Modified CAIX targeting cyclic peptide as well as nuclide marker and application thereof

Abstract

The invention belongs to the technical field of nuclear medicine molecular diagnosis and treatment, and discloses a modified CAIX targeting cyclic peptide and a nuclide marker and application thereof. According to the CAIX targeted cyclic peptide, a rigid bifunctional chelating agent RESCA is introduced to replace a traditional chelating agent, and a sulfonated or alkylated amino acid connector is combined, so that the enzymolysis resistance and in-vivo stability of the probe are remarkably improved. By optimizing the structure of the connector, the lipophilicity is reduced, liver and gall metabolism and non-specific uptake are reduced, and the high uptake rate of tumor target tissues is maintained. The cyclic peptide can label a diagnostic nuclide for PET imaging, or a therapeutic nuclide for intra-tumor irradiation therapy, using DOTA or NOTA in combination with an alkylated amino acid linker or an albumin ligand. Experiments show that the marker has high radiochemical purity, excellent pharmacokinetic characteristics and low kidney retention, and is suitable for precise diagnosis and targeted therapy of CAIX high expression tumors such as kidney cancer, colorectal cancer and brain glioma.

Description

Technical Field

[0001] The present invention belongs to the technical field of nuclear medicine molecular diagnosis and treatment, and specifically relates to a modified CAIX-targeted cyclic peptide, its radionuclide label, and applications thereof. Background Art

[0002] Carbonic anhydrase IX (CAIX), as a membrane protein, has attracted much attention in the field of tumor research. In many tumor types with hypoxia or specific tumor suppressor gene mutations, CAIX shows a high expression state, while in normal healthy tissues, its expression is relatively limited, mainly concentrated in the gastrointestinal epithelium. This differential expression characteristic between tumors and normal tissues makes CAIX a highly potential tumor diagnosis and treatment target. Previous studies have shown that the high expression of CAIX is closely related to tumor progression, poor prognosis, and the occurrence of metastasis. In clear cell renal cell carcinoma (ccRCC), due to the impaired function of the von Hippel - Lindau tumor suppressor factor, the expression of hypoxia-inducible factor 1α is dysregulated, which in turn promotes the continuous high expression of CAIX. In a hypoxic environment, and in some solid tumors such as colorectal cancer (CRC), breast cancer, and pancreatic ductal adenocarcinoma (PDAC), the transcriptional regulation of CAIX by hypoxia-inducible factor 1α is abnormal, leading to overexpression of CAIX.

[0003] In recent years, the application of radionuclide probes in tumor diagnosis and treatment has become increasingly widespread. Radionuclide probes can utilize the characteristics of radionuclides to achieve precise diagnosis and treatment of tumors. Among them, 68 Ga and 18 F-labeled probes can be used for positron emission tomography (PET) imaging. With their excellent imaging characteristics, they play a key role in early tumor detection, staging, and evaluation of treatment effects. 177 Lu and 225 Probes labeled with therapeutic radionuclides such as Ac can kill tumor cells by the rays released by the radionuclides, achieving the purpose of treating tumors. However, existing radionuclide probes for CAIX still have many deficiencies. Some probes have low uptake efficiency at the tumor site, making it difficult to achieve precise tumor localization and effective treatment. At the same time, some probes have high non-specific uptake in normal tissues, easily causing radiation damage to normal tissues and triggering adverse reactions. In addition, the performance of the probes such as stability, affinity, and specificity also needs to be further improved.

[0004] Developing efficient and safe CAIX cyclic peptide radionuclide probes has become a research hotspot in the current field of tumor diagnosis and treatment. The new CAIX cyclic peptide radionuclide probes can not only improve the accuracy and effectiveness of tumor diagnosis and treatment, but also provide more accurate diagnostic information for clinicians and formulate more targeted treatment plans for tumor patients, having important clinical significance and broad market prospects. Summary of the Invention

[0005] The object of the present invention is to provide a modified CAIX-targeting cyclic peptide, its radionuclide label and applications.

[0006] To achieve the object of the present invention, in a first aspect, the present invention provides a modified CAIX-targeting cyclic peptide, the structure of which is shown in Formula I, II, III, IV or V:

[0007] In a second aspect, the present invention provides a radionuclide label of the cyclic peptide, which is the cyclic peptide labeled with a radionuclide, and the radionuclide includes a diagnostic radionuclide and a therapeutic radionuclide.

[0008] Further, the diagnostic radionuclide may be selected from 68 Ga or 18 F, etc., preferably the cyclic peptide shown in Formula I, II, III or IV labeled with a radionuclide.

[0009] Further, the therapeutic radionuclide may be selected from 90 Y, 177 Lu, 225 Ac or 213 Bi, etc., preferably the cyclic peptide shown in Formula IV or V labeled with a radionuclide.

[0010] In a third aspect, the present invention provides a preparation method of the radionuclide label. When the radionuclide is 68 Ga, the preparation method of the radionuclide label includes the following steps: (1) Prepare 68 Ga nuclide by using a column-type germanium-gallium generator; (2) Elute 68 Ga with 2 - 4 mL of 0.03 - 0.06 M HCl solution and mix with 0.8 - 1.2 M NaAc; (3) Add the cyclic peptide to the 68 Ga solution obtained in step (2), mix well and heat at 37 °C for reaction for 10 - 20 min; (4) Purify the product obtained in step (3), and elute the product with absolute ethanol to obtain.

[0011] When the radionuclide is 18 F, the preparation method of the radionuclide label includes the following steps: 1) Prepare 18 F solution by using a cyclotron; 2) The18 H2 of F 18 O passes through the QMA ion exchange column to adsorb 18 F on the QMA column; rinse the above QMA column with 0.45 - 0.55 mL of physiological saline to elute 18 F; 3) Take the 18 physiological saline of F, mix it with KHP (potassium hydrogen phthalate) and AlCl3 solution, shake well and let it stand at room temperature for 4 - 6 min, then add the cyclic peptide, react at 37 °C for 10 - 20 min. After the reaction solution is cooled, load the product onto a C18 separation column, wash it with physiological saline and then elute it with ethanol to obtain the product.

