Protein binding group modified dimeric PSMA diagnosis and treatment integrated probe

Abstract

The invention belongs to the technical field of medicine, and particularly relates to a protein binding group modified dimeric PSMA diagnosis and treatment integrated probe. According to the probe, on the basis of PSMA-617, PSMA structure small molecules are subjected to dimerization, albumin binding groups are added into the molecules so that the residence time of the molecules in plasma can be prolonged, tumor uptake can be increased, meanwhile, in order to optimize pharmacokinetics of the whole molecules, PEG linking groups are added so that the T / NT ratio can be increased, and the probe is used for diagnosis and treatment of prostate cancer. Diagnosis and treatment integration is realized. A cellular uptake internalization result shows that compared with the 177Lu-PSMA-617, the 177Lu-PSMA-NAI, the 177Lu-PSMA-I, which is optimized by the dimerization structure, has the advantages that the combining capacity of a medicine and tumor cells is obviously improved; according to an albumin binding experiment 177Lu-PSMA-NAI, the binding rate of 177Lu-PSMA-I albumin is higher than that of 177Lu-PSMA-617, and it is proved that the binding rate of the medicine and albumin is really increased through a bimerization structure and an albumin binding group, and the expected effect is basically achieved.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a dual-functional PSMA diagnostic and therapeutic probe modified with a protein-binding group. Technical Background

[0002] Global cancer data released by IARC shows that in 2020, the number of newly diagnosed prostate cancer cases worldwide exceeded 1,400,000, seriously affecting men's health and becoming the second most common malignancy after lung cancer. Early diagnosis of the disease is crucial for the treatment of prostate cancer patients. Conventional imaging examinations such as CT / MRI have obvious limitations in detecting the primary focus and local staging of prostate cancer. Radiopharmaceuticals have been applied clinically in the early diagnosis and treatment of prostate cancer due to their sensitivity and specificity. Prostate-specific membrane antigen (PSMA) is significantly overexpressed on the surface of prostate cancer cells, reaching 100-1000 times that of normal cells, and is closely related to the occurrence, development, and treatment prognosis of tumors. This characteristic makes PSMA an ideal biomarker target for prostate cancer diagnosis and treatment research. Commonly used radiopharmaceuticals in clinical practice are mainly 177 Lu-PSMA-617 and 177 Lu-PSMA-I&T. However, the above drugs have deficiencies in actual application: high uptake in non-target organs (salivary glands, kidneys, intestines, etc.); certain blood toxicity; poor efficacy in about 30% of patients. Therefore, the research aims to study and solve the blood toxicity of radiopharmaceuticals, reduce the uptake of non-target organs in prostate cancer patients, and improve the diagnostic and therapeutic effects. Summary of the Invention

[0003] To solve the problems existing in the above-mentioned prior art, the present invention is implemented by the following technical solutions:

[0004] The main content of the present invention is to construct and synthesize a PSMA small molecule modified with PEG. To improve the affinity of the molecule for PSMA, on the basis of the PSMA-617 structure, the PSMA structure small molecule is dimerized, and a tetrahydronaphthalene ring albumin-binding group is added to the molecule to increase the residence time of the molecule in plasma, enhance tumor uptake, and at the same time, a PEG linking group is added to optimize the pharmacokinetics of the molecule and improve the drug T / NT ratio, for the diagnosis and treatment of prostate cancer, realizing the integration of diagnosis and treatment.

[0005] Furthermore, the above synthesized drug is labeled with the radionuclide 177 Lu. Compared with other radionuclides, 177Lu is easily obtainable and can emit γ-rays with energies of 112.95 keV (6.40%), 208 keV (11.00%), 321.3 keV (0.219%), 249.7 keV (0.212%), and 71.65 keV (0.15%). Through the use of traditional SPECT imaging, lesions can be located and measured. It can emit three β-particles with energies of 498 keV (79.3%), 380 keV (9.1%), and 176 keV (12.2%) respectively, and can be used for the treatment of tumors.

