CN114796535A - Targeting G-quadruplex polypeptide PET (polyethylene terephthalate) imaging agent as well as preparation method and application thereof - Google Patents
Targeting G-quadruplex polypeptide PET (polyethylene terephthalate) imaging agent as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a targeting G-quadruplex polypeptide PET imaging agent and a preparation method and application thereof, the PET imaging agent is an excellent pharmacokinetic novel probe, the structure of the PET imaging agent comprises polypeptide, metal chelating group and radioactive metal nuclide, the preparation method is simple, convenient and quick, the yield is higher, the PET imaging agent has good stability, strong water solubility and quick in vivo blood clearance, can be specifically absorbed in tumor parts mainly through metabolism of kidney and liver, and can be widely applied to biological function imaging.
Description
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 tumor (cancer) is one of the biggest public health problems in the world, and the traditional cancer treatment methods mainly comprise surgical treatment, chemotherapy, radiation therapy and the like. The traditional Chinese medicine composition can not be used for effectively performing operative treatment and radiotherapy on non-solid tumors and cancers widely metastasized throughout the body, and meanwhile, the chemotherapy method also has the problems of large side effect, poor specificity, low curative effect and the like, so that the requirement of clinical treatment is difficult to meet. Compared with common chemotherapy, molecular targeted therapy aiming at specific oncogenes, proteins or receptors has the advantages of strong specificity, obvious curative effect and obvious small side effect, and is gradually one of important means for clinical tumor treatment. In recent years, research and development of new anti-tumor drugs have been greatly advanced, and a large number of new target drugs and new target drugs are developed and applied, wherein research on anti-tumor drugs taking a G-quadruplex as a target has attracted extensive attention.
The G-quadruplex (G-quadruplex) is an atypical structure which can be formed in a nucleic acid sequence rich in guanine (G), a DNA single strand forms a planar G-quadruplex through hydrogen bonding between G bases, and two or more layers of the quadruplex form a G-quadruplex spiral structure through pi-pi accumulation. About 70 million gene sequences capable of forming G-quadruplexes are arranged in a human body, mainly comprise telomere ends, mutation hot spots of genes, promoter regions of genes (such as Bcl-2, c-myc, c-myb, c-kit, Kras, VEGF and VEGFR), cover genes related to senescence, apoptosis, growth factors and receptors thereof, transcription regulatory factors and signal transduction factors, and play 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, corresponding biochemical processes are changed (such as the activity of telomerase is inhibited, the transcription level of oncogenes is reduced, and the like), so that the anti-tumor effect is achieved.
Positron emission tomography (positron emission tomography) has been widely used in diagnosis and differential diagnosis of various diseases, treatment effect evaluation, organ function research, new drug development and other methodsAnd (5) kneading. Broad-spectrum tumor imaging agent with most clinical applications 18 The F-FDG has the problems of false positive, incapability of distinguishing tumors and inflammations and the like in tumor detection, and although the current PET imaging agents of various novel specific targets (such as PSMA, FAP, EGFR and the like) are developed, researched and applied, and more new targets are also tried to develop related PET imaging agents, the PET imaging agents still have the problems of false positive, incapability of distinguishing tumors and inflammations and the like. The target property of the G-quadruplex determines that the specific PET/CT imaging of the targeted G-quadruplex has the potential of real-time, noninvasive, in-vivo definite tumor focus distribution, early diagnosis of solid tumor, tumor staging, preoperative positioning, postoperative contrast and curative effect monitoring, and can be used for establishing a personalized treatment scheme and guiding operation or targeted treatment. Meanwhile, the PET imaging taking the G-quadruplex as a target has important research and application prospects in the aspects of guiding the research and development of the novel tumor drugs, evaluating the drug effect, screening sensitive people and the like, so that the development of a novel related PET probe targeting the G-4 quadruplex has important significance for biological function imaging.
