Radioactive polypeptide probe as well as preparation method and application thereof

Abstract

The invention discloses a radioactive polypeptide probe as well as a preparation method and application thereof. The structural formula of the radioactive polypeptide probe is as shown in formula I in the specification. The radioactive polypeptide probe is good in radiostability and high in radiochemical purity, and can be used as a radionuclide probe in the field of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a radioactive polypeptide probe and a preparation method and application thereof. BACKGROUND

[0002] Hepatocyte growth factor receptor (c-Met) plays a key role in the occurrence and development of various tumors. In recent years, targeted inhibitors against c-Met have been widely studied in clinical trials, and some drugs have achieved significant results in clinical transformation. Studies have shown that the abnormal expression level and activation state of c-Met are closely related to the response to molecular targeted therapy, treatment effect and patient prognosis. Therefore, accurate evaluation of the abnormal expression and activation state of c-Met is of great significance for guiding individualized treatment.

[0003] c-Met is a member of the receptor tyrosine kinase (RTK) family encoded on the long arm of human chromosome 7 (7q21-q31), and its natural ligand is hepatocyte growth factor (HGF) secreted by interstitial cells, which together constitute the HGF / c-Met signaling pathway. The mature c-Met receptor is a heterodimeric complex containing a transmembrane beta chain (145 kDa) and an extracellular alpha chain (50 kDa), in which the sema domain and IPT domain of the beta chain are involved in HGF binding, and the intracellular region contains functional domains such as Juxtamembrane (JM) and tyrosine kinase domain. When HGF binds to c-Met, the key tyrosine residues in the intracellular region of c-Met are phosphorylated, further recruiting downstream adaptor proteins (such as Gab-1, Grb-2, Shc, c-Cbl, etc.), and then activating multiple signaling pathways such as PI3K / Akt, ERK1 / 2, PLC-γ, STATs, FAK, etc., regulating various physiological activities of cells such as proliferation, survival, migration, differentiation, etc.

[0004] In non-small cell lung cancer (NSCLC) and other tumors, the HGF / c-Met pathway is often abnormally activated, showing forms such as receptor overexpression and gene amplification. This abnormal activation promotes DNA synthesis, proliferation, anti-apoptosis, invasion and metastasis, and neovascularization of tumor cells, and is one of the important mechanisms leading to acquired resistance to EGFR-targeted therapy. Therefore, c-Met has become an important target for targeted therapy of NSCLC and other tumors.

[0005] At present, the methods for detecting c-Met abnormal expression in clinic mainly include molecular pathology and serological detection. Molecular pathological methods such as immunohistochemistry (IHC), FISH, Western blot, real-time fluorescent quantitative PCR and DNA sequencing, although high accuracy, are considered as the 'gold standard', but belong to invasive examination, sampling is difficult to repeat, and cannot fully reflect tumor heterogeneity. Although serological detection is simple and repeatable, its sensitivity is low, cannot accurately locate the tumor, and the results lag behind the disease progression. Therefore, these traditional methods are difficult to meet the needs of individualized precise treatment of tumors.

[0006] Molecular imaging as a new technology can monitor the expression and activation state of tumor-related molecular targets in vivo, real-time, dynamic and quantitative, and provide important auxiliary information for clinic. PET molecular imaging has been widely used in imaging research of tumor-related targets due to its high sensitivity, high specificity, high imaging resolution and non-invasive advantages. c-Met molecular imaging can realize the whole-process management of early diagnosis of tumors, screening of targeted therapy patients, evaluation of curative effect and prognosis judgment, and has a wide clinical application prospect. However, the current c-Met related molecular imaging research is mainly based on monoclonal antibody or fluorescent labeled probe. The molecular weight of monoclonal antibody probe is large (>150 kDa), the in vivo clearance is slow, the half-life is long, and the immunogenicity is high, resulting in high background signal; the penetration ability of fluorescent probe is weak, which is only suitable for superficial organs, and it is difficult to realize clinical transformation. Therefore, developing new type, high specificity, easy to clear, strong clinical applicability of c-Met targeted molecular imaging probe, especially based on small molecule or polypeptide radioactive probe, has become one of the important directions of current research. The probe based on small molecule compound has fast in vivo clearance, but the retention time is short, and the radioactive labeling process is relatively complex, which increases the preparation difficulty and cost. SUMMARY

[0007] The present application aims at at least solving one of the above technical problems existing in the prior art. To this end, the object of the present application is to provide a radioactive polypeptide probe and a preparation method and application thereof.

