CN114796535B - Targeting G-quadruplex polypeptide PET imaging agent and preparation method and application thereof - Google Patents

Targeting G-quadruplex polypeptide PET imaging agent and preparation method and application thereof Download PDF

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CN114796535B
CN114796535B CN202210402612.8A CN202210402612A CN114796535B CN 114796535 B CN114796535 B CN 114796535B CN 202210402612 A CN202210402612 A CN 202210402612A CN 114796535 B CN114796535 B CN 114796535B
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imaging agent
pet imaging
quadruplex
polypeptide
kr12c
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CN114796535A (en
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黄顺
吴湖炳
石大志
唐刚华
韩彦江
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Southern Hospital Southern Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • 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 discloses a targeting G-quadruplex polypeptide PET imaging agent, a preparation method and application thereof, wherein the PET imaging agent is an excellent novel pharmacokinetic probe, the structure of the PET imaging agent comprises polypeptide, metal chelating groups and radionuclides, the preparation method is simple, convenient and quick, the yield is high, the PET imaging agent has good stability and strong water solubility, blood in a body is cleared quickly, the PET imaging agent is mainly metabolized by kidneys and livers, and can be specifically absorbed at tumor sites, and the PET imaging agent can be widely applied to biological functional imaging.

Description

Targeting G-quadruplex polypeptide PET imaging agent and preparation method and application thereof
Technical Field
The invention belongs to the field of pharmacy, and particularly relates to a targeting G-quadruplex polypeptide PET imaging agent, and a preparation method and application thereof.
Background
Malignant tumors (cancers) are one of the greatest public health problems worldwide, and traditional cancer treatment methods mainly comprise operation treatment, chemotherapy, radiation treatment and the like. For non-solid tumors and cancers widely transferred in the whole body, effective operation treatment and radiotherapy cannot be performed, and meanwhile, the chemotherapy method also has the problems of large side effect, poor specificity, low curative effect and the like, so that the requirements of clinical treatment are difficult to meet. Compared with common chemotherapy, the molecular targeting therapy for specific oncogenes, proteins or receptors has the advantages of strong specificity, remarkable curative effect and obvious small side effect, and is gradually one of the important means for clinical tumor treatment. In recent years, development of new antitumor drugs has been greatly advanced, a large number of new targets and new target drugs are developed and applied, and research on antitumor drugs with G-quadruplex as targets has attracted a great deal of attention.
G-quadruplex (G-quadruplex) is an atypical structure that can be formed in guanine (G) -rich nucleic acid sequences, with single strands of DNA forming planar G-quadruplexes by hydrogen bonding between G bases, and two or more of the quadruplexes forming a G-quadruplex helix by pi-pi stacking. The human body has about 70 ten thousand gene sequences capable of forming G-quadruplex, mainly comprising telomere ends, mutation hot spot areas of genes and promoter areas of genes (such as Bcl-2, c-myc, c-myb, c-kit, kras, VEGF and VEGFR), covering genes related to senescence, apoptosis, growth factors and receptors thereof, transcription regulating factors and signal transduction factors, and playing important roles in cell senescence, proliferation, apoptosis and tumor formation. In tumor tissues, when a specific ligand is combined with a G-quadruplex formed by the gene sequences, the corresponding biochemical process is changed (such as telomerase activity is inhibited, the transcription level of an oncogene is down-regulated, and the like), so that the anti-tumor effect is achieved, and the possible biological functions and the unique structural characteristics of the G-quadruplex make the G-quadruplex an important target point of an anti-tumor drug.
Positron emission computed tomography (positron emission tomography PET) has been widely used for diagnosis and differential diagnosis of various diseases, efficacy evaluation, organ function research, new drug development, and the like. Broad-spectrum tumor imaging agent with largest clinical application 18 F-FDG has the problems that false positive is frequently generated in tumor detection, tumors and inflammations cannot be distinguished, and the like, while a plurality of novel PET imaging agents with specific targets (such as PSMA, FAP, EGFR and the like) are developed, researched and applied at present, more novel targets are tried to develop related PET imaging agents, the PET imaging agents still have the problems that false positive is easily generated, tumors and inflammations cannot be distinguished, and the like. The target property of the G-quadruplex determines that the specific PET/CT imaging of the targeted G-quadruplex has real-time, noninvasive and in-vivo clear tumor focus distribution, can perform early diagnosis, tumor staging, preoperative positioning, postoperative contrast and curative effect monitoring of solid tumors, and can be used for preparing personalized treatment schemes and guiding the potential of surgery or targeted treatment. Meanwhile, the PET imaging taking the G-quadruplex as the target has important research and application prospects in the aspects of guiding the research and development of the novel tumor drugs, drug effect evaluation, sensitive crowd screening and the like, so that a novel G-4-strand-targeted related PET probe pair is developedFunctional imaging is of great significance.
