CN112043839A - Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof - Google Patents

Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof Download PDF

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CN112043839A
CN112043839A CN202010851049.3A CN202010851049A CN112043839A CN 112043839 A CN112043839 A CN 112043839A CN 202010851049 A CN202010851049 A CN 202010851049A CN 112043839 A CN112043839 A CN 112043839A
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polypeptide
radioisotope
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樊卫
王瑞敏
张晓飞
张旭
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Sun Yat Sen University Cancer Center
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Abstract

The invention discloses a radioactive isotope labeled polypeptide imaging agent of a targeted transferrin receptor and application thereof. Two polypeptide precursors specifically disclosed therein68Ga chelation reaction labeling method and application thereof as imaging agent in transferrin receptor over-expression tumor positron emission tomography PET. The radiocontolont labeled product has the radiocontolont purity of over 95 percent, good in vivo stability, can be rapidly gathered at a tumor part within 5 minutes, has obvious contrast of tumor muscles, can realize clear development of tumor tissues and edges, has certain difference between pharmacokinetics and biological distribution due to structural modification of different connecting groups, and can be used as a tumor specific developer of a targeted transferrin receptor.

Description

Radioisotope-labeled polypeptide imaging agent targeting transferrin receptor and application thereof
Technical Field
The invention belongs to the field of preparation and application of biomedical imaging agents, and particularly relates to a transferrin receptor targeted radioisotope labeled polypeptide imaging agent and application thereof.
Background
Positron Emission Tomography (PET) is the most advanced medical molecular imaging technology at present, and is widely applied to early diagnosis, curative effect evaluation and the like of serious diseases such as malignant tumors, cardiovascular diseases, nervous systems and the like. PET has significant advantages: firstly, three-dimensional functional imaging of the radioactive probe can be obtained, and the spatial distribution of biomolecular activities in a human body model is displayed; secondly, the noninvasive in vivo imaging mode realizes the real-time visualization of the cancer related process and promotes the understanding of the tumor evolution process; and combining with other imaging modes such as Magnetic Resonance Imaging (MRI), Computed Tomography (CT) and the like to form a multi-mode, and obtaining structural and functional imaging in the same examination. In accordance with these advantages, PET/MR, PET/CT, and whole-body PET dynamic parametric imaging enable multiple dimensions of biological signaling to be evaluated in a single examination.
The key to the development of the PET molecular imaging technology is to develop an effective PET radioactive imaging agent for disease-specific biomarkers. The PET molecular image realizes functional imaging, has clinical significance far higher than structural imaging of CT and MRI, and is also better than Single-photon emission computed tomography (SPECT), and an imaging agent used by the SPECT is a Single-photon radioisotope99mTc、131I, and the like. The two imaging principles are different, PET imaging depends on electronic collimation, and a collimator is not needed, so that the sensitivity of PET is more than 10 times higher than that of SPECT, the resolution of a SPECT system is 8-16mm, the resolution of the PET system is 2-8mm, the image is clear, and the diagnosis accuracy is high.
Transferrin receptor (TFRC) is a type II transmembrane glycoprotein, and its primary function is to mediate internalization of iron-containing serum Transferrin (tff) into cells, providing iron for cell proliferation. Current studies indicate that TFRC has been identified as one of the clinically relevant tumor imaging markers.
The existing research is based on a TFRC-Tf mechanism, and TF is used as a targeting ligand to design an imaging agent, however, the mechanism has 3 defects: firstly, endogenous Tf of an organism and a Tf ligand developer generate competitive combination effect on TFRC; ② the Tf protein has large molecular weight (76-81 kDa), long time consumption for in vivo distribution, and needs radioactive isotope with extremely long half-life (such as89Zr t1/278.4h) labeling for PET imaging, adding extra bodyThe radiation dose of (c); ③ the change of the spatial conformation of the Tf protein easily leads to the inactivation of the function of the Tf protein and is not beneficial to the targeting effect.
Peptide drugs have many advantages such as good tissue distribution pharmacokinetics, good permeability, low toxicity, no immunogenicity, chemical modification, and easy radiolabeling, and are therefore increasingly being developed as imaging agents. However, the polypeptide drug also has the defects of rapid metabolism of the proto-drug in vivo, short elimination half-life, low bioavailability and the like.
Therefore, how to obtain an imaging agent which can solve the defects of the prior art, has the functions of tumor PET imaging, strong specificity and high sensitivity, and can ensure the radiation dose safety under different metabolic rates has extremely high research value for the prior isotope tracing and imaging technology.
