NTSR1 and PSMA double-target probe as well as preparation method and application thereof

Abstract

The invention relates to a double-target probe of NTSR1 and PSMA as well as a preparation method and application of the double-target probe. The structure of the double-target probe is as follows: # imgabs0 #. The double-target probe has high affinity and targeting property for NTSR1 and PSMA at the same time, has high stability in vivo and in vitro, has high nuclide labeling rate and radiochemical purity, and has excellent in-vivo metabolism performance. In addition, the double-target probe can quickly remove blood and main organs (brain, heart, lung, liver, muscle and skeleton), and is mainly metabolized through kidney.

Description

Technical Field

[0001] The present invention relates to the field of nuclear medicine imaging agents, and more specifically, to a dual-target probe of NTSR1 and PSMA and a preparation method and application thereof. Background Art

[0002] Prostate cancer (PCa) is the most common malignancy of the male urinary system. In China, most PCa patients are often diagnosed with advanced stage at the time of initial diagnosis. Androgen deprivation therapy (ADT) is the main intervention for the treatment of advanced prostate cancer. However, 20%-40% of patients will progress to castration-resistant prostate cancer (CRPC) 18-24 months after ADT treatment, leading to the death of PCa patients. Monitoring the course of ADT treatment is an effective measure to change the treatment plan in time and improve the prognosis of patients. Positron emission tomography / computed tomography (PET / CT) has the advantages of high sensitivity and large field of view whole body imaging and is often used for ADT treatment monitoring. Prostate-specific membrane antigen (PSMA) PET / CT imaging can change the treatment plan of 28% of PCa patients by detecting the recurrence site, lymph nodes or distant metastasis after ADT treatment. However, due to the uneven expression or complete lack of expression of PSMA in some PCa patients' lesions, and the imaging flickering after ADT treatment due to androgen dependence, (PSMA) PET / CT is still challenging to guide prostate cancer treatment. Therefore, it is imperative to explore advanced nuclear medicine probes for accurate use in ADT treatment monitoring.

[0003] Using bispecific radioligands linked to fibroblast activation protein α (FAP) or gastrin-releasing peptide (GRPR) and PSMA, initial results have been achieved in enhancing tumor targeting and improving prostate cancer imaging sensitivity, such as a Chinese patent titled A dual-target inhibitor, molecular probe and application targeting FAP and PSMA. However, simultaneous imaging of FAP as a tumor microenvironment marker and PSMA on the surface of tumor cells is challenging. Although GRPR can make up for the limitations of PSMAPET in diagnosing low-grade tumors, its expression is limited to androgen-dependent tumors, and hormone therapy can also lead to downregulation of GRPR expression, and the problem of imaging being interfered with by androgens has not been solved. Summary of the invention

[0004] The primary purpose of the present invention is to overcome the problem that the existing probes for ADT treatment monitoring are androgen-dependent, and to provide a dual-target probe of NTSR1 and PSMA.

[0005] A further object of the present invention is to provide a method for preparing the dual-target probe.

[0006] A further object of the present invention is to provide the use of the above dual-target probe in the preparation of radionuclide imaging diagnostic reagents.

[0007] The above-mentioned object of the present invention is achieved by the following technical solutions:

[0008] A dual-target probe of NTSR1 and PSMA, the structure of the dual-target probe is as follows:

[0009]

[0010] In the structure, n is an integer of 4 to 7; M is a radionuclide, and Chelater is a radionuclide chelating group.

[0011] The dual-target probe of the present invention comprises a structure DUPA-6-Ahx targeting PSMA (prostate-specific membrane antigen), a polypeptide structure NT20.3 targeting NTSR1 (neurotensin receptor 1), a linking group derived from lysine, and a central structure chelated with a radionuclide M.

[0012] Studies have shown that NTSR1 is a peptide receptor located on the plasma membrane and a member of the G protein-coupled receptor family. It is expressed in approximately 91% of prostate cancer tissues and is associated with tumor progression and proliferation. In advanced prostate cancer, especially in the absence of androgens, the NTSR1 pathway acts as an alternative growth pathway to promote continued tumor growth. In patients with prostate cancer who have undergone long-term anti-androgen treatment, neuroendocrine cell clusters that bind to NTSR1 are also enriched around the tumor; moreover, NTSR1 antagonists can sensitize prostate cancer cells to radiotherapy, regardless of androgen dependence or androgen receptor expression status.

