Polypeptide boron carrying agent as well as preparation method and application thereof

Abstract

The invention provides a polypeptide boron carrying agent. The polypeptide boron carrying agent comprises a polypeptide and at least one boron-containing group 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 boron carrying agent provided by the invention has better targeting property and cell penetrability to tumor cells, has high boron loading amount, and meets the requirements of boron neutron capture therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of boron neutron capture therapy in cancer treatment, and in particular to a polypeptide boron carrier, a preparation method and use thereof. Background Art

[0002] How to treat cancer accurately and effectively has become a scientific research difficulty that needs to be overcome urgently. Among the many cancer patients, more than 70% choose combined radiotherapy. With the substantial development of medical research in countries around the world, the technology of radiotherapy is constantly improving, from the initial non-selective X-ray and gamma radiotherapy to heavy ion radiotherapy with longitudinal energy selection, tumor-targeted neutron capture therapy and photodynamic therapy.

[0003] Boron neutron capture therapy (BNCT) is a dual-targeted radiotherapy that first allows a boron carrier with high affinity and high targeting to accumulate in the tumor, then uses a neutron beam to locally irradiate the tumor site, and uses the particles and energy generated by the nuclear reaction between boron-10 and neutrons to destroy the tumor tissue. 10 The particles generated by the B reaction have a short range and cause little damage to normal tissues, which can provide a new treatment option for patients who are not suitable for traditional surgical treatment and radiotherapy. Boron neutron capture therapy is currently one of the best means to achieve precision radiotherapy, and has an excellent killing effect on locally invasive malignant tumors such as melanoma, glioma and recurrent head and neck tumors.

[0004] Ideally, requirements for boron-containing carriers for BNCT include: strong tumor targeting, 10 B concentration ratio (T / N) is high, at least T / N greater than 3-4:1; tumor uptake is high and retention time is long, alone or in combination, each tumor cell should contain 10 9 indivual 10 B atoms or per gram of tumor 10 B is about 20-50μg, and this concentration can be maintained during neutron irradiation (usually neutron irradiation takes 30-60 minutes); it is quickly cleared from the blood and has low toxicity in the body. However, the most mature BNCT drugs in clinical application are boronophenylalanine (BPA) and sodium borocaptate (BSH), which fail to fully meet the requirements of an ideal boron carrier. There is only one boron atom in the BPA molecule, and its boron carrying capacity is extremely limited; although BSH has a higher boron carrying capacity, its targeting and toxicity are not ideal.

[0005] 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.

[0006] Therefore, it is of great significance in the field of tumor radiotherapy to develop a polypeptide boron carrier that has a strong penetrating effect on cell membranes, good selectivity in vivo, and a high boron loading amount. Summary of the invention

[0007] In view of this, the main purpose of the present invention is to provide a polypeptide boron carrier and a preparation method thereof. The polypeptide boron carrier of the present invention has a strong penetrating effect on cell membranes, has a strong targeting effect on tumor cells, and has a high boron loading capacity.

[0008] The first aspect of the present invention provides a polypeptide boron carrier, which comprises a polypeptide and at least one boron-containing 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, and 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.

[0009] In some embodiments, the at least one boron-containing group is selected from C2B 10 H 11 -R1-CO-, CB5H8-R1-CO-, C2B4H7-R1-CO-, C3B3H6-R1-CO-, C4B2H5-R1-CO-, C2B3H6-R1-CO-, C2B3H4-R1-CO-, C2B4H5-R1-CO-, C2B5H6-R1-CO- and CB5H6-R1-CO-, wherein R1 is (CH2) n , n=0-2.

[0010] In some embodiments, the polypeptide boron carrier comprises 1-5, preferably 1-3 boron-containing groups.

[0011] 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, and the hydrophilic group is selected from one or more of NH2-PEG4-CO-, NH2-PEG2000-CO-, NH2-PEG-5000-CO-, and NH2-miniPEG-CO-.

[0012] In some embodiments, the polypeptide further comprises a chelating group labeled with a radionuclide, and the chelating group is connected to the hydrophilic group or to the main chain or side chain amino group of the at least one MAP peptide.

[0013] 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.

[0014] 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.

[0015] The present invention also provides use of the polypeptide boron carrier 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 carrier provided by the present invention includes a polypeptide and at least one boron-containing group, wherein the polypeptide uses c(RGD-DPhe-K) cyclic peptide as a tumor targeting molecule and is coupled to 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. In addition, the polypeptide also includes a plurality of connection sites for boron-containing groups, and the boron loading of the boron carrier can be effectively increased by connecting boron-containing groups with higher boron content. Therefore, the boron carrier provided by the present invention has stronger targeting and membrane permeability, and at the same time has a high boron loading, which can meet the requirements of the boron carrier in BNCT treatment. Furthermore, the polypeptide boron carrier of the present invention can also be labeled with radionuclides to obtain a radionuclide-labeled polypeptide boron carrier. In BNCT treatment, radionuclide labeling can be used to achieve integrated diagnosis and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the nuclear magnetic resonance spectrum of compound I during the preparation process of Example 1 of the present invention.

