Modified CAIX targeting cyclic peptide, nuclide marker and application of modified CAIX targeting cyclic peptide in tumor diagnosis and treatment

Abstract

The invention belongs to the technical field of nuclear medicine molecular diagnosis and treatment, and discloses a modified CAIX targeting cyclic peptide, a nuclide marker and application of the modified CAIX targeting cyclic peptide in tumor diagnosis and treatment. 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 therapeutic nuclide marker of the cyclopeptide can be used for intratumoral 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

[0001] This invention is a divisional application of the invention patent with application number 202510705190.5 entitled "Modified CAIX-targeting cyclic peptide and its radionuclide labeling and application". Technical Field

[0002] This invention belongs to the field of nuclear medicine molecular diagnostic and therapeutic technology, specifically, it relates to modified CAIX-targeting cyclic peptides, radionuclide markers and their applications in tumor diagnosis and treatment. Background Technology

[0003] Carbonic anhydrase IX (CAIX), a membrane protein, has attracted considerable attention in the field of tumor research. CAIX is highly expressed in many hypoxic tumor types or those carrying specific tumor suppressor gene mutations, while its expression is more limited in normal healthy tissues, mainly concentrated in the gastrointestinal epithelium. This differential expression between tumor and normal tissues makes CAIX a highly promising target for tumor diagnosis and treatment. Previous studies have shown that high CAIX expression is closely related to tumor progression, poor prognosis, and metastasis. In clear cell renal cell carcinoma (ccRCC), impaired function of von Hippel-Lindau tumor suppressor leads to dysregulation of hypoxia-inducible factor 1α (HIF1α), which in turn promotes persistent high CAIX expression. Under hypoxic conditions, and in some solid tumors such as colorectal cancer (CRC), breast cancer, and pancreatic ductal adenocarcinoma (PDAC), abnormal transcriptional regulation of CAIX by HIF1α leads to CAIX overexpression.

[0004] In recent years, the application of radionuclide probes in tumor diagnosis and treatment has become increasingly widespread. Radionuclide probes can utilize the properties of radioactive isotopes to achieve precise diagnosis and treatment of tumors. Among these, 68 Ga and 18 F-labeled probes can be used in positron emission tomography (PET) imaging, and with their excellent imaging properties, they play a key role in early tumor detection, staging, and treatment efficacy evaluation. 177 Lu and 225 Radiolabeled probes, such as Ac, used in therapeutics, can kill tumor cells through the radiation released by the radioactive isotopes, thus 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 achieve precise tumor localization and effective treatment. At the same time, some probes have high non-specific uptake in normal tissues, which can easily cause radiation damage to normal tissues and trigger adverse reactions. In addition, the stability, affinity, and specificity of the probes need further improvement.

[0005] Developing efficient and safe CAIX cyclic peptide nucleoside probes has become a research hotspot in the field of tumor diagnosis and treatment. Novel CAIX cyclic peptide nucleoside probes can not only improve the accuracy and effectiveness of tumor diagnosis and treatment, but also provide clinicians with more precise diagnostic information and enable the development of more targeted treatment plans for cancer patients, thus possessing significant clinical value and broad market prospects. Summary of the Invention

[0006] The purpose of this invention is to provide modified CAIX-targeting cyclic peptides, radionuclide markers, and their applications in tumor diagnosis and treatment.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides a modified CAIX-targeting cyclic peptide, with a structure as shown in Formula IV or V:

[0008]

[0009]

[0010] Secondly, the present invention provides a radionuclide label for the cyclic peptide, wherein the cyclic peptide is labeled with a radionuclide, the radionuclide including a diagnostic radionuclide or a therapeutic radionuclide.

[0011] Furthermore, the radionuclide can be selected from... 90 Y、 177 Lu、 225 Ac or 213 Bi, preferably a cyclic peptide of formula IV or V labeled with a radionuclide.

[0012] When the radionuclide is 177 Lu, the method for preparing the radionuclide label includes the following steps:

[0013] (1) Add [ under aseptic conditions] 177 Lu]LuCl3 solution (usually from a reactor or generator);

[0014] (2) Mix with 0.8-1.2M sodium acetate buffer. 177 Lu]LuCl3 solution is used to maintain a pH of 3.8-5.5;

[0015] (3) Add the cyclic peptide to the product obtained in step (2). 177 In Lu solution, mix well and heat to 95℃ for 10-20 min;

[0016] (4) The product obtained in step (3) is purified by eluting the product with anhydrous ethanol to obtain the final product.

[0017] When the radionuclide is 225When Ac is used, the method for preparing the radionuclide label includes the following steps:

[0018] (1) Add [ under aseptic conditions] 225 Ac]AcCl3 solution (usually from a reactor or generator);

[0019] (2) Mix with 0.8-1.2M sodium acetate buffer. 225 The solution of Ac]AcCl3 is used to make the pH 5;

[0020] (3) Add the cyclic peptide to the product obtained in step 2). 225 In Ac solution, mix well and heat to 95℃ for 10-30 min;

[0021] (4) The product obtained in step (3) is purified by eluting the product with anhydrous ethanol to obtain the final product.

