A cyclic polypeptide targeting CD36 protein and its derived radioactive tracer

Abstract

The present invention relates to the field of biomedical technologies, and particularly to a cyclic polypeptide targeting CD36 protein and a derived radioactive tracer. In the present invention, the cyclic polypeptide targeting CD36 protein is condensed with diglycol as a linker, and then chelator NOTA is coupled to form a labeling precursor NOTA-ZL01 targeting CD36 protein. After the labeling precursor NOTA-ZL01 targeting CD36 protein is labeled with a radionuclide, a radioactive tracer [68Ga]Ga-ZL01 is formed. Through in vitro radiochemical property studies and in vivo stability studies, it is found that the radioactive tracer [68Ga]Ga-ZL01 has strong stability and is hydrophilic; through cell uptake studies and receptor binding force studies, it is found that the radioactive tracer [68Ga]Ga-ZL01 has a high affinity for the CD36 protein expressed by U87 cells; through small animal PET / CT to investigate the tumor imaging effect of the radioactive tracer [68Ga]Ga-ZL01, it is found that it can be used for in vivo imaging of CD36-positive glioblastoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a cyclic polypeptide targeting CD36 protein and a radioactive tracer derived therefrom. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the overall understanding of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Glioma is one of the most common brain tumors, accounting for 30% of brain tumors in the central nervous system. According to the grading criteria of the World Health Organization (WHO), gliomas are divided into grades I to IV. Among them, grade IV glioma with the highest malignancy is also called glioblastoma multiforme (GBM). Glioblastoma often occurs in the brain parenchyma and is diffusely distributed. It is the most common and invasive type of glioma in clinical practice. The median survival period of patients is only 12 - 18 months, and the 5-year survival rate is less than 5%. Therefore, early diagnosis and intervention are crucial for improving the prognosis of patients.

[0004] CD36 is a transmembrane glycoprotein that participates in various cellular functions, including lipid metabolism, inflammation, and the occurrence and development of tumors. In recent years, due to its key role in fatty acid uptake and its close association with tumor growth and metastasis, CD36 has received extensive attention as a therapeutic target. Preliminary studies have shown that elevated CD36 expression significantly promotes the metastasis of highly invasive tumors, such as melanoma and breast cancer. In addition, studies have also found that in various cancers including gastric cancer, oral cancer, and ovarian cancer, CD36-mediated lipid uptake significantly accelerates tumor progression and metastasis. In glioblastoma, CD36 plays a more subtle role in the occurrence and progression of tumors. Cancer stem cell populations gain a metabolic advantage by selectively utilizing CD36, enhancing tumor survival and energy metabolism. Therefore, CD36 can be used as a diagnostic target for glioblastoma.

[0005] PET imaging, with its high sensitivity and target specificity, can achieve non-invasive, rapid, and systemic visual assessment of lesions and has become an important tool in clinical diagnosis. Among them, polypeptide-based radiopharmaceuticals play an important role in PET imaging. With the characteristics of high affinity, low immunogenicity, and easy synthesis and modification, they exhibit excellent targeting performance. Therefore, developing polypeptide-based radiopharmaceuticals targeting CD36 protein and using PET imaging to evaluate the expression level of CD36 protein in vivo can provide guidance for the diagnosis and treatment of glioblastoma. Summary of the Invention

[0006] To overcome the above problems, the present invention provides a cyclic polypeptide targeting CD36 protein and a radio - tracer derived therefrom.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a cyclic polypeptide targeting CD36 protein is provided, and its structural formula is shown as the following formula (Ⅰ):

[0009]

[0010] Formula (Ⅰ).

[0011] In the second aspect of the present invention, a labeling precursor targeting CD36 protein is provided, which is composed of the following components: a cyclic polypeptide targeting CD36 protein, diethylene glycol (PEG2), and a chelating agent NOTA.

[0012] In the third aspect of the present invention, a preparation method of the labeling precursor targeting CD36 protein described in the second aspect is provided, including the following steps:

[0013] Using solid - phase synthesis method to synthesize the cyclic polypeptide targeting CD36 protein;

[0014] Condense the cyclic polypeptide targeting CD36 protein with diethylene glycol (PEG2), and then couple the chelating agent NOTA to obtain the labeling precursor targeting CD36 protein.

