Cyclic polypeptide radiopharmaceuticals for PD-L1 targeting and preparation method and use thereof

Abstract

The present invention discloses a cyclic peptide radiopharmaceutical for targeting PD-L1 and its preparation method. The radiopharmaceutical is a radionuclide-bifunctional chelator-PDLP12. The radionuclide is labeled with a peptide via the bifunctional chelator. The PDLP12 peptide is a cyclic structure composed of 12 amino acids. In vivo, the radiopharmaceutical uses the cyclic peptide PDLP12 to specifically recognize PD-L1, enabling specific nuclear medicine positron emission tomography (PET) imaging diagnosis of PD-L1-positive tumors or lesions, such as autoimmune diseases and cardiovascular and cerebrovascular diseases.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a cyclic peptide radiopharmaceutical for targeting PD-L1, and a preparation method and use thereof. Background Art

[0002] In the existing technology, immune checkpoints are signaling pathways that play an important role in mediating immune tolerance and preventing excessive immune responses. Among them, the programmed death receptor-1 (PD-1) / programmed death ligand 1 (PD-L1) signaling pathway is highly expressed in the microenvironments of various malignant tumors, affecting the transmission of antigen signals. Tumor cells in the tumor microenvironment highly express PD-L1, which mediates the inhibition and apoptosis of specific effector T cells by binding to PD-1 on the surface of immune cells, thereby promoting the immune escape of tumor cells and inducing unlimited proliferation of tumor cells. Therefore, immune checkpoint inhibition therapy can activate the immune system and kill tumor cells. Currently, PD-L1 immune blocking therapy is the fastest-developed and most widely clinically used immune checkpoint inhibition therapy.

[0003] Although PD-L1 immunoblocking therapy has achieved satisfactory results in different types of tumors, not all patients are suitable for this treatment. Several clinical studies have shown that the tumor treatment response rate of patients using PD-L1 antibodies alone is only 20%. The prognosis of PD-L1 immunotherapy is related to the in vivo expression of relevant biomarkers. Its accurate detection can be used to screen patients who are most likely to respond to PD-L1 immunotherapy, and in the early stages of the disease, responsive tumors can be distinguished from refractory tumors through detection. In existing technologies, conventional biopsy, immunohistochemistry staining, and hematological biomarkers have been widely used to detect PD-L1 expression. However, due to the high heterogeneity of PD-L1 expression in primary tumors and metastases and the dynamic changes in spatiotemporal expression, these conventional detection methods cannot fully and comprehensively evaluate the true expression changes of PD-L1. In recent years, with the rapid development of nuclear medicine PET molecular imaging technology, this technology can non-invasively, quantitatively and visually monitor the expression of relevant immune checkpoints at the living molecular level, providing more accurate, real-time and comprehensive information on PD-L1 expression levels, thereby screening patients suitable for PD-L1 immunotherapy and estimating its efficacy.

[0004] In recent years, PET imaging probes targeting PD-L1 have emerged, but they have numerous drawbacks. For example, some probes require prolonged patient exposure; or, due to their high lipophilicity, some probes result in high uptake in normal organs and a low target / non-target ratio. Therefore, the development of new PET imaging probes targeting PD-L1 with improved physicochemical properties and imaging performance has important clinical and societal implications. Summary of the Invention

[0005] The present invention aims to provide a cyclic peptide radiopharmaceutical for PD-L1 specific imaging, a preparation method and use thereof, which has low preparation cost, good in vitro and in vivo stability and good tumor imaging effect.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] The first aspect of the present invention provides a cyclic peptide radiopharmaceutical for targeting PD-L1, wherein the drug is formed by labeling a cyclic peptide with a radionuclide via a bifunctional chelating agent.

