Polypeptide nuclide probe, preparation method and application thereof

Abstract

The invention provides a polypeptide nuclide probe, the polypeptide nuclide probe comprises a polypeptide and a chelating group labeled by a radionuclide and connected with a main chain or side chain amino group of the polypeptide, the polypeptide comprises at least one MAP peptide and a c (RGD-DPhe-K) cyclic peptide, the C end of the at least one MAP peptide is connected with the N end of the c (RGD-DPhe-K) cyclic peptide, and the MAP peptide is a KLALKLALKAALKLA continuous sequence. The polypeptide nuclide probe provided by the invention can improve tumor targeting and uptake.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor targeting polypeptide research, and in particular to a polypeptide nuclide probe, a preparation method and use thereof. Background Art

[0002] Cancer is currently one of the biggest threats to human health, and how to treat cancer accurately and effectively has become a scientific research difficulty that urgently needs to be overcome. Radioactive drugs can use the specific binding of targeting molecules and receptors to carry radioactive nuclides to the tumor site, and accurately locate or kill tumors through the radiation energy of radioactive nuclides. It has now become a powerful means of early diagnosis and precise treatment of diseases.

[0003] Peptides are condensed from several to dozens of natural or unnatural amino acids and can be obtained through natural product extraction, gene recombination and chemical synthesis. Peptide drugs have the advantages of low immunogenicity, good tissue permeability, easy synthesis and modification, good safety, and not easy to accumulate in tissues. They have shown significant therapeutic effects in anti-tumor, anti-bacterial, chronic metabolic diseases, cardiovascular diseases, and immune diseases. Peptides can bind to receptors or transporters overexpressed in tumor cells with high affinity and selectivity. At present, several peptides containing tight carbon chains have been shown to have high binding affinity to their receptors, such as neuropeptide Y (NPY), somatostatin, epidermal growth factor, integrin α v β3, etc. Among them, integrin α v β3 is expressed in various tumor cells and is a very attractive therapeutic target.

[0004] To this end, a novel integrin α v β3 is a highly selective polypeptide radionuclide probe and is of great significance in the field of tumor radiotherapy. Summary of the invention

[0005] In view of this, the main object of the present invention is to provide a polypeptide radionuclide probe, which includes a polypeptide and a radionuclide-labeled chelating group connected to the main chain or side chain amino group of the polypeptide, the polypeptide includes at least one MAP peptide and a c(RGD-DPhe-K) cyclic peptide, the C-terminus of the at least one MAP peptide is connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is a continuous sequence of KLALKLALKALKAALKLA.

[0006] In some embodiments, the chelating group is a group formed by a condensation reaction between a chelating agent and the main chain or side chain amino group of the polypeptide, and the chelating agent is selected from one or more of DOTA, HYNIC, NOTA, and DTPA.

[0007] In some embodiments, the radionuclide is selected from 177 Lu, 68 Ga, 89 Zr, 90 Y. 64 Cu, 67 Cu, 170 Tm, 161 Tb, 211 At 18 F. 111 In, 99m Tc, 131 I. 125 I. 123 I. 124 I. 225 Ac, 212 Pb, 223 Ra, 153 Sm, 188 One or more of Re.

[0008] In some embodiments, the polypeptide further comprises at least one hydrophilic group attached to the main chain or side chain amino group of the at least one MAP peptide.

[0009] In some embodiments, the hydrophilic group is selected from one or more of NH2-PEG-4-CO-, NH2-PEG-2000-CO-, NH2-PEG-5000-CO-, and NH2-miniPEG-CO-.

[0010] The present invention also provides a method for preparing the above-mentioned polypeptide nuclide probe, comprising the following steps:

[0011] S1, preparing a polypeptide, wherein the polypeptide comprises a c(RGD-DPhe-K) cyclic peptide and at least one MAP peptide connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the at least one MAP peptide is a continuous sequence of KLALKLALKALKAALKLA;

[0012] S2, connecting a chelating group labeled with a radionuclide to the amino group of the main chain or side chain of at least one MAP peptide of the polypeptide to obtain a polypeptide radionuclide probe.

