CN116999580A - Preparation method and application of polypeptide boron carrier radiopharmaceutical - Google Patents
Preparation method and application of polypeptide boron carrier radiopharmaceutical Download PDFInfo
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- CN116999580A CN116999580A CN202310038675.4A CN202310038675A CN116999580A CN 116999580 A CN116999580 A CN 116999580A CN 202310038675 A CN202310038675 A CN 202310038675A CN 116999580 A CN116999580 A CN 116999580A
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 58
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 58
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 58
- 229910052796 boron Inorganic materials 0.000 title claims abstract description 43
- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 239000012217 radiopharmaceutical Substances 0.000 title description 2
- 229940121896 radiopharmaceutical Drugs 0.000 title description 2
- 230000002799 radiopharmaceutical effect Effects 0.000 title description 2
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- ZOXJGFHDIHLPTG-BJUDXGSMSA-N Boron-10 Chemical compound [10B] ZOXJGFHDIHLPTG-BJUDXGSMSA-N 0.000 claims abstract description 5
- SIAVMDKGVRXFAX-UHFFFAOYSA-N 4-carboxyphenylboronic acid Chemical group OB(O)C1=CC=C(C(O)=O)C=C1 SIAVMDKGVRXFAX-UHFFFAOYSA-N 0.000 claims abstract description 4
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/009—Neutron capture therapy, e.g. using uranium or non-boron material
- A61K41/0095—Boron neutron capture therapy, i.e. BNCT, e.g. using boronated porphyrins
-
- 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
- A61K51/0474—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group
- A61K51/0482—Organic compounds complexes or complex-forming compounds, i.e. wherein a radioactive metal (e.g. 111In3+) is complexed or chelated by, e.g. a N2S2, N3S, NS3, N4 chelating group chelates from cyclic ligands, e.g. DOTA
-
- 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
-
- 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
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a nuclide probe of a polypeptide boron carrying agent, a preparation method and application thereof, and belongs to the leading edge field of Positron Emission Tomography (PET) technology-guided Boron Neutron Capture Therapy (BNCT). The probe is a cell penetrating peptide modified by a nuclide-marked chelating group; the sequence of the polypeptide is 4-carboxyphenylboronic acid amide-TFFYGGSRGKRNNFKTEEYCK. The invention can determine the distribution of the boron-10 modified cell penetrating peptide by PET imaging through a radioactive drug, and can guide the accurate application of BNCT and carry out nuclide treatment by switching lutetium-177 when reaching a certain tumor enrichment degree. In addition, the cell penetrating peptide is connected through different bifunctional chelating agents, so that matching of different diagnostic or therapeutic radionuclides is an important way for further developing the diagnosis and treatment value of the polypeptide drug.
Description
Technical Field
The invention relates to the technical field of PET molecular medical imaging, in particular to a radionuclide probe of a boron-10 modified cell penetrating peptide boron carrying agent ANG-B, and a preparation method and application thereof.
Background
Boron Neutron Capture Therapy (BNCT) utilizes a high enrichment of boron-10 element and nuclear reaction with thermal neutrons to treat tumors. Tumor targeting and enrichment of boron-carrying agents are key factors in determining the efficacy of BNCT. 10 B-Paraphthaloyl phenylalanine (BPA) is a fat-soluble compound that has a structure similar to phenylalanine. Because tumor cells are metabolized vigorously and require more phenylalanine than normal cells, tumor cells can take up BPA in large amounts, and are one of the most commonly used boron carriers in clinic. With the development of BNCT application, people find that the targeting of BPA is insufficient, the in vivo metabolism speed of BPA is too fast, the residence time at the tumor part is short, and the treatment requirement of BNCT cannot be met.
The cell penetrating peptide is a possible boron atom carrier, can improve the targeting property of tumor cells and the residence time in the cells, can penetrate the blood brain barrier, and can become the synthesis foundation of the brain glioma boron carrier. ANG is a tumor-targeted cell penetrating peptide that can be prepared by solid phase peptide synthesis. The applicant connects a boron-containing compound at the N end of ANG through 4-carboxyphenylboronic acid, and the generated boron-10 connected cell penetrating peptide is a boron carrying agent with excellent properties, and has good treatment effect in BNCT treatment. Can penetrate the blood brain barrier, directly transport into cells, and enrich in brain glioma cells. The preclinical experimental result shows that the boron carrying agent has good tumor targeting and treatment effects when being applied to Boron Neutron Capture Therapy (BNCT) of brain glioma.