[0022] When the radionuclide is 213 When Bi is used, the method for preparing the radionuclide label includes the following steps:

[0023] (1) Rinse with pre-cooled 0.1M HCl containing 0.1% ascorbic acid. 225 Ac / 213 Bi generator, collecting... 213 Bi's rinsing solution;

[0024] (2) Impurities were removed by passing the solution through an anion exchange column, followed by rinsing with 0.1M HCl and elution with 0.5M HNO3. 213 Bi was dried under nitrogen and then redissolved in 0.1M HCl. Then NaAc-HAc buffer was added, and the mixture was stirred until the pH was controlled at 3.5-4.0. The reaction was then terminated by heating and stirring and then cooling in an ice bath.

[0025] (3) The reaction product obtained in (2) is purified, eluted with 70% ethanol / 30% physiological saline, and filtered to obtain the final product.

[0026] When the radionuclide is 90 When Y, the method for preparing the radionuclide label includes the following steps:

[0027] (1) Rinse with 0.05M HCl containing 0.1% ascorbic acid. 90 Sr / 90 Y generator, collecting... 90 Y's rinsing solution;

[0028] (2) containing 90 The eluent of Y was purified by removing residues using a strontium-selective resin column, and then... 90 NH4OAc buffer solution was added to solution Y to stabilize the pH of the reaction system at 4.5-5.0;

[0029] (3) Add CAIX-DOTA-cyclic peptide to the (2) system, stir at 80℃, and cool in an ice bath to terminate the reaction;

[0030] (4) The reaction solution in (3) is purified, washed with ethanol and eluted to obtain the final product.

[0031] Thirdly, the present invention provides the use of the radionuclide marker in the preparation of tumor radionuclide diagnostic and / or therapeutic drugs.

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

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

[0034] (II) Diagnostic radioactive molecular probes 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 assess a patient's response to CAIX-targeted therapy and determine whether the patient is a suitable candidate for therapeutic radionuclide therapy. 90 Y / 177 Lu / 225 Ac / 213 Bi substitution 68 Ga / 18 F undergoes radionuclide-targeted therapy.

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

[0037] (V) The present invention 68 Ga / 18 F / 177 Lu / 225 The preparation method of Ac-CAIX cyclic peptide has a high labeling rate. 68 Ga labeling rate can reach over 95%. 18 The F labeling rate can reach over 30%. 177The Lu labeling rate can reach over 70%. 225 Ac labeling rate can reach over 80%. Attached Figure Description

[0038] Figures 1a-1e The results are HPLC and mass spectrometry quality control results for 5 structures. Figure 1a For the quality control results of Equation I, Figure 1b For the quality control results of Formula II, Figure 1c For the quality control results of Formula III, Figure 1d For the quality control results of Formula IV, Figure 1e This is the quality control result for 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 analysis and parameter statistics of F-CAIX-RESCA-cyclic peptide (Formula III).

[0041] Figure 4 The numbers 'ac' represent the Micro-PET / CT imaging results of CAIX cyclic peptide I in the HT-29 mouse model and the imaging results of the Block group, as well as the SUVmax statistics, respectively, in the preferred embodiment of the present invention.

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

[0043] Figure 6 The numbers 'ac' represent the Micro-PET / CT imaging results of CAIX cyclic peptide III in the HT-29 mouse model and the imaging results of the Block group, as well as the SUVmax statistics, respectively, in the preferred embodiment of the present invention.

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

[0045] Figures 8a-8d The comparison of block biodistribution and 4-hour biodistribution of CAIX cyclic peptide III in the HT-29 mouse model in the preferred embodiment of the present invention, as well as specific values ​​and human dose estimation, are respectively.

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

[0047] Figure 10 In the examples a and b, respectively, are the results of radionuclide therapy experiments using CAIX cyclic peptides IV and V in the HT-29 mouse model, as described in the preferred embodiments of the present invention. 177 Lu-CAIX-cyclic peptide IV / V and 225 Ac-CAIX-cyclic peptide IV / V. Detailed Implementation