[0013] In some embodiments, the step S1 further includes:

[0014] At least one hydrophilic group is linked to the amino group of the main chain or the side chain of at least one MAP peptide in the prepared polypeptide.

[0015] The present invention also provides use of the polypeptide nuclide probe in preparing a drug for treating tumors or cancers.

[0016] In some embodiments, the tumor or cancer has integrin α v β3 overexpression.

[0017] In some embodiments, the tumor is selected from one or more of melanoma, glioma, head and neck tumor, lung cancer, breast cancer, prostate cancer, liver cancer, and angiosarcoma.

[0018] The polypeptide nuclide probe provided by the present invention comprises a polypeptide and a chelating group labeled with a radionuclide, wherein the polypeptide uses c(RGD-DPhe-K) cyclic peptide as a tumor targeting molecule and is coupled with at least one MAP peptide, so that the polypeptide has both the excellent cell membrane penetration ability of MAP peptide and the high targeting of c(RGD-DPhe-K) cyclic peptide. Furthermore, a novel targeted radiopharmaceutical molecule is obtained by labeling the polypeptide with a radionuclide. The polypeptide nuclide probe improves tumor targeting and uptake, and can accurately and efficiently diagnose and treat a variety of tumors, making up for the shortcomings of existing RGD polypeptide radiopharmaceutical molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the chemical structure of the polypeptide P-RM prepared in Example 1 of the present invention.

[0020] Figure 2 This is the mass spectrum of the polypeptide P-RM prepared in Example 1 of the present invention.

[0021] Figure 3 This is a high performance liquid chromatogram of the polypeptide P-RM prepared in Example 1 of the present invention.

[0022] Figure 4 Schematic diagram of the chemical structure of the polypeptide nuclide probe prepared in Example 1 of the present invention.

[0023] Figure 5 The polypeptide nuclide probe of Example 2 of the present invention 68 Tumor target cell targeting results of Ga-P-RM are shown in the bar graph. DETAILED DESCRIPTION

[0024] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in combination with the embodiments of the present invention and the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0025] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0026] It should be noted that, in the embodiments of the present invention, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a method or device including a series of elements includes not only the elements explicitly stated, but also other elements not explicitly listed, or also includes elements inherent to the implementation of the method or device. In the absence of further restrictions, an element defined by the sentence "includes a ..." does not exclude the existence of other related elements in the method or device including the element.

[0027] It should be noted that the terms "first\second\third" involved in the embodiments of the present invention are only used to distinguish similar objects, and do not represent a specific order for the objects. It is understandable that the specific order or sequence of "first\second\third" can be interchanged where permitted. It should be understood that the objects distinguished by "first\second\third" can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein.

[0028] The term "targeting" mentioned herein refers to the property of a compound (such as a polypeptide) that can enter and / or bind to a specific tissue or organ (such as a tumor) with high selectivity.

[0029] The term "cell permeability" mentioned herein refers to a compound (such as a polypeptide) having a strong penetrating effect on the cell membrane and being able to break through the natural barrier of the cell membrane and enter the interior of the cell.

[0030] The term "MAP peptide" mentioned herein refers to a polypeptide having a sequence structure of [SEQ ID NO.: 1], i.e., KLALKLALKALKAALKLA. The polypeptide is composed of alanine (A), lysine (K) and leucine (L) connected in a specific order. It usually has a strong penetrating effect on the cell membrane and can carry a variety of biologically active molecules into the cell through endocytosis, but its selectivity for tumor cells is poor. In this sequence, both lysine and leucine have side chain amino groups, thereby providing multiple sites for connecting functional groups. The term "c(RGD-DPhe-K) cyclic peptide" mentioned herein refers to a polypeptide having a sequence structure of [SEQ ID NO.: 2]. The polypeptide is a cyclic peptide formed by arginine (R), glycine (G), aspartic acid (D), phenylalanine (DPhe), and lysine (K) connected in a specific order. It is a kind of integrin α vThe amino acid sequence that specifically binds to integrin α v β3 has high affinity and selectivity.

