Modified CAIX-targeted cyclic peptides and their radionuclide labels and applications

Abstract

The present invention belongs to the field of nuclear medicine molecular diagnosis and treatment technology, and discloses a modified CAIX-targeting cyclic peptide and its radionuclide label and application. The CAIX-targeting cyclic peptide significantly improves the probe's resistance to enzymatic degradation and in vivo stability by introducing a rigid bifunctional chelator, RESCA, in place of a traditional chelator, combined with a sulfonated or alkylated amino acid linker. By optimizing the linker structure, lipophilicity is reduced, liver and gallbladder metabolism and nonspecific uptake are reduced, and a high uptake rate in the tumor target tissue is maintained. The cyclic peptide can be labeled with diagnostic radionuclides for PET imaging, or therapeutic radionuclides for intratumoral irradiation therapy, using DOTA or NOTA in combination with an alkylated amino acid linker or an albumin ligand. Experiments have shown that the label has high radiochemical purity, excellent pharmacokinetic properties, and low renal retention, making it suitable for the precise diagnosis and targeted treatment of CAIX-high-expressing tumors such as renal cancer, colorectal cancer, and brain gliomas.

Description

Technical Field

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

[0002] Carbonic anhydrase IX (CAIX), a membrane protein, has garnered significant attention in oncology research. CAIX is highly expressed in many tumor types that are hypoxic or harbor specific tumor suppressor gene mutations. In normal healthy tissues, its expression is more restricted, primarily in the gastrointestinal epithelium. This differential expression between tumors and normal tissues makes CAIX a promising target for oncology diagnosis and therapy. Previous studies have shown that elevated CAIX expression is closely associated with tumor progression, poor prognosis, and the development of metastasis. In clear cell renal cell carcinoma (ccRCC), impaired von Hippel-Lindau tumor suppressor function leads to dysregulated expression of hypoxia-inducible factor 1a, which in turn promotes sustained high CAIX expression. Under hypoxic conditions and in some solid tumors, such as colorectal cancer (CRC), breast cancer, and pancreatic ductal adenocarcinoma (PDAC), CAIX overexpression is aberrantly regulated by hypoxia-inducible factor 1a.

[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 accurate diagnosis and treatment of tumors. 68 Ga and 18 F-labeled probes can be used for positron emission tomography (PET) imaging. With their excellent imaging properties, they play a key role in early tumor detection, staging, and treatment effect evaluation. 177 Lu and 225 Probes labeled with therapeutic radionuclides, such as Ac, can kill tumor cells through the radiation released by the radionuclides, achieving the goal of tumor treatment. However, existing radionuclide probes targeting CAIX still have many shortcomings. Some probes have low uptake efficiency at the tumor site, making it difficult to accurately locate and effectively treat the tumor. Furthermore, some probes have high nonspecific uptake in normal tissues, which can easily cause radiation damage to normal tissues and trigger adverse reactions. Furthermore, the probe's stability, affinity, and specificity need to be further improved.

[0004] The development of efficient and safe CAIX cyclic peptide radionuclide probes has become a research hotspot in the field of cancer diagnosis and treatment. These novel CAIX cyclic peptide radionuclide probes not only improve the accuracy and effectiveness of cancer diagnosis and treatment, but also provide clinicians with more precise diagnostic information and tailor treatment plans for cancer patients. These probes hold significant clinical significance and hold broad market potential. Summary of the Invention

[0005] The purpose of the present invention is to provide a modified CAIX-targeted cyclic peptide and a radionuclide label and application thereof.

[0006] To achieve the purpose 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]

[0008] In a second aspect, the present invention provides a radionuclide label of the cyclic peptide, which is the cyclic peptide labeled with a radionuclide, wherein the radionuclide includes a diagnostic radionuclide and a therapeutic radionuclide.

[0009] Furthermore, the diagnostic radionuclide may be selected from 68 Ga or 18 F, etc., preferably a cyclic peptide represented by formula I, II, III or IV labeled with a radionuclide.

[0010] Furthermore, the therapeutic radionuclide may be selected from 90 Y. 177 Lu, 225 Ac or 213 Bi, etc., preferably a cyclic peptide represented by formula IV or V labeled with a radionuclide.

[0011] In a third aspect, the present invention provides a method for preparing the radionuclide marker, wherein the radionuclide is 68 When Ga is used, the preparation method of the radionuclide marker comprises the following steps:

[0012] (1) Prepared by using a columnar germanium gallium generator 68 Ga nuclide;

[0013] (2) Elute with 2-4 mL of 0.03-0.06 M HCl solution 68 Ga, and mixed with 0.8-1.2 M NaAc;

[0014] (3) Add the cyclic peptide obtained in step (2) 68 Ga solution, mix well and heat to 37℃ for 10-20min;

[0015] (4) Purify the product obtained in step (3) and elute the product with anhydrous ethanol to obtain the product.

[0016] When the radionuclide is 18 When F, the preparation method of the radionuclide marker comprises the following steps:

[0017] 1) Preparation using cyclotron 18 F solution;

[0018] 2) The cyclotron produced 18 F's H2 18 O passes through the QMA ion exchange column and 18 F is adsorbed on the QMA column; the QMA column is washed with 0.45-0.55 mL of saline and eluted. 18 F;

[0019] 3) Take the result from step 2) 18 The physiological saline of F is mixed with KHP (potassium hydrogen phthalate) and AlCl3 solution, shaken and placed at room temperature for 4-6 minutes, and then the cyclic peptide is added and reacted at 37°C for 10-20 minutes. After the reaction solution is cooled, the product is loaded onto a C18 separation column, washed with physiological saline and eluted with ethanol to obtain the product.