[0006] Based on the above technical solutions, the beneficial effects of the present invention are as follows:

[0007] Prostate-specific membrane antigen (PSMA) is the most commonly used and important target in the current diagnosis of prostate cancer. Therefore, the present invention designed and synthesized a PSMA small molecule for the diagnosis and treatment of prostate cancer to achieve the integration of diagnosis and treatment. A dimeric PSMA small molecule was constructed to increase the binding ability of the targeting ligand to improve its targeting and imaging effects in a prostate cancer-bearing mouse model; an albumin-binding group was added to the molecule to increase the residence time of the molecule in plasma, extend the plasma half-life, and increase tumor uptake. At the same time, in order to optimize the pharmacokinetics of the whole molecule, a PEG linking group was added.

[0008] After the molecule was successfully prepared, radionuclide 177 Lu labeling was carried out with PSMA-NAI as the premise. After purification, the drug labeling rate and radiochemical purity should both be >95%; the drug should have certain stability and hydrophilicity in vivo and in vitro, and the binding ability to human serum albumin is significantly improved compared with PSMA-617.

[0009] The probe affinity was measured using the PC3 PIP cell line with high expression of PSMA after transfection. Small animal SPECT / CT imaging and biodistribution were used to evaluate its tumor targeting and distribution in important organs. A 30-day treatment study was carried out on PC3 PIP-bearing mice. The targeting of the PSMA-NAI probe in a human prostate tumor-bearing mouse model should be significantly improved. At the same time, the plasma half-life was increased, showing higher tumor uptake, promoting drug metabolism, and shortening the residence time in other tissues and organs. Compared with the treatment results of 177 Lu-PSMA-617, the survival rate of mice treated with the PSMA-NAI small molecule inhibitor was significantly increased, showing significant treatment advantages and more favorable safety.

[0010] The PSMA-NAI probe should have the potential for clinical translation, or could become a promising tumor diagnosis and treatment integrated targeting molecular probe to achieve early diagnosis and treatment of prostate cancer patients, improve the pharmacokinetics in patients, reduce drug toxicity, and improve the prognosis and quality of life of patients. Description of the Drawings

[0011] The drawings herein are used to illustrate specific embodiments of the present experiment and form a part of the application, but do not limit the embodiments of the present invention.

[0012] Figure 1 It is the mass spectrum of PSMA-NAI.

[0013] Figure 2 It is the ultraviolet spectrum obtained by HPLC detection of PSMA-NAI.

[0014] Figure 3 It is 177 the Radio-HPLC spectrum of Lu-PSMA-NAI.

[0015] Figure 4 It is 68 the in vitro stability data of Ga-PSMA-NAI.

[0016] Figure 5 It is 177 the uptake data of Lu-PSMA-NAI on PC3-PIP cells.

[0017] Figure 6 It is 177 the in vivo distribution data of Lu-PSMA-NAI in the PC3-PIP model.

[0018] Figure 7 It is 177 the SPETCT / CT imaging map of Lu-PSMA-617 in the PC3-PIP model.

[0019] Figure 8 It is 177 the SPETCT / CT imaging map of Lu-PSMA-NAI in the PC3-PIP model.

[0020] Figure 9 It is the tumor inhibition rate of the PC3-PIP model after injecting 1mCi 177 Lu-PSMA-617 and 177 Lu-PSMA-NAI drugs respectively.

[0021] Figure 10 It is the survival rate of the PC3-PIP model after injecting different doses (0.5 / 1 / 2mCi) of 177 Lu-PSMA-617 and 177 Lu-PSMA-NAI respectively.

[0022] Specific Embodiment

[0023] The content of the present invention will be further described below in conjunction with specific embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

[0024] Example 1

[0025] 177 Preparation of Lu-PSMA-NAI

[0026] Using PSMA-NAI as the reaction precursor, configure it into a solution of 1 μg / μl. Analyze the purified product by analytical HPLC and MS. The mass spectrometry diagram is shown in Figure 1 , and the HPLC diagram is shown in Figure 2 . Take 20 μl of the above solution and add it to 37 MBq - 1000 MBq of 177 LuCl3 solution, and adjust the pH = 4 - 5 with 0.25 M sodium acetate buffer solution. React at 90 °C for 15 min. Add 5 ml of normal saline to the reaction solution and pass it through a C18light column, and rinse the residual 177 LuCl3 on the C18 column with 5 ml of normal saline. Elute the product in the C18 column with 2 ml of 50% ethanol solution and finally dilute the ethanol concentration to less than 10%. The radiochemical purity detected by HPLC > 98%, as shown in Figure 3 .