[0008] Wherein, the cyclic peptide structure is PDLP12, as shown in formula (I):

[0009]

[0010] Wherein, the radionuclide is 68 Ga, 64 Cu or 18 F;

[0011] The bifunctional chelating agent is any one or more of DOTA, NOTA or their derivatives;

[0012] Further, the bifunctional chelating agent is DOTA-NHS, NOTA-NHS, DOTAGA-NHS, NOTAGA-NHS, p-SCN-Bn-DOTA or p-SCN-Bn-NOTA;

[0013] A second aspect of the present invention provides a method for preparing the cyclic peptide radiopharmaceutical targeting PD-L1, comprising the following steps:

[0014] S1 Preparation of bifunctional chelating agent - PDLP12: Dissolve the cyclic peptide PDLP12 in a suitable solvent, then add an appropriate amount of alkaline reagent to adjust the pH to a weak base, add 1.2 to 30 times the mass of the bifunctional chelating agent, mix well, and react at room temperature for 2 to 24 hours. Separate and purify the reaction mixture by HPLC, collect the product peak, and lyophilize the collected product peak liquid to obtain a white powder, namely the bifunctional chelating agent - PDLP12;

[0015] S2 Preparation of radionuclide-bifunctional chelator-PDLP12: dissolve the bifunctional chelator-PDLP12 obtained in step S1 in an appropriate amount of sterile water for injection, add a weak acidic buffer to adjust the pH to weak acidity, then add 5mCi to 2Ci of radionuclide thereto, and heat in a water bath at 80 to 120°C for 10 to 30 minutes to prepare the radionuclide-bifunctional chelator-PDLP12;

[0016] Furthermore, in step S1, the suitable solvent is water for injection, DMSO or DMF;

[0017] Furthermore, in step S1, the alkaline reagent is triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA);

[0018] Furthermore, in step S1, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: mobile phase: phase A organic phase is acetonitrile containing 0.1% v / v trifluoroacetic acid; phase B aqueous phase is 0.1% w / w trifluoroacetic acid aqueous solution; elution conditions: at 0 minutes: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes;

[0019] Furthermore, in step S1, the retention time of the product peak is in the range of 10 to 13 minutes;

[0020] Furthermore, in step S2, the weakly acidic reagent is a NaAc buffer solution with a concentration of 0.5 to 2 mol / L;

[0021] Furthermore, when the radionuclide is 68 Ga or 64 Cu, said step S2 comprises the following steps: dissolving the bifunctional chelating agent - PDLP12 obtained in step S1 in an appropriate amount of sterile water for injection, adding a 0.5-2 mol / L NaAc buffer solution to adjust the pH to 4.0-6.5, then adding 5 mCi-2 Ci of radioactive nuclide ions thereto, heating in a water bath at 80-120° C. for 10-30 min, and cooling to room temperature to prepare a radioactive nuclide 68 Ga or 64 Cu-bifunctional chelator-PDLP12;

[0022] Or further, when the radionuclide is 18 F, said step S2 comprises the following steps: mixing the bifunctional chelating agent - PDLP12 obtained in S1 with AlCl3 solution and 5mCi to 2Ci radioactive nuclide ions, adjusting the pH to 4.0 to 6.5, reacting at 80 to 120°C for 10 to 30 minutes, cooling to room temperature, and preparing the radioactive nuclide 18 F- bifunctional chelating agent - PDLP12, wherein the bifunctional chelating agent - PDLP12 and AlCl3 solution are mixed in a ratio of 20-300 μg bifunctional chelating agent - PDLP12: 0.004-0.04 mmol AlCl3;

[0023] The third aspect of the present invention provides a detection reagent comprising any one of the above-mentioned cyclic peptide radiopharmaceuticals for targeting PD-L1;

[0024] A fourth aspect of the present invention provides use of any of the above-mentioned cyclic peptide radiopharmaceuticals for targeting PD-L1 in the preparation of a PET imaging probe for treating and targeting PD-L1.

