A cyclic peptide targeting CAIX and preparation method and application thereof

Abstract

The application belongs to the technical field of nuclear medicine, and relates to a CAIX-targeting cyclic peptide as well as a preparation method and application thereof. The structure of the CAIX-targeting cyclic peptide is shown as formula 1. The Linker is a ring-forming linker. The Chelator is a radionuclide chelating group. The CAIX-targeting cyclic peptide provided by the application has high affinity with CAIX and low affinity with CAIX isozyme CAII, so that the CAIX-targeting cyclic peptide has high selective affinity with CAIX. The radionuclide-labeled radioactive probe has high labeling rate and excellent in-vitro stability, has excellent pharmacokinetic characteristics in tumor-bearing model mice of human renal clear cell carcinoma, has extremely high tumor uptake, and can meet the diagnosis requirement of tumor muscle ratio and tumor kidney ratio. The long-time retention characteristics of the probe in the tumor also make the probe have high tumor treatment application value.

Description

Technical Field

[0001] This invention belongs to the field of nuclear medicine technology and relates to a cyclic peptide targeting CAIX, its preparation method and application. Background Technology

[0002] Carbonic anhydrase 9 (CAIX) is a zinc-containing metalloenzyme mediated by hypoxia-inducible factor 1-α (HIF1-α) and highly upregulated in hypoxic tumors. Overexpression of CAIX maintains intracellular pH homeostasis by catalyzing the interconversion between CO2 and HCO3-, thus contributing to tumor cell survival. Besides regulating the microenvironment pH, CAIX also plays a crucial role in mediating tumor progression, invasion, and metastasis. CAIX is overexpressed in various solid tumors, such as clear cell renal cell carcinoma, colorectal cancer, glioma, bladder cancer, cervical cancer, head and neck cancer, breast cancer, lung cancer, and pancreatic cancer, while its expression is low in normal tissues.

[0003] CAIX, as a hypoxia-induced tumor-specific biomarker, can be used for imaging and treatment of malignant solid tumors using radionuclide molecular probes. Radionuclide probe imaging offers advantages such as non-invasiveness and high sensitivity, while radionuclide probe therapy boasts advantages such as precise targeting, minimal side effects, and non-invasiveness, making it suitable for the diagnosis and treatment of primary and metastatic tumors with high CAIX expression.

[0004] While a number of CAIX-targeting nuclide probes have entered preclinical research, most suffer from drawbacks such as low tumor uptake and high uptake in non-target organs like the kidneys and liver. DPI-4452 is a publicly disclosed CAIX-targeting compound, radiolabeled with a nuclide. 68 Ga-DPI-4452 and 177 Lu-DPI-4452 shows high uptake in tumors with high CAIX expression, demonstrating promising application prospects, but 68 Ga-DPI-4452 exhibits high gastrointestinal uptake, which can affect its imaging performance and also make the gastrointestinal tract a potential therapeutic probe. 177 The Lu-DPI-4452 probe, with its dose limit on major organs, suffers from high non-target uptake in clinical applications. Its pharmacokinetics still have room for optimization to further improve tumor uptake and retention time, thereby achieving better tumor treatment outcomes. Therefore, there remains an urgent clinical need for radiopharmaceuticals targeting CAIX with better drug-like properties. Summary of the Invention

[0005] The main objective of this invention is to overcome the deficiencies in the prior art and provide a cyclic peptide targeting CAIX, its preparation method, and its application.

[0006] To achieve the above objectives, the specific technical solution is as follows:

[0007] This invention provides a cyclic peptide targeting CAIX, the structure of which is shown in Formula 1:

[0008] Formula 1;

[0009] Wherein, the Linker is a loop connector, and the loop connector is selected from one of the following structures:

[0010] ;

[0011] The chelator is a nuclide chelating group.

[0012] This invention constructs a CAIX-targeting cyclic peptide with high affinity for CAIX through spatial configuration optimization and pharmacokinetic regulation. The constructed CAIX-targeting cyclic peptide significantly reduces its affinity for CAII (an isoenzyme of CAIX, which is highly expressed in the human gastrointestinal tract).

[0013] Furthermore, the ring connector is selected from one of the following structures:

[0014] .

[0015] Furthermore, the radionuclide chelating group is a bifunctional chelating agent, which is selected from one of DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, AAZTA, DOTAGA, NODA-MPAA, HBED, THP, and Macropa.

[0016] Furthermore, the cyclic peptide targeting CAIX has the structure shown in Formula 2:

[0017] Equation 2.

[0018] Furthermore, the cyclic peptide targeting CAIX has the structure shown in Formula 3:

[0019] Formula 3.

[0020] This invention also provides a method for preparing a CAIX-targeting cyclic peptide, comprising the following steps:

[0021] (1) The Fmoc solid-phase synthesis method was adopted, and the Rink Amide MBHA Resin resin was used as a carrier to sequentially couple amino acids to synthesize peptide chains. The synthesis sequence was: Cys → Ser → Trp → Thr → Leu → Phe (3-NH-(4-sulfonylbutanylamino)) → Asp → DPro → Glu → Cys → Gln → 2-(piperazin-1-yl)acetic acid;

[0022] (2) After the peptide chain is synthesized, the radionuclide chelating group is attached to the N-terminus of the peptide chain, and the linear peptide is obtained by cleavage and purification;

[0023] (3) The linear peptide was cyclized using a halogenated linker to form a cyclic peptide, which was then purified to obtain a cyclic peptide targeting CAIX.

[0024] Further, in step (1), 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIC) are used as condensing agents to attach the 9-fluorenemethoxycarbonyl (Fmoc) protected amino acid to the resin; the Fmoc protecting group is removed using an N,N-dimethylformamide (DMF) solution containing piperidine.