[0012] When the radionuclide is 177 Lu, the preparation method of the radionuclide-labeled compound includes the following steps: (1) Add 177 Lu] LuCl3 solution (usually from a reactor or a generator) under sterile conditions; (2) Mix 177 Lu] LuCl3 solution with 0.8 - 1.2 M sodium acetate buffer solution to make the pH 3.8 - 5.5; (3) Add the cyclic peptide to the 177 Lu solution obtained in step (2), mix well and heat at 95 °C for 10 - 20 min; (4) Purify the product obtained in step (3), elute the product with absolute ethanol to obtain the product.

[0013] When the radionuclide is 225 Ac, the preparation method of the radionuclide-labeled compound includes the following steps: 1) Add 225 Ac] AcCl3 solution (usually from a reactor or a generator) under sterile conditions; 2) Mix 225 Ac] AcCl3 solution with 0.8 - 1.2 M sodium acetate buffer solution to make the pH 5; 3) Add the cyclic peptide to the 225 Ac solution obtained in step 2), mix well and heat at 95 °C for 10 - 30 min; 4) Purify the product obtained in step 3), elute the product with absolute ethanol to obtain the product.

[0014] Fourthly, the present invention provides the application of the diagnostic radionuclide-labeled compound in the preparation of tumor PET imaging reagents.

[0015] Fifthly, the present invention provides the application of the therapeutic radionuclide-labeled compound in the preparation of tumor radionuclide therapy drugs.

[0016] With the above technical solutions, the present invention has at least the following advantages and beneficial effects: (1) The CAIX cyclic peptide structure of the present invention has good in vivo stability, pharmacokinetic properties, affinity and specificity.

[0017] (2) The diagnostic radioactive molecular probe can be used for 68 Ga / 18 PET imaging of F-CAIX cyclic peptide; 68 Ga / 18 F-CAIX cyclic peptide can be used for screening, treatment prediction and efficacy monitoring of tumor patients such as CAIX-positive renal cell carcinoma (ccRCC), colorectal cancer (CRC), breast cancer and pancreatic ductal adenocarcinoma (PDAC).

[0018] (3) 68 Ga / 18 PET imaging of F-CAIX cyclic peptide can judge the efficacy response of patients to CAIX-targeted therapy, and judge whether the patients are suitable for using therapeutic radionuclides 90 Y / 177 Lu / 225 Ac / 213 Bi substitution 68 Ga / 18 for radionuclide targeted therapy.

[0019] (4) 90 Y / 177 Lu / 225 Ac / 213 Radionuclide targeted therapies such as Bi-CAIX cyclic peptide can provide new treatment means for drug-resistant CAIX tumor patients.

[0020] (5) The preparation method of 68 Ga / 18 F / 177 Lu / 225 Ac-CAIX cyclic peptide of the present invention has a high labeling rate. 68 The Ga labeling rate can be over 95%. 18 The F labeling rate can reach over 30%. 177 The Lu labeling rate can reach over 70%. 225 The Ac labeling rate can reach over 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figures 1a - 1e are the HPLC and mass spectrometry quality control results of 5 structures. Figure 1a are the quality control results of Formula I. Figure 1b are the quality control results of Formula II. Figure 1c are the quality control results of Formula III. Figure 1d are the quality control results of Formula IV.Figure 1e It is the quality control result of Formula V.

[0022] Figure 2 It is the optimized 18 Detection results of labeling rate and radiochemical purity of F-CAIX-RESCA-cyclic peptide (Formula III).

[0023] Figure 3 It is the optimized 18 Pharmacokinetic detection and parameter statistics of F-CAIX-RESCA-cyclic peptide (Formula III).

[0024] Figures 4a - 4c They are respectively the Micro-PET / CT imaging results of CAIX cyclic peptide Formula I in the HT-29 mouse model, the imaging results of the Block group, and the SUVmax statistics in the preferred embodiment of the present invention.

[0025] Figure 5 They are the Micro-PET / CT imaging results of CAIX cyclic peptide Formula II in the HT-29 mouse model and the imaging results of the Block group in the preferred embodiment of the present invention.

[0026] Figures 6a - 6c They are respectively the Micro-PET / CT imaging results of CAIX cyclic peptide Formula III in the HT-29 mouse model, the imaging results of the Block group, and the SUVmax statistics in the preferred embodiment of the present invention.

[0027] Figure 7 They are the Micro-PET / CT imaging results of CAIX cyclic peptide Formula IV in the HT-29 mouse model and the imaging results of the Block group in the preferred embodiment of the present invention.

[0028] Figures 8a - 8d They are respectively the Block biodistribution comparison, 4-hour biodistribution, specific values, and human dose estimation of CAIX cyclic peptide Formula III in the HT-29 mouse model in the preferred embodiment of the present invention.

[0029] Figures 9a - 9b They are respectively the blood routine analysis, body weight monitoring, and blood biochemical test results of the acute toxicity experiment of CAIX cyclic peptide Formula III in the preferred embodiment of the present invention.

[0030] Figures 10a - 10b They are respectively the radionuclide therapy experiment results of CAIX cyclic peptides Formula IV and V in the HT-29 mouse model in the preferred embodiment of the present invention, including 177 Lu-CAIX-cyclic peptide IV / V and 225 Ac-CAIX-cyclic peptide IV / V. Specific implementation manners

[0031] The present invention aims to provide a modified CAIX cyclic peptide radionuclide probe molecular structure, as well as a further obtained diagnostic and therapeutic radionuclide label, and its preparation method and application. Specifically, it relates to the preparation and application of a carbonic anhydrase IX (CAIX)-targeted cyclic peptide based on a metabolically optimized linker and its diagnostic and therapeutic radionuclide label. The CAIX-targeted cyclic peptide replaces the traditional chelator by introducing a rigid bifunctional chelator RESCA (Rigid Ethylene-bridged Cyclam Chelator Assembly), and combines with a sulfonated or alkylated amino acid linker (such as PPAc, Cys(SO3H)-His, Leu-Thr), significantly improving the anti-enzymatic degradation performance and in vivo stability of the probe. By optimizing the linker structure, the lipophilicity is reduced (the logP value is optimized from -6.51 to -3.06), reducing hepatobiliary metabolism and non-specific uptake (liver uptake is reduced to 0.54% ID / g), while maintaining a high uptake rate in tumor target tissues (92.6% ID / g). The cyclic peptide can be labeled with diagnostic radionuclides (such as 68 Ga, 18 F) for PET imaging, or therapeutic radionuclides (such as 177 Lu, 225 Ac) for tumor internal radiotherapy, using DOTA or NOTA combined with an alkylated amino acid linker or an albumin ligand (such as Leu-Thr-Leu-Leu, ABM). Experiments show that the label has a high radiochemical purity (>95%), excellent pharmacokinetic properties and low renal retention, and is suitable for the precise diagnosis and targeted therapy of CAIX-highly expressed tumors such as colorectal cancer, renal cancer, and breast cancer. The present invention provides an efficient and safe molecular probe solution for tumor diagnosis and treatment.