[0025] The present invention is further described in detail. The radionuclide is labeled with the PDLP12 polypeptide (PDLP12 polypeptide is a cyclic structure composed of 12 amino acids) through a bifunctional chelator. The radiopharmaceutical realizes specific nuclear medicine positron emission tomography (PET) imaging diagnosis of PD-L1-positive tumors or PD-L1-positive lesions (autoimmune diseases, cardiovascular and cerebrovascular diseases, etc.) through the specific recognition of PD-L1 by the cyclic peptide PDLP12 in vivo. The schematic diagram of the structure of the formed radionuclide-bifunctional chelator labeled PDLP12 polypeptide is shown in FIG. Figure 1 As shown, wherein the polypeptide PDLP12 polypeptide shown in formula (I) is obtained by reacting DOTA, NOTA or their derivatives and radionuclide, represents a DOTA or NOTA derivative, Represents radionuclide.

[0026] Beneficial effects

[0027] The PD-L1 targeting cyclic peptide PDLP12 of the present invention is a disulfide bond cyclized polypeptide composed of 12 amino acids. Through structural modification, an amino group that can condense with a bifunctional chelating group is reserved to achieve functional changes, transforming conventional chemotherapy drugs into nuclear medicine molecular imaging probes, thereby expanding its application field. The cyclic peptide PDLP12 is coupled with DOTA, NOTA or its derivatives through the reserved amino group and uses them as bifunctional chelating agents to bind radionuclides. 68 Ga or 64 Cu or 18 F is labeled onto the cyclic peptide molecule, and thus the radionuclide is carried to the lesion site with high expression of PD-L1, such as the site of tumor, inflammation, trauma, fibrosis, etc., through the specific recognition of the cyclic peptide and PD-L1 in the body, and the lesion is diagnosed non-invasively and in real time using nuclear medicine positron emission tomography technology.

[0028] Through this impact diagnosis, patients with high expression of PD-L1 can be screened out, which has a positive guiding role in the use of antibody drugs to treat the disease. It not only reduces the blindness of patients in using drugs, but also collects case data of patients with high expression of PD-L1 from a clinical perspective, which has positive guiding significance for further research on this target and development of new drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of a cyclic peptide radiopharmaceutical targeting PD-L1 formed by labeling PDLP12 polypeptide with a radionuclide through a bifunctional chelator.

[0030] Figure 2 Prepared in Example 1 68 TLC results of Ga-DOTA-PDLP12.

[0031] Figure 3 Prepared in Example 2 68 TLC results of Ga-p-SCN-Bn-DOTA-PDLP12.

[0032] Figure 4 Prepared in Example 3 64 TLC results of Cu-NOTA-PDLP12.

[0033] Figure 5 for 18 F TLC results of Al-NOTA-PDLP12.

[0034] Figure 6 Prepared in Example 1 68 In vitro stability of Ga-DOTA-PDLP12.

[0035] Figure 7 For injection 68 Pharmacokinetic profile and half-life of Ga-DOTA-PDLP12.

[0036] Figure 8 For injection 68 PET / CT images of B16F10 tumor-bearing (PD-L1 positive) C57BL / 6 mice at 30, 60, and 90 min after Ga-DOTA-PDLP12.

[0037] Figure 9 For injection 68 PET / CT images of BALB / c-nu nude mice bearing NCI-H292 tumors (PD-L1 positive) at 30, 60, and 90 minutes after Ga-DOTA-PDLP12. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] Embodiment 1: 68 Synthesis and labeling of Ga-DOTA-PDLP12

[0040] (1) A cyclic peptide radiopharmaceutical for targeting PD-L1, wherein the bifunctional chelating agent (DOTA)-PDLP12 is DOTA-NHS, and its structure is shown in Formula (II):

[0041]

[0042] (2) Preparation of cyclic peptide radiopharmaceuticals for PD-L1 targeting:

[0043] S1: Weigh 1.0 mg of cyclic peptide PDLP12 and 1.2-30 equivalents of a bifunctional chelating agent and dissolve them in water for injection, wherein the bifunctional chelating agent is DOTA-NHS. Add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to 8.5-9.0, mix well, and react at room temperature for 12 hours. After the reaction, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC to collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C 18 Column (specifications: 250×4.6 mm, 5 μm); mobile phase: organic phase A is acetonitrile containing 0.1% v / v trifluoroacetic acid; aqueous phase B is an aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at 0 minutes: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes; the flow rate is 1.0 mL / min. The retention time t R The total weight was 11.23, which was concentrated by rotary evaporation and freeze-dried into a white powder, namely the bifunctional chelating agent - PDLP12;

