CN116712571A - Application of novel cyclic peptide in preparation of tumor diagnosis and/or treatment reagent - Google Patents
Application of novel cyclic peptide in preparation of tumor diagnosis and/or treatment reagent Download PDFInfo
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- CN116712571A CN116712571A CN202310726679.1A CN202310726679A CN116712571A CN 116712571 A CN116712571 A CN 116712571A CN 202310726679 A CN202310726679 A CN 202310726679A CN 116712571 A CN116712571 A CN 116712571A
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- 229960003105 metformin Drugs 0.000 description 1
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- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention relates to the technical fields of fluorescent contrast agents, radiopharmaceuticals and nuclear medicine, and particularly discloses application of a novel cyclic peptide in preparation of tumor diagnosis and/or treatment reagents, wherein the novel cyclic peptide comprises the following components: beta-Ala-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp. The molecular probe constructed by the thymic pentapeptide derivative can be specifically targeted to a tumor part, has good uptake and retention capacity at the tumor part, has high target/non-target ratio, is suitable for preparing an image navigation reagent in tumor operation and preparing a radioactive drug, and is used for tumor nuclear medicine diagnosis and accurate radiotherapy.
Description
Technical Field
The invention is a divisional application with the application number of 2021115423051, and the application date is 2021, 12, 16.
The invention relates to the technical fields of fluorescent contrast agents, radiopharmaceuticals and nuclear medicine, in particular to application of a novel cyclic peptide in preparation of tumor diagnosis and/or treatment reagents.
Background
Malignant tumors have become the "first killer" threatening human health and life, and needless to say, diagnosis of malignant tumors and effective treatment against tumors are imminent. Molecular imaging probes targeted to tumors are well known as an advantageous tool for tumor diagnosis, staging and intra-operative navigation. Wherein, the ligand of the tumor specific targeting is the key point of the tumor targeting molecular probe. The current method for designing and screening the target ligand mainly comprises the steps of computer-aided drug design, modification and reconstruction of lead compounds, discovery from metabolites, discovery from drug synthesis intermediates, combinatorial chemistry and high-throughput screening, separation and extraction from natural compounds, phage display library screening and 'old drug new use'. 1988 Nobel's physiological or medical prize, james Blacks, suggested that the best path for new drug discovery starts with the old drug. The "old drug" refers to a drug which has definite pharmacokinetic information and toxicology information and is marketed or in clinic, and the most obvious characteristic is high safety. The nonselective beta receptor antagonists propranolol and cimetidine, the first histamine H2 receptor antagonist, developed by Zhan S Blacks, are typical examples of "new use of the old". Propranolol is a classical drug for treating coronary heart disease and hypertension, and is now used for the treatment of osteoporosis and melanoma; cimetidine is a revolutionary drug for treating peptic gastric ulcer, and proved to be suitable for treating chronic obstructive pulmonary disease, HIV virus infection and the like; nature indicates that metformin in combination with another "new use of old drug" heme can be used to treat triple negative breast cancer; arsenic trioxide, commonly known as "arsenic trioxide", is a highly toxic agent, and has been recently discovered to be useful in the treatment of acute promyelocytic leukemia; therefore, the strategy of 'old medicine new use' has important guiding significance in medicine development, so that the application of the strategy of 'old medicine new use' for screening the targeted medicine of the tumor is a rapid and effective method.
Thymopentin is an important bioactive substance in mammals, and plays an important role in maintaining the balance of the immune system, resisting tumors, resisting microbial infection and the like. The thymic pentapeptide is the fragment of amino acid residues at the 32 th-36 th positions in thymic hormone II, retains the biological activity of thymic hormone, and has the same physiological function and medicinal effect as thymic extract thymosin and thymic peptide. Based on the "old drug new use" strategy, no prior art has been reported as to what effect the thymopentin has.
Disclosure of Invention
In view of the above, the present invention aims to provide the application of the thymopentin and the derivatives thereof in preparing tumor diagnosis and/or treatment reagents, and provides the novel application of the thymopentin derivatives.
The invention provides an application of a novel cyclic peptide in preparing a tumor diagnosis and/or treatment reagent, wherein the novel cyclic peptide is beta-Ala-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp, and the side chain carboxyl of N-terminal amino and C-terminal Asp form an amide ring;
wherein: d represents an unnatural D-type amino acid, and L represents an L-type natural amino acid.
Further, the tumor comprises one or more of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer and breast cancer
Further, the agents include fluorescent imaging agents including tumor boundary pinpoint and/or intra-operative image navigation optical imaging agents and/or radioactive agents.
Further, the reagent has the following general formula: M-L-G;
the M represents a photo-label, a metal chelator and a metal radionuclide complex, a nonmetallic radionuclide 18 F and F 11 C;
l is a linking group;
g is a novel cyclic peptide;
the optical label comprises one or more of an organic chromophore, an organic fluorophore, a light absorbing compound, a light reflecting compound, a light scattering compound, and a bioluminescent molecule;
the metal chelator is selected from the group consisting of modification of dihydrazineimide, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, 7- [ (4-hydroxypropyl) methylene ] -1,4, 7-triazacyclononane-1, 4-diacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, mercaptoacetyltriglycine, diethylenetriamine pentaacetic acid, or combinations thereof.
Further, the optical label includes a near infrared one-region fluorescent dye and/or a near infrared two-region fluorescent dye, the near infrared one-region fluorescent dye including one or more of MPA, IRDye800, cy7.5, ICG, and cy 5.5.
Further, the linking group comprises 6-aminocaproic acid, NH 2 -PEG 3 -COOH、NH 2 -PEG 4 -COOH、NH 2 -PEG 6 -COOH and NH 2 -one or more of GGGGGG-COOH.
Further, the agents include novel cyclic peptides.
Further, the reagent comprises 99m Tc-HYNIC-PEG 4 -TP-8MPA-TP-8 and MPA-PEG 4 -TP-8。
Compared with the prior art, the invention has the beneficial effects that:
1. the invention provides an application of a novel cyclic peptide in preparation of tumor diagnosis and/or treatment reagents, a molecular probe constructed by the novel cyclic peptide can specifically target to a tumor part, has good uptake and detention capacity at the tumor part, has high target/non-target ratio, is suitable for preparing image navigation reagents in tumor operation and preparing radiopharmaceuticals, and is used for tumor nuclear medicine diagnosis and accurate radiotherapy.