[0020] Figure 2 This is the nuclear magnetic resonance spectrum of compound II during the preparation process of Example 1 of the present invention.

[0021] Figure 3 This is the mass spectrum of the carborane polypeptide P-RMB-1 prepared in Example 1 of the present invention.

[0022] Figure 4 This is a high performance liquid chromatogram of the carborane polypeptide P-RMB-1 prepared in Example 1 of the present invention.

[0023] Figure 5 This is the mass spectrum of the carborane polypeptide P-RMB-2 prepared in Example 2 of the present invention.

[0024] Figure 6 This is a high performance liquid chromatogram of the carborane polypeptide P-RMB-2 prepared in Example 2 of the present invention.

[0025] Figure 7 This is the mass spectrum of the carborane polypeptide P-RMB-4 prepared in Example 3 of the present invention.

[0026] Figure 8 This is a high performance liquid chromatogram of the carborane polypeptide P-RMB-4 prepared in Example 3 of the present invention.

[0027] Fig. 9 The distribution of P-RMB-1 in various tissues of U87MG tumor-bearing mice measured in Example 7 of the present invention.

[0028] Fig.10The distribution of P-RMB-1 in various tissues of 4T1 tumor-bearing mice measured in Example 7 of the present invention.

[0029] Fig.11 The measured value of Example 8 of the present invention is 177 SPECT imaging images of Lu-P-RMB in model animals.

[0030] Fig.12 The measured value of Example 9 of the present invention is 89 PET imaging images of Zr-P-RMB in model animals.

[0031] Fig.13 This is a bar graph of the boron content in the cell solution after different concentrations of targeted inhibitors and P-RMB-1 were co-incubated for 24 hours, measured in Example 10 of the present invention.

[0032] Fig.14 This is a diagram of the clone formation of 4T1 cells after treatment measured in Example 11 of the present invention.

[0033] Fig.15 This is the tumor growth curve of 4T1 tumor-bearing mice after tumor treatment measured in Example 11 of the present invention. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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 v The amino acid sequence that specifically binds to integrin α v β3 has high affinity and selectivity.

[0041] The term "boron-containing group" as used herein refers to a group containing 10 The boron-containing group is an organic or inorganic group of atom B. For example, the boron-containing group may be a group derived from p-dihydroxyborylphenylalanine, disodium mercapto dodecaborane, carborane, borane, alkyl boron, borohydride anion compounds, and the like.

[0042] The term "carborane" mentioned herein refers to a product in which some of the boron atoms in a polyhedral borane molecule are replaced by carbon atoms, such as monocarbon carborane B n C (n is 4-11), dicarbonyl carborane, such as closo-C2B n H n+2(n is 3-10), nido-C2B3H7, arachno-C2B7H 13 , nested cages CB5H9, C2B4H8, C3B3H7, C4B2H6 and C2B3H7, closed cages C2B3H5, ​​C2B4H6, C2B5H7 and CB5H7, etc. Among them, B 10 C2H 12 It and its derivatives have special thermal and chemical stability and are widely used in radiopharmaceuticals.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] Boron neutron capture therapy gathers boron carriers with high affinity and high targeting to tumors in tumors, and then uses the particles and energy generated by the nuclear reaction of boron-10 and neutrons to destroy tumor tissues. One of the hot topics in the research of third-generation boron carriers is to couple boron-containing groups with peptide molecules to prepare different tumor-targeting molecules.

[0047] 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 powerful tools 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, but lacks specificity as a drug carrier. 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, the histidine was protonated and became positively charged, resulting in the release of the electrostatic interaction between the glutamate residues and the cationic residues on MAP, thereby activating the cell transmembrane activity of MAP.

[0048] Integrin α v β3 is highly expressed on the surface of various solid cancer cells. RGD is a 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 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.

[0049] In view of this, the present invention provides a polypeptide boron carrier, which comprises a polypeptide and at least one boron-containing 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

[0050] KLALKLALKALKAALKLA continuous sequence.

[0051] In the present invention, MAP peptide has a strong penetrating effect on cell membranes.

[0052] 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. v β3 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.