[0013] The inventors of the present invention attempted to combine the structure DUPA-6-Ahx and the polypeptide structure NT20.3 to achieve dual targeting of the probe to NTSR1 and PSMA. However, if a common linking group (such as a polyethylene glycol segment, etc.) is used to connect the two, the affinity of the structure DUPA-6-Ahx in the molecule will be reduced, thereby reducing the uptake of the probe by tumor cells expressing PSMA, and high-contrast imaging cannot be achieved.

[0014] The inventors of the present invention further discovered that using a shorter fragment derived from lysine as a linker group can maintain the affinity of the entire molecule for the two targets and avoid a reduction in the amount of probe uptake by tumor cells; further, a highly hydrophilic radionuclide chelating group is used to compensate for the insufficient hydrophilicity of the linker group, thereby improving the metabolism of the probe. Due to the maintenance of uptake and the improvement of metabolism, the dual-target probe can still perform high-contrast imaging in cells expressing PSMA while performing high-contrast imaging in cells expressing NTSR1, that is, the dual-target probe can be used for imaging of androgen-dependent and androgen-independent tumors at the same time, providing a nuclear medicine imaging tool that is not interfered by anti-androgens for the response evaluation after ADT (androgen deprivation therapy).

[0015] In addition, the dual-target probe of the present invention has good in vitro and in vivo stability, high radionuclide labeling rate and radiochemical purity, excellent in vivo metabolic performance, and is quickly cleared from the blood and major organs (brain, heart, lungs, liver, muscles and bones), mainly metabolized by the kidneys.

[0016] Preferably, M is 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I or 124 At least one of I.

[0017] Preferably, the Chelater is DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.

[0018] Preferably, n is an integer of 5-6.

[0019] Preferably, the structure of the dual-target probe is as follows:

[0020]

[0021] The preparation method of the dual-target probe comprises the following steps:

[0022] The polypeptide structure NT20.3 targeting NTSR1 is synthesized by solid phase synthesis, lysine is modified at the N-terminus of the polypeptide structure NT20.3, and amino acids are modified at the N-terminus of the lysine close to the carboxyl group to synthesize the structure DUPA-6-Ahx targeting PSMA; a radioactive nuclide chelating group is bonded to the lysine; cleavage and purification are performed; and the radioactive nuclide is labeled to obtain the dual-target probe.

[0023] The polypeptide structure NT20.3 is as follows: The structure of DUPA-6-Ahx is as follows:

[0024] Preferably, the composition of the lysis buffer used for the lysis is: 90-95wt% trifluoroacetic acid, 1-3wt% triisopropylsilane, and the rest is water.

[0025] The use of the dual-target probe as a nuclear medicine probe for treatment monitoring in androgen deprivation therapy is also within the protection scope of the present invention.

[0026] The application of the above dual-target probe in the preparation of radionuclide imaging diagnostic reagents is also within the protection scope of the present invention.

[0027] Preferably, the radionuclide imaging diagnostic agent is a diagnostic agent for tumors with high expression of NTSR1 and / or PSMA.

[0028] Preferably, the diagnostic reagent is an early detection reagent, a prognosis determination reagent or a therapeutic efficacy evaluation reagent.

[0029] Preferably, the tumor is a prostate tumor, a breast tumor or a pancreatic tumor.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The present invention improves the affinity and hydrophilicity of the dual-target probe by combining a specific PSMA-targeting structure (DUPA-6-Ahx), a specific linking group and a specific central structure, thereby improving the uptake and metabolism of the dual-target probe in tumor cells, and achieving the dual-target probe's simultaneous targeting of NTSR1 and PSMA, thereby being simultaneously used for androgen-dependent and androgen-independent tumor imaging.

[0032] In addition, the dual-target probe of the present invention has high stability in vivo and in vitro, high radionuclide labeling rate and radiochemical purity, and excellent in vivo metabolic performance. In addition, the dual-target probe is rapidly cleared in the blood and major organs (brain, heart, lung, liver, muscle and bone), mainly metabolized by the kidneys. High-contrast imaging in three model mice confirmed that the dual-target probe can be used for androgen-dependent and androgen-independent tumor imaging at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Schematic diagram of the synthesis route of DOTA-NT-PSMA, the precursor of the dual-target probe of Example 1.