[0031] The term "hydrophilic group", also known as oleophobic group, mentioned herein refers to a group having an atomic group that is soluble in water or easily affinity with water, such as hydroxyl groups, ether bonds, block polyethers, carboxylic acid groups, sulfonic acid groups, phosphoric acid groups, amino groups, quaternary ammonium groups, etc., which can provide hydrogen or accept hydrogen for the formation of hydrogen bonds.

[0032] The term "chelating group" mentioned herein refers to a group having two or more coordinating atoms and capable of combining with the same central atom to form a ring structure.

[0033] The term "isotope labeling" mentioned in this article refers to the use of isotopes to track the laws of the movement and change of substances. Compounds labeled with isotopes have the same chemical properties as non-isotope labeled compounds, but different nuclear physical properties. By using the nuclear physical properties of radioactive isotopes that continuously emit characteristic rays, the location, quantity and transformation of isotope-labeled substances in the body or outside the body can be tracked through nuclear detectors.

[0034] Based on literature research, the inventors found that cell membrane penetrating peptides (Cell Penetrate Peptides, CPPs) are a class of short peptides with strong penetrating effects on cell membranes, which can be used to modify targeted drugs, break the natural barrier of cell membranes, and promote cell uptake. They have received extensive attention in the field of drug delivery and are a powerful tool for improving the effect of targeted drug delivery. For example, the MAP peptide with the sequence KLALKLALKALKAALKLA is an amphipathic membrane penetrating peptide that can carry a variety of bioactive molecules into cells through endocytosis. Zaro et al. used biotechnology to fuse oligopeptide HE (histidine-glutamic acid copolymer) with cell membrane penetrating peptide MAP to obtain a recombinant HE-MAP, which was expressed as a fusion protein GST-HE-MAP by glutathione-S-transferase (glutathione-S-transferase, GST). The results of this study showed that when recombinant HE-MAP was exposed to a slightly acidic environment, histidine was protonated and became positively charged, which resulted in the release of the electrostatic interaction between the glutamic acid residues and the cationic residues on MAP, thereby activating the cell transmembrane activity of MAP. However, the poor selectivity of CPPs has limited their application in vivo.

[0035] Integrin α v β3 is highly expressed on the surface of various solid cancer cells. RGD is a vThe amino acid sequence that specifically binds to β3 is composed of arginine, glycine, and aspartic acid, which is specific to integrin α v β3 has become the focus of targeted drug research due to its high affinity and selectivity. In recent years, great progress has been made in using RGD peptide coupling materials to improve drug tumor targeting and tumor penetration, improve the drug's solid cancer treatment effect, and improve safety. Recent studies have also found that RGD peptides not only have the ability to assist drug tumor targeting and tumor penetration, but also have potential tumor growth inhibition effects. However, due to the small molecular weight of RGD and its short retention time in tumors, it is not conducive to tumor treatment. Therefore, in order to develop radioactive drugs labeled with radionuclides, the RGD molecule needs to be modified accordingly.

[0036] In view of this, the present invention provides a polypeptide nuclide probe, which comprises a polypeptide and a radioactive nuclide-labeled chelating group connected to the main chain or side chain amino group of the polypeptide, wherein the polypeptide comprises at least one MAP peptide and a c(RGD-DPhe-K) cyclic peptide, wherein the C-terminus of the at least one MAP peptide is connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is

[0037] KLALKLALKALKAALKLA continuous sequence.

[0038] In the present invention, MAP peptide has a strong penetrating effect on cell membranes.

