Cyclic polypeptide targeting CD36 protein and radioactive tracer derived from cyclic polypeptide

Abstract

The invention relates to the technical field of biomedicine, in particular to cyclic polypeptide targeting CD36 protein and a radioactive tracer derived from the cyclic polypeptide. The preparation method comprises the following steps: condensing cyclic polypeptide targeting CD36 protein and dipolyethylene glycol serving as a connexon, coupling a chelating agent NOTA to form a labeled precursor NOTA-ZL01 targeting the CD36 protein, labeling radionuclide with the labeled precursor NOTA-ZL01 targeting the CD36 protein to form a radioactive tracer [68Ga] Ga-ZL01, and carrying out a reaction on the radioactive tracer [68Ga] Ga-ZL01 and the labeled precursor NOTA-ZL01 and the labeled precursor NOTA-ZL01 targeting the CD36 protein. Through in-vitro radiochemical property research and in-vivo stability research, the radioactive tracer [68Ga] Ga-ZL01 is found to be high in stability and hydrophilic; through cell uptake research and receptor binding force research, it is found that the radioactive tracer [68Ga] Ga-ZL01 has high affinity to CD36 protein expressed by U87 cells; the tumor imaging effect of the radioactive tracer [68Ga] Ga-ZL01 is investigated through small animal PET / CT, and it is found that the radioactive tracer [68Ga] Ga-ZL01 can be used for in-vivo imaging of CD36 positive glioblastoma.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a cyclic polypeptide targeting CD36 protein and a radioactive tracer derived therefrom. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled 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 standards of the World Health Organization (WHO), gliomas are divided into grades I to IV. Among them, the most malignant grade IV glioma is also called glioblastoma multiforme (GBM). Glioblastoma often occurs in the brain parenchyma and is diffusely dispersed. It is the most common and most invasive type of glioma in clinical practice. The median survival time of patients is only 12 to 18 months, and the 5-year survival rate is less than 5%. Therefore, early diagnosis and intervention are crucial to improving the prognosis of patients.

[0004] CD36 is a transmembrane glycoprotein involved in a variety of cellular functions, including lipid metabolism, inflammation, and tumor initiation and progression. In recent years, CD36 has received extensive attention as a therapeutic target due to its key role in fatty acid uptake and its close association with tumor growth and metastasis. Preliminary studies have shown that elevated CD36 expression significantly promotes the metastasis of highly aggressive tumors, such as melanoma and breast cancer. In addition, studies have also found that CD36-mediated lipid uptake significantly accelerates tumor progression and metastasis in a variety of cancers, including gastric cancer, oral cancer, and ovarian cancer. In glioblastoma, CD36 plays a more subtle role in tumor initiation and progression, and cancer stem cell populations gain metabolic advantages 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 targeting specificity, can achieve non-invasive, rapid, and systemic visual assessment of lesions, and has become an important tool in clinical diagnosis. Among them, peptide radiopharmaceuticals occupy an important position in PET imaging, and exhibit excellent targeting performance due to their high affinity, low immunogenicity, and easy synthesis and modification. Therefore, the development of peptide radiopharmaceuticals targeting CD36 protein and the use of 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] In order to overcome the above problems, the present invention provides a cyclic polypeptide targeting CD36 protein and a radioactive tracer derived therefrom.

[0007] In order to achieve the above technical objectives, the present invention adopts the following technical solutions: The first aspect of the present invention provides a cyclic polypeptide targeting CD36 protein, the structural formula of which is shown in the following formula (I):

[0008] Formula (I).

[0009] The second aspect of the present invention provides a marker precursor targeting CD36 protein, which is composed of the following components: a cyclic polypeptide targeting CD36 protein, di-polyethylene glycol (PEG2) and a chelating agent NOTA.

[0010] The third aspect of the present invention provides a method for preparing a marker precursor targeting CD36 protein according to the second aspect, comprising the following steps: The solid phase synthesis method was used to synthesize a cyclic peptide targeting CD36 protein; The cyclic peptide targeting CD36 protein was condensed with polyethylene glycol (PEG2) and then coupled with the chelating agent NOTA to obtain a labeled precursor targeting CD36 protein.