[0044] S2: Dissolve the bifunctional chelating agent-PDLP12 obtained in S1 in an appropriate amount of sterile water for injection, wherein the mass ratio of the bifunctional chelating agent-PDLP12 to sterile water for injection is 1:1, take 50-100 μL of the bifunctional chelating agent-PDLP12 aqueous solution, add NaAc buffer to adjust the pH to 4.0-6.5, and then add 5 mCi-1 Ci of radioactive nuclide to it. 68 Ga ions are heated at 100°C for 10 to 30 minutes to prepare a radionuclide-bifunctional chelating agent-cyclic peptide.

[0045] The labeling rate was determined by the instant thin layer chromatography (iTLC) method: the stationary phase was glass fiber silica gel chromatography paper, the mobile phase was a mixture of 1 mol / L ammonium acetate and methanol in a volume ratio of 1:1, and the detection equipment was a Mini-Scan radioactive TLC thin layer scanner. The radiochemical purity was 98.5%, see Figure 2 .

[0046] Example 2: 68 Synthesis and labeling of Ga-p-SCN-Bn-DOTA-PDLP12

[0047] S1: Weigh 1.0 mg of cyclic peptide PDLP12 and 3-30 equivalents of a bifunctional chelating agent and dissolve them in water for injection, wherein the bifunctional chelating agent is p-SCN-Bn-DOTA. Add an appropriate amount of triethanolamine (TEA) or triethylenediamine TEAB to adjust the pH to a weak alkaline pH of 8.5-9.0. Mix well and react at 60°C for 60 minutes. After the reaction, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC to collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C18 column (250×4.6mm, 5μm); mobile phase: organic phase A is acetonitrile containing 0.1% v / v trifluoroacetic acid; aqueous phase B is aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at 0 minutes: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes; the flow rate is 1.0 mL / min. The retention time t R The total weight of the product was 11.23, which was concentrated by rotary evaporation and freeze-dried to a white powder, namely the bifunctional chelating agent - PDLP12, whose structure is shown in formula (III):

[0048]

[0049] S2: Dissolve the bifunctional chelating agent-PDLP12 obtained in S1 in an appropriate amount of sterile water for injection, wherein the mass ratio of the bifunctional chelating agent-PDLP12 to sterile water for injection is 1:1, take 50-100 μL of the bifunctional chelating agent-PDLP12 aqueous solution, add NaAc buffer to adjust the pH to 4.0-6.5, and then add 5 mCi-1 Ci of radioactive nuclide to it. 68 Ga ions are heated at 100°C for 10 to 30 minutes to prepare a radionuclide-bifunctional chelating agent-cyclic peptide.

[0050] The labeling rate was determined by the instant thin layer chromatography (iTLC) method: the stationary phase was glass fiber silica gel chromatography paper, the mobile phase was a mixture of 1 mol / L ammonium acetate and methanol in a volume ratio of 1:1, and the detection equipment was a Mini-Scan radioactive TLC thin layer scanner. The radiochemical purity was 97.66%, see Figure 3 .

[0051] Example 3: 64 Synthesis and labeling of Cu-NOTA-PDLP12

[0052] (1) A cyclic polypeptide radiopharmaceutical for targeting PD-L1, which is a radionuclide-bifunctional chelator-cyclic polypeptide. The bifunctional chelator-PDLP12 has the structural formula (III):

[0053]

[0054] (2) Preparation of cyclic peptide radiopharmaceuticals for PD-L1 targeting:

[0055] S1: Weigh 1.0 mg of cyclic peptide PDLP12 and 1.2 to 3 times the mass of the bifunctional chelating agent of the cyclic peptide and dissolve them in water for injection, wherein the bifunctional chelating agent is DOTA-NHS, add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to a weak alkaline pH of 8.5 to 9.0, mix well, and react at room temperature for 12 hours. After the reaction, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC, and collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C18 column (250×4.6 mm, 5 μm); mobile phase: organic phase A is acetonitrile containing 0.1% v / v trifluoroacetic acid; aqueous phase B is an aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at 0 minutes: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes; the flow rate is 1.0 mL / min. The retention time t R The result was 10.89 points, which was concentrated by rotary evaporation and freeze-dried into a white powder, namely the bifunctional chelating agent - PDLP12;