[0008] In order to achieve the above object, the technical scheme adopted by the present application is: The first aspect of the present application provides a radioactive polypeptide probe, the structural formula of which is shown as formula I:

[0009] In the formula, Linker is selected from amino acid, polyethylene glycol structural unit and aliphatic chain; M is an imaging agent; and D is a sugar analogue.

[0010] In some embodiments, the amino acid includes at least one of lysine, aspartic acid, glutamic acid, glycine, alanine, and serine.

[0011] In some embodiments, the polyethylene glycol structural unit is PEG. n , where n is a natural number between 1 and 10.

[0012] In some embodiments, the fatty chain includes -(CH2) n - The structure is a unit, n=2-12.

[0013] In some embodiments, the imaging agent is at least one of FB groups, radionuclides, biotin, fluorophores, fluorescent proteins, antibodies, horseradish peroxidase, and alkaline phosphatase.

[0014] In some embodiments, the radionuclide is linked by a chelating agent. The method of linking the radionuclide to the chelating agent in this invention is a conventional technique in the art.

[0015] In some embodiments, the radionuclide is 18 F, 68 Ga、 64 Cu、 89 Zr、 99 mTc, 177 Lu、 131 I, 90 Y、 51 Cr 67 Cu、 67 Ga、 111 In、 186 Re、 188 Re、 139 La、 140 La、 175 Yb、 153 Sm、 166 Ho、 86 Y、 149 Pm, 165 Dy、 169 Er、 47 Sc、 142 Pr, 159 Gd, 212 Bi、 213 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101 mRh、 119 Sb, 128 Ba、 11 C123 I, 124 I, 125 I, 197 Hg, 211 At、 223 Ra、 151 Eu、 153 Eu、 169 Eu、 201 Tl、 203 Pb, 212 Pb, 161 Tb, 198 Au、 225 Ac、 227 Th or 199 Any one of Ag.

[0016] In some embodiments, the chelating agent includes HYNIC, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-N,N',N'',N'''-tetraacetic acid), DOTAM, DOTAGA, DO2A, DO3A, DOTP, NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid), Dar (2,2',2'',2''-(52,132-dihydroxy-55,135-dimethyl-3, At least one of 7,11,15-tetraaza-1,9(2,6)-dipyridin-5,13(1,3)-dibenzocyclohexanedione-3,7,1,11,15-tetrayl)tetraacetic acid, NO2A, NOTP, DTPA (dimethyltriaminepentaacetic acid), NODA, DTPA, PCTA, TETA, CB-TE2A, Cyclam, DFO (deferroamine), MAG3 (mercaptoacetyltriglycine), EC, EDTA, DADT, HYNIC (hydrazinenic acid amide), NS3, 3,2-HOPO, macropa and its derivatives.

[0017] In some embodiments, the fluorophore includes a fluorescent dye.

[0018] In some embodiments, the chelating agent is selected from structures shown in formulas (1)-(9):

[0019] In some embodiments, formula (1) is 1,4,7-triazacyclononadecanyl-4,7-diacetyl-1-acetyl(-NOTA).

[0020] In some embodiments, formula (2) is 1-(4-isothiocyanobenzyl)-1,4,7-triazacyclononadecanyl-4,7-diacetic acid (-NCS-MP-NODA).

[0021] In some embodiments, formula (3) is 2-(4-isothiocyanobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA).

[0022] In some embodiments, formula (4) is 1-(2-(2-(2,5-dioxo-1-pyrrolidinyl)ethylamino)acylethyl)-1,4,7-tetraazacyclononane-4,7-diacetic acid (-Mal-NOTA).