[0011] The fourth aspect of the present invention provides a radioactive tracer, comprising the labeled precursor targeting CD36 protein described in the second aspect or the labeled precursor targeting CD36 protein prepared by the preparation method described in the third aspect and a radioactive nuclide.

[0012] A fifth aspect of the present invention provides a method for preparing the radioactive tracer according to the fourth aspect, comprising the following steps: The radioactive tracer is obtained by mixing and reacting the radioactive nuclide with the labeling precursor targeting CD36 protein.

[0013] The sixth aspect of the present invention provides the use of the cyclic polypeptide targeting CD36 protein described in the first aspect, the labeled 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 detecting the expression level of CD36 protein in tumors.

[0014] The seventh aspect of the present invention provides the use of the cyclic polypeptide targeting CD36 protein described in the first aspect, the labeled 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.

[0015] The beneficial effects of the present invention are: The present invention relates to the field of biomedical technology, and in particular to a cyclic polypeptide targeting CD36 protein and a radioactive tracer derived therefrom. In the present invention, the cyclic polypeptide targeting CD36 protein is condensed with diethylene glycol (PEG2) as a linker, and then coupled with a chelating agent NOTA to form a labeled precursor NOTA-ZL01 targeting CD36 protein. The labeled precursor NOTA-ZL01 targeting CD36 protein is labeled with a radioactive nuclide to form a radioactive tracer [ 68 Ga]Ga-ZL01, through in vitro radiochemical properties studies and in vivo stability studies, it was found that the radioactive tracer [ 68 Ga]Ga-ZL01 is highly stable and hydrophilic; through cell uptake studies and receptor binding studies, it was found that the radioactive tracer [ 68 Ga]Ga-ZL01 has a high affinity for CD36 protein expressed by U87 cells; the radiotracer [ 68 Ga]Ga-ZL01 has a tumor imaging effect and was found to be useful for in vivo imaging of CD36-positive glioblastoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0017] Figure 1 is the mass spectrum of the labeled precursor NOTA-ZL01 targeting CD36 protein; Figure 2 For radioactive tracers 68 The radiochemical purity test results of Ga]Ga-ZL01; Figure 3 For radioactive tracers 68 Ga]Ga-ZL01 and CD36 protein cellular uptake experimental results; Figure 4 is a radioactive tracer 68 Saturation experimental curve of Ga]Ga-ZL01 and CD36 protein; Figure 5 is a radioactive tracer 68 Blood clearance curve of Ga]Ga-ZL01; Figure 6 is a radioactive tracer 68 In vivo biodistribution of Ga]Ga-ZL01 in U87MG tumor-bearing mice; Figure 7 PET / CT images of U87MG tumor-bearing mice in the experimental group and the inhibition group in Example 8; Figure 8 The immunohistochemical images of CD36 expression in tumors of U87MG tumor-bearing mice in Example 9, wherein a is magnified 200 times; b is magnified 400 times; Fig. 9 These are the results of tumor IgG immunohistochemistry of U87MG tumor-bearing mice in Example 9, wherein a is magnified 200 times; b is magnified 400 times. DETAILED DESCRIPTION

[0018] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation 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 skilled in the art to which the present invention belongs.

[0019] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0020] A first typical embodiment of the present invention provides a cyclic polypeptide targeting CD36 protein, the structural formula of which is shown in the following formula (I):

[0021] Formula (I).

[0022] A second typical embodiment of the present invention provides a labeling precursor targeting CD36 protein, which is composed of the following components: a cyclic polypeptide targeting CD36 protein, di-polyethylene glycol (PEG2) and a chelating agent NOTA.

[0023] In one or more embodiments, the structural formula of the marker precursor targeting CD36 protein is shown in the following formula (II):

[0024] Formula (II).