2. The novel cyclopeptide in-vivo molecular probe has unique advantages in the aspect of safety, and can obviously reduce the research and development cost and risk of the medicine;
by means of the property that the thymus pentapeptide can specifically target the tumor, a fluorescent probe is constructed through a novel cyclic peptide coupled fluorescent dye, so that a doctor can be assisted in accurately positioning the tumor boundary in an operation, and the purpose of accurately cutting the tumor is achieved. In addition, the thymus pentapeptide and the derivative thereof can be coupled with radionuclides with diagnosis/treatment functions to construct corresponding radiopharmaceuticals, so that the purposes of tumor diagnosis and accurate radiotherapy can be achieved.
3. The molecular probe constructed based on the novel cyclic peptide has excellent targeting effect on various tumors, including liver cancer, lung cancer, colorectal cancer, breast cancer, pancreatic cancer, gastric cancer and the like through in vivo optics and radionuclide imaging results. The characteristic that the probe can specifically target a tumor part can possibly realize nuclear medicine diagnosis, treatment and optical operation navigation of malignant tumors.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is MPA-PEG 3 -TP-1-NH 2 Is a mass spectrum of (3).
FIG. 2 shows the prepared monomeric fluorescent compound MPA-PEG 3 -TP-1-NH 2 2h fluorescence imaging in tumor-bearing mice; wherein A is lung cancerFluorescence imaging in A549 tumor-bearing mice; b is fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice; c is fluorescence imaging in a pancreatic cancer CFPAC-1 tumor-bearing mouse; d is fluorescence imaging in lung cancer H1299 tumor-bearing mice; e is fluorescence imaging in colorectal carcinoma HCT116 tumor-bearing mice; f is fluorescence imaging in a liver cancer HepG2 tumor-bearing mouse; g is fluorescence imaging in colorectal carcinoma HT29 tumor-bearing mice; h is fluorescence imaging in a breast cancer MAD-MB-231 tumor-bearing mouse; i is fluorescence imaging in gastric cancer MGC-803 tumor-bearing mice; j is fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice; k is fluorescence imaging in breast cancer MCF-7 tumor-bearing mice; l is fluorescence imaging in pancreatic cancer MiaPaPc-2 tumor-bearing mice;
FIG. 3 shows the prepared monomeric fluorescent compound MPA-PEG 4 -TP-2、MPA-PEG 4 -TP-3、MPA-PEG 4 -TP-4、MPA-PEG 4 -TP-5、MPA-PEG 4 -TP-6、MPA-PEG 4 -TP-8、MPA-PEG 4 -2 h fluorescence imaging of TP-9 in tumor bearing mice; wherein A is MPA-PEG 4 -fluorescence imaging of TP-2 in lung cancer a549 tumor-bearing mice; b is MPA-PEG 4 -fluorescence imaging of TP-3 in gastric cancer SGC-803 tumor-bearing mice; c is MPA-PEG 4 -fluorescent imaging of TP-4 in breast cancer MDA-MB-468 tumor bearing mice; d is MPA-PEG 4 -fluorescence imaging of TP-5 in breast cancer MCF-7 tumor-bearing mice; e is MPA-PEG 4 -fluorescence imaging of TP-6 in pancreatic cancer AsPC-1 tumor-bearing mice; f is MPA-PEG 4 -fluorescence imaging of TP-8 in lung cancer H1299 tumor-bearing mice; g is MPA-PEG 4 -fluorescence imaging of TP-9 in colon cancer HCT116 tumor-bearing mice;
FIG. 4 shows the prepared monomeric fluorescent compound MPA-PEG 4 -2 h fluorescence imaging of TP-7 in tumor bearing mice; wherein A is fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice; b is fluorescence imaging in colorectal carcinoma HCT116 tumor-bearing mice; c is fluorescence imaging in breast cancer MDA-MB-231 tumor-bearing mice; d is fluorescence imaging in pancreatic cancer MiaPaCa-2 tumor-bearing mice; e is fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice;
FIG. 5 shows the prepared monomeric fluorescent compound MPA-PEG 4 2h fluorescence imaging of TP-10 in tumor-bearing mice The method comprises the steps of carrying out a first treatment on the surface of the Wherein A is fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice; b is fluorescence imaging in colorectal carcinoma HCT116 tumor-bearing mice; c is fluorescence imaging in breast cancer MDA-MB-231 tumor-bearing mice; d is fluorescence imaging in pancreatic cancer MiaPaCa-2 tumor-bearing mice; e is fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice;
FIG. 6 shows the monomeric radioactive compound prepared 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging in tumor-bearing mice for 1 h; wherein A is SPECT-CT imaging in pancreatic cancer AsPC-1 tumor-bearing mice; b is SPECT-CT imaging in a gastric cancer SGC-7901 tumor-bearing mouse body; c is SPECT-CT imaging in pancreatic cancer MiaPaPc-2 tumor-bearing mice; d is SPECT-CT imaging in breast cancer MCF-7 tumor-bearing mice; e is SPECT-CT imaging in colorectal cancer HT29 tumor-bearing mice;
FIG. 7 shows the monomeric radioactive compound prepared 99m Tc-HYNIC-PEG 4 -SPECT-CT imaging of TP-7 in tumor-bearing mice for 1 h; wherein A is SPECT-CT imaging in breast cancer MCF-7 tumor-bearing mice; b is SPECT-CT imaging in pancreatic cancer AsPC-1 tumor-bearing mice; c is SPECT-CT imaging in pancreatic cancer MiaPaPc-2 tumor-bearing mice; d is SPECT-CT imaging in a colorectal cancer HT29 tumor-bearing mouse; e is SPECT-CT imaging in the stomach cancer MGC-803 tumor-bearing mice; e is SPECT-CT imaging in gastric cancer SGC-7901 tumor-bearing mice;
FIG. 8 shows the dimeric radioactive compounds prepared 99m Tc-HYNIC-2Aca-(TP-1-NH 2 ) 2 SPECT-CT imaging in pancreatic cancer AsPC-1 and colorectal cancer HT29 tumor-bearing mice, respectively.
Detailed Description
The following detailed description of specific embodiments of the invention is, but it should be understood that the invention is not limited to specific embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The experimental methods described in the examples of the present invention are conventional methods unless otherwise specified, and materials, reagents, etc. used in the examples described below are commercially available.