Disclosure of Invention
In order to overcome the problems of the prior art, one of the purposes of the present invention is to provide a targeting G-quadruplex polypeptide PET imaging agent; the second purpose of the invention is to provide a preparation method of the targeting G-quadruplex polypeptide PET imaging agent; the invention also aims to provide the application of the targeted G-quadruplex polypeptide PET imaging agent.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides a targeting G-quadruplex polypeptide PET imaging agent, the structure of which is shown as a formula (I) or a formula (II):
Figure BDA0003600863900000021
in the formula (I), R 1 Represents a metal ion chelating group, M represents a radiometal nuclide, and n is selected from positive integers;
in the formula (II), R 2 Represents a metal ion chelating group, A represents a radiometal nuclide, and b is selected from positive integers.
Preferably, the radionuclides M n+ Or A b+ Are respectively and independently selected from 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 、 177 Lu 3 + The method comprises the steps of carrying out a first treatment on the surface of the Further preferably, the radionuclide M n+ Or A b+ Are respectively and independently selected from 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 。
Preferably, the metal ion chelating group R 1 Or R is 2 Each independently selected from the structures represented by the formulae (1) to (9):
Figure BDA0003600863900000031
preferably, the formula (1) is 1,4, 7-triazacyclononalkyl-4, 7-diacetoxy-1-acetyl (-NOTA).
Preferably, the formula (2) is 1- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononalkyl-4, 7-diacetic acid (p-SCN-Bn-NODA).
Preferably, the formula (3) is 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA).
Preferably, the formula (4) is 1- (2- (2- (2, 5-dioxo-1-pyrrolidinyl) ethylamino) acyl-ethyl) -1,4, 7-tetraazacyclononane-4, 7-diacetic acid (MaI-NODA).
Preferably, the formula (5) is 1- (2, 5-dioxo-1-pyrrolidinyl) acyl ethyl) -1,4, 7-tetraazacyclononane-4, 7-diacetic acid.
Preferably, the formula (6) is 2- ((4, 7-dicarboxymethyl) -1,4,7 triazacyclononane) yl glutarate.
Preferably, the formula (7) is 1,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA).
Preferably, the formula (8) is 1- (2- (2- (2, 5-dioxo-1-pyrrolidinyl) ethylamino) ethyl) -1,4,7, 10-tetraazacyclononane-4, 7, 10-triacetic acid (MaI-NODA).
Preferably, the formula (9) is 2- (4-isothiocyanatobenzyl) -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (p-SCN-Bn-DOTA).
Preferably, the targeted G-quadruplex polypeptide PET imaging agent comprises a compound with the structure shown in the specification;
Figure BDA0003600863900000041
/>
Figure BDA0003600863900000051
preferably, the amino acid in the polypeptide structure is an L-type amino acid.
Preferably, the amino acid sequence of the polypeptide is KRIVKLIKKWLR.
According to a second aspect of the invention, there is provided a method for preparing a targeted G-quadruplex polypeptide PET imaging agent according to the first aspect of the invention, comprising the steps of:
and (3) reacting the PET imaging agent precursor with the radionuclide in a buffer solution to obtain the targeted G-quadruplex polypeptide PET imaging agent.
Preferably, the buffer solution comprises at least one of sodium acetate, ammonium acetate, potassium acetate, hydrochloric acid and acetic acid; further preferably, the buffer solution is sodium acetate.
Preferably, the concentration of the buffer solution is 0.05mol/L to 0.7mol/L; further preferably, the concentration of the buffer solution is 0.1mol/L to 0.6mol/L.
Preferably, the reaction is carried out at a pH of 3.5 to 7.0; further preferably, the reaction is carried out at a pH of 4.0 to 5.5.
Preferably, the temperature of the reaction is 80-120 ℃; further preferably, the temperature of the reaction is from 90 ℃ to 110 ℃.
Preferably, the reaction time is 8min-40min; further preferably, the reaction time is 10min to 30min.