[0025] Further, in step (2), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) are used as condensing agents to connect the radionuclide chelating group to the N-terminus of the peptide chain.

[0026] Furthermore, in steps (2) and (3), reversed-phase high-performance liquid chromatography is used for purification.

[0027] The present invention also provides a cyclic peptide radioactive probe targeting CAIX, comprising the above-mentioned cyclic peptide targeting CAIX and a labeled radionuclide.

[0028] Furthermore, the radionuclide used for labeling is a diagnostic radionuclide or a therapeutic radionuclide.

[0029] Specifically, the diagnostic radionuclide is selected from... 68 Ga、 64 Cu、 18 F, 86 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I; the therapeutic radionuclide is selected from...177 Lu、 125 I, 131 I, 211 At、 90 Y、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

[0030] The probes of the present invention targeting CAIX cyclic peptides, after being labeled with radionuclides, have better targeting selectivity, while not changing the affinity between the labeled probes and CAIX proteins.

[0031] The present invention further provides the application of the above-mentioned CAIX-targeting cyclic peptide and the above-mentioned CAIX-targeting cyclic peptide radioactive probe in the preparation of diagnostic and therapeutic tumors.

[0032] Specifically, the tumors include, but are not limited to, clear cell renal cell carcinoma, colorectal cancer, glioma, bladder cancer, cervical cancer, head and neck cancer, breast cancer, lung cancer, and pancreatic cancer, especially clear cell renal cell carcinoma.

[0033] Compared with the prior art, the present invention has the following significant advantages:

[0034] The cyclic peptide targeting CAIX provided by this invention has a high affinity for CAIX, while having a low affinity for CAIX isoenzyme CAII, resulting in a high selective affinity for CAIX. The radionuclide-labeled probe has a high labeling rate and excellent in vitro stability, exhibiting excellent pharmacokinetic characteristics in human clear cell renal cell carcinoma-bearing mouse models. The probe has extremely high tumor uptake, and the tumor-to-muscle ratio and tumor-to-kidney ratio meet diagnostic requirements. Its long-term retention in tumors also gives it high value for tumor treatment applications. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is the HPLC chromatogram for WF1 purity detection according to the present invention;

[0037] Figure 2This is the LC-MS spectrum of WF1 in this invention;

[0038] Figure 3 This is the HPLC chromatogram for WF2 purity detection according to the present invention;

[0039] Figure 4 This is the LC-MS spectrum of WF2 in this invention;

[0040] Figure 5 This is the SPR binding kinetic curve of WF1 and human CAII protein in this invention;

[0041] Figure 6 This is the SPR binding kinetic curve of WF2 and human CAII protein in this invention;

[0042] Figure 7 This is the SPR binding affinity curve of WF2 and human CAII protein of this invention;

[0043] Figure 8 This is the SPR binding kinetic curve of the present invention's DPI-4452 and human CAII protein;

[0044] Figure 9 This is the SPR binding affinity curve of the present invention's DPI-4452 and human CAII protein;

[0045] Figure 10 This is the present invention. 68 Chromatogram of Ga-WF1 radiochemical purity determination;

[0046] Figure 11 This is the present invention. 68 Chromatogram of Ga-WF2 radiochemical purity determination;

[0047] Figure 12 This is the present invention. 68 In vitro stability graph of Ga-WF1;

[0048] Figure 13 This is the present invention. 68 In vitro stability graph of Ga-WF2;

[0049] Figure 14 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled DPI-4452 probe and human CAIX protein;

[0050] Figure 15 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled WF1 probe and human CAIX protein;

[0051] Figure 16 This is the present invention. 68Diagram of the dissociation constant (Kd) of Ga-labeled WF2 probe and human CAIX protein;

[0052] Figure 17 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled DPI-4452 probe and human CAII protein;

[0053] Figure 18 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled WF1 probe and human CAII protein;

[0054] Figure 19 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled WF2 probe and human CAII protein;

[0055] Figure 20 This is the present invention. 68 Diagram showing the dissociation constant (Kd) of Ga-labeled DPI-4452 probe and mouse CAII protein;

[0056] Figure 21 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled WF1 probe and mouse CAII protein;

[0057] Figure 22 This is the present invention. 68 Diagram of the dissociation constant (Kd) of Ga-labeled WF2 probe and mouse CAII protein;

[0058] Figure 23 This is the present invention. 68 PET / CT images of Ga-labeled probes;

[0059] Figure 24 This is the present invention. 68 Figure showing the uptake of Ga-WF1 in major organs of OS-RC-2 CDX model mice;

[0060] Figure 25 This is the present invention. 68 Figure showing the uptake of Ga-WF2 in major organs of OS-RC-2 CDX model mice;

[0061] Figure 26 This is the present invention. 68 Ga-DPI-4452 uptake in major organs of OS-RC-2 CDX model mice;

[0062] Figure 27 This is the present invention. 68 Uptake maps of Ga-labeled probes in the stomach of OS-RC-2 CDX model mice were obtained using GraphPadPrism 9.5.1 software and independent samples.t The test involves comparing the two groups. P A value <0.05 is considered statistically significant. In the figure, ns represents no statistically significant difference. represent P <0.05, represent P <0.01, represent P <0.001, represent P <0.0001;

[0063] Figure 28 This is the present invention. 68 Uptake maps of Ga-labeled probes in the gut of OS-RC-2 CDX model mice were obtained using GraphPadPrism 9.5.1 software and independent samples. t The test involves comparing the two groups. P A value <0.05 is considered statistically significant. In the figure, ns represents no statistically significant difference. represent P <0.05, represent P <0.01, represent P <0.001;