[0034] Figure 2 This is the mass spectrum of DOTA-NT-PSMA, the precursor of the dual-target probe in Example 1.

[0035] Figure 3 The precursor of the dual-target probe of Example 1 68 Schematic diagram of the Ga labeling process.

[0036] Figure 4 The HPLC purity of DOTA-NT-PSMA in Example 1 and 68 Radio-HPLC analysis results of Ga-NT-PSMA and in vivo and in vitro stability results.

[0037] Figure 5 For Example 1 68 Uptake and inhibition results of Ga-NT-PSMA in PC3 and 22Rv1 cells. ns, not significant. *P<0.05, ***P<0.001.

[0038] Figure 6 For Example 1 68 IC of Ga-NT-PSMA in PC3 cells expressing NTSR1 and 22Rv1 cells expressing PSMA 50 Value result graph.

[0039] Figure 7 For Example 1 68 Endogenization and efflux results of Ga-NT-PSMA in PC3 and 22Rv1 cells.

[0040] Figure 8 For Example 1 68 Micro-PET / CT imaging results of Ga-NT-PSMA in PC3 and 22Rv1 tumor model mice. ns, not significant.**P<0.01,***P<0.001.

[0041] Fig. 9 For Example 1 68 Ga-NT-PSMA and single-target probes used in clinical practice 68 Ga-DOTA-NT20.3 and 68Micro-PET / CT imaging results of Ga-PSMA-11 in PC3, 22Rv1 and DU145 tumor model mice. ns, not significant. *P<0.05, **P<0.01, ***P<0.001.

[0042] Fig.10 For Example 1 68 In vitro biodistribution diagram of Ga-NT-PSMA 90 minutes after injection into PC3, 22Rv1 and DU145 tumor model mice and the ratio of tumor to non-target organs (n=3).

[0043] Fig.11 The immunohistochemical results of NTSR1 and PSMA target proteins in tumor tissues of PC3, 22Rv1 and DU145 tumor model mice (Scale bar, 200μm; ×40.). DETAILED DESCRIPTION

[0044] In order to more clearly and completely describe the technical solution of the present invention, the present invention is further described in detail through specific embodiments below. It should be understood that the specific embodiments described herein are only used to explain the present invention, and are not used to limit the present invention. Various changes can be made within the scope of the rights of the present invention.

[0045] Example 1

[0046] This embodiment provides a dual-target probe of NTSR1 and PSMA, the structure of which is as follows:

[0047]

[0048] The preparation method thereof comprises the following steps:

[0049] 1) Add DCM (dichloromethane) to a container containing Wang resin (1.0 mmol, 100-200 mesh, sub: 1.0 mmol / g) and swell for 12 hours overnight. Drain and wash with DCM three times, 30 seconds each time.

[0050] 2) Solid-phase peptide synthesis 9-Fmoc chemistry method coupling leucine: Add Fmoc-leucine solution to the Wang resin obtained in step 1), mix for 30 seconds, then add coupling agent (4-dimethylaminopyridine and N,N'-diisopropylcarbodiimide, molar ratio 1:1), stir at room temperature for 1.5 hours; 20% piperidine / DMF, mix for 15 minutes; wash the resin beads with DMF (dimethylformamide; 3 times), DCM (3 times) and DMF (3 times) in sequence;

[0051] 3) Referring to the solid phase peptide synthesis 9-fluorenylmethoxycarbonyl chemistry method in step 2), tert-leucine, tyrosine, proline, arginine, N-methylarginine, proline, lysine and acetic acid were coupled to the N-terminus of the leucine of the resin beads obtained in step 2) to obtain compound 10 (see Figure 1 ); then, lysine was coupled to compound 10 as a linking group, and then 6-aminocaproic acid and glutamic acid were coupled to the N-terminus of lysine in sequence to obtain compound 13; in this step, the coupling agent was 1-hydroxybenzotriazole and N,N'-diisopropylcarbodiimide (molar ratio 1:1);