[0015] In the fourth aspect of the present invention, a radio - tracer is provided, which includes the labeling precursor targeting CD36 protein described in the second aspect or the labeling precursor targeting CD36 protein prepared by the preparation method described in the third aspect and a radionuclide.

[0016] In the fifth aspect of the present invention, a preparation method of the radio - tracer described in the fourth aspect is provided, including the following steps:

[0017] Mix and react the radionuclide with the labeling precursor targeting CD36 protein to obtain the radio - tracer.

[0018] In the sixth aspect of the present invention, an application of the cyclic polypeptide targeting CD36 protein described in the first aspect or the labeling precursor targeting CD36 protein described in the second aspect or the radio - tracer described in the fourth aspect in the preparation of a preparation for detecting the expression level of CD36 protein in tumors is provided.

[0019] In a seventh aspect of the present invention, there is provided an application of the cyclic polypeptide targeting CD36 protein described in the first aspect, or the labeling precursor targeting CD36 protein described in the second aspect, or the radioactive tracer described in the fourth aspect in the preparation of a preparation for diagnosing and / or treating tumors with high expression of CD36 protein.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention relates to the field of biomedical technologies, and specifically relates to a cyclic polypeptide targeting CD36 protein and a radioactive tracer derived therefrom. In the present invention, after condensing the cyclic polypeptide targeting CD36 protein with diethylene glycol (PEG2) as a linker, the chelating agent NOTA is then coupled to form a labeling precursor NOTA-ZL01 targeting CD36 protein. After the labeling precursor NOTA-ZL01 targeting CD36 protein is labeled with a radionuclide, a radioactive tracer 68 Ga]Ga-ZL01 is formed. Through in vitro radiochemical property research and in vivo stability research, it is found that the radioactive tracer 68 Ga]Ga-ZL01 has strong stability and is hydrophilic; through cell uptake research and receptor binding force research, it is found that the radioactive tracer 68 Ga]Ga-ZL01 has a high affinity for the CD36 protein expressed by U87 cells; through small animal PET / CT to investigate the tumor imaging effect of the radioactive tracer 68 Ga]Ga-ZL01, it is found that it can be used for in vivo imaging of CD36-positive glioblastoma. Description of the Drawings

[0022] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0023] Figure 1 It is the mass spectrum of the labeling precursor NOTA-ZL01 targeting CD36 protein;

[0024] Figure 2 It is for 68 the radiochemical purity detection result of the radioactive tracer

[0025] Figure 3 It is for 68 the cell uptake experiment result of the radioactive tracer

[0026] Figure 4 It is for 68 the saturation experiment curve of the radioactive tracer

[0027] Figure 5 is a radioactive tracer 68 Blood clearance curve of Ga]Ga-ZL01;

[0028] Figure 6 is a radioactive tracer 68 In vivo biodistribution map of Ga]Ga-ZL01 in U87MG tumor-bearing mice;

[0029] Figure 7 PET / CT imaging maps of U87MG tumor-bearing mice in the experimental group and the inhibition group in Example 8;

[0030] Figure 8 Immunohistochemical map of tumor CD36 expression in U87MG tumor-bearing mice in Example 9, where a is magnified 200 times; b is magnified 400 times;

[0031] Figure 9 Immunohistochemical results of tumor IgG in U87MG tumor-bearing mice in Example 9, where a is magnified 200 times; b is magnified 400 times. Detailed implementation manners

[0032] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary implementation manners of the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms are also intended to include the plural forms. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or their combinations.

[0034] The first typical implementation manner of the present invention provides a cyclic polypeptide targeting CD36 protein, and its structural formula is shown as the following formula (Ⅰ):

[0035]

[0036] Formula (Ⅰ).

[0037] The second typical implementation manner of the present invention provides a labeling precursor targeting CD36 protein, which is composed of the following components: a cyclic polypeptide targeting CD36 protein, diethylene glycol (PEG2), and a chelating agent NOTA.

[0038] In one or more embodiments, the structural formula of the labeling precursor targeting the CD36 protein is as shown in the following formula (II):

[0039]

[0040] Formula (II).

[0041] The third typical embodiment of the present invention provides a method for preparing the labeling precursor targeting the CD36 protein described in the second aspect, comprising the following steps:

[0042] Synthesize a cyclic polypeptide targeting the CD36 protein by solid-phase synthesis;

[0043] Condense the cyclic polypeptide targeting the CD36 protein with diethylene glycol (PEG2), and then couple the chelator NOTA to obtain the labeling precursor targeting the CD36 protein.

