A radioactive molecular probe for targeting CSPG4 / NG2 and preparation method and application thereof

Abstract

This invention discloses a radioactive molecular probe for targeting CSPG4 / NG2, its preparation method, and its application. The radioactive molecular probe comprises the structure shown in Formula I or Formula II. The technical solution of this invention uses a radioactive molecular probe to specifically recognize CSPG4 / NG2 in vivo through the target polypeptide portion, thereby achieving specific nuclear medicine positron emission tomography (PET) or single-photon emission computed tomography (SPECT) imaging diagnosis of CSPG4 / NG2 positive tumors or CSPG4 / NG2 positive lesions, such as autoimmune diseases and cardiovascular and cerebrovascular diseases, thereby realizing early diagnosis and efficacy evaluation of malignant tumors.

Description

Technical Field

[0001] This invention belongs to the field of radiochemistry technology, and particularly relates to a radioactive molecular probe for targeting CSPG4 / NG2, its preparation method and application. Background Technology

[0002] Chondroitin sulfate proteoglycan 4 (CSPG4) is a single-pass type I transmembrane protein encoded by the CSPG4 gene on chromosome 15. It consists of a core protein and chondroitin sulfate side chains, and its murine homolog is Nerve / glial antigen 2 (NG2). CSPG4 / NG2 is not expressed or is expressed at low levels in most normal tissues, but is highly expressed in various human malignant tumor cells, the extracellular matrix of the tumor microenvironment, and tumor progenitor cells, such as malignant melanoma, glioblastoma, triple-negative breast cancer, pancreatic tumors, undifferentiated thyroid cancer, and head and neck tumors. Overexpression of CSPG4 / NG2 in tumor tissues not only promotes tumor cell proliferation but also enhances the adhesion and invasiveness of cancer cells, promotes tumor angiogenesis, increases drug resistance, promotes immune escape, and enhances resistance to radiotherapy. A growing body of research confirms that CSPG4 / NG2 has the potential to serve as a novel molecular marker for early tumor diagnosis, prognosis prediction, and immunotherapy, and is expected to become a new target for broad-spectrum nuclear medicine molecular diagnosis and treatment of tumors.

[0003] CSPG4 / NG2 plays a crucial biological role in early tumor development, metastasis, targeted therapy, and prognostic assessment, making it a highly promising diagnostic and therapeutic target. However, CSPG4 expression exhibits significant heterogeneity, varying considerably across different tumor types and subtypes, and even within the same tumor at different sites. Furthermore, CSPG4 expression is dynamic throughout tumor progression and treatment. Currently, common methods for detecting CSPG4 / NG2 expression include flow cytometry, immunohistochemistry, immunofluorescence, Western blotting, and quantitative PCR. These methods are invasive, ex vivo, and localized, and cannot provide real-time, dynamic, and comprehensive visualization at the in vivo level. Therefore, rapid, real-time, dynamic, and comprehensive visualization of CSPG4 / NG2 expression during tumorigenesis is crucial for early tumor diagnosis, molecular subtyping, determining the timing of malignant transformation, timely adjustment of treatment plans, and accurate prognostic assessment. This aligns with the requirements of the national "Major Research Plan for Molecular Functional Visualization of Tumor Evolution and Diagnosis."

[0004] Nuclear medicine molecular imaging studies the function and metabolism of drugs in vivo at the molecular level, enabling rapid, non-invasive, and real-time imaging of physiological and pathological processes. This provides new methods and tools for truly early diagnosis and timely treatment, and offers possible pathways to preventive medicine, translational medicine, and personalized medicine. However, China lacks innovative radiopharmaceuticals with independent intellectual property rights, and research and development is slow. Therefore, exploring new biomarkers as new targets for nuclear medicine imaging and developing targeted radiotherapeutic drugs with independent intellectual property rights is an important goal of the "Medium- and Long-Term Development Plan for Medical Isotopes (2021-2035)".

[0005] Based on this, it is hoped that by targeting CSPG4 / NG2, a key molecule in tumor progression, and using reported CAQK-targeting peptides as lead compounds, a radioactive molecular probe targeting CSPG4 / NG2 will be developed. Nuclear medicine molecular imaging techniques will be used to achieve real-time, dynamic visualization of CSPG4 / NG2 signals, and ultimately, real-time dynamic evaluation of CSPG4 / NG2 expression will be achieved to demonstrate the scientific value, social benefits, and economic benefits of this patent. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, this invention provides a radioactive molecular probe for targeting CSPG4 / NG2, its preparation method, and its applications. The technical solution described in this invention has advantages such as simple preparation process, good in vitro and in vivo stability, rapid clearance from normal organs, and a high target / non-target ratio between tumors and other normal organs or tissues, and also exhibits excellent tumor imaging effects.