Preferably, the reaction further comprises the step of separating and purifying the targeting G-quadruplex polypeptide PET imaging agent.
Preferably, the separation and purification comprises separation and purification of the targeted G-quadruplex polypeptide PET imaging agent by using an HLB (HLB) column or a Sep-pak C18 column.
In a third aspect, the invention provides the use of a targeted G-quadruplex polypeptide PET imaging agent according to the first aspect of the invention in imaging biological functions.
Preferably, the biological function imaging comprises disease biological function imaging.
Preferably, the disease comprises a tumor.
Preferably, the tumor comprises at least one of brain glioma, lung cancer or breast cancer.
The beneficial effects of the invention are as follows:
the invention discloses a targeting G-quadruplex polypeptide PET imaging agent, which is a novel excellent pharmacokinetic probe, the structure of the PET imaging agent comprises polypeptide, metal chelating groups and radiometal nuclides, the preparation method is simple, convenient and quick, the yield is high, the PET imaging agent has good stability and strong water solubility, the in vivo blood is cleared quickly, the targeting G-quadruplex polypeptide PET imaging agent is mainly metabolized by kidneys and livers, and can be specifically ingested at tumor sites in a targeting way, and the PET imaging agent can be widely applied to biological function imaging.
Drawings
FIG. 1 is an HPLC chromatogram of precursor NOTA-KR 12C.
FIG. 2 is a high resolution mass spectrum of precursor NOTA-KR 12C.
FIG. 3 shows targeting of G-quadruplex polypeptides as PET imaging agents 18 F]Radioactive HPLC profile of AlF-NOTA-KR 12C.
FIG. 4 shows targeting of G-quadruplex polypeptides as PET imaging agents 68 Ga]Radioactivity HPLC profile of NOTA-KR 12C.
FIG. 5 is [ 18 F]Radioactive HPLC profile of AlF-NOTA-KR12C injection in PBS 2h in vitro.
FIG. 6 is [ 18 F]Radioactive HPLC profile of AlF-NOTA-KR12C injection in serum 1h in vivo.
FIG. 7 is [ 18 F]AlF-NOTA-KR12C in vivo in Kunming mice for 60 minutes.
FIG. 8 is [ 18 F]AlF-NOTA-KR12C images from Mico-PET/CT at various time points within 120 minutes in U87 tumor-bearing mice.
FIG. 9 is [ 18 F]AlF-NOTA-KR12C time plot of% ID/g values at various time points of the viscera.
FIG. 10 is [ 18 F]AlF-NOTA-KR12C images and competitive inhibition images in U87 tumor-bearing mice for 60 minutes Mico-PET/CT.
FIG. 11 is [ 18 F]AlF-NOTA-KR12C in HeLa tumor-bearing mice for 60 minutes Mico-PET/CT imaging and competitive inhibition imaging.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but are not intended to limit the practice and protection of the invention. It should be noted that the following processes, if not specifically described in detail, can be realized or understood by those skilled in the art with reference to the prior art. The reagents or instruments used did not identify the manufacturer and were considered conventional products available commercially.
Example 1
The precursor NOTA-KR12C was prepared as follows:
the method is characterized in that L-type amino acid is used as a raw material, KR12C polypeptide is synthesized through a solid-phase polypeptide synthesis method, a metal ion chelating group is 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA), the side chain amino group of lysine (Lys) at the N end is connected with NOTA, and a preparation type HPLC is utilized to separate, purify and collect product peaks, and then freeze-dried to obtain a NOTA-KR12C precursor. FIG. 1 is an HPLC profile of precursor NOTA-KR 12C; FIG. 2 is a high resolution mass spectrum of precursor NOTA-KR 12C. HPLC profile retention time Rt is 11.708, mass spectrum MS (M/z) molecular weight [ M+2H]2H + The purity of the precursor structure of NOTA-KR12C was greater than 97% by HPLC and high resolution mass spectrometry at 1016.75.