[0044] The fourth typical embodiment of the present invention provides a radioactive tracer, comprising the labeling precursor targeting the CD36 protein described in the second aspect or the labeling precursor targeting the CD36 protein prepared by the preparation method described in the third aspect and a radionuclide.

[0045] In one or more embodiments, the radionuclide includes 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd or 166 Dy; preferably 68 Ga.

[0046] Preferably, the structural formula of the radioactive tracer is as shown in formula (III),

[0047]

[0048] Formula (III).

[0049] The fifth typical embodiment of the present invention provides a preparation method of the radioactive tracer described in the fourth aspect, including the following steps:

[0050] After mixing and reacting the radioactive nuclide with the labeling precursor targeting the CD36 protein, the radioactive tracer is obtained.

[0051] The sixth typical embodiment of the present invention provides the application of the cyclic polypeptide targeting the CD36 protein described in the first aspect, or the labeling precursor targeting the CD36 protein described in the second aspect, or the radioactive tracer described in the fourth aspect in the preparation of a preparation for detecting the expression level of CD36 protein in tumors.

[0052] The seventh typical embodiment of the present invention provides the application of the cyclic polypeptide targeting the CD36 protein described in the first aspect, or the labeling precursor targeting the CD36 protein described in the second aspect, or the radioactive tracer described in the fourth aspect in the preparation of a preparation for diagnosing and / or treating tumors with high expression of CD36 protein.

[0053] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with specific examples.

[0054] Example 1

[0055] Prepare the labeling precursor NOTA-ZL01 targeting the CD36 protein:

[0056] (1) Using Rink amide-MBHA resin as the starting resin and Fmoc-amino acids as raw materials, a polypeptide shown in formula (IV) is synthesized by solid-phase method with a condensation system of N,N-diisopropylethylamine (DIPEA) / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT).

[0057]

[0058] Formula (IV).

[0059] Specifically: Remove the amino protecting group on the Rink amide-MBHA resin with a piperidine DMF solution, wash the resin, and block the unreacted sites with a dichloromethane blocking solution containing DIPEA and acetic anhydride; remove the solvent, wash the resin, and couple Lys with a protecting group, using the Lys bound to the resin as the starting point for synthesis; remove the Fmoc protecting group, and repeat the deprotection and condensation reactions in sequence according to the amino acid sequence from the carboxyl terminus to the amino terminus until all amino acids are coupled to the resin to obtain the polypeptide shown in formula (IV).

[0060] Among them, the concentration of the piperidine DMF solution is 20% (volume percentage);

[0061] In the blocking solution, the concentration of DIPEA in dichloromethane is 0.2 M, and the concentration of acetic anhydride in dichloromethane is 0.2 M.

[0062] (2) Remove the Fmoc protecting group of the last amino acid proline in the polypeptide sequence, condense the carboxyl group of lysine and the amino group of proline to form a peptide bond, complete cyclization, and form a cyclic polypeptide targeting the CD36 protein; the cyclization conditions are: at room temperature, mix the resin coupled with the polypeptide with dimethyl sulfoxide (DMSO), and maintain the reaction for 24 hours.

[0063] (3) Condense the cyclic polypeptide targeting the CD36 protein with diethylene glycol (PEG2), then couple the chelating agent NOTA, cleave the polypeptide from the resin, and further purify it by high performance liquid chromatography to obtain the labeling precursor NOTA-ZL01 targeting the CD36 protein.

[0064] Among them, the method for condensing the cyclic polypeptide targeting the CD36 protein with diethylene glycol (PEG2) is: solid-phase synthesis using an N,N-diisopropylethylamine (DIPEA) / benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) condensation system; during the condensation reaction, based on the molar ratio of the reactants, diethylene glycol:DIPEA:HOBT:HBTU:resin grafted with polypeptide = 3.5:7:4:4:1.