In the BNCT treatment process, the real-time and accurate measurement of the boron concentration of the tumor part directly influences the grasp of the optimal treatment time window. BNCT needs to set neutron radiation dose according to the difference of boron concentration in tumor tissues, and provides a basis for preparing reasonable BNCT treatment schemes. However, in the conventional method, the patient needs to collect a sample every 15 minutes or half an hour for detection, and the process is complex and time-consuming. Therefore, a more intuitive technology is needed for detecting in vivo distribution and metabolism of the polypeptide boron carrying agent ANG-B.
Positron emission computed tomography (PET) is an important medical imaging modality for detecting tumors, and generally the radionuclide tracers used have a short half-life. Gallium-68% 68 Ga) is one of the positron nuclides originally used in clinical medicine and surrounds 68 Research on Ga species is also increasing. To be commercialized 68 The Ga generator is based on the fact that, 68 the marking strategy and the purification method of Ga are continuously improved, and the method for marking Ga by Ga is characterized in that 68 Ga species are linked to different polypeptides through bifunctional chelators, which confer different functions to the probe.
Macrocyclic bifunctional chelating agent DOTA capable of modifying and linking polypeptides to prepare [ 68 Ga]The safety and effectiveness of the polypeptide probes, which have been successfully tested by clinical trials, are confirmed. DOTA is known as a "universal chelator" and forms thermodynamically and kinetically stable metal complexes with a wide variety of divalent and trivalent metals.
Disclosure of Invention
The invention aims to provide a radionuclide probe of a polypeptide boron carrying agent ANG-B based on a cell penetrating peptide ANG, and a preparation method and application thereof, which can be used for noninvasively, in vivo and specifically detecting in-vivo distribution and metabolism of the polypeptide boron carrying agent ANG-B and provide a basis for accurately applying BNCT to cancer treatment.
The technical scheme of the invention is realized as follows:
the invention provides a nuclide probe of a polypeptide boron carrying agent ANG-B, wherein the probe is a nuclide-labeled chelate group modified polypeptide; the sequence of the polypeptide is 4-carboxyphenylboronic acid amide-TFFYGGSRGKRNNFKTEEYCK.
As a further development of the invention, the chelating group is selected from among DOTA (1, 4,7, 10-Tetraazacyclododecane-tetraacetic acid), NOTA (2- (4-isothiocyanatophenyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid), NODA (2, 2' - (7- (1-carboxy-4- ((2, 5-dioxarridin-1-yl) oxy) -4-oxazinyl) -1,4, 7-triazocine-1, 4-diyl diacetic acid), DOTP (1, 4,7, 10-tetraazacyclodacene-1, 4,7, 10-tetraacetic acid), HBEDCC (3- [3- [4- [5- (2-carboxyyl) -2-hydroxyphenolyl ] -1,4-bis (carboxymethylamino) butyl ] -4-dioxazine ] procyanic acid), DTPA (2-diaminodiacetic acid), and at least one of the amino acids DTPA.
The structures of DOTA and NOTA are shown below, respectively:
as a further improvement of the present invention, the nuclide comprises a radionuclide or a therapeutic radionuclide, the radionuclide comprising 68 Ga、 64 Cu、 18 F、 86 Y、 90 Y、 89 Zr、 111 In、 99 mTc、 123 I、 124 One of I; the therapeutic nuclides include 177 Lu、 125 I、 131 I、 211 At、 223 Ra、 225 Ac、 67 Cu、 212 Pb、 213 One of Bi.
As a further improvement of the invention, the chelating group is DOTA and the nuclide is 68 Ga, at this time, the structure of the probe is shown as formula I:
formula I.
The invention further provides a preparation method of the radionuclide probe of the polypeptide boron carrying agent ANG-B, which comprises the following steps:
s1, preparing polypeptide;
s2, connecting a bifunctional chelating agent;
s3, labeling nuclides.