Disclosure of Invention
The object of the present invention is to provide a polypeptide precursor;
it is another object of the present invention to provide a radioisotope-labeled polypeptide targeting transferrin receptor;
another object of the present invention is to provide a method for preparing the above-mentioned radioisotope-labeled polypeptide;
it is another object of the present invention to provide a PET imaging agent;
it is another object of the present invention to provide the use of the above imaging agent for evaluating the expression level of transferrin receptor.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a polypeptide precursor is characterized in that the structure of the polypeptide precursor is a targeting polypeptide of which the NOTA-R-amino acid sequence is HAIYPRH, and the structural formula of the polypeptide precursor is as shown in formula I:
Figure BDA0002644744400000021
wherein A is CO;
b is NH;
r is selected from a group represented by formula II or formula III:
Figure BDA0002644744400000022
Figure BDA0002644744400000031
formula II or # in formula III represents connection with A in formula I, and formula II or # in formula III represents connection with B in formula I.
The polypeptide sequence HAIYPRH (namely His-Ala-Ile-Tyr-Pro-Arg-His) can be specifically combined with TFRC, the combination constant reaches 10nM, and the receptor affinity is extremely high. And endogenous Tf protein does not inhibit the specific binding of the vector modified with HAIYPRH polypeptide to TFRC on the surface of tumor cells. At present, no study on the preparation of Positron Emission Tomography (PET) agents by using the HAIYPRH polypeptide as a targeting ligand and the application of the PET agents in malignant tumor receptor imaging exists in China.
The invention utilizes the connecting group to change the charge and the hydrophilicity of the prototype polypeptide, thereby changing the pharmacokinetics and the biodistribution of the prototype drug to a certain extent so as to adapt to different indications and various clinical requirements.
The bifunctional metal chelating agent NOTA is a new bifunctional chelating agent after DOTA, Desferrioxamine (DFO) and the like, is a ring ligand with the smallest volume, and has higher selectivity of NOTA triazo ring ligand on metal cations, particularly Ga3+The ionic radius is 0.76 angstroms, the chelate is very suitable for a trinitrogen ring ligand cavity, the formed chelate has higher thermodynamic and kinetic stability, and the traditional bifunctional chelating agent, such as DOTA chelate, has lower thermodynamic stability.
Wherein formula II is also referred to as 4-amino- (1-carboxymethyl) piperidine (4-amino- (1-carboxymethy) piperidine, Pip); formula III is also known as polyethylene glycol (AEEP).
Pip can increase the lipid solubility of the compound and AEEP can increase the water solubility of the compound. The improvement of lipid solubility can promote the drug to penetrate through the blood brain barrier, and the development and the application of the drug to malignant brain tumor imaging are realized.
Further, the structural formula of the polypeptide precursor is formula IV or formula V:
Figure BDA0002644744400000032
Figure BDA0002644744400000041
the invention takes a tumor imaging marker TFRC as a target spot, takes HAIYPRH as a targeting polypeptide ligand, modifies the nitrogen end of a targeting peptide HAIYPRH through 2 connecting groups (Pip and AEEP), and is connected with a bifunctional metal chelating agent 1,4, 7-triazacyclononane-N, N' -triacetic acid (NOTA) to form the target peptide for being used for tumor imaging68Ga、64Cu and Al18F, and the like, and a plurality of radioisotope-labeled polypeptide precursors 1 (formula IV) and 2 (formula V). Indeed, a positron isotope metal coordinating moiety can be any moiety that is used to complex (also referred to as "coordinate") one or more metal positron isotopes under chemical or physiological conditions. The positron isotope may include68Ga、64Cu、86Y、89Metal such as Zr and Al18And metal complexes of nonmetallic positron isotopes such as F. The positron isotope metal coordinating moiety forms a thermodynamically and kinetically stable complex with the positron isotope to maintain the complex intact under physiological conditions; otherwise, systemic release of the coordinating positron isotope may occur.
Generally, the positron isotope metal coordinating moiety can be acyclic or cyclic. For example, the coordinating moiety comprises a polycarboxylic acid such as NOTA, DOTA, DTPA, DFO, or an analog or homolog thereof. In order to obtain greater stability under physiological conditions, macrocyclic moieties (e.g., triaza and tetraazamacrocycles) are generally preferred. In this embodiment, the macrocyclic metal coordinating moiety is a NOTA.