[0064] Figure 29 This is the present invention. 68 Uptake maps of Ga-labeled probes in OS-RC-2 CDX model mouse tumors were obtained using GraphPadPrism 9.5.1 software and independent samples. t The test involves comparing the two groups. P A value <0.05 is considered statistically significant. In the figure, ns represents no statistically significant difference. represent P <0.05;

[0065] Figure 30 This is the present invention. 68 The tumor uptake inhibition analysis of Ga-labeled probes at 1 hour was performed using GraphPadPrism 9.5.1 software with independent samples. t The test involves comparing the two groups. P A value <0.05 is considered statistically significant. (See figure) represent P <0.001, represent P <0.0001;

[0066] Figure 31 This is the present invention. 177Chromatogram of Lu-WF1 radiochemical purity determination;

[0067] Figure 32 This is the present invention. 77 Chromatogram of Lu-WF2 radiochemical purity determination;

[0068] Figure 33 This is the present invention. 177 In vitro stability diagram of Lu-WF1 probe;

[0069] Figure 34 This is the present invention. 177 In vitro stability diagram of Lu-WF2 probe;

[0070] Figure 35 This is the present invention. 177 A study of Lu-WF1 and human CAIX protein;

[0071] Figure 36 This is the present invention. 177 A study of Lu-WF2 and human CAIX protein;

[0072] Figure 37 This is the present invention. 177 A study of Lu-DPI-4452 and human CAIX protein;

[0073] Figure 38 This is the present invention. 177 Dissociation constant (Kd) of Lu-labeled DPI-4452 probe and human CAII protein;

[0074] Figure 39 This is the present invention. 177 Dissociation constant (Kd) of Lu-labeled WF1 probe with human CAII protein;

[0075] Figure 40 This is the present invention. 177 Dissociation constant (Kd) of Lu-labeled WF2 probe and human CAII protein;

[0076] Figure 41 This is the present invention. 177 SPECT / CT images of Lu-labeled probes;

[0077] Figure 42 This is the present invention. 77 Image showing uptake of Lu-labeled probes at tumor sites in OS-RC-2 CDX model mice;

[0078] Figure 43 This is the present invention. 177 Area under the curve of Lu-labeled probes at tumor sites in OS-RC-2 CDX model mice. Detailed Implementation

[0079] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0080] Unless otherwise specified in the embodiments of the present invention, the techniques or conditions described in the literature in this field or the product instructions shall be followed; if the manufacturers of the reagents or instruments used are not specified, they are all conventional products that can be purchased through legitimate channels.

[0081] Example 1

[0082] The synthesis of a CAIX-targeting cyclic peptide WF1 (as shown in structure 2) includes the following steps:

[0083] Step 1: First, place Rink Amide MBHA Resin into a reaction tube and add the first amino acid. Then, add a solution containing Fmoc-Cys(Trt)-OH (1.17 g, 2 mmol, 2 eq) and DIPEA (523 μL, 3 mmol, 6 eq), and stir at room temperature for 1 hour. Then, filter off the solvent and remove the Fmoc protecting group using a 20% piperidine DMF solution (20 mL). Then, perform additional washing with DMF (wash 5 times).

[0084] Step 2: Introduce the second amino acid. Then, add Fmoc-Ser(Trt)-OH (1.15 g, 3 mmol, 3 eq), HOBt (405 mg, 3 mmol, 3 eq), and DIC (465 μL, 3 mmol, 3 eq) from 20 mL of DMF to the resin and stir at room temperature for 2 hours. Perform additional washing with DMF (wash 3 times), filter the solvent, and deprotect the Fmoc group with a 20% piperidine DMF solution (20 mL) for 30 minutes, followed by additional washing with DMF (wash 5 times).

[0085] Step 3: Repeat step 2, then sequentially couple the following amino acids: Fmoc-Trp(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Phe(3-NH-(4-sulfonylbutanylamino))-OH, Fmoc-Asp(OtBu)-OH, Fmoc-DPro-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-2-(piperazin-1-yl)acetic acid-OH. The procedure is the same: add 20 mL of Fmoc-amino acids (3 mmol × molecular weight, 3 eq), HOBt (405 mg, 3 mmol, 3 eq), and DIC (465 μL, 3 mmol, 3 eq) from DMF to the resin, and stir at room temperature for 2 hours. After filtering the solvent, the Fmoc group was deprotected in a 20 mL solution of 20% piperidine DMF, followed by additional washing with DMF (wash 5 times).

[0086] Step 4: Coupling DOTA using DMF, HATU, and DIPEA. Add DOTA (1.15 g, 2 mmol, 2 eq), HATU (760 mg, 2 mmol, 2 eq), and DIPEA (697 μL, 4 mmol, 4 eq) from 20 mL of DMF to the resin and stir at room temperature for 2 hours. Then perform additional washing with DMF (3 times), followed by 2 washes with DCM and 1 wash with MeOH, repeating once.

[0087] Step 5: The resin peptide was dried under high vacuum, 20 mL of solution E (activator) was added, and the reaction was carried out at room temperature for 2 hours. Then 200 mL of diethyl ether was added, and a large amount of solid precipitated out. The solid was centrifuged and dried to obtain crude product, which was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain high-purity peptide. After lyophilization, a white lyophilized powder P241719-Peptide (Yield: 24.9%) was obtained.