[0032] The present invention adopts the following technical solutions: The present invention provides a modified CAIX cyclic peptide, and the sequence of the modified CAIX cyclic peptide is (N-terminus - C-terminus): RESCA-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula I); RESCA-Cys(SO3H)-His-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula II); RESCA-Leu-Thr-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula III); DOTA - Leu - Thr - Leu - Leu - PPAc - Gln - [Cys(3MeBn) - Glu - Dpro - Asp - Af3(Cpsu) - Leu - Thr - Trp - Ser - Cys - Cys] - NH2 (Formula Ⅳ); ABM - DOTA - PPAc - Gln - [Cys(3MeBn) - Glu - Dpro - Asp - Af3(Cpsu) - Leu - Thr - Trp - Ser - Cys - Cys] - NH2 (Formula Ⅴ); The N - terminus or C - terminus of the cyclic peptide is modified by a bifunctional linker; The chelating agent contained in the cyclic peptide is RESCA, and this RESCA is connected to a sulfonated or alkylated amino acid linker; The linker is coupled with the chelating agent RESCA. Meanwhile, the linker can also be coupled with other chelating agents selected from DOTA, NOTA, HBED - CC, DTPA, or 3pC - NETA - NCS, etc.

[0033] The structure of the CAIX cyclic peptide is as shown in Formulas I, II, Ⅲ, Ⅳ, and Ⅴ:

[0034] The present invention provides a structural optimization scheme for a cyclic peptide - based radionuclide probe targeting carbonic anhydrase IX (CAIX).

[0035] Specifically, the modified CAIX - targeting cyclic peptides shown in Formulas I, II, and III are particularly suitable for preparing diagnostic radionuclide markers. Formulas I, II, and III show a progressive structural design, specifically as follows: Its molecular structural feature is that Formula Ⅰ is based on the DOTA chelation system of the prototype probe 68 Ga]Ga - DPI - 4452. The structure of the probe molecule is improved by replacing the traditional DOTA coordination system with a rigid - structure bifunctional chelating agent RESCA (Rigid Ethylene - bridged Cyclam Chelator Assembly). Compared with large - polarity chelating agents such as DOTA and NOTA, RESCA has significantly enhanced anti - enzymatic hydrolysis performance. Its rigid cyclic structure can effectively reduce the in - vivo metabolic degradation rate, thereby significantly extending the systemic circulation half - life of the probe.

[0036] In terms of the structural optimization of the molecular linker, through structure-activity relationship studies, the present invention found that: when using the PPAc (Pyridine-Propionic Acid) linker, the non-specific uptake rates of the probe in the gastrointestinal tract and liver parenchyma reached (28.4 ± 3.1)%ID / g and (18.7 ± 2.6)%ID / g, respectively. Based on the design strategy of reducing the overall lipophilicity of the molecule (the logP value was optimized from -6.51 to -3.06), the His-CA (Histidine-Carbonic Anhydrase) sequence (Formula II) was added to the PPAc linker. This novel linker achieved a dual optimization mechanism through molecular modification with sulfonic acid groups: on the one hand, the capture rate of the reticuloendothelial system (RES) was reduced through charge modification (the uptake by Kupffer cells decreased by 67%), and on the other hand, the hepatobiliary metabolic clearance rate decreased to (1.4 ± 1.9)%ID / g by means of electrostatic repulsion, thereby increasing the target-to-background ratio.

[0037] Although the RESCA-sulfonation design reduced hepatobiliary metabolism, the probe was partially cleared through the glomerular filtration pathway and at a slow rate. Further structural optimization showed that after changing to the Leu-Thr (leucine-threonine) dipeptide sequence linker (Formula III), the probe showed significant advantages in pharmacokinetics: 4 hours after injection, the non-specific uptake rates in the liver, kidney, and gastrointestinal tract decreased to (0.54 ± 0.11)%ID / g, (1.5 ± 0.1)%ID / g, and (1.14 ± 0.26)%ID / g, respectively, while the uptake in the tumor target tissue remained at (92.6 ± 3.8)% of the initial value. This improvement in metabolic characteristics was mainly attributed to the β-sheet secondary structure formed by the Leu-Thr linker, which effectively reduced kidney uptake.

[0038] In the field of radionuclide therapy, the present invention optimized the metabolic characteristics of the labeling ligands based on the DOTA or NOTA chelator systems. The modified CAIX-targeted cyclic peptides shown in Formula IV or V are particularly suitable for the preparation of radionuclide-labeled therapeutic agents, and their structural design is as follows: By introducing a specific polypeptide sequence Leu-Thr-Leu-Leu-PPAc (Formula IV) or ABM-PPAc (Formula V) as a linker structure, the renal metabolic properties of the drug were significantly optimized (the renal 4-hour uptake rate was reduced by ≥90%), effectively reducing the risk of radionuclide accumulation in non-target organs. Further, to improve the long-term stability of the drug in the circulatory system, the present invention for the first time covalently coupled an albumin-binding ligand (as shown in Formula V) to the end of the polypeptide sequence. Through the active binding mechanism targeting plasma albumin, the metabolic clearance rate was significantly slowed down while maintaining the tumor targeting efficiency (the target / non-target ratio was increased by more than 1.8 times). This design achieved the synergistic optimization of the metabolic clearance rate and the circulatory retention ability, providing a solution with both high efficiency and safety for the precise delivery of highly toxic α / β-radionuclides (such as 225 Ac, 177 Lu).