In a second aspect of the present invention, there is provided:
the radioactive isotope labeled polypeptide targeting transferrin receptor consists of the polypeptide precursor and radioactive isotope.
The radioisotopic labeled polypeptide of the targeted transferrin receptor is distributed in vivo through a normal KM mouse and a nude mouse human malignant brain glioma model, and the in vivo imaging research of a small animal PET/CT discovers that the imaging agents are mainly eliminated through liver and kidney metabolism, can be rapidly concentrated at a tumor part after entering the body, are long in retention time at the tumor part, increase the tumor/muscle ratio along with the time extension, can be stable within about 30 minutes, can effectively identify the tumor part through images, has ideal radioactive uptake value within 30-70 minutes, is expected to be used as a malignant tumor imaging agent with abnormal TFRC expression, and has market development potential.
Further, the radioisotope-labeled polypeptide is targeted to bind to transferrin receptor.
Further, the above radioactive isotope comprises68Ga、64Cu、86Y and Al18F. Preferably, the radioisotope is68Ga。
In current PET imaging agents, positron emitting radioisotopes68Ga is one of the most attractive diagnostic isotopes of PET for three reasons:
①68the half-life of Ga is 67.7min, which is close to18The physical half-life of F, the abundance of positrons, is 89%18The ideal substitute nuclide has lower radiation dose to organisms under the same diagnostic image standard;
② because the polypeptides and small molecule drugs diffuse rapidly in vivo,68the half-life of Ga is matched with the pharmacokinetics of the molecules, so that the imaging agent can realize better tumor localization and faster blood clearance, can realize fast imaging after the imaging agent enters into the body, can also realize continuous dynamic imaging from 0s, and has long half-life89The Zr marked macromolecule or antibody developer needs to circulate for many hours or even 2 to 3 days to reach a steady state;
③68ga is mainly commercialized68Ge/68Ga hairThe generator can be prepared and can also be produced by utilizing a cyclotron, so that the cost can be effectively controlled, the continuous production can be realized, and the feasibility of the production market is higher than that of the production market18F、89Zr and the like are completely dependent on isotopes produced by a cyclotron.
Further, in the present invention68The Ga labeling method is simple, short in time and high in labeling rate, and chemical synthesis conditions and basic procedures in manual labeling can be directly converted into the conventional industrialized radiopharmaceutical production module to realize industrialized production, such as
Figure BDA0002644744400000053
Ga-68 labels the module.
In the present example, two examples of the radioisotope-labeled polypeptide are shown, respectively68Ga-NOTA-Pip-HAIYPRH and68Ga-NOTA-AEEP-HAIYPRH。
as described above68The structural formula of Ga-NOTA-Pip-HAIYPRH is shown as formula VI:
Figure BDA0002644744400000051
as described above68The structural formula of Ga-NOTA-AEEP-HAIYPRH is shown as formula VII:
Figure BDA0002644744400000052
in a third aspect of the present invention, there is provided:
the preparation method of the radioactive isotope labeled polypeptide comprises the following steps:
(1) mixing the polypeptide precursor with a weak acid salt buffer solution to obtain a polypeptide precursor weak acid salt mixed solution;
(2) leaching the radioactive isotope with HCl to obtain radioactive isotope leacheate;
(3) mixing the polypeptide precursor weak acid salt mixed solution in the step (1) and the radioisotope eluent in the step (2);
(4) adjusting pH to 3.8-4.2, and heating to obtain radioisotope labeled polypeptide.
Wherein, the weak acid salt buffer solution can be selected from weak acid salt solution, including one or more of sodium acetate, gentisic acid and sodium phosphate, and the buffer salt solution familiar to those skilled in the art can also be used.
In a fourth aspect of the present invention, there is provided:
a PET imaging agent, wherein the imaging agent component comprises the radioisotope-labeled polypeptide.
The polypeptide PET imaging agent has great market value, related researches in China are more and more active along with the popularization of PET/CT large-scale medical equipment, and the polypeptide PET imaging agent is utilized compared with the traditional nuclear medicine99mThe Tc imaging agent is used for SPECT imaging, the PET imaging agent has higher imaging accuracy and sensitivity, is one of important ways for accurate tumor diagnosis in clinic, and is an important development direction of tumor molecular imaging drugs and medical technology.