[0025] A third typical embodiment of the present invention provides a method for preparing a marker precursor targeting CD36 protein according to the second aspect, comprising the following steps: The solid phase synthesis method was used to synthesize a cyclic peptide targeting CD36 protein; The cyclic peptide targeting CD36 protein was condensed with polyethylene glycol (PEG2) and then coupled with the chelating agent NOTA to obtain a labeled precursor targeting CD36 protein.

[0026] A fourth typical embodiment of the present invention provides a radioactive tracer, comprising the labeled precursor targeting CD36 protein described in the second aspect or the labeled precursor targeting CD36 protein prepared by the preparation method described in the third aspect and a radioactive nuclide.

[0027] In one or more embodiments, the radionuclide comprises 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 One of Dy; preferably 68 Ga.

[0028] Preferably, the structural formula of the radioactive tracer is as shown in formula (III),

[0029] Formula (III).

[0030] A fifth typical embodiment of the present invention provides a method for preparing the radioactive tracer according to the fourth aspect, comprising the following steps: The radioactive tracer is obtained by mixing and reacting the radioactive nuclide with the labeling precursor targeting CD36 protein.

[0031] A sixth typical embodiment of the present invention provides the use of the cyclic polypeptide targeting CD36 protein described in the first aspect, the labeled 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 detecting the expression level of CD36 protein in tumors.

[0032] A seventh typical embodiment of the present invention provides the use of the cyclic polypeptide targeting CD36 protein described in the first aspect, the labeled 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.

[0033] 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 in conjunction with specific embodiments.

[0034] Example 1 Preparation of the labeled precursor NOTA-ZL01 targeting CD36 protein: (1) Using Rink amide-MBHA resin as the starting resin and Fmoc-amino acid as the raw material, the polypeptide represented by formula (IV) was synthesized by solid phase method using N,N-diisopropylethylamine (DIPEA) / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) condensation system.

[0035]

[0036] Formula (IV).

[0037] Specifically: using piperidine DMF solution to remove the amino protecting group on Rink amide-MBHA resin, washing the resin, and blocking the unreacted sites with a dichloromethane blocking solution containing DIPEA and acetic anhydride; removing the solvent, washing the resin, connecting Lys with a protecting group, and using the Lys bound to the resin as the starting point for synthesis; removing the Fmoc protecting group, and repeating the deprotection and condensation reactions in sequence according to the amino acid sequence from the carboxyl end to the amino end until all the amino acids are coupled to the resin, thereby obtaining a polypeptide represented by formula (IV).

[0038] Wherein, the concentration of piperidine DMF solution is 20% (volume percentage); The blocking solution contained 0.2 M DIPEA in dichloromethane and 0.2 M acetic anhydride in dichloromethane.

[0039] (2) The Fmoc protecting group of the last amino acid proline in the peptide sequence is removed, and the carboxyl group of lysine and the amino group of proline are condensed to form a peptide bond, completing the cyclization to form a cyclic peptide targeting the CD36 protein; the cyclization conditions are: at room temperature, the resin coupled with the peptide is mixed with dimethyl sulfoxide (DMSO) and the reaction is maintained for 24 hours.

[0040] (3) The cyclic peptide targeting CD36 protein was condensed with polyethylene glycol (PEG2) and then coupled with the chelating agent NOTA. The peptide was cleaved from the resin and further purified by high performance liquid chromatography to obtain the labeled precursor NOTA-ZL01 targeting CD36 protein.

[0041] Among them, the method for condensing the cyclic peptide targeting CD36 protein with polyethylene glycol (PEG2) is: solid phase synthesis using N,N-diisopropylethylamine (DIPEA) / benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) / 1-hydroxybenzotriazole (HOBT) condensation system; during the condensation reaction, the reactants are calculated by the molar ratio of polyethylene glycol: DIPEA: HOBT: HBTU: resin grafted with polypeptide = 3.5:7:4:4:1.