[0023] In some embodiments, formula (5) is 1-(2-(2,5-dioxo-1-pyrrolidinyl)ethyl)-1,4,7-tetraazacyclononane-4,7-diacetic acid.

[0024] In some embodiments, formula (6) is 2-((4,7-dicarboxymethyl)-1,4,7-triazacyclononane) glutaric acid (-NODAGA).

[0025] In some embodiments, formula (7) is 1,4,7,10-tetraazacyclododecyl-4,7,10-triacetyl-1-acetyl(-DOTA).

[0026] In some embodiments, formula (8) is 1-(2-(2-(2,5-dioxo-1-pyrrolidinyl)ethylamino)acylethyl)-1,4,7,10-tetraazacyclononane-4,7,10-triacetic acid (-Mal-DOTA).

[0027] In some embodiments, formula (9) is 2-(4-isothiocyanobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA).

[0028] In some embodiments, the polysaccharide analogue includes at least one of 2-acetamido-2-deoxy-β-D-glucosamine (Glc), 2-amino-2-deoxy-β-D-glucose pyranose, and 1-deoxy-1-azido-beta-D-maltose.

[0029] In some embodiments, the sugar analogue includes , , At least one of them.

[0030] In some embodiments, the compound of formula I is selected from the following compounds: , , .

[0031] A second aspect of the present invention provides a method for preparing the aforementioned radioactive polypeptide probe, comprising the following steps: After removing the Fmoc protecting group from the compound of formula II, the linker, Glu, developer and sugar analog were sequentially attached by condensation reaction to obtain the compound of formula I. .

[0032] In some embodiments, when M is a radionuclide, the method for preparing the radioactive polypeptide probe includes the following steps: after removing the Fmoc protecting group from the compound of formula II, a linker, Glu, a chelating agent and a sugar analog are sequentially attached through a condensation reaction, and then radionuclide labeling is performed to obtain the compound of formula I.

[0033] In some embodiments, the condensation reaction uses a condensing agent including 1-hydroxybenzotriazole (HOBt) / N,N'-diisopropylcarbodiimide (DIC).

[0034] In some embodiments, the condensation reaction takes 30 min to 4 h.

[0035] In some embodiments, the condensation reaction includes an amide condensation reaction.

[0036] A third aspect of the invention provides a pharmaceutical composition comprising the aforementioned radioactive polypeptide probe, and optionally, pharmaceutically acceptable excipients.

[0037] In some embodiments, the pharmaceutically acceptable excipients include any carrier, diluent, adjuvant, or others, such as preservatives or antioxidants, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. These media and agents are well known in the art for use with pharmaceutically active substances. Any conventional media or agent, unless incompatible with the active ingredient, may also be considered for use in therapeutic compositions. Supplemental active ingredients may also be incorporated into the composition to create a suitable therapeutic combination.

[0038] In some embodiments, the pharmaceutical composition includes a tracer.

[0039] In a fourth aspect, the present invention provides a kit comprising the radioactive polypeptide probe or the pharmaceutical composition described herein.

[0040] A fifth aspect of the invention provides the use of the radioactive polypeptide probe or the pharmaceutical composition described herein in the preparation of diagnostic and / or therapeutic drugs.

[0041] In some implementations, the diagnosis includes PET imaging diagnosis.

[0042] In some embodiments, the disease includes agents for diseases characterized by overexpression of hepatocyte growth factor receptor (c-Met).

[0043] In some embodiments, the disease includes squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, liver cancer, kidney cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, esophageal cancer, head and neck cancer, nasopharyngeal carcinoma, oral cancer, salivary gland cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, leukemia, lymphoma, glioma, neuroblastoma, melanoma, sarcoma, endometrial cancer, testicular cancer, or thyroid cancer, or subtypes thereof; more preferably lung cancer; more preferably non-small cell lung cancer.