Intravenous injection of the imaging agent of the invention (comprising68Ga-NOTA-Pip-HAIYPRH or68Ga-NOTA-AEEP-HAIYPRH), the in vivo biological distribution characteristics and the PET/CT imaging effect. The pharmacokinetics and biodistribution of the two imaging agents have some common characteristics. Both imaging agents are mainly metabolized by the liver and excreted by the kidney of the mouse, and secondly, the spleen also has partial physiological uptake, but generally, the physiological uptake and the metabolic pathway of the PET imaging agent do not influence the diagnosis of tumor tissues, and the problem can be solved by delayed scanning. The PET/CT imaging and in vivo biological distribution results of the tumor-bearing nude mouse living body show that besides physiological uptake of the liver, the kidney, the spleen and the like, the tumor part also has obvious radioactive uptake, and radioactive concentration appears over time, so that the tumor part can be effectively identified.
In a fifth aspect of the present invention, there is provided:
the use of the above imaging agent in the assessment of transferrin receptor expression levels.
In a sixth aspect of the present invention, there is provided:
the application of the imaging agent in preparing malignant tumor imaging diagnostic reagent with transferrin receptor expression abnormality.
Furthermore, the malignant tumor with abnormal transferrin receptor expression comprises glioma mediated by c-Myc oncogene, prostate cancer, hepatocellular carcinoma, lymphoma, triple negative breast cancer, lung cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, bladder cancer and colorectal cancer.
In a tumor cell signaling pathway, activating the c-Myc oncogene can stimulate TFRC transcription in a system, so that TFRC is generally highly expressed in various malignant tumors such as glioma, prostate cancer, hepatocellular carcinoma, lymphoma, triple negative breast cancer, lung cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, bladder cancer, colorectal cancer and the like mediated by the c-Myc oncogene, and the imaging agent mainly aims at the expression level of a transferrin receptor, so that clinical early diagnosis, staging and curative effect evaluation can be performed on various malignant tumors such as glioma, prostate cancer, hepatocellular carcinoma, lymphoma, triple negative breast cancer, lung cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, bladder cancer, colorectal cancer and the like with abnormal expression of the transferrin receptor.
The invention has the beneficial effects that:
1. the invention firstly uses the HAIYPRH polypeptide as a targeting ligand to prepare a positron radioactive developer, and uses the positron radioactive developer for the research of malignant tumor receptor imaging, the HAIYPRH polypeptide sequence has small molecular weight and low immunogenicity, does not generate competitive action with in vivo endogenous transferrin, and can be specifically combined with transferrin receptor;
2. the imaging agent is mainly eliminated through metabolism of liver and kidney, can be rapidly concentrated on a tumor part after entering a body, has long retention time at the tumor part, increases the ratio of tumor to muscle along with the time extension, can be stable within about 30 minutes, can effectively identify the tumor part by an image, has ideal radioactive uptake value between 30min and 70min, is expected to be used as a malignant tumor imaging agent with abnormal TFRC expression, and has market development potential;
3. the invention also discloses a structure improvement method of the HAIYPRH sequence polypeptide, which can increase the lipid solubility of the HAIYPRH sequence polypeptide by using the connecting group Pip, increase the water solubility of the HAIYPRH sequence polypeptide by using the connecting group AEEP, promote the drug to penetrate through the blood brain barrier by improving the lipid solubility, and develop and apply the HAIYPRH sequence polypeptide to malignant brain tumor imaging.
Drawings
FIG. 1 is a drawing of example 168PET imaging of Ga-NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice;
FIG. 2 is a drawing showing a structure of example 168The radioactive uptake metabolic curve of tumor, liver and muscle tissues after Ga-NOTA-Pip-HAIYPRH administration;
FIG. 3 is a drawing showing a modified example of example 268PET imaging of Ga-NOTA-AEEP-HAIYPRH in tumor-bearing nude mice;
FIG. 4 shows the results of example 268The radioactive uptake metabolic profile of tumor, liver and muscle tissues after Ga-NOTA-AEEP-HAIYPRH administration.
Detailed Description
In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to specific embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.
The laboratory reagents and instruments used are, unless otherwise specified, consumables and reagents which are conventionally available from commercial sources.