[0048] This invention aims to provide a modified CAIX cyclic peptide nuclide probe molecular structure, as well as the resulting diagnostic and therapeutic nuclide markers, preparation methods, and applications. Specifically, it relates to the preparation and application of a carbonic anhydrase IX (CAIX)-targeting cyclic peptide based on a metabolically optimized linker and its diagnostic and therapeutic nuclide markers. The CAIX-targeting cyclic peptide, by introducing the rigid bifunctional chelator RESCA (Rigid Ethylene-bridged Cyclam Chelator Assembly) instead of traditional chelators, and combining it with sulfonated or alkylated amino acid linkers (such as PPAc, Cys(SO3H)-His, Leu-Thr), significantly improves the probe's resistance to enzymatic degradation and in vivo stability. By optimizing the linker structure, lipophilicity is reduced (logP value optimized from -6.51 to -3.06), hepatobiliary metabolism and non-specific uptake are reduced (liver uptake reduced to 0.54% ID / g), while maintaining a high uptake rate in tumor target tissue (92.6% ID / g). The cyclic peptide can label diagnostic nuclides (such as... 68 Ga、 18 F) Used for PET imaging, or therapeutic radionuclides (such as...) 177 Lu、 225 Ac) is used for intratumoral irradiation therapy, employing DOTA or NOA bound to alkylated amino acid linkers or albumin ligands (such as Leu-Thr-Leu-Leu, ABM). Experiments show that the labeling agents possess high radiochemical purity (>95%), excellent pharmacokinetic properties, and low renal retention, making them suitable for the precise diagnosis and targeted therapy of CAIX-highly expressed tumors such as colorectal cancer, renal cell carcinoma, and breast cancer. This invention provides a highly efficient and safe molecular probe solution for tumor diagnosis and treatment.

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

[0050] This invention provides a modified CAIX cyclic peptide, the sequence of which 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 cyclic peptide contains a chelating agent called RESCA, and the RESCA is linked to a sulfonated or alkylated amino acid linker.

[0058] The linker is coupled with the chelating agent RESCA, and can also be coupled with other chelating agents selected from DOTA, NOA, HBED-CC, DTPA, or 3pC-NETA-NCS.

[0059] The structures of the CAIX cyclic peptides are shown in Formulas I, II, III, IV, and V:

[0060]

[0061]

[0062] This invention provides a structural optimization scheme for a cyclic peptide radionuclide probe based on carbonic anhydrase IX (CAIX) targeting.

[0063] 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 exhibit a progressive structural design, as detailed below:

[0064] Its molecular structure is characterized by: Formula I using a prototype probe [ 68 Based on the DOTA chelating system of Ga-DPI-4452, the structure of the probe molecule is modified by replacing the traditional DOTA coordination system with the rigid bifunctional chelating agent RESCA (Rigid Ethylene-bridged Cyclam Chelator Assembly). Compared with highly polar chelating agents such as DOTA and Nota, RESCA has significantly enhanced anti-enzymatic properties, and its rigid cyclic structure can effectively reduce the rate of metabolic degradation in vivo, thereby significantly extending the systemic cycling half-life of the probe.

[0065] Regarding the structural optimization of the linker, this invention, through structure-activity relationship studies, 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 a design strategy that reduces 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 achieves a dual optimization mechanism through molecular modification of the sulfonic acid group: on the one hand, it reduces the capture rate of the reticuloendothelial system (RES) through charge modification (Kupffer cell uptake decreased by 67%); on the other hand, it reduces the hepatobiliary metabolic clearance rate to (1.4±1.9)%ID / g through electrostatic repulsion, thereby improving the target-to-probe ratio.

[0066] Although the RESCA-sulfonation design reduced hepatobiliary metabolism, the probe was partially cleared via glomerular filtration at a slow rate. Further structural optimization revealed that changing to a Leu-Thr (leucine-threonine) dipeptide linker (Formula III) resulted in significant pharmacokinetic advantages for the probe: 4 hours post-injection, nonspecific uptake by the liver, kidneys, 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 by the tumor target tissue remained at the initial value of (92.6±3.8)%. This improved metabolic profile is primarily attributed to the β-sheet secondary structure formed by the Leu-Thr linker, which effectively reduces renal uptake.

[0067] In the field of radionuclide therapy, this invention optimizes the metabolic properties of labeled ligands based on DOTA or Nota chelating agent systems. The modified CAIX-targeting cyclic peptides shown in Formula IV or V are particularly suitable for preparing therapeutic radionuclide labels, and their structural designs are as follows:

[0068] By introducing specific polypeptide sequences Leu-Thr-Leu-Leu-PPAc (Formula IV) or ABM-PPAc (Formula V) as linker structures, the renal metabolic characteristics of the drug were significantly optimized (renal 4-hour uptake rate reduced by ≥90%), effectively reducing the risk of radionuclide accumulation in non-target organs. Furthermore, to improve the long-term stability of the drug in the circulatory system, this invention, for the first time, covalently coupled an albumin-binding ligand (as shown in Formula V) to the end of the polypeptide sequence. Through targeting the active binding mechanism of plasma albumin, the metabolic clearance rate was significantly slowed down while maintaining tumor targeting efficiency (target / non-target ratio increased by more than 1.8 times). This design achieves synergistic optimization of metabolic clearance rate and circulatory retention capacity, providing a safer environment for highly toxic α / β-nuclides (such as...). 225 Ac、 177 Lu's precise delivery provides a solution that combines efficiency and security.

[0069] The present invention also provides a diagnostic radionuclide marker, which is the modified CAIX cyclic peptide labeled with a radionuclide.