[0053] Furthermore, the main chain end of the MAP peptide and the side chains provided by leucine and lysine also have multiple amino sites that can be connected to boron-containing groups, thereby being able to connect more boron-containing groups and increase the boron loading capacity of the boron carrier. The polypeptide boron carrier formed by connecting the above polypeptide and at least one boron-containing group has high membrane permeability and high targeting, and at the same time has a high boron loading capacity, meeting the requirements of neutron capture therapy (BNCT) technology for boron-containing carriers.

[0054] In some embodiments, the at least one boron-containing group is selected from C2B 10 H 11 -R1-CO-, CB5H8-R1-CO-, C2B4H7-R1-CO-, C3B3H6-R1-CO-, C4B2H5-R1-CO-, C2B3H6-R1-CO-, C2B3H4-R1-CO-, C2B4H5-R1-CO-, C2B5H6-R1-CO- and CB5H6-R1-CO-, wherein R1 is (CH2) n, n=0-2. The above-mentioned boron-containing groups have stable structures and no biological toxicity, and can be used for the preparation of radioactive therapeutic drugs. In some preferred embodiments, the at least one boron-containing group is C2B 10 H 11 CH2CO-、C2B 10 H 11 CH2CH2CO-、C2B 10 H 11 CO-、C2B 10 H 11 CH2CH2CO-. These boron-containing groups are derived from carborane C2B 10 H 12 It has a high boron content, is both hydrophilic and lipophilic, and has special thermal and chemical stability. It is easy to couple with polypeptide molecules, which is beneficial for the preparation of polypeptide boron carriers and increasing the boron loading capacity of boron carriers.

[0055] In some embodiments, the polypeptide boron carrier includes 1-5 boron-containing groups, preferably 1-3 boron-containing groups. In the polypeptide boron carrier of the present invention, the main chain and the measurement of the polypeptide have multiple amino groups, which can be used to connect the boron-containing groups. By making the polypeptide boron carrier include the boron-containing groups in the above-mentioned number range, it is beneficial to further increase the boron loading capacity of the polypeptide boron carrier, thereby improving the effect on tumor cells. Exemplarily, the polypeptide boron carrier includes 1, 2, 3, 4, and 5 boron-containing groups. In some preferred embodiments, the polypeptide boron carrier includes 1-3 boron-containing groups.

[0056] 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, and 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-. 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 obtain the case of 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 PEG groups with larger molecular weight can increase the water solubility of the polypeptide and increase the hydrophilicity. Exemplarily, the polypeptide comprises 1, 2, 3, 4, and 5 hydrophilic groups. In some embodiments, the polypeptide comprises 1-5 hydrophilic groups, preferably 1-3 hydrophilic groups, and more preferably 1 hydrophilic group. By making the polypeptide also include a hydrophilic group, it is beneficial to improve the hydrophilicity of the polypeptide, thereby facilitating the increase of the water solubility of the polypeptide boron carrier, promoting drug metabolism, and improving bioavailability and solubility. Exemplarily, the polypeptide includes in a preferred embodiment, the hydrophilic group is NH2-PEG-4-CO-.

[0057] In some embodiments, the polypeptide further comprises a chelating group labeled with a radionuclide, and the chelating group is connected to the hydrophilic group or cascaded with the main chain or side chain amino group of the at least one MAP peptide. By connecting the chelating group labeled with a radionuclide to the polypeptide, the biodistribution and targeting of the boron carrier in the animal body can be visualized by SPECT or PET imaging, which can be used to guide the selection of neutron irradiation time and facilitate the evaluation of the therapeutic effect. When the radionuclide used for labeling is a therapeutic nuclide, the boron carrier can also be combined with neutron capture therapy and radiotherapy.

[0058] 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 groups have amino or carboxyl side chains and can be connected to amino or carboxyl groups on polypeptides or hydrophilic groups through condensation reactions, thereby providing radionuclide labeling sites for polypeptide boron carriers. 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.

[0059] 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 boron carrier 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.

[0060] The present application also provides a method for preparing a polypeptide boron carrier, comprising the following steps:

[0061] 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;

[0062] S2, connecting at least one boron-containing group to the amino group of the main chain or side chain of at least one MAP peptide of the polypeptide to obtain a polypeptide carrier.

[0063] 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.

[0064] In the present invention, at least one boron-containing group can be attached to the polypeptide by a method known to those skilled in the art, and is not particularly limited thereto. For example, a boron-containing molecule having a carboxyl group is subjected to an amidation reaction with an amino group on the main chain or side chain of the polypeptide, so that the boron-containing group is attached to the amino group.

[0065] In some embodiments, step S2 further comprises cutting the synthesized polypeptide boron carrier P-RMB 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.

[0066] 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.

[0067] 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-.