As a further improvement of the present invention, the specific steps in step S1 are as follows:
adding 2-chlorotrityl chloride resin into dichloromethane, oscillating for 20-40min, and then connecting with the first amino acid; removing the solvent by suction filtration, adding 0.5-1.5 times of Fmoc-Lys (Dde) -OH, adding 8-12 times of N, N-diisopropylethylamine, finally adding dimethylformamide for dissolution, and oscillating to obtain a methanol seal head; removing the solvent, and adding a dimethylformamide solution containing 20% of piperidine for deprotection in two times; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; dissolving 2-4 times molar excess Fmoc-Cys (Trt) -OH and 2-4 times molar excess HBTU in dimethylformamide, mixing with the obtained product, adding 8-12 times molar excess N, N-diisopropylethylamine, and performing condensation reaction; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; repeating the operation steps, and sequentially connecting amino acids in the sequence until the 4-carboxyphenylboronic acid pinacol ester is finally connected to prepare the polypeptide;
the molar excess is the molar amount relative to the first amino acid.
As a further improvement of the present invention, the specific steps in step S2 are as follows: weighing polypeptide, 2-4 times molar excess DOTA-tris (tbu) ester and 2-4 times molar excess peptide coupling reagent HBTU, dissolving in dimethylformamide, and then adding 8-12 times molar excess N, N-diisopropylethylamine for condensation reaction; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; pumping the resin, cutting polypeptide from the resin under the dark condition, drying the lysate by nitrogen, washing by diethyl ether, volatilizing at normal temperature to obtain a crude product; purifying with high performance liquid chromatography, and lyophilizing the purified solution to obtain DOTA-ANG-B;
the molar excess is the molar amount relative to the polypeptide.
As a further improvement of the present invention, the specific steps in step S3 are as follows: mixing gallium-68 ion stock solution and NaOAc buffer solution dissolved with DOTA-ANG-B, and reacting for 10-20min at 95-100 ℃; then adding water for dilution, adsorbing the product on activated C18 column, eluting the column with purified water, pressing to dry the column, eluting the C18 column with product eluent, diluting the eluent with normal saline and filtering with sterile filter membrane to obtain the final product 68 Ga]-DOTA-ANG-B。
As a further improvement of the invention, the eluent is obtained by uniformly mixing ethanol and water according to the volume ratio of 0.5-1.5:0.5-1.5.
Preferably, the method specifically comprises the following steps:
step 1: preparation of the polypeptide
Firstly, resin swelling is carried out, 2-chlorotrityl chloride resin is put into a reaction tube, DCM is added, and the first amino acid is connected after shaking for 30min; filtering out the solvent through a sand core, adding 1-time molar excess Fmoc-Lys (Dde) -OH, adding 10-time molar excess DIEA, finally adding DMF for dissolution, and oscillating to obtain a methanol seal head; removing the solvent, and adding 20% piperidine/DMF solution for deprotection in two times; washing twice by using DMF, methanol and DMF solution respectively; the three times excess of Fmoc-Cys (Trt) -OH, the three times excess of HBTU and the minimum DMF are used for dissolution, then the reaction tube is added, and the ten times excess of DIEA is immediately added for condensation reaction; washing twice by using DMF, methanol and DMF solution respectively; the procedure is repeated, connecting the amino acids in the sequence in sequence until the final connection of 4-carboxyphenylboronic acid pinacol ester.
Step 2: linking bifunctional chelators
Weighing three times excess DOTA-tris (tbu) ester and three times excess HBTU, dissolving with a small amount of DMF, and adding into a reaction tube; then adding the DIEA with ten times excess to carry out condensation reaction; washing three times with DMF, methanol and DMF solution respectively; pumping the resin, cutting polypeptide from the resin (avoiding light), blowing the lysate to dry with nitrogen as much as possible, washing with diethyl ether for several times, and volatilizing at normal temperature to obtain a crude product; purifying by HPLC, and lyophilizing the purified solution by vacuum lyophilizing machine to obtain DOTA-ANG-B.