[0088] Step 6: P241719-Peptide (100 mg / 0.05 mmol / 1 eq) was dissolved in 120 mL of 0.05 M NH4HCO3 / ACN (1:1). Dibromo-p-xylene (18.5 mg 0.07 mmol 1.4 eq) was added under nitrogen protection. The reaction was carried out at room temperature for 2 h. LC-MS was used to monitor the completion of the reaction. After reverse-phase purification, the CAIX-targeting cyclic peptide WF1 (Yield: 10.3%) was obtained. LC-MS determined its molecular weight to be 2106.37, and HPLC determined its purity to be 98.81%. The HPLC chromatogram for WF1 purity determination is shown below. Figure 1 The LC-MS spectrum of WF1 is as follows: Figure 2 .

[0089] Example 2

[0090] Synthesis of a CAIX-targeting cyclic peptide WF2 (as shown in structure 3):

[0091] In this embodiment, the synthesis steps one through five of the CAIX-targeting cyclic peptide WF2 are the same as those of WF1. The difference lies in the sixth step, where the cyclization linker for WF2 differs from that of WF1. The sixth step is as follows: P241719-Peptide (100 mg / 0.05 mmol / 1 eq) was dissolved in 120 mL of 0.05 M NH4HCO3 / ACN (1:1). Under nitrogen protection, dibromo-o-xylene (18.5 mg / 0.07 mmol / 1.4 eq) was added, and the reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until completion. After reverse-phase purification, WF2 (Yield: 8%) was obtained. LC-MS determined its molecular weight to be 2106.37, and HPLC determined its purity to be 96.78%. The HPLC chromatogram for WF1 purity detection is shown below. Figure 3 The LC-MS spectrum of WF1 is as follows: Figure 4 .

[0092] Comparative Example 1

[0093] Synthesis of a CAIX-targeting cyclic peptide DPI-4452:

[0094] The synthesis steps one through five of the CAIX-targeting peptide DPI-4452 in this comparative example are the same as those in WF1, except for the sixth step. The sixth step is as follows: P241719-Peptide (100 mg 0.05 mmol 1 eq) was dissolved in 120 mL of 0.05 M NH4HCO3 / ACN (1:1). Under nitrogen protection, dibromo-m-xylene Compd 1 (15.8 mg 0.06 mmol 1.2 eq) was added. The reaction was carried out at room temperature for 2 h. The reaction was monitored by LC-MS until completion. After purification by reverse-phase chromatography, DPI-4452 (Yield: 11.5%) was obtained. The structural formula of DPI-4452 is as follows:

[0095]

[0096] 1.1 Affinity test of CAIX-targeting cyclic peptides with human CAII protein

[0097] CAII protein is a widely expressed carbonic anhydrase isoenzyme in the human body, highly expressed in various tissues and cells such as gastric parietal cells, lungs, kidneys, and erythrocytes. The affinity of CAIX-targeting cyclic peptides for CAII protein suggests that non-specific uptake may occur in the human body. Surface plasmon resonance (SPR) technology was used to detect the binding affinity of three CAIX-targeting cyclic peptides (WF1, WF2, and DPI-4452) to carbonic anhydrase II (CAII) to assess the non-specific binding of CAIX-targeting cyclic peptides in vivo.

[0098] Dissolve the CAII protein in PBS containing trehalose, and dissolve the cyclic peptides targeting CAIX (DOTA-DPI-4452, WF1, and WF2) in DMSO. Activate the CM5 sensor chip for 420 seconds at a flow rate of 10 μL / min using freshly prepared activation buffer. Dilute the CAII protein to 20 μg / mL using fixation buffer, and then inject it into the sample channel Fc2 at a flow rate of 10 μL / min to achieve a protein fixation level of 12600 RU. No ligand fixation is required for the reference channel Fc1. Inactivate the chip by rinsing it with blocking buffer at a flow rate of 10 μL / min for 420 seconds.

[0099] A multi-cycle method was used to detect the analyte. First, the CAIX-targeting cyclic peptide was diluted to different concentration gradients using analyte buffer. Then, the CAIX-targeting cyclic peptide was injected into channels Fc1 and Fc2 at a flow rate of 20 μL / min for 100 seconds of binding, followed by 180 seconds of dissociation. Eight cycles of analysis were performed sequentially, with the CAIX-targeting cyclic peptide concentration increasing from lowest to highest. After each cycle, the chip was regenerated. The binding and dissociation processes were recorded by real-time monitoring of the resonance unit (RU). The kinetic parameters were analyzed using BIAevaluation software, and the dissociation constant Kd was calculated.

[0100] The test results are as follows Figures 5 to 9 As shown, the results indicate that DPI-4452 binds to CAII in a concentration-dependent manner and exhibits significant binding saturation. The calculated dissociation constant Kd is 1.4 μM, indicating a micromolar level binding affinity between the two. WF2 shows some degree of concentration-dependent binding to CAII, but it is much lower than that of DPI-4452, with a calculated dissociation constant Kd of 2.7 μM. WF1, however, shows no binding to CAII protein, with a Kd > 1000 μM. These findings suggest that compounds WF1 and WF2 of this invention have lower binding rates to CAII protein compared to DPI-4452, resulting in lower non-specific uptake in the human body.

[0101] Application Example 1:

[0102] Targeting CAIX cyclic peptide probes 68 Ga nuclide labeling

[0103] Probe marking: Take gallium chloride [ 68 0.5 mL of Ga solution was placed in a 1.5 mL centrifuge tube, and a certain volume of WF1 or WF2 or DPI-4452 targeting CAIX cyclic peptide solution (1 mg / mL) was added. An appropriate amount of sodium acetate solution (pH=4.0) was added to adjust the pH of the system, and an appropriate amount of gentian acid solution (10 mg / mL) was added as a stabilizer. The mixture was mixed and placed in a dry heater. After heating at a fixed temperature for a certain period of time, the mixture was cooled.