[0052] 4) Continue to couple glutamate di-tert-butyl ester hydrochloride at the N-terminus of glutamate to obtain compound 14, and the coupling agent is N,N'-diisopropylcarbodiimide, N,N-diisopropylethylamine and 4-dimethylaminopyridine (molar ratio of 1:1:1); then bond the chelate ring 1,4,7,10-tetraazacyclononane-1,4,7,10-tetraacetic acid (DOTA) to the lysine linker to obtain a side chain protected dual-target probe precursor, and the coupling agent used to bond the chelate ring is benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N-diisopropylethylamine (molar ratio of 1:1);

[0053] 5) Add lysis buffer (95% trifluoroacetic acid, 2.5% triisopropylsilane, 2.5% H2O) to the flask containing the side chain protected dual-target probe precursor and stir at room temperature for 3 hours; then precipitate with ice ether and centrifuge (3000rpm, 5min); then wash twice with ice ether; vacuum dry to obtain a white solid; use preparative high performance liquid chromatography (A: H2O (0.05% trifluoroacetic acid), B: acetonitrile) to purify the white solid to obtain a dual-target labeled probe precursor (denoted as DOTA-NT-PSMA) with a purity of >95.0%. The mass spectrometry identification results of DOTA-NT-PSMA are shown in the following figure. Figure 2 As shown;

[0054] 6) 68 GaCl3 from 68 Ge- 68 The Ga generator was eluted with 4 mL of 0.05 M HCl. 68 GaCl3 (4 mL), sodium acetate (1000 μL, pH 8.0) and DOTA-NT-PSMA (25 μL) were mixed and labeled at 95 °C for 15 min, and then 5 mL of H2O was passed through a Sep-Pak C18-Light column (pre-treated with 10 mL of ethanol and 10 mL of H2O), eluted with 1 mL of 50% ethanol and diluted with 5 mL of saline to obtain a dual-target probe of NTSR1 and PSMA (denoted as 68 Ga-NT-PSMA).

[0055] The schematic diagram of the synthetic route of steps 1) to 5) is as follows Figure 1 As shown; Step 2) The mass spectrometry identification results of DOTA-NT-PSMA are as shown Figure 2 Schematic diagram of the radionuclide labeling process in step 6) is shown as Figure 3 shown.

[0056] Example 2

[0057] This embodiment is a dual-target probe of NTSR1 and PSMA. The preparation method thereof is different from that of embodiment 1 in that, in step 6), the chelated radionuclide is 64 Cu.

[0058] Example 3

[0059] This embodiment is a dual-target probe of NTSR1 and PSMA. The preparation method thereof is different from that of embodiment 1 in that, in step 6), the chelated radionuclide is 111 In.

[0060] Performance Testing

[0061] 1. Stability

[0062] The radiochemical purity of the final product of Example 1 was analyzed by radio-HPLC, and a radiochemical purity of greater than 95.00% was obtained. 68 The radiolabeled yield of Ga-NT-PSMA products was 71.27% ± 1.58%. The HPLC purity and 68 The Radio-HPLC analysis results of Ga-NT-PSMA are as follows Figure 4 The cells were analyzed by incubation in saline or 5% human serum for 1 h or 2 h. 68 In vitro stability of Ga-NT-PSMA. In vivo stability was analyzed in urine and blood of KM mice (male, 18-20 g, n=3) 30 min after injection. Radiochemical purity was analyzed by radio-HPLC. After incubation in saline and human serum, the radiochemical purity reached more than 95% within 2 h. The mice were injected within 1 h. 68 Ga-NT-PSMA, blood was taken for analysis, and the radiochemical purity was higher than 90%, such as Figure 4 As shown, the results indicate that the dual-target probe of the present invention has good in vivo and in vitro stability.

[0063] 2. Fat-water partition coefficient

[0064] Example 1 68The octanol-water partition coefficient of Ga-NT-PSMA was obtained by vortexing the mixture for 3 min and centrifuging at 10000 rpm for 5 min in a 1.5 mL test tube (n=5), then taking 3 samples (60 μL) from each phase and counting the radioactivity using a γ counter (Hidex, Turku, Finland). The partition coefficient was calculated as the counts in octanol divided by the counts in phosphate buffered saline. 68 The lipid-water partition coefficient of Ga-NT-PSMA is logD 7.4 =-3.54±0.08, indicating that the dual-target probe of the present invention has excellent hydrophilicity.