Disclosure of Invention
In order to overcome the problems of the prior art, the invention aims to provide a targeting G-quadruplex polypeptide PET imaging agent; the invention also aims to provide a preparation method of the targeting G-quadruplex polypeptide PET imaging agent; the invention also aims to provide the application of the targeting G-quadruplex polypeptide PET imaging agent.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a targeting G-quadruplex polypeptide PET imaging agent, which has a structure shown in formula (I) or formula (II):
in the formula (I), R 1 Represents a metal ion chelating group, M represents a radioactive metal nuclide, and n is selected from positive integers;
in the formula (II), R 2 Represents a metal ion chelating group, A represents a radioactive metal nuclide, and b is selected from positive integers.
Preferably, the radioactive metal species M n+ Or A b+ Are each independently selected from 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 、 177 Lu 3 + (ii) a Further preferably, the radioactive metal species M n+ Or A b+ Are each independently selected from 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 。
Preferably, the metal ion chelating group R 1 Or R 2 Are respectively and independently selected from structures shown in formulas (1) to (9):
preferably, the formula (1) is 1,4, 7-triazacyclononaalkyl-4, 7-diacetoxy-1-acetyl (-NOTA).
Preferably, the compound of formula (2) is 1- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononanyl-4, 7-diacetic acid (p-SCN-Bn-NODA).
Preferably, the compound of formula (3) is 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononaalkane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA).
Preferably, the compound of formula (4) is 1- (2- (2- (2, 5-dioxo-1-pyrrolidinyl) ethylamino) acylethyl) -1,4, 7-tetraazacyclononalkane-4, 7-diacetic acid (MaI-NODA).
Preferably, the compound of formula (5) is 1- (2- (2, 5-dioxo-1-pyrrolidinyl) acylethyl) -1,4, 7-tetraazacyclononalkane-4, 7-diacetic acid.
Preferably, the compound of formula (6) is 2- ((4, 7-dicarboxymethyl) -1,4,7 triazacyclononane) glutaric acid.
Preferably, the compound of formula (7) is 1,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA).
Preferably, the compound of formula (8) is 1- (2- (2- (2, 5-dioxo-1-pyrrolidinyl) ethylamino) acylethyl) -1,4,7, 10-tetraazacyclononalkane-4, 7, 10-triacetic acid (MaI-NODA).
Preferably, the compound of formula (9) is 2- (4-isothiocyanatobenzyl) -1,4,7, 10-tetraazacyclododecane-1, 4,7, 10-tetraacetic acid (p-SCN-Bn-DOTA).
Preferably, the G-quadruplex polypeptide-targeted PET imaging agent comprises a compound with the structure shown in the specification;
preferably, the amino acid in the polypeptide structure is an L-type amino acid.
Preferably, the amino acid sequence of the polypeptide is KRIVKLIKKWLR.
In a second aspect, the invention provides a method for preparing the targeting G-quadruplex polypeptide PET imaging agent according to the first aspect, comprising the following steps:
and (3) reacting the PET imaging agent precursor with radioactive metal nuclide 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-0.7 mol/L; further preferably, the concentration of the buffer solution is 0.1mol/L to 0.6 mol/L.
Preferably, the reaction is carried out at a pH value 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 between 90 ℃ and 110 ℃.
Preferably, the reaction time is 8min-40 min; further preferably, the reaction time is 10min to 30 min.
Preferably, the reaction further comprises a step of separating and purifying the targeting G-quadruplex polypeptide PET imaging agent.
Preferably, the separation and purification comprises the separation and purification of the targeting G-quadruplex polypeptide PET imaging agent by using an HLB column or a Sep-pak C18 column.
In a third aspect, the invention provides the use of a targeting G-quadruplex polypeptide PET imaging agent according to the first aspect of the invention for 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 invention has the beneficial effects that:
the invention discloses a targeting G-quadruplex polypeptide PET imaging agent, which is an excellent pharmacokinetic novel probe, the structure of the PET imaging agent comprises polypeptide, metal chelating group and radioactive metal nuclide, the preparation method is simple, convenient and quick, the yield is higher, the PET imaging agent has good stability, strong water solubility and quick in vivo blood clearance, can be specifically absorbed in tumor parts by mainly metabolism of kidney and liver, and 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 is the PET imaging agent of targeting G-quadruplex polypeptide 18 F]Radioactive HPLC profile of AlF-NOTA-KR 12C.