Targeting G-quadruplex polypeptides PET imaging agent 18 F]The preparation steps of AlF-NOTA-KR12C are as follows:
by cyclotrons 18 O(p,n) 18 Produced by F nuclear reaction 18 F - Under helium loading, the enrichment was carried out in a Sep-Pak QMA anion column. The QMA column is filled with 0.3 mL-0.4 mL of physiological saline (the mass fraction is 0.9 percent) 18 F - Eluting into small bottle for use. In a reaction flask containing NOTA-KR12C (1. Mu.g/. Mu.L, 50. Mu.L) prepared in step 1, 2mmol/L AlCl was added sequentially 3 mu.L of the solution, 5. Mu.L of glacial acetic acid and 300. Mu.L of acetonitrile were mixed. 50 mu L of the above 18 F - Adding the eluent into a reaction bottle, stirring and uniformly mixing, and heating at 100 ℃ for reaction for 15min. Cooling, adding 6-8 mL of water for injection into a reaction bottle, uniformly mixing, and transferring to a Sep-pak C-18 column. The Sep-pak C-18 column was then rinsed with 10mL×3 water for injection and blow dried. Finally, eluting the product with 1mL of ethanol, passing through a sterile filter membrane, collecting the product in a receiving bottle, diluting the product into a product solution containing 5% of ethanol by using physiological saline to obtain the product solution meeting the requirements 18 F]AlF-NOTA-KR12C injection. Uncorrected radiochemical yields of 30% -45% for total radiosynthesisThe interval is 30min. FIG. 3 shows targeting of G-quadruplex polypeptides as PET imaging agents 18 F]The radioactive HPLC spectrum of AlF-NOTA-KR12C shows the peak time of 12.317min for the target developer, and the radiochemical purity of the product is more than 99%.
Example 2
Targeting G-quadruplex polypeptides PET imaging agent 68 Ga]The preparation steps of NOTA-KR12C are as follows:
into a reaction tube containing 50. Mu.L of the precursor NOTA-KR12C prepared in example 1 (1. Mu.g/. Mu.L) was added 900. Mu.L of a sodium acetate solution having a concentration of 0.25 mol/L. From the slave 68 Ge/ 68 The Ga generator is eluted with 4mL hydrochloric acid with the concentration of 0.05mol/L 68 GaCl 3 Mixing the materials in the reaction tube, and heating at 100deg.C for reaction for 10-30min, wherein pH of the solution is about 4.0. Cooled and added with 4mL of water for injection, mixed evenly and transferred to an HLB column. The HLB column was rinsed with 10mL 2 water for injection and dried. Then eluting the product with 1mL of ethanol and diluting it with physiological saline to a product solution containing 5% ethanol to obtain the desired product 68 Ga]NOTA-KR12C injection. FIG. 4 shows targeting of G-quadruplex polypeptides as PET imaging agents 68 Ga]The peak time of the target imaging agent of the radioactive HPLC spectrum of NOTA-KR12C is 12.217min. [ 68 Ga]The uncorrected radiochemical yield of NOTA-KR12C is 70% -95% and the total radiosynthesis time is 30min.
Example 3
Targeting G-quadruplex polypeptides PET imaging agent 64 Cu]The preparation steps of NOTA-KR12C are as follows:
into a reaction tube containing 100. Mu.L of the precursor NOTA-KR12C prepared in example 1 (1. Mu.g/. Mu.L) was charged 1.0mL 64 CuCl 2 The pH of the solution (about 5 mCi) was adjusted to 4.0-6.5 with 0.1mol/L sodium acetate buffer solution, and the reaction was heated at 100℃for 10min-30min. Cooled and added with 4mL of water for injection, mixed evenly and transferred to an HLB column. The HLB column was rinsed with 10mL 2 water for injection and dried. Then eluting the product with 1mL of ethanol and diluting it with physiological saline to a product solution containing 5% ethanol to obtain the desired product 64 Cu]NOTA-KR12C (R is 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA)). [ 64 Cu]NOTA-KR12C uncorrected amplificationThe radiochemical yield is 70-95%, and the total radiosynthesis time is 30min.
Example 4
The precursor DOTA-KR12C is prepared as follows:
the method is characterized in that L-type amino acid is used as a raw material, KR12C polypeptide is synthesized through a solid-phase polypeptide synthesis method, a metal ion chelating group is 1,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), the side chain amino group of lysine (Lys) at the N end is connected with DOTA, and the DOTA-KR12C precursor is obtained after separation, purification and collection of product peaks through preparative HPLC.