[0039] KLALKLALKALKAALKLA continuous sequence. c(RGD-DPhe-K) cyclic peptide is a kind of integrin α v The amino acid sequence that specifically binds to β3 is composed of arginine, glycine, and aspartic acid, which is specific to integrin α v β3 has high affinity and selectivity. The carboxyl group at one end of the MAP peptide can bind to the amino group on the lysine side chain in the c(RGD-DPhe-K) cyclic peptide. Thus, the polypeptide formed by connecting at least one MAP peptide and the c(RGD-DPhe-K) cyclic peptide has strong penetration into the cell membrane and high targeting to tumor cells, which can improve the efficiency of the peptide carrier entering tumor cells and bind to integrin α highly expressed by various tumor cells. vβ3 specific binding. It is understandable that the amino group at the other end of the MAP peptide can continue to connect amino acids repeatedly according to the sequence of KLALKLALKALKAALKLA, thereby connecting one or more MAP peptides to the c(RGD-DPhe-K) cyclic peptide. It should be noted that the embodiments of the present invention only show the case where one MAP peptide is included, but those skilled in the art can obtain polypeptides including multiple MAP peptides based on conventional technical means in the art and the teachings of the present invention, and the effects of polypeptides including multiple MAP peptides can be expected. Exemplarily, the polypeptide includes 1, 2, 3, 4, and 5 MAP peptides. In some embodiments, the polypeptide includes 1-5 MAP peptides. In some preferred embodiments, the polypeptide includes 1-2 MAP peptides, and more preferably, includes 1 MAP peptide.

[0040] Furthermore, the main chain end of the MAP peptide and the side chains provided by, for example, leucine and lysine also have multiple amino sites that can be connected to a chelating group labeled with a radionuclide. By connecting a chelating group labeled with a radionuclide to the polypeptide, the biodistribution and targeting of the polypeptide radionuclide in an animal body can be visualized by, for example, SPECT or PET imaging. When the radionuclide used for labeling is a therapeutic nuclide, tumors or cancers can also be treated by radiotherapy and other means to achieve integrated diagnosis and treatment. The polypeptide nuclide probe obtained by radionuclide labeling of the above-mentioned polypeptide improves the targeting and cellular uptake of tumor cells, and can accurately and efficiently diagnose and treat a variety of tumors, providing a new method for the diagnosis and treatment of tumors.

[0041] In some embodiments, the chelating group is a group formed by a chelating agent through a condensation reaction with the main chain or side chain amino group of the polypeptide, and the chelating agent is selected from one or more of 1,4,7,10-tetraacetic acid-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid diamide (DOTA), hydrazine nicotinamide (HYNIC), methyl tetrazine undecyl polyethylene glycol (NOTA), and diethylenetriamine pentaacetic acid (DTPA). The above-mentioned chelating agents have amino or carboxyl side chains, and can react with amino sites on the main chain or side chain of the polypeptide through their carboxyl groups, thereby achieving radionuclide labeling of polypeptides. Among them, DOTA has excellent thermodynamic stability and kinetic inertness. NOTA has a strong affinity for metal ions, stable binding, and is harmless to biological molecules. DTPA combines stably with metal ions to reduce biological toxicity and increase water solubility. In a preferred embodiment, the chelating agent is DOTA.

[0042] In some embodiments, the radionuclide is selected from 177 Lu, 68 Ga, 89 Zr,90 Y. 64 Cu, 67 Cu, 170 Tm, 161 Tb, 211 At 18 F. 111 In, 99m Tc, 131 I. 125 I. 123 I. 124 I. 225 Ac, 212 Pb, 223 Ra, 153 Sm, 188 Re or more. The above-mentioned nuclides can be used for PET imaging or SPECT imaging, thereby realizing the visualization study of the biodistribution and targeting of the polypeptide nuclide probe in the animal body and guiding the selection of neutron irradiation time. In addition, some radionuclides, such as Lu-177, can also emit beta rays to destroy tumor tissue.