[0056] S2: Dissolve the bifunctional chelating agent-PDLP12 obtained in S1 in an appropriate amount of sterile water for injection, wherein the mass ratio of the bifunctional chelating agent-PDLP12 to sterile water for injection is 1:1, take 50-100 μL of the bifunctional chelating agent-PDLP12 aqueous solution, add 1 mol / L NaAc buffer, adjust the pH to 4.0-6.5, and then add 5 mCi-1 Ci of radioactive nuclide thereto.64 Cu, heated at 100°C for 10 to 30 minutes to prepare a radionuclide-bifunctional chelating agent-cyclic peptide.

[0057] The labeling rate was determined by the instant thin layer chromatography (iTLC) method: the stationary phase was glass fiber silica gel chromatography paper, the mobile phase was a mixed solution of 1 mol / L ammonium acetate and methanol in a volume ratio of 1:1, and the detection equipment was a Mini-Scan radioactive TLC thin layer scanner. The radiochemical purity was 99.0% (see Figure 4 ).

[0058] Embodiment 4: 18 Synthesis and labeling of FAl-NOTA-PDLP12

[0059] (1) A cyclic polypeptide radiopharmaceutical for targeting PD-L1, wherein the bifunctional chelating agent is NOTA or a derivative thereof, for example, a compound represented by formula (III), and the radionuclide is 18 F:

[0060]

[0061] (2) Preparation of cyclic peptide radiopharmaceuticals for PD-L1 targeting:

[0062] S1: Weigh 1.0 mg of cyclic peptide PDLP12 and 1.2 to 3 times the mass of the bifunctional chelating agent of the cyclic peptide and dissolve them in water for injection, wherein the bifunctional chelating agent is DOTA-NHS, add an appropriate amount of triethanolamine (TEA) or N,N'-diisopropylethylamine (DIEA) to adjust the pH to a weak alkaline pH of 8.5 to 9.0, mix well, and react at room temperature for 12 hours. After the reaction, dilute the reaction solution with water for injection and separate and purify it by semi-preparative HPLC, and collect the product peak. The chromatographic conditions include: chromatographic column: Phenomenex Gemini C18 column (250×4.6 mm, 5 μm); mobile phase: organic phase A is acetonitrile containing 0.1% v / v trifluoroacetic acid; aqueous phase B is an aqueous solution containing 0.1% w / w trifluoroacetic acid; elution conditions: at 0 minutes: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes; the flow rate is 1.0 mL / min. The retention time t R The result was 10.89 points, which was concentrated by rotary evaporation and freeze-dried into a white powder, namely the bifunctional chelating agent - PDLP12;

[0063] S2: The bifunctional chelating agent PDLP12 obtained in S1 is dissolved in an appropriate amount of sterile water for injection, wherein the mass ratio of the bifunctional chelating agent PDLP12 to sterile water for injection is 1:1. 50 μL of the bifunctional chelating agent PDLP12 aqueous solution is mixed with 3 μL of a 0.1 mol / L AlCl3 solution and 5 mCi to 2 Ci radioactive nuclide ions. The pH is adjusted to 4.0 to 6.5 using 100 μL of 0.1 mol / L acetate buffer, and the mixture is reacted at 95°C for 30 minutes. The mixture is cooled to room temperature to prepare a radioactive nuclide. 18 F-Bifunctional chelator-PDLP12.

[0064] The labeling rate was determined by thin layer chromatography (TLC): the stationary phase was a glass silica gel chromatography plate, the mobile phase was a mixed solution of acetonitrile and water in a volume ratio of 95:5, and the detection equipment was a Mini-Scan radioactive TLC thin layer scanner. The radiochemical purity was 98.53% (see Figure 5 ).