[0027] Example 2

[0028] 68 In vitro stability of Ga-PSMA-NAI

[0029] Take 5 mCi of 68 Ga-PSMA-NAI, add 1 ml of PBS solution, place it at 37 °C, and take samples at 0.5 h, 1 h, and 2 h respectively, and measure the radiochemical purity by Radio-HPLC.

[0030] Take 5 mCi of 68 Ga-PSMA-NAI, add it to 1 ml of 10% FBS solution, place it at 37 °C, and take samples at 0.5 h, 1 h, and 2 h respectively, and measure the radiochemical purity by Radio-HPLC. The obtained data are shown in Figure 4 .

[0031] Example 3

[0032] 177 Cell uptake and internalization of Lu-PSMA-NAI

[0033] Culture PC3-PIP cells in 1640 medium containing 10% FBS. After the cells grow to 70 - 80%, digest them with trypsin and inoculate them into a 24-well plate (about 1.5*10 5Cells / Well), and cultured overnight at 37°C with 5% CO2. The next day, the cells were starved with pure medium 1640, and then about 50 μl / 1 μCi / well of the radiolabeled product was added. The cells were cultured in an incubator for 30, 60, and 120 min, and the uptake values of the cells at each time point were measured using a γ counter (n≥3). The obtained data are shown in Figure 5 or Table 1.

[0034] Table 1 PC3-PIP Cell Uptake and Internalization

[0035]

[0036] Example 4

[0037] 177 Detection of the Albumin Binding Rate of Lu-PSMA-NAI

[0038] 177 Lu-labeled PSMA-NAI and PSMA-617 were co-incubated with human serum albumin, counted and measured using a γ counter, and the albumin binding rates of the two drugs at 30 min and 2 h were calculated. The obtained data are shown in Table 2.

[0039] Table 2 Drug Albumin Binding Rate

[0040]

[0041] Example 5

[0042] 177 In Vivo Distribution of Lu-PSMA-NAI

[0043] Take 100 - 200 μci of 177 Lu-PSMA-NAI, and inject it into PC3-PIP tumor-bearing mice with high PSMA expression via the tail vein. After 4 h, 1 d, 3 d, 5 d, and 8 d, the mice were sacrificed by cervical dislocation, and the heart, blood, lung, liver, stomach, spleen, kidney, muscle, small intestine, bone, salivary gland, brain, and tumor of the mice were immediately collected for γ counting. And the percentage of the injected dose per gram of tissue (%ID / g) was calculated. The obtained data are shown in Figure 6 or Table 3.

[0044] Table 3 177 Tissue Distribution of Lu-PSMA-NAI in Tumor-Bearing Mice

[0045]

[0046] Example 5

[0047] 177 SPECT / CT Imaging of Lu-PSMA-617

[0048] Take 200 μCi of 177 Lu-PSMA-617 and inject it into PC3-PIP tumor-bearing mice with high PSMA expression via the tail vein. Perform SPECT / CT imaging at 4H, 1D, 3D, and 5D after injection. The results are as Figure 7 shown.

[0049] Example 6

[0050] 177 SPETCT / CT imaging of Lu-PSMA-NAI

[0051] Take 200 μCi of 177 Lu-PSMA-NAI and inject it into PC3-PIP tumor-bearing mice with high PSMA expression via the tail vein. Perform SPETCT / CT imaging at 4H, 1D, 3D, and 5D after injection. The results are as Figure 8 shown.

[0052] Example 7

[0053] 177 Treatment with Lu-PSMA-NAI

[0054] Take 177 Lu-PSMA-NAI and inject it into PC3-PIP tumor-bearing mice with high PSMA expression via the tail vein. After injecting 1 mCi of the drug into the tumor-bearing mice via the tail vein, perform a 30-day treatment. Measure the tumor and body weight of the mice every 2 days, record and analyze the tumor inhibition rate and the survival of the mice. The changes in tumor volume are shown in Table 4, and the tumor inhibition rate is as Figure 9 shown, and the overall survival rate of mice at different doses (0.5 / 1 / 2 mCi) is as Figure 10 shown.