Step 3: labeling nuclides
Mixing gallium-68 ion hydrochloric acid solution and NaOAc buffer solution dissolved with DOTA-ANG-B and having pH of 6-7 in a mass ratio of 0.5-1.5:0.5-1.5, and reacting for 10-20min at 95-100 ℃; then adding 5mL of water for dilution, and adsorbing the product on a C18 column through the activated C18 column; eluting the column with purified water, and press drying the columnSub-to purify the product; slowly eluting the C18 column with the product eluent (ethanol/water=1/1), diluting the eluent with physiological saline, and filtering with sterile filter membrane to obtain the final product [ 68 Ga]-DOTA-ANG-B。
The invention further protects application of the nuclide probe of the polypeptide boron carrying agent ANG-B in preparation of PET diagnostic imaging agents.
The invention has the following beneficial effects: the invention adopts DOTA to connect with polypeptide boron carrying agent ANG-B, and guides the ANG-B to be used in BNCT treatment through gallium-68 labeling and PET imaging technology. DOTA can also form ligand action with the therapeutic radionuclide lutetium-177, and is a diagnosis and treatment integrated chelating agent through clinical verification. PET nuclides 68 The combination of Ga and the ANG-B of BNCT can observe the boron concentration and the distribution in the patient body through PET images in BNCT treatment, thereby being beneficial to setting neutron radiation dose more accurately, making a reasonable BNCT irradiation scheme and reducing blindness and side effects of BNCT irradiation. In addition, the distribution of the ANG-B living body can be determined after PET diagnosis, and when a certain tumor enrichment degree is reached, the nuclide treatment can be performed by switching lutetium-177. In addition, the ANG-B is connected through different bifunctional chelating agents, so that matching of different diagnostic nuclides or therapeutic nuclides is an important way for further developing the polypeptide drug.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions of the prior art, the drawings which are used in the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention, and that other drawings can be obtained according to these drawings without inventive faculty for a person skilled in the art.
FIG. 1 is the flow chart of example 1 68 Ga]-synthetic route to DOTA-ANG-B.
FIG. 2 shows the results of mass spectrometry (ESI-MS) of DOTA-ANG-B drug in examples.
FIG. 3 shows the results of high performance liquid chromatography (Agilent 1260 HPLC) of the drug of example DOTA-ANG-B.
FIG. 4 shows the results of an absorption distribution experiment of the nuclide drug polypeptide boron carrier ANG-B prepared in the example in a mouse model.
FIG. 5 is a comparison of tumor volumes before and after BNCT treatment with the polypeptide boron-carrying agent ANG-B in the example mouse model.
FIG. 6 is a gallium-68 nuclide labeled product of the example polypeptide boron carrier [ 68 Ga]DOTA-ANG-B test results.
FIG. 7 is a schematic illustration of an example polypeptide boron carrier 68 Ga]Imaging quality test results of DOTA-ANG-B probe.
Description of the embodiments
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all 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.
EXAMPLE 1 preparation of polypeptide boron Carrier ANG-B
As shown in FIG. 1A, firstly, 2-chlorotrityl chloride resin is put into a reaction tube, DCM (15 ml/g) is added and oscillated for 30min, and the first amino acid is connected through resin swelling; filtering out the solvent through a sand core, adding Fmoc-Lys (Dde) -OH with 1-time molar excess, adding DIEA with 10-time molar excess, finally adding DMF for dissolution, oscillating for 30min, and sealing with methanol; removing the solvent, and adding 20% piperidine/DMF solution (15 ml/g) in two times for deprotection; washing with DMF (10 ml/g), methanol (10 ml/g), DMF (10 ml/g) solution twice each; taking three times of excessive Fmoc-Cys (Trt) -OH and three times of excessive HBTU, dissolving through 0.5ml of DMF, adding into a reaction tube, immediately adding ten times of excess DIEA, and carrying out condensation reaction for 30min; washing twice by using DMF, methanol and DMF solution respectively; repeating the operation, sequentially connecting amino acids in the sequence, and finally connecting 4-carboxyphenylboronic acid pinacol ester to obtain the ANG-B polypeptide.