[0104] Probe purification: Take a Sep-Pak LightC18 solid-phase extraction column, and activate it by sequentially drawing 1 mL of 50% ethanol-water (V / V) solution and rinsing with 2 mL of ultrapure water using a syringe. Then, use a syringe to extract the labeled sample and pass it through the column, allowing the sample to be adsorbed onto the column. Finally, rinse the column with 3 mL of ultrapure water to remove any residue. 68 Ga ions. The purified product was obtained by eluting the column with 0.5 mL of 50% ethanol-water (v / v) solution. 2-4 mL of physiological saline and 0.1-0.2 mL of gentian acid solution (10 mg / mL) were added to the eluent to dilute the solution to less than 10% (v / v) ethanol. Samples for animal testing should be filtered through a 0.22 μm microporous membrane.

[0105] 2.1 68 Ga-WF1 and 68 Quality control and in vitro stability of Ga-WF2 probe

[0106] The labeled probes were visually observed, their pH values ​​were measured using pH test paper, and the radiochemical purity of the product was determined using Radio-HPLC. The results showed that... 68 Ga-WF1 and 68 Ga-WF2 probes were all colorless, clear liquids with a pH between 3.0 and 3.5. After purification, HPLC analysis showed that the initial radiochemical purity of the probes was ≥95%. Figure 10 and Figure 11 As shown.

[0107] Take the mark 68 Ga-WF1 or 68500 μL of Ga-WF2 probe was added to either 500 μL of physiological saline or 500 μL of 5% HSA (human serum albumin) and incubated at 37 °C. Samples were taken at 0 h, 1 h, 2 h, 3 h, and 4 h, and the radiochemical purity of the probe was determined by Radio-HPLC to assess its in vitro stability. For samples incubated in 5% HSA, protein precipitation was required first. The specific procedure was as follows: 100 μL of sample was mixed with 100 μL of acetonitrile, centrifuged at 3000 r / min for 3 min, and the supernatant was aspirated with a syringe, filtered through a 0.22 μm filter membrane, and then injected for analysis.

[0108] Stability testing results showed that the initial radiochemical purity of the probe was >95%. With increasing in vitro co-incubation time, the decrease in radiochemical purity of the probe was not significant within 4 hours, indicating that the labeled probe exhibited excellent in vitro stability. Figure 12 and Figure 13 As shown.

[0109] 2.2 68 Protein binding affinity assay of Ga-labeled probes

[0110] Investigation 68 The binding affinity of Ga-labeled CAIX targeting probes to human CAIX, human CAII protein, and mouse CAII protein.

[0111] Each protein to be tested was diluted to 1 μg / mL with protein coating buffer (sodium carbonate-sodium bicarbonate buffer, pH≈9.5), and then added to a 96-well plate at a volume of 100 μL per well. The plate was sealed and incubated overnight at 4°C. The next day, the solution in the plate was discarded, and then 200 μL of 5% skim milk was added to each well after washing five times with PBST. The plate was sealed and incubated at 37°C for 2 h, washed five times with PBST, and dried before use. Each labeled CAIX targeting probe was diluted to different concentrations with PBS and added to a 96-well plate (n=5) coated with CAIX protein. After incubation at 37°C for 2 h, the plate was washed five times with PBST, dried, and cut open. The radioactivity of each well was measured using a gamma counter. The measured data were processed using GraphPadPrism software, and the Kd value was calculated through nonlinear fitting (One site -- Specific binding).

[0112] like Figures 14-16 As shown, the equilibrium dissociation constants of the three probes with human CAIX protein are all on the order of nM. 68 The Kd of Ga-DPI-4452 and CAIX protein is 6.82 nM; 68 Ga-WF1 and 68 Ga-WF2 has a higher affinity for CAIX.

[0113] like Figures 17-19 , 68 The Kd values ​​for Ga-DPI-4452 and human CAII protein are 430.0 nM, while 68 Ga-WF1 and 68 Ga-WF2 and human CAII protein do not have a significant affinity (Kd > 600 nM), therefore 68 Ga-WF1 and 68 Ga-WF2 has advantages in patient PET / CT imaging compared to... 68 Ga-DPI-4452 has lower uptake in non-target organs.

[0114] like Figures 20-22 , 68 The Kd of Ga-DPI-4452 and mouse CAII protein is 197.0 nM. 68 The Kd of Ga-WF1 with mouse CAII protein is 364.8 nM, and both probes exhibit micromolar affinity for mouse CAII protein. However, under the same experimental conditions, 68 Ga-WF2 has no significant affinity for mouse CAII protein. This demonstrates... 68 Ga-WF1 and 68 Ga-WF2 has a lower affinity for mouse CAII protein. The core structure and functional regions of mouse CAII protein and human CAII protein are almost identical, and their amino acid sequences are highly conserved with about 80% homology. However, there are still some substitutions of key amino acids. Therefore, the affinity of the above probe for mouse CAII is not completely consistent with its affinity for human CAII protein.

[0115] 2.3 68 Ga-WF1 and 68 PET / CT imaging with Ga-WF2 probe

[0116] OS-RC-2 human clear cell renal cell carcinoma model mice were induced to develop tumors with a volume of 200-400 mm. 3 Each animal was given a tail vein injection (100-200 μCi / animal). 68 Ga-WF1, 68 Ga-WF2 probe was administered to mice simultaneously. 68To compare differences in imaging effects, Ga-DPI-4452 probe injection was performed at different time points after injection (30, 60, 120, and 240 min) for 10 min and CT for 4 min. After the scans, VivoQuant software was used to iteratively reconstruct the scan data (OSEM 3D) and delineate the tumor, kidney, stomach, and muscle tissues as regions of interest (ROIs). The percentage of total radioactive dose per gram of tissue in each ROI (%ID / g) was calculated, and tumor uptake and metabolism in probe model mice were observed.