The invention provides application of thymic pentapeptides and derivatives thereof in preparing tumor diagnosis and/or treatment reagents, wherein the thymic pentapeptides are selected from one or more of the following polypeptides:
TP-1:L-Arg-L-Lys-L-Asp-L-Val-L-Tyr(Thymopentin);
TP-2:L-homo-Arg-L-Lys-L-Asp-L-Nva-L-Tyr;
TP-3:D-Arg-L-Lys-L-Asp-L-Val-L-Tyr;
TP-4:D-Arg-D-Lys-L-Asp-L-Val-L-Tyr;
TP-5:D-Arg-L-Lys-L-Asp-L-Val-D-Tyr;
TP-6:D-Arg-L-Lys-L-Asp-L-Nva-D-Tyr;
TP-7: L-Cys-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Cys, wherein the Cys-Cys disulfide bond forms a ring;
TP-8: beta-Ala-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp in which the side chain carboxyl groups of the N-terminal amino group and the C-terminal Asp form an amide ring (novel cyclic peptide);
TP-9: D-Lys-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Glu in which the N-terminal backbone amino group and the side chain carboxyl group of the C-terminal Glu form an amide ring;
TP-10: D-Lys-L-Gly-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp, wherein the N-terminal backbone amino group and the C-terminal Asp backbone carboxyl group form an amide ring;
wherein: d represents an unnatural D-type amino acid, L represents an L-type natural amino acid; homoArg is homoarginine; nva is norvaline.
In the present invention, the tumor preferably includes one or more of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, stomach cancer and breast cancer.
Preferably, the invention couples the thymic pentapeptide and the derivative thereof with the imaging group to obtain the reagent. In the present invention, the agent preferably comprises a fluorescent imaging agent and/or a radioactive agent, and the fluorescent imaging agent preferably comprises an optical imaging agent for tumor border accurate localization and/or intra-operative image navigation.
In the present invention, the reagent has the following general formula: M-L-G; the M represents a light label, a metal chelator and a metal radionuclide complexPhysical, nonmetallic radionuclides 18 F and F 11 C; l is a linking group; g is thymic pentapeptide and its derivative. In the present invention, the optical label preferably includes one or more of an organic chromophore, an organic fluorophore, a light absorbing compound, a light reflecting compound, a light scattering compound, and a bioluminescent molecule. In the present invention, the metal chelating agent is preferably selected from the group consisting of dihydrazinonenamide, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, 7- [ (4-hydroxypropyl) methylene ]-1,4, 7-triazanonane-1, 4-diacetic acid, 1,4,7, 10-tetraazacyclotetraazacyclododecane-1, 4,7, 10-tetraacetic acid, mercaptoacetyltriglycine, diethylenetriamine pentaacetic acid or a combination thereof. In the present invention, the optical label preferably comprises a near infrared one-region fluorescent dye and/or a near infrared two-region fluorescent dye, the near infrared one-region fluorescent dye comprising one or more of MPA, IRDye800, cy7.5, ICG, and cy 5.5. In the present invention, the linking group preferably comprises 6-aminocaproic acid, NH 2 -PEG 3 -COOH、NH 2 -PEG 4 -COOH、NH 2 -PEG 6 -COOH and NH 2 -one or more of GGGGGG-COOH.
In the invention, the thymus pentapeptide and the derivative thereof and the near infrared fluorescent probe based on the thymus pentapeptide are synthesized by Hangzhou solid-state biotechnology limited company through a solid phase method, and the thymus pentapeptide comprises the following components:
1) Synthesis of near infrared fluorescent dye MPA
Mixing glacial acetic acid, p-hydrazinobenzenesulfonic acid, methyl isopropyl ketone and sodium acetate for reaction, and purifying to obtain a product 2, 3-trimethyl [3H ] -indole-5-sulfonic acid; adding o-dichlorobenzene into the mixture of 2, 3-trimethyl [3H ] -indole-5-sulfonic acid and 1, 3-propane sulfonic acid lactone to prepare 2, 3-trimethyl-5-sulfonic acid-1- (3-sulfonic acid-propyl) - [3H ] -indole. And then reacting the product with N- [ (3- (anilinometer) -2-chloro-1-cyclopen-1-yl) methyl ] -aniline monohydrochloride to obtain green carbocyanine dye, and finally reacting the carbocyanine dye with mercaptopropionic acid and triethylamine to prepare the liquid phase separation and purification to obtain the water-soluble near infrared dye MPA.
2) Synthesis of MPA-L-TP-X (X=1-10)
A Ramage Amide AM resin resin with Loading of 0.45mmol/g was selected and Fmoc protecting groups were removed after swelling. According to the polypeptide sequence, coupling is carried out from the C end to the N end in sequence until Fmoc-L-carboxyl, and after small sample cutting, the molecular weight of the polypeptide is detected by mass spectrometry, wherein the side chains of Tyr, asp, lys, arg are respectively protected by tBu, otBu, boc, arg. The amino acid used is Fmoc-protected alpha amino; determination of the polypeptide Fmoc-L-TP-X-NH 2 After the mass spectrum is correct, fmoc protecting group is removed, near infrared dye MPA with the molar multiple of 1.2 times is added for solid phase reaction, and after ninhydrin detection is negative, the reaction is finished. Reaction of the cleavage solution (TFA: triisopropylsilane: water=95:2.5:2.5) with a linear peptide resin gives MPA-L-TP-X-NH with all side chain protecting groups removed 2 The method comprises the steps of carrying out a first treatment on the surface of the MPA-L-TP-X-NH 2 Dissolving in water, purifying by semi-preparative chromatography, separating to obtain qualified liquid, and collecting rotary-evaporated and freeze-dried product.
The radionuclide probe monomer form constructed based on the thymic pentapeptide and the derivative thereof is simpler to prepare than the dimer form, the dimer structure of the radionuclide probe monomer form contains the thymic pentapeptide and the derivative thereof for targeting tumors and a bifunctional chelating agent bishydrazinium amide (HYNIC) for radiolabeling, and a connector L for increasing the distance between the thymic pentapeptide or the analogue thereof and radionuclide ligand N-tris (hydroxymethyl) methylglycine (Tricine) and triphenylphosphine sodium trisulphonate (TPPTS) and regulating in vivo pharmacokinetics is selected from 6-aminocaproic acid and NH 2 -PEG 3 -COOH、NH 2 -PEG 4 -COOH、NH 2 -PEG 6 -COOH or NH 2 -any one or more of GGGGGG-COOH.
The purpose of coupling different radionuclides can be achieved by changing the bifunctional chelating agent. For example, the substitution of the dihydrazinonenamide for the bifunctional chelating agent 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, 7- [ (4-hydroxypropyl) methylene]-any one of 1,4, 7-triazacyclononane-1, 4-diacetic acid, 1,4,7, 10-tetraazacyclotetraazacyclododecane-1, 4,7, 10-tetraacetic acid, mercaptoacetyltriglycine or diethyltriaminopentaacetic acid, a radionuclide 99m Tc can be replaced by 68 Ga、 64 Cu、 67 Ga、 90 Y、 111 In、 89 Zr or 177 Lu. Or to radionuclides 124 I、 125 I、 131 I is directly marked on Tyr in the structure of free peptide TP-X to realize the function of disease diagnosis/treatment.