EXAMPLE 2 preparation of DOTA-ANG-B
As shown in FIG. 1B, first, the three times excess DOTA-tris (tbu) ester and the three times excess HBTU are weighed, dissolved by a small amount of DMF, added into a reaction tube, immediately added with the ten times excess DIEA, and subjected to condensation reaction for 30min; washing 3 times with DMF, methanol and DMF solution respectively; pumping the resin, cutting polypeptide from the resin in a dark place, blowing the lysate to dry with nitrogen as much as possible, washing with diethyl ether for 6 times, and volatilizing at normal temperature to obtain a crude product; purifying by HPLC, and lyophilizing the purified solution by vacuum lyophilizing machine to obtain DOTA-ANG-B.
Example 3 preparation [ 68 Ga]-DOTA-ANG-B
As shown in fig. 1C, a gallium-68 ion hydrochloric acid solution is placed in a 10mL penicillin bottle, naOAc buffer solution (the pH of normal reaction solution is 3.5-4) with the ph=6.5 of 100nM DOTA-ANG-B reaction precursor is added, the two solutions are mixed in a mass ratio of 1:1, and a gland is sealed; the reaction bottle is placed at 95 ℃ for reaction for 10min; after the reaction is finished, adding 4 mL water into a reaction bottle for dilution, passing the diluted solution through an activated C18 column, adsorbing the product on the C18 column, and air-drying the column; eluting the column with 10mL purified water, and drying the column by pressing to purify the product; slowly eluting the C18 column with 2 mL product eluent (ethanol/water=1/1), diluting the eluate with physiological saline, and filtering with sterile filter membrane to obtain final product [ 68 Ga]-DOTA-ANG-B polypeptide probe.
Example 4 [ 68 Ga]Full process of-DOTA-ANG-B synthesis
And (1) swelling of the resin: the resin 2-Chlorotrityl Chloride Resin was placed in the reaction tube and DCM (15 ml/g) was added and the mixture was shaken for 30min.
(II) connecting the first amino acid: the solvent was filtered off with suction through a sand core, 5mM Fmoc-Lys (Dde) -OH was added, 50mM DIEA was added, and finally DMF was added for dissolution, and shaking was performed for 30min. Methanol seal head for 30min.
(III) deprotection: the solvent was removed, 20% piperidine/DMF solution (15 ml/g) was added for 5min, and a further 20% piperidine/DMF solution (15 ml/g) was removed for 15min.
(IV) detection: pumping off piperidine solution, taking more than ten resin particles, washing with ethanol for three times, adding Kaiser reagent, heating at 105-110 ℃ for 5min, and turning deep blue to be positive reaction.
(V) washing: DMF (10 ml/g) was twice, methanol (10 ml/g) was twice, and DMF (10 ml/g) was twice.
(VI) condensation: the protected amino acids Fmoc-Cys (Trt) -OH and HBTU were added to 15mM, dissolved in as little DMF as possible, added to the reaction tube, immediately added DIEA to 50mM and reacted for 30min.
And (seventh) detection: the solution is pumped out, more than ten pieces of resin are taken, ethanol is used for three times, kaiser reagent is added, heating is carried out for 5min at the temperature of 105-110 ℃, and the reaction is colorless and negative, which indicates that the reaction is complete.
(eight) washing: DMF (10 ml/g) was taken once, methanol (10 ml/g) was taken twice, and DMF (10 ml/g) was taken twice.
And (nine) repeating the three to eight steps, and sequentially connecting the amino acids in the sequence until the 4-carboxyphenylboronic acid pinacol ester is finally connected.
(ten) detection and washing: removing Dde by using 3% hydrazine hydrate/DMF, pumping out the solution, taking more than ten resin particles, washing the resin particles with ethanol for three times, adding Kaiser reagent, heating for 5min at 105-110 ℃, and turning deep blue to be positive reaction. DMF (10 ml/g) was twice, methanol (10 ml/g) was twice, and DMF (10 ml/g) was twice.
(eleven) condensation: DOTA-tris (tbu) ester and HBTU were dissolved to 15mM with minimum DMF, added to the reaction tube, immediately added DIEA to 50mM and reacted for 30min.
(twelve) washing: DMF (10 ml/g) was twice, methanol (10 ml/g) was three times, DCM (10 ml/g) was three times. The resin was drained.