[0043] In some embodiments, the polypeptide further comprises at least one hydrophilic group connected to the main chain or side chain amino group of the at least one MAP peptide. Modification of the polypeptide by a hydrophilic group is beneficial to improving the water solubility of the polypeptide, promoting the metabolism of the polypeptide radionuclide probe, and improving the bioavailability and solubility. It is understandable that the embodiments of the present invention only show the case of one hydrophilic group, but those skilled in the art can obtain the case including multiple hydrophilic groups based on the teachings of the present invention, and it can be expected that the inclusion of multiple hydrophilic groups or the selection of a PEG group with a larger molecular weight can increase the water solubility of the polypeptide and increase the hydrophilicity. Exemplarily, the polypeptide comprises 1, 2, 3, 4, or 5 hydrophilic groups. In some embodiments, the polypeptide comprises 1-5 hydrophilic groups, preferably 1-3 hydrophilic groups, and more preferably 1 hydrophilic group.

[0044] In some embodiments, the hydrophilic group is selected from one or more of NH2-PEG-4-CO-, NH2-PEG-2000-CO-, NH2-PEG-5000-CO-, and NH2-miniPEG-CO-. By making the polypeptide further include a hydrophilic group, it is beneficial to improve the hydrophilicity of the polypeptide, thereby increasing the water solubility of the polypeptide nuclide probe, promoting drug metabolism, and improving bioavailability and solubility. Exemplarily, the polypeptide includes in a preferred embodiment, the hydrophilic group is NH2-PEG-4-CO-.

[0045] In some embodiments, when the polypeptide further comprises at least one hydrophilic group connected to the main chain or side chain amino group of the at least one MAP peptide, the chelating group is connected to the amino group at the end of the hydrophilic group. The chelating group is more likely to react with the amino group at the end of the hydrophilic group, which is more conducive to connecting the chelating group to the polypeptide.

[0046] The present application also provides a method for preparing a polypeptide nuclide probe, comprising the following steps:

[0047] S1, preparing a polypeptide, wherein the polypeptide comprises a c(RGD-DPhe-K) cyclic peptide and at least one MAP peptide connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the at least one MAP peptide is a continuous sequence of KLALKLALKALKAALKLA;

[0048] S2, connecting a chelating group labeled with a radionuclide to the amino group of the main chain or side chain of at least one MAP peptide of the polypeptide to obtain a polypeptide radionuclide probe.

[0049] In the present invention, the polypeptide can be prepared by methods known to those skilled in the art, and there is no particular limitation on this. Exemplarily, step S1 may include first synthesizing a c(RGD-DPhe-K) cyclic peptide or directly using a prepared or commercially available c(RGD-DPhe-K) cyclic peptide, and then sequentially connecting at least one MAP peptide to the N-terminus of the c(RGD-DPhe-K) cyclic peptide according to the sequence of KLALKLALKALKAALKLA. In an exemplary embodiment, the synthesis of c(RGD-DPhe-K) cyclic peptide can be carried out by a conventional solid-liquid synthesis method, including connecting Fmoc-protected aspartic acid to a DCM dichloromethane swollen resin, verifying whether the amino acid is connected to the resin with ninhydrin, adding Fmoc-protected arginine after color development, reacting with aspartic acid under the catalysis of Pd(PPh3)4, shaking at room temperature for 4 hours, and connecting by condensation reaction. After washing with DMF, lysine and phenylalanine are connected to the resin in sequence according to the amino acids in the polypeptide sequence. After confirming that all amino acids are connected, the solvent is filtered after washing with DMF, and the condensation agent benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate (O-Benzotriazol-1-yl-tetramethyluronium, HBTU), DIEA and DMF are added to oscillate for 45 minutes to form a ring, and c(RGD-DPhe-K) cyclic peptide is obtained. The method of sequentially connecting each amino acid of the MAP sequence to the cyclic peptide is a conventional method.