[0039] The present invention also provides a diagnostic and therapeutic radionuclide marker, which is a radionuclide-labeled modified CAIX cyclic peptide.

[0040] According to a specific embodiment of the present invention, the radionuclide can be a diagnostic radionuclide, and the diagnostic radionuclide is preferably a positron radionuclide 68 Ga or 18 F.

[0041] When the radionuclide is 68 Ga, the preparation method of the radionuclide-labeled CAIX cyclic peptide may include the following steps: 1) Prepare 68 Ga nuclide by using a columnar germanium-gallium generator; 2) Elute 68 Ga with 2-4 mL of 0.03-0.06 M HCl solution and mix with 0.8-1.2 M NaAc; 3) Add the modified CAIX cyclic peptide to the 68 Ga solution obtained in step 2), mix well and heat at 85-100 °C for 10-20 min; 4) Purify the product obtained in step 3), elute the product with absolute ethanol, and the obtained product is 68 Ga-labeled CAIX cyclic peptide.

[0042] Specifically, taking the modification with the bifunctional chelating agent RESCA as an example, 68 The labeling of the modified CAIX cyclic peptide with After the CAIX cyclic peptide was modified with the bifunctional coupling agent RESCA, the sequence RESCA-Leu-Thr-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula III) (RESCA-CAIX cyclic peptide) was obtained; the prepared RESCA-CAIX cyclic peptide was 68 Ga (T 1 / 2 = 68 min; β + : 89%; E = 511 keV) nuclide labeling, 68 Ga was used 68 Ge- 68 Ga generator for preparation. Take 3 mL of 0.05 M HCl solution to elute 68 Ga into 195 μL of 1 M NaAc; add 0.1 mL (60 μg) of the RESCA-CAIX cyclic peptide precursor to the above system, mix well, react at 95 °C for 10 min, elute radioactive impurities with 3 mL of physiological saline, and then elute the target compound with 0.8 mL of 80% ethanol 68 Ga-RESCA-CAIX cyclic peptide, and the labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. The obtained 68 Ga-RESCA-CAIX cyclic peptide had a radiochemical purity greater than 95%. When the labeling rate was less than 90%, it was separated and purified by a Sep-pak C18 column. The Sep-pak column needed to be activated with 5 mL of anhydrous ethanol and 5 mL of high-purity water for standby. Take an appropriate amount of the product preparation that has been sterile filtered for quality control inspection, and conduct subsequent research after all items are qualified.

[0043] When the radionuclide is 18 F, the preparation method of the radionuclide-labeled CAIX cyclic peptide may include the following steps: 1) Prepare 18 F solution using a cyclotron; 2) Pass the H2 18 O containing 18 F produced by the cyclotron through a QMA ion exchange column, and adsorb 18 F on the QMA column; rinse the above QMA column with 0.45 - 0.55 mL of physiological saline to elute 18 F; 3) Take the product obtained in step 2 18Mix the physiological saline of F with the KHP and AlCl3 solutions, shake well and let stand at room temperature for 4 - 6 min, then add the modified CAIX cyclic peptide, react at 37 °C for 10 - 20 min. After the reaction solution is cooled, load the product onto a C18 separation column, wash with physiological saline and elute with ethanol to obtain the product 18 F-labeled CAIX cyclic peptide

[0044] Specifically, taking the modification with the bifunctional chelator H3RESCA as an example, 18 The labeling of the modified CAIX cyclic peptide with F can be carried out by the following method: The sequence of the CAIX cyclic peptide modified with the bifunctional coupling agent H3RESCA is RESCA-Leu-Thr-PPAcCys(SO3H)-His-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula III) (RESCA-CAIX cyclic peptide); carry out 18 F (T 1 / 2 = 109.8 min; β + : 96.7%; E = 511 keV) nuclide labeling, 18 F is prepared using a cyclotron. The H2 18 F - containing 18 produced by the accelerator passes through a QMA ion exchange column, 18 F - is adsorbed into the QMA column, and the above QMA column is rinsed with 0.5 mL of physiological saline; take 0.1 mL of physiological saline containing 18 F - and place it in a reaction tube containing 11 µL of 10-fold KHP and 6 µL of 2 mM AlCl3 solution, mix well and let stand at room temperature for 5 min; add 10 µL of 10 mg / mL labeling precursor H3RESCA-CAIX cyclic peptide, react at 100 °C for 15 min; after the reaction solution is cooled to room temperature, purify the product with a C18 separation column, wash with 5 mL of physiological saline, and elute the product with 0.6 mL of 80% ethanol and pass through a 0.22 µm sterile filter membrane; after drying the solvent with N2, dilute with physiological saline to obtain the product preparation; measure the labeling rate and radiochemical purity using radio-HPLC or radio-TLC. The 18The radiochemical purity of the F-CAIX cyclic peptide is greater than 95% after separation and purification. When the labeling rate is less than 90%, it is separated and purified by a Sep-pak C18 column. The Sep-pak column needs to be activated with 5 mL of absolute ethanol and 5 mL of high-purity water for standby. An appropriate amount of the sterile-filtered product preparation is taken for quality control inspection, and subsequent research is carried out after all items are qualified.

[0045] According to the present invention, the radionuclide can also be a therapeutic radionuclide, in order to achieve the purpose of targeted molecular imaging therapy for tumors with high CAIX expression. The preferred therapeutic radionuclide is 90 Y, 177 Lu, 225 Ac and 213 at least one of Bi.

[0046] The CAIX cyclic peptide modified by the bifunctional coupling agent DOTA, the bifunctional coupling agent NOTA, the bifunctional coupling agent DTPA, and the bimodal bifunctional ligand 3pC-NETA-NCS can be used for the therapeutic radionuclide 90 Y, 177 Lu, 225 Ac or 213 Bi labeling to obtain 90 Y-CAIX cyclic peptide, 177 Lu-CAIX cyclic peptide, 225 Ac-CAIX cyclic peptide, 213 Bi-CAIX cyclic peptide therapeutic molecular probe.