Targeting G-quadruplex polypeptides PET imaging agent 68 Ga]The DOTA-KR12C was prepared as follows:
into a reaction tube containing 50. Mu.L of a precursor DOTA-KR12C solution (metal ion chelating group is 2,4,7,10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), 1. Mu.g/. Mu.L) was charged 900. Mu.L of a sodium acetate solution at 0.25 mol/. Mu.L. From the slave 68 Ge/ 68 Elution with 0.05mol/L hydrochloric acid 4mL in Ga generator 68 GaCl 3 Mixing the materials in the reaction tube, and heating and reacting at 100 ℃ for about 10-30min, wherein the pH value of the solution is 4.0. Cooled and added with 5mL of water for injection, mixed evenly and transferred to an HLB column. The HLB column was rinsed with 10mL 2 water for injection and dried. Then eluting the product with 1mL of ethanol and diluting it with physiological saline to a product solution containing 5% ethanol to obtain the desired product 68 Ga]DOTA-KR12C (R is 1,4,7, 10-triazacyclododecyl-4, 7, 10-triacetoxy-N-acetyl) injection. [ 68 Ga]The uncorrected radiochemical yield of DOTA-KR12C is 70% -95% and the total radiosynthesis time is 30min.
Example 5
Targeting G-quadruplex polypeptides PET imaging agent 64 Cu]The DOTA-KR12C was prepared as follows:
into a reaction tube were sequentially added 50. Mu.L of DOTA-KR12C prepared in example 4 (metal ion chelating group is 2,4,7,10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), 1. Mu.g/. Mu.L) and 1.0mL 64 CuCl 2 The solution (about 5 mCi) was adjusted to pH 4.0-6.5 with 0.1mol/L sodium acetate buffer and reacted at 100℃for 10-30 min. Cooling and addingMixing with 4mL of injectable water, and transferring to HLB column. The HLB column was rinsed with 10mL 2 water for injection and dried. Then eluting the product with 1mL of ethanol and diluting it with physiological saline to a product solution containing 5% ethanol to obtain the desired product 64 Cu]DOTA-KR12C injection. [ 64 Cu]The uncorrected radiochemical yield of DOTA-KR12C is 80-95%.
Performance detection
1. Determination of radiochemical purity and stability
The radiochemical purity and stability of the drug injection were determined by High Performance Liquid Chromatography (HPLC). HPLC analysis conditions: the analytical column is ZORBAX Eclipse XDB-C18 column; mobile phase: phase a was 0.1% trifluoroacetic acid (TFA) in water and phase B was 0.1% TFA in acetonitrile. The leaching gradient is as follows: 0-2min, 85% of phase A, 15% of phase B and 1mL/min of flow rate; 2min-20min, the phase A is reduced to 5%, the phase B is increased to 95%, and the flow rate is 1mL/min;20min-22min, the phase A is raised to 85%, the phase B is lowered to 15%, and the flow rate is 1mL/min;22min-30min, 85% of phase A, 15% of phase B and 1mL/min of flow rate. The ultraviolet detection wavelength is 220nm, and the radioactivity detector is LabLogic Systems Ltd.
To test the stability of the imaging agent in vitro, test separately 18 F]Stability of AlF-NOTA-KR12C in PBS (ph=7.4) and mouse serum.
2mL of PBS (pH=7.4) buffer was mixed well with 50. Mu. Ci imaging agent, incubated in an incubator at 37℃for 2h, a small amount of solution was taken, and the stability of the imaging agent was checked by HPLC, and the above experiment was repeated 4 times. FIG. 5 is [ 18 F]Radioactive HPLC profile of AlF-NOTA-KR12C injection in PBS 2h in vitro.
Taking Kunming mouse with weight of about 22g and tail vein injection 18 F]300 μCi of AlF-NOTA-KR12C injection is taken normally for 1h, 1.5mL of blood is collected from eyes, centrifugation is carried out for 5min under 12000 r/min, supernatant is taken for HPLC analysis, a tube is collected every 0.5 min of mobile phase, radioactivity count is measured by a gamma counter, and a stability HPLC image of the blood in the probe body is obtained by Origin mapping. FIG. 6 is [ 18 F]Radioactive HPLC profile of AlF-NOTA-KR12C injection in serum 1h in vivo.
FIGS. 5 and 6 result surfaces [ 18 F]AlF-NOTA-KR12C was found to be 100% stable in prototype in both in vitro (PBS) and in vivo (serum).
2. Ester water distribution coefficient test
10 mu L of the prepared [ 18 F]AlF-NOTA-KR12C injection is put into a 2.5mL centrifuge tube filled with 1mL of n-octanol and 990 mu L of water, and is placed in a dry type thermostat in a sealed manner, and is oscillated for 10min at normal temperature, and is kept stand for 10min to separate two phases, 500 mu L of each of the two phases is taken out of the two phases by a liquid transfer device and placed into a gamma counter tube, and the gamma counter is used for measuring and counting. Two experiments were performed in parallel, each repeated three times. The Log P value is calculated according to equation 1.
