Polypeptide nuclide probes, methods of making and uses thereof

Abstract

The present application provides a polypeptide nuclide probe, which comprises a polypeptide and a radionuclide-labeled chelating group connected with an amino group of a main chain or a side chain of the polypeptide, the polypeptide comprising at least one MAP peptide and a c(RGD-DPhe-K) cyclic peptide, a C-terminal of the at least one MAP peptide being connected with an N-terminal of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is a continuous sequence of KLALKLALKALKAALKLA. The polypeptide nuclide probe of the present application can improve tumor targeting and uptake.

Description

Technical Field

[0001] This invention relates to the technical field of tumor-targeting peptide research, specifically to a peptide nuclide probe, its preparation method, and its uses. Background Technology

[0002] Cancer is currently one of the greatest threats to human health, and how to treat cancer precisely and effectively has become a pressing scientific challenge. Radiopharmaceuticals utilize the specific binding of target molecules to receptors to deliver radionuclides to tumor sites. The radiation energy of the radionuclides accurately locates or kills tumors, and this has become a powerful tool for early diagnosis and precision treatment of diseases.

[0003] Peptides are composed of several to dozens of natural or non-natural amino acids condensed together and can be obtained through natural product extraction, gene recombination, and chemical synthesis. Peptide drugs have advantages such as low immunogenicity, good tissue penetration, ease of synthesis and modification, good safety, and low accumulation in tissues, demonstrating significant efficacy in anti-tumor, antibacterial, chronic metabolic, cardiovascular, and immune disease treatments. Peptides can bind to overexpressed receptors or transporters in tumor cells, exhibiting high affinity and selectivity. Currently, 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, and integrin α. v β3, etc. Among them, integrin α v β3 is expressed in various tumor cells, making it a very attractive therapeutic target.

[0004] Therefore, a method for treating integrin α was developed. v β3-dependent polypeptide nuclide probes with good selectivity are of great significance in the field of tumor radiotherapy. Summary of the Invention

[0005] In view of this, the main objective of the present invention is to provide a polypeptide nuclide probe, the polypeptide nuclide probe comprising a polypeptide and a radiolabeled chelating group linked to an amino group of the main chain or side chain of the polypeptide, the polypeptide comprising 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 linked to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is a continuous sequence KLALKLALKALKAALKLA.

[0006] In some embodiments, the chelating group is a group formed by a chelating agent through a condensation reaction with an amino group of the polypeptide backbone or side chain, 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 includes at least one hydrophilic group linked to an amino group on the main chain or side chain 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, Prepare a polypeptide comprising a c(RGD-DPhe-K) cyclic peptide and at least one MAP peptide linked to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the at least one MAP peptide is a KLALKLALKALKAALKLA continuous sequence.

[0012] S2, a radionuclide-labeled chelating group is attached to the amino group of at least one MAP peptide backbone or branch of the polypeptide to obtain a polypeptide nuclide probe.

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

[0014] At least one hydrophilic group is attached to the amino group of at least one MAP peptide backbone or branch chain of the prepared polypeptide.

[0015] The present invention also provides the use of the above-mentioned polypeptide nuclide probe in the preparation of medicaments for treating tumors or cancer.

[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 tumors, lung cancer, breast cancer, prostate cancer, liver cancer, and angiosarcoma.

[0018] The polypeptide-nucleoside probe provided by this invention comprises a polypeptide and a radiolabeled chelating group, wherein the polypeptide uses a c(RGD-DPhe-K) cyclic peptide as a tumor-targeting molecule and is coupled with at least one MAP peptide. Thus, the polypeptide combines the excellent cell membrane penetration ability of the MAP peptide with the high targeting specificity of the c(RGD-DPhe-K) cyclic peptide. Furthermore, novel targeted radiopharmaceutical molecules are obtained by radiolabeling the polypeptide. This polypeptide-nucleoside probe improves tumor targeting and uptake, enabling precise and efficient diagnosis and treatment of various tumors, thus overcoming the shortcomings of existing RGD polypeptide-based radiopharmaceutical molecules. Attached Figure Description

[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 The image shows a high-performance liquid chromatogram of the polypeptide P-RM prepared in Example 1 of this invention.

[0022] Figure 4 This is a 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 Bar chart of tumor target cell targeting results for Ga-P-RM. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0026] It should be noted that, in the embodiments of the present invention, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a method or apparatus that includes a list of elements includes not only the elements expressly stated, but also other elements not expressly listed, or elements inherent to implementing the method or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other related elements in the method or apparatus that includes that element.

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

[0028] The term “targeting” as used in this article refers to the property of a compound (such as a polypeptide) to enter and / or bind to a specific tissue or organ (such as a tumor) with high selectivity.