In the present invention, the method for preparing the monomeric radionuclide probe comprises the following steps:
1) Synthesis of bifunctional chelating agent HYNIC-L-NHS
Adding 6-chloronicotinic acid and 80% hydrazine hydrate into ethanol, heating and refluxing for reaction, decompressing and steaming the solvent after the reaction is completed, adding the obtained sticky substance into distilled water, adjusting the PH value to be 5.5, separating out solid, filtering and drying to obtain yellow solid, and determining the product to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. Adding the obtained 6-dihydrazide nicotinic acid and para-aminobenzaldehyde into dimethyl sulfoxide (DMSO), heating for reaction for 5-6 hours, adding into water for precipitation after the reaction is completed, filtering to obtain a solid, drying the solid, adding the solid, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and N-hydroxysuccinimide (NHS) into the DMSO for reaction at room temperature, adding into water for precipitation after the reaction is completed, purifying the solid through a silica gel column, determining the solid as an intermediate HYNIC-NHS through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum, then reacting the intermediate with a connector L under alkaline condition, activating with an activator EDCI and NHS, and purifying to obtain the HYNIC-L-NHS solid for later use.
2) Synthesis of HYNIC-L-TP-X (X=1-10)
Purified intermediate HYNIC-L-NHS is dissolved in DMSO, 1-1.5 molar amount of TP-X is added, then 2-3 molar amount of DIPEA is added, reaction is carried out for 1-2 hours at room temperature, and separation and purification are carried out by preparing liquid phase after the reaction is completed and confirmed by mass spectrum.
3) 99m Synthesis of Tc-HYNIC-L-TP-X (X=1-10)
Preparing TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution with concentration of 100.0-120mg/mL, tricine (trimethylglycine) with concentration of 130.0-150mg/mL, succinic acid-sodium succinate buffer solution with concentration of 102.4-110mg/mL (77.0-88.8 mg succinic acid, 25.4-29.3mg sodium succinate), respectively taking 10.0uL TPPTS solution and 10.0uL TPPTS solutionTricine solution, 10.0uL succinic acid-sodium succinate buffer, and 10.0uL (1.0 mg/mL) of each of the HYNIC-L-TP-X were mixed in a penicillin bottle, and then 10mCi Na was added 99m TcO 4 Heating in a metal bath at 100 ℃ for 20-30 minutes, cooling to room temperature after the reaction is finished, and obtaining the product after HPLC analysis and identification.
In the present invention, the method for preparing the dimeric radionuclide probe preferably comprises:
1) Synthesis of bifunctional chelating agent HYNIC-L-NHS
The method is the same as the preparation step 1) of the monomeric radionuclide probe.
2) Synthesis of scaffold (2L-glutamic acid)
Dissolving a proper amount of Boc-glutamic acid in DMSO, adding 2-3 times of EDCI and NHS, heating at 60 ℃ for reaction for 0.5-1 hour, analyzing that the glutamic acid double-activated ester is generated by HPLC, adding 2-3 times of connecting agent L and 2-3 times of DIPEA into the solution, heating at 60 ℃ for reaction for 0.5-1 hour, analyzing that 2 molecules of connecting agent L are connected with glutamic acid by HPLC, adding equal volume of TFA, reacting at room temperature overnight for removing Boc protection, and finally separating the crude product by a preparation liquid phase, and freeze-drying for later use.
3) Synthesis of intermediate 2L-E-HYNIC-NHS
Dissolving the prepared stent 2L-glutamic acid in DMSO, then adding the same molar quantity of HYNIC-L-NHS, adding 2-3 times of DIPEA, reacting for 1-2 hours at room temperature, preparing liquid phase separation and purification after the reaction is finished, confirming the target compound by mass spectrum, activating the purified product with EDCI and NHS, and purifying to obtain 2L-E-HYNIC-NHS for later use.
4)(TP-X) 2 -synthesis of 2L-E-HYNIC (x=1-10)
Purified intermediate 2L-E-HYNIC-NHS is dissolved in DMSO, 1-1.5 molar amount of TP-X is added, then 2-3 molar amount of DIPEA is added, reaction is carried out for 1-2 hours at room temperature, and separation and purification are carried out by preparing liquid phase after the reaction is completed and confirmed by mass spectrum.
5) Radioactive probe 99m Tc-HYNIC-2L-E-(TP-X) 2 Synthesis of (X=1-10)
Respectively preparing the concentration of100.0-120 mg/mL of TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, tricine (trimethylglycine) with the concentration of 130.0-150mg/mL, succinic acid-sodium succinate buffer with the concentration of 102.4-110mg/mL (77.0-88.8 mg of succinic acid and 25.4-29.3mg of sodium succinate), 10.0uL of TPPTS solution, 10.0uL of Tricine solution, 10.0uL of succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) of HYNIC-2L-E- (TP-X) respectively are taken 2 Mix in a penicillin bottle and then add 10mCi Na 99m TcO 4 Heating in a metal bath at 100 ℃ for 20-30 minutes, cooling to room temperature after the reaction is finished, and obtaining the product through HPLC analysis and identification.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1: MPA-PEG 3 -TP-1-NH 2 Is a synthesis of (a).
A Ramage Amide AM resin resin with Loading of 0.45mmol/g was selected and Fmoc protecting groups were removed after swelling. According to the thymus pentapeptide sequence: arg-Lys-Asp-Val-Tyr, coupling from C-terminal to N-terminal in sequence until Fmoc-PEG 3 Propionic acid, wherein the side chains of Tyr, asp, lys, arg are protected with tBu, otBu, boc, arg, respectively, and the amino acids used are Fmoc protected alpha amino groups. And (3) detecting the molecular weight of the polypeptide by mass spectrometry after cutting a small sample, removing Fmoc protecting groups after determining that the Fmoc-PEG3-TP-X-NH2 mass spectrum of the polypeptide is correct, adding near infrared dye MPA with the molar multiple of 1.2 for solid phase reaction, and ending the reaction after detecting ninhydrin is negative. Reaction of the cleavage solution (TFA: triisopropylsilane: water=95:2.5:2.5) with a linear peptide resin gave MPA-PEG with all side chain protecting groups removed 3 -TP-1-NH 2 MPA-PEG 3 -TP-1-NH 2 Dissolving in water, purifying with semi-preparative chromatography, separating to obtain qualified liquid, collecting, spin-evaporating, lyophilizing, and determining as target compound MPA-PEG by ESI-MS mass spectrometry 3 -TP-1-NH 2 ,ESI-MS:[M-2H] 2- = 895.96 and [ M-3H] 3- = 597.26 (fig. 1).
Example 2: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in lung cancer a549 tumor-bearing mice.