[0007] To achieve the above objectives, the present invention proposes the following technical solution:

[0008] In a first aspect, the present invention proposes a radioactive molecular probe for targeting CSPG4 / NG2, the radioactive molecular probe comprising the structure shown in Formula I or Formula II:

[0009]

[0010] Wherein, R1 is the structure shown in Formula III; R2 and R4 are radiolabeled nuclides or radiolabeled nuclides including bifunctional chelating groups; R3 is the structure shown in Formula IV or Formula V;

[0011] Equation III is shown below:

[0012]

[0013] Equation IV or Equation V is shown below:

[0014]

[0015] In equation IV or equation V, n is an integer between 0 and 10.

[0016] Preferably, the bifunctional chelating group is one of the structures shown in Formulas VI to XIII:

[0017]

[0018]

[0019]

[0020]

[0021]

[0022] Preferably, the radiolabeled nuclide is selected from one of the following:

[0023] 18 F-Al, 67 Ga、 68 Ga、 47 Sc、 64 Cu、 67 Cu、 89 Zr、 86 Y、 89 Sr、 90 Y、 99 mTc, 105 Rh、 109 Pd, 111 In、 119 Sb、 149 Tb, 153 Sm、 157 Gd, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 Any one or more of Ac;

[0024] Furthermore, the radionuclide is 18 F-Al, 68 Ga、 64 Cu、 177 Any one or more of Lu.

[0025] Secondly, the present invention provides a method for preparing the above-mentioned radioactive molecular probe, characterized in that the preparation method includes the following steps:

[0026] Step S1: Mix the polypeptide molecules with the bifunctional chelating agent to obtain the bifunctional chelating agent-polypeptide molecule;

[0027] Step S2: Mix the bifunctional chelating agent-peptide molecule with the radiolabeled nuclide to obtain the radionuclide-bifunctional chelating agent-peptide molecule.

[0028] Preferably, the polypeptide molecule is CSPG4 / NG2.

[0029] Preferably, the specific operation of step S1 is as follows:

[0030] The polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 7.5-9.5, and a bifunctional chelating agent with a molecular weight of 1.5-20 times the polypeptide is added. After mixing, the mixture is reacted at room temperature for 0.5-24 hours. The reaction mixture is separated and purified by HPLC, the product peak is collected, and the collected product peak liquid is lyophilized to obtain a white powder, which is the bifunctional chelating agent-polypeptide molecule.

[0031] The suitable solvent is one or more of water for injection, ethanol, DMSO, and DMF.

[0032] Preferably, according to the preparation method of claim 6, the HPLC is a semi-preparative HPLC method, and the chromatographic conditions include: the mobile phase is: phase A (i.e., organic phase) is acetonitrile containing 0.1 v / v% trifluoroacetic acid; phase B (i.e., aqueous phase) is 0.1 v / v% trifluoroacetic acid; the elution conditions are: 0-3 minutes: phase A 5 v / v%, phase B 95 v / v%; 3-25 minutes: phase A 5%-95 v / v%, phase B 95-5 v / v%; 25.5 minutes: phase A 5 v / v%, phase B 95 v / v%; elution is stopped at 30 minutes, and the flow rate of the mobile phase is 3 mL / min.

[0033] Preferably, the specific operation of step S2 is as follows:

[0034] The bifunctional chelating agent-peptide molecule is dissolved in a suitable solvent, the pH is adjusted to 4.0-4.5, and then 5 MCI-2 Ci of radionuclide is added. The mixture is heated in a water bath at 60-120°C for 10-30 min to prepare the radionuclide-bifunctional chelating agent-peptide molecule.

[0035] The suitable solvent is one or more of water for injection, ethanol, DMSO, and DMF.

[0036] Preferably, the alkaline reagent used to adjust the pH is N,N'-diisopropylethylamine (DIEA) or triethanolamine (TEA).

[0037] Preferably, the acidic reagent used to adjust the pH is a NaAc solution or a NaAc / HAc buffer solution with a concentration of 0.1–5 mmol / L.