[0065] The method for coupling the chelating agent NOTA is as follows

[0066] Place the condensation product of the above resin grafted with polypeptide and diethylene glycol (PEG2) in dichloromethane (DCM), add trifluoroacetic acid (TFA), the concentration of TFA is 0.2 M, remove TFA and DCM after the reaction ends, add DMF, NOTA-NHS, and DIPEA, and the final product can be obtained after the reaction ends. When coupling NOTA, based on the molar ratio of the reactants, resin grafted with polypeptide:NOTA-NHS:DIPEA = 2:8:1;

[0067] When the polypeptide is cleaved, by mass ratio, the resin grafted with the polypeptide: the cleavage solution = 1:10; in the cleavage solution, by volume ratio, trifluoroacetic acid (TFA): triisopropylsilane (TIS): phenol (PhOH): methyl phenyl sulfide (Thioanisole): H2O = 30:2:2:1:1.

[0068] The mass spectrum of the labeled precursor NOTA-ZL01 targeting the CD36 protein obtained in this example is as Figure 1 shown. The experimental results show that the molecular weight of NOTA-ZL01 is consistent with the theoretical value, and its purity is higher than 98%.

[0069] Example 2

[0070] Preparation of the radiotracer 68 Ga]Ga-ZL01:

[0071] Dissolve NOTA-ZL01 in NaOAc buffer solution, and add 68 Ga]GaCl3 solution to the NOTA-ZL01 solution, and react at 95 °C for 15 minutes to obtain the radiotracer 68 Ga]Ga-ZL01.

[0072] Among them, the concentration of the NaOAc buffer solution is 0.1 M, and the pH is 4.0 - 4.6; after NOTA-ZL01 is dissolved in the NaOAc buffer solution, the concentration is 20 μM; 68 The 68 Ga]GaCl3 solution is the solution eluted with 0.01N HCl, and is 18 ± 1 mCi; 68 When

[0073] labeling with 68 Ga, the volume ratio of the NOTA-ZL01 solution and Figure 2 Ga]GaCl3 solution is 1:1.

[0074] The radiochemical purity of the radiotracer

[0075] Table 1 HPLC elution conditions

[0076]

[0077] As can be seen from Figure 2 it, the radiotracer68 The radiochemical purity of Ga]Ga-ZL01 is greater than 98%.

[0078] Example 3

[0079] Radioactive tracer 68 Study on the in vitro radiochemical properties of Ga]Ga-ZL01:

[0080] The radioactive tracer prepared in Example 2 68 Ga]Ga-ZL01 was mixed with PBS buffer at pH = 7.4 and human serum (volume ratio 1:2) and incubated at 37 °C for 30 minutes and 60 minutes respectively. After incubation, the PBS sample was taken out and injected into Radio-HPLC for detection; anhydrous ethanol was added to the serum sample, shaken, centrifuged, filtered, and then detected by Radio-HPLC; the experimental results were expressed as 68 the percentage of the radionuclide present in the chemical form of Ga]Ga-ZL01 in the total radionuclide. The results are shown in Table 2.

[0081] Table 2 In vitro stability

[0082]

[0083] As can be seen from Table 2, the radioactive tracer 68 Ga]Ga-ZL01 has good stability in vitro.

[0084] Example 4

[0085] Hydrophilic-lipophilicity test:

[0086] 10 μL (about 0.1 MBq) of the radioactive tracer prepared in Example 2 68 Ga]Ga-ZL01 was diluted to 495 μL with HEPES buffer at pH = 7.4, and then 495 μL of n-octanol was added and shaken vigorously. After centrifugation, 200 μL of liquid was taken from each of the aqueous and organic phases for measurement. Subsequently, it was centrifuged again, 100 μL of liquid was taken from each of the aqueous and organic phases, and its radioactivity count was measured.

[0087] The partition coefficient is calculated by the formula Log D = [Log 10 (radioactivity count in the organic phase / radioactivity count in the aqueous phase)].

[0088] The experimental results show that the partition coefficient Log D of the radioactive tracer 68 Ga]Ga-ZL01 is -3.48 ± 0.22, indicating good hydrophilicity.

[0089] Example 5

[0090] Radioactive tracer 68 Cell uptake study and receptor binding affinity study of [68Ga]Ga-ZL01:

[0091] Cell culture:

[0092] Human glioblastoma cell line U87MG was cultured in MEM (containing NEAA) basal medium with 10% fetal bovine serum in a 5% CO2, 37 °C cell incubator.

[0093] Cell uptake experiment:

[0094] After culturing U87MG cells in a 12-well plate to 2×10 5 cells / well, they were washed with PBS.