[0044] The beneficial effects of this invention are: The radioactive polypeptide probe of this invention has good radiostability and high radiochemical purity, and can be used as a radionuclide probe in the field of oncology.

[0045] After radioactive metal nuclides are chelated in the radiopeptide probe of the present invention, PET / CT imaging results show that it has high uptake in tumors and very low accumulation in other normal tissues, achieving an excellent tumor / background ratio. It is expected to be applied in clinical practice and developed into a new type of c-Met receptor-targeted PET imaging agent. Attached Figure Description

[0046] Figure 1 In Experimental Example 2 of the present invention [ 68 Ga]Ga-NOTA-PEG n Graphs of radiochemical stability of Glc-MetP (n=2, 4, 6) in PBS and FBS.

[0047] Figure 2 In Example 3 of the present invention [ 68 Ga]Ga-NOTA-PEG n Figure of in vitro cellular uptake experiment of Glc-MetP (n=2, 4, 6).

[0048] Figure 3 In embodiment 3 of the present invention, [ 68 Ga]Ga-NOTA-PEG n -Glc-MetP (n=2, 4, 6) PET / CT images of tumor-bearing mouse models. Detailed Implementation

[0049] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0050] The term “amino acid” means L- or D-amino acid, amino acid analog (e.g., naphthylalanine), or amino acid mimic (which may be naturally occurring or of entirely synthetic origin) and may be optically pure, i.e., a single enantiomer (and therefore chiral), or a mixture of enantiomers. The conventional three-letter or single-letter abbreviation for amino acids is used herein. The amino acids of the present invention are preferably optically pure. The term “amino acid mimic” means a synthetic natural amino acid analog that is an isosteric compound, i.e., designed to mimic the stereo and electronic structure of a natural compound. These isosteres are well known to those skilled in the art and include, but are not limited to, condensates, retro-inverso peptides, thioamides, cycloalkanes, or 1,5-disubstituted tetrazolium [see M. Goodman, Biopolymers, 24, 137, (1985)].

[0051] The term "sugar analogue" refers to a sugar analogue of a monosaccharide, disaccharide, or trisaccharide. Suitable monosaccharides, disaccharides, or trisaccharides include glucose, galactose, maltose, mannose, and lactose. Sugar analogues may optionally be functionalized to allow easy coupling to amino acids. Thus, glucosamine derivatives of amino acids, for example, can be conjugated to other amino acids via peptide bonds.

[0052] Example 1 In this embodiment, Nota-PEG2-Glc-MetP was prepared. The specific process is as follows: Pre-peptide PEG n Glc-MetP (n=2,4,6) was synthesized using a solid-phase peptide synthesis method on a dichloroethylene resin, with Fmoc-protected amino acids sequentially elongating the peptide chain. The resin was pre-swollen with N,N-dimethylformamide (DMF), and then stirred at room temperature for 45–60 minutes using 1-hydroxybenzotriazole (HOBt) / N,N'-diisopropylcarbodiimide (DIC) as a condensing agent, following the target peptide sequence Tyr-Leu-Phe-Ser-Val-His-Trp-Pro-Pro-Leu-Lys (PEG). n-Glu(Glc))-Ala was used to progressively couple Fmoc-protected amino acids onto the resin from the C-terminal Ala to the N-terminal Tyr. His, Ser, Trp, Tyr, and Lys were protected using side chains. After each coupling step, the Fmoc protecting group was removed using 20% ​​piperidine / DMF at room temperature (10 min × 2), followed by 5 washes with DMF. The Kaiser test was used to confirm the completion of amino condensation. The Lys side chain Dde was removed by adding 10 mL of 2% hydrazine hydrate to the peptide synthesis tube. N,N The deprotection reaction was carried out in three separate steps using a dimethylformamide solution, with each reaction lasting 5 minutes. After the Dde protecting group was removed, 4 times the resin equivalent of NH2-PEG was added. n The amide condensation reaction was carried out with -COOH (n=2, 4, 6), HOBt, DIC, and DMF for 1-2 hours. After the reaction, the mixture was washed 5 times with DMF. Then, Fmoc-Glu(OAll)-OH was added, and the reaction was carried out under the above condensation conditions (HOBt / DIC as condensing agent, stirred at room temperature for 45-60 minutes). After the reaction, the mixture was washed 5 times with DMF. The Fmoc protecting group was removed by using 20% ​​piperidine / DMF at room temperature (10 minutes × 2). Then, 4 resin equivalents of 2-(4,7-bis(2-(tert-butoxy)-2-oxoethyl)-1,4,7-triazacyclononane-1-yl)acetic acid (NOTA) was added, and the amide condensation reaction was carried out with HOBt, DIC, and DMF for 1-2 hours. After the reaction, the peptide was washed five times with DMF, followed by OALL protection removal in anhydrous DCM in the presence of Pd(PPh3)4 (0.2 equivalents) and phenylsilane (22 equivalents) for 4 hours. After the reaction, the peptide was washed five times with DMF. Then, 4 resin equivalents of 2-amino-2-deoxy-β-D-pyranose, HOBt, DIC, and DMF were added for amide condensation for 1-2 hours. After the reaction, the peptide was washed five times with DMF. After peptide synthesis, the resin was immersed in a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIPS) / 1,2-ethylenedithiol (EDT) / water and stirred vigorously at room temperature for 2.5 hours to cleave the peptide chain and remove side chain protecting groups. The reaction solution was precipitated three times with frozen diethyl ether to obtain crude peptide by centrifugation. Subsequently, the crude peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the pure product was collected and lyophilized for later use.