[0065] Example 5 68 In vitro stability assay of Ga-DOTA-PDLP12

[0066] The prepared 68 Ga-DOTA-PDLP12 probes were added to 200 μL of physiological saline solution (about 100 μCi) and placed in a 37°C shaking incubator for different time periods to determine the radiochemical purity (iTLC analysis). 68 The Ga-DOTA-PDLP12 probe solution was added to 0.2 mL of serum and incubated at 37°C for different time periods to determine the radiochemical purity (iTLC analysis). The probe's in vitro stability in saline and serum was observed (see Figure 6 ). The results show: 68 The radiochemical purity of the Ga-DOTA-PDLP12 probe was greater than 95% after incubation in physiological saline and serum for 180 minutes, indicating that the probe maintained good stability in physiological saline and serum.

[0067] Example 6 68 In vivo pharmacokinetic analysis of Ga-DOTA-PDLP12

[0068] Each healthy mouse was injected with about 0.1-0.4 mCi of 68Ga-DOTA-PDLP12 was used for pharmacokinetic studies. Blood was collected by cutting the tail vein at 1, 3, 5, 10, 15, 30, 60, and 90 minutes after administration. The blood was weighed and gamma counted. After correction for radioactive decay, the results were calculated and expressed as the percentage of radioactive counts per gram of blood to the total injected radioactive counts (%ID / g). The blood half-life was calculated using DAS2.0 software (see results). Figure 7 ). The results show: 68 Ga-DOTA-PDLP12 probe in T 1 / 2(fast) 0.9min, T 1 / 2(slow) The time taken for the probe to be cleared from the blood was 37.0 min, indicating that the probe was cleared quickly from the blood.

[0069] Example 7 68 Biological evaluation of Ga-DOTA-PDLP12

[0070] The following is a probe targeting PD-L1 prepared according to the method of Example 1 of the present invention. 68 The PET / CT imaging performance of Ga-DOTA-PDLP12 is described as follows:

[0071] (1) Preparation of mouse B16F10 melanoma model

[0072] Melanoma B16F10 cells were used as an example to establish a mouse tumor model. The cells were digested with a digestion solution (0.25wt% trypsin / 0.02wt% EDTA), rinsed with sterile PBS, and resuspended in sterile saline to make 5×10 4 / μL cell suspension. Take 4-5 week old C57BL / 6 black mice and inoculate 5×10 6 The animals were cultured in an SPF animal room with 100 μL of cells per animal. After 2 to 3 weeks, the animals were used for experiments when the average tumor diameter reached 0.8 to 1.0 cm.

[0073] (2) Preparation of mouse NCI-H292 (human lung cancer cell (lymph node metastasis)) tumor model

[0074] NCI-H292 cells were digested with a digestion solution (0.25 wt% trypsin / 0.02 wt% EDTA), washed with sterile PBS, and resuspended in sterile saline to make 8 × 10 4 4-6 week old BALB / c-nu nude mice were subcutaneously inoculated with 8×10 6 Cells were added to each animal (approximately 100 μL) and maintained in an SPF animal room. After 3-5 weeks, the animals were used for experiments when the average tumor diameter reached 0.8-1.0 cm.

[0075] (3) 68 MicroPET / CT imaging of Ga-DOTA-PDLP12 in a melanoma model

[0076] Tumor-bearing mice (n=3) cultured in (1) were anesthetized and placed prone on a PET / CT bed. 0.1 mL of a probe (7.4 MBq) was injected via the tail vein. Static acquisition was performed for 10 minutes 30, 60, and 90 minutes after the injection of the probe. Figure 8 ) and tumor models and NCI-H292 (human lung cancer cells (lymph node metastasis)) (see Figure 9 ) image clarity, tumor uptake rate, retention time in tumors, and uptake in normal tissues, especially kidney, liver, and muscle (see Figure 7 ). The results show: 68 In both tumor models, the Ga-DOTA-PDLP12 probe was taken up by tumors 30 minutes after injection, reaching maximum uptake 60 minutes after injection, and exhibiting the highest target / non-target ratio compared to contralateral muscle tissue. Furthermore, the probe was primarily cleared via the kidneys. This suggests that the probe binds well to tumor PD-L1 and can be used for molecular imaging monitoring of tumor PD-L1 expression.