Taking prepared fluorescent compound MPA-PEG 3 -TP-1-NH 2 And a physiological saline solution (100 nmol/mL) was prepared, 0.1mL (about 10 nmol) was injected into the tail vein of 3 lung cancer A549 tumor-bearing nude mice (body weight about 22 g) respectively, and optical signal collection was performed 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The distribution of the fluorescent drug in the model mice and the enrichment of the tumor area were observed. The results are shown in FIG. 2A, which shows that the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target the lung cancer (A549) site.
Example 3: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in pancreatic cancer AsPC-1 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 3 pancreatic cancer AsPC-1 tumor-bearing nude mice were injected respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 2B, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target pancreatic cancer (AsPC-1) sites.
Example 4: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in pancreatic cancer CFPAC-1 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 The fluorescent signals were collected by injecting into 3 pancreatic cancer CFPAC-1 tumor-bearing nude mice, respectively, and 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The results are shown in FIG. 2C, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target pancreatic cancer (CFPAC-1) sites.
Example 5: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in lung cancer H1299 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 Respectively injecting into 3 lung cancer H1299 tumor-bearing nude mice, and collecting fluorescent signals at 1H, 2H, 4H, 6H, 8H, 10H and 12H after administration. The results are shown in the graph2, the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target the lung cancer (H1299) part.
Example 6: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in colorectal carcinoma HCT116 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 3 colorectal cancer HCT116 tumor-bearing nude mice were injected respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h post-administration. The distribution of the fluorescent drug in the model mice and the enrichment of the tumor area were observed. The results are shown in FIG. 2, which shows the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target colorectal cancer (HCT 116) sites.
Example 7: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in hepatoma HepG2 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 Respectively injecting into 3 liver cancer HepG2 tumor-bearing nude mice, and collecting fluorescence signals at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The results are shown as F in FIG. 2, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target liver cancer (HepG 2) parts.
Example 8: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in colorectal carcinoma HT29 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 3 colorectal cancer HT29 tumor bearing nude mice were injected separately and fluorescent signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h and 12h post-dose. The results are shown in FIG. 2, G, and the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target colorectal cancer (HT 29) sites.
Example 9: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in breast cancer MAD-MB-231 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 The fluorescent signals are collected by injecting into 3 breast cancer MAD-MB-231 tumor-bearing nude mice respectively, and carrying out fluorescent signal collection at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The results are shown in FIG. 2, H, and the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target the breast cancer (MAD-MB-231) site.
Example 10: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in gastric cancer MGC-803 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 Respectively injecting the fluorescent signals into 3 gastric cancer MGC-803 tumor-bearing nude mice, and collecting fluorescent signals 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The results are shown in FIG. 2, I, and the fluorescent probe MPA-PEG 3 -TP-1-NH 2 Can specifically target the stomach cancer (MGC-803) part.
Example 11: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in gastric cancer SGC-7901 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 Respectively injecting the fluorescent signals into 3 gastric cancer SGC-7901 tumor-bearing nude mice, and collecting fluorescent signals at 1h, 2h, 4h, 6h, 8h, 10h and 12h after administration. The results are shown in J in FIG. 2, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target the gastric cancer (SGC-7901) site.
Example 12: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in breast cancer MCF-7 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 3 breast cancer MCF-7 tumor-bearing nude mice were injected separately and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h and 12h post-dose. The results are shown in K in FIG. 2, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target the breast cancer (MCF-7) site.
Example 13: the monomeric fluorescent compound MPA-PEG prepared in example 1 3 -TP-1-NH 2 Fluorescence imaging in pancreatic cancer MiaPaPc-2 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 -TP-1-NH 2 3 pancreatic cancer MiaPaPc-2 tumor-bearing nude mice were injected respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in L of FIG. 2, and the fluorescent probe MPA-PEG is seen 3 -TP-1-NH 2 Can specifically target pancreatic cancer (MiaPaPc-2) sites.
Example 14: the prepared monomeric fluorescent compound MPA-PEG 4 TP-2 (preparation method same as in example 1, TP-1-NH 2 Is replaced by TP-2) in lung cancer A549 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-2 was injected into 3 lung cancer A549 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3A, and the fluorescent probe MPA-PEG is seen 4 TP-2 can specifically target lung cancer (A549) sites.
Example 15: the prepared monomeric fluorescent compound MPA-PEG 4 TP-3 (preparation method same as in example 1, TP-1-NH 2 Is replaced by TP-3) in gastric cancer SGC-803 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-3 was injected into 3 gastric cancer SGC-803 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3B, and the fluorescent probe MPA-PEG is seen 4 TP-3 can specifically target gastric cancer (SGC-803) site.
Example 16: the prepared monomeric fluorescent compound MPA-PEG 4 TP-4 (preparation method same as in example 1, TP-1-NH 2 Can be replaced by TP-4) in the breast cancer MDA-MB-468 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-4 was injected into 3 breast cancer MDA-MB-468 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3C, and the fluorescent probe MPA-PEG is seen 4 TP-4 can specifically target the site of breast cancer (MDA-MB-468).
Example 17: preparationMonomeric fluorescent compound MPA-PEG of (C) 4 TP-5 (preparation method same as in example 1, TP-1-NH 2 Is replaced by TP-5) in the breast cancer MCF-7 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-5 was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3D, and the fluorescent probe MPA-PEG is seen 4 TP-5 can specifically target the breast cancer (MCF-7) site.
Example 18: the prepared monomeric fluorescent compound MPA-PEG 4 TP-6 (preparation method same as in example 1, TP-1-NH 2 Replacement with TP-6) in pancreatic cancer AsPC-1 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-6 was injected into 3 pancreatic cancer AsPC-1 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3, which shows the fluorescent probe MPA-PEG 4 TP-6 can specifically target pancreatic cancer (AsPC-1) sites.
Example 19: the prepared monomeric fluorescent compound MPA-PEG 4 TP-8 (preparation method same as in example 1, TP-1-NH 2 Is replaced by TP-8) in lung cancer H1299 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-8 was injected into 3 lung cancer H1299 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed at 1H, 2H, 4H, 6H, 8H, 10H, and 12H after administration. The results are shown as F in FIG. 3, and the fluorescent probe MPA-PEG is seen 4 TP-8 can specifically target the lung cancer (H1299) site.
Example 20: the prepared monomeric fluorescent compound MPA-PEG 4 TP-9 (preparation method same as in example 1, TP-1-NH 2 Replacement with TP-9) in colon cancer HCT116 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 4 TP-8 was injected into 3 colon cancer HCT116 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 3, G, and fluorescence probeNeedle MPA-PEG 4 TP-9 can specifically target colon cancer (HCT 116) sites.