Thirteen cleavage of polypeptide from resin (protected from light): preparing a cutting fluid (10/g) TFA 95%; 1% of water; EDT 2%; TIS 2%; cutting time: 180min.
Blowing and washing: drying the lysate with nitrogen as much as possible, washing with diethyl ether for six times, and volatilizing at normal temperature.
(fifteen) purification: purification of the product was performed by HPLC. High performance liquid chromatography column using C18 column (Kromasil 100-5C18,20mmX250mm,10 μm) was purified using 24% -34% acetonitrile in water 0.1% TFA. And freeze-drying the purified solution by a vacuum freeze dryer to obtain a finished product.
Sixteenth nuclide labeling: the gallium-68 ion hydrochloric acid solution was placed in a 10mL penicillin bottle, and NaOAc buffer solution (normal reverse) with ph=6.5 in which 100nM DOTA-ANG-B reaction precursor was dissolved was addedThe pH of the reaction liquid is 3.5-4), and the two are mixed in a mass ratio of 1:1, and the pressure cover is sealed; the reaction bottle is placed at 95 ℃ for reaction for 10min; after the reaction is finished, adding 4 mL water into a reaction bottle for dilution, passing the diluted solution through an activated C18 column, adsorbing the product on the C18 column, and air-drying the column; eluting the column with 10mL purified water, and drying the column by pressing to purify the product; slowly eluting the C18 column with 2 mL product eluent (ethanol/water=1/1), diluting the eluate with physiological saline, and filtering with sterile filter membrane to obtain [ 68 Ga]-DOTA-ANG-B polypeptide probe.
EXAMPLE 5 HPLC purity identification of polypeptide probes
The product is subjected to purity identification through an Agilent 1260 HPLC analysis system and is provided with a radioactive detection module and an ultraviolet detector. Sample composition analysis was performed using a Kromasil 100-5C18 chromatography column (4.6 mmX250mm,5 mm) at 25℃and a flow rate of 1.0. 1.0 mL/min, and isocratic elution was performed for 20 min. Mobile phase a was acetonitrile (0.1% tfa) and B was ultrapure water (0.1% tfa). The elution gradient was set as: 25% A and 75% B at 0min, 50% A and 50% B at 20min, followed by 100% A and 0% B.
The results are shown in FIG. 2. The first peak of the HPLC test result chart is the peak of the buffer solution, the 2 nd peak is the peak of the target object, the peak area is the detected purity, namely 96.87%, and the 3 rd peak is the impurity peak. The peak time of the radioactivity detector and the peak time of the polypeptide probe occur simultaneously, and the success of the gallium-68 labeling is determined; nuclide probe by peak area calculation 68 Ga]Radiochemical purity of DOTA-ANG-B>95%。
Example 6 mass spectrometric molecular weight characterization of DOTA-ANG-B
FIG. 3 is an MS spectrum of DOTA-ANG-B polypeptide, which is detected as the molecular weight of the polypeptide. According to the data of mass spectrum, whether the obtained product is a target object can be judged. The peaks shown in this figure are the number of scored polypeptide products. Since the ESI-MS instrument may have several charge states of the generated gaseous ions during detection, the detection value may have several peaks. For positive ions of molecular mass M, the mass-to-charge ratio is as follows: x1= (m+n)/n. As can be seen from the comparison of the formulas, the 4 values are peak values of the target.
EXAMPLE 7 absorption Profile experiment results of ANG-B boron Carrier in mouse model
As shown in fig. 4, ANG-B was mainly accumulated in the liver and kidney at 1 hour by analyzing the change of boron content in each tissue of tumor-bearing nude mice (HS 683 glioma cancer cells), indicating that the compound was mainly metabolized by the liver and kidney. Analysis of variance shows that the boron concentration in the brain glioma tissue of the ANG-B group is obviously higher than that of the BPA group (P < 0.001), which indicates that the ANG-B can transmit more boron to the tumor part and increase the BNCT irradiation effect. Furthermore, boron distribution in other tissues suggests that ANG-B accumulates mainly in the liver and kidneys at 1 hour. No significant differences were observed in drug distribution in other organs of the different groups, such as spleen, heart and lung. The contents of the polypeptide boron carrier ANG-B in tumor tissues in 2 hours, 4 hours and 6 hours are 17.9+/-2.41 mu g B/g, 26.5+/-3.51 mu g B/g and 16.1+/-1.67 mu g B/g respectively, which shows that the stay time of the ANG-B in the tumor tissues is longer, and the method for continuously intravenous injection of the boron carrier in BNCT irradiation can be effectively avoided.