[0047] The labeling of the therapeutic radionuclide can adopt various conventional methods in the art. According to a preferred embodiment of the present invention, the 90 Y, 177 Lu, 225 Ac or 213 Bi labeling method of CAIX cyclic peptide can adopt the following method: Taking 177 Lu as an example, the CAIX cyclic peptide modified by the bifunctional chelating agents DOTA, NOTA, DTPA, and 3pC-NETA-NCS is obtained to get the corresponding labeling precursor. Prepare the labeling buffer according to 3 mL of 0.05 M HCl corresponding to 195 µL of 1 M NaAc for standby; in 100 µL (60 µg) of the labeling precursor (DOTA-CAIX cyclic peptide, NOTA-CAIX cyclic peptide, DTPA-CAIX cyclic peptide, 3pC-NETA-CAIX cyclic peptide), add 100 - 150 µL of the labeling buffer, and then add 177 Lu; adjust the pH to 5.5, react at 70 - 95 o °C for 10 - 15 min. When the labeling rate is less than 90%, it is separated and purified by a Sep-pak C18 column to obtain 177Lu-CAIX cyclic peptide. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. The prepared 177 Lu-CAIX cyclic peptide had a radiochemical purity greater than 95% after separation and purification.

[0048] The present invention also provides the use of a radioisotope-labeled CAIX cyclic peptide for diagnosis in the preparation of a CAIX-targeted tumor PET imaging reagent.

[0049] The present invention also provides the use of a radioisotope-labeled CAIX cyclic peptide for treatment in the preparation of a CAIX-targeted tumor radionuclide therapy drug.

[0050] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0051] Example 1 Synthesis and Characterization of a CAIX-Targeted Cyclic Peptide with a Metabolically Optimized Linker 1. Experimental Purpose: To synthesize a carbonic anhydrase IX (CAIX)-targeted cyclic peptide based on a metabolically optimized linker and verify its structure.

[0052] 2. Experimental Procedures: (1) Design of the Cyclic Peptide Sequence: The target cyclic peptide sequences are as follows: RESCA-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula I); RESCA-Cys(SO3H)-His-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula II); RESCA-Leu-Thr-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula III); DOTA-Leu-Thr-Leu-Leu-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula IV); ABM-DOTA-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys-Cys]-NH2 (Formula V);

[0053] (2)Solid-phase synthesis: Using the Fmoc solid-phase synthesis method, with Rink Amide resin as the carrier, amino acids and linkers were coupled in sequence, and finally a cyclic structure was formed through a thiol-maleimide cyclization reaction.

[0054] (3)Purification and characterization: HPLC analysis: Chromatographic column: Kromasil 100-5C18 (4.6×250mm); Mobile phase A: 0.1% TFA acetonitrile, mobile phase B: 0.1% TFA water; Gradient: 0-20 min, 30% A → 60% A; 20-25 min, 60% A → 100% A; Flow rate: 1 mL / min; Detection wavelength: 220 nm.

[0055] Mass spectrometry (MS) verification: The molecular weights measured by MALDI-TOF MS were 2183.41 (theoretical value 2183.86); 2424.66 (theoretical value 2183.86); 2350.78 (theoretical value 2350.99), confirming the sequence correctness.

[0056] The results were as Figures 1a - 1e shown, proving that all 5 cyclic peptides were successfully synthesized and had high chemical purity.

[0057] Example 2 68 Preparation of Ga-labeled CAIX-targeted cyclic peptide The prepared CAIX-RESCA-cyclic peptide (the cyclic peptide shown in Formula I, II or III) was 68 labeled with the Ga radionuclide (T 1 / 2 = 68 min; β + : 96.7%; E = 511 keV) radionuclide, and was prepared by using a column-type germanium-gallium generator 68Ga nuclide, elute with 4 mL of 0.05 MhCl solution 68 Ga, and mixed with 1 M NaAc, the cyclic peptide was added to the obtained 68 Ga solution, mixed and heated at 37°C for 10-20 minutes. The product was purified by C18 separation column, washed with 5 mL of saline, eluted with 0.6 mL of 80% ethanol and filtered through a 0.22 μm sterile filter; the solvent was dried with N2 and diluted with saline to obtain the product preparation. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. 68 The labeling rate of Ga-CAIX-RESCA-cyclic peptide was about 92%, and the radiochemical purity was greater than 95%.

[0058] Example 3 18 Preparation of F-labeled CAIX-targeting cyclic peptide The prepared CAIX-RESCA-cyclic peptide (cyclic peptide shown in formula I, II or III) is subjected to 18 F(T 1 / 2 =109.8min; β + :96.7%; E=511 keV) nuclide labeling, 18 F is prepared using a cyclotron. Accelerator-produced 18 F - H2 18 O passes through a QMA ion exchange column, 18 F - was adsorbed onto the QMA column, and the QMA column was rinsed with 0.5 mL of saline; 0.1 mL of 18 F - The saline solution was placed in a reaction tube containing 11µL 10x KHP and 6µL 2mm AlCl3 solution, mixed and placed at room temperature for 5 minutes; 10µL 10mg / mL labeled precursor CAIX-RESCA-cyclic peptide was added and reacted at 100℃ for 15 minutes; after the reaction solution was cooled to room temperature, the product was purified by C18 separation column, washed with 5mL saline, 0.6mL 80% ethanol was used to elute the product and pass it through a 0.22μm sterile filter membrane; the solvent was blown dry by N2 and diluted with saline to obtain the product preparation, and the labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC.

[0059] Figure 2 Shown 18 The Radio-TLC test results of F-CAIX-RESCA-cyclic peptide (Formula III) were obtained by determination. 18 The labeling rate of F-CAIX-RESCA-cyclic peptide (Formula III) is about 92%, and the radiochemical purity is greater than 99%.