[0095] Experimental group: Incubated with the radioactive tracer 68 [68Ga]Ga-ZL01 (20 nM) prepared in Example 2 at 37 °C for 5 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes respectively.

[0096] Inhibition group: The binding of CD36 protein was blocked by simultaneously adding 25-fold concentration of NOTA-ZL01 (500 nmol), and then the radioactive tracer 68 [68Ga]Ga-ZL01 (20 nM) was added to further incubate the cells for 15 minutes, 30 minutes, 60 minutes, and 120 minutes at 37 °C.

[0097] After incubation, the cells were washed 3 times with cold PBS, 400 µL of NaOH was added to each well to lyse the cells, the cells were collected and the intracellular radioactivity count was detected by a gamma counter, and then the cell uptake histogram was calculated, as Figure 3 shown.

[0098] As Figure 3 shown, the experimental results showed that the radioactive uptake of the radioactive tracer 68 [68Ga]Ga-ZL01 by the cells gradually increased within 60 minutes and tended to be stable after 60 minutes; the radioactive uptake in the inhibition group was significantly lower than that in the experimental group, indicating that the uptake of the radioactive tracer 68 [68Ga]Ga-ZL01 by U87MG cells could be significantly inhibited by unlabeled NOTA-ZL01, thus indicating its binding specificity.

[0099] Saturation experiment:

[0100] After culturing U87MG cells in a 12-well plate to 2×10 5Cells / well, and different concentrations of the radioactive tracer prepared in Example 2 were added to U87MG cells 68 Ga]Ga-ZL01, the radioactive tracer 68 Ga]Ga-ZL01 had final concentrations of: 0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM, and 40 nM. Incubate at 37 °C for 60 minutes, and measure the radioactive counts on the cell surface and inside the cells to obtain the binding capacity (specific binding + non-specific binding) of the radioactive tracer 68 Ga]Ga-ZL01 to CD36 protein.

[0101] Non-specific binding was obtained by co-incubating the radioactive tracer 68 Ga]Ga-ZL01 and 25-fold concentration of unlabeled NOTA-ZL01 (500 nmol). The saturation binding curve was fitted by Graphpad Prism software to obtain the maximum specific binding capacity ( 68 Ga]Ga-ZL01 to the receptor B max ), and the binding affinity ( K d ). The results are shown in Table 3 and Figure 4 (saturation experimental curve).

[0102] Table 3 Maximum specific binding capacity ( B max ), and binding affinity ( K d )

[0103]

[0104] The saturation experiment results showed that the binding affinity of the radioactive tracer 68 Ga]Ga-ZL01 to the CD36 protein receptor in U87MG cells K d value reached 8.15 ± 2.06 nM, and the maximum binding capacity of the radioactive tracer 68 Ga]Ga-ZL01 to the CD36 cell receptor B max was approximately 1131 ± 58.53 fmol / mg protein.

[0105] Example 6

[0106] Pharmacokinetic study of the radioactive tracer 68 Ga]Ga-ZL01:

[0107] Three healthy female Balb / c mice were used to evaluate the pharmacokinetic properties of the radioactive tracer 68 Ga]Ga-ZL01 prepared in Example 2. Each mouse was injected with approximately 3.7 MBq of the radioactive tracer 68 Ga]Ga-ZL01 prepared in Example 2 via the tail vein. Blood samples were collected at 1 minute, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 60 minutes, 90 minutes, and 120 minutes after injection. After weighing and measuring the radioactivity counts, the percentage injected dose per gram of tissue (%ID / g) was calculated after radioactive decay correction. The pharmacokinetic curve was fitted using DAS 2.0 software, and the results are as Figure 5 . The change in drug concentration obtained by fitting with DAS software reflects the summary of the distribution phase (α phase) and the elimination phase (β phase). In the distribution phase (α phase), the blood drug concentration drops rapidly after administration, which is the fast elimination stage. In the elimination phase (β phase), the drug concentration decreases proportionally following the law of dynamic equilibrium, which is the slow elimination stage. Through analysis with DAS software, it was found that the blood clearance curve of the radioactive tracer 68 Ga]Ga-ZL01 conforms to the two-compartment model. The half-life of the distribution phase (T1 / 2α) is 0.72 ± 0.02 minutes, and the half-life of the elimination phase (T1 / 2β) is 21.6 ± 0.6 minutes, indicating a moderate blood metabolism rate.