Example 8 comparison of tumor volumes in murine models before and after BNCT treatment with the polypeptide boron-carrying agent ANG-B
As shown in FIG. 5, the mouse tumor model was divided into BPA group, ANG-B low dose group and ANG-B high dose group. Based on PET/CT imaging results of in vivo neutron irradiation studies using a nude mouse brain tumor model, BNCT treatment with high dose ANG-B was more effective than BPA (P < 0.001). The average reduction volume percentages of tumors after BNCT treatment of the BPA group, the ANG-B high dose group and the ANG-B low dose group are 23.0%, 62.9% and 29.2%, respectively, which shows that the ANG-B has good BNCT treatment effect.
Example 9[ 68 Ga]Nuclide labeling of-DOTA-ANG-B polypeptide probe and HPLC test result
DOTA-ANG-B reaction precursor (100 mg) was dissolved in 50 mL ultrapure water, 25 mL liquid was sucked up by a 10mL pipette and placed in a 10mL penicillin bottle, followed by the addition of 1mL NaOAc buffer salt (0.25M); rinsing with 0.05M HCl 4 mL 68 Ge- 68 Ga generator, obtain 4 mL 68 Ga 3+ Placing the stock solution in a 10mL penicillin bottle; dissolving the above in the solvent to reactNaOAc buffer solution of precursor is added to 68 Ga 3+ Sealing the raw liquid by a gland; the reaction flask was placed at room temperature for 15min, and 40. Mu.l of sample was introduced by HPLC to test the nuclide labeling efficiency. The mobile phase adopts acetonitrile water solution gradient (10% -80%), liquid phase column specification: luna 5mm c18 100a, flow rate: 1 ml/min, detection wavelength: 254 nm/190 nm, equilibrium column pressure: 110-130 mPa; radiometric detection modules and HPLC were used in combination to test the radiometric purity of the nuclidic labeled products. As shown in FIG. 6, in the ultraviolet detection chart, the liquid phase peak time of DOTA-ANG-B is 7.99min; in the radioactivity detection diagram, 2-7min is used as nuclide raw material 68 Ga for 8-9min 68 Ga]DOTA-ANG-B (target radioproduct). Since the UV detector is positioned in front of the radioactive detection probe, the results confirm 68 Ga]The labeling of the-DOTA-ANG-B nuclide is successful.
Example 10[ 68 Ga]PET/CT mouse tumor model imaging effect of DOTA-ANG-B polypeptide probe
As shown in FIG. 7, the mouse tumors were divided into gallium-68 ion control group and [ [ V.E.) 68 Ga]DOTA-ANG-B mouse tumor PET imaging group. Nude mouse tumor models were prepared from brain cancer glioma cells HS 683. [ 68 Ga]The DOTA-ANG-B polypeptide probe was prepared as a 3mCi/mL physiological saline solution, 0.1mL of the drug was taken and injected through the tail vein of the mouse, and PET/CT imaging was performed 30min after the administration, and enrichment of the polypeptide probe in the mouse and in the tumor area (circular dotted line area in FIG. 7) was observed. FIG. 7A shows the results of cross-sectional PET/CT imaging of two groups of mice; figure 7B shows the longitudinal PET/CT imaging results for two groups of mice. The dotted circular areas are tumor sites and the non-gray areas show PET probe signals. From the figure, it can be seen that 68 Ga]DOTA-ANG-B has obvious tumor targeting.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. A boron-10 modified nuclear probe of the cell penetrating peptide boron carrying agent ANG-B, characterized in that the probe is a radionuclide-labeled chelating group modified polypeptide; the sequence of the polypeptide is 4-carboxyphenylboronic acid amide-TFFYGGSRGKRNNFKTEEYCK.