[0060] Example 4 In vitro stability analysis of purified 18 F-CAIX-RESCA-cyclic peptide Take 10 μL of the purified product containing 1.11 MBq (30 μCi) 18 Add F-CAIX-RESCA-cyclic peptide to 200 μL of normal saline (or 5% hSA solution) and incubate at 4°C. Take out 37-74 kBq (1-2 μCi) samples at 0 h, 2 h, 12 h, 24 h, 36 h, and 60 h of incubation for radio-TLC analysis. Analysis method: Take 2 μL of the 18 F-CAIX-RESCA-cyclic peptide normal saline solution or 18 F-CAIX-RESCA-cyclic peptide 5% hSA solution and add it to 20 μL of saturated EDTA, mix well, and perform radio-TLC analysis. Drop 2 μL of the sample 1 cm from the bottom of the No. 1 Whatman filter paper, place it in the normal saline developing system. After complete development, take out the filter paper and dry it, and perform radio-TLC detection. The Rf values of free 18 F and 18 F-CAIX-RESCA-cyclic peptide are 0.9-1 and 0-0.1 respectively; the results show that 18 F-CAIX-RESCA-cyclic peptide has good stability in normal saline solution or 5% hSA solution within 4 h.

[0061] The above experiments show that 18 F-CAIX-RESCA-cyclic peptide structure has good stability.

[0062] Example 5 Pharmacokinetic analysis of optimized 18 F-CAIX-RESCA-cyclic peptide (Formula III) in normal KM mice Prepare 5 KM mice (female, 5-6 weeks old, 18-20 g). Use the optimized 18 F-CAIX-RESCA-cyclic peptide (Formula III) diluted with normal saline to 18.5 MBq / mL (0.5 mCi / ml). Inject 3.7 MBq (0.1 mCi, 200 μL) of the labeled product into each mouse via the tail vein. At the corresponding time points (1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h) after injecting the labeled product, take blood from the periorbital venous plexus of the mice using a capillary tube and place it in a radioimmunoassay tube. Take the 181% of the radioactivity of the F-CAIX-RESCA-cyclic peptide (Formula III), namely 0.037 MBq (1 μCi, 2 μL), was used as the reference activity for determination. The reference activity was measured together with the collected blood samples using a Gamma counter. After attenuation correction, data analysis was performed using Prism 6.0 software, and the percentage injection dose rate per gram of blood sample was calculated. The results were expressed as %ID / g ± SD.

[0063] The specific results are as Figure 3 shown. It can be seen from the figure that 18 the F-CAIX-RESCA-cyclic peptide (Formula III) has good pharmacokinetic properties and is suitable for in vivo imaging studies.

[0064] Example 6 18 PET / CT imaging study of F-CAIX-RESCA-cyclic peptides (Formulas I, II, III, and IV) in HT29 tumor-bearing mouse models Seven HT29 tumor-bearing mouse models were taken and divided into four groups (n = 2, n = 2, n = 2, n = 1) for imaging studies. 7.4 MBq (0.2 mCi, 200 μL) of 18 F-CAIX-RESCA-cyclic peptides (Formulas I, II, III, and IV) were injected into the tail vein respectively. For blocking imaging, 0.1 mg of non-radioactively labeled CAIX-RESCA-cyclic peptides (Formulas I, II, III) was injected simultaneously. Among them, 18 the mice in the F-CAIX-RESCA-cyclic peptide (Formula I) group were subjected to PET / CT imaging at 30 min, 60 min, 120 min, and 240 min after injection. The image results are as Figure 4a shown. Obvious uptake was visible in the tumor sites of the experimental group, showing a significant difference from the following inhibition imaging group. At the same time, there was relatively high uptake in the kidneys and gastrointestinal tract. The statistical results of SUVmax of important organs in the experimental group are as Figure 4b shown, and the statistical results of SUVmax of important organs in the inhibition imaging group are as Figure 4c shown.

[0065] 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula II) group were subjected to PET / CT imaging at 30 min, 60 min, and 120 min after injection. The results are shown in Figure 5 and relatively high uptake in the tumors and kidneys was still visible, and the uptake in the gastrointestinal tract was reduced compared with that of Formula I.

[0066] 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula III) group were subjected to PET / CT imaging and statistical analysis of the delineation of important organs at 40 min, 60 min, 120 min, 240 min, and 360 min after injection. The results are shown in Figure 6a ,Figure 6b and Figure 6c After optimization, the probe showed slightly increased uptake in the liver and kidneys in the early imaging stage compared to Formulas I and II. However, in the late imaging stage, the uptake in the liver, kidneys, stomach, and intestines decreased significantly, while the uptake in the tumor site did not show a significant decline and maintained a strong retention effect. The late imaging results were superior to those of Formulas I and II, showing greater prospects for clinical translation.

[0067] 18 For the mice in the F-CAIX-RESCA-cyclic peptide (Formula IV) group, PET / CT imaging was performed at 2 h, 3 h, and 4 h after injection. The results are shown in Figure 7 , indicating that as time elapsed, the uptake in the tumor site did not show a significant decline, which can be used to guide subsequent radionuclide therapy research.

[0068] Example 7 18 Distribution and inhibition distribution experiments of F-CAIX-RESCA-cyclic peptide (Formula III) in tumor-bearing mice, and estimation of human radiation dose Fifteen HT29 tumor-bearing mice (female, 5 - 6 weeks old, 18 - 20 g) were prepared and randomly divided into 5 time groups, with 3 mice in each group. After tail vein injection of 7.4 MBq (0.2 mCi, 200 μL) 18 of F-CAIX-RESCA-cyclic peptide, the mice were anesthetized and sacrificed at 5 min, 30 min, 60 min, 120 min, and 240 min respectively. Blood, heart, liver, spleen, lung, kidney, stomach, intestines, muscle, bone, and brain were taken for weighing, and the radioactivity counts of the organs were detected using a γ-counter. After attenuation correction, the in vivo biodistribution of each organ at different time points was statistically analyzed. For the blocking group, 0.1 mg of non-radioactive CAIX-RESCA-cyclic peptide was injected simultaneously.

[0069] As Figure 8a , Figure 8b , Figure 8c The results all showed that 18 F-CAIX-RESCA-cyclic peptide (Formula III) was mainly metabolized through the kidneys in animals, with low non-specific uptake in the whole body tissues, extremely high uptake and long retention in the tumor site, and could be blocked by the precursor. The human radiation dose was estimated using the biodistribution results as shown in Figure 8d , and the effective dose was significantly lower than the radioactive dose limit, demonstrating the radiation safety of the probe for clinical application.