[0053] Purified PEG nThe Glc-MetP peptide was then added to a mixture of N,N-diisopropylethylamine (DIPEA) and DMF in a 1:5 molar ratio with NOTA-NHS ester, and the mixture was stirred for 5 hours to ensure complete coupling. The coupling product was purified by RP-HPLC, and its molecular weight was confirmed by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HPLC purity of NOTA-PEG2-Glc-MetP, NOTA-PEG4-Glc-MetP, and NOTA-PEG6-Glc-MetP was greater than 99%. The theoretical mass of NOTA-PEG2-Glc-MetP was 2232.15, and the molecular weight [M+2H] was determined by MS (m / z). + / 2 is 1117.8; the theoretical mass of NOTA-PEG4-Glc-MetP is 2320.20, and the molecular weight [M+2H] is determined by mass spectrometry (MS / z). + / 2 is 1162.2; the theoretical mass of NOTA-PEG4-Glc-MetP is 2408.25, and the molecular weight [M+2H] is determined by mass spectrometry (MS / z). + / 2 is 1206.41.

[0054] Example 2 This embodiment prepared [ 68 Ga]Ga-NOTA-PEG n -Glc-MetP (n=2, 4, 6), the specific process is as follows:

[0055] First, 20 μg NOTA-PEG n -Glc-MetP (n=2, 4, 6) was dissolved in 13 μL of 1.5 M sodium acetate buffer solution; from 68 Ge / 68 Ga was obtained by elution with 0.1 M hydrochloric acid in a Ga generator. 68 100 μL (88 MBq) of GaCl3 solution was added to the precursor solution, the pH was adjusted to 4.5, and the reaction was carried out at 95℃ for 10 minutes. After the reaction was completed, a small amount of the reaction solution was taken for radioactive thin-layer chromatography analysis (developing solvent: methanol / 1 M ammonium acetate = 1:1) to determine the radiochemical yield. After purification by C18 column, the product was analyzed for radiochemical purity using an HPLC system equipped with a γ detector (Agilent 1260 Infinity II, column: Shimadzu C18 reversed-phase column 250 mm × 4.6 mm, 5 μm).