[0029] The term "cell permeability" mentioned in this article refers to the ability of a compound (such as a polypeptide) to strongly penetrate the cell membrane, breaking through this natural barrier and entering the cell.

[0030] The term "MAP peptide" used in this article refers to a polypeptide with the sequence structure [SEQ ID NO.:1], namely KLALKLALKALKAALKLA. This polypeptide consists of alanine (A), lysine (K), and leucine (L) linked in a specific sequence. It typically exhibits strong cell membrane penetration and can carry various bioactive molecules into cells via endocytosis, but it has poor selectivity for tumor cells. In this sequence, both lysine and leucine have side-chain amino groups, providing multiple sites for linking functional groups. The term "c(RGD-DPhe-K) cyclic peptide" used in this article refers to a polypeptide with the sequence structure [SEQ ID NO.:2]. This polypeptide is a cyclic peptide formed by linking arginine (R), glycine (G), aspartic acid (D), phenylalanine (DPhe), and lysine (K) in a specific sequence. It is a peptide that can bind to integrin α. vThe amino acid sequence that β3 specifically binds to, for integrin α v β3 has high affinity and selectivity.

[0031] The term "hydrophilic group" mentioned in this article, also known as an oleophobic group, refers to a group that has atomic groups that are soluble in water or readily affinity for water. These groups include hydroxyl, ether, block polyether, carboxylic acid, sulfonic acid, phosphate, amino, and quaternary ammonium groups, which can provide or accept hydrogen for the formation of hydrogen bonds.

[0032] The term "chelating group" as used in this article refers to a group having two or more coordinating atoms that can combine with the same central atom to form a ring structure.

[0033] The term "isotope labeling" used in this article refers to the use of isotopes to track the movement and changes of matter. Compounds labeled with isotopes have the same chemical properties as non-isotope-labeled compounds, but their nuclear physical properties are different. By utilizing the nuclear physical property that radioactive isotopes continuously emit characteristic rays, nuclear detectors can be used to track the location, quantity, and transformation of isotope-labeled substances inside or outside the body.

[0034] Based on literature review, the inventors discovered that cell penetrate peptides (CPPs) are a class of short peptides with strong cell membrane penetration capabilities. They can be used to modify targeted drugs, breaking down the natural barrier of the cell membrane and promoting cellular uptake. Currently, they have received widespread attention in the field of drug delivery and are a powerful tool for improving targeted drug delivery efficacy. For example, the MAP peptide with the sequence KLALKLALKALKAALKLA is an amphiphilic membrane-penetrating peptide that can carry various bioactive molecules into cells via endocytosis. Zaro et al. used biotechnology to fuse the oligopeptide HE (histidine-glutamic acid copolymer) with the cell penetrate peptide MAP to obtain a recombinant HE-MAP, which was then expressed as the fusion protein GST-HE-MAP by glutathione-S-transferase (GST). The results of this study show that when recombinant HE-MAP is exposed to a slightly acidic environment, histidine undergoes protonation and becomes positively charged, leading to the dissociation of the electrostatic interaction between glutamate residues and cationic residues on MAP, thereby activating the cell membrane-penetrating activity of MAP. However, the poor selectivity of CPPs limits their application in vivo.

[0035] Integrin α v β3 is highly expressed on the surface of various solid tumor cells. RGD is a cell that can interact with integrin α. vThe amino acid sequence that specifically binds to β3 consists of arginine, glycine, and aspartic acid, and it is associated with integrin α. v β3's high affinity and selectivity have made it a focus of targeted drug research. In recent years, significant progress has been made in utilizing RGD peptide conjugates to improve drug targeting and penetration in tumors, thereby enhancing the therapeutic efficacy and safety of drugs for solid tumors. Recent studies have also discovered that RGD peptides not only assist in drug targeting and penetration but also have potential tumor growth inhibition effects. However, due to the small molecular weight and short residence time of RGD in tumors, it is not conducive to tumor treatment. Therefore, in order to develop radionuclide-labeled radiopharmaceuticals, RGD molecules need to be modified accordingly.

[0036] In view of this, the present invention provides a polypeptide nuclide probe, the polypeptide nuclide probe comprising a polypeptide and a radiolabeled chelating group linked to an amino group of the polypeptide backbone or side chain, the polypeptide comprising 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 linked to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the MAP peptide is

[0037] KLALKLALKALKAALKLA continuous sequence.

[0038] In this invention, MAP peptide has a strong penetrating effect on cell membranes, and it is...