Example 21: the prepared monomeric fluorescent compound MPA-PEG 3 TP-7 (preparation method same as in example 1, TP-1-NH 2 Replacement with TP-7) in pancreatic cancer AsPC-1 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-7 was injected into 3 pancreatic cancer AsPC-1 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 4A, and the fluorescent probe MPA-PEG is seen 3 TP-7 can specifically target pancreatic cancer (AsPC-1) sites.
Example 22: monomeric fluorescent compound MPA-PEG prepared in example 21 3 Fluorescence imaging of TP-7 in colorectal carcinoma HCT116 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-7 was injected into 3 colorectal cancer HCT116 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 4B, and the fluorescent probe MPA-PEG is seen 3 TP-7 specifically targets colorectal cancer (HCT 116) sites.
Example 23: monomeric fluorescent compound MPA-PEG prepared in example 21 3 Fluorescent imaging of TP-7 in breast cancer MAD-MB-231 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-7 was injected into 3 breast cancer MAD-MB-231 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 4C, and the fluorescent probe MPA-PEG is seen 3 TP-7 can specifically target the breast cancer (MAD-MB-231) site.
Example 24: monomeric fluorescent compound MPA-PEG prepared in example 21 3 Fluorescent imaging of TP-7 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-7 was injected into 3 pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 4, D, and the fluorescent probe MPA-PEG 3 TP-7 can specifically target pancreatic cancer (MiaPaPc-2) sites.
Example 25: monomeric fluorescent compound MPA-PEG prepared in example 21 3 Fluorescence imaging of TP-7 in gastric cancer SGC-7901 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-7 was injected into 3 tumor-bearing nude mice with SGC-7901 stomach cancer, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration, respectively. The results are shown in FIG. 4, which shows the fluorescent probe MPA-PEG 3 TP-7 can specifically target the gastric cancer (SGC-7901) site.
Example 26: the prepared monomeric fluorescent compound MPA-PEG 3 TP-10 (preparation method same as in example 1, TP-1-NH 2 Is replaced by TP-10) in vivo in pancreatic cancer AsPC-1 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-10 was injected into 3 pancreatic cancer AsPC-1 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 5A, and the fluorescent probe MPA-PEG is seen 3 TP-10 can specifically target pancreatic cancer (AsPC-1) sites.
Example 27: the prepared monomeric fluorescent compound MPA-PEG 3 Fluorescence imaging of TP-10 in colorectal carcinoma HCT116 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-10 was injected into 3 colorectal cancer HCT116 tumor-bearing nude mice, respectively, and fluorescent signal acquisition was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 5B, and the fluorescent probe MPA-PEG is seen 3 TP-10 specifically targets colorectal cancer (HCT 116) sites.
Example 28: the prepared monomeric fluorescent compound MPA-PEG 3 Fluorescence imaging of TP-10 in breast cancer MAD-MB-231 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-10 was injected into 3 breast cancer MAD-MB-231 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 5C, and the fluorescent probe MPA-PEG is seen 3 TP-10 can specifically target breast cancerMAD-MB-231).
Example 29: the prepared monomeric fluorescent compound MPA-PEG 3 Fluorescent imaging of TP-10 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-10 was injected into 3 pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, respectively, and fluorescent signal collection was performed at 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 5, D, and the fluorescent probe MPA-PEG 3 TP-10 can specifically target pancreatic cancer (MiaPaPc-2) sites.
Example 30: the prepared monomeric fluorescent compound MPA-PEG 3 Fluorescence imaging of TP-10 in gastric cancer SGC-7901 tumor-bearing mice.
MPA-PEG was prepared in the same manner as in example 2 3 TP-10 was injected into 3 tumor-bearing nude mice with SGC-7901 stomach cancer, and fluorescent signal collection was performed 1h, 2h, 4h, 6h, 8h, 10h, and 12h after administration. The results are shown in FIG. 5, which shows the fluorescent probe MPA-PEG 3 TP-10 can specifically target the gastric cancer (SGC-7901) site.
Example 31: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging in pancreatic cancer AsPC-1 tumor-bearing mice.
1) Bifunctional chelating agent HYNIC-PEG 4 Synthesis of NHS
1g of 6-chloronicotinic acid and 2.0mL of 80% hydrazine hydrate are added into 10mL of ethanol, the mixture is heated and refluxed for 4 hours, the solvent is distilled under reduced pressure after the reaction is completed, the obtained sticky substance is added into distilled water, the pH value is regulated to about 5.5, solids are separated out, 0.86g of yellow solid is obtained through suction filtration and drying, and the product is determined to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. The obtained 0.86g of 6-dihydrazide nicotinic acid and 0.61g of p-aminobenzaldehyde are added into 3.0mL of dimethyl sulfoxide (DMSO), the mixture is heated and reacted for 5 to 6 hours, and the mixture is added into water to be separated out after the reaction is finished, filtered by suction, and dried to obtain 1.2g of solid. 1.2g of the dried solid is added into DMSO together with 2.5g of EDCI and 1.5g of NHS to react at room temperature, water is added after the reaction is finished to separate out solid, the solid is purified by a silica gel column and then dried, 1.3g of the solid is weighed, and the solid is determined to be HYNIC-NHS by ESI-MS mass spectrum and nuclear magnetic resonance hydrogen spectrum, and ESI-MS: [ M+H ] ]=382.1508。The product was purified and added to PEG containing DIPEA 4 In the method, the reaction is carried out for 2 hours at room temperature, EDCI and NHS with 2 times of molar weight are added into the solution after the reaction is finished, the solution is separated and purified through preparation liquid phase after the reaction is finished, and the target product HYNIC-PEG is verified by mass spectrum after freeze drying 4 -NHS,ESI-MS:[M+H]= 630.3 and [ m+na ]] + =652.3。
2) Purified 5mg of intermediate HYNIC-PEG 4 NHS was dissolved in 0.3mL DMSO, 3mg TP-1 and 5.6mg DIPEA were added to the mixture and reacted at room temperature for 2 hours. After the reaction is finished, separating and purifying the product by preparing a liquid phase, finally obtaining 2.8mg of yellow solid, and confirming the product as a target product HYNIC-PEG by mass spectrum 4 -TP-1-NH 2 ,ESI-MS:[M+2H] 2+ = 596.81 and [ m+3h] 3+ =398.18。
3) Radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Is synthesized by (a)
Preparing 100.0mg/mL TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, 130.0mg/mL Tricine (trimethylglycine) solution, 102.4mg/mL succinic acid-sodium succinate buffer (77.0 mg succinic acid and 25.4mg sodium succinate), respectively taking 10.0uL TPPTS solution, 10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) and HYNIC-PEG 4 -TP-1-NH 2 Mix in a penicillin bottle and then add 10mCi Na 99m TcO 4 Heating in metal bath at 100deg.C for 20 min, cooling to room temperature after reaction, and making into radiopharmaceuticals 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 The product was identified by HPLC analysis.