[0020] When the radionuclide is 177 When Lu, the preparation method of the radionuclide marker comprises the following steps:

[0021] (1) Add [ 177 Lu]LuCl3 solution (usually from a reactor or generator);

[0022] (2) Mix with 0.8-1.2M sodium acetate buffer [ 177 Lu]LuCl3 solution to a pH of 3.8-5.5;

[0023] (3) Add the cyclic peptide obtained in step (2) 177 Lu solution, mix well and heat to 95℃ for 10-20min;

[0024] (4) Purify the product obtained in step (3) and elute the product with anhydrous ethanol to obtain the product.

[0025] When the radionuclide is 225 When Ac, the preparation method of the radionuclide marker comprises the following steps:

[0026] 1) Add [ 225 Ac]AcCl3 solution (usually from a reactor or generator);

[0027] 2) Mix with 0.8-1.2M sodium acetate buffer [ 225 Ac]AcCl3 solution to a pH of 5;

[0028] 3) Add the cyclic peptide obtained in step 2) 225 Ac solution, mix well and heat to 95℃ for 10-30min;

[0029] 4) Purify the product obtained in step 3) and elute the product with anhydrous ethanol to obtain the product.

[0030] In a fourth aspect, the present invention provides use of the diagnostic radionuclide marker in the preparation of a tumor PET imaging reagent.

[0031] In a fifth aspect, the present invention provides the use of the therapeutic radionuclide marker in the preparation of tumor radionuclide therapeutic drugs.

[0032] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0033] (1) The CAIX cyclic peptide structure of the present invention has good in vivo stability, pharmacokinetic properties, affinity and specificity.

[0034] (II) Radioactive molecular probes for diagnosis 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 patients with CAIX-positive renal cell carcinoma (ccRCC), colorectal cancer (CRC), breast cancer and pancreatic ductal adenocarcinoma (PDAC).

[0035] (three) 68 Ga / 18 F-CAIX cyclic peptide PET imaging can determine the patient's response to CAIX targeted therapy and determine whether the patient is suitable for therapeutic radionuclide. 90 Y / 177 Lu / 225 Ac / 213 Bi replacement 68 Ga / 18 F for radionuclide targeted therapy.

[0036] (Four) 90 Y / 177 Lu / 225 Ac / 213 Radionuclide targeted therapy such as Bi-CAIX cyclic peptide can provide new treatment options for patients with drug-resistant CAIX tumors.

[0037] (V) The present invention 68 Ga / 18 F / 177 Lu / 225The preparation method of Ac-CAIX cyclic peptide has a high labeling rate. 68 Ga labeling rate can be above 95%, 18 The F marking rate can reach more than 30%, 177 Lu labeling rate can reach more than 70%, 225 The Ac labeling rate can reach over 80%. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1a-Figure 1e HPLC and mass spectrometry quality control results of 5 structures, Figure 1a is the quality control result of formula Ⅰ, Figure 1b This is the quality control result of formula II, Figure 1c is the quality control result of formula III, Figure 1d This is the quality control result of formula IV, Figure 1e This is the quality control result of Formula V.

[0039] Figure 2 For optimized 18 Radio-TLC detection results of labeling rate and radiochemical purity of F-CAIX-RESCA-cyclic peptide (Formula III).

[0040] Figure 3 For optimized 18 Pharmacokinetic testing and parameter statistics of F-CAIX-RESCA-cyclic peptide (Formula III).

[0041] Figure 4a-4c They are respectively the Micro-PET / CT imaging results of the CAIX cyclic peptide Formula I in the preferred embodiment of the present invention in the HT-29 mouse model and the imaging results of the Block group and the SUVmax statistics.

[0042] Figure 5 The Micro-PET / CT imaging results of CAIX cyclic peptide Formula II in the preferred embodiment of the present invention in the HT-29 mouse model and the Block group imaging results are shown.

[0043] Figure 6a-6c They are respectively the Micro-PET / CT imaging results of CAIX cyclic peptide formula III in the preferred embodiment of the present invention in the HT-29 mouse model and the imaging results of the Block group and the SUVmax statistics.

[0044] Figure 7 The Micro-PET / CT imaging results of CAIX cyclic peptide Formula IV in the preferred embodiment of the present invention in the HT-29 mouse model and the Block group imaging results are shown.

[0045] Figure 8a-8dThe block biodistribution comparison and 4h biodistribution of CAIX cyclic peptide formula III in the HT-29 mouse model, as well as the specific values ​​and human dosage estimation are shown respectively.

[0046] Figure 9a-9b They are respectively the results of routine blood analysis, body weight monitoring and blood biochemical test of the acute toxicity experiment of CAIX cyclic peptide formula III in the preferred embodiment of the present invention.