[0039] KLALKLALKALKAALKLA continuous sequence. The c(RGD-DPhe-K) cyclic peptide is a peptide that can interact with integrin α. v The amino acid sequence that specifically binds to β3 consists of arginine, glycine, and aspartic acid, and it is associated with integrin α. v β3 exhibits 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 of the c(RGD-DPhe-K) cyclic peptide. Therefore, peptides formed by linking at least one MAP peptide and the c(RGD-DPhe-K) cyclic peptide possess strong cell membrane penetration and high targeting specificity to tumor cells, enhancing the efficiency of peptide carriers entering tumor cells and interacting with integrin α, which is highly expressed in various tumor cells. vβ3-specific binding. It is understood that the amino group at the other end of the MAP peptide can continue to be linked with repeating amino acids according to the sequence KLALKLALKALKAALKLA, thereby linking 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 of including one MAP peptide, but those skilled in the art can obtain polypeptides including multiple MAP peptides based on conventional techniques 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, or 5 MAP peptides. In some embodiments, the polypeptide includes 1-5 MAP peptides. In some preferred embodiments, the polypeptide includes 1-2 MAP peptides, more preferably, 1 MAP peptide.

[0040] Furthermore, the main chain of the MAP peptide and its side chains, provided by, for example, leucine and lysine, have multiple amino sites at their ends that can be linked to chelating groups labeled with radionuclides. By linking radionuclides labeled with chelating groups to the peptide, the biodistribution and targeting of the peptide radionuclides in animals can be visualized using imaging techniques such as SPECT or PET. When the radionuclides used for labeling are therapeutic radionuclides, tumors or cancers can also be treated through radiotherapy, achieving integrated diagnosis and treatment. The peptide radionuclide probes obtained by radionucliding the above-mentioned peptides improve the targeting and cellular uptake of tumor cells, enabling precise and efficient diagnosis and treatment of various tumors, and providing a new method for tumor diagnosis and treatment.

[0041] In some embodiments, the chelating group is a group formed by a condensation reaction between a chelating agent and an amino group on the main chain or side chain of the polypeptide. 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), hydrazinenic acid amide (HYNIC), methyltetraazine undecylethylene glycol (NOTA), and diethylenetriaminepentaacetic acid (DTPA). These chelating agents have amino or carboxyl side chains and can undergo a condensation reaction between their carboxyl groups and amino sites on the main chain or side chain of the polypeptide, thereby achieving radiolabeled polypeptides. DOTA exhibits excellent thermodynamic stability and kinetic inertness. NOTA has a strong affinity for metal ions, binds stably, and is harmless to biomolecules. DTPA binds stably to metal ions, reducing biotoxicity and increasing 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 One or more of Re are used. These radionuclides can be used for PET or SPECT imaging, enabling visualization studies of the biodistribution and targeting of peptide radionuclide probes in animals, guiding the selection of neutron irradiation time. Furthermore, some radionuclides, such as Lu-177, can also emit beta rays to destroy tumor tissue.

[0043] In some embodiments, the polypeptide further includes at least one hydrophilic group linked to an amino group on the main chain or side chain of the at least one MAP peptide. Modifying the polypeptide with a hydrophilic group improves its water solubility, promotes the metabolism of the polypeptide nuclide probe, and enhances bioavailability and solubility. It is understood that the embodiments of the present invention only show the case of one hydrophilic group, but those skilled in the art can deduce the case of multiple hydrophilic groups based on the teachings of the present invention, and can anticipate that including multiple hydrophilic groups or selecting a PEG group with a larger molecular weight can increase the water solubility and hydrophilicity of the polypeptide. Exemplarily, the polypeptide includes 1, 2, 3, 4, or 5 hydrophilic groups. In some embodiments, the polypeptide includes 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-. Including a hydrophilic group in the polypeptide enhances its hydrophilicity, thereby increasing the water solubility of the polypeptide nuclide probe, promoting drug metabolism, and improving bioavailability and solubility. Exemplarily, in a preferred embodiment, the polypeptide includes NH2-PEG-4-CO- as the hydrophilic group.

[0045] In some embodiments, when the polypeptide further includes at least one hydrophilic group linked to an amino group on the main chain or side chain of the at least one MAP peptide, the chelating group is linked to the amino group at the end of the hydrophilic group. The chelating group reacts more readily with the amino group at the end of the hydrophilic group, making it more advantageous to attach the chelating group to the polypeptide.

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

[0047] S1, Prepare a polypeptide comprising a c(RGD-DPhe-K) cyclic peptide and at least one MAP peptide linked to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, wherein the at least one MAP peptide is a KLALKLALKALKAALKLA continuous sequence.