[0056] Experimental Example 1 This experimental example measures [68 Ga]Ga-NOTA-PEG n The radioactive lipid water coefficient of -Glc-MetP (n=2, 4, 6) is determined as follows: The determination was performed using an octanol-buffered saline biphasic system. 68 Ga]Ga-NOTA-PEG n Lipid-water partition coefficient of Glc-MetP (n=2,4,6) (Log D) 7.4 The specific method is as follows: First, the test system is prepared by mutual saturation treatment, that is, 1-n-octanol is saturated with phosphate buffer (PBS, 1×, pH=7.4), and the same volume of PBS buffer is saturated with 1-n-octanol. Take 100 μL containing 37 kBq (1 μCi) [ 68 Ga]Ga-NOTA-PEG n Add 1× PBS buffer (pH=7.4) of Glc-MetP to a 15 mL centrifuge tube containing 2.9 mL PBS buffer (1×, pH=7.4) and 3 mL 1-n-octanol. Vortex the mixture for 2 minutes and centrifuge at 3000 rpm for 5 minutes at room temperature. After the two phases recrystallize, take 200 μL of the organic phase and aqueous phase samples respectively, and determine the radioactivity using an automated gamma counter. Log D 7.4 The values ​​were calculated as the logarithm of the ratio of radioactivity in the organic phase to that in the aqueous phase. All experiments were repeated three times.

[0057] The results are shown in the table below. When n=2, Log D 7.4 The value was -1.389±0.004; -1.765±0.074 when n=4; and decreased to -2.150±0.022 when n=6. This data trend clearly indicates that the hydrophilicity of the compound is significantly enhanced with the increase of PEG chain length.

[0058] Table 1 [ 68 Ga]Ga-NOTA-PEG n The lipid-water partition coefficient (Log D) of -Glc-MetP (n=2, 4, 6) 7.4 )

[0059] Experimental Example 2 This experimental example measures [ 68 Ga]Ga-NOTA-PEG n The radiostability of -Glc-MetP (n=2, 4, 6) is determined by the following process: Get 3.70-5.55 MBq (100 150 μCi) [ 68Ga]Ga-NOTA-PEG n Glc-MetP (n=2,4,6) was incubated with 200 μL of PBS buffer (1×, pH=7.4) at room temperature; simultaneously, 7.40–11.10 MBq (200–300 μCi) of the same label was incubated with fetal bovine serum (FBS) at 37°C. After 60 and 120 minutes of incubation, 10 μL of PBS or FBS samples were collected for in vitro stability analysis using radio-HPLC. All experiments were independently repeated three times.

[0060] The results are as follows Figure 1 As shown, [ 68 Ga]Ga-NOTA-PEG n The Glc-MetP (n=2, 4, 6) probes exhibited excellent stability under both conditions: after incubation in room temperature PBS buffer for 120 minutes, over 90% of the probes retained their intact structure; after incubation in fetal bovine serum (FBS) at 37°C for the same period, the probe integrity remained above 90%. These results demonstrate that this series of probes possesses good in vitro stability and meets the requirements for subsequent biological applications.

[0061] Experimental Example 3 This experimental example measures [ 68 Ga]Ga-NOTA-PEG n The in vitro cellular uptake of Glc-MetP (n=2, 4, 6) is as follows: MKN45 and A549 cells were fed at a rate of 1×10⁻⁶. 5 Tumor cells were seeded at a density of approximately 37 kBq (1 µCi) per well in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator. After washing with PBS buffer (1×, pH=7.4), tumor cells were seeded in PBS buffer containing approximately 37 kBq (1 µCi) per well. 68 Ga]Ga-NOTA-PEG n Cells were incubated in serum-free Glc-MetP (n=2, 4, 6) medium at 37°C for 15, 30, 60, and 120 minutes. Parallel controls were included, with / without excess unlabeled peptides. After incubation, cells were washed three times with ice-cold PBS, lysed with 1 M NaOH, and intracellular residual radioactivity was measured using a gamma counter. Free […] was also included. 68 Ga]Ga-NOTA-PEG n -Glc-MetP (~37 kBq [1 µCi]) was used as a decay correction control. Cell uptake was expressed as the percentage of radioactivity in the lysate relative to the control radioactivity (% applied radioactivity). Each experiment had three replicates.