[0038] Preferably, in step S1, the retention time of the product peak is in the range of 8 to 12 minutes.

[0039] Preferably, when the radionuclide is 68 Ga、 64 Cu or 177 Lu, step S2 includes the following steps: dissolving the bifunctional chelating agent-peptide molecule obtained in step S1 in any one or more of water for injection, DMSO, and ethanol, adding a NaAc solution or NaAc / HAc buffer solution with a concentration of 0.1-0.5 mmol to adjust the pH to 3.5-6.5, then adding 5 mCi-2 Ci of radionuclide ions, heating in a water bath at 60-120°C for 5-30 min, and cooling to room temperature to obtain the radionuclide-bifunctional chelating agent-peptide molecule;

[0040] Or when the radionuclide is 18 F, step S2 includes the following steps: mixing the bifunctional chelating agent-CSPG4 / NG2 obtained in S1 with AlCl3 solution and 5mCi to 2Ci radionuclide ions, adjusting the pH to 3.5 to 6.5, reacting at 60 to 120°C for 5 to 30 minutes, and cooling to room temperature to prepare radionuclide-18F-bifunctional chelating agent-peptide molecules, wherein the bifunctional chelating agent-peptide molecules and AlCl3 solution are mixed in a ratio of 20 to 300 μg of bifunctional chelating agent-peptide molecules to 0.004 to 0.04 mmol of AlCl3 to obtain radionuclide-bifunctional chelating agent-peptide molecules.

[0041] Thirdly, the present invention provides the application of the above-mentioned polypeptide molecular probe in the preparation of PET / CT tumor imaging diagnostic reagents.

[0042] Fourthly, this invention proposes the application of the above-mentioned polypeptide molecular probe in the preparation of diagnostic reagents targeting CSPG4 / NG2.

[0043] Preferably, the diagnostic reagents targeting CSPG4 / NG2 are indicated for autoimmune diseases or cardiovascular diseases.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] This invention innovatively constructs a radioactive molecular probe targeting CSPG4 / NG2. The radionuclide and the CSPG4 / NG2 targeting peptide are coupled through a linker and a bifunctional chelating agent. This series of probes has advantages such as simple preparation and good stability. Specific nuclear medicine imaging of CSPG4 / NG2 positive tumors or CSPG4 / NG2 positive lesions can be achieved using positron emission tomography (PET) or single-photon emission computed tomography (SPECT) techniques.

[0046] This invention has promising applications not only in PET / SPECT imaging agents for diseases with high CSPG4 / NG2 expression, but also in radiotherapy for tumors with high CSPG4 / NG2 expression. Specifically, the diagnostic probe serves as a PET or SPECT imaging agent for specific identification of CSPG4 / NG2-positive tumors or CSPG4 / NG2-positive lesions; the therapeutic effect is radiotherapy for CSPG4 / NG2-positive tumors or CSPG4 / NG2-positive lesions. Attached Figure Description

[0047] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments will be briefly described below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0048] Figure 1 The [Prepared in Example 2] 68 Radiochemical purity of Ga]Ga-NOTA-C2-Mal-CK4 (Radio-HPLC chromatogram);

[0049] Figure 2 The [prepared in Example 4] 68 Ga]Ga-DOTA-cycle CA6 radiochemical purity Radio-HPLC chromatogram;

[0050] Figure 3 The [prepared for Example 2] 68 Stability of Ga]Ga-NOTA-C2-Mal-CK4 in physiological saline, mouse plasma, and mouse urine;

[0051] Figure 4 The [prepared for Example 4] 68Stability of Ga]Ga-DOTA-cycle CA6 in physiological saline, mouse plasma, and mouse urine;

[0052] Figure 5 The [prepared for Example 2] 68 MicroPET / CT image of Ga]Ga-NOTA-C2-Mal-CK4 in a B16F10 tumor-bearing model of C57BL / 6 black mice;

[0053] Figure 6 The [prepared in Example 4] 68 microPET / CT image of Ga]Ga-DOTA-cycle CA6 in a C57BL / 6 mouse MC38 colorectal cancer peritoneal metastasis model. Detailed Implementation

[0054] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0055] The following specific examples illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of them. This application can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of this application. It should be noted that, in the absence of conflict, the following embodiments and features in the embodiments can be combined with each other. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0056] It should be noted that various aspects of embodiments within the scope of the appended claims are described below. It will be apparent that the aspects described herein can be embodied in a wide variety of forms, and any particular structure and / or function described herein is merely illustrative. Based on this application, those skilled in the art will understand that one aspect described herein can be implemented independently of any other aspect, and two or more of these aspects can be combined in various ways. For example, any number and aspects set forth herein can be used to implement the device and / or practice the method. Additionally, this device and / or method can be implemented using structures and / or functionalities other than one or more of the aspects set forth herein.