[0070] By introducing metabolically optimized linkers (such as PPAc-Gln; Cys(SO3H)-His-PPAc-Gln and Leu-Thr-PPAc-Gln), the present invention significantly improved the pharmacokinetic properties of the CAIX-targeted cyclic peptide, endowing it with the advantages of high tumor targeting, low non-specific uptake, and rapid renal clearance. In combination with diagnostic and therapeutic radionuclides (such as 68Ga, 177 Lu) labeled, can simultaneously achieve accurate tumor imaging and efficient internal radiotherapy, and has broad clinical application prospects.

[0071] Example 8 optimized 18 Safety detection (toxicity experiment) of 18F-CAIX-RESCA-cyclic peptide (Formula III) Prepare 10 normal Kunming mice, divide them into two groups for experiments, namely the experimental group and the control group, n = 5. Each mouse in the experimental group was injected with 10 times the use dose 18 18F-CAIX-RESCA-cyclic peptide (Formula III) 74 MBq (2 mCi, 200 μL), and the control group was injected with the same volume of physiological saline solution. After a certain time, blood was taken respectively for routine blood test and liver and kidney function test of mice, and the body weight change of mice was continuously counted (16 days). The results are as Figure 9a , Figure 9b , there was no significant difference in the routine blood test results of the mice in the experimental group compared with the control group, the liver and kidney functions were within the normal range, and there was no difference in the body weight change of the mice in the experimental group compared with the control group. The experiment proved the high safety of the probe and is expected to conduct clinical translational research.

[0072] Example 9 177 Preparation of Lu-labeled CAIX-targeted cyclic peptide Using CAIX-targeted cyclic peptides of Formula IV and Formula V as precursors, the direct labeling method was adopted. Under sterile conditions, add 177 [177Lu]LuCl3 solution (specific activity ≥ 50 GBq / mg), and mix with 0.8 - 1.2 M sodium acetate buffer solution 177 [177Lu]LuCl3 solution to make the pH 3.8 - 5.5; add the cyclic peptide to 177 [177Lu]Lu solution, mix well and heat at 95 °C for 10 - 20 min; use a C-18 column to purify the obtained product, and elute the product with absolute ethanol to obtain the radionuclide therapy probe 177 [177Lu]-CAIX-DOTA-cyclic peptide (radiochemical purity > 98%, specific activity 22.5 MBq / μg).

[0073] Example 10 225 Preparation of 68Ga-labeled CAIX-targeted cyclic peptide Using CAIX-targeted cyclic peptides of Formula IV and Formula V as precursors, the direct labeling method was adopted. Under sterile conditions, add 225 [68Ga]GaCl3 solution (specific activity ≥ 50 GBq / mg), and mix with 0.8 - 1.2 M sodium acetate buffer solution 225 [68Ga]GaCl3 solution to make the pH 5; add the cyclic peptide to 225In an Ac solution, mix well and heat at 95 °C for reaction for 10 - 30 min; use a C-18 column to purify the obtained product, and elute the product with absolute ethanol to obtain the radionuclide therapy probe. 225 Ac-CAIX-DOTA-cyclic peptide (radiochemical purity > 98%, specific activity 22.5 MBq / μg).

[0074] Example 11 177 Lu-CAIX-DOTA-cyclic peptide and 225 Therapeutic effect evaluation of Ac-CAIX-DOTA-cyclic peptide on HT-29 renal cancer model mice Select 6 - 8-week-old BALB / c nude mice, and subcutaneously inoculate HT-29 human renal cancer cells (5×10 6 cells / mouse). After the tumor volume reaches 100 - 150 mm³, randomly divide them into seven groups (n = 5): treatment group: single intravenous injection of 177 Lu / 225 Ac-CAIX-DOTA-cyclic peptide-3 (Formula IV) (dose: 3.7 MBq and 1.85 MBq) and inject an equal dose of 177 Lu / 225 Ac-CAXI-DOTA-cyclic peptide-4 (Formula V) (linked with albumin ligand); blank control group: inject normal saline.

[0075] The efficacy evaluation adopts dynamic monitoring of tumor volume (calculated according to the formula V = 0.5×L×W²) and survival analysis (the end point is defined as tumor volume ≥ 1000 mm³ or body weight loss > 20%); The experimental results are as Figure 10a 、 Figure 10b shown. The data show that the 177 Lu / 225 Ac-CAXI-DOTA-cyclic peptide shows significant advantages in the HT-29 renal cancer model: the tumor volume of the treatment group increased by ≤ 30% compared with the baseline within 28 days (the positive control group increased by 85%, p < 0.001), and the median survival period was extended to 45 days (the control group ≤ 31 days); the biodistribution analysis shows that the tumor / kidney uptake ratio (T / K = 2.16) of the treatment group is significantly higher than that of the unmodified ligand group (T / K = 1.2), confirming that the optimized linker and albumin ligand can synergistically reduce kidney uptake and prolong circulation retention; in terms of safety, the body weight fluctuation of the treatment group < 5%, and there is no statistical difference in kidney function indicators (creatinine, urea nitrogen) between the treatment group and the blank group (p > 0.05), and no radioactive damage is seen in histopathology, verifying the clinical applicability of the present invention.