[0062] The results are as follows Figure 2 As shown, all three radioactive probes exhibited significant cell-selective uptake characteristics at 37°C: in MKN45 gastric cancer cells, all probes showed rapid binding within 15 minutes of culture, followed by a time-dependent increasing trend, maintaining stable uptake during 120 minutes of culture; in contrast, the uptake levels in A549 lung cancer cells were significantly lower (P<0.01). Notably, when an excess of unlabeled competitive agent was added, the uptake rates of the three probes in MKN45 cells decreased by 75.70±3.21% (PEG2), 65.48±2.87% (PEG4), and 78.51±3.45% (PEG6) at 60 minutes. This dose-dependent inhibition (P<0.001, n=6) clearly confirms that the uptake mechanism of this series of probes is mediated by specific receptors. These results not only validate the targeting of the probe design but also provide important experimental evidence for subsequent in vivo imaging studies.

[0063] Example 3 This embodiment adopts [ 68 Ga]Ga-NOTA-PEG n -Glc-MetP (n=2, 4, 6) were used for in vivo PET / CT imaging in tumor-bearing mice. The specific procedure was as follows: MKN45 and A549 tumor-bearing mice (n=3 per group) were injected via tail vein with 3.70-5.55 MBq (100-150 μCi) of [ 68 Ga]Ga-NOTA-PEG n -Glc-MetP (n=2, 4, 6) radiotracers. Mice were anesthetized with isoflurane and PET scans were performed at 30, 60, and 120 minutes post-injection. To verify specific binding, an MKN45 tumor-bearing mouse blocking group was established, which received 300 μg of unlabeled NOA-PEG concurrently with the radiotracer injection. n Competitive inhibition experiments were conducted using Glc-MetP (n=2,4,6).

[0064] The results are as follows Figure 3As shown, all probes clearly visualized MKN45 tumor tissue. At 30 minutes, tumor uptake rates were 1.65±0.29, 1.50±0.06, and 1.55±0.11 %ID / g, respectively, decreasing to 1.00±0.15, 0.77±0.22, and 1.08±0.08 %ID / g at 60 minutes, exhibiting typical time-dependent clearance characteristics. Notably, all three probes showed significant accumulation in the kidneys (from 3.71±0.23 to 5.10±1.35 %ID / g at 30 minutes), indicating that they are primarily excreted through the urinary system. Specificity validation experiments confirmed that tumor uptake was significantly reduced in the unlabeled peptide co-injection group (P<0.01), and the A549 tumor uptake rate was significantly lower than that of MKN45 tumors, highly consistent with the results of in vitro cell experiments. Furthermore, probe accumulation in other normal tissues was extremely low, achieving an excellent tumor / background ratio. These results not only confirm the specific binding ability of this series of probes to c-Met targets, but also demonstrate their promising application prospects as tumor imaging agents.

[0065] Experimental Example 3 This experimental example measures [ 68 Ga]Ga-NOTA-PEG n The biodistribution of Glc-MetP (n=2, 4, 6) in MKN45 tumor-bearing mice was as follows: The experimental setup was as follows: (1) MKN45 tumor-bearing mice (n=3 per group) were injected with 3.70-5.55 MBq (100-150 μCi) [ 68 After Ga-NOTA-PEG2-Glc-MetP, mice were sacrificed at 30 and 60 minutes, respectively; (2) MKN45 tumor-bearing mice in the blocking group (n=3 per group) were simultaneously injected with an equal amount of radiotracer and 300 μg of unlabeled NOTA-PEG2-Glc-MetP and sacrificed at 60 minutes. Immediately after sacrifice, tumors and healthy tissues were separated, accurately weighed, and radioactivity was measured using a gamma counter. Data are expressed as the percentage of the dose taken up per gram of tissue relative to the injected dose (%ID / g), calculated as: (tissue radioactivity count / tissue weight) / (injected dose radioactivity count) × 100%. All operations were performed under the same experimental conditions to ensure data comparability.