[0065] 3. Cellular uptake assay

[0066] Example 1 68 The cellular uptake experiment of Ga-NT-PSMA was tested on two cells, PC3 with high expression of NTSR1 and 22Rv1 with high expression of PSMA. PC3 and 22Rv1 cells were trypsinized, centrifuged and resuspended in RPMI-1640, and then aliquoted into 12-well cell culture plates at a concentration of 2×10^5 cells / ml. 68 In the presence of Ga-NT-PSMA, the cells were incubated at 37°C for 5, 30, 60 and 120 min. 68 Ga-DOTA-NT20.3 (Synthesized by reference: 68 Ga-DOTA-NT-20.3Neurotensin Receptor 1PET Imaging as a Surrogate for Neuroendocrine Differentiation of Prostate Cancer. doi:10.2967 / jnumed.121.263132) or 68Ga-PSMA-11 (synthesized according to the literature: PET imaging with a [68Ga] gallium-labelled PSMA ligand for the diagnosis of prostate cancer: biodistribution in humans and first evaluation of tumour lesions. doi: 10.1007 / s00259-012-2298-2) was used for single blocking, and 33μg DOTA-NT-PSMA was used for double blocking. After each time point, the culture medium was removed and the cells were washed with cold PBS (pH 7.40.01M, 1mL×2). After incubation, the samples were washed three times with ice-cold PBS and the radioactivity (cpm) was measured using a calibrated γ counter. The uptake was expressed as the percentage of the total radioactivity of the radiotracer added to the cells (%ID / 10^ 6 cells). Figure 5 As shown, 68 Ga-NT-PSMA effectively bound to PC3 and 22Rv1 cells in a time-dependent manner. At 120 min, the uptake in PC3 cells reached 5.02±0.44ID% / 1 million cells, and the uptake in 22Rv1 cells reached 2.06±0.44ID% / 1 million cells (see Figure 5 A). 68 The binding of Ga-NT-PSMA to NTSR1 or PSMA was significantly inhibited by the corresponding blockers (P < 0.001) (see Figure 5 B), which indicates that the dual-target probe of the present invention has high affinity to NTSR1 and PSMA at the in vitro cell level and the inhibitor specifically blocks them.

[0067] 4. IC 50 Determination

[0068] The competitive binding assay was used to determine the 68 IC of Ga-NT-PSMA relative to PC3 and 22Rv1 cells 50 Unlabeled ligand with different concentrations (0.01 nM to 1 μM) was added to the radiolabeled 68Ga-NT-PSMA (74 kBq / well) was fixed in active cells. After incubation at 37 ° C for 60 minutes, the cells were washed with cold PBS (pH 7.4, 0.01M, 1 mL×2), and then 200 μL NaOH (0.1M) was added for cell lysis. The lysate was collected and measured by a γ counter. The experiment was repeated 3 times for each concentration. The experiment was repeated 3 times for each concentration. The relative IC was obtained by nonlinear curve fitting using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) 50 .like Figure 6 As shown, the experimental results 68 IC of Ga-NT-PSMA in PC3 cells 50 39.32nM (see Figure 6 A), IC in 22Rv1 cells 50 is 63.47nM (see Figure 6 B), the results show that the dual-target probe of the present invention has high affinity for both NTSR1 and PSMA.

[0069] 5. Cell internalization and efflux

[0070] For endogenization experiments, after incubation for 60 min in a CO2 incubator at 37°C, the medium was discarded and the cells were washed twice with PBS and then twice with glycine (0.2 M). Subsequently, the cells were lysed with NaOH (1 M) at 37°C for 10 min. For efflux experiments, the radioactive medium was removed after 60 min of incubation and the cells were incubated with non-radioactive medium for 0-180 min. Figure 7 As shown, the internalization test showed 68 Ga-NT-PSMA interacts with cell surface proteins primarily through membrane binding (see Figure 7 A); outflow experiments showed that 68 Ga-NT-PSMA showed moderate cellular efflux in vitro, retaining approximately 73% and 67% of the initial accumulated radioactivity in PC3 and 22Rv1 cells, respectively, after incubation in radioactive-free medium for 180 min (see Figure 7 B), which indicates that the dual-target probe of the present invention has a strong retention ability in both cells and the receptor-ligand binding is relatively stable.