FIG. 4 is a PET imaging agent of a target G-quadruplex polypeptide 68 Ga]A radioactive HPLC profile of NOTA-KR 12C.
FIG. 5 is a schematic view of a term 18 F]Radioactivity HPLC profile of AlF-NOTA-KR12C injection at 2h in PBS in vitro.
FIG. 6 is [ 2 ] 18 F]Serum of AlF-NOTA-KR12C injection in vivo for 1hRadioactivity HPLC profile of time.
FIG. 7 is [ 2 ] 18 F]Biodistribution of AlF-NOTA-KR12C in Kunming mice for 60 min.
FIG. 8 is a cross section of a word "2 18 F]Mico-PET/CT images of AlF-NOTA-KR12C were visualized at different time points within 120min in U87 tumor-bearing mice.
FIG. 9 is [ 2 ] 18 F]AlF-NOTA-KR12C is a graph showing the change in% ID/g value with time at different time points in the organ.
FIG. 10 is a cross section of [ 2 ] 18 F]The Mico-PET/CT visualization and the competitive inhibition visualization were performed at 60 minutes in U87 tumor-bearing mice in AlF-NOTA-KR 12C.
FIG. 11 is [ 2 ] 18 F]The 60-minute Mico-PET/CT visualization and the competitive inhibition visualization of AlF-NOTA-KR12C in HeLa tumor-bearing mice.
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available through commercial purchase.
Example 1
The precursor NOTA-KR12C was prepared as follows:
the method comprises the steps of synthesizing KR12C polypeptide by using L-type amino acid as a raw material through a solid-phase polypeptide synthesis method, enabling a metal ion chelating group to be 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononaalkane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA), connecting the side chain amino of lysine (Lys) at the N end with NOTA, separating, purifying and collecting a product peak by using preparative HPLC (high performance liquid chromatography), and freeze-drying to obtain the NOTA-KR12C precursor. FIG. 1 is an HPLC chromatogram of precursor NOTA-KR 12C; FIG. 2 is a high resolution mass spectrum of precursor NOTA-KR 12C. Retention time Rt of HPLC chromatogram is 11.708, mass spectrum MS (M/z) molecular weight [ M +2H]2H + 1016.75, purity greater than 97% by HPLC, NOTA-KR12C precursor structure determined by HPLC and high resolution mass spectrometry.
Targeting G-quadruplex polypeptide PET imaging agent [ 2 ] 18 F]The preparation steps of AlF-NOTA-KR12C are as follows:
by a cyclotron 18 O(p,n) 18 Produced by reaction of F nuclei 18 F - On helium loading, enriched in a Sep-Pak QMA anion column. Adding 0.3-0.4 mL of physiological saline (mass fraction is 0.9%) into the QMA column 18 F - Elute into a vial for use. 2mmol/L AlCl was added sequentially to a reaction flask containing NOTA-KR12C (1. mu.g/. mu.L, 50. mu.L) prepared in step 1 3 mu.L of the solution, 5. mu.L of glacial acetic acid and 300. mu.L of acetonitrile were mixed well. Collecting 50 μ L of the above 18 F - Adding the eluent into a reaction bottle, stirring uniformly, and heating to react for 15min at 100 ℃. And cooling, adding 6-8 mL of water for injection into the reaction bottle, uniformly mixing, and transferring to a Sep-pak C-18 column. The Sep-pak C-18 column was then washed with 10mL of X3 water for injection and blown dry. Finally, the product eluted by 1mL of ethanol is collected in a receiving bottle after passing through a sterile filter membrane, and is diluted into a product solution containing 5 percent of ethanol by using normal saline to obtain the product solution meeting the requirement 18 F]AlF-NOTA-KR12C injection. Uncorrected radiochemical yield is 30-45% and total radiosynthesis time is 30 min. FIG. 3 is a PET imaging agent of a target G-quadruplex polypeptide 18 F]And the radioactive HPLC pattern of AlF-NOTA-KR12C shows that the peak time of the target imaging agent is 12.317min, and the radiochemical purity of the product is more than 99%.