[0068] In some embodiments, the step S1 further comprises connecting a chelating group to the hydrophilic group; the preparation method further comprises: step S3, labeling the chelating group in the polypeptide with a radionuclide. After the hydrophilic group is connected to the polypeptide, the chelating group is then connected to the hydrophilic group to facilitate the subsequent labeling of the polypeptide boron carrier with a radionuclide. In the present invention, the connection of the chelating group can be carried out by methods known in the art, and this is not particularly limited. After the chelating group is connected, a boron-containing group is connected to the polypeptide to obtain a polypeptide boron carrier, and then the chelating group in the polypeptide is labeled with a radionuclide to obtain a radiolabeled polypeptide boron carrier.

[0069] It is understood that the radionuclide labeling of the chelating agent in the polypeptide 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 S3 comprises: dissolving the polypeptide carrier 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.

[0070] In some embodiments, the radionuclide salt solution is selected from hydrochloride, sulfate, and nitrate solutions of radionuclides, preferably a hydrochloride solution of radionuclides.

[0071] 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.

[0072] The present invention also provides a use of the polypeptide carrier of the present invention in preparing a drug for treating tumors or cancer. After being absorbed in vivo, the polypeptide boron carrier of the present invention targets the tumor site, is specifically taken up by the tumor tissue, and then neutron irradiates, and after a neutron capture reaction occurs, high-LET alpha particles are released, which accurately kill the tumor tissue in cell size.

[0073] 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 boron carrier can be targeted to the tumor site after being absorbed in the body.

[0074] 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.

[0075] Example

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Unless otherwise specified, the reagents used in the following examples are all commercially available conventional reagents.

[0080] Example 1

[0081] Synthesis of Carborane Acetic Acid

[0082] Carborane acetic acid was prepared according to the following reaction pathway:

[0083]

[0084] Specifically, 757 mg of o-carborane (5.18 mmol, 1 eq) was dissolved in 20 mL of tetrahydrofuran, 3.28 mL of 1.6 M butyl lithium solution (5.25 mmol, 1 eq) was added at -78 °C, and the mixture was stirred at 0 °C under argon for 1 h. 784 μL of 6.4 M benzyl bromoacetate (5.02 mmol, 1 eq) was added, and after stirring at room temperature for 3 h, water was added to quench the reaction. The mixture was extracted twice with ethyl acetate, washed with brine, dried with sodium sulfate, filtered, and concentrated. Purification by silica gel column (ethyl acetate: hexane 1:9) gave 691 mg of colorless oily product compound I with a yield of 47%. The structural confirmation spectral data of compound I are shown below, and the nuclear magnetic spectrum is shown below Figure 1 :

[0085] 1 HNMR (400MHz, CDCl3): δ7.40-7.33(m,5H),5.14(s,2H),4.35(s,1H),3.27(s,2H),3.00-1.60(m,10H); 13 CNMR (125MHz, CDCl3): δ167.3,134.6,129.1,128.1,128.8,67.9,58.7,41.7; 11 BNMR (160MHz, CDCl3): δ-2.15,-5.0,-9.2,-10.5,-12.0,-12.8.

[0086] 207 mg of compound I (0.7 mmol, 2 eq) was dissolved in 10 mL of methanol, 37 mg of Pd / C (0.35 mmol, 1 eq) was added under nitrogen, and hydrogen was introduced to react for 15 h. After the reaction was completed, the crude product was concentrated to obtain a crude product. The crude product was dissolved in aqueous solution and washed three times with hexane. The aqueous layer was acidified with hydrochloric acid, extracted with ethyl acetate, dried with sodium sulfate, filtered, and concentrated. 123 mg of product compound II was obtained with a yield of 86%. The structure confirmation spectral data of compound II are shown below, and the NMR spectrum is shown below Figure 2 :

[0087] 1HNMR (400MHz, CDCl3): δ8.08(s,0.94),7.00-6.98(m,2H),6.77-6.75(m,2H),3.74-3.73(m,2H),1.20(s,9H). 13 CNMR (125MHz, CDCl3): δ167.3,134.6,129.1,128.1; 11 BNMR (160MHz, CD3CN): δ-2.9,-5.5,-9.6,-10.6,-11.8,-12.9.