[0070] According to a specific embodiment of the present invention, the radionuclide can be a diagnostic radionuclide, preferably a positron-emitting radionuclide. 68 Ga or 18 F.

[0071] According to the present invention, the radionuclide can also be a therapeutic radionuclide, with the aim of achieving CAIX-overexpressing tumor-targeting molecular imaging therapy. The therapeutic radionuclide is preferably... 90 Y、 177 Lu、 225 Ac and 213 At least one of Bi.

[0072] CAIX cyclic peptides modified with the bifunctional conjugates DOTA, NOTA, DTPA, and the bimodal bifunctional ligand 3pC-NETA-NCS can be used as therapeutic radionuclides. 90 Y, 177 Lu, 225 Ac or 213 Bi marker, obtain 90 Y-CAIX cyclic peptide, 177 Lu-CAIX cyclic peptide,225 Ac-CAIX cyclic peptide, 213 Bi-CAIX cyclic peptide therapeutic molecular probe.

[0073] The labeling of therapeutic radionuclides can be performed using various methods conventional 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 adopted as follows: 177 Taking Lu as an example, CAIX cyclic peptides modified with bifunctional chelators DOTA, NOA, DTPA, and 3pC-NETA-NCS were used to obtain the corresponding labeling precursors. Labeling buffers were prepared according to the ratio of 3 mL of 0.05 M HCl to 195 μL of 1 M NaAc and set aside. 100 μL (60 μg) of labeling precursors (DOTA-CAIX cyclic peptide, NOA-CAIX cyclic peptide, DTPA-CAIX cyclic peptide, 3pC-NETA-CAIX cyclic peptide) were added to 100-150 μL of labeling buffer, and then... 177 Lu; Adjust the pH to 5.5, react at 70-95℃ for 10-15 min. When the labeling rate is less than 90%, separate and purify using a Sep-pak C18 column to obtain... 177 Lu-CAIX cyclic peptide. Labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC. The prepared... 177 The Lu-CAIX cyclic peptide was isolated and purified to a radiochemical purity greater than 95%.

[0074] The present invention also provides the use of diagnostic radionuclide-labeled CAIX cyclic peptides in the preparation of CAIX-targeted tumor PET imaging reagents.

[0075] The present invention also provides the use of therapeutically radiolabeled CAIX cyclic peptides in the preparation of CAIX-targeted tumor radionuclide therapeutic drugs.

[0076] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0077] Example 1: Synthesis and Characterization of CAIX-Targeting Cyclic Peptide for Metabolic Optimization Linker

[0078] 1. Experimental objective: To synthesize a carbonic anhydrase IX (CAIX)-targeting cyclic peptide based on a metabolically optimized linker and to verify its structure.

[0079] 2. Experimental steps:

[0080] (1) Cyclic peptide sequence design:

[0081] The cyclic peptide sequences and structures are shown in Formulas I, II, III, IV, and V above.

[0082] (2) Solid-phase synthesis:

[0083] The Fmoc solid-phase synthesis method was adopted, using Rink Amide resin as a carrier, to sequentially couple amino acids and linkers, and finally form a cyclic structure through a thiol-maleimide cyclization reaction.

[0084] (3) Purification and characterization:

[0085] HPLC analysis:

[0086] Column: Kromasil 100-5C18 (4.6×250mm);

[0087] Mobile phase A: 0.1% TFA acetonitrile, Mobile phase B: 0.1% TFA water;

[0088] Gradient: 0-20 min, 30% A → 60% A; 20-25 min, 60% A → 100% A;

[0089] Flow rate: 1 mL / min; Detection wavelength: 220 nm.

[0090] Mass spectrometry (MS) validation:

[0091] 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.

[0092] The results are as follows Figures 1a-1e As shown, all five cyclic peptides were successfully synthesized and possessed high chemical purity.

[0093] Example 2 68 Preparation of Ga-labeled CAIX-targeting cyclic peptide

[0094] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula I, II or III) was subjected to... 68 Ga nuclide labeling (T) 1 / 2 =68min; β + :96.7%; E=511keV) Nuclide labeling, prepared using a columnar germanium-gallium generator. 68 Ga nuclide was rinsed with 4 mL of 0.05 M HCl solution. 68 Ga, mixed with 1M NaAc, and the cyclic peptide was added to the resulting68 In Ga solution, mix well and heat to 37℃ for 10-20 min. Purify the product using a C18 column, wash with 5 mL of physiological saline, elute 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 formulation. The labeling rate and radiochemical purity are determined by radio-HPLC or radio-TLC. 68 The Ga-CAIX-RESCA-cyclic peptide labeling rate is approximately 92%, and the radiochemical purity is greater than 95%.