Example 2
Targeting G-quadruplex polypeptide PET imaging agent [ 2 ] 68 Ga]NOTA-KR12C was prepared by the following steps:
to a reaction tube containing 50. mu.L of the precursor NOTA-KR12C (1. mu.g/. mu.L) prepared in example 1 was added 900. mu.L of a sodium acetate solution having a concentration of 0.25 mol/L. From 68 Ge/ 68 The Ga generator is eluted by 4mL of hydrochloric acid with the concentration of 0.05mol/L 68 GaCl 3 Mixing the above materials in the reaction tube, adjusting pH to 4.0, and heating at 100 deg.C for 10-30 min. Cooled and mixed with 4mL of water for injection, and transferred to an HLB column. The HLB column was rinsed with 10mL of 2 water for injection and blown dry. Then, the product was eluted with 1mL of ethanol and diluted with physiological saline to give a product solution containing 5% ethanol 68 Ga]NOTA-KR12C injection. FIG. 4 is a PET imaging agent of a target G-quadruplex polypeptide 68 Ga]Radioactivity HPLC profile of NOTA-KR12C, targetingThe peak time of the imaging agent is 12.217 min. [ 68 Ga]The uncorrected radiochemical yield of NOTA-KR12C was 70% to 95% and the total radiosynthesis time was 30 min.
Example 3
Targeting G-quadruplex polypeptide PET imaging agent [ 2 ] 64 Cu]NOTA-KR12C was prepared by the following steps:
to a reaction tube containing 100. mu.L of the precursor NOTA-KR12C (1. mu.g/. mu.L) prepared in example 1 was added 1.0mL 64 CuCl 2 The solution (about 5mCi) is adjusted to pH value of 4.0-6.5 by 0.1mol/L sodium acetate buffer solution, and is heated and reacted for 10min-30min at 100 ℃. Cooled and mixed with 4mL of water for injection, and transferred to an HLB column. The HLB column was rinsed with 10mL of 2 water for injection and blown dry. Then, the product was eluted with 1mL of ethanol and diluted with physiological saline to give a product solution containing 5% ethanol 64 Cu]NOTA-KR12C (R is 2- (4-isothiocyanatobenzyl) -1,4, 7-triazacyclononane-1, 4, 7-triacetic acid (p-SCN-Bn-NOTA)) injection. [ 64 Cu]The uncorrected radiochemical yield of NOTA-KR12C was 70% to 95% and the total radiosynthesis time was 30 min.
Example 4
The precursor DOTA-KR12C was prepared as follows:
the KR12C polypeptide is synthesized by a solid phase polypeptide synthesis method by using L-type amino acid as a raw material, a metal ion chelating group is 1,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), a side chain amino group of lysine (Lys) at the N end is connected with the DOTA, and a product peak is separated, purified and collected by preparative HPLC (high performance liquid chromatography), and then is lyophilized to obtain the DOTA-KR12C precursor.
Targeting G-quadruplex polypeptide PET imaging agent [ 2 ] 68 Ga]DOTA-KR12C was prepared as follows:
to a reaction tube containing 50. mu.L of a precursor DOTA-KR12C solution (metal ion chelating group: 2,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), 1. mu.g/. mu.L) was added 900. mu.L of a 0.25mol/L sodium acetate solution. From 68 Ge/ 68 Eluting with 4mL of 0.05mol/L hydrochloric acid in a Ga generator 68 GaCl 3 Mixing the above materials in the reaction tube, adjusting pH to 4.0, and heating at 100 deg.C for 10-30 min. Cooling and addingThe mixture was mixed with 5mL of water for injection and transferred to an HLB column. The HLB column was rinsed with 10mL of 2 water for injection and blown dry. Then, the product was eluted with 1mL of ethanol and diluted with physiological saline to give a product solution containing 5% ethanol 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 was 70% to 95% and the total radiosynthesis time was 30 min.