[0050] In the present invention, the connection of the chelating group in step S2 can be carried out by methods known in the art, and is not particularly limited. After the chelating group is connected, the chelating group in the polypeptide is then labeled with a radioactive nuclide to obtain a polypeptide nuclide probe.

[0051] It is understood that the radionuclide labeling of the chelating group in the polypeptide in step S2 can be carried out by methods known in the art, and the present invention is not particularly limited to this. In one embodiment, the step S2 comprises: dissolving the polypeptide in PBS, adding a buffer to adjust the pH to 5-9, adding a radionuclide salt solution and reacting at a constant temperature of 37-40°C for 0.5-1.5h. In some embodiments, the buffer is selected from ammonium acetate solution or sodium bicarbonate solution. In some embodiments, the concentration of the buffer is 0.25M-0.1M.

[0052] In some embodiments, the radionuclide salt solution is selected from hydrochloride, sulfate, and nitrate solutions of radionuclides, preferably a hydrochloride solution of radionuclides.

[0053] In some embodiments, the radionuclide is selected from 177 Lu, 68 Ga, 89 Zr, 90 Y. 64 Cu, 67 Cu, 170 Tm, 161 Tb, 211 At 18 F. 111 In, 99m Tc, 131 I. 125 I. 123 I. 124 I. 225 Ac, 212 Pb, 223 Ra, 153 Sm, 188 One or more of Re.

[0054] In some embodiments, step S1 further comprises: connecting at least one hydrophilic group to the amino group of the main chain or side chain of at least one MAP peptide on the prepared polypeptide. In the present invention, the hydrophilic group can be connected to the polypeptide by methods known to those skilled in the art, and is not particularly limited thereto.

[0055] In some embodiments, the hydrophilic group is selected from one or more of NH2-PEG-4-CO-, NH2-PEG-2000-CO-, NH2-PEG-5000-CO-, and NH2-miniPEG-CO-. By making the polypeptide further include a hydrophilic group, it is beneficial to improve the hydrophilicity of the polypeptide, thereby increasing the water solubility of the polypeptide boron carrier, promoting drug metabolism, and improving bioavailability and solubility. In a preferred embodiment, the hydrophilic group is NH2-PEG-4-CO-.

[0056] In the embodiment where at least one hydrophilic group is connected to the amino group of the main chain or the branched chain of at least one MAP peptide on the prepared polypeptide, the step S2 comprises connecting a chelating group to the hydrophilic group.

[0057] In some embodiments, step S2 further comprises cutting the synthesized polypeptide nuclide probe from the solid resin using a cutting solution. In some embodiments, the cutting solution comprises, based on the total volume of the cutting solution, 95% trifluoroacetic acid (TFA), 2% ethylenediaminetetraacetic acid (EDT), 2% triisopropylsilane (TIS) and 1% H2O. The cutting time is 120-140 min.

[0058] The present invention also provides a use of the polypeptide radionuclide probe of the present invention in the preparation of a drug for treating tumors or cancer. After being absorbed in the body, the polypeptide radionuclide probe of the present invention targets the tumor site, and after being specifically taken up by the tumor tissue, SPECT or PET imaging can be used to visualize the biodistribution and targeting of the polypeptide radionuclide in the animal body. When the radionuclide used for labeling is a therapeutic radionuclide, tumors or cancers can also be treated by radiotherapy and other means to achieve integrated diagnosis and treatment.

[0059] In some embodiments, the tumor or cancer has integrin α v β3 overexpression. The c(RGD-DPhe-K) cyclic peptide in the polypeptide boron carrier of the present invention has an effect on integrin α overexpression on tumor cells. v β3 has specific binding, so that the polypeptide radionuclide probe is absorbed by the body and targeted to the tumor site.

[0060] In some embodiments, the tumor or cancer is selected from one or more of melanoma, glioma, head and neck tumor, lung cancer, breast cancer, prostate cancer, liver cancer, and angiosarcoma.