[0042] The method for coupling the chelating agent NOTA is: The condensation product of the above-mentioned resin grafted with polypeptide and diethylene glycol (PEG2) was placed in dichloromethane (DCM), and trifluoroacetic acid (TFA) was added, and the concentration of TFA was 0.2 M. After the reaction, TFA and DCM were removed, and DMF, NOTA-NHS, and DIPEA were added. The final product was obtained after the reaction was completed. During NOTA coupling, the reactants were calculated by the molar ratio of the resin grafted with polypeptide: NOTA-NHS:DIPEA = 2:8:1; When the polypeptide is cleaved, the mass ratio of the resin grafted with the polypeptide: the cleavage solution is 1:10; in the cleavage solution, the volume ratio of trifluoroacetic acid (TFA): triisopropylsilane (TIS): phenol (PhOH): methyl phenyl sulfide (Thioanisole): H2O is 30:2:2:1:1.

[0043] The mass spectrum of the labeled precursor NOTA-ZL01 targeting CD36 protein obtained in this example is as follows Figure 1 As 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%.

[0044] Example 2 Radioactive tracers 68 Preparation of Ga]Ga-ZL01: Dissolve NOTA-ZL01 in NaOAc buffer and add [ 68 Ga]GaCl3 solution, reacted at 95 °C for 15 minutes, to obtain the radioactive tracer [ 68 Ga]Ga-ZL01.

[0045] The concentration of NaOAc buffer was 0.1 M and the pH was 4.0-4.6; after NOTA-ZL01 was dissolved in NaOAc buffer, the concentration was 20 μM; [ 68 Ga]GaCl3 solution is the solution eluted with 0.01N HCl, which is 18 ± 1mCi; 68 When Ga is labeled, NOTA-ZL01 solution and [ 68 The volume ratio of Ga]GaCl3 solution is 1:1.

[0046] Radioactive tracers 68 The radiochemical purity of Ga]Ga-ZL01 was tested by analytical HPLC with a radioactivity detector. Figure 2 shown.

[0047] Among them, HPLC mobile phase (A = 0.1% TFA / water, B = 0.1% TFA / acetonitrile), Zorbax 5 μ C18 100 Å (250 × 4.6 mm, 5 μm), see Table 1 for details.

[0048] Table 1 HPLC elution conditions

[0049] Depend on Figure 2 It is known that radioactive tracers [ 68 The radiochemical purity of Ga]Ga-ZL01 is greater than 98%.

[0050] Example 3 Radioactive tracers 68 Study on the radiochemical properties of Ga]Ga-ZL01 in vitro: The radioactive tracer prepared in Example 2 [ 68 Ga]Ga-ZL01 was mixed with PBS buffer (pH = 7.4) and human serum (volume ratio of 1:2) and incubated at 37 °C for 30 minutes and 60 minutes respectively. After the incubation, the PBS sample was taken out and injected into Radio-HPLC for detection; the serum sample was added with anhydrous ethanol, centrifuged and filtered, and then detected by Radio-HPLC; the experimental results are [ 68 The results are shown in Table 2.

[0051] Table 2 In vitro stability

[0052] As can be seen from Table 2, the radiotracer [ 68 Ga]Ga-ZL01 has good stability in vitro.

[0053] Example 4 Hydrophilicity and lipophilicity test: 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, centrifugation was repeated, and 100 µL of liquid was taken from each of the aqueous and organic phases, and their radioactivity counts were measured.

[0054] The lipid-water partition coefficient is given by the formula Log D = [Log 10 (radioactivity count of organic phase / radioactivity count of aqueous phase)].

[0055] The experimental results showed that the radioactive tracer [ 68 The lipid-water partition coefficient Log D of Ga]Ga-ZL01 is -3.48 ± 0.22, indicating good hydrophilicity.

[0056] Example 5 Radioactive tracers 68 Cellular uptake and receptor binding studies of Ga]Ga-ZL01: Cell culture: The human glioblastoma cell line U87MG was cultured in MEM (containing NEAA) basal medium containing 10% fetal bovine serum in a 5% CO2, 37°C cell culture incubator.