[0117] Blocking experiment: 0.5 h before the injection of the radioactive probe, OS-RC-2 model mice were injected with 100 μL of each CAIX-targeting cyclic peptide (the chemical amount is about 100 times that of the CAIX-targeting probe). Imaging was performed 1 h later to conduct the blocking experiment.

[0118] PET / CT imaging results are as follows Figure 23 As shown. Within the range of 0.5h to 4h, 68 Ga-WF1 and 68 The Ga-WF2 probe showed high uptake at tumor sites in mice. 68 The Ga-WF2 probe showed no significant radioactive uptake in the mouse stomach, while 68 Ga-WF1 and 68 Ga-DPI-4452 exhibits strong radioactive uptake in the stomach of mice because... 68 The Ga-WF2 probe has no affinity for mouse CAII protein, while 68 Ga-WF1 and 68 Ga-DPI-4452 exhibits sub-micromolar affinity for mouse CAII protein. Furthermore, the probe of this invention shows no significant affinity for human CAII protein, which greatly reduces non-target uptake of the probe in human gastrointestinal tract and other organs. One-hour imaging in the blocking experiment showed no significant uptake of the probe at the tumor site, demonstrating the specificity of the probe's uptake at the tumor site.

[0119] like Figure 24 , 68The uptake of Ga-WF1 in tumors increased progressively with time after injection, from (23.83±2.00)%ID / g at 30 minutes to a maximum of (29.84±2.53)%ID / g at 4 hours. Uptake in the stomach decreased progressively with time, from a maximum of (14.80±0.86)%ID / g at 30 minutes to (8.05±0.75)%ID / g at 4 hours, a decrease of approximately 45% compared to 30 minutes. Uptake in the intestines reached a maximum of (4.31±0.12)%ID / g at 1 hour and decreased to a minimum of (2.66±0.32)%ID / g at 4 hours. Uptake in the kidneys was highest at 1 hour (3.71±0.39)%ID / g), while uptake in muscle was low, below 1%ID / g at all four time points. The tumor-to-muscle ratio (T / M) of probe uptake was analyzed. 68 The T / M ratio of Ga-WF1 was highest at 1 h (67.22±10.67) and lowest at 4 h (44.33±7.99). The ratios at 0.5 h and 2 h were 49.99±9.20 and 53.39±5.64, respectively. The tumor-to-kidney ratio (T / K) of the probe uptake was analyzed. 68 The T / K of Ga-WF1 was highest at 4 h (15.09±2.02) and lowest at 1 h (6.87±0.81), with values ​​of 7.63±0.76 and 13.55±1.31 at 0.5 h and 2 h, respectively. Analysis of tumor uptake values ​​in normal and blocked imaging 1 h after probe injection showed a significant difference between the blocked and unblocked tumor uptake, with a difference of 24.63±0.99%ID / g vs. 2.72±0.16%ID / g. P <0.0001), the above results indicate that the probe has very strong specific uptake at the tumor site.

[0120] like Figure 25 , 68 The uptake of Ga-WF2 in tumors showed a slow decreasing trend with increasing time after injection, decreasing from (34.34±1.74)%ID / g at 30 minutes to (29.77±1.56)%ID / g at 4 hours. Uptake in the stomach was low, <1.5%ID / g in the first 2 hours, slightly increasing to (2.12±0.14)%ID / g at 4 hours. Uptake in the intestines reached its maximum at 1 hour (2.33±0.16)%ID / g. Uptake in the kidneys did not change significantly with time, ranging from (2.77±0.11)%ID / g to (3.28±0.62)%ID / g. Uptake in muscle was low, approximately 0.6%ID / g from 0.5 to 2 hours, increasing slightly to approximately 1%ID / g at 4 hours. The tumor-to-muscle ratio (T / M) of probe uptake was analyzed. 68The T / M ratio of Ga-WF2 was highest at 1 h (59.44±3.03) and lowest at 4 h (32.08±7.69), with values ​​of 56.61±2.76 and 51.86±8.83 at 0.5 h and 2 h, respectively. The tumor-to-kidney ratio (T / K) of the probe uptake was analyzed. 68 The T / K ratio of Ga-WF2 was highest at 0.5 h, at 12.52 ± 1.03. The T / K ratios at other time points did not decrease significantly compared to 0 h and remained >10. Analysis of tumor uptake values ​​in normal and blocked imaging 1 h after probe injection showed a significant difference in tumor uptake between blocked and unblocked images. 68 Ga-WF2 is 34.74±2.50 %ID / g vs 2.23±0.29%ID / g ( P <0.001), the above results indicate that 68 Ga-WF2 probes exhibit very strong specific uptake at tumor sites.

[0121] like Figure 26 , 68 The uptake of Ga-DPI-4452 in tumors increased continuously with time after injection, from (32.13±3.21)%ID / g at 30 minutes to a maximum of (40.03±2.53)%ID / g at 4 hours; the uptake in the stomach decreased continuously with time, from a maximum of (12.57±0.85)%ID / g at 30 minutes to (6.33±1.29)%ID / g at 4 hours; and the uptake behavior in the intestines... Similar to that in the stomach, the uptake decreased from a maximum of (4.66±0.22)%ID / g at 30 min to (2.46±0.22)%ID / g at 4 h. The uptake in the kidneys did not change significantly over time, ranging from (2.83±0.23)%ID / g to (3.64±0.67)%ID / g. The uptake in muscle was lower, approximately 0.5%ID / g from 0.5 h to 2 h, and increased slightly at 4 h, but remained below 1%ID / g.