Example 5
Targeting G-quadruplex polypeptide PET imaging agent [ 2 ] 64 Cu]DOTA-KR12C was prepared as follows:
to a reaction tube were added 50. mu.L of DOTA-KR12C (metal ion chelating group: 2,4,7, 10-tetraazacyclododecyl-4, 7, 10-triacetoxy-1-acetyl (-DOTA), prepared in example 4, and 1.0mL in this order 64 CuCl 2 The solution (about 5mCi) is adjusted to pH 4.0-6.5 with 0.1mol/L sodium acetate buffer solution and reacted at 100 ℃ for 10-30 min. Cooled and mixed with 4mL of water for injection, and transferred to an HLB column. The HLB column was rinsed with 10mL of 2 water for injection and blown dry. Then, the product was eluted with 1mL of ethanol and diluted with physiological saline to give a product solution containing 5% ethanol 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 pharmaceutical injection is determined by High Performance Liquid Chromatography (HPLC). Conditions for HPLC analysis: the analytical column is a 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 elution gradient is as follows: 0-2min, 85% of phase A and 15% of phase B, and the flow rate is 1 mL/min; 2min-20min, reducing the phase A to 5%, increasing the phase B to 95%, and enabling the flow rate to be 1 mL/min; 20min-22min, the phase A is increased to 85%, the phase B is decreased to 15%, and the flow rate is 1 mL/min; 22min-30min, 85% of phase A and 15% of phase B, and the flow rate is 1 mL/min. The ultraviolet detection wavelength is 220nm, and the radioactivity detector is LabLogic Systems Ltd.
For detecting the imaging agent in vivoExternal stability, respectively test [, ] 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 μ Ci of the imaging agent, incubated in an incubator at 37 ℃ for 2h, a small amount of the solution was taken, and the imaging agent stability was checked by HPLC, and the above experiment was repeated 4 times. FIG. 5 is a schematic view of a term 18 F]Radioactivity HPLC profile of AlF-NOTA-KR12C injection at 2h in PBS in vitro.
The Kunming mouse with the weight of about 22g is injected into the tail vein 18 F]300 mu Ci of AlF-NOTA-KR12C injection, after normal ingestion for 1h, 1.5mL of blood is obtained by eye picking, centrifugation is carried out at 12000 rpm for 5min, the supernatant is taken for HPLC analysis, one tube is collected every 0.5 min by a mobile phase, then radioactivity counting is measured by a gamma counter, and a probe in-vivo blood stability HPLC chart is obtained by Origin mapping. FIG. 6 is [ 2 ] 18 F]Radioactivity HPLC pattern of AlF-NOTA-KR12C injection at 1h in serum.
FIG. 5 and FIG. 6 result surface [ 2 ] 18 F]AlF-NOTA-KR12C was 100% stably present in vitro (PBS) and in vivo (serum) as the prototype.
2. Ester Water partition coefficient test
Taking 10 mu L of prepared 18 F]Placing the AlF-NOTA-KR12C injection in a 2.5mL centrifuge tube containing 1mL n-octanol and 990 μ L water, sealing, placing in a dry thermostat, shaking at normal temperature for 10min, standing for 10min to allow two phases to separate, placing 500 μ L of the two phases in a γ counting tube by a pipette, and counting by a γ counter. Two batches of experiments were performed in parallel, three replicates per batch. And calculating according to a formula 1 to obtain a Log P value.
In equation 1, counts in water represent the number of radioactivity counts in the aqueous phase; counts in 1-octanol represent the radioactivity counts in the n-octanol phase; log takes the base 10 logarithm.
Measured by a radioactive technique 18 F]The lipid-water partition coefficient log P of AlF-NOTA-KR12C is-2.409 +/-0.173, which indicates that the imaging agent is water-soluble substanceThe contrast medium has good hydrophilic property, and is predicted to be mainly metabolized by the kidney with the imaging agent in vivo, the possibility of lower uptake by other soft tissues is possibly lower, and the imaging background uptake is lower.
