Rv1 cells reached 64.14 ± 1.47% and 57.43 ± 3.76% respectively. After 2 h, the internalization rate gradually increased, reaching 80.33 ± 5.43% and 66.73 ± 2.60% respectively. In contrast, the internalization rate of PC-3 cells was only 28.46 ± 6.89% at 15 min and increased to 57.26 ± 4.81% after 2 h, which indicates that 68 Ga]Ga-PSMA-DIM has good internalization ability in PSMA-positive cells. As Figure 17As shown in B of , after 1 hour of incubation, the internalization rates of LNCaP and 22Rv1 cells reached 68.37±0.78% and 31.38±1.45% respectively. With the increase of time, the internalization rates gradually increased, reaching 83.01±1.13% and 69.97±1.57% respectively at the highest. In contrast, the highest internalization rate of PC-3 cells was only 46.67±0.01%, indicating that 68 Ga]Ga-PSMA-DIM has good internalization ability in PSMA-positive cells.

[0102] Experimental Example 6: In vivo stability experiment of PSMA-targeted molecular probe

[0103] This experimental example provides an in vivo stability experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0104] The molecular probe 68 Ga]Ga-PSMA-DIM prepared in Example 2, dissolved in 100 μL of physiological saline and with 2 mCi, was injected into male BALB / C mice (5 weeks old, purchased from Changzhou Cavens Laboratory Animal Co., Ltd.) through the tail vein; after the injection of the probe, blood was taken from the tail vein of the mice at different time points (0.5, 1, and 2 h), an equal volume of acetonitrile was added, and the serum was separated from the protein by high-speed centrifugation at 12,000 g for 5 min. The supernatant was aspirated and analyzed by radioactive HPLC using a Gabi Nova radioactive detector. The analysis results are shown in Figure 7 .

[0105] The results are as shown in Figure 7 It can be seen that 68 Ga]Ga-PSMA-DIM shows excellent stability in mice, and its radioactive signal is maintained for a long time, and no significant decomposition or metabolism is observed.

[0106] Experimental Example 7: Mouse pharmacokinetic analysis experiment of PSMA-targeted molecular probe

[0107] This experimental example provides a mouse pharmacokinetic analysis experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0108] The molecular probe 68 Ga]Ga-PSMA-DIM prepared in Example 2, dissolved in 100 μL of physiological saline and with 200 μCi, and the control probe 68Ga]Ga-PSMA-617 was injected into male BALB / C mice (5 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.) via the tail vein; after the probe injection, blood was collected from the tail vein of the mice at different time points (1, 2, 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 drug 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. The plotted results are shown in Figure 8 and Figure 9 .

[0109] As Figure 8 shown, by measuring the blood drug concentration in mice, the pharmacokinetic parameters of 68 Ga]Ga-PSMA-DIM were obtained, and its metabolic curve conforms to the two-compartment model. According to the analysis of the compartment model parameters and statistical moment parameters, 68 Ga]Ga-PSMA-DIM had an average distribution half-life (t 1 / 2α ) of 0.76 min, indicating that 68 Ga]Ga-PSMA-DIM could be rapidly targeted and transported to the tumor site along with the blood circulation. In addition, 68 Ga]Ga-PSMA-DIM had an average elimination half-life (t 1 / 2β ) of 99.55 min. Compared with the control probe 68 Ga]Ga-PSMA-617 ( Figure 9 , t 1 / 2β = 41.82 min), it was extended by 57.73 min, indicating that the probe had a longer half-life in vivo, which could improve the imaging quality and extend the imaging time window.