Radioactive compounds 999m Tc-HYNIC-PEG 4 -TP-1-NH 2 Physiological saline solution (3 mCi/mL) was prepared, 0.1mL (about 300 μCi) was injected into the tail vein of 3 pancreatic cancer AsPC-1 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, 3h, and 4h after administration. The distribution of radionuclide probes in mice and the enrichment of tumor areas were observed. The results are shown in FIG. 6A, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Can be specificTargeting pancreatic cancer (AsPC-1) sites.
Example 32: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging in gastric cancer SGC-7901 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Physiological saline solution (3 mCi/mL) was prepared, and 0.1mL (about 300 μCi) was injected into 3 tumor-bearing nude mice (SGC-7901) with gastric cancer, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 6B, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Can specifically target the gastric cancer (SGC-7901) site.
Example 33: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging in pancreatic cancer MiaPaPc-2 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Physiological saline solution (3 mCi/mL) was prepared, 0.1mL (about 300 μCi) was injected into 3 pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 6C, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Can specifically target pancreatic cancer (MiaPaPc-2) sites.
Example 34: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging of breast cancer MCF-7 tumor-bearing mice in vivo.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Physiological saline solution (3 mCi/mL) was prepared, and 0.1mL (about 300 μCi) was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 6, D, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Can specifically target the breast cancer (MCF-7) site.
Example 35: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 SPECT-CT imaging of colorectal cancer HT29 tumor-bearing mice in vivo.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Physiological saline solution (3 mCi/mL) was prepared, and 0.1mL (about 300 μCi) was injected into 3 colorectal cancer HT29 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 6, E, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 -TP-1-NH 2 Can specifically target colorectal cancer (HT 29) sites.
Example 36: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 TP-7 (preparation method same as in example 31, TP-1-NH 2 Is replaced by TP-7) in the tumor-bearing mice of the breast cancer MCF-7.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated as a physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 breast cancer MCF-7 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 7A, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 can specifically target the breast cancer (MCF-7) site.
Example 37: monomeric radioactive compounds prepared 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of TP-7 in pancreatic cancer AsPC-1 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated as a physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 pancreatic cancer AsPC-1 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 7B, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 can specifically target pancreatic cancer (AsPC-1) sites.
Example 38: monomeric radioactive compounds prepared 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of TP-7 in pancreatic cancer MiaPaPc-2 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated as a physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 pancreatic cancer MiaPaPc-2 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 7C, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 can specifically target pancreatic cancer (MiaPaPc-2) sites.
Example 39: monomeric radioactive compounds 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of TP-7 in colorectal cancer HT29 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated as a physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 colorectal cancer HT29 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 7, D, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 specifically targets colorectal cancer (HT 29) sites.
Example 40: monomeric radioactive compounds prepared 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of TP-7 in gastric cancer MGC-803 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated as a physiological saline solution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 gastric cancer MGC-803 tumor-bearing nude mice, respectively, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 7, E, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 can specifically target the gastric cancer (MGC-803) site.
Example 41: monomeric radioactive compounds prepared 99m Tc-HYNIC-PEG 4 SPECT-CT imaging of TP-7 in gastric cancer SGC-7901 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-PEG 4 TP-7 was formulated into physiological saline solution (3 mCi/mL), 0.1mL (about 300. Mu. Ci) was taken and injected into 3 gastric cancer MGC-803 tumor-bearing nude mice, respectively, and givenSPECT-CT signal acquisition is carried out at 0.5h, 1h, 2h and 4h after the medicine. The results are shown in F in FIG. 7, from which the nuclide probe can be seen 99m Tc-HYNIC-PEG 4 TP-7 can specifically target gastric cancer (SGC-7901) site.
Example 42: dimer 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 Is disclosed.
1) Synthesis of bifunctional chelating agent HYNIC-NHS
1g of 6-chloronicotinic acid and 2.0mL of 80% hydrazine hydrate are added into 10mL of ethanol, the mixture is heated and refluxed for 4 hours, the solvent is distilled under reduced pressure after the reaction is completed, the obtained sticky substance is added into distilled water, the pH value is regulated to about 5.5, solids are separated out, 0.86g of yellow solid is obtained through suction filtration and drying, and the product is determined to be 6-dihydrazide nicotinic acid through ESI-MS mass spectrum and nuclear magnetic hydrogen spectrum. The obtained 0.86g of 6-dihydrazide nicotinic acid and 0.61g of p-aminobenzaldehyde are added into 3.0mL of dimethyl sulfoxide (DMSO), the mixture is heated and reacted for 5 to 6 hours, and the mixture is added into water to be separated out after the reaction is finished, filtered by suction, and dried to obtain 1.2g of solid. The dried 1.2g of solid was then reacted with 2.5g of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 1.5g N-hydroxysuccinimide (NHS) together in DMSO at room temperature, after the reaction was completed, water was added to precipitate a solid, which was purified by a silica gel column, dried, weighed 1.3g, and identified as the target product HYNIC-NHS by ESI-MS mass spectrometry and nuclear magnetic hydrogen spectrometry, ESI-MS: m+h= 382.15.
2) Support (Aca) 2 Synthesis of E
5.0g of tert-butyloxycarbonyl (carbony) protected glutamic acid (E), 8.3g of Dicyclohexylcarbodiimide (DCC) and 4.6g of NHS are added to 100mL of Tetrahydrofuran (THF) as an organic solvent, the mixture is stirred at room temperature overnight for dicarboxylic activation, the mixture is filtered with suction after the reaction is completed, the filtrate is washed with THF, no further purification is performed after the washing is completed, the mixture is directly added to 50mL of dimethyl sulfoxide (DMSO) for dissolution, then 10g of aminocaproic acid (Aca) is added, finally 14.6g of DIPEA is added for reaction at room temperature for 2 hours, 3.0mL of trifluoroacetic acid (TEA) is added to the reaction for Boc protecting group removal after the reaction is detected, the separation and purification are performed by preparing a liquid phase after the reaction is completed, and finally 7.8g of thick solid is obtained after drying, and the solid is verified by mass spectrum to be the expected target (Aca) 2 -E。
3) Intermediate HYNIC-E- (Aca) 2 Synthesis of-2 NHS
The prepared 0.5g bracket (Aca) 2 Dissolving E in DMSO, adding 0.28g HYNIC-NHS, adding 0.32g DIPEA, reacting at room temperature for 2 hours, adding EDCI and NHS for activation, preparing liquid phase for separation and purification, and freeze drying to obtain yellow solid 0.34g, and verifying by mass spectrum to be expected target compound HYNIC-E- (Aca) 2 -2NHS。
4)HYNIC-2Aca-E-(TP-1-NH 2 ) 2 Is synthesized by (a)
Purified 5mg of intermediate HYNIC-E- (Aca) 2 -2NHS was dissolved in 0.3mL DMSO, 5mg TP-1 was added after the reaction was completed, then 5.6mg DIPEA was added, the reaction was carried out at room temperature for 2 hours, and after the reaction was completed, separation and purification were carried out by preparing a liquid phase, and finally 3.5mg of a yellow solid was obtained, which was confirmed by mass spectrometry as the target product.