[0055] Example 8

[0056] 177 Treatment with Lu-PSMA-617

[0057] Take 177 Lu-PSMA-617 and inject it into PC3-PIP tumor-bearing mice with high PSMA expression via the tail vein. After injecting 1 mCi of the drug into the tumor-bearing mice via the tail vein, perform a 30-day treatment. Measure the tumor and body weight of the mice every 2 days, record and analyze the tumor inhibition rate. The changes in tumor volume are shown in Table 4, and the tumor inhibition rate is as Figure 9 shown, and the overall survival rate of mice at different doses (0.5 / 1 / 2 mCi) is as Figure 10 shown.

[0058] Table 4 Changes in tumor volume of tumor-bearing mice after injecting the drug

[0059]

[0060] Result:

[0061] It can be seen from Figure 4 that 177 Lu-PSMA-NAI has good in vitro stability, and the radiochemical purity is still >95% after 2 h.

[0062] It can be seen from Figure 5 and Table 1 that 177 Lu-PSMA-NAI has high uptake and internalization in PC3-PIP cells with high PSMA expression, and the blocking experiment also proves that PSMA-NAI targets the PSMA target.

[0063] It can be seen from Table 1 that the small molecule drug of PSMA with dimerized structure and albumin-binding group modification does improve the drug's binding ability to albumin. This result can extend the drug's half-life and enhance its biological stability, etc.

[0064] It can be seen from Figure 6 and Table 3 that 177 the uptake of Lu-PSMA-NAI in mice, where the T / NT value can reach up to 14 at most, having a relatively high T / NT value.

[0065] It can be seen from Figure 7 and Figure 8 that 177 Lu-PSMA-617, 177 Lu-PSMA-NAI has specific uptake in the tumor site of PC3-PIP with high PSMA expression. 177 The albumin-binding group of Lu-PSMA-NAI increases the lipophilicity of the drug, resulting in increased kidney uptake. However, there is no uptake in the kidney during 3D imaging, and there is no obvious uptake in other organs.

[0066] It can be seen from Table 4 and Figure 9 that 177 Both Lu-labeled PSMA-617 and PSMA-NAI have therapeutic effects in tumor-bearing mice; the VT / V0 values at 30 D with 1 mCi treatment are respectively: for PSMA-NAI: 0.14 ± 0.07, and for PSMA-617: 0.04 ± 0.04).

[0067] It can be seen from Figure 10 that after treating mice with the PSMA-NAI drug, the survival rate of the mice at 30 days is better than that of PSMA-617, reducing the drug toxicity.

[0068] Although the main inventive examples affecting the factors of this experiment have been described and listed, those of ordinary skill in the art can understand that various changes, explorations, modifications, and combinations can be made to the embodiments without departing from the principles and purposes of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A PSMA-DOTA-NAI compound modified with a protein-binding group, and its structural formula is as follows:

2. A radiolabeled ligand, characterized in that: The ligand includes a radionuclide and the PSMA-DOTA-NAI compound or a salt thereof described in claim 1.

3. The ligand according to claim 2, characterized in that: The radionuclide of the ligand is selected from 68 Ga,[[]] 177 Lu,[[]] 99m Tc,[[]] 111 In,[[]] 67 Ga,[[]] 86 Y,[[]] 90 Y,[[]] 161 Tb,[[]] 186 Re,[[]] 188 Re,[[]] 64 Cu,[[]] 67 Cu,[[]] 211 At,[[]] 225 Ac,[[]] 18 F,[[]] 123 I,[[]] 124 I,[[]] 125 at least one of I.

4. The radionuclide of the ligand according to claim 3 is selected from 68 Ga, 177 Lu, or 225 at least one of Ac.

5. Use of the PSMA-DOTA-NAI compound or its ligand according to claim 1 or 2, characterized in that: Use of the compound or its ligand in a prostate cancer diagnosis and treatment integrated pharmaceutical composition or kit.

6. Use of the PSMA-DOTA-NAI compound or its ligand according to claim 1 or 2, characterized in that: Use in the preparation of a product for detecting and / or treating cells and / or tissues expressing prostate-specific membrane antigen.

7. The application according to claim 5, wherein: The cells and / or tissues expressing prostate-specific membrane antigen include prostate cancer, pre-metastatic prostate cancer, colon cancer, breast cancer, kidney cancer or bladder cancer.

8. The application according to claim 5, wherein: The product includes a prostate cancer diagnostic tracer or a therapeutic preparation.

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