[0088] Synthesis of Carborane Peptide P-RMB

[0089] The carborane peptide P-RMB-1 was prepared according to the following reaction pathway:

[0090]

[0091] Specifically, dichlororesin was swollen with dimethylacetamide (DCM) for 30 minutes, washed three times with N,N-dimethylformamide (DMF), and then an excess of aspartic acid (3eq) protected by fluorenemethoxycarbonyl (Fmoc) was added after the solution was filtered, and N,N-diisopropylethylamine (DIEA) (10eq) was added, and methanol was capped for 30 minutes. After filtering the solvent, a small amount of resin was washed with ethanol, and one drop of ninhydrin, potassium cyanide (KCN) and phenol was added, and heated at 105°C for 5 minutes. A positive reaction occurred, and the color changed from colorless to dark blue. After washing with DMF three times, Fmoc-protected arginine, lysine, and phenylalanine were linked in sequence. Add tetrakistriphenylphosphine palladium (Pd(PPh3)4) catalyst (0.1eq) and dissolve in DMF to react for 4h. After washing with DMF and filtering, 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 the 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, polyethylene glycol-4 (PEG4) and DOTA are connected in sequence, where A in the polypeptide sequence represents alanine, L represents leucine, and K represents lysine. The last lysine was protected by dimethylbenzoyl (Dde), and the remaining amino acids were protected by Fmoc. After DMF washing and filtration, 4% hydrazine hydrate DMF solution (15 ml / g) was added for 30 min to remove the Dde protecting group.

[0092] Add the prepared carborane acetic acid (3 eq) and dissolve it in DMF together with HBTU (3 eq) and DIEA (10 eq), and shake at room temperature for 45 min. Prepare the cutting solution: trifluoroacetic acid (TFA) 95%, ethylenediaminetetraacetic acid (EDT) 2%, triisopropylsilane (TIS) 2%, H2O 1%, and the cutting time is 120 min. After cutting, blow dry as much as possible with nitrogen, ether precipitates, centrifuge to remove the supernatant, wash the precipitate with ether six times, evaporate at room temperature, and obtain the crude carborane polypeptide P-RMB.

[0093] purification

[0094] Take a small amount of the crude carborane peptide P-RMB and dissolve it in H2O / acetonitrile (ACN). Take a small amount of sample and analyze it on a high performance liquid chromatography (HPLC) analyzer to determine the peak time corresponding to the target peak. Use a C18 reverse phase chromatography preparation system (Wavelength: 220nm FlowRate: 15ml / min; Inj.Vol: 20mL Column Temp: 25℃ Buffer A: 0.1% TFA in water Buffer

[0095] B: 0.1% TFA in Acetonitrile) to collect the target peak solution. A small amount of the target peak solution was taken in a 1.5ml centrifuge tube for mass spectrometry confirmation and purity detection. After detection, the purity was 92.3%. The mass spectrum of the carborane peptide P-RMB is shown in Figure 3 , wherein the structure confirmation spectrum data are as follows: M+3H: 1094.5, M+4H: 821.33, M+5H: 657.20, M+6H: 548.01. The HPLC of the carborane polypeptide P-RMB is shown in Figure 4 , among which, the elution time of P-RMB-1 is 11.947min.

[0096] In order to facilitate subsequent SPECT and PET imaging tests and accelerate the metabolism of drugs in cells, the polypeptide boron carrier prepared in this embodiment includes a hydrophilic group -NH-PEG-4-CO- and a -DOTA chelating group connected to the hydrophilic group. When the polypeptide boron carrier does not need to increase hydrophilicity and radioactive labeling and is only used for BNCT neutron capture therapy, the polypeptide boron carrier may not be connected to the hydrophilic group and the chelating group.

[0097] Example 2

[0098] The carborane peptide P-RMB-2 was prepared according to the following reaction pathway:

[0099] The specific preparation method is similar to that of Example 1, except that the amount of the carborane acetic acid prepared above is adjusted.

[0100]

[0101] The prepared carborane peptide P-RMB-2 was subjected to mass spectrometry and HPLC tests. The mass spectrum of the carborane peptide P-RMB is shown in Figure 5 The HPLC test results are shown in Figure 6 The elution time of the carborane peptide P-RMB-2 was 6.658 min.

[0102] Example 3

[0103] The carborane peptide P-RMB-4 was prepared according to the following reaction pathway:

[0104] The specific preparation method is similar to that of Example 1, except that the amount of the carborane acetic acid prepared above is adjusted.

[0105]

[0106]

[0107] The prepared carborane peptide P-RMB-4 was subjected to mass spectrometry and HPLC tests. The mass spectrum of the carborane peptide P-RMB is shown in Figure 7 The HPLC test results are shown in Figure 8 , the elution time of the carborane peptide P-RMB-4 was 10.993 min.