[0061] Example

[0062] The present invention will be more readily understood by referring to the following examples, which are intended to illustrate the present invention and should not be construed to limit the scope of the present invention in any way.

[0063] Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific data used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

[0064] The polypeptide provided by the present invention can be prepared by the above conventional method, and the HPLC data of the corresponding polypeptide is shown in the attached figure.

[0065] Unless otherwise specified, the reagents used in the following examples are all commercially available conventional reagents.

[0066] Example 1

[0067] Synthesis of Peptide P-RM

[0068] Use dimethylacetamide (DCM) to swell the dichlororesin for 30 minutes, wash it three times with N,N dimethylformamide (DMF), filter the solution, add excess fluorenylmethoxycarbonyl (Fmoc) protected aspartic acid (3eq), add N,N diisopropylethylamine (DIEA) (10eq), cap it with methanol for 30 minutes, filter the solvent and add 20% piperidine DMF solution (15ml / g) for 15 minutes. After filtering the solvent, take a small amount of resin and wash it with ethanol, add one drop each of ninhydrin, potassium cyanide (KCN) and phenol, heat it at 105℃ for 5 minutes, and a positive reaction occurs, changing from colorless to dark blue. After washing with DMF three times, Fmoc protected arginine, lysine, and phenylalanine are linked in sequence. Add tetrakistriphenylphosphine palladium (Pd(PPh3)4) catalyst (0.1eq) dissolved in DMF to react for 4h, wash with DMF and filter, add 20% piperidine DMF solution to react for 5min, add 20% piperidine DMF solution to the filtered solution and react for 15min to remove the Fmoc protecting group. Take a small amount of resin ethanol to wash the filtered solution, add one drop of ninhydrin, KCN and phenol, heat at 105℃ for 5min, and a positive reaction blue occurs. Wash with DMF three times. After filtering the solvent, add condensing agent benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (3eq), DIEA (10eq) and DMF to oscillate for 45min to form a ring. According to the polypeptide sequence KLALKLALKALKAALKLA, amino acids, PEG4 and DOTA are connected in sequence, where A represents alanine, L represents leucine, and K represents lysine in the polypeptide sequence. Among them, the last lysine is connected to Dde protection, and the remaining amino acids are protected by Fmoc. After DMF washing and filtration, 2%-5% hydrazine hydrate DMF solution (15ml / g) was added for 30min to remove the Dde protecting group to obtain the polypeptide P-RM, whose structural formula is shown in Figure 1 .

[0069] The prepared polypeptide P-RM was subjected to mass spectrometry and high performance liquid chromatography (HPLC). The mass spectrometry confirmed that the structure of the prepared polypeptide P-RM was correct. The mass spectrum is shown in Figure 2 , wherein the structure confirmation spectrum data are as follows: M+3H: 1033.05, M+4H: 775.68, M+5H: 620.72, M+6H: 517.42, M+7H: 443.68. The chemical purity of the prepared polypeptide P-RM was 97.34% as determined by HPLC. The HPLC spectrum is shown in Figure 3 , among which, the elution time of polypeptide P-RM is: 13.26min.

[0070] Radiolabeling of P-RM peptides

[0071] Take a 1.5 mL EP tube, add 100 μL of the above prepared polypeptide P-RM (10 mg / mL), dissolve it in phosphate buffer solution (PBS), then add 150 μL of pH=8.0 CH3COONH4 buffer solution (0.25 M), according to the polypeptide: 68 GaCl3 is added in a volume ratio of 1:4 68 The GaCl3 solution was vortexed and mixed. The solution was placed in a constant temperature incubator and reacted at 37°C for 1 hour to obtain a polypeptide nuclide probe. 68 Ga-P-RM, whose structural formula is shown in Figure 4 .