Figure BDA0003600863900000101
In equation 1, counts in water represents the radioactivity count in the aqueous phase; counts in 1-octanol represents the radioactive counts in the n-octanol phase; log takes the base 10 logarithm.
Determination by means of a radioactive technique [ 18 F]The lipid water distribution coefficient log P of AlF-NOTA-KR12C is-2.409+/-0.173, which shows that the imaging agent is a water-soluble substance and has good hydrophilic property, and the in-vivo uptake and the imaging agent are predicted to be mainly metabolized by the kidney, other soft tissues can be lower in uptake, and the imaging background uptake is lower.
3.[ 18 F]AlF-NOTA-KR12C in vivo biodistribution test
EGFR-targeting polypeptide PET imaging agent prepared in example 1 18 F]After AlF-NOTA-KR12C, after 30 mu Ci imaging agent is respectively injected into 4 normal Kunming mice through tail veins, the mice are sacrificed after normal feeding and ingestion for 1h, main organs and tissues such as blood, brain, heart, lung, liver, kidney and the like are weighed and gamma counting is carried out, and the biodistribution of the imaging agent in the mice is studied. FIG. 7 is [ 18 F]AlF-NOTA-KR12C in vivo in Kunming mice for 60 minutes. The results show that the medicine is mainly metabolized by the kidney, the blood clearance speed is high, the radioactivity in the bone is not high, and the medicine is not defluorinated in the body.
Micro PET/CT imaging test
Micro-PET/CT-imaging studies utilized Siemens Inveon Micro-PET/CT with an acquisition workstation of Inveon Acquirision Workplace (IAW) 2.2 and a data analysis workstation of Inveon Research Workplace (IRW). Taking brain glioma U87 and cervical cancer cell HeLa at a ratio of 5×10 6 Density of nude mice was inoculated subcutaneously and when the tumor diameter had grown to 10mm-15mm, the imaging agent study was performed.
Dynamic imaging: taking U87 tumor-bearing mice, fixing the mice on a scanning bed after being anesthetized by 10 percent chloral hydrate, establishing a method to ensure that the bed is positioned in a PET visual field, taking [ 18 F]AlF-NOTA-KR12C injection is about 200 μCi, and is injected through tail vein, and the start button is clicked at the same time of injection, and the scanning is continued for 120 minutes.
Static imaging: fetch [ 18 F]About 200 μCi of AlF-NOTA-KR12C injection is injected into U87 or HeLa tumor-bearing mice via tail vein, and after conventional ingestion for 40 minutes, 0.1mL of 10% chloral hydrate is injected, followed by conventional PET/CT imaging.
Inhibition of development: other procedures were followed as "dynamic imaging" and "static imaging", with the addition of 250 μg KR12C polypeptide to the injected drug.
FIG. 8 is [ 18 F]The AlF-NOTA-KR12C is in a U87 tumor-bearing mouse at different time points Mico-PET/CT imaging diagram within 120 minutes, wherein a CT diagram, a 15min PET diagram, a 30min PET diagram, a 60min PET diagram, a 90min PET diagram and a 120min PET diagram of the cross section of a tumor part are sequentially arranged above the diagram 8; the lower part of FIG. 8 is a CT image, a 15min PET image, a 30min PET image, a 60min PET image, a 90min PET image and a 120min PET image of the coronary surface of the tumor part in sequence. FIG. 9 is [ 18 F]AlF-NOTA-KR12C time plot of% ID/g values at various time points of the viscera. FIG. 10 is [ 18 F]AlF-NOTA-KR12C was shown in 60-minute Mico-PET/CT and competitive inhibition images in U87 tumor-bearing mice, wherein the upper left was the uptake CT, the upper right was the uptake PET, the lower left was the inhibition CT, and the lower right was the inhibition PET. FIG. 11 is [ 18 F]AlF-NOTA-KR12C was shown in 60-minute Mico-PET/CT and competitive inhibition images in HeLa tumor-bearing mice, wherein the upper left was the uptake CT, the upper right was the uptake PET, the lower left was the inhibition CT, and the lower right was the inhibition PET.