[0048] S2, a radionuclide-labeled chelating group is attached to the amino group of at least one MAP peptide backbone or branch of the polypeptide to obtain a polypeptide nuclide probe.

[0049] In this invention, the polypeptide can be prepared using methods known to those skilled in the art, and there are no particular limitations. 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 linking at least one MAP peptide to the N-terminus of the c(RGD-DPhe-K) cyclic peptide according to the sequence KLALKLALKALKAALKLA. In one exemplary embodiment, the synthesis of the c(RGD-DPhe-K) cyclic peptide can be performed using a conventional solid-liquid synthesis method, including linking Fmoc-protected aspartic acid to a DCM dichloromethane-swollen resin, verifying the amino acid linkage to the resin with ninhydrin, adding Fmoc-protected arginine after color development, reacting with aspartic acid under Pd(PPh3)4 catalysis, shaking at room temperature for 4 hours, and performing a condensation reaction. After washing with DMF, lysine and phenylalanine were sequentially linked to the resin according to the amino acid sequence of the peptide. Once all amino acids were linked, the peptide was washed with DMF again, filtered, and the solvent was added along with the condensing agent benzotriazol-N,N,N',N'-tetramethylurea hexafluorophosphate (O-Benzotriazol-1-yl-tetramethyluronium, HBTU), DIEA, and DMF. The mixture was shaken for 45 min to form a cyclic peptide, yielding the c(RGD-DPhe-K) cyclic peptide. The method for sequentially linking each amino acid of the MAP sequence to the cyclic peptide is a standard procedure.

[0050] In this invention, the chelating group linkage in step S2 can be performed using methods known in the art, and is not particularly limited thereto. After linkage, the chelating group in the polypeptide is then labeled with a radionuclide to obtain a polypeptide radionuclide probe.

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

[0052] In some embodiments, the radionuclide salt solution is selected from the hydrochloride, sulfate, and nitrate solutions of radionuclides, and is preferably the 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 includes attaching at least one hydrophilic group to an amino group on the main chain or branch chain of at least one MAP peptide of the prepared polypeptide. In this invention, the attachment of the hydrophilic group to the polypeptide can be performed using 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-. Including a hydrophilic group in the polypeptide enhances its hydrophilicity, 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 an embodiment where at least one hydrophilic group is attached to the amino group of the main chain or branch chain of at least one MAP peptide on the prepared polypeptide, step S2 includes attaching a chelating group to the hydrophilic group.

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

[0058] This invention also provides the use of the polypeptide radionuclide probe of this invention in the preparation of drugs for treating tumors or cancer. After being absorbed in vivo, the polypeptide radionuclide probe of this invention targets the tumor site and is specifically taken up by tumor tissue. SPECT or PET imaging can be used to visualize the biodistribution and targeting of the polypeptide radionuclide in animals. When the radionuclide used for labeling is a therapeutic radionuclide, tumors or cancer can also be treated through radiotherapy and other means, achieving 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 boron-carrying peptide of this invention targets integrin α overexpressed on tumor cells. v β3 has a specific binding ability, which allows the polypeptide nuclide probe to be absorbed by the body and target the tumor site.

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

[0061] Example

[0062] The invention will be more readily understood by referring to the following embodiments, which are used to illustrate the invention and should not be construed as limiting the scope of the invention in any way.

[0063] Unless otherwise defined or the context clearly specifies 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 this invention pertains. It should be understood that any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention.

[0064] The polypeptides provided by this invention can be prepared by the conventional methods described above, and the HPLC data of the corresponding polypeptides are shown in the attached figures.

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

[0066] Example 1

[0067] Synthesis of peptide P-RM

[0068] Dichloropolymer resin was swollen with dimethylacetamide (DCM) for 30 min, washed three times with N,N-dimethylformamide (DMF), and the solution was filtered. Excess fluorenemethyloxycarbonyl acyl (Fmoc)-protected aspartic acid (3 eq) was added, followed by N,N-diisopropylethylamine (DIEA) (10 eq). The resin was capped with methanol for 30 min, and the solvent was filtered. 20% piperidine DMF solution (15 ml / g) was added for 15 min. After filtration, a small amount of resin was washed with ethanol, and one drop each of ninhydrin, potassium cyanide (KCN), and phenol were added. The mixture was heated at 105 °C for 5 min, resulting in a positive reaction that changed the color from colorless to deep blue. After three washes with DMF, Fmoc-protected arginine, lysine, and phenylalanine were sequentially linked. A tetraphenylphosphine palladium (Pd(PPh3)4) catalyst (0.1 eq) was dissolved in DMF and reacted for 4 h. After washing with DMF and filtration, a 20% piperidine DMF solution was added and reacted for 5 min. The filtered solution was then reacted with another 20% piperidine DMF solution for 15 min to remove the Fmoc protecting group. The filtered solution was washed with a small amount of resin ethanol, and one drop each of ninhydrin, KCN, and phenol were added. The mixture was heated at 105 °C for 5 min, resulting in a positive blue reaction. The solution was washed three times with DMF. After filtration, the condensing agent benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU) (3 eq), DIEA (10 eq), and DMF were added and shaken for 45 min to form a cyclization. The amino acids, PEG4, and DOTA were sequentially linked according to the polypeptide sequence KLALKLALKALKAALKLA, where A represents alanine, L represents leucine, and K represents lysine. The last lysine was protected by Dde, and the remaining amino acids were protected by Fmoc. After washing and filtration with DMF, 2%-5% hydrazine hydrate DMF solution (15 ml / g) was added for 30 min to remove the Dde protecting group, yielding the polypeptide P-RM, the structural formula of which is shown below. Figure 1 .