[0108] Example 4

[0109] Carborane Peptide P-RMB 68 Ga labeling preparation

[0110]

[0111] Take a 1.5 mL EP tube, add 100 μL of P-RMB (10 mg / mL) prepared in Example 1, dissolve it in phosphate buffer solution (PBS), then add 150 μL of CH3COONH4 buffer solution (0.25 M) at pH = 8.0, and adjust the peptide according to: 68 GaCl3 (v = 1:4) added 68 The GaCl3 solution was vortexed and placed in a constant temperature incubator at 37°C for 1 hour.

[0112] The labeling rate was determined by radio-iTLC, wherein the developing system used was 1% ammonium acetate: methanol = 50:50 system. 68Ga-P-RMB was spotted on instant thin layer scanning (iTLC-SG) chromatographic paper (1cm×10cm), developed upward, the iTLC-SG chromatographic paper was taken out and dried, and scanned with a radioactive thin layer scanner, and the labeling rate was measured to be 96.8%. After separation and purification with a Sep-pak C18 column, the radiochemical purity was measured to be 99.1%.

[0113] Example 5: Carborane polypeptide P-RMB 177 Lu labeling preparation

[0114]

[0115] Take a 1.5 mL EP tube and add 100 μL of P-RMB (10 mg / mL) prepared in Example 1, dissolve it in PBS, add 145 μL of CH3COONH4 buffer solution (0.25 M) at pH = 5.0, and finally add 2 μL 177 LuCl3 solution (1.80 mCi) was vortexed and mixed. The solution was placed in a constant temperature incubator and reacted at 40°C.

[0116] The labeling rate was determined by radio-thin layer chromatography (radio-iTLC), using 10 mM (pH = 5.5) diethylenetriaminepentaacetic acid (DTPA) as the developing agent. The sample was spotted on iTLC-SG chromatographic paper (1 cm × 10 cm), developed in a developing system of 10 mM DTPA solution (pH = 5.5), the iTLC-SG chromatographic paper was taken out and dried, and scanned with a radio-thin layer scanner, and the labeling rate was measured to be 95.2%.

[0117] Sep-pak C18 column separation and purification: Add the labeled sample solution to the pretreated chromatographic column and add pure water to elute to remove free 177 Lu, and drain the liquid in the chromatographic column. Then add 80% ethanol solution for elution, and collect the eluent, which is 177 Lu-P-RMB, the radiochemical purity of the labeled substance was determined by iTLC method to be 99.2%.

[0118] Example 6: Carborane polypeptide P-RMB 89 Zr labeling preparation

[0119]

[0120] Take a 1.5 mL EP tube and add deferoxamine chelator-isothiocyanate (DFO-NCS) (1.5 mg), add 100 μL of P-RMB (10 mg / mL) solution prepared in Example 1, adjust the pH to about 9 with 0.1 mol / L sodium bicarbonate solution, leave at room temperature for 1 hour, and purify with C18 column to obtain DFO-P-RMB conjugate. Take 20 μL 89 The pH of Zr oxalic acid solution (1.44 mCi) was adjusted to 7-7.5, and DFO-P-RMB solution (P-RMB was 1 mg) was added thereto, and the reaction was shaken at 40° C. for 1 hour. After the reaction was completed, it was purified by ultrafiltration centrifugation.

[0121] The labeling rate was determined by radio-thin layer chromatography (radio-iTLC), and ethanol was used as the mobile phase in the development system. The sample was spotted on iTLC-SG chromatographic paper (1 cm × 10 cm), and developed in a 10 mM DTPA solution (pH = 5.5) development system. The iTLC-SG chromatographic paper was taken out and dried, and scanned with a radio-thin layer scanner, and the labeling rate was measured to be 96.6%.

[0122] After the reaction is completed, the product is purified by ultrafiltration centrifugation. Take a 3KDA (3000 Dalton molecular weight) ultrafiltration centrifuge tube and add unpurified 89 The Zr-DFO-P-RMB solution was centrifuged at 8000 g for 5 min to obtain the target retentate, and the radiochemical purity of the sample was determined by a radioactive thin-layer scanner to be 98.9%.

[0123] Example 7

[0124] Distribution of P-RMB in tumor models

[0125] In U87MG tumor-bearing mice (purchased from Spefoc, quality certificate number

[0126] No.11032421101174467) was injected with P-RMB (150 mg / kg) through the tail vein 2h, 4h, 8h, 12h and 24h, and the blood, heart, liver, spleen, lung, kidney, muscle and tumor tissue homogenate were collected and the volume was fixed to 25mL. After nitration digestion, the boron concentration of each tissue solution was detected by ICP-MS. The distribution of P-RMB in various tissues of tumor-bearing mice was obtained. The results are shown in Fig. 9 .