[0071] 6. Micro-PET / CT imaging of model mice

[0072] The model mice inoculated with PC3 and 22Rv1 were injected with 7.4 MBq of Example 1 into the tail vein. 68After the injection of Ga-NT-PSMA, the model mice were subjected to 2-h dynamic micro-PET / CT imaging. The PET images were iteratively reconstructed using the three-dimensional ordered subset expectation maximum (OSEM) algorithm and converted into the percentage of injected dose per gram of tissue (%ID / g) by delineating the region of interest. Figure 8 As shown, within 120 minutes after injection 68 The corresponding time-activity curves of Ga-NT-PSMA and representative maximum intensity projection (MIP) images taken 90 min after injection showed rapid biodistribution and specific uptake of both tumor xenografts in vivo, mainly through renal metabolism. 68 Ga-NT-PSMA rapidly accumulated in the tumor 15 min after injection, with 4.37 ± 0.54% ID / g in PC3 xenografts (see Figure 8 A), 22Rv1 xenografts were 5.06±0.33%ID / g (see Figure 8 B). The accumulation remained relatively stable until 120 min after injection, with PC3 xenografts at 3.07±0.51%ID / g and 22Rv1 xenografts at 1.39±0.20%ID / g. 68 The uptake of Ga-NT-PSMA was significantly inhibited by the corresponding unlabeled reagent (P < 0.01) (see Figure 8 C and Figure 8 D) The above indicates that the dual-target probe of the present invention is specifically taken up by tumors in mice.

[0073] In mice bearing AI and AD tumors 68 Ga-NT-PSMA and corresponding monomers 68 Ga-PSMA-11 and 68 The tumor uptake of Ga-DOTA-NT20.3 was compared and analyzed. To further demonstrate that the dual-target probe imaging of the present invention is independent of androgen levels, a DU145 xenograft model was included. All of these radiotracers were performed on the same mouse at 24h intervals. Fig. 9 As shown, PET imaging shows 68 Ga-NT-PSMA can be imaged with high contrast in both PC3 and 22Rv1 xenografts, whereas the corresponding monomer can only be used for a single xenograft (see Fig. 9 A) Among them, PC3 xenografts 68 The uptake of Ga-NT-PSMA was 3.64±0.49%ID / g. 68 The uptake of Ga-DOTA-NT20.3 was 5.60±1.42%ID / g(ns); in 22Rv1 xenografts, 68The uptake of Ga-NT-PSMA was 1.94±0.29%ID / g. 68 The uptake of Ga-PSMA-11 was 3.94±0.26%ID / g (P<0.001) (see Fig. 9 B), it can be seen that the introduction of the structure targeting NTSR1 will reduce the affinity of the entire molecule to the PSMA structure ( 68 The uptake ratio of Ga-NT-PSMA 68 Ga-PSMA-11 low), but 68 The uptake of Ga-NT-PSMA is still at a high level, and high-contrast imaging can be achieved; this is because: using a shorter lysine as the linker can maintain the affinity of the entire molecule for the two targets, thereby maintaining a higher level of uptake; further combined with the hydrophilic chelating ring DOTA, 68 High contrast imaging of Ga-NT-PSMA. In DU145 xenografts, 68 The uptake of Ga-NT-PSMA also varies with the monomer 68 Ga-DOTA-NT20.3 is comparable (2.49±0.20%ID / g vs. 2.34±0.18%ID / g, ns) (see Fig. 9 C and Fig. 9 D), further confirming the in vivo specificity of the dual-target probe of the present invention to NTSR1 and PSMA.