[0047] Figure 10a-Figure 10b The results of the radionuclide treatment experiments of CAIX cyclic peptides IV and V in the HT-29 mouse model are respectively the preferred embodiments of the present invention, including 177 Lu-CAIX-cyclic peptide Ⅳ / Ⅴ and 225 Ac-CAIX-cyclic peptide IV / V. DETAILED DESCRIPTION

[0048] The present invention aims to provide a modified CAIX cyclic peptide radionuclide probe molecular structure, as well as the further obtained diagnostic radionuclide marker, preparation method and application. Specifically, it relates to a carbonic anhydrase IX (CAIX) targeted cyclic peptide based on a metabolically optimized linker and the preparation and application of its diagnostic radionuclide marker. The CAIX targeted cyclic peptide introduces a rigid bifunctional chelator RESCA (Rigid Ethylene-bridged Cyclam Chelator Assembly) to replace the traditional chelator, and combines a sulfonated or alkylated amino acid linker (such as PPAc, Cys(SO3H)-His, Leu-Thr) to significantly improve the probe's anti-enzymatic performance and in vivo stability. By optimizing the linker structure, the lipophilicity is reduced (logP value is optimized from -6.51 to -3.06), the hepatobiliary metabolism and nonspecific uptake are reduced (liver uptake is reduced to 0.54% ID / g), while maintaining a high uptake rate in the tumor target tissue (92.6% ID / g). The cyclic peptide can label diagnostic radionuclides (such as 68 Ga, 18 F) for PET imaging, or therapeutic radionuclides (such as 177 Lu, 225 Ac) is used for intratumoral irradiation therapy, using DOTA or NOTA in conjunction with an alkylated amino acid linker or albumin ligand (e.g., Leu-Thr-Leu-Leu, ABM). Experimental studies have shown that this marker exhibits high radiochemical purity (>95%), excellent pharmacokinetic properties, and low renal retention, making it suitable for the precise diagnosis and targeted treatment of tumors with high CAIX expression, such as colorectal cancer, renal cancer, and breast cancer. This invention provides an efficient and safe molecular probe solution for tumor diagnosis and treatment.

[0049] The present invention adopts the following technical solutions:

[0050] The present invention provides a modified CAIX cyclic peptide, wherein the sequence of the modified CAIX cyclic peptide is (N-terminus-C-terminus):

[0051] RESCA-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula I);

[0052] RESCA-Cys(SO3H)-His-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula II);

[0053] RESCA-Leu-Thr-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula III);

[0054] DOTA-Leu-Thr-Leu-Leu-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula IV);

[0055] ABM-DOTA-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula V);

[0056] The N-terminus or C-terminus of the cyclic peptide is modified by a bifunctional linker;

[0057] The chelating agent contained in the cyclic peptide is RESCA, and the RESCA is connected to a sulfonated or alkylated amino acid linker;

[0058] 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.

[0059] The structure of the CAIX cyclic peptide is shown in Formula I, II, III, IV, and V:

[0060]

[0061] The present invention provides a structural optimization scheme for a cyclic peptide radionuclide probe targeting carbonic anhydrase IX (CAIX).

[0062] Specifically, the modified CAIX targeting cyclic peptides represented by Formulas I, II, and III are particularly suitable for preparing diagnostic radionuclide labels. Formulas I, II, and III exhibit a progressive structural design, as follows:

[0063] Its molecular structure is characterized by: Formula I is a prototype probe [ 68 Based on the DOTA chelation system of Ga]Ga-DPI-4452, the probe's structure was modified by replacing the traditional DOTA coordination system with the rigid bifunctional chelator RESCA (Rigid Ethylene-bridged Cyclam Chelator Assembly). Compared to highly polar chelators such as DOTA and NOTA, RESCA exhibits significantly enhanced resistance to enzymatic degradation. Its rigid cyclic structure effectively reduces metabolic degradation in vivo, significantly extending the probe's systemic circulation half-life.

[0064] Regarding the structural optimization of the molecular linker, structure-activity relationship studies revealed that when using a PPAc (Pyridine-Propionic Acid) linker, the probe achieved nonspecific uptake rates of (28.4±3.1)%ID / g in the gastrointestinal tract and (18.7±2.6)%ID / g in the liver parenchyma. Based on a design strategy to reduce the overall lipophilicity of the molecule (logP value optimized from -6.51 to -3.06), a His-CA (Histidine-Carbonic Anhydrase) sequence (Formula II) was added to the PPAc linker. This novel linker, through molecular modification of the sulfonic acid group, achieves dual optimization mechanisms: charge modification reduces the reticuloendothelial system (RES) capture rate (Kupffer cell uptake decreased by 67%), while electrostatic repulsion reduces hepatobiliary clearance to (1.4±1.9)%ID / g, thereby improving the target-to-substance ratio.

[0065] Although the RESCA-sulfonation design reduces hepatobiliary metabolism, the probe is partially cleared via glomerular filtration at a slow rate. Further structural optimization revealed that switching to a Leu-Thr (leucine-threonine) dipeptide linker (Formula III) exhibited significant pharmacokinetic advantages: Four hours after injection, nonspecific uptake 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 uptake in tumor target tissues remained at the initial value of (92.6±3.8)%. This improved metabolic profile is primarily attributed to the β-pleated secondary structure formed by the Leu-Thr linker, which effectively reduces renal uptake.

[0066] In the field of radionuclide therapy, this invention optimizes the metabolic properties of labeling ligands based on DOTA or NOTA chelating systems. The modified CAIX-targeting cyclic peptides represented by Formula IV or V are particularly suitable for preparing therapeutic radionuclide labels. Their structural design is as follows:

[0067] By introducing a specific polypeptide sequence Leu-Thr-Leu-Leu-PPAc (Formula IV) or ABM-PPAc (Formula V) as a connecting structure, the drug's renal metabolic characteristics are significantly optimized (the 4-hour renal uptake rate is reduced by ≥90%), effectively reducing the risk of accumulation of radionuclides in non-target organs. Furthermore, in order to improve the long-term stability of the drug in the circulatory system, the present invention covalently couples an albumin-binding ligand (as shown in Formula V) to the end of the polypeptide sequence for the first time, and through the active binding mechanism targeting plasma albumin, the metabolic clearance rate is significantly slowed down while maintaining tumor targeting efficiency (the target / non-target ratio is increased by more than 1.8 times). This design achieves the synergistic optimization of metabolic clearance rate and circulation retention capacity, which is a promising target for highly toxic α / β-nuclides (such as 225 Ac, 177 Lu) provides a solution that is both efficient and safe.