[0057] Cellular uptake assay: U87MG cells were cultured in 12-well plates to a volume of 2 × 10 5 After 10 cells / well, wash with PBS.

[0058] Experimental group: at 37°C with the radioactive tracer prepared in Example 2 [ 68 Ga]Ga-ZL01 (20 nM) was incubated for 5 min, 30 min, 60 min, 90 min, and 120 min, respectively.

[0059] Inhibition group: CD36 protein binding was blocked by adding 25-fold concentration of NOTA-ZL01 (500 nmol) at the same time, and then radioactive tracers were added at 37 °C. 68 The cells were further incubated with Ga]Ga-ZL01 (20 nM) for 15 min, 30 min, 60 min, and 120 min.

[0060] After the incubation, the cells were washed three 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 counts were measured by a gamma counter to calculate the cell uptake histogram, as shown in Figure 3 shown.

[0061] like Figure 3 As shown, the experimental results show that within 60 minutes, the cells respond to the radioactive tracer [ 68 The radioactive uptake of Ga]Ga-ZL01 gradually increased and stabilized after 60 minutes; the radioactive uptake of the inhibition group was significantly lower than that of the experimental group, indicating that the radioactive tracer [ 68 The uptake of Ga]Ga-ZL01 in U87MG cells was significantly inhibited by unlabeled NOTA-ZL01, indicating its binding specificity.

[0062] Saturation experiment: U87MG cells were cultured in 12-well plates to a volume of 2 × 10 5 cells / well, and different concentrations of the radioactive tracer prepared in Example 2 were added to U87MG cells. 68 Ga]Ga-ZL01, radioactive tracer [ 68 The final concentrations of Ga]Ga-ZL01 were 0.3125 nM, 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, 20 nM and 40 nM. The cells were incubated at 37 °C for 60 min, and the radioactivity counts on the cell surface and inside the cells were measured to obtain the radioactive tracer [ 68 Binding capacity of Ga]Ga-ZL01 to CD36 protein (specific binding + nonspecific binding).

[0063] Nonspecific binding was detected by co-incubation with a radioactive tracer [ 68 Ga]Ga-ZL01 and 25-fold concentration of unlabeled NOTA-ZL01 (500 nmol) were obtained, and the saturation binding curve was fitted by Graphpad Prism software to obtain the radiotracer [ 68 Ga]Ga-ZL01 maximum specific binding capacity to receptors ( B max ) and binding force ( K d ), the results are shown in Table 3 and Figure 4 (Saturation experimental curve) shown.

[0064] Table 3 Maximum specific binding capacity ( B max ) and binding force ( K d )

[0065] The results of saturation experiments showed that the radioactive tracer [ 68 Receptor binding ability of Ga]Ga-ZL01 to CD36 protein in U87MG cells K d The value reached 8.15±2.06 nM, and the radiotracer [ 68 Maximum binding capacity of Ga]Ga-ZL01 to the receptor of CD36 cells B max Approximately 1131 ± 58.53 fmol / mg protein.

[0066] Example 6 Radioactive tracers 68 Pharmacokinetic study of Ga]Ga-ZL01: Three healthy female Balb / c mice were used to evaluate the radioactive tracer prepared in Example 2. 68 Pharmacokinetic properties of Ga]Ga-ZL01. Each mouse was injected via the tail vein with about 3.7 MBq of the radioactive tracer prepared in Example 2 [ 68 Blood samples were collected, weighed and radioactive counts were determined after 1, 3, 5, 10, 15, 30, 60, 90 and 120 minutes of injection of Ga]Ga-ZL01. The percentage injected dose rate per gram of tissue (%ID / g) was calculated after correction for radioactive decay. The pharmacokinetic curves were fitted using DAS 2.0 software. The results are shown in Figure 5 The drug concentration changes obtained by DAS software fitting reflect the summary of the distribution phase (α phase) and the elimination phase (β phase). The blood drug concentration in the distribution phase (α phase) drops rapidly after administration, which is the fast elimination phase. The drug concentration in the elimination phase (β phase) decreases proportionally according to the dynamic equilibrium law, which is the slow elimination phase. The radioactive tracer [ 68 The blood clearance curve of Ga]Ga-ZL01 conformed to the two-compartment model, with the distribution phase half-life (T1 / 2α) of 0.72±0.02 minutes and the elimination phase half-life (T1 / 2β) of 21.6±0.6 minutes, indicating a moderate blood metabolic rate.