3.[ 18 F]AlF-NOTA-KR12C in vivo biodistribution assay
Example 1 the EGFR-targeting polypeptide PET imaging agent prepared 18 F]After AlF-NOTA-KR12C, after injecting 30 μ Ci of imaging agent into 4 normal Kunming mice respectively via tail vein, after feeding and ingesting for 1h, the mice were sacrificed, blood and major organs and tissues such as brain, heart, lung, liver, kidney and the like were taken and weighed and subjected to gamma counting, and the biodistribution condition of the imaging agent in the mice was studied. FIG. 7 is [ 2 ] 18 F]Biodistribution of AlF-NOTA-KR12C in Kunming mice for 60 min. The results show that the drug is mainly metabolized by the kidney, the blood clearance speed is high, the radioactivity in the bone is not high, and the drug is not defluorinated in the body.
Micro PET/CT imaging test
Micro-PET/CT imaging study Using Siemens Inveon Micro-PET/CT, the acquisition workstation was Inveon Acquisition Workplace (IAW)2.2 and the data analysis workstation was Inveon Research Workplace (IRW). Taking glioma U87 and cervical cancer cell HeLa at 5 × 10 6 The density of the mice is used for subcutaneous inoculation of the nude mice, and the developer research is carried out when the tumor grows to 10mm-15mm in diameter.
Dynamic imaging: the U87 tumor-bearing mouse is anesthetized with 10% chloral hydrate and then fixed on a scanning bed, and a method is established to make the bed position be positioned in the PET visual field, and the [ 2 ], [ is ] is selected 18 F]AlF-NOTA-KR12C injection (about 200. mu. Ci) was injected via tail vein, and the "start" button was clicked during injection, and scanning was continued for 120 minutes.
Static development: 2 [ 2 ] 18 F]About 200 mu Ci of AlF-NOTA-KR12C injection is injected into U87 or HeLa tumor-bearing mice through tail vein, 0.1mL of 10% chloral hydrate is injected after 40 minutes of routine ingestion, and then routine PET/CT imaging is carried out.
Inhibiting development: other operations are the same as dynamic imaging and static imaging, and 250 mu g of KR12C polypeptide is added into the injection medicine.
FIG. 8 is a cross section of a word "2 18 F]AlF-NOTA-KR12C atMico-PET/CT images at different time points within 120 minutes in U87 tumor-bearing mice, wherein 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 cross section of a tumor part are sequentially arranged above the image 8; in the lower part of FIG. 8, there are sequentially CT, 15min PET, 30min PET, 60min PET, 90min PET and 120min PET images of the tumor part coronal plane. FIG. 9 is [ 2 ] 18 F]AlF-NOTA-KR12C is a graph showing the change in% ID/g value with time at different time points in the organ. FIG. 10 is a cross section of [ 2 ] 18 F]The 60-minute Mico-PET/CT and competitive inhibition visualizations of AlF-NOTA-KR12C in U87 tumor-bearing mice, wherein the upper left is the uptake CT image, the upper right is the uptake PET image, the lower left is the inhibition CT image, and the lower right is the inhibition PET image. FIG. 11 is [ 2 ] 18 F]The 60-minute Mico-PET/CT and competitive inhibition visualizations of AlF-NOTA-KR12C in HeLa tumor-bearing mice, wherein the upper left is the uptake CT image, the upper right is the uptake PET image, the lower left is the inhibition CT image, and the lower right is the inhibition PET image.