Reagent and apparatus
Reagent:
ultrapure hydrochloric acid (Merck, Germany), pharmaceutical grade absolute ethanol, sodium acetate and sodium citrate, Chelex-100 resin (100-200 mesh, Sigma, USA), 0.9% physiological saline, MEM medium and fetal bovine serum (Gibco, USA), 0.25% trypsin-EDTA digest and 0.01M PBS buffer, isoflurane, 6 Fmoc-protected amino acids, fluorenylmethoxychloride (Fmoc-Cl), tert-butanol, 1,4, 7-triazacyclononane-N, N ', N "-triacetic acid (NOTA), 4-amino- (1-carboxymethyl) piperidine, 3- [2- (2-aminoethoxy) ethoxy ] -propionic acid, Fmoc-Leu-Wang resin, Dimethylformamide (DMF), piperidine, 4-amino- (1-carboxymethyl) piperidine, N-N, N' -triacetic acid (NOTA), N, N-Diisopropylethylamine (DIEA), N-methylpyrrolidone (NMP), benzotriazole-N, N' -tetramethyluronium Hexafluorophosphate (HBTU), trifluoroacetic acid (TFA), Triisopropylsilane (TIS);
wherein the structural formulas of the 6 Fmoc-protected amino acids are respectively as follows:
Fmoc-His-OH, having the structural formula:
Figure BDA0002644744400000081
Fmoc-Arg (Pbf) -OH, formula:
Figure BDA0002644744400000082
Fmoc-Pro-OH, having the structural formula:
Figure BDA0002644744400000083
Fmoc-Tyr (tBu) -OH, having the formula:
Figure BDA0002644744400000091
Fmoc-Ile-OH, having the structural formula:
Figure BDA0002644744400000092
Fmoc-Ala-OH, structural formula:
Figure BDA0002644744400000093
the instrument comprises the following steps:
68Ge/68ga generator (ITG, Germany), radionuclide activity meter CRC-15PET, thin-layer radioactivity scanner (Radio-TLC), SN-695 gamma radioimmunoassay, analytical balance, multipurpose incubator MH2800D (Tianjin Athens instruments, Ltd.), carbon dioxide incubator, animal anesthesia machine, and water bathTemperature system (Shanghai Yuyan scientific instruments Co., Ltd.), small animal PET/CT (Siemens Enveono PET/CT, Germany), polypeptide solid phase synthesizer (Symphony, U.S. PTI), high performance liquid chromatograph, electrospray ionization mass spectrometer (ESI-MS, Bruker Optics, USA).
Preparation of polypeptide precursors
Preparation of Fmoc-Pip-OH
Fmoc-Pip-OH can be obtained by protecting 4-amino- (1-carboxymethyl) piperidine with t-butanol, Fmoc-Cl, although other methods familiar to those skilled in the art can be used.
The Fmoc-Pip-OH finally prepared in this example has the following structural formula:
Figure BDA0002644744400000101
preparation of Fmoc-AEEP-OH
Fmoc-AEEP-OH can be prepared by sequential protection of 3- [2- (2-aminoethoxy) ethoxy ] -propionic acid with t-butanol and Fmoc-Cl, although other methods familiar to those skilled in the art can be used.
The Fmoc-AEEP-OH prepared finally in this example has the following structural formula:
Figure BDA0002644744400000102
preparation of NOTA-tBu (2)
NOTA-tBu (2) can be prepared by reacting NOTA with t-butanol under a triflic anhydride catalyst system, or by reacting NOTA with potassium t-butoxide, although other methods familiar to those skilled in the art can be used.