PET imaging results show that: [ 18 F]AlF-NOTA-KR12C has higher uptake in U87 and HeLa tumor-bearing mice tumors, and the uptake of tumor sites is obviously higher than that of organs or tissues such as muscles, lungs, intestines and the like. Dynamic imaging shows that the probe has fast in vivo blood clearance, is mainly metabolized by kidneys and livers, and the time dynamic graph of each organ discovers that the tumor/muscle ratio is higher about 60 minutes, so the optimal imaging time is selected to be 60 minutes, the imaging result is inhibited for 60 minutes, the tumor/muscle ratio is obviously reduced, and the probe is shown to be specifically ingested at the tumor part. The imaging result shows that the imaging agent can be specifically absorbed at the tumor part, and has better application prospect.
In this embodiment, KR12C polypeptide structure is used as a pharmacophore, and a metal ion chelating group is connected to construct a precursor, and a radioactive metal nuclide M is used n+ Marker constructs such as [ M ] n+ ]NOTA-KR12C or [ M n+ ]The structure of the DOTA-KR12C probe is shown as a formula (I) to obtain a specific targeted G-quadruplex polypeptide PET imaging agent;
Figure BDA0003600863900000111
in the formula (I), R 1 Represents a metal ion chelating group, M represents a radiometal nuclide, and n is selected from positive integers.
In the embodiment of the application, after a cysteine (Cys) is modified at the N end of KR12C polypeptide lysine (Lys), a sulfhydryl side chain of the cysteine is reacted with a metal ion chelating group (such as NOTA or DOTA analogue) to construct a precursor, and then the precursor is reacted with a radioactive metal nuclide A b+ Reacting in buffer solution to obtain [ A ] b+ ]NOTA-KR12C or [ A ] b+ ]DOTA-KR12C, a targeting G-quadruplex polypeptide PET imaging agent shown as a formula (II);
Figure BDA0003600863900000121
in the formula (II), R 2 Represents a metal ion chelating group, A b+ And b represents a radionuclide, b being selected from positive integers.
[M n+ ]NOTA-KR12C、[M n+ ]DOTA-KR12C、[A b+ ]NOTA-KR12C or [ A ] b+ ]The DOTA-KR12C probe has the condition of being a useful tool for diagnosing malignant tumors, and provides powerful imaging evidence for the determination of treatment schemes and the monitoring of curative effects.
The foregoing examples are illustrative of the present invention and are not intended to be limiting, but rather, the invention is intended to be limited to the specific embodiments shown, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent substitutes and modifications within the scope of the invention.

Claims (9)

1. A targeted G-quadruplex polypeptide PET imaging agent, characterized in that: the structure of the targeting G-quadruplex polypeptide PET imaging agent is shown as a formula (I):
Figure FDA0004174671620000011
in the formula (I), R 1 Represents a metal ion chelating group, M represents a radiometal nuclide, and n is selected from positive integers;
the radiometal nuclides M n+ Selected from the group consisting of 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 、 177 Lu 3+ 。
2. The targeted G-quadruplex polypeptide PET imaging agent of claim 1, wherein: the metal ion chelating group R 1 Selected from the structures shown in the following formulas (1) - (2):
Figure FDA0004174671620000012
3. the targeted G-quadruplex polypeptide PET imaging agent of any one of claims 1-2, wherein: the targeted G-quadruplex polypeptide PET imaging agent comprises a compound with a structure shown as the following;
Figure FDA0004174671620000021
Figure FDA0004174671620000031
4. a method for preparing a targeted G-quadruplex polypeptide PET imaging agent according to any one of claims 1 to 3, characterized in that: the method comprises the following steps:
and (3) reacting the PET imaging agent precursor with the radionuclide in a buffer solution to obtain the targeted G-quadruplex polypeptide PET imaging agent.
5. The method of manufacturing according to claim 4, wherein: the buffer solution comprises at least one of sodium acetate, ammonium acetate, potassium acetate, hydrochloric acid and acetic acid.
6. The method of manufacturing according to claim 4, wherein: the reaction is carried out at a pH of 3.5 to 7.0.
7. The method of manufacturing according to claim 5, wherein: the concentration of the buffer solution is 0.05mol/L to 0.7mol/L.
8. The method of manufacturing according to claim 4, wherein: the temperature of the reaction is 80-120 ℃; the reaction time is 8min-40min.
9. Use of a targeted G-quadruplex polypeptide PET imaging agent according to any one of claims 1-3 for the preparation of a biological functional diagnostic reagent.
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