[0057] Example 1: Synthesis of Nota-C2-Mal-CK4

[0058] Weigh 2.0 mg (4.5 μmol) of CK4 peptide and 5.2 mg (9.0 μmol) of NOTA-C2-Mal and dissolve them in 0.1 mL of DMSO. Add 10 μL of triethylamine (TEA) and react overnight at room temperature. Perform semi-preparative HPLC purification. Chromatographic conditions included: mobile phase: Phase A (organic phase) was acetonitrile (v / v) containing 0.1% trifluoroacetic acid; Phase B (aqueous phase) was 0.1% trifluoroacetic acid (v / v); elution conditions: 0–3 minutes: Phase A 5%, Phase B 95%; 3–25 minutes: Phase A 5%–95%, Phase B 95%–5%; 25.5 minutes: Phase A 5%, Phase B 95%; elution stopped at 30.0 minutes, with a mobile phase flow rate of 3 mL / min. Then perform mass spectrometry analysis [M+H]. + :874.2256, molecular formula C 36 H 60 N 10 O 13 S, with a theoretically calculated value of 872.9930, yielded the compound NOA-C2-Mal-CK4.

[0059] The structural formula of the NOTA-C2-Mal-CK4 compound is shown below:

[0060]

[0061] Example 2: [ 68 The mark Ga]Ga-NOTA-C2-Mal-CK4

[0062] Dissolve NOTA-C2-Mal-CK4 in sterile water for injection (1 mg / mL), take 50 μL of the aqueous solution and add it to NaAc (1 mol / L) buffer, adjust the pH to about 4.0, and then add 1-30 mCi of radionuclide. 68Ga eluent (0.1M HCl) was heated at 100°C for 15 minutes to prepare a radiopharmaceutical. The labeling rate was determined using radio-HPLC: Phase A (organic phase) consisted of acetonitrile (v / v) containing 0.1% trifluoroacetic acid; Phase B (aqueous phase) consisted of 0.1% trifluoroacetic acid (v / v); elution conditions: 0–3 minutes: Phase A 5% v / v, Phase B 95% v / v; 3–10 minutes: Phase A 5%–95% (v / v), Phase B 95%–5% (v / v); 10–13 minutes: Phase A 95% (v / v), Phase B 5% (v / v); 13–14 minutes: Phase A 95%–5% (v / v), Phase B 5%–95% (v / v); 14–15 minutes: Phase A 5% (v / v), Phase B 95% (v / v); elution was stopped, and the flow rate of the mobile phase was 1 mL / min. Gradient HPLC was used with a Kinetex 5μm EVO C18 column (250.0mm × 4.6mm); UV detection wavelength was 214nm; flow rate was 1.0mL / min; and injection volume was 20μL. Radio-HPLC results are shown (see attached results). Figure 1 ), [ 68 The radiochemical purity of Ga]Ga-NOTA-C2-Mal-CK4 is greater than 98%, and it can be used directly in subsequent experiments.

[0063] [ 68 The structural formula of Ga]Ga-NOTA-C2-Mal-CK4 is as follows:

[0064]

[0065] Example 3: Synthesis of DOTA-cycle CA6

[0066] Weigh 2.0 mg (4.5 μmol) of CK4 peptide and 5.2 mg (9.0 μmol) of DOTA-NHS and dissolve them in 0.1 mL of DMSO. Add 10 μL of triethylamine (TEA) and react overnight at room temperature. Perform semi-preparative HPLC purification. Chromatographic conditions included: mobile phase: Phase A (organic phase) was acetonitrile (v / v) containing 0.1% trifluoroacetic acid; Phase B (aqueous phase) was 0.1% trifluoroacetic acid (v / v); elution conditions: 0–3 minutes: Phase A 5%, Phase B 95%; 3–25 minutes: Phase A 5%–95%, Phase B 95%–5%; 25.5 minutes: Phase A 5%, Phase B 95%; stop elution at 30.0 minutes, with a mobile phase flow rate of 3 mL / min. Then perform mass spectrometry analysis for identification. Mass spectrometry analysis [M+H] + :1177.50, molecular formula C 55 H 76 N 12 O15 S, with a theoretically calculated value of 1176.5274, yields the compound DOTA-cycleCA6, whose structural formula is as follows:

[0067]

[0068] Furthermore, the structural formula of DOTA-NHS is shown below:

[0069]

[0070] Example 4: [ 68 The tag Ga]Ga-DOTA-cycle CA6

[0071] DOTA-cycle CA6 was dissolved in sterile water for injection (1 mg / mL). 50 μL of this aqueous solution was added to a NaAc (1 mol / L) buffer solution, and the pH was adjusted to 4.0. Then, 1-30 mCi of radionuclide 68Ga (0.1 M HCl) was added, and the mixture was heated at 100 °C for 15 min to prepare the radiopharmaceutical. The labeling rate was determined using radio-iTLC (see [link to documentation]). Figure 3 a) The target labeled product was sampled using a capillary tube and spotted onto a plate using ammonium acetate (1.0 M) / methanol (V / V = 1 / 1) as the developing solvent. The detection equipment was a Mini-Scan radioactive TLC thin-layer scanner with a radiochemical purity of 99.01%. The labeling rate was determined using radio-HPLC: Phase A (organic phase) consisted of acetonitrile (v / v) containing 0.1% trifluoroacetic acid; Phase B (aqueous phase) consisted of 0.1% trifluoroacetic acid (v / v); elution conditions: 0–3 minutes: Phase A 5% v / v, Phase B 95% v / v; 3–10 minutes: Phase A 5%–95% (v / v), Phase B 95%–5% (v / v); 10–13 minutes: Phase A 95% (v / v), Phase B 5% (v / v); 13–14 minutes: Phase A 95%–5% (v / v), Phase B 5%–95% (v / v); 14–15 minutes: Phase A 5% (v / v), Phase B 95% (v / v); elution was stopped, and the flow rate of the mobile phase was 1 mL / min. Gradient HPLC was used with a Kinetex 5μm EVO C18 column (250.0mm × 4.6mm); UV detection wavelength was 214nm; flow rate was 1.0mL / min; injection volume was 20μL. Radio-HPLC results are shown (see...). Figure 2 The radiochemical purity of [68Ga]Ga-DOTA-cycle CA6 is greater than 98%, and it can be directly used for subsequent experiments.

[0072] [ 68The structural formula of Ga]Ga-DOTA-cycle CA6 is as follows:

[0073]

[0074] Example 5: [ 68 In vitro and in vivo stability of Ga]Ga-NOTA-C2-Mal-CK4

[0075] Prepared in Example 2 [68 The Ga-NOTA-C2-Mal-CK4 probe was added to 200 μL of physiological saline and approximately 200 μCi of mouse plasma solution, respectively. After incubation in a metal bath at 37°C for 2 h, the radiochemical purity was determined (Radio-HPLC). The results are shown below. Figure 3 a and b in the text Figure 3 show[ 68 Ga]Ga-NOTA-C2-Mal-CK4 maintained good stability after incubation in physiological saline and mouse plasma for 2 hours in vitro.

[0076] Will[ 68 Ga-NOTA-C2-Mal-CK4 (200 μCi) was injected into normal mice via the tail vein. Approximately 2 hours later, urine samples were collected and radiochemical purity was determined (Radio-HPLC). The Radio-HPLC results are as follows: Figure 3 As shown in Figure c, the probe maintains good stability in vivo.

[0077] Example 6: [ 68 In vitro and in vivo stability of Ga-DOTA-cycle CA6

[0078] Prepare the [in Example 4] 68 The Ga-DOTA-cycle CA6 probe was added to 200 μL of physiological saline and approximately 200 μCi of mouse plasma solution, respectively. After incubation in a metal bath at 37°C for 2 h, the radiochemical purity was measured (Radio-HPLC). The results are as follows: Figure 4 As shown in a and b, it displays [ 68 Ga]Ga-DOTA-cycle CA6 maintained good stability after incubation in physiological saline and mouse plasma for 2 hours in vitro.