[0076] Example 12 213 Preparation of Bi-labeled CAIX-targeted cyclic peptide The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula IV or V) is subjected to 213 Bi nuclide labeling (T 1 / 2 =45.6min; α; E=8.4 MeV) nuclide labeling. Elute with 5 mL of pre-cooled 0.1 MhCl (containing 0.1% ascorbic acid) 225 Ac / 213 Bi generator, collects 213 Bi eluent. Remove impurities (such as 225 Ac leakage), washed with 0.1 MhCl and eluted with 0.5 MhNO3 213 Bi, dried with nitrogen, and then redissolved in 0.1 M HCl for later use. 213 Add 200µL 1 M NaAc-HAc buffer (pH 4.0) to the Bi solution, and the pH should be controlled at 3.5-4.0 after mixing. Add 50-100μg CAIX-DOTA-cyclic peptide IV or V (dissolved in 0.1 M NH4OAc, pH 5.0), and adjust the final volume to 2mL. Heat and stir at 95℃ for 10 minutes (sealed to prevent evaporation), and terminate the reaction by sudden cooling in an ice bath. Load the reaction solution onto a pre-activated (5mL ethanol 10mLh2O) C18 column, and wash with 10mL saline to remove free 213 Bi. Elute with 1 mL 70% ethanol / 30% saline 213 Bi-CAIX-DOTA-cyclopeptide, filtered through a 0.22μm sterile filter. Dry the ethanol with nitrogen, reconstitute with saline to a final volume of 1mL, and use immediately after aliquoting. Radio-HPLC analysis (flow rate 1mL / min, acetonitrile: 0.1% TFA gradient, detection of α radioactivity); purity > 95%.

[0077] Embodiment 13 90 Preparation of Y-labeled CAIX-targeting cyclic peptide The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula IV or V) is subjected to 90 Y-nuclides labeling (T 1 / 2 =64.1h; β - ; E = 2.28 MeV) nuclide labeling. Elute with 8 mL 0.05 MhCl (containing 0.1% ascorbic acid) 90 Sr / 90 Y generator, collects 90 The residual Y is removed by passing it through a strontium selective resin column (such as Sr-Resin®). 90 Sr, ensure that the radioactivity purity is > 99.9%. 90Add 500 μL of 1 M NH4OAc buffer (pH 5.0) to the Y solution to stabilize the pH of the reaction system at 4.5 - 5.0. Add 100 - 200 μg of CAIX-DOTA-cyclic peptide (dissolved in 0.1 M NH4OAc), and adjust the final volume to 3 mL. Stir at 80 °C for 30 minutes (sealed and protected from light), and terminate the reaction by cooling in an ice bath. Load the reaction solution onto a pre-activated C18 column (5 mL ethanol + 10 mL H2O), and wash with 10 mL of normal saline to remove free 90 Y. Elute with 1.5 mL of 60% ethanol 90 Y-CAIX-DOTA-cyclic peptide, and pass through a 0.22 μm sterile filter membrane. Blow dry the ethanol with nitrogen, and re-dissolve with normal saline to a final volume of 2 mL. Analyze by radio-HPLC (flow rate 1 mL / min, acetonitrile: 0.1% TFA gradient, detect β radioactivity); the expected purity is > 90%.

[0078] Although the present invention has been described in detail with general descriptions and specific embodiments above, based on the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A modified CAIX-targeting cyclic peptide, characterized in that, The structure is as shown in Formula I, II, III, IV or V: 。 2. The radionuclide-labeled compound of the cyclic peptide according to claim 1, wherein, It is the cyclic peptide labeled with a radionuclide, and the radionuclide includes a diagnostic radionuclide and a therapeutic radionuclide.

3. The radionuclide marker according to claim 2, wherein The diagnostic radionuclide is selected from 68 Ga or 18 F.

4. The radionuclide marker according to claim 3, wherein It is the cyclic peptide shown in Formula I, II, III or IV labeled with a radionuclide.

5. The radionuclide marker according to claim 2, wherein The radioactive nuclides for treatment are selected from 90 Y, 177 Lu, 225 Ac or 213 Bi.

6. The radionuclide marker according to claim 5, wherein It is the cyclic peptide shown in Formula IV or V labeled with a radionuclide.

7. The preparation method of the radionuclide marker according to claim 3 or 4, characterized in that, When the radionuclide is 68 Ga, the preparation method of the radionuclide marker comprises the following steps: (1) Prepared by using a columnar germanium-gallium generator 68 Ga nuclide; (2) Rinse with 2 - 4 mL of 0.03 - 0.06 M HCl solution 68 Ga, and mix with 0.8 - 1.2 M NaAc; (3) Add the cyclic peptide to the 68 Ga solution obtained in step (2), mix well and heat at 37 °C for 10 - 20 min; (4) Purify the product obtained in step (3), and elute the product with absolute ethanol to obtain the product. When the radionuclide is 18 F, the preparation method of the nuclide marker comprises the following steps: 1) Prepared using a cyclotron 18 F solution; 2) Pass the H2 18 O containing 18 F produced by the cyclotron through a QMA ion exchange column, and adsorb 18 F on the QMA column; rinse the above QMA column with 0.45 - 0.55 mL of normal saline to elute 18 F; 3) Take what is obtained in step 2) 18 Mix the physiological saline of F with the KHP and AlCl₃ solutions, shake well and let stand at room temperature for 4 - 6 min, then add the cyclic peptide, react at 37 °C for 10 - 20 min. After the reaction solution is cooled, load the product onto a C18 separation column, wash with physiological saline and elute with ethanol to obtain the product.

8. The preparation method of the radionuclide marker according to claim 5 or 6, characterized in that, When the radionuclide is 177 Lu, the preparation method of the radionuclide marker comprises the following steps: (1) Add 177 Lu] LuCl3 solution under sterile conditions; (2) Mix with sodium acetate buffer solution of 0.8 - 1.2M 177 and LuCl3 solution to make the pH 3.8 - 5.5; (3) Add the cyclic peptide to the 177 Lu solution obtained in step (2), mix well and heat at 95 °C for 10 - 20 min; (4) Purify the product obtained in step (3), and elute the product with absolute ethanol to obtain the product. When the radionuclide is 225 Ac, the preparation method of the nuclide marker comprises the following steps: 1) Add 225 Ac] AcCl3 solution under sterile conditions; 2) Mix with a sodium acetate buffer solution of 0.8 - 1.2 M 225 Ac]AcCl3 solution to make the pH 5; 3) Add the cyclic peptide to the 225 Ac solution obtained in step 2), mix well and heat at 95 °C for 10 - 30 min; (4) Purify the product obtained in step 3), and elute the product with absolute ethanol to obtain the product.

9. Use of the diagnostic radionuclide label described in claim 3 or 4 in the preparation of a tumor PET imaging reagent.

10. Use of the therapeutic radionuclide label described in claim 5 or 6 in the preparation of a tumor radionuclide therapeutic drug.

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