[0095] Example 3 18 Preparation of F-labeled CAIX-targeting cyclic peptide

[0096] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide represented by formula I, II or III) was subjected to... 18 F(T 1 / 2 =109.8min; β + :96.7%; E=511keV) labeling of nuclides, 18 F is prepared using a cyclotron accelerator. The accelerator produces [a substance containing...]. 18 F - H2 18 O is passed through a QMA ion exchange column. 18 F - Adsorbed onto a QMA column, the column was washed with 0.5 mL of physiological saline; 0.1 mL containing... 18 F - Physiological saline was placed in a reaction tube containing 11 μL of 10-fold KHP and 6 μL of 2 mM AlCl3 solution, mixed well, and incubated at room temperature for 5 min. 10 μL of the 10 mg / mL labeled precursor CAIX-RESCA-cyclic peptide was added, and the reaction was carried out at 100 °C for 15 min. After the reaction solution was cooled to room temperature, the product was purified using a C18 separation column. After washing with 5 mL of physiological saline, the product was eluted with 0.6 mL of 80% ethanol and passed through a 0.22 μm sterile filter membrane. The solvent was dried with N2, and the product was diluted with physiological saline to obtain the product formulation. The labeling rate and radiochemical purity were determined by radio-HPLC or radio-TLC.

[0097] Figure 2 Showing 18 Radio-TLC detection results of F-CAIX-RESCA-cyclic peptide (Formula III), obtained by measurement 18 The labeling rate of F-CAIX-RESCA-cyclic peptide (Formula III) is approximately 92%, and the radiochemical purity is greater than 99%.

[0098] Example 4: Purified 18In vitro stability analysis of F-CAIX-RESCA-cyclic peptide

[0099] 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 physiological saline (or 5% HSA solution) and incubated at 4°C. Samples containing 37-74 kBq (1-2 μCi) of radioactivity were collected at 0 h, 2 h, 12 h, 24 h, 36 h, and 60 h for radio-TLC analysis. Analytical method: 2 μL of sample containing 37-74 kBq (1-2 μCi) of radioactivity was taken. 18 F-CAIX-RESCA-cyclic peptide saline solution or 18 Add 5% HSA solution of F-CAIX-RESCA-cyclic peptide to 20 μL of saturated EDTA and mix well. Perform radio-TLC analysis. Place 2 μL of sample 1 cm from the bottom of Xinhua No. 1 filter paper in the physiological saline developing system. After complete development, remove the filter paper and let it dry. Perform radio-TLC detection to detect free peptides. 18 F and 18 The Rf values ​​of the F-CAIX-RESCA-cyclic peptide were 0.9–1 and 0–0.1, respectively; the results showed that... 18 The F-CAIX-RESCA-cyclic peptide exhibits good stability within 4 hours in physiological saline solution or 5% HSA solution.

[0100] The above experiment shows that, 18 The F-CAIX-RESCA-cyclic peptide structure exhibits good stability.

[0101] Optimized Example 5 18 Pharmacokinetic analysis of F-CAIX-RESCA-cyclic peptide (Formula III) in normal KM mice

[0102] Prepare 5 female KM mice (5-6 weeks old, 18-20g) and use optimized... 18 F-CAIX-RESCA-cyclic peptide (Formula III) was diluted with physiological saline to 18.5 MBq / mL (0.5 mCi / ml). Each mouse was injected with 3.7 MBq (0.1 mCi, 200 μL) of the labeled product via the tail vein. Blood was collected via capillary tube from the periorbital venous plexus at corresponding time points after injection (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) and placed in a radioimmunoassay tube. The blood sample injected via the tail vein of each mouse was collected. 18One percent of the radioactivity of F-CAIX-RESCA-cyclic peptide (Formula III), i.e., 0.037 MBq (1 μCi, 2 μL), was used as the reference activity for measurement. The reference activity was measured together with the collected blood sample using a gamma counter. After attenuation correction, the data were analyzed using Prism 6.0 software to calculate the percentage injection dose rate per gram of blood sample. The results are expressed as %ID / g±SD.

[0103] Specific results are as follows Figure 3 As shown in the figure, 18 F-CAIX-RESCA-cyclic peptide (Formula III) has good pharmacokinetic properties and is suitable for in vivo imaging studies.

[0104] Example 6 18 Study of F-CAIX-RESCA-cyclic peptides (Formulas I, II, III, and IV) in PET / CT imaging of HT29 tumor-bearing mouse model

[0105] Seven HT29 tumor-bearing mice were used and divided into four groups (n=2, n=2, n=2, n=1) for imaging studies. Each group was injected with 7.4 MBq (0.2 mCi, 200 μL) via the tail vein. 18 For F-CAIX-RESCA-cyclic peptides (Formulas I, II, III, and IV), imaging blocking is performed by simultaneously injecting 0.1 mg of non-radioactively labeled CAIX-RESCA-cyclic peptides (Formulas I, II, and III). 18 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 image results are as follows: Figure 4 a. Significant uptake was observed at the tumor site in the experimental group, showing a clear difference from the inhibition imaging group below. High uptake was also observed in the kidneys and gastrointestinal tract. The SUVmax statistical results for important organs in the experimental group are as follows: Figure 4 As shown in b, the statistical results of SUVmax of important organs in the suppressed imaging group are as follows: Figure 4 As shown in c.