[0108] Example 7

[0109] Biological distribution study of the radioactive tracer 68 Ga]Ga-ZL01 in vivo:

[0110] Establishment of NSG subcutaneous tumor model:

[0111] U87MG cells were cultured in vitro, washed and centrifuged with serum-free culture medium. After counting the viable cells, NSG immunodeficient mice were anesthetized with oxygen containing 2% isoflurane. 150 µL of serum-free suspension containing 3×10 6 glioblastoma cells was injected into the left axilla of the anterior limb of the mice. The general condition of the mice and the tumor volume were monitored. When the tumor diameter grew to 5 - 8 mm, subsequent experiments could be carried out.

[0112] Six U87MG tumor-bearing mice were randomly divided into two groups. Both groups were injected with approximately 3.7 MBq of the radioactive tracer 68 Ga]Ga-ZL01 via the tail vein. Three mice in one group were sacrificed 30 minutes after injection, and three mice in the other group were sacrificed 60 minutes after injection. Blood, tumors, and other major organs and tissues were collected, weighed, and the radioactivity counts were measured. After radioactive decay correction, the percentage injected dose per gram of tissue (%ID / g) was calculated. The results are shown in Table 5 and Figure 6 .

[0113] The tumor / muscle ratio (T / M) is defined as the ratio of the radioactivity count in the tumor to the radioactivity count in the muscle.

[0114] Table 5 Radiotracer 68 In vivo biodistribution of [68Ga]Ga-ZL01

[0115]

[0116] The experimental results showed that in the U87MG tumor-bearing mouse model, after injecting the radiotracer 68 [68Ga]Ga-ZL01 for 30 minutes and 60 minutes, the tumor uptakes were 3.65 ± 0.35 and 1.73 ± 0.11 respectively, indicating that the radiotracer 68 [68Ga]Ga-ZL01 has tumor targeting in CD36-positive tumors. After injecting for 30 minutes and 60 minutes, the T / M ratios were 9.37 ± 0.98 and 6.22 ± 1.52 respectively, and the tracer had a good tumor-to-background ratio at 30 minutes.

[0117] Example 8

[0118] Radiotracer 68 [68Ga]Ga-ZL01 PET / CT imaging in tumor-bearing mice:

[0119] Six U87MG tumor-bearing mice from Example 7 were randomly divided into two groups, one group was the experimental group and the other was the inhibition group, with 3 mice in each group.

[0120] The experimental group was directly injected with approximately 7.4 MBq of the radiotracer 68 [68Ga]Ga-ZL01 via the tail vein. The inhibition group was co-injected with approximately 7.4 MBq of the radiotracer 68 [68Ga]Ga-ZL01 and NOTA-ZL01 into the mice via the tail vein. The mice were induced and maintained under anesthesia with oxygen containing 2% isoflurane, and PET / CT imaging was performed to dynamically acquire images at 30 and 60 minutes in three-dimensional mode.

[0121] Reconstruction was performed using Nucline NanoScan 3.00 to obtain the attenuation-corrected PET / CT fusion images, and image analysis was performed using InterView FUSION 3.0.

[0122] The imaging conditions of the experimental group and the inhibition group were observed, and the results are as Figure 7 shown.

[0123] The experimental results showed that there was an obvious 68Radioactive uptake of 68 Ga]Ga-ZL01 in the inhibition group showed a significant decrease in radioactive accumulation in tumors, indicating that the radioactive tracer

[0124] Example 9

[0125] Tumor CD36 and IgG immunohistochemical staining in tumor-bearing mice:

[0126] The excised tumor tissues of U87MG tumor-bearing mice in Example 7 were placed in 4% paraformaldehyde for fixation for 24 h, then embedded and paraffin sections were prepared. The paraffin sections were placed in an oven at 60 °C for baking for 30 minutes, and then the sections were immersed in xylene I and xylene II for 10 minutes each in turn; the sections were placed in ethanol with decreasing concentration for 2 minutes each in turn (ethanol gradient: 100%, 95%, 90%, 80%, 70%); then the sections were placed in a citric acid antigen retrieval solution, heated in a microwave oven at high power until boiling, then turned off after 15 minutes, and cooled naturally to room temperature;