[0067] Example 7 Radioactive tracers 68Study on the biodistribution of Ga]Ga-ZL01 in vivo: Establishment of NSG subcutaneous tumor model: U87MG cells were cultured in vitro, washed with serum-free culture medium and centrifuged. After counting the viable cells, NSG immunodeficient mice were anesthetized by oxygen induction with 2% isoflurane. 150 μL of 3×10 6 The serum-free suspension of glioblastoma cells was injected into the left armpit of the mouse forelimb, and the general condition of the mouse and the size of the tumor were monitored. When the tumor grew to 5-8 mm in diameter, subsequent experiments could be carried out.

[0068] Six U87MG tumor-bearing mice were randomly divided into two groups. Both groups were injected with about 3.7 MBq of radioactive tracer via the tail vein. 68 Ga]Ga-ZL01. Three mice were killed 30 minutes after injection in one group, and three mice were killed 60 minutes after injection in the other group. Blood, tumors and other major organs and tissues were collected, weighed and radioactive counts were measured. After correction for radioactive decay, the percentage injected dose rate per gram of tissue (%ID / g) was calculated. The results are shown in Table 5 and Figure 6 shown.

[0069] The tumor / muscle ratio (T / M) was defined as the ratio of radioactivity counts in the tumor to those in the muscle.

[0070] Table 5 Radioactive tracers [ 68 Biodistribution of Ga]Ga-ZL01 in vivo

[0071] The experimental results showed that in the U87MG tumor-bearing mouse model, injection of radioactive tracer [ 68 After 30 and 60 minutes, the tumor uptake of Ga]Ga-ZL01 was 3.65±0.35 and 1.73±0.11, respectively, indicating that the radiotracer [ 68 Ga]Ga-ZL01 has tumor targeting in CD36 positive tumors. The T / M ratios were 9.37 ± 0.98 and 6.22 ± 1.52 at 30 and 60 minutes after injection, respectively, and the tracer had a good tumor-to-background ratio at 30 minutes.

[0072] Example 8 Radioactive tracers 68 PET / CT imaging of Ga]Ga-ZL01 in tumor-bearing mice: Six U87MG tumor-bearing mice in Example 7 were randomly divided into two groups, one as an experimental group and the other as an inhibition group, with 3 mice in each group.

[0073] The experimental group was injected directly into the tail vein with approximately 7.4 MBq of radioactive tracer [68 Ga]Ga-ZL01, the inhibition group transferred about 7.4 MBq of radioactive tracer [ 68 Ga]Ga-ZL01 and NOTA-ZL01 were co-injected into mice through the tail vein. The mice were anesthetized by induction and maintenance of oxygen containing 2% isoflurane. PET / CT imaging was performed, and images were dynamically collected in three-dimensional mode for 30 and 60 minutes.

[0074] Nucline NanoScan 3.00 was used to reconstruct and obtain attenuation-corrected PET / CT fusion images, and InterView FUSION 3.0 was used for image analysis.

[0075] Observe the imaging of the experimental group and the inhibition group. The results are as follows Figure 7 shown.

[0076] The experimental results showed that the tumor sites in the experimental group had obvious [ 68 Ga]Ga-ZL01 radioactive uptake, and the radioactive accumulation of tumors in the inhibition group was significantly reduced; this indicates that the radiotracer [ 68 Ga]Ga-ZL01 specifically targets CD36 protein and can be used for imaging and efficacy monitoring of CD36 protein-expressing glioblastoma.