[0068] The present invention also provides a diagnostic and therapeutic nuclide marker, which is the modified CAIX cyclic peptide labeled with a radionuclide.

[0069] According to a specific embodiment of the present invention, the radionuclide can be a diagnostic radionuclide, and the diagnostic radionuclide is preferably a positron-emitting radionuclide. 68 Ga or 18 F.

[0070] When the radionuclide is 68 When Ga is used, the preparation method of the radionuclide-labeled CAIX cyclic peptide may include the following steps:

[0071] 1) Prepared by using a columnar germanium gallium generator 68 Ga nuclide;

[0072] 2) Elute with 2-4 mL of 0.03-0.06 M HCl solution 68 Ga, and mixed with 0.8-1.2 M NaAc;

[0073] 3) Add the modified CAIX cyclic peptide obtained in step 2) 68 Ga solution, mix well and heat to 85-100℃ for 10-20min;

[0074] 4) Purify the product obtained in step 3) and elute the product with anhydrous ethanol to obtain 68 Ga-labeled CAIX cyclic peptide.

[0075] Specifically, taking the modification of the bifunctional chelator RESCA as an example, 68 The following methods can be used to label the modified CAIX cyclic peptide with Ga:

[0076] 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 subjected to 68 Ga(T 1 / 2 =68 min; β + :89%; E=511 keV) nuclide labeling, 68 Ga use 68 Ge- 68 Prepare the Ga generator. Take 3 mL of 0.05 M HCl solution to elute 68 Ga was added to 195 μL 1M NaAc; 0.1 mL (60 μg) of RESCA-CAIX cyclic peptide precursor was added to the above system, mixed, reacted at 95°C for 10 min, radioactive impurities were eluted with 3 mL of normal saline, and the target compound was eluted with 0.8 mL of 80% ethanol. 68 The labeling rate and radiochemical purity of Ga-RESCA-CAIX cyclic peptide were determined by radio-HPLC or radio-TLC. 68The radiochemical purity of the Ga-RESCA-CAIX cyclic peptide should be greater than 95%. If the labeling efficiency is less than 90%, separate and purify using a Sep-pak C18 column. The Sep-pak column should be activated with 5 mL of anhydrous ethanol and 5 mL of high-purity water before use. An appropriate amount of the sterile-filtered product preparation should undergo quality control testing. Only after all parameters pass must further research be performed.

[0077] When the radionuclide is 18 When F, the preparation method of the radionuclide-labeled CAIX cyclic peptide may include the following steps:

[0078] 1) Preparation using cyclotron 18 F solution;

[0079] 2) The cyclotron produced 18 F's H2 18 O passes through the QMA ion exchange column and 18 F is adsorbed on the QMA column; the QMA column is washed with 0.45-0.55 mL of saline and eluted. 18 F;

[0080] 3) Take the result from step 2) 18 The physiological saline of F was mixed with KHP and AlCl3 solution, shaken and placed at room temperature for 4-6 minutes, and then the modified CAIX cyclic peptide was added and reacted at 37°C for 10-20 minutes. After the reaction solution was cooled, the product was loaded onto a C18 separation column, washed with physiological saline and eluted with ethanol to obtain the product. 18 F-labeled CAIX cyclic peptide.

[0081] Specifically, taking the modification of the bifunctional chelator H3RESCA as an example, 18 The following methods can be used to label the modified CAIX cyclic peptide:

[0082] The sequence of the CAIX cyclic peptide modified by 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); the prepared H3RESCA-CAIX cyclic peptide was subjected to 18 F(T 1 / 2 =109.8 min; β + :96.7%; E=511 keV) nuclide labeling, 18 F is prepared using a cyclotron. 18 F - H218 O passes through a QMA ion exchange column, 18 F - was adsorbed onto the QMA column, and the QMA column was washed with 0.5 mL of normal saline; 0.1 mL of 18 F - The saline solution was placed in a reaction tube containing 11 μL 10-fold KHP and 6 μL 2 mM AlCl3 solution, mixed and placed at room temperature for 5 minutes; 10 μL 10 mg / mL labeled precursor H3RESCA-CAIX cyclic peptide was added and reacted at 100°C for 15 minutes; after the reaction solution was cooled to room temperature, the product was purified using a C18 separation column, washed with 5 mL of saline, and then eluted with 0.6 mL of 80% ethanol and passed through a 0.22 μm sterile filter membrane; the solvent was blown dry 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. 18 After isolation and purification, the radiochemical purity of the F-CAIX cyclic peptide is greater than 95%. If the labeling efficiency is less than 90%, separate and purify using a Sep-pak C18 column. The Sep-pak column needs to be activated with 5 mL of anhydrous ethanol and 5 mL of high-purity water before use. An appropriate amount of the sterile-filtered product preparation is then subjected to quality control testing. Further studies are performed only after all parameters pass.

[0083] According to the present invention, the radionuclide may also be a therapeutic radionuclide, in order to achieve the purpose of targeted molecular imaging therapy for tumors with high CAIX expression. The therapeutic radionuclide is preferably 90 Y. 177 Lu, 225 Ac and 213 At least one of Bi.