Claims

1. A cyclic peptide radiopharmaceutical for targeting PD-L1, characterized in that: The drug is formed by labeling a cyclic peptide with a radionuclide through a bifunctional chelating agent. Wherein, the cyclic peptide structure is PDLP12, as shown in formula (I): Wherein, the radionuclide is 68 Ga, 64 Cu or 18 F, the bifunctional chelating agent is DOTA, NOTA, DOTA-NHS, NOTA-NHS, DOTAGA-NHS, NOTAGA-NHS, p-SCN-Bn-DOTA or p-SCN-Bn-NOTA.

2. A method for preparing the cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 1, characterized in that: The following steps are involved: S1 Preparation of bifunctional chelating agent - PDLP12: Dissolve the cyclic peptide PDLP12 in a suitable solvent, then add an appropriate amount of alkaline reagent to adjust the pH to a weak base, add 1.2 to 30 times the mass of the bifunctional chelating agent, mix well, and react at room temperature for 2 to 24 hours. Separate and purify the reaction mixture by HPLC, collect the product peak, and lyophilize the collected product peak liquid to obtain a white powder, namely the bifunctional chelating agent - PDLP12; S2 Preparation of radionuclide-bifunctional chelator-PDLP12: dissolve the bifunctional chelator-PDLP12 obtained in step S1 in an appropriate amount of sterile water for injection, add a weak acidic reagent to adjust the pH to weak acidity, then add 5mCi~2Ci of radionuclide thereto, heat in a water bath at 80~120℃ for 10~30min to prepare radionuclide-bifunctional chelator-PDLP12.

3. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S1, the suitable solvent is water for injection, DMSO or DMF.

4. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S1, the alkaline reagent is triethanolamine or N,N'-diisopropylethylamine.

5. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S1, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: mobile phase: phase A organic phase is acetonitrile containing 0.1% v / v trifluoroacetic acid; phase B aqueous phase is 0.1% w / w trifluoroacetic acid aqueous solution; elution conditions: at 0 minute: phase A is 20% v / v, phase B is 80% v / v; at 25 minutes: phase A is 45% v / v, phase B is 55% v / v; at 25.1 minutes: phase A is 100% v / v, phase B is 0% v / v; elution is stopped at 30.0 minutes.

6. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S1, the retention time of the product peak is in the range of 10 to 13 minutes.

7. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S2, the weakly acidic reagent is NaAc buffer or HAc.

8. The method for preparing a cyclic peptide radiopharmaceutical targeting PD-L1 according to claim 2, characterized in that: In step S2, When the radionuclide is 68 Ga or 64 Cu, said step S2 comprises the following steps: dissolving the bifunctional chelating agent - PDLP12 obtained in step S1 in an appropriate amount of sterile water for injection, adding a 0.5-2 mol / L NaAc buffer solution to adjust the pH to 4.0-6.5, then adding 5 mCi-2 Ci of radioactive nuclide ions thereto, heating in a water bath at 80-120°C for 10-30 minutes, and cooling to room temperature to prepare a radioactive nuclide 68 Ga or 64 Cu-bifunctional chelator-PDLP12; Or when the radionuclide is 18 F, said step S2 comprises the following steps: mixing the bifunctional chelating agent - PDLP12 obtained in S1 with AlCl3 solution and 5mCi to 2Ci radioactive nuclide ions, adjusting the pH to 4.0 to 6.5, reacting at 80 to 120°C for 10 to 30 minutes, cooling to room temperature, and preparing the radioactive nuclide 18 F- bifunctional chelating agent - PDLP12, wherein the bifunctional chelating agent - PDLP12 and AlCl3 solution are mixed in a ratio of 20 to 300 μg bifunctional chelating agent - PDLP12: 0.004 to 0.04 mmol AlCl3.

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