[0127] like Fig. 9As shown, the boron content of the tumor gradually increased with time. At 24h, the highest boron concentration in the tumor tissue solution was 20.54ppm, which was higher than the boron content in other tissues, and the T / N was 5.24. This proves that P-RMB has good targeting in the U87MG mouse tumor model. At the same time, we found that the uptake of P-RMB in the liver was always high, proving that P-RMB molecules are large and are mainly metabolized by the liver in mice.

[0128] After 2h, 4h, 8h, 12h, 24h and 48h of tail vein injection of P-RMB (150mg / kg) into 4T1 tumor-bearing mice (purchased from Sibeifu, quality certificate No. 11032421100110383), blood, heart, liver, spleen, lung, kidney, muscle and tumor tissue homogenate were collected, and the boron concentration of each tissue solution was detected by ICP-MS after nitration digestion to 25mL. The distribution of P-RMB in various tissues of tumor-bearing mice was obtained. The results are shown in Fig.10 .

[0129] like Fig.10 As shown, the boron content of the tumor gradually increased with time. At 24 hours, the highest boron concentration in the tumor tissue solution was 19.45 ppm, which was higher than the boron content in other tissues, and the T / N was 5.15. This proves that P-RMB has good targeting in the 4T1 mouse tumor model. At the same time, we found that P-RMB has always had a high uptake in the liver, proving that P-RMB molecules are large and are mainly metabolized by the liver in mice.

[0130] Embodiment 8: 177 SPECT imaging of Lu-P-RMB in model animals

[0131] Three female 4T1 tumor model mice weighing about 20 g were purchased from Spike (quality certificate number: 110324241101363461). Each tumor-bearing mouse was injected with the prepared in Example 5 via the tail vein. 177 Lu-P-RMB, injection dose 800μCi. Static micro-SPECT imaging was performed at 2h, 4h, 12h, 24h and 48h after injection, with an acquisition time of 40min. 177 Targeting and biodistribution of Lu-P-RMB in 4T1 tumor model.

[0132] 4T1 tumor-bearing mice were injected with 800 μCi of 177 After Lu-P-RMB, the images were collected by SPECT imaging at 24 hours. The images are shown in Fig.11 After attenuation correction, the 4T1 tumor-bearing mouse static imaging images were reconstructed by computer. 17724 hours after Lu-P-RMB, the tumor enrichment was the highest (the green circle in the figure is the tumor location), and there was a good contrast compared with the contralateral side of the tumor.

[0133] Embodiment 9: 89 PET imaging of Zr-P-RMB in model animals

[0134] Three 4T1 tumor model mice were taken, and each tumor-bearing mouse was injected with the 89 Zr-P-RMB, injection dose 100μCi. Static micro-PET imaging was performed at 2h, 4h, 12h, 24h and 48h after injection, with an acquisition time of 10min. 89 Targeting and biodistribution of Zr-P-RMB in 4T1 tumor model.

[0135] 4T1 tumor-bearing mice were injected with 100 μCi of 89 After Zr-P-RMB, PET images were collected at 2, 4, 12, 24 and 48 hours, with peak energy of 350-650 Kev. Static images of 4T1 tumor-bearing mice were obtained by computer reconstruction after attenuation correction. 89 24h after Zr-P-RMB, the tumor enrichment was the highest, the imaging effect was the best, and there was good contrast compared with the contralateral side of the tumor. The static micro-PET images at 24h and 48h are shown in Fig.12 Among them, the bright spot on the left is the tumor site, the right side is the drug that has been labeled for a long time and has been metabolized, the blue represents the residual labeled drug, and there is no obvious bright spot.

[0136] Example 10

[0137] P-RMB targeting experiment

[0138] Glioma (U87MG cells purchased from Prosai) and 4T1 tumor cells (purchased from Prosai) were selected as cell models. During the culture, DMEM medium containing 10% FBS was prepared, and 1% penicillin and streptomycin were added at a concentration of 1*10 4 Pieces / cm 2 The cells were cultured at a density of 100 μg / mL in a 37°C cell culture incubator.

[0139] The experimental group was incubated with 300 μg / mL P-RMB-1 prepared in Example 1 and cells at concentrations of 0, 50, 100 and 500 μM RGD targeting inhibitors, respectively, for 24 h. The culture medium was discarded and the cells were washed 3 times with PBS. Subsequently, 1 mL of 2 mg / mL pronase solution was added to each well and incubated at 4 ° C for 4 h to remove the compounds bound to the cell surface. The pronase solution was discarded, the cells were washed 3 times with PBS, and the cells were lysed with RIPA lysis buffer (the formula includes 50 mM Tris-HCl (trishydroxymethylamine hydrochloric acid solution) (pH 7.4), 150 mM NaCl, 1 mM EDTA-2Na (ethylenediaminetetraacetic acid disodium salt), 1% Triton X-100 (Triton X100), 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate), and the cell lysate was collected, and the supernatant was collected by centrifugation at 10000 rpm. The supernatant was detected by ICP-MS (Agilent Technologies, Agilent 7800). 11 The results of P-RMB targeting tumor cells are shown in Fig.13 As shown in the figure, as the concentration of the targeted inhibitor increases, the boron content in the cells gradually decreases, proving that P-RMB has good targeting properties for glioma cells and 4T1 tumor cells.