[0074] 7. In vitro biodistribution in PC3, 22Rv1 and DU145 tumor model mice

[0075] For biodistribution studies, administration was via the lateral tail vein. 68 Ga-NT-PSMA (0.74-2.96 MBq in 200 μL saline). Mice were sacrificed by CO2 asphyxiation 90 min after injection, and the radioactivity in target tissues (blood, muscle, bone, liver, spleen, kidney, heart, lung, brain, intestine, and stomach) was measured using a gamma counter. Organ weights were recorded, and the radioactivity of each sample was calculated as the percentage of injected dose per gram of tissue (%ID / g), corrected for radioactive decay. The biodistribution and tumor uptake of the radiotracer in vivo were evaluated. Fig.10 As shown, 90 min after injection, significant tumor radioactivity accumulation was observed in the three models, with 3.64±0.54%ID / g in PC3 xenografts, 1.78±0.09%ID / g in 22Rv1 xenografts, and 2.53±0.66%ID / g in DU145 xenografts (see Fig.10A). PC3 xenografts had the greatest ratio of tumor to non-tumor organ uptake, tumor / muscle = 6.46 ± 0.99, tumor / blood = 6.29 ± 0.79. 22Rv1 xenografts had lower tumor uptake, but a higher ratio of tumor uptake to non-target organs, tumor / muscle = 4.34 ± 0.87, compared with 2.48 ± 0.66 for DU145 xenografts (see Fig.10 B) These findings together demonstrate that the dual-target probe imaging of the present invention is not affected by androgen dependence.

[0076] 8. Immunohistochemistry of tumor model mice

[0077] Tumor tissues of PC3, 22Rv1 and DU145 tumor-bearing mice were taken, fixed with formalin and embedded in paraffin, and then incubated with NTSR1 antibody (ab133579, Abcam, China) and PSMA antibody (ab207178, Abcam, China) overnight, sliced ​​and stained, and image processed. Fig.11 As shown, the results of immunohistochemical staining showed that NTSR1 was highly expressed in PC3 and DU145 tumor-bearing mice, and was lowly expressed in 22Rv1 tumor-bearing mice, and the expression in PC3 tumor-bearing mice was higher than that in DU145. PSMA was highly expressed in 22Rv1 tumor-bearing mice, and was lowly expressed in PC3 and DU145 tumor-bearing mice. The results proved that the probe of the present invention is suitable for imaging of the three selected tumor model mice, and tumor uptake is positively correlated with receptor expression.

[0078] The above experimental results indicate that the dual-target probe provided by the present invention can target NTSR1 and PSMA tumor-specific uptake in an androgen-independent manner, and can be imaged with high contrast in androgen-dependent 22Rv1 tumor-bearing mice, and androgen-independent PC3 and DU145 tumor-bearing mice, and can be rapidly metabolized out of the body through the kidneys, thus providing a nuclear medicine imaging tool that is not interfered by anti-androgens for the response evaluation after androgen deprivation therapy.

[0079] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. 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 embodiments here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A dual-target probe of NTSR1 and PSMA, characterized in that: The structure of the dual-target probe is as follows: In the structure, n is an integer of 4 to 7; M is a radionuclide, and Chelater is a radionuclide chelating group.

2. The dual-target probe according to claim 1, characterized in that: The M is 68 Ga, 64 Cu, 18 F. 86 Y. 90 Y. 89 Zr, 111 In, 99m Tc, 11 C. 123 I. 125 I or 124 At least one of I.

3. The dual-target probe according to claim 1, characterized in that: The Chelater is DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3 or isonitrile.

4. The dual-target probe according to claim 1, characterized in that: The structure of the dual-target probe is as follows:

5. The method for preparing the dual-target probe according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: synthesizing a polypeptide structure NT20.3 targeting NTSR1 by a solid phase synthesis method, modifying the N-terminus of the polypeptide structure NT20.3 with lysine, and modifying the N-terminus of the lysine close to the carboxyl group with an amino acid to synthesize a structure DUPA-6-Ahx targeting PSMA; bonding a radionuclide chelating group to the lysine; cleavage and purification; and labeling the radionuclide to obtain the dual-target probe.

6. Use of the dual-target probe according to any one of claims 1 to 4 as a nuclear medicine probe for treatment monitoring in androgen deprivation therapy.

7. Use of the dual-target probe according to any one of claims 1 to 4 in the preparation of radionuclide imaging diagnostic reagents.

8. The use according to claim 7, characterized in that: The nuclear imaging diagnostic reagent is a diagnostic reagent for tumors with high expression of NTSR1 and / or PSMA.

9. The use according to claim 8, characterized in that: The diagnostic reagent is an early detection reagent, a prognosis judgment reagent or a therapeutic effect evaluation reagent.

10. The use according to claim 8, characterized in that: The tumor is a prostate tumor, a breast tumor or a pancreatic tumor.

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