[0110] Experimental Example 8: Mouse PET Imaging Experiment of PSMA-Targeted Molecular Probe

[0111] This experimental example provides a mouse PET imaging experiment of a PSMA-targeted molecular probe. The specific process is as follows:

[0112] LNCaP cells (5×10 6 cells) were implanted subcutaneously into the right anterior axilla of male NOD / SCID mice (7 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.). 22Rv1 and PC-3 cells (5×10 6 cells) were implanted subcutaneously into the right anterior axilla of male NOD / SCID mice (5 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.); when the tumor volume reached 200.0 ± 25.0 mm3 (Tumor volume calculation formula: 1 / 2 × long diameter × short diameter 2 ) when the experiment was carried out. The tumor-bearing mice were anesthetized with oxygen containing 2% (v / v) isoflurane at a flow rate of 2 L / min; after fixing the limbs and tail of the mice, 150 μCi of the molecular probe prepared in Example 2 dissolved in 100 μL of physiological saline 68 [

[68] ]Ga]Ga-PSMA-DIM was injected through the tail vein. Immediately after the injection of the probe, a 60-min dynamic PET scan was performed, and the PET imaging results are shown in Figure 10 . For the PSMA-positive model, after the 60-min dynamic scan, static scans were continued for 90, 120, 150, and 180 min, and the PET imaging results are shown in Figure 12 A in. At the same time, other tumor-bearing mice were injected with a control probe 68 [

[68] ]Ga]Ga-PSMA-617 (200 μCi) for static scanning, and the results are shown in Figure 13 A in. After the scan was completed, the 60-min PET imaging results were segmented into 12 frames of images using the OSEM3D / MAP algorithm, with one frame every 5 min, to achieve real-time analysis of the in-vivo imaging of the mice; the region of interest (ROI) technique in the ASIPRO software was used to outline and analyze the distribution of the probe in the tumor site and other organ tissues, and the analysis results are shown in Figure 11 , Figure 12 B in~ Figure 12 C in and Figure 13 B in~ Figure 13 C in. The uptake values of the molecular probe in various tissues in vivo were expressed as %ID / mL (percentage of the injection dose per milliliter of tissue).

[0113] As Figures 10 - 11 shown, 68 [

[68] ]Ga]Ga-PSMA-DIM had a high accumulation in the tumor sites of LNCaP and 22Rv1 tumor-bearing mice, reaching the maximum uptake of 4.19 ± 0.65%ID / mL and 4.08 ± 0.47%ID / mL at 45 min and 20 min, respectively. In contrast, there was no obvious tracer uptake in the tumor sites of PC-3 tumor-bearing mice. The experiment proved that 68 [

[68] ]Ga]Ga-PSMA-DIM had the effect of targeting PSMA. In addition, 68 [

[68] ]Ga]Ga-PSMA-DIM in LNCaP and 22Rv1 tumor-bearing mice had a continuously increasing tumor-to-muscle ratio and remained above 2.3, significantly higher than that of the PC-3 model. As Figure 12 shown, as time went by, the tracer was gradually cleared from non-tumor tissues, while a large amount of radioactive tracer remained in the tumor sites. 180 min after injection,68 The uptake values of 68 Ga]Ga-PSMA-DIM in LNCaP and 22Rv1 tumors were still high, being 3.85 ± 0.05% ID / mL and 3.50 ± 0.20% ID / mL respectively. Compared with the control probe Figure 13 B, LNCaP: 2.29 ± 0.05% ID / mL, 22Rv1: 1.39 ± 0.05% ID / mL), it showed better retention effect and uptake rate. At the same time, the tumor-to-muscle ratio of LNCaP remained above 3, and the ratio of 22Rv1 could reach 5.53 ± 0.28 at 180 min. This indicates that 68 Ga]Ga-PSMA-DIM has good tumor retention.

[0114] Experimental Example 9: Mouse SPECT Imaging Experiment of PSMA-Targeted Molecular Probe

[0115] This experimental example provides a mouse SPECT imaging experiment of a PSMA-targeted molecular probe, and the specific process is as follows:

[0116] LNCaP cells (5 × 10 6 cells) were subcutaneously implanted into the right anterior axilla of male NDG mice (7 weeks old, purchased from Changzhou Cavens Experimental Animal Co., Ltd.). When the tumor volume reached 200.0 ± 25.0 mm 3 (tumor volume calculation formula: 1 / 2 × long diameter × short diameter 2 ), 500 μCi of the molecular probe prepared in Example 4 dissolved in 100 μL of physiological saline 177 Lu]Lu-PSMA-DIM was injected through the tail vein; the mice were anesthetized with oxygen containing 2 vol% isoflurane at a flow rate of 2 L / min, and static SPECT scans were performed at 1, 4, 12, and 24 h. After the scans were completed, the imaging results were processed in the form of MIP maps, and the region of interest (VOI) technique in PMOD software was used to outline and analyze the probe at the tumor site. The results are shown in Figure 18 A to Figure 18 B in. The uptake values of the molecular probe in various tissues in vivo were expressed as % ID / mL (percentage of injection dose per milliliter of tissue).

[0117] The results are as Figure 18 shown, 177 the uptake of Lu]Lu-PSMA-DIM at the tumor site of LNCaP mice increased with time and continued to rise within 24 h, indicating that it can effectively and stably accumulate in tumor cells, enhance the radioactive irradiation effect, and may reduce the damage to normal tissues at the same time, showing its potential in targeted radiotherapy.

[0118] Experimental Example 10: Mouse Biodistribution Experiment of PSMA-Targeted Molecular Probe

[0119] This experimental example provides a mouse biodistribution experiment of a PSMA-targeted molecular probe. The specific process is as follows:

[0120] The PSMA-targeted molecular probe 68 Ga]Ga-PSMA-DIM (5.5 - 7.4 MBq, 200 μL) was injected into LNCaP, 22Rv1, and PC-3 tumor-bearing mice via the tail vein. The mice were sacrificed and dissected 1 h after injection. The main organs and tissues were removed, weighed, and the radioactivity of each tissue and organ was measured using a γ counter, and decay correction was performed. 68 The uptake of Ga]Ga-PSMA-DIM in tumors and other tissues was expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0121] The PSMA-targeted molecular probe prepared in Example 4 177 Lu]Lu-PSMA-DIM (5.5 - 7.4 MBq, 200 μL) was injected into LNCaP tumor-bearing mice via the tail vein. The mice were sacrificed and dissected 12 h after injection. The main organs and tissues were removed, weighed, and the radioactivity of each tissue and organ was measured using a γ counter, and decay correction was performed. 177 The uptake of Lu]Lu-PSMA-DIM in tumors and other tissues was expressed as the percentage of the injected dose per gram of tissue (%ID / g).

[0122] The results are as Figure 14 shown, 68 For Ga]Ga-PSMA-DIM in normal organs, high radioactivity was mainly observed in the kidneys. As for tumor tissues, the uptake in LNCaP and 22Rv1 tumors was significantly higher than that in PC-3 tumors. 68 The biodistribution curve of Ga]Ga-PSMA-DIM in most organs was comparable to the biodistribution curve observed in PET imaging. The results are as Figure 19 shown, 177 For Lu]Lu-PSMA-DIM in normal organs, high radioactivity was mainly observed in the kidneys, but good uptake was also shown at the tumor site, indicating that it can effectively target tumor cells while reducing radiation damage to healthy tissues.

[0123] Obviously, the above embodiments are merely examples for clear illustration and 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 implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.

Claims

1. A molecular probe targeting prostate-specific membrane antigen, 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, 64 Cu, 90 Y. 177 Lu, 161 Tb, 225 Ac or 111 In.

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 177 Lu, 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: synthesizing a molecular probe labeling precursor PSMA-DIM; labeling the molecular probe labeling precursor PSMA-DIM with radioactive nuclides to obtain the molecular probe; The labeling precursor PSMA-DIM has the following structure:

7. Use of the molecular probe according to any one of claims 1 to 5 in the preparation of a prostate-specific membrane antigen imaging agent, a tumor imaging agent or an anti-tumor drug.

8. A prostate-specific membrane antigen imaging agent, characterized in that: The imaging agent comprises the molecular probe according to any one of claims 1 to 5.

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

10. An anti-tumor drug, characterized in that: The anti-tumor drug comprises the molecular probe according to any one of claims 1 to 5.

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