[0122] like Figures 27-29 , 68 Ga-WF2 was the least absorbed in the stomach and intestines of OS-RC-2 CDX model mice, due to its lowest affinity for mouse CAII protein. Meanwhile... 68 Ga-WF2 compared to 68 Ga-DPI-4452 showed no significant difference in uptake in tumors. 68 Although Ga-WF1 has slightly lower uptake in tumors, it can still reach up to 20% ID / g. It also has a low affinity for human CAII protein and low non-target uptake in the human body.

[0123] like Figure 30The tumor uptake values ​​of the probe in normal imaging and blocked imaging 1 hour after injection were analyzed. A significant difference in tumor uptake was observed between the blocked and unblocked images, indicating... 68 Ga-WF1 and 68 Ga-WF2 probes exhibit very strong specific uptake at tumor sites.

[0124] The above results indicate that 68 Ga-WF1 and 68 Ga-WF2 exhibits excellent tumor diagnostic performance, with high uptake in tumors and low uptake in normal organs. It is rapidly metabolized by the kidneys. The radioactive molecular probe targeting CAIX provided by this invention has extremely high specific uptake in tumors with high CAIX expression. Furthermore, it can achieve good imaging results within 0.5h to 4h after injection, without the need for a long waiting time. This will bring convenience to patients during examination, reduce the dosage, and decrease radiation exposure.

[0125] Application Example 2:

[0126] Targeting CAIX cyclic peptide probes 177 Lu nuclide labeling

[0127] Take lutetium chloride [ 177 Take an appropriate amount of Lu solution, dilute it to 0.5 mL with 0.05 mol / L hydrochloric acid, add a certain volume of WF1 or WF2 or DPI-4452 targeting CAIX cyclic peptide solution (1 mg / mL), add an appropriate amount of sodium acetate solution (1 mol / L) to adjust the pH of the system, add an appropriate amount of gentian acid solution (10 mg / mL) and vitamin C sodium solution (10 mg / mL) as stabilizers, mix well, place in a dry heater, heat at 100℃ for 10 min and then cool to obtain the product.

[0128] 3.1 177 Lu-WF1 and 177 Quality control and in vitro stability of Lu-WF2 probe

[0129] Take the mark 177 Lu-WF1 and 177 The Lu-WF2 probes were visually observed, their pH values ​​were measured using pH test paper, and their radiochemical purity was determined using Radio-HPLC. The results showed that both probes were colorless, clear liquids with a pH range of 4.5–5.5, and their initial radiochemical purity was ≥95%. 177 The typical Radio-HPLC chromatogram of Lu-WF1 shows a main peak retention time of approximately 12.5 min and a radiochemical purity of 96.86%. 177The typical Radio-HPLC chromatogram of Lu-WF2 shows a main peak retention time of approximately 12.6 min and a radiochemical purity of 98.03%. Figures 31-32 .

[0130] Pick 177 Lu-WF1 or 177 500 μL of Lu-WF2 probe was added to either 500 μL of physiological saline or 500 μL of 5% HSA (human serum albumin) and incubated at room temperature. Samples were taken at 0 h, 12 h, 24 h, 48 h, and 72 h, and the radiochemical purity of the probe was determined by Radio-HPLC to assess its in vitro stability. For samples incubated in 5% HSA, protein precipitation was required. The specific procedure was as follows: 100 μL of sample was mixed with 100 μL of acetonitrile, centrifuged at 3000 r / min for 3 min, and the supernatant was aspirated with a syringe, filtered through a 0.22 μm filter membrane, and then placed in a HPLC vial for injection.

[0131] 177 Lu-WF1 and 177 The Lu-WF2 probe exhibits excellent in vitro stability, with initial radiochemical purity >95% in physiological saline and 5% HSA. Within 72 hours, the radiochemical purity of the probe gradually decreases over time. At the end of the stability test period, 177 Lu-WF1 can still maintain a radiochemical purity greater than 92% in physiological saline and 5% HSA, such as Figures 33-34 .

[0132] 3.2 177 Protein binding affinity assay of Lu-labeled probe

[0133] Investigation 177 The binding affinity of Lu-labeled CAIX-targeting probes to human CAIX and CAII proteins. 177 Lu-labeled CAIX targeting probes were diluted with purified water to different concentrations and added to 96-well plates (n=4) coated with CAIX protein. After incubation at 37°C for 2 hours, the plates were washed four times with PBST, dried, and cut open. The radioactivity of each well was measured using a gamma counter. The measured data were processed using GraphPad Prism software, and the Kd value was calculated through nonlinear fitting (One site -- Specific binding).

[0134] like Figures 35-37 Both probes showed affinity for human CAIX protein in the nM range, among which... 177 Lu-WF2 has the highest affinity for human CAIX, with a Kd of 5.23 nM; 177Lu-WF1 has an affinity of 13.74 nM for human CAIX protein. 177 Lu-DPI-4452 has a similar affinity to human CAIX. This demonstrates that the probe of this invention has a high affinity for human CAIX protein.

[0135] like Figures 38-40 , 177 Lu-DPI-4452 has a micromolar affinity for human CAII protein, with a Kd value of 0.481 μM. No detectable levels were observed within the tested concentration range. 177 Lu-WF1 and 177 Lu-WF2 exhibits significant affinity, demonstrating 177 Lu-WF1 and 177 Lu-WF2 has no significant affinity for human CAII protein, while the two probes exhibit higher specificity in binding to CAIX target proteins.