[0084] CAIX cyclic peptide modified with bifunctional coupling agent DOTA, bifunctional coupling agent NOTA, bifunctional coupling agent DTPA, and bimodal bifunctional ligand 3pC-NETA-NCS can be used for therapeutic radionuclide 90 Y, 177 Lu, 225 Ac or 213 Bi mark, get 90 Y-CAIX cyclic peptide, 177 Lu-CAIX cyclic peptide, 225 Ac-CAIX cyclic peptide, 213 Bi-CAIX cyclic peptide molecular probe for therapeutics.

[0085] The labeling of therapeutic radionuclides can be carried out by various conventional methods in the art. According to a preferred embodiment of the present invention, the 90 Y,177 Lu, 225 Ac or 213 The Bi-labeled CAIX cyclic peptide method can be used as follows: 177 Lu was used as an example. The CAIX cyclic peptide modified with bifunctional chelating agents DOTA, NOTA, DTPA, and 3pC-NETA-NCS was used to obtain the corresponding labeled precursors. The labeling buffer was prepared by 3 mL of 0.05M HCl to 195 µL of 1M NaAc and set aside. 100 µL (60 µg) of labeled precursors (DOTA-CAIX cyclic peptide, NOTA-CAIX cyclic peptide, DTPA-CAIX cyclic peptide, 3pC-NETA-CAIX cyclic peptide) was added with 100-150 µL of labeling buffer and then added. 177 Lu; adjust pH to 5.5, 70-95 o C, react for 10-15 min, when the labeling rate is less than 90%, separate and purify with Sep-pak C18 column to obtain 177 Lu-CAIX cyclic peptide. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. 177 The radiochemical purity of Lu-CAIX cyclic peptide after separation and purification is greater than 95%.

[0086] The present invention also provides the use of a CAIX cyclic peptide labeled with a diagnostic radionuclide in the preparation of a CAIX-targeted tumor PET imaging agent.

[0087] The present invention also provides the use of a CAIX cyclic peptide labeled with a therapeutic radionuclide in the preparation of a CAIX-targeted tumor radionuclide therapeutic drug.

[0088] The following examples are used to illustrate the present invention but are not intended 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.

[0089] Example 1 Synthesis and Characterization of CAIX-Targeted Cyclic Peptides with Metabolism-Optimized Linkers

[0090] 1. Objective: To synthesize a carbonic anhydrase IX (CAIX)-targeted cyclic peptide based on a metabolically optimized linker and verify its structure.

[0091] 2. Experimental steps:

[0092] (1) Cyclic peptide sequence design:

[0093] The target cyclic peptide sequence is:

[0094] RESCA-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula I);

[0095] RESCA-Cys(SO3H)-His-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula II);

[0096] RESCA-Leu-Thr-PPAc-Gln-[Cys(3MeBn)-Glu-DPro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula III);

[0097] DOTA-Leu-Thr-Leu-Leu-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula IV);

[0098] ABM-DOTA-PPAc-Gln-[Cys(3MeBn)-Glu-Dpro-Asp-Af3(Cpsu)-Leu-Thr-Trp-Ser-Cys]-NH2 (Formula V);

[0099] (2) Solid-phase synthesis:

[0100] The Fmoc solid-phase synthesis method was adopted, 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.

[0101] (3) Purification and characterization:

[0102] HPLC analysis:

[0103] Chromatographic column: Kromasil 100-5C18 (4.6 × 250 mm);

[0104] Mobile phase A: 0.1% TFA acetonitrile, mobile phase B: 0.1% TFA water;

[0105] Gradient: 0-20 min, 30% A→60% A; 20-25 min, 60% A→100% A;

[0106] Flow rate: 1 mL / min; detection wavelength: 220 nm.

[0107] Mass spectrometry (MS) verification:

[0108] The molecular weights measured by MALDI-TOF MS were 2183.41 (theoretical value 2183.86); 2424.66 (theoretical value 2183.86); and 2350.78 (theoretical value 2350.99), confirming the correctness of the sequence.

[0109] The results are as follows Figure 1a-Figure 1e As shown, it was proved that the five cyclic peptides were successfully synthesized with high chemical purity.

[0110] Example 2 68 Preparation of Ga-labeled CAIX-targeting cyclic peptide

[0111] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide shown in formula I, II or III) is subjected to 68 Ga nuclide labeling (T 1 / 2 =68 min; β + :96.7%; E=511 keV) nuclides were prepared by using a column-type germanium gallium generator 68 Ga nuclide, elute with 4 mL of 0.05 M HCl 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 normal saline, eluted with 0.6 mL of 80% ethanol and filtered through a 0.22 μm sterile filter membrane; the solvent was blown dry with N2 and diluted with normal 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%.

[0112] Example 3 18 Preparation of F-labeled CAIX-targeting cyclic peptide

[0113] 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. 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 washed with 0.5 mL of normal saline; 0.1 mL of 18 F - The normal saline was placed in a reaction tube containing 11 μL 10x KHP and 6 μL 2 mM AlCl3 solution, mixed and allowed to stand at room temperature for 5 min; 10 μL 10 mg / mL labeled precursor CAIX-RESCA-cyclic peptide was added and reacted at 100°C for 15 min; after the reaction solution was cooled to room temperature, the product was purified using a C18 separation column, washed with 5 mL of normal saline, eluted with 0.6 mL of 80% ethanol and passed through a 0.22 μm sterile filter membrane; the solvent was blown dry with N2 and diluted with normal saline to obtain the product preparation, which was then determined by radio-HPLC or radio-TLC for labeling rate and radiochemical purity.