[0079] Will[ 68 Ga-DOTA-cycle CA6 (approximately 200 μCi) was injected into normal mice via the tail vein. Approximately 2 hours later, urine samples were collected and radiochemical purity was determined (Radio-HPLC). The Radio-HPLC results are as follows: Figure 4As shown in c, the probe maintains good stability in vivo.

[0080] Example 7: [ 68 Biological evaluation of Ga]Ga-NOTA-C2-Mal-CK4

[0081] The following describes the CSPG4 / NG2-targeted probe prepared according to Example 2 of the present invention. 68 The PET / CT imaging performance of Ga-NOTA-C2-Mal-CK4 is described below:

[0082] (1) Preparation of the C57BL / 6 black mouse B16F10 model

[0083] A mouse tumor model was established using B16F10 cells as an example. Cells were digested with a digestive solution (2.5 g / L trypsin and 0.38 g / L EDTA-4Na), washed with sterile PBS, and then resuspended in sterile HBSS to prepare 5 × 10⁶ cells / day. 4 / μL of cell suspension. Six-week-old (1) C57BL / 6 black mice were subcutaneously inoculated with 5×10⁹ cells per inch of cell suspension in the right forelimb axilla. 6 100 μL of cells per animal were housed in an SPF-grade animal facility. After 2–3 weeks, when the average tumor diameter reached 0.8–1.0 cm, the tumors were used for experiments.

[0084] (2) 68 MicroPET / CT imaging of Ga-NOTA-C2-Mal-CK4 in a C57BL / 6 mouse B16F10 tumor-bearing model

[0085] (1) Three C57BL / 6 black mice and B16F10 tumor-bearing mice (n=3) were injected via tail vein with 0.2 mL of approximately 200 μCi of [ 68 The Ga-NOTA-C2-Mal-CK4 probe was administered. Approximately 20 minutes later, the patient was anesthetized with isoflurane (3%) gas and placed prone on the MicroPET / CT bed. Static PET and CT scans were performed for 10 minutes and 5 minutes respectively, 30 minutes after probe injection. [Discussion] 68 The Ga-NOTA-C2-Mal-CK4 probe was evaluated in tumor model image clarity, tumor uptake rate, tumor retention time, and uptake in normal tissues, particularly the heart, kidneys, liver, and muscle (see [link to relevant documentation]). Figure 5 The results show: [ 68 The Ga]Ga-NOTA-C2-Mal-CK4 probe can be taken up by the tumor after injection in the tumor model, and has a significant target / non-target ratio with the contralateral muscle tissue.

[0086] Example 8: [ 68Ga]Ga-DOTA-cycle CA6 biological evaluation

[0087] The following describes the CSPG4 / NG2-targeting probe prepared according to Example 4 of the present invention. 68 The PET / CT imaging performance of Ga]Ga-DOTA-cycleCA6 is described as follows:

[0088] (1) Preparation of C57BL / 6 mouse MC38 colorectal cancer peritoneal metastasis model

[0089] MC38 cells were digested with a digestion solution (2.5 g / L trypsin and 0.38 g / L EDTA-4Na), washed with sterile PBS, and then resuspended in sterile HBSS to prepare 5 × 10⁶ cells / day. 4 / μL of cell suspension. Six-week-old (1) C57BL / 6 black mice were injected intraperitoneally with 5×10⁹ / μL of cell suspension. 6 100 μL of cells per animal were housed in an SPF-grade animal facility. After 2–3 weeks, once the tumor metastasis model was successfully established, the cells were used for subsequent MicroPET / CT imaging experiments.

[0090] (2) 68 Ga-DOTA-cycle CA6 in a C57BL / 6 mouse B16F10 tumor-bearing model: MicroPET / CT imaging

[0091] (1) C57BL / 6 black mice with MC38 colorectal cancer peritoneal metastasis model were injected via the tail vein with 0.2 mL of approximately 200 μCi of [ 68 Ga]Ga-DOTA-cycle CA6 probe, approximately 20 minutes later, anesthesia with isoflurane (3%) gas was administered, and the patient was placed prone on the MicroPET / CT bed. Static PET and CT acquisitions were performed for 10 minutes and 5 minutes respectively, 30 and 60 minutes after probe injection. [Discussion] 68 The image clarity, tumor uptake rate, tumor retention time, and uptake in normal tissues, particularly the heart, kidneys, liver, and muscle, of the Ga-DOTA-cycle CA6 probe in a peritoneal metastatic tumor model (see Ga-DOTA-cycle CA6 probe image clarity, tumor uptake rate, tumor retention time, and uptake in normal tissues, particularly the heart, kidneys, liver, and muscle). Figure 6 The results show: [ 68 The Ga]Ga-DOTA-cycle CA6 probe can be taken up by the tumor after injection in the tumor model, and has a significant target / non-target ratio with the contralateral muscle tissue.