2. The nuclide probe of the polypeptide boron carrier ANG-B of claim 1, wherein the chelating group is selected from at least one of DOTA, NOTA, NODA, DOTP, HBEDCC, DTPA, DFO, PTSM.
3. The nuclear species probe of the polypeptide boron carrier ANG-B of claim 1, wherein the radionuclide comprises a diagnostic or therapeutic nuclear species comprising 68 Ga、 64 Cu、 18 F、 86 Y、 90 Y、 89 Zr、 111 In、 99 mTc、 123 I、 124 One of I; the therapeutic nuclides include 177 Lu、 125 I、 131 I、 211 At、 223 Ra、 225 Ac、 67 Cu、 212 Pb、 213 One of Bi.
4. The nuclide probe of the polypeptide boron carrier ANG-B of claim 1, wherein the chelating group is DOTA and the nuclide is 68 Ga, at this time, the structure of the probe is shown as formula I:
formula I.
5. A method for preparing a nuclide probe of the polypeptide boron carrier ANG-B of claim 4, comprising the steps of:
s1, preparing polypeptide;
s2, connecting a bifunctional chelating agent;
s3, labeling nuclides.
6. The preparation method according to claim 5, wherein the specific steps in step S1 are as follows:
adding 2-chlorotrityl chloride resin into dichloromethane, oscillating for 20-40min, and then connecting with the first amino acid; removing the solvent by suction filtration, adding 0.5-1.5 times of Fmoc-Lys (Dde) -OH, adding 8-12 times of N, N-diisopropylethylamine, finally adding dimethylformamide for dissolution, and oscillating to obtain a methanol seal head; removing the solvent, and adding a dimethylformamide solution containing 20% of piperidine for deprotection in two times; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; dissolving 2-4 times molar excess Fmoc-Cys (Trt) -OH and 2-4 times molar excess HBTU in dimethylformamide, mixing with the obtained product, adding 8-12 times molar excess N, N-diisopropylethylamine, and performing condensation reaction; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; repeating the operation steps, and sequentially connecting amino acids in the sequence until the 4-carboxyphenylboronic acid pinacol ester is finally connected to prepare the polypeptide;
the molar excess is the molar amount relative to the first amino acid.
7. The preparation method according to claim 5, wherein the specific steps in step S2 are as follows: weighing polypeptide, 2-4 times molar excess DOTA-tris (tbu) ester and 2-4 times molar excess peptide coupling reagent HBTU, dissolving in dimethylformamide, and then adding 8-12 times molar excess N, N-diisopropylethylamine for condensation reaction; sequentially washing with dimethylformamide, methanol and dimethylformamide solution; pumping the resin, cutting polypeptide from the resin under the dark condition, drying the lysate by nitrogen, washing by diethyl ether, volatilizing at normal temperature to obtain a crude product; purifying with high performance liquid chromatography, lyophilizing the purified solution to obtain the final product, named DOTA-ANG-B;
the molar excess is the molar amount relative to the polypeptide.
8. The preparation method according to claim 5, wherein the specific steps in step S3 are as follows: hydrochloric acid solution of gallium-68 ion and DOTA-ANG-B dissolved thereinNaOAc buffer solution is mixed according to the mass ratio of 0.5-1.5:0.5-1.5, and reacts for 10-20min at the temperature of 95-100 ℃; then adding water for dilution, adsorbing the product on activated C18 column, eluting the column with purified water, pressing to dry the column, eluting the C18 column with product eluent, diluting the eluent with normal saline and filtering with sterile filter membrane to obtain the final product, named as [ 68 Ga]-DOTA-ANG-B。
9. The preparation method of claim 8, wherein the eluent is obtained by uniformly mixing ethanol and water according to a volume ratio of 0.5-1.5:0.5-1.5.
10. Use of a nuclide probe of the polypeptide boron carrier ANG-B of any one of claims 1-4 for the preparation of a PET diagnostic imaging agent.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112724196A (en) * | 2020-12-30 | 2021-04-30 | 中国原子能科学研究院 | Radionuclide labeled CD206 receptor targeting peptide and preparation method thereof |
| CN112972679A (en) * | 2021-03-18 | 2021-06-18 | 北京大学 | Polypeptide-coupled boron carrier, preparation method thereof and pharmaceutical preparation |
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