[0114] Figure 2 Shown 18 The results of Radio-TLC detection of F-CAIX-RESCA-cyclic peptide (Formula III) were obtained by 18 The labeling rate of F-CAIX-RESCA-cyclic peptide (Formula III) is about 92%, and the radiochemical purity is greater than 99%.

[0115] Example 4 Purified 18 In vitro stability analysis of F-CAIX-RESCA-cyclic peptide

[0116] Take 10 µL of the purified product containing 1.11 MBq (30 µCi) 18 F-CAIX-RESCA-cyclic peptide was added to 200 μL of normal saline (or 5% HSA solution) and incubated at 4°C. 37-74 kBq (1-2 μCi) samples were taken at 0 h, 2 h, 12 h, 24 h, 36 h, and 60 h of incubation for radio-TLC analysis. Analysis method: 2 μL of the sample containing 37-74 kBq (1-2 μCi) of radioactivity was taken and the sample was analyzed by radio-TLC. 18 F-CAIX-RESCA-cyclic peptide saline solution or 18Add 5% HSA solution of F-CAIX-RESCA-cyclic peptide to 20 μL saturated EDTA and mix well. Perform radio-TLC analysis. Take 2 μL sample and drop it 1 cm from the bottom of Xinhua No. 1 filter paper. Place it in physiological saline development system. After complete development, remove the filter paper and dry it. Perform radio-TLC detection. 18 F and 18 The Rf values ​​of F-CAIX-RESCA-cyclic peptide were 0.9-1 and 0-0.1 respectively; the results showed 18 F-CAIX-RESCA-cyclic peptide showed good stability in normal saline solution or 5% HSA solution within 4 hours.

[0117] The above experiments show that 18 The F-CAIX-RESCA-cyclic peptide structure has good stability.

[0118] Example 5 Optimized 18 Pharmacokinetic Analysis of F-CAIX-RESCA-Cyclic Peptide (Formula III) in Normal KM Mice

[0119] Prepare 5 KM mice (female, 5-6 weeks old, 18-20 g) using an optimized 18 F-CAIX-RESCA-cyclic peptide (Formula III) was diluted with physiological saline to 18.5 MBq / mL (0.5 mCi / ml), and each mouse was injected with 3.7 MBq (0.1 mCi, 200 μL) of the labeled product through the tail vein. At the corresponding time points (1 min, 3 min, 5 min, 10 min, 15 min, 30 min, 45 min and 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 18 h, 24 h, 36 h) after the injection of the labeled product, blood was collected from the periocular venous plexus of the mouse using a capillary tube and placed in a radioimmunoassay tube. 18 A reference activity of 0.037 MBq (1 µCi, 2 µL), representing 1% of the radioactivity of F-CAIX-RESCA-cyclic peptide (Formula III), was used for the assay. This reference activity was measured along with the collected blood sample using a gamma counter. Data were analyzed using Prism 6.0 software after decay correction, and the percentage injected dose per gram of blood was calculated. Results are expressed as %ID / g ± SD.

[0120] Specific results such as Figure 3 As shown in the figure, 18 The F-CAIX-RESCA-cyclic peptide (Formula III) has good pharmacokinetic properties and is suitable for in vivo imaging studies.

[0121] Example 618 PET / CT imaging study of F-CAIX-RESCA-cyclic peptide (Formula Ⅰ, Ⅱ, Ⅲ, Ⅳ) in HT29 tumor-bearing mouse model

[0122] Seven HT29 tumor-bearing mice were 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 peptide (Formula I, II, III, IV), blocking imaging is done by injecting 0.1 mg of non-radioactive labeled CAIX-RESCA-cyclic peptide (Formula I, II, III). 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula I) group underwent PET / CT imaging at 30 min, 60 min, 120 min, and 240 min after injection. The imaging results are shown in Figure 2. Figure 4a The experimental group had obvious uptake in the tumor, which was significantly different from the suppression imaging group below. At the same time, the kidneys and gastrointestinal tract had higher uptake. The statistical results of SUVmax of important organs in the experimental group were as follows: Figure 4b As shown, the statistical results of SUVmax of important organs in the inhibition imaging group are as follows Figure 4c shown.

[0123] 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula II) group underwent PET / CT imaging at 30 min, 60 min, and 120 min after injection. Figure 5 , high uptake was still observed in tumors and kidneys, while gastrointestinal uptake was somewhat lower than that of Formula I.

[0124] 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula III) group underwent PET / CT imaging and important organ delineation statistics at 40 min, 60 min, 120 min, 240 min, and 360 min after injection. The results are shown in the figure. Figure 6a 、 Figure 6b and Figure 6c After optimization, the probe showed a slight increase in liver and kidney uptake compared to Formulas I and II in early imaging. However, in the late imaging phase, uptake in the liver, kidneys, stomach, and intestines decreased significantly, while uptake in the tumor site did not decrease significantly, maintaining a strong retention effect. This late imaging result was superior to Formulas I and II, and has greater prospects for clinical translation.

[0125] 18 The mice in the F-CAIX-RESCA-cyclic peptide (Formula IV) group underwent PET / CT imaging at 2h, 3h, and 4h after injection. Figure 7 It can be seen that as time goes on, the uptake in the tumor site does not decrease significantly, which can be used to guide subsequent radionuclide therapy research.