[0092] The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made based on the essence of the content of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A radioactive molecular probe for targeting CSPG4 / NG2, characterized in that, The radioactive molecular probe comprises the structure shown in Formula I or Formula II as follows: Wherein, R1 is the structure shown in Formula III; R2 and R4 are radiolabeled nuclides or radiolabeled nuclides including bifunctional chelating groups; R3 is the structure shown in Formula IV or Formula V; Equation III is shown below: Equation IV or Equation V is shown below: In equation IV or equation V, n is an integer between 0 and 10.

2. The radioactive molecular probe according to claim 1, characterized in that, The bifunctional chelating group is one of the structures shown in Formulas VI to XIII below:

3. The radioactive molecular probe according to claim 1, characterized in that, The radiolabeled nuclide is selected from one of the following: 18 F-Al, 67 Ga、 68 Ga、 47 Sc、 64 Cu、 67 Cu、 89 Zr、 86 Y、 89 Sr、 90 Y、 99 mTc, 105 Rh、 109 Pd, 111 In、 119 Sb, 149 Tb, 153 Sm、 157 Gd, 161 Tb, 166 Ho、 177 Lu、 186 Re、 188 Re、 201 Tl、 203 Pb, 212 Pb, 212 Bi、 213 Bi、 223 Ra、 227 Th and 225 Any one or more of Ac; Furthermore, the radioactive nuclide is 18 F-Al, 68 Ga、 64 Cu、 177 Any one or more of Lu.

4. A method for preparing the radioactive molecular probe according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: Step S1: Mix the polypeptide molecules with the bifunctional chelating agent to obtain the bifunctional chelating agent-polypeptide molecule; Step S2: Mix the bifunctional chelating agent-peptide molecule with the radiolabeled nuclide to obtain the radionuclide-bifunctional chelating agent-peptide molecule.

5. The preparation method according to claim 4, characterized in that, The polypeptide molecule is CSPG4 / NG2.

6. The preparation method according to claim 4 or 5, characterized in that, The specific operation of step S1 is as follows: The polypeptide molecule is dissolved in a suitable solvent, the pH is adjusted to 7.5-9.5, and a bifunctional chelating agent with a molecular weight of 1.5-20 times the polypeptide is added. After mixing, the mixture is reacted at room temperature for 0.5-24 hours. The reaction mixture is separated and purified by HPLC, the product peak is collected, and the collected product peak liquid is lyophilized to obtain a white powder, which is the bifunctional chelating agent-polypeptide molecule. The suitable solvent is one or more of water for injection, ethanol, DMSO, and DMF.

7. The preparation method according to claim 6, characterized in that, The HPLC method described is a semi-preparative HPLC method. The chromatographic conditions include: mobile phase A is acetonitrile containing 0.1 v / v% trifluoroacetic acid; phase B is 0.1 v / v% trifluoroacetic acid; elution conditions are: 0-3 minutes: phase A 5 v / v%, phase B 95 v / v%; 3-25 minutes: phase A 5%-95 v / v%, phase B 95-5 v / v%; 25.5 minutes: phase A 5 v / v%, phase B 95 v / v%; elution is stopped at 30 minutes, and the flow rate of the mobile phase is 3 mL / min.

8. The preparation method according to claim 4 or 5, characterized in that, The specific operation of step S2 is as follows: The bifunctional chelating agent-peptide molecule is dissolved in a suitable solvent, the pH is adjusted to 4.0-4.5, and then 5 MCI-2 Ci of radionuclide is added. The mixture is heated in a water bath at 60-120°C for 10-30 min to prepare the radionuclide-bifunctional chelating agent-peptide molecule. The suitable solvent is one or more of water for injection, ethanol, DMSO, and DMF.

9. The use of the radioactive molecular probe as described in any one of claims 1 to 3 in the preparation of PET / CT tumor imaging diagnostic reagents.

10. The use of the radioactive molecular probe as described in any one of claims 1 to 3 in the preparation of diagnostic reagents targeting CSPG4 / NG2.

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