The NOTA-tBu (2) ultimately prepared in this example has the following structural formula:
Figure BDA0002644744400000103
synthesis of NOTA-Pip-HAIYPRH polypeptide precursor
The NOTA-Pip-HAIYPRH polypeptide precursor in this example was passed through N-channel using Fmoc-Leu-Wang resinα-Fmoc solid phase polypeptide synthesis strategy synthesis, comprising the following steps:
the resin was treated with 20% piperidine in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) were subsequently coupled according to the polypeptide sequence, including Fmoc-His-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Ala-OH, and Fmoc-Pip-OH and NOTA-tBu (2). Coupling was carried out with 3 equivalents of the condensing agent O- (benzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) in N-methylpyrrolidinone (NMP) under 6 equivalents of N, N-Diisopropylethylamine (DIEA). After the extension was completed, the mixture was cooled at room temperature using a volume ratio of 95: 2.5: trifluoroacetic acid (TFA)/H of 2.52The O/Triethylsilane (TIS) mixture was treated for 4h to deprotect while cleaving from the resin. After filtration, cold ether was added to the TFA solution to precipitate the peptide. The precipitated crude peptide was collected by centrifugation and purified by semi-preparative column HPLC in 47% yield. The purified product was 96.8% pure by routine HPLC analysis. Calculation of NOTA-Pip-HAIYPRH peptide (C)60H91N19O15) Molecular weight of 1318.5, ESI-MS mass spectrometry: results [ M + H]+1319.8。
Synthesis of NOTA-AEEP-HAIYPRH polypeptide precursors
The NOTA-AEEP-HAIYPRH polypeptide precursor in this example was prepared by using Fmoc-Leu-Wang resin general Nα-Fmoc solid phase polypeptide synthesis strategy synthesis, comprising the following steps:
the resin was treated with 20% piperidine in DMF to remove the Nα-Fmoc protecting group. The following Fmoc-protected amino acids (3 equivalents) were subsequently coupled according to the polypeptide sequence, including Fmoc-His-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Tyr (tBu) -OH, Fmoc-Ile-OH, Fmoc-Ala-OH, and Fmoc-AEEP-OH and NOTA-tBu (2). Coupling was performed with 3 equivalents of condensing agent HBTU in NMP under 6 equivalents DIEA. After the extension was completed, the mixture was cooled at room temperature using a volume ratio of 95: 2.5: 2.5 TFA/H2And treating the O/TIS mixed solution for 4h for deprotection and simultaneously cracking from the resin. After filtration, cold ether was added to the TFA solution to precipitate the peptide. Separation deviceThe precipitated crude peptide was collected by heart and purified by semi-preparative column HPLC with a yield of 30%. The purified product was 97.4% pure by conventional HPLC analysis. Calculation of NOTA-AEEP-HAIYPRH peptide (C)60H92N18O17) Molecular weight of 1337.5, ESI-MS mass spectrometry: results [ M + H]+1338.6。
The structure of the polypeptide precursor can be summarized as a target polypeptide of which the NOTA-R-amino acid sequence of the bifunctional metal chelating agent is HAIYPRH, and the general structural formula of the polypeptide precursor is shown as a formula I:
Figure BDA0002644744400000111
wherein A is CO;
b is NH;
and R is selected from a group shown in formula II or formula III:
Figure BDA0002644744400000112
Figure BDA0002644744400000121
the expression of the formula II or the expression of the formula III indicates the connection with the A in the formula I, and the expression of the formula II or the expression of the formula III indicates the connection with the B in the formula I.
R is 4-amino- (1-carboxymethyl) piperidine (4-amino- (1-carboxymethyl) piperidine, Pip) (formula I) or polyethylene glycol (AEEP) (formula II).
Example 168Preparation of Ga-NOTA-Pip-HAIYPRH
1.68Ga-labeled NOTA-Pip-HAIYPRH
20ug of the above-mentioned polypeptide precursor NOTA-Pip-HAIYPRH was dissolved in 200ul of 1M sodium acetate solution, placed in a reaction flask, and 0.05mol/L of 5ml HCl solution was extracted with a 5ml syringe68Ge/68The Ga generator is leached in sections with the flow rate of 1ml/min, and 1.5ml of middle high activity is collected68And adding the Ga leacheate into a reaction bottle, and adjusting the pH value of the reaction liquid to 3.8-4.2, sealing, putting into a multifunctional heater for reaction for 10min at 90 ℃.
Measuring the radiochemical purity of the product, and if the radiochemical purity of the product is more than or equal to 80 percent, indicating that the labeling is successful.
2. Purification of the product
Sucking the reacted labeled product solution with a syringe, adding into an activated Sep-pak C-18 column, and eluting with 5ml of normal saline to remove impurities: (68Ga and68ge) to a waste liquid bottle, and finally washing the Sep-pak C-18 column by using 0.5ml of 60% ethanol to obtain the purified product68Ga-NOTA-Pip-HAIYPRH in 60% ethanol.
Detecting the radiochemical purity by using paper chromatography, and carrying out the next step when the radiochemical purity is more than or equal to 95 percent, or continuously repeating the purification. The radiochemical purity measuring method comprises the following steps: detecting the reaction solution by Radio-TLC paper chromatography with 0.1M sodium citrate buffer solution as developing agent68The Ga is close to the origin of the beam,68ga-labeled NOTA-Pip-HAIYPRH was distributed at the solvent front and the radiochemical purity of the product was calculated by% area of the emission peak.
Diluting with normal saline about 10 times, and filtering with 0.22um sterile filter membrane to obtain the final product for in vivo experiment.