[0126] Example 7 18 Distribution and inhibition of F-CAIX-RESCA-cyclic peptide (Formula III) in tumor-bearing mice, and estimation of human radiation dose

[0127] Fifteen HT29 tumor-bearing mice (female, 5-6 weeks old, 18-20 g) were randomly divided into five time groups, with 3 mice in each group. 7.4 MBq (0.2 mCi, 200 µL) was injected into the tail vein. 18 After F-CAIX-RESCA-cyclic peptide was added, the mice were anesthetized and killed at 5 min, 30 min, 60 min, 120 min, and 240 min, respectively. The blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, and brain were weighed, and the organ radioactivity counts were detected using a γ-counter. The in vivo biodistribution of each organ at different time points was statistically analyzed after attenuation correction. The blocking group was simultaneously injected with 0.1 mg of non-radioactive CAIX-RESCA-cyclic peptide.

[0128] like Figure 8a 、 Figure 8b 、 Figure 8c The results showed 18 F-CAIX-RESCA-cyclic peptide (Formula III) is mainly metabolized by the kidneys in animals. It has low nonspecific uptake in systemic tissues, extremely high uptake in tumors, and can be retained for a long time, and can be blocked by precursors. The biodistribution results are used to estimate the radiation dose to humans. Figure 8d As shown, the effective dose is significantly lower than the radioactive dose limit, proving the radiation safety of the probe in clinical application.

[0129] The present invention significantly improves the pharmacokinetic properties of CAIX-targeted cyclic peptides by introducing metabolically optimized linkers (such as PPAc-Gln; Cys(SO3H)-His-PPAc-Gln and Leu-Thr-PPAc-Gln), making it have the advantages of high tumor targeting, low nonspecific uptake and rapid renal clearance. 68 Ga, 177 Lu) labeling can simultaneously achieve accurate tumor imaging and efficient internal irradiation therapy, and has broad clinical application prospects.

[0130] Example 8 is optimized 18 Safety testing (toxicology test) of F-CAIX-RESCA-cyclic peptide (Formula III)

[0131] Ten normal Kunming mice were prepared and divided into two groups for the experiment, namely the experimental group and the control group, with n=5. Each mouse in the experimental group was injected with 10 times the dose of 18F-CAIX-RESCA-cyclic peptide (Formula III) 74 MBq (2 mCi, 200 µL) was injected into the control group with the same volume of saline solution. After a certain period of time, blood samples were collected for routine blood tests and liver and kidney function tests, and the weight changes of the mice were continuously recorded (16 days). The results are shown in the figure below. Figure 9a 、 Figure 9b The blood routine test results of the experimental group mice were not significantly different from those of the control group. The liver and kidney functions were within the normal range. The weight changes of the experimental group mice were not different from those of the control group. The experiment demonstrated the high safety of the probe and is expected to be used for clinical translation research.

[0132] Example 9 177 Preparation of Lu-labeled CAIX-targeting cyclic peptide

[0133] The CAIX targeting cyclic peptides of formula IV and formula V were used as precursors and the direct labeling method was adopted. 177 Lu]LuCl3 solution (specific activity ≥50 GBq / mg) was mixed with 0.8-1.2 M sodium acetate buffer [ 177 Lu] LuCl3 solution to make pH 3.8-5.5; add the cyclic peptide 177 Lu solution, mixed well and heated at 95℃ for 10-20min; the obtained product was purified using C-18 column and eluted with anhydrous ethanol to obtain radionuclide therapeutic probe. 177 Lu-CAIX-DOTA-cyclic peptide (radiochemical purity >98%, specific activity 22.5 MBq / μg).

[0134] Example 10 225 Preparation of Ac-labeled CAIX-targeting cyclic peptide

[0135] The CAIX targeting cyclic peptides of formula IV and formula V were used as precursors and the direct labeling method was adopted. 225 Ac]AcCl3 solution (specific activity ≥50 GBq / mg) was mixed with 0.8-1.2 M sodium acetate buffer [ 225 Ac]AcCl3 solution to pH 5; add the cyclic peptide 225 Ac solution, mixed and heated at 95℃ for 10-30min; the obtained product was purified using C-18 column and eluted with anhydrous ethanol to obtain radionuclide therapeutic probe. 225 Ac-CAIX-DOTA-cyclic peptide (radiochemical purity >98%, specific activity 22.5 MBq / μg).

[0136] Example 11 177 Lu-CAIX-DOTA-cyclic peptide and 225Evaluation of the therapeutic effect of Ac-CAIX-DOTA-cyclic peptide on HT-29 renal cancer model mice

[0137] BALB / c nude mice aged 6-8 weeks were subcutaneously inoculated with HT-29 human renal cancer cells (5×10 6 cells / mouse), and the mice were randomly divided into seven groups (n=5) after the tumor volume reached 100-150 mm³: Treatment group: single tail vein injection 177 Lu / 225 Ac-CAIX-DOTA-cyclic peptide-3 (Formula IV) (doses: 3.7 MBq and 1.85 MBq) and injection of equal doses 177 Lu / 225 Ac-CAXI-DOTA-cyclic peptide-4 (Formula V) (linked to albumin ligand); blank control group: injected with normal saline.

[0138] Efficacy was evaluated by dynamic monitoring of tumor volume (calculated by the formula V=0.5×L×W²) and survival analysis (endpoint defined as tumor volume ≥1000 mm³ or weight loss >20%);

[0139] The experimental results are as follows Figure 10a 、 Figure 10b As shown, the data show that the present invention 177 Lu / 225 Ac-CAXI-DOTA-cyclic peptide demonstrated significant advantages in the HT-29 renal cancer model: the tumor volume in the treatment group increased by only ≤30% compared with baseline within 28 days (85% increase in the positive control group, p<0.001), and the median survival was extended to 45 days (≤31 days in the control group); biodistribution analysis showed that the tumor / kidney uptake ratio (T / K=2.16) in the treatment group was significantly improved compared with the unmodified ligand group (T / K=1.2), confirming that the optimized linker and albumin ligand can synergistically reduce renal uptake and prolong circulatory retention; in terms of safety, the weight fluctuation in the treatment group was <5%, and there was no statistical difference in renal function indicators (creatinine, urea nitrogen) compared with the blank group (p>0.05), and no radiation damage was observed in histopathology, verifying the clinical applicability of the present invention.