[0136] 3.3 177 Lu-WF1 and 177 SPECT / CT imaging with Lu-WF2 probe

[0137] OS-RC-2 human clear cell renal cell carcinoma model mice were induced to develop tumors with a volume of 200-400 mm. 3 Each animal was given a tail vein injection of approximately 150 μCi. 177 Lu-WF1 probe, simultaneously administered to mice 177 To compare differences, Lu-DPI-4452 probe injections were performed at different time points after injection (4h, 1d, 2d, 4d, and 7d) followed by 20-minute SPECT static scans and 4-minute CT static scans. After the scans, VivoQuant software was used to iteratively reconstruct the scan data (OSEM 3D) and delineate the tumor tissue as the region of interest (ROI). The percentage of total radioactive dose per gram of tissue in the ROI (%ID / g) was calculated, and tumor uptake and metabolism in the probe model mice were observed.

[0138] Experimental results are as follows Figures 41-43 The arrow points to the tumor site; the probe... 177 Lu-WF1 exhibited extremely high tumor uptake and prolonged retention in tumors of the OS-RC-2 human renal clear cell carcinoma model in mice. At 4 hours, tumor uptake was 285.4 ± 0.4% ID / g. Tumor-specific uptake decreased continuously over 7 days post-injection, remaining at 64.3 ± 9.2% ID / g on day 7. Simultaneously, due to the rapid pharmacokinetics of this probe, no significant radioactive uptake was observed in major metabolic organs such as the liver and kidneys at any time point. The area under the curve (AUC) of the probe at the tumor site in tumor-bearing mice over 7 days was 23813% ID / g·h.177 The area under the curve (AUC) of the Lu-DPI-4452 probe at the tumor site in tumor-bearing mice within 7 days was 12179 %ID / g·h. 177 Lu-WF1 compared to 177 The area under the curve for Lu-DPI-4452 increased by approximately 100%, which will significantly improve its therapeutic effect on tumors. (Probe) 177 Lu-WF2 exhibited high tumor uptake and prolonged retention in the OS-RC-2 human renal clear cell carcinoma model mouse tumors. At 4 hours post-injection, tumor uptake was 197.7 ± 12.8% ID / g, and at 48 hours post-injection, it remained at 44.5 ± 3.7% ID / g. Furthermore, due to the rapid pharmacokinetics of this probe, no significant radioactive uptake was observed in major metabolic organs such as the liver and kidneys at any time point.

[0139] The above results indicate that 177 Lu-WF1 and 177 Lu-WF2 has significant potential applications in tumor treatment. It exhibits high uptake and long retention in tumors, while showing extremely low uptake in major organs such as the liver and kidneys. The radioactive molecular probe targeting CAIX provided in this invention shows promising therapeutic potential in tumors with high CAIX expression.

[0140] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A cyclic peptide targeting CAIX, characterized in that, Its structure is shown in Equation 1: Formula 1 Wherein, the Linker is a loop connector, and the loop connector is selected from one of the following structures: The chelator is a nuclide chelating group.

2. The CAIX-targeting cyclic peptide according to claim 1, characterized in that, The radionuclide chelating group is a bifunctional chelating agent, which is selected from one of DOTA, NOTA, NODA, NODAGA, DOTP, TETA, ATSM, PTSM, EDTA, EC, HBEDCC, DTPA, SBAD, BAPEN, Df, DFO, TACN, NO2A / NOTAM, CB-DO2A, Cyclen, NOOTA-AA, DO3A, DO3AP, HYNIC, MAS3, MAG3, AAZTA, DOTAGA, NODA-MPAA, HBED, THP, and Macropa.

3. The CAIX-targeting cyclic peptide according to claim 1 or 2, characterized in that, The cyclic peptide targeting CAIX has the structure shown in Formula 2: Equation 2.

4. The CAIX-targeting cyclic peptide according to claim 1 or 2, characterized in that, The cyclic peptide targeting CAIX has the structure shown in Formula 3: Formula 3.

5. A method for preparing a CAIX-targeting cyclic peptide as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) The Fmoc solid-phase synthesis method was adopted, and the Rink Amide MBHA Resin resin was used as a carrier to sequentially couple amino acids to synthesize peptide chains. The synthesis sequence was: Cys → Ser → Trp → Thr → Leu → Phe (3-NH-(4-sulfonylbutanylamino)) → Asp → DPro → Glu → Cys → Gln → 2-(piperazin-1-yl)acetic acid; (2) After the peptide chain is synthesized, the radionuclide chelating group is attached to the N-terminus of the peptide chain, and the linear peptide is obtained by cleavage and purification; (3) The linear peptide was cyclized using a halogenated linker to form a cyclic peptide, which was then purified to obtain a cyclic peptide targeting CAIX.

6. A cyclic peptide radioactive probe targeting CAIX, characterized in that, Includes the CAIX-targeting cyclic peptide and the labeling radionuclide as described in any one of claims 1-4.

7. The CAIX-targeting cyclic peptide radioactive probe according to claim 6, characterized in that, The radionuclide used for labeling is a diagnostic radionuclide or a therapeutic radionuclide.

8. The CAIX-targeting cyclic peptide radioactive probe according to claim 7, characterized in that, The diagnostic radionuclide is selected from... 68 Ga、 64 Cu、 18 F, 86 Y、 89 Zr、 111 In、 99m Tc, 11 C 123 I, 125 I and 124 At least one of I; the therapeutic radionuclide is selected from... 177 Lu、 125 I, 131 I, 211 At、 90 Y、 153 Sm、 186 Re、 188 Re、 67 Cu、 212 Pb, 225 Ac、 213 Bi、 212 Bihe 212 At least one of Pb.

9. The use of the CAIX-targeting cyclic peptide as described in any one of claims 1 to 4 or the CAIX-targeting cyclic peptide radioactive probe as described in any one of claims 7 to 8 in the preparation of diagnostic and therapeutic tumor probes.

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