[0106] 18 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. Results are shown below. Figure 5 It is still observed that tumors and kidneys have relatively high uptake, while gastrointestinal uptake is reduced compared to Formula I.

[0107] 18 Mice in the F-CAIX-RESCA-cyclic peptide (Formula III) group underwent PET / CT imaging and delineation of important organs at 40 min, 60 min, 120 min, 240 min, and 360 min after injection. The results are shown in the table below. Figure 6 a, Figure 6 b and Figure 6 c. After optimization, the probe showed a slight increase in uptake in the liver and kidneys in the early imaging stages compared to Formulas I and II. However, in the later imaging stages, the uptake in the liver, kidneys, stomach, and intestines decreased significantly, while the uptake in the tumor site did not decrease significantly, maintaining a strong retention effect. This later imaging result is superior to Formulas I and II, and has greater potential for clinical translation.

[0108] 18 Mice in the F-CAIX-RESCA-cyclic peptide (Formula IV) group underwent PET / CT imaging at 2h, 3h, and 4h post-injection. Results are shown below. Figure 7 It can be seen that the uptake at the tumor site did not decrease significantly over time, which can be used to guide subsequent radionuclide therapy research.

[0109] Example 7 18 Distribution and inhibition experiments of F-CAIX-RESCA-cyclic peptide (Formula III) in tumor-bearing mice, and estimation of human radiation dose.

[0110] Fifteen HT29 tumor-bearing mice (female, 5-6 weeks old, 18-20g) were randomly divided into 5 time groups, with 3 mice in each group. 7.4 MBq (0.2 mCi, 200 μL) was injected via the tail vein. 18 Following the administration of F-CAIX-RESCA-cyclic peptide, mice were anesthetized and euthanized at 5 min, 30 min, 60 min, 120 min, and 240 min. Blood, heart, liver, spleen, lung, kidney, stomach, intestine, muscle, bone, and brain were collected and weighed. The radioactivity counts of the organs were detected using a γ-counter. After attenuation correction, the biodistribution of each organ at different time points was statistically analyzed. The blocking group was simultaneously injected with 0.1 mg of non-radioactive CAIX-RESCA-cyclic peptide.

[0111] like Figure 8a , Figure 8b , Figure 8c The results all show 18 F-CAIX-RESCA-cyclic peptide (Formula III) is primarily metabolized by the kidneys in animals, exhibiting low non-specific uptake in systemic tissues, but showing extremely high uptake and prolonged retention at tumor sites, and can be blocked by precursors. Human radiation dose can be estimated using biodistribution results. Figure 8d As shown, the effective dose is significantly lower than the radiation dose limit, demonstrating the radiation safety of the probe in clinical applications.

[0112] This invention significantly improves the pharmacokinetic properties of the CAIX-targeting cyclic peptide by introducing metabolically optimized linkers (such as PPAc-Gln; Cys(SO3H)-His-PPAc-Gln and Leu-Thr-PPAc-Gln), giving it the advantages of high tumor targeting, low non-specific uptake, and rapid renal clearance. It can also be used in conjunction with therapeutic radionuclides (such as...) 68 Ga、 177 Lu) labeling can simultaneously achieve precise tumor imaging and efficient internal irradiation therapy, and has broad clinical application prospects.

[0113] Optimized Example 8 18 Safety testing (toxicological experiments) of F-CAIX-RESCA-cyclic peptide (Formula III)

[0114] Ten normal Kunming mice were prepared and divided into two groups for the experiment: an experimental group and a control group, n=5. Each mouse in the experimental group was injected with 10 times the recommended dose. 18 F-CAIX-RESCA-cyclic peptide (Formula III) 74 MBq (2 mCi, 200 μL) was administered to mice, while the control group received the same volume of physiological saline solution. After a certain period, blood samples were collected for routine blood tests, liver and kidney function tests, and body weight changes were continuously recorded (for 16 days). Results are as follows: Figure 9a , Figure 9b The experimental group mice showed no significant differences in blood routine results compared to the control group, and their liver and kidney functions were within the normal range. The weight changes in the experimental group mice were also similar to those in the control group. This experiment demonstrates the high safety of the probe and holds promise for clinical translation studies.

[0115] Example 9 177 Preparation of Lu-labeled CAIX-targeting cyclic peptide

[0116] Using CAIX-targeted cyclic peptides IV and V as precursors, a direct labeling method was employed. [Under aseptic conditions, […]] 177 LuCl3 solution (specific activity ≥ 50 GBq / mg) was mixed with 0.8-1.2 M sodium acetate buffer. 177 The pH of the Lu]LuCl3 solution was adjusted to 3.8-5.5; the cyclic peptide was then added. 177 The product was mixed in Lu solution and heated to 95°C for 10-20 min. The resulting product was purified using a C-18 column, eluted with anhydrous ethanol, to obtain the radionuclide therapy probe. 177 Lu-CAIX-DOTA-cyclic peptide (radiochemical purity > 98%, specific activity 22.5 MBq / μg).