[0140] Example 12 213 Preparation of Bi-labeled CAIX-targeting cyclic peptide

[0141] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula IV or V) is subjected to 213 Bi nuclide labeling (T 1 / 2 =45.6 min; α; E = 8.4 MeV) nuclides. Elute with 5 mL of pre-cooled 0.1 M HCl (containing 0.1% ascorbic acid) 225 Ac / 213Bi generator, collects 213 Bi eluent. Remove impurities (such as 225 Ac leakage), washed with 0.1 M HCl, and eluted with 0.5 M HNO3 213 Bi, dried under nitrogen, was redissolved in 0.1 M HCl for later use. 213 Add 200 μL of 1 M NaAc-HAc buffer (pH 4.0) to the Bi solution and mix thoroughly. The pH should be controlled between 3.5 and 4.0. Add 50-100 μg of CAIX-DOTA-cyclic peptide Formula IV or V (dissolved in 0.1 M NH4OAc, pH 5.0) to adjust the final volume to 2 mL. Heat and stir at 95°C for 10 minutes (sealed to prevent evaporation), then cool in an ice bath to terminate the reaction. Load the reaction solution onto a pre-activated (5 mL ethanol, 10 mL H2O) C18 column and wash with 10 mL of saline to remove free 213 Bi. Elute with 1 mL of 70% ethanol / 30% saline 213 Bi-CAIX-DOTA-cyclic peptide was sterile filtered through a 0.22 μm membrane. Ethanol was dried with nitrogen purging, and the resulting solution was reconstituted in saline to a final volume of 1 mL. Aliquoted and used immediately. Analysis by radio-HPLC (flow rate 1 mL / min, acetonitrile:0.1% TFA gradient, α-radioactivity detection) was performed; purity was >95%.

[0142] Example 13 90 Preparation of Y-labeled CAIX-targeting cyclic peptide

[0143] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula IV or V) is subjected to 90 Y-nuclides labeling (T 1 / 2 =64.1 h; β - ; E = 2.28 MeV) nuclides. Elute with 8 mL of 0.05 M HCl (containing 0.1% ascorbic acid) 90 Sr / 90 Y generator, collects 90 The eluent is passed through a strontium selective resin column (such as Sr-Resin®) to remove residual 90 Sr, ensure the radioactivity purity> 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 cool in an ice bath to terminate the reaction. Load the reaction solution onto a pre-activated (5 mL ethanol 10 mL H2O) C18 column and wash with 10 mL of saline to remove free 90 Y. Elute with 1.5 mL of 60% ethanol 90 Y-CAIX-DOTA-cyclic peptide was sterile filtered through a 0.22 μm membrane. The ethanol was dried with nitrogen and reconstituted in saline to a final volume of 2 mL. Analyzed by radio-HPLC (flow rate 1 mL / min, acetonitrile:0.1% TFA gradient, detection of beta radioactivity); expected purity >90%.

[0144] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A modified CAIX-targeting cyclic peptide, characterized in that: The structure is shown in Formula III: 。 2. The radionuclide label of the cyclic peptide according to claim 1, characterized in that The cyclic peptide is labeled with radionuclides, and the radionuclides include diagnostic radionuclides and therapeutic radionuclides.

3. The radionuclide label according to claim 2, characterized in that The diagnostic radionuclide is selected from 68 Ga or 18 F.

4. The radionuclide label according to claim 2, characterized in that The therapeutic radionuclide is selected from 90 Y. 177 Lu, 225 Ac or 213 Bi.

5. The method for preparing the radionuclide label according to claim 3, characterized in that: When the radionuclide is 68 When Ga is used, the preparation method of the radionuclide marker comprises the following steps: (1) Prepared by using a columnar germanium gallium generator 68 Ga nuclide; (2) Elute with 2-4 mL of 0.03-0.06 M HCl solution 68 Ga, and mixed with 0.8-1.2 M NaAc; (3) Add the cyclic peptide obtained in step (2) 68 Ga solution, mix well and heat to 37℃ for 10-20min; (4) Purifying the product obtained in step (3) and eluting the product with anhydrous ethanol to obtain; When the radionuclide is 18 When F, the preparation method of the radionuclide marker comprises the following steps: 1) Preparation using cyclotron 18 F solution; 2) The cyclotron produced 18 F's H2 18 O passes through the QMA ion exchange column and 18 F is adsorbed on the QMA column; the QMA column is washed with 0.45-0.55 mL of saline and eluted. 18 F; 3) Take the result from step 2) 18 The physiological saline of F is mixed with KHP and AlCl3 solution, shaken and placed at room temperature for 4-6 minutes, and then the cyclic peptide is added and reacted at 37°C for 10-20 minutes. After the reaction solution is cooled, the product is loaded onto a C18 separation column, washed with physiological saline and eluted with ethanol to obtain the product.

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

7. Use of the diagnostic radionuclide marker according to claim 3 in the preparation of tumor PET imaging reagents.

8. Use of the therapeutic radionuclide marker according to claim 4 in the preparation of tumor radionuclide therapeutic drugs.

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