Prepared as in this example68The structural formula of Ga-NOTA-Pip-HAIYPRH is shown as follows:
Figure BDA0002644744400000131
example 268Preparation of Ga-NOTA-AEEP-HAIYPRH
1.68Ga-labeled NOTA-AEEP-HAIYPRH
20ug of the above-mentioned polypeptide precursor NOTA-AEEP-HAIYPRH was dissolved in 200ul of 1M sodium acetate solution, placed in a reaction flask, and 0.05mol/L of 5ml HCl was extracted by a 5ml syringe68Ge/68The Ga generator is leached in sections with the flow rate of 1ml/min, and 1.5ml of middle high activity is collected68Adding Ga leacheate into a reaction bottle, adjusting the pH of the reaction liquid to 3.8-4.2, sealing, and placing the reaction liquid into a multifunctional heater for reaction for 10min at 90 DEG C。
Measuring the radiochemical purity of the product, and if the radiochemical purity of the product is more than or equal to 80 percent, indicating that the labeling is successful.
2. Purification of the product
Sucking the reacted labeled product solution with a syringe, adding into an activated Sep-pak C-18 column, and eluting with 5ml of normal saline to remove impurities: (68Ga and68ge) to a waste liquid bottle, and finally washing the Sep-pak C-18 column by using 0.5ml of 60% ethanol to obtain the purified product68Ga-NOTA-AEEP-HAIYPRH in 60% ethanol.
Detecting the radiochemical purity by using paper chromatography, and carrying out the next step when the radiochemical purity is more than or equal to 95 percent, or continuously repeating the purification.
Diluting with normal saline about 10 times, and filtering with 0.22um sterile filter membrane to obtain the final product for in vivo experiment.
Radiochemical purity measurement method: detecting the reaction solution by Radio-TLC paper chromatography with 0.1M sodium citrate buffer solution as developing agent68The Ga is close to the origin of the beam,68ga-labeled NOTA-AEEP-HAIYPRH was distributed at the solvent front and the radiochemical purity of the product was calculated by% area of the emission peak.
Prepared as in this example68The structural formula of Ga-NOTA-AEEP-HAIYPRH is shown as follows:
Figure BDA0002644744400000132
Figure BDA0002644744400000141
biodistribution test of the polypeptide imaging agent of the present invention
In example 1 of the present invention68Biodistribution of Ga-NOTA-Pip-HAIYPRH in KM mice
Taking 12 KM mice, female, randomly grouping, 3 KM mice in each group, fasting overnight for 12h, injecting 68Ga-NOTA-Pip-HAIYPRH 3.7MBq by tail vein, taking blood from eyeball 5, 30, 60 and 120min after injection, dislocating cervical vertebra, killing KM mice, taking organs or tissues such as heart, lung, liver, spleen and kidney, weighing by an analytical balance, measuring radioactivity count, and calculating percent injection dosage rate per gram of tissue (percent ID/g) after attenuation correction. The results are shown in Table 1.
TABLE 1 biodistribution of Ga-NOTA-Pip-HAI in KM mice (% ID/g, mean + -SD, n ═ 3)
Figure BDA0002644744400000142
The results of in vivo distribution experiments show that,68after entering into the internal circulation, Ga-NOTA-Pip-HAIYPRH is rapidly distributed to each tissue and organ, the physiological uptake of the liver and the spleen is higher, the brain uptake is very low, and the Ga-NOTA-Pip-HAIYPRH is difficult to permeate the blood brain barrier. The medicine is water soluble and is mainly cleared by renal metabolism.
In example 2 of the present invention68Biodistribution of Ga-NOTA-AEEP-HAIYPRH in KM mice
Taking 12 KM mice, female, randomly grouping, 3 mice in each group, fasting overnight for 12h, and injecting via tail vein68Ga-NOTA-AEEP-HAIYPRH 3.7MBq, blood is taken from eyeballs at 5min, 30min, 60min and 120min after injection, KM mice are killed by dislocation of cervical vertebrae, organs or tissues such as heart, lung, liver, spleen, kidney and the like are taken, a balance is analyzed and weighed, radioactivity counting is measured, and percent injection dosage rate per gram of tissue (% ID/g) is calculated after attenuation correction. The results are shown in Table 2.
Table 2.68Biodistribution of Ga-NOTA-AEEP-HAIYPRH in KM mice (% ID/g, mean. + -. SD, n ═ 3)
Figure BDA0002644744400000151
The results of in vivo distribution experiments show that,68after entering into the internal circulation, Ga-NOTA-AEEP-HAIYPRH is rapidly distributed to various tissues and organs, wherein the physiological uptake of the liver and the spleen is higher, and the brain uptake is extremely low, which indicates that the Ga-NOTA-AEEP-HAIYPRH does not easily permeate the blood brain barrier. The medicine is water soluble and is mainly cleared by renal metabolism.