The PET imaging result shows that: [ 18 F]AlF-NOTA-KR12C has high uptake in U87 and HeLa tumor-bearing mice tumors, and the uptake at tumor parts is obviously higher than that of organs or tissues such as muscles, lungs, intestines and the like. Dynamic imaging shows that the blood in the probe body is quickly cleared, the probe is mainly metabolized by the kidney and the liver, and the time dynamic graph of each organ shows that the tumor/muscle ratio is higher when about 60 minutes, so that the optimal imaging time is selected to be 60 minutes, and the imaging inhibition result of 60 minutes shows that the tumor/muscle ratio is obviously reduced, which indicates that the probe is specifically absorbed at the tumor part. The imaging result shows that the imaging agent can be specifically absorbed in the tumor part, and has better application prospect.
This example uses KR12C polypeptide as the pharmacophore, connects a metal ion chelating group to construct the precursor, and utilizes radioactive metal nuclide M n+ The marker is constructed as [ M n+ ]NOTA-KR12C or [ M ] n+ ]The structure of the probe of DOTA-KR12C is shown in the formula (I), so that a specific targeting G-quadruplex polypeptide PET imaging agent is obtained;
in the formula (I), R 1 Represents a metal ion chelating group, M represents a radioactive metal nuclide, and n is selected from positive integers.
In the embodiment of the application, after one cysteine (Cys) is modified at the N-terminal of the lysine (Lys) of KR12C polypeptide, the 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 metallic nuclide A b+ Reaction in a buffer solution to obtain [ A ] b+ ]NOTA-KR12C or [ A ] b+ ]DOTA-KR12C, a targeting G-quadruplex polypeptide PET imaging agent shown in formula (II);
in the formula (II), R 2 Represents a metal ion chelating group, A b+ Represents a radioactive metallic species, b is selected from positive integers.
[M n+ ]NOTA-KR12C、[M n+ ]DOTA-KR12C、[A b+ ]NOTA-KR12C or [ A ] b+ ]The DOTA-KR12C probe provides a useful tool for diagnosing malignant tumors and provides a powerful imaging adjuvant for determining a treatment regimen and monitoring the efficacy of the treatment.
The above examples are preferred embodiments of the present invention, but the present invention is not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and they are included in the scope of the present invention.
Claims (10)
1. A targeting G-quadruplex polypeptide PET imaging agent is characterized in that: the structure of the targeting G-quadruplex polypeptide PET imaging agent is shown as a formula (I) or a formula (II):
in the formula (I), R 1 Represents goldA metal ion chelating group, M represents a radioactive metal nuclide, and n is selected from positive integers;
in the formula (II), R 2 Represents a metal ion chelating group, A represents a radioactive metal nuclide, and b is selected from positive integers.
2. The targeted G-quadruplex polypeptide PET imaging agent of claim 1, wherein: the radioactive metal nuclide M n+ Or A b+ Are each independently selected from 68 Ga 3+ 、[Al 18 F] 2+ 、 64 Cu 2+ 、 177 Lu 3+ 。
3. The G-quadruplex-targeted polypeptide PET imaging agent of claim 1, wherein: the metal ion chelating group R 1 Or R 2 Are respectively and independently selected from structures shown in formulas (1) to (9):
4. the G-targeted quadruplex polypeptide PET imaging agent according to any one of claims 1 to 3, wherein: the targeted G-quadruplex polypeptide PET imaging agent comprises a compound with the structure shown as the following formula;
5. the process for preparing a PET imaging agent of the targeted G-quadruplex polypeptides as claimed in any one of claims 1 to 4, wherein: the method comprises the following steps:
and (3) reacting the PET imaging agent precursor with radioactive metal nuclide in a buffer solution to obtain the targeted G-quadruplex polypeptide PET imaging agent.
6. The method of claim 5, wherein: the buffer solution comprises at least one of sodium acetate, ammonium acetate, potassium acetate, hydrochloric acid and acetic acid.
7. The method of claim 6, wherein: the reaction is carried out at a pH of 3.5 to 7.0.
8. The method of claim 6, wherein: the concentration of the buffer solution is 0.05mol/L-0.7 mol/L.
9. The method of claim 5, wherein: the reaction temperature is 80-120 ℃; the reaction time is 8min-40 min.
10. Use of the PET imaging agent of any one of claims 1 to 4 for imaging biological functions.
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