[0140] Embodiment 11

[0141] Neutron irradiation therapy experiment

[0142] Cell cloning experiments

[0143] 4T1 cells (1×10 6 ) were inoculated in a 100 mm culture dish and cultured at 37°C for 24 hours. P-RMB (200 μg / mL) prepared in Example 1 was added and neutron irradiated in a cyclotron for 40 minutes, which was recorded as the P-RMB+N group. In addition, a group of 4T1 cells (1×10 6 ) were not added with P-RMB solution and were not irradiated with neutrons. They were cultured in a similar way as above and served as blank control group. Another group of 4T1 cells (1×10 6 The cells of the three groups were digested with trypsin and inoculated into 90 mm culture dishes at 2,000 cells per dish and cultured at 37°C for 10 days. When cell clones were observed, the culture medium was discarded, the cells were washed with PBS, fixed with anhydrous methanol, and stained with 0.2% crystal violet. The results are shown in Fig.14 .

[0144] The long-term cytotoxicity of BNCT treatment to cells was evaluated by cell cloning experiments. Cell cloning experiments are often used in cancer radiotherapy research to determine the effect of radiation on tumor cell proliferation. Fig.14 As shown in the figure, the number of cell clones in the cells treated with P-RMB+neutrons was significantly reduced compared with the control group, indicating that BNCT effectively inhibited the proliferation of 4T1 tumor cells. The number of cell clones in the P-RMB-treated group was also reduced compared with the control group, which was mainly due to the fact that P-RMB can induce cell apoptosis, causing some cancer cells to lose their cell proliferation ability. Through the quantification of cell clones, the cell survival rate after P-RMB incubation was 28.5%, while the survival rate of cells treated with P-RMB+neutrons dropped sharply to 0.24%, indicating that P-RMB has an excellent killing effect on tumor cells as a BNCT boron carrier.

[0145] Animal treatment experiments

[0146] When the 4T1 tumor volume grows to 600mm 3 , and started the in vivo treatment experiment. 4T1 tumor-bearing mice were divided into two groups (n=7), namely (1) neutron group (no injection of P-RMB-1) and (2) P-RMB-1 intravenous injection group (150 mg / kg). 24 hours after P-RMB-1 intravenous injection, neutron irradiation was performed for 40 minutes under a cyclotron. Tumor size and body weight were recorded every two days. The results are shown in Fig.15 . Fig.15 Figure 2 is the tumor growth curve of 4T1 tumor-bearing mice after tumor treatment. Fig.15 As shown, tumor growth was significantly inhibited in mice irradiated with neutrons after intravenous injection of P-RMB.

[0147] 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 boron carrier, characterized in that: The polypeptide boron carrier includes a polypeptide and at least one boron-containing 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.

2. The polypeptide boron carrier according to claim 1, characterized in that: The at least one boron-containing group is selected from C2B 10 H 11 -R1-CO-, CB5H8-R1-CO-, C2B4H7-R1-CO-, C3B3H6-R1-CO-, C4B2H5-R1-CO-, C2B3H6-R1-CO-, C2B3H4-R1-CO-, C2B4H5-R1-CO-, C2B5H6-R1-CO- and CB5H6-R1-CO-, wherein R1 is (CH2) n , n=0-2.

3. The polypeptide boron carrier according to claim 1, characterized in that: The polypeptide boron carrier comprises 1 to 5, preferably 1 to 3 boron-containing groups.

4. The polypeptide boron carrier according to claim 1, wherein: 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, wherein the hydrophilic group is selected from NH2-PEG-4-CO-, NH2-PEG-2000-CO-, NH2-PEG-5000-CO-, One or more of NH2-miniPEG-CO-.

5. The polypeptide boron carrier according to claim 4, wherein: The polypeptide further comprises a chelating group labeled with a radionuclide, wherein the chelating group is connected to the hydrophilic group or to the main chain or side chain amino group of the at least one MAP peptide.

6. The polypeptide boron carrier according to claim 5, 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.

7. The polypeptide boron carrier according to claim 6, 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.

8. Use of the polypeptide boron carrier according to any one of claims 1 to 7 in the preparation of a medicament 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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