[0077] Example 9 Tumor CD36 and IgG immunohistochemical staining in tumor-bearing mice: The ex vivo tumor tissue of the U87MG tumor-bearing mouse in Example 7 was fixed in 4% paraformaldehyde for 24 h, and then embedded and paraffin sections were made to complete the preparation; the paraffin sections were placed in a 60°C oven for 30 minutes, and then the sections were immersed in xylene I and xylene II for 10 minutes each; the sections were placed in ethanol from high to low for 2 minutes each (ethanol gradient: 100%, 95%, 90%, 80%, 70%); then the sections were placed in citric acid antigen repair solution, heated in a microwave oven at high heat until boiling, and then turned off the heat for 15 minutes, and naturally cooled to room temperature; Endogenous peroxidase blocker was dripped on the tissue, incubated at room temperature for 15 minutes, and washed with double distilled water; normal goat serum for blocking was dripped and incubated at room temperature for 20 minutes; CD36 antibody or IgG antibody (rabbit anti-mouse, 1:200) was dripped on the slices, and incubated at 4 ℃ overnight. After taking out, it was rewarmed at room temperature for 30 minutes, and washed with PBS for 5 minutes × 3 times to complete the primary antibody incubation; secondary antibody (goat anti-rabbit, 1:200) was dripped, incubated at 37 ℃ for 30 minutes, and washed with PBS for 5 minutes × 3 times to complete the secondary antibody incubation; DAB color developing solution was dripped, incubated for 10 seconds and then washed with water; Mayor's hematoxylin was dripped and incubated at room temperature for 2 minutes; hydrochloric acid alcohol differentiation, alkaline solution turned blue, and washed with water; the slices were placed in ethanol from high to low for a few seconds (ethanol gradient: 80%, 90%, 95%), anhydrous ethanol for 10 minutes, and then immersed in xylene for 10 minutes, and 1 drop of neutral resin was added to seal the slices, and the results were observed under a microscope and photographed.

[0078] The results of CD36 immunohistochemistry in tumors of U87MG tumor-bearing mice were as follows: Figure 8 In a and b, the results of IgG immunohistochemistry in tumors of U87MG tumor-bearing mice are shown in Fig. 9 As shown in a and b. According to the results of immunohistochemistry, it was confirmed that CD36 receptor was positively expressed in U87MG tumor tissue and negatively expressed in IgG tumor tissue staining.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in 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 marker precursor targeting CD36 protein, characterized in that: The invention is composed of the following components: the cyclic polypeptide targeting CD36 protein according to claim 1, diethylene glycol and a chelating agent NOTA.

3. The marker precursor targeting CD36 protein according to claim 2, characterized in that: The structural formula of the marker precursor targeting CD36 protein is shown in the following formula (II): Formula (II).

4. The method for preparing a labeled precursor targeting CD36 protein according to claim 2, characterized in that: The following steps are involved: The solid phase synthesis method was used to synthesize a cyclic peptide targeting CD36 protein; The cyclic polypeptide targeting CD36 protein is condensed with di-polyethylene glycol, and then coupled with a chelating agent NOTA to obtain a labeled precursor targeting CD36 protein.

5. A radioactive tracer, characterized in that It comprises the labeled precursor targeting CD36 protein as claimed in claim 2 or the labeled precursor targeting CD36 protein prepared by the preparation method as claimed in claim 4 and a radioactive nuclide.

6. The radioactive tracer according to claim 5, characterized in that 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 One of Dy.

7. The radioactive tracer according to claim 6, characterized in that 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: The following steps are involved: The radioactive tracer is obtained by mixing and reacting the radioactive nuclide with the labeling precursor targeting CD36 protein.

9. Use of the cyclic polypeptide targeting CD36 protein according to claim 1, the labeled precursor targeting CD36 protein according to claim 2, or the radioactive tracer according to 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 according to claim 1, the labeled precursor targeting CD36 protein according to claim 2, or the radioactive tracer according to any one of claims 5 to 7 in the preparation of a preparation for diagnosing and / or treating tumors with high expression of CD36 protein.

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