[0117] Example 10 225 Preparation of Ac-labeled CAIX-targeting cyclic peptide

[0118] Using CAIX-targeted cyclic peptides IV and V as precursors, a direct labeling method was employed. [Under aseptic conditions, […]] 225 Ac]AcCl3 solution (specific activity ≥ 50 GBq / mg), mixed with 0.8-1.2 M sodium acetate buffer. 225 The pH was adjusted to 5 using an AcCl3 solution; the cyclic peptide was then added. 225 In Ac solution, mix well and heat to 95℃ for 10-30 min; purify the obtained product using a C-18 column, eluting the product with anhydrous ethanol to obtain the radionuclide therapy probe. 225 Ac-CAIX-DOTA-cyclic peptide (radiochemical purity > 98%, specific activity 22.5 MBq / μg).

[0119] Example 11 177 Lu-CAIX-DOTA-cyclic peptide and 225 Evaluation of the therapeutic effect of Ac-CAIX-DOTA-cyclic peptide on HT-29 renal cell carcinoma model mice

[0120] 6-8 week old BALB / c nude mice were subcutaneously inoculated with HT-29 human renal cell carcinoma cells (5×10⁻⁶). 6 (cells / each), until the tumor volume reaches 100-150mm. 3 They were then randomly divided into seven groups (n=5): Treatment group: single tail vein injection 177 Lu / 225 Ac-CAIX-DOTA-cyclic peptide-3 (Formula IV) (dosage: 3.7 MBq and 1.85 MBq) and injectable equivalent doses 177 Lu / 225 Ac-CAXI-DOTA-cyclic peptide-4 (Formula V) (linked to albumin ligand); Blank control group: injected with physiological saline.

[0121] The efficacy was evaluated by dynamically monitoring tumor volume (using the formula V = 0.5 × L × W). 2 Calculation) and survival analysis (endpoint defined as tumor volume ≥1000 mm) 3 Or a weight loss of >20%;

[0122] Experimental results are as follows Figure 10 a, Figure 10 As shown in b, the data indicates that the present invention 177 Lu / 225Ac-CAXI-DOTA-cyclic peptide demonstrated significant advantages in the HT-29 renal cell carcinoma model: the tumor volume in the treatment group increased by only ≤30% from baseline within 28 days (compared to 85% 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 / renal uptake ratio (T / K = 2.16) in the treatment group was significantly higher than that in the unmodified ligand group (T / K = 1.2), confirming that the optimized linker arm 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 were no statistically significant differences in renal function indicators (creatinine, blood urea nitrogen) compared with the control group (p>0.05), and no radiation damage was observed in histopathology, verifying the clinical applicability of the present invention.

[0123] Example 12 213 Preparation of Bi-labeled CAIX-targeting cyclic peptide

[0124] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide shown in formula IV or V) was subjected to... 213 Bi nuclide labeling (T) 1 / 2 Labeling of nuclides (α; E = 8.4 MeV) at 45.6 min. Elute with 5 mL of pre-cooled 0.1 M HCl (containing 0.1% ascorbic acid). 225 Ac / 213 Bi generator, collecting... 213 The eluent for Bi is used to remove impurities (such as Bi) through an anion exchange column (e.g., AG1-X8). 225 Ac leakage was washed with 0.1M HCl and then eluted with 0.5M HNO3. 213 Bi, dried under nitrogen, was redissolved in 0.1M HCl for later use. 213 Add 200 μL of 1M NaAc-HAc buffer (pH 4.0) to the Bi solution, and mix well. The pH should be controlled at 3.5-4.0. Add 50-100 μg of CAIX-DOTA-cyclic peptide IV or V (dissolved in 0.1M NH4OAc, pH 5.0), and adjust the final volume to 2 mL. Heat and stir at 95°C for 10 minutes (sealed to prevent volatilization), and terminate the reaction by rapid cooling in an ice bath. Load the reaction solution into a pre-activated (5 mL ethanol 10 mL H2O) C18 column, and wash with 10 mL physiological saline to remove free... 213 Bi was eluted with 1 mL of 70% ethanol / 30% physiological saline. 213 Bi-CAIX-DOTA-cyclic peptide, filtered through a 0.22 μm sterile membrane. Ethanol was dried under nitrogen, reconstituted with physiological saline to a final volume of 1 mL, aliquoted, and used immediately. Radio-HPLC analysis (flow rate 1 mL / min, acetonitrile: 0.1% TFA gradient, alpha radioactivity detection); purity >95%.