[0127] An endogenous peroxidase blocker was added dropwise to the tissue and incubated at room temperature for 15 minutes, then washed with double-distilled water; normal goat serum for blocking was added dropwise and incubated at room temperature for 20 minutes; CD36 antibody or IgG antibody (rabbit anti-mouse, 1:200) was added dropwise to the sections and incubated overnight at 4 °C, then taken out and rewarmed at room temperature for 30 minutes, washed with PBS for 5 minutes × 3 times to complete the incubation of the primary antibody; secondary antibody (goat anti-rabbit, 1:200) was added dropwise and incubated at 37 °C for 30 minutes, washed with PBS for 5 minutes × 3 times to complete the incubation of the secondary antibody; DAB chromogenic solution working solution was added dropwise and incubated for 10 seconds, then washed with water; Mayer's hematoxylin was added dropwise and incubated at room temperature for 2 minutes; differentiated with hydrochloric acid alcohol, blued with an alkaline solution, and washed with water; the sections were placed in ethanol with decreasing concentration for several seconds in turn (ethanol gradient: 80%, 90%, 95%), anhydrous ethanol for 10 minutes, then immersed in xylene for 10 minutes, and sealed with 1 drop of neutral resin, and the results were observed and photographed under a microscope.

[0128] The immunohistochemical results of CD36 in tumors of U87MG tumor-bearing mice are shown in Figure 8 a and b in, and the immunohistochemical results of IgG in tumors of U87MG tumor-bearing mice are shown in Figure 9 a and b in. According to the immunohistochemical detection results, it was confirmed that the CD36 receptor was positively expressed in U87MG tumor tissues and negatively expressed in IgG tumor tissue staining.

[0129] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A cyclic polypeptide targeting CD36 protein, characterized in that, Its structural formula is shown in the following formula (I): Formula (I).

2. A labeling precursor targeting CD36 protein, characterized in that, The cyclic polypeptide targeting CD36 protein described in claim 1 is condensed with diethylene glycol and then coupled with the chelator NOTA to obtain a labeling precursor of the CD36 protein-targeting agent.

3. The labeling precursor targeting the CD36 protein according to claim 2, wherein, The structural formula of the labeling precursor of the CD36 protein-targeting agent is shown in the following formula (II): Formula (II).

4. The preparation method of the labeling precursor targeting the CD36 protein according to claim 2, characterized in that, It includes the following steps: Using solid-phase synthesis method, synthesize the cyclic polypeptide targeting CD36 protein; The cyclic polypeptide targeting CD36 protein is condensed with diethylene glycol and then coupled with the chelator NOTA to obtain a labeling precursor of the CD36 protein-targeting agent.

5. A radioactive tracer, characterized in that, It includes the labeling precursor of the CD36 protein-targeting agent described in claim 2 or the labeling precursor of the CD36 protein-targeting agent prepared by the preparation method described in claim 4 and a radionuclide.

6. The radioactive tracer according to claim 5, wherein, The radionuclides include 18 F, 94 Tc, 99m Tc, 90 In, 111 In, 67 Ga, 68 Ga, 86 Y, 90 Y, 177 Lu, 151 Tb, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co, 57 Co, 43 Sc, 44 Sc, 47 Sc, 225 Ac, 213 Bi, 212 Bi, 212 Pb, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd or 166 Dy.

7. The radioactive tracer according to claim 6, wherein, The structural formula of the radioactive tracer is shown in Formula (III): Formula (III).

8. The method for preparing a radioactive tracer according to any one of claims 5 to 7, characterized in that, It includes the following steps: After mixing and reacting the radionuclide with the labeling precursor of the CD36 protein-targeting agent, the radioactive tracer is obtained.

9. Use of the cyclic polypeptide targeting CD36 protein described in claim 1 or the labeling precursor of the CD36 protein-targeting agent described in claim 2 or the radioactive tracer described in any one of claims 5 to 7 in the preparation of a preparation for detecting the expression level of CD36 protein in tumors.

10. Use of the cyclic polypeptide targeting CD36 protein described in claim 1 or the labeling precursor of the CD36 protein-targeting agent described in claim 2 or the radioactive tracer described in any one of claims 5 to 7 in the preparation of a preparation for diagnosing tumors with high expression of CD36 protein.

Images