5) 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 Is prepared from
Preparing 100.0mg/mL TPPTS (triphenylphosphine sodium tri-m-sulfonate) solution, 130.0mg/mL Tricine (trimethylglycine) solution, 102.4mg/mL succinic acid-sodium succinate buffer (77.0 mg succinic acid and 25.4mg sodium succinate), respectively taking 10.0uL TPPTS solution, 10.0uL Tricine solution, 10.0uL succinic acid-sodium succinate buffer and 10.0uL (1.0 mg/mL) and HYNIC-2Aca-E- (TP-1-NH) 2 ) 2 Mix in a penicillin bottle and then add 10mCi Na 99m TcO 4 Heating in metal bath at 100deg.C for 20 min, cooling to room temperature after reaction, and making into radiopharmaceuticals 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 The product was identified by HPLC analysis.
Example 43: dimeric emissive compound prepared in example 42 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 SPECT-CT imaging in pancreatic cancer AsPC-1 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-2Aca-E-(TP-1-NH2) 2 Is prepared into physiological saline solutionSolution (3 mCi/mL), 0.1mL (about 300 μCi) was injected into 3 mice bearing tumors at pancreatic cancer AsPC-1, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 8A, from which the nuclide probe can be seen 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 Can specifically target pancreatic cancer (AsPC-1) sites.
Example 44: dimeric radioactive compounds prepared 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 SPECT-CT imaging in colorectal cancer HT29 tumor-bearing mice.
The radioactive compound was prepared in the same manner as in example 31 99m Tc-HYNIC-2Aca-E-(TP-1-NH2) 2 Physiological saline solution (3 mCi/mL) was prepared, 0.1mL (about 300 μCi) was injected into 3 tumor-bearing nude mice (HT 29) at colorectal cancer, and SPECT-CT signal acquisition was performed at 0.5h, 1h, 2h, and 4h after administration. The results are shown in FIG. 8B, from which the nuclide probe can be seen 99m Tc-HYNIC-2Aca-E-(TP-1-NH 2 ) 2 Can specifically target colorectal cancer (HT 29) sites.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. It is therefore intended that the following claims be interpreted as including the preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (8)
1. The application of a novel cyclic peptide in preparing a tumor diagnosis and/or treatment reagent is characterized in that the novel cyclic peptide is beta-Ala-L-Arg-L-Lys-L-Asp-L-Val-L-Tyr-L-Asp, wherein the side chain carboxyl groups of N-terminal amino group and C-terminal Asp form an amide ring;
wherein: d represents an unnatural D-type amino acid, and L represents an L-type natural amino acid.
2. The use of claim 1, wherein the tumor comprises one or more of lung cancer, pancreatic cancer, colorectal cancer, liver cancer, gastric cancer, and breast cancer.
3. The use according to claim 1, wherein the novel cyclic peptide is coupled to an imaging group to obtain the agent.
4. The use according to claim 3, wherein the agent comprises a fluorescent imaging agent and/or a radioactive agent, the fluorescent imaging agent comprising an optical imaging agent for tumor boundary pinpointing and/or intra-operative image navigation.
5. Use according to claim 3, wherein the agent has the general formula: M-L-G;
the M represents a photo-label, a metal chelator and a metal radionuclide complex, a nonmetallic radionuclide 18 F and F 11 C;
l is a linking group;
G is a novel cyclic peptide;
the optical label comprises one or more of an organic chromophore, an organic fluorophore, a light absorbing compound, a light reflecting compound, a light scattering compound, and a bioluminescent molecule;
the metal chelator is selected from the group consisting of modification of dihydrazineimide, 1,4, 7-triazacyclononane-1, 4, 7-triacetic acid, 7- [ (4-hydroxypropyl) methylene ] -1,4, 7-triazacyclononane-1, 4-diacetic acid, 1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid, mercaptoacetyltriglycine, diethylenetriamine pentaacetic acid, or combinations thereof.
6. The use according to claim 5, wherein the optical label comprises a near infrared one-region fluorescent dye and/or a near infrared two-region fluorescent dye, the near infrared one-region fluorescent dye comprising one or more of MPA, IRDye800, cy7.5, ICG and cy 5.5.
7. The use according to claim 5, wherein the linking group comprises 6-aminocaproic acid, NH 2 -PEG 3 -COOH、NH 2 -PEG 4 -COOH、NH 2 -PEG 6 -COOH and NH 2 -one or more of GGGGGG-COOH.
8. The use according to any one of claims 5 to 7, wherein the agent comprises 99m Tc-HYNIC-PEG 4 -TP-8MPA-TP-8 and MPA-PEG 4 -TP-8。
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US4629723A (en) * | 1984-06-27 | 1986-12-16 | Ortho Pharmaceutical Corporation | Potent thymopentin analogs |
CN107805282A (en) * | 2017-11-01 | 2018-03-16 | 中国药科大学 | A kind of targeted therapies and the united polypeptide of immunotherapy |
CN110845572A (en) * | 2019-11-28 | 2020-02-28 | 中国药科大学 | Tumor-targeted GRP (glass-fiber reinforced Polypropylene) analogue and application thereof |
CN111558050A (en) * | 2020-05-11 | 2020-08-21 | 中国药科大学 | Tumor targeting polypeptide and application thereof |
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US4629723A (en) * | 1984-06-27 | 1986-12-16 | Ortho Pharmaceutical Corporation | Potent thymopentin analogs |
CN107805282A (en) * | 2017-11-01 | 2018-03-16 | 中国药科大学 | A kind of targeted therapies and the united polypeptide of immunotherapy |
CN110845572A (en) * | 2019-11-28 | 2020-02-28 | 中国药科大学 | Tumor-targeted GRP (glass-fiber reinforced Polypropylene) analogue and application thereof |
CN111558050A (en) * | 2020-05-11 | 2020-08-21 | 中国药科大学 | Tumor targeting polypeptide and application thereof |
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