[0066] The results are shown in the table below. 68The Ga-NOTA-PEG2-Glc-MetP probe exhibited good MKN45 tumor uptake characteristics and an excellent tumor / tissue ratio. Quantitative analysis showed that the tumor uptake rate was 2.56±0.91%ID / g at 30 minutes post-injection, decreasing to 0.99±0.20%ID / g at 60 minutes. Significant accumulation was observed in the kidneys at 60 minutes (3.41±0.88%ID / g), while hepatic uptake remained at a low level (0.98±0.21%ID / g), clearly confirming that the probe is primarily excreted through the kidneys. Particularly noteworthy was the significant decrease in tumor uptake at 60 minutes in the blocking group (P<0.01), a result that strongly validates the specific accumulation mechanism of the probe in tumor tissue.

[0067] Table 2 [ 68 Biodistribution of Ga-NOTA-PEG2-Glc-MetP in MKN45 tumor-bearing mice

[0068] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A radioactive polypeptide probe, characterized in that: Its structural formula is shown in Formula I: Linker is selected from amino acids, polyethylene glycol structural units, and fatty acid chains; M is a developer; and D is a sugar analog.

2. The radioactive polypeptide probe according to claim 1, characterized in that: The imaging agent is at least one of FB group, radionuclide, biotin, fluorophore, fluorescent protein, antibody, horseradish peroxidase and alkaline phosphatase; preferably, the radionuclide is linked by a chelating agent.

3. The radioactive polypeptide probe according to claim 1, characterized in that: The radioactive nuclide is 18 F, 68 Ga、 64 Cu、 89 Zr、 99 mTc, 177 Lu、 131 I, 90 Y、 51 Cr 67 Cu、 67 Ga、 111 In、 186 Re、 188 Re、 139 La、 140 La、 175 Yb、 153 Sm、 166 Ho、 86 Y、 149 Pm, 165 Dy、 169 Er、 47 Sc、 142 Pr、 159 Gd, 212 Bi、 213 Bi、 72 As、 72 Se、 97 Ru、 109 Pd, 105 Rh、 101 mRh、 119 Sb, 128 Ba、 11 C 123 I, 124 I, 125 I, 197 Hg, 211 At、 223 Ra、 151 Eu、 153 Eu、 169 Eu、 201 Tl、 203 Pb, 212 Pb, 161 Tb, 198 Au、 225 Ac、 227 Th or 199 Any one of Ag.

4. The radioactive polypeptide probe according to claim 2, characterized in that: The chelating agent includes at least one of HYNIC, DOTA, DOTAM, DOTAGA, DO2A, DO3A, DOTP, NOTA, NODAGA, Dar, NO2A, NOTP, DTPA, NODA, DTPA, PCTA, TETA, CB-TE2A, Cyclam, DFO, MAG3, EC, EDTA, DADT, HYNIC, NS3, 3,2-HOPO, macropa, and their derivatives.

5. The radioactive polypeptide probe according to claim 1, characterized in that: The polysaccharide analogues include at least one of 2-acetamido-2-deoxy-β-D-glucosamine, 2-amino-2-deoxy-β-D-glucose pyranose, and 1-deoxy-1-azido-beta-D-maltose.

6. The radioactive polypeptide probe according to claim 1, characterized in that: The compound of formula I is selected from the following compounds: 、 、 。 7. A method for preparing a radioactive polypeptide probe according to any one of claims 1-6, characterized in that: Includes the following steps: After removing the Fmoc protecting group from the compound of formula II, the linker, Glu, developer and sugar analog were sequentially attached by condensation reaction to obtain the compound of formula I. 。 8. A pharmaceutical composition, characterized in that: It includes the radioactive polypeptide probe as described in any one of claims 1-6; and optionally, pharmaceutically acceptable excipients.

9. A reagent kit, characterized in that: Includes the radioactive polypeptide probe according to any one of claims 1-6 or the pharmaceutical composition according to claim 8.

10. The use of a radioactive polypeptide probe according to any one of claims 1-6 or the pharmaceutical composition according to claim 8 in the preparation of diagnostic and / or therapeutic drugs for diseases.

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