[0125] Example 13 90 Preparation of Y-labeled CAIX-targeting cyclic peptide

[0126] The prepared CAIX-RESCA-cyclic peptide (cyclic peptide shown in formula IV or V) was subjected to... 90 Y-nucleolabeled (T) 1 / 2 =64.1h; β - Labeling of nuclides (E = 2.28 MeV). Elute with 8 mL of 0.05 M HCl (containing 0.1% ascorbic acid). 90 Sr / 90 Y generator, collecting... 90 Y's eluent. Passed through a strontium-selective resin column (e.g.) Remove residue 90 Sr, ensuring radioactive purity > 99.9%. To the purified... 90 Add 500 μL of 1M NH4OAc buffer (pH 5.0) to solution Y to stabilize the pH of the reaction system at 4.5-5.0. Add 100-200 μg of CAIX-DOTA-cyclic peptide (dissolved in 0.1M NH4OAc), adjusting the final volume to 3 mL. Stir at 80°C for 30 minutes (sealed and protected from light), then terminate the reaction by cooling in an ice bath. Load the reaction solution into a pre-activated (5 mL ethanol 10 mL H2O) C18 column and wash with 10 mL physiological saline to remove free peptides. 90 Y. Elute with 1.5 mL of 60% ethanol. 90 Y-CAIX-DOTA-cyclic peptide, filtered through a 0.22 μm sterile membrane. Ethanol was dried under nitrogen and reconstituted with physiological saline to a final volume of 2 mL. Radio-HPLC analysis was performed (flow rate 1 mL / min, acetonitrile: 0.1% TFA gradient, detecting β-radioactivity); expected purity >90%.

[0127] The above description is only a preferred embodiment of the present invention and does not limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A modified CAIX-targeting cyclic peptide, characterized in that, The structure is shown in Equation IV or Equation V.

2. The radionuclide label of the cyclic peptide according to claim 1, characterized in that, It is a cyclic peptide labeled with a radionuclide, the radionuclide including diagnostic or therapeutic radionuclides.

3. The radionuclide marker according to claim 2, characterized in that: The radionuclides are selected from 90 Y、 177 Lu、 225 Ac or 213 Bi.

4. The method for preparing the radionuclide marker according to claim 3, characterized in that, When the radionuclide is 177 Lu, the method for preparing the radionuclide label includes the following steps: (1) Add [ under aseptic conditions] 177 Lu]LuCl3 solution; (2) Mix with 0.8-1.2M sodium acetate buffer. 177 Lu]LuCl3 solution is used to maintain a pH of 3.8-5.5; (3) Add the cyclic peptide to the product obtained in step (2). 177 In Lu solution, mix well and heat to 95℃ for 10-20 min; (4) The product obtained in step (3) is purified by eluting the product with anhydrous ethanol to obtain the final product.

5. The method for preparing the radionuclide label according to claim 3, characterized in that, When the radionuclide is 225 When Ac is used, the method for preparing the radionuclide label includes the following steps: (1) Add [ under aseptic conditions] 225 Ac]AcCl3 solution; (2) Mix with 0.8-1.2M sodium acetate buffer. 225 The solution of Ac]AcCl3 is used to make the pH 5; (3) Add the cyclic peptide to the product obtained in step (2). 225 In Ac solution, mix well and heat to 95℃ for 10-30 min; (4) The product obtained in step (3) is purified by eluting the product with anhydrous ethanol to obtain the final product.

6. The method for preparing the radionuclide marker according to claim 3, characterized in that, When the radionuclide is 213 When Bi is used, the method for preparing the radionuclide label includes the following steps: (1) Rinse with pre-cooled 0.1M HCl containing 0.1% ascorbic acid. 225 Ac / 213 Bi generator, collecting... 213 Bi's rinsing solution; (2) Impurities were removed by passing the solution through an anion exchange column, followed by rinsing with 0.1M HCl and elution with 0.5M HNO3. 213 Bi was dried under nitrogen and then redissolved in 0.1M HCl. Then NaAc-HAc buffer was added, and the mixture was stirred until the pH was controlled at 3.5-4.

0. The reaction was then terminated by heating and stirring and then cooling in an ice bath. (3) The reaction product obtained in (2) is purified, eluted with 70% ethanol / 30% physiological saline, and filtered to obtain the final product.

7. The method for preparing the radionuclide marker according to claim 3, characterized in that, When the radionuclide is 90 When Y, the method for preparing the radionuclide label includes the following steps: (1) Rinse with 0.05M HCl containing 0.1% ascorbic acid. 90 Sr / 90 Y generator, collecting... 90 Y's rinsing solution (2) containing 90 The eluent of Y was purified by removing residues using a strontium-selective resin column, and then... 90 NH4OAc buffer solution was added to solution Y to stabilize the pH of the reaction system at 4.5-5.0; (3) Add CAIX-DOTA-cyclic peptide to the (2) system, stir at 80℃, and cool in an ice bath to terminate the reaction; (4) The reaction solution in (3) is purified, washed with ethanol and eluted to obtain the final product.

8. The use of the radionuclide marker according to any one of claims 2-7 in the preparation of tumor radionuclide diagnostic and / or therapeutic drugs.

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