[0069] The prepared peptide P-RM was detected by mass spectrometry and high-performance liquid chromatography (HPLC). Mass spectrometry confirmed the correct structure of the prepared peptide P-RM, and the mass spectrum is shown in the figure. Figure 2 The structural confirmation chromatographic 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, as determined by HPLC, is 97.34%. The HPLC chromatogram is shown below. Figure 3 The efflux time of the peptide P-RM was 13.26 min.

[0070] Radiolabeling of P-RM peptides

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

[0072] The labeling rate was determined using radio-iTLC, with a developing system of 1% ammonium acetate:methanol = 50:50. Product 68 Ga-P-RM was spotted onto iTLC-SG chromatographic paper (1cm × 10cm), developed upwards, and the instantaneous thin-layer scanning (iTLC-SG) chromatographic paper was removed, dried, and scanned using a radiometric thin-layer scanner. The labeling efficiency was calculated to be 94.8%. After separation and purification on a Sep-pak C18 column, the radiochemical purity of the labeled analyte was determined by iTLC to be 98.6%.

[0073] Example 2

[0074] Tumor cell uptake experiment

[0075] RGD targeting inhibitors at concentrations of 0, 50, 100, or 500 μM and peptide nuclide probes at 2 μCi were respectively applied. 68 Ga-P-RM was co-incubated for 2 hours with 4T1 breast cancer cells (purchased from Pronosei) and U87MG glioma cells (purchased from Pronosei). Cell lysis supernatants were collected, and the concentrations of certain substances in the supernatants were detected using a radioactive counter. 68 The radioactivity of Ga-P-RM was obtained. 68 The targeting results of Ga-P-RM on tumor cells are shown in... Figure 5 .like Figure 5 As shown, with increasing concentration of the targeted inhibitor, tumor cells take up [the inhibitor]. 68 The amount of Ga-P-RM gradually decreases, proving that 68 Ga-P-RM exhibits excellent targeting properties for 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 transformations made under the inventive concept of the present invention using the contents of the present invention specification and drawings, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A polypeptide nuclide probe, characterized in that, The polypeptide nuclide probe consists of a polypeptide, a hydrophilic group, and a radionuclide-labeled chelating group. The polypeptide comprises a MAP peptide and a c(RGD-DPhe-K) cyclic peptide, connected as shown in the figure below. The MAP peptide sequence is KLALKLALKALKAALKLA. The hydrophilic group is connected to the N-terminus of the MAP peptide and the radionuclide-labeled chelating group at both ends, respectively. The hydrophilic group is NH2-PEG4-CO-. 。 2. The polypeptide nuclide probe according to claim 1, wherein, The chelating group is selected from one or more of DOTA, HYNIC, NOTA, and DTPA.

3. The polypeptide nuclide probe according to claim 1, wherein, The radionuclides are 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. A method for preparing a polypeptide nuclide probe according to any one of claims 1-3, comprising the following steps: S1, Prepare a polypeptide, the polypeptide consisting of a c(RGD-DPhe-K) cyclic peptide and a MAP peptide linked to the N-terminus of the c(RGD-DPhe-K) cyclic peptide, the MAP peptide sequence being KLALKLALKALKAALKLA. S2, in the polypeptide, a hydrophilic group is attached to the N-terminus of the MAP peptide, and the hydrophilic group is NH2-PEG4-CO-; S3, a radionuclide-labeled chelating group is attached to the amino group of the hydrophilic group to obtain a polypeptide nuclide probe.

5. Use of the polypeptide nuclide probe according to any one of claims 1-3 in the preparation of a medicament for treating breast cancer and glioma.

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