[0072] The labeling rate was determined by radio-iTLC, and the developing system used was 1% ammonium acetate: methanol = 50:50 system. 68 Ga-P-RM was spotted on iTLC-SG chromatographic paper (1cm×10cm), developed upward, and the instant thin layer scanning (iTLC-SG) chromatographic paper was taken out and dried, and scanned with a radioactive thin layer scanner to calculate the labeling rate, which was 94.8%. After separation and purification with a Sep-pak C18 column, the radiochemical purity of the labeled substance was determined by iTLC method to be 98.6%.

[0073] Example 2

[0074] Tumor cell uptake assay

[0075] 0, 50, 100 or 500 μM of RGD targeting inhibitor and 2 μCi of peptide radionuclide probe were added to the cells. 68 Ga-P-RM was incubated with 4T1 breast cancer cells (purchased from Prosac) and U87MG glioma cells (purchased from Prosac) for 2 h. The cell lysis supernatant was collected and the radioactivity in the supernatant was detected by a radioactivity counter. 68 The radioactivity of Ga-P-RM is obtained 68 The results of Ga-P-RM targeting tumor cells are shown in Figure 5 .like Figure 5 As shown, as the concentration of targeted inhibitors increases, tumor cells take up 68 The amount of Ga-P-RM gradually decreased, proving 68 Ga-P-RM has good targeting ability to tumor cells.

[0076] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A polypeptide nuclide probe, characterized in that: The polypeptide radionuclide probe comprises a polypeptide and a radionuclide-labeled chelating group connected to the main chain or side chain amino group of the polypeptide, wherein the polypeptide comprises at least one MAP peptide and a c(RGD-DPhe-K) cyclic peptide, wherein the C-terminus of the at least one MAP peptide is connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is a continuous sequence of KLALKLALKALKAALKLA.

2. The polypeptide nuclide probe according to claim 1, wherein The chelating group is a group formed by a condensation reaction between a chelating agent and the main chain or side chain amino group of the polypeptide, and the chelating agent is selected from one or more of DOTA, HYNIC, NOTA, and DTPA.

3. The polypeptide boron carrier according to claim 1, wherein: The radionuclide is selected from 177 Lu, 68 Ga, 89 Zr, 90 Y. 64 Cu, 67 Cu, 170 Tm, 161 Tb, 211 At 18 F. 111 In, 99m Tc, 131 I. 125 I. 123 I. 124 I. 225 Ac, 212 Pb, 223 Ra, 153 Sm, 188 One or more of Re.

4. The polypeptide nuclide probe according to claim 1, wherein The polypeptide further comprises at least one hydrophilic group attached to the main chain or side chain amino group of the at least one MAP peptide.

5. The polypeptide nuclide probe according to claim 4, wherein: The hydrophilic group is selected from one or more of NH2-PEG-4-CO-, NH2-PEG-2000-CO-, NH2-PEG-5000-CO-, and NH2-miniPEG-CO-.

6. A method for preparing a polypeptide nuclide probe according to any one of claims 1 to 5, comprising the following steps: S1, preparing a polypeptide, wherein the polypeptide comprises a c(RGD-DPhe-K) cyclic peptide and at least one MAP peptide connected to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the at least one MAP peptide is a continuous sequence of KLALKLALKALKAALKLA; S2, connecting a chelating group labeled with a radionuclide to the amino group of the main chain or side chain of at least one MAP peptide of the polypeptide to obtain a polypeptide radionuclide probe.

7. The preparation method according to claim 6, wherein: The step S1 further comprises: At least one hydrophilic group is linked to the amino group of the main chain or the side chain of at least one MAP peptide in the prepared polypeptide.

8. Use of the polypeptide nuclide probe according to any one of claims 1 to 5 in the preparation of a drug for treating tumors or cancer.

9. The use according to claim 8, wherein The tumor or cancer has integrin alpha v β3 overexpression.

10. The use according to claim 8 or 9, wherein The tumor is selected from one or more of melanoma, glioma, head and neck tumor, lung cancer, breast cancer, prostate cancer, liver cancer, and angiosarcoma.

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