From tables 1 and 2, it can be seen that in normal KM mice, there is also some difference in pharmacokinetics and biodistribution of the two imaging agents,68Ga-NOTA-Pip-HAIYPRH has a blood radioactivity uptake% ID/g higher than that of68Ga-NOTA-AEEP-HAIYPRH, the former has longer in vivo circulation time, slower metabolic clearance and higher brain uptake, and has important relation with the modification of chemical structure of a linker. The LogD7.0 predicted value of the linker Pip is-2.57 and the LogD7.0 predicted value of the linker AEEP is-3.66, therefore, compared with the AEEP modified imaging agent, the lipid solubility of the Pip modification is improved, the binding rate of the imaging agent and serum albumin is probably increased, the renal clearance is reduced, the in vivo circulation time is prolonged, and the binding probability with the target TFRC is increased.
Targeted testing of the polypeptide imaging agents of the invention
U87MG tumor-bearing nude mouse model establishment
Balb/c nununuu nude mice, SPF grade, female, 4-5 weeks old, 8. Selecting a human malignant glioblastoma cell line U87MG for culture, wherein the culture conditions are as follows: MEM culture medium containing penicillin 100U/ml and streptomycin 0.1mg/ml, adding 10% fetal calf serum, carbon dioxide incubator at 37 deg.C, and CO25%, humidity 90%, and culturing for 48 hours. Adding 0.25% trypsin-EDTA digestive juice to digest and collect cells, centrifuging and washing 2 times at 1000rpm PBS buffer solution, adjusting the cell concentration of MEM culture medium cell suspension to 2.5 × 107One/ml, the cell suspension was aspirated by a 1ml syringe and the right axilla of nude mice was inoculated with 5X 10 cells6Seed volume 0.2 ml/seed. 2 to 3 weeks after inoculation, when the tumor volume is 300 to 500m3And is used for in vivo biodistribution and imaging experiments.
In example 168Biodistribution of Ga-NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice
The U87MG tumor-bearing nude mice were used as female mice, fasted overnight for 12h, and injected via tail vein into the mice of example 168Ga-NOTA-Pip-HAIYPRH 3.7MBq, blood is taken from eyeball 60min after injection, U87MG tumor-bearing nude mouse is killed by dislocation of cervical vertebra, organs or tissues such as heart, lung, liver, spleen, kidney and the like are taken, weighed by an analytical balance, radioactivity counting is measured, and the percentage is calculated after attenuation correctionID/g. Statistical methods statistical analysis was performed using SPSS10.0 statistical software, with variance analysis, P < 0.05 being statistically significant for differences. The results are shown in Table 3.
Table 3.68Biodistribution (% ID/g, mean + -SD, n-3) of Ga-NOTA-Pip-HAIYPRH 60min after injection in U87MG tumor-bearing nude mice
Figure BDA0002644744400000161
The results show that the physiological uptake of the liver, the spleen and the kidney is higher, the brain uptake is extremely low, and the tissues are paired68The physiological uptake of Ga-NOTA-pip-HAIYPRH was substantially consistent with the in vivo biodistribution profile of normal KM mice at the same time. The% ID/g ratio of tumor/muscle was 3.08. + -. 0.45, indicating significant uptake by tumor tissue.
In example 268Biodistribution of Ga-NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice
The U87MG tumor-bearing nude mice were taken 3, female, fasted overnight for 12h, and injected via tail vein into the mice of example 268Ga-NOTA-AEEP-HAIYPRH 3.7MBq, blood is taken from eyeball 60min after injection, U87MG tumor-bearing nude mice are killed by dislocation of cervical vertebra, organs or tissues such as heart, lung, liver, spleen, kidney and the like are taken, weighed by an analytical balance, radioactivity counting is measured, and% ID/g is calculated after attenuation correction. Statistical methods statistical analysis was performed using SPSS10.0 statistical software, with variance analysis, P < 0.05 being statistically significant for differences. The results are shown in Table 4.
Table 4.68Biodistribution (% ID/g, mean + -SD, n-3) of Ga-NOTA-AEEP-HAIYPRH 60min after injection in U87MG tumor-bearing nude mice
Figure BDA0002644744400000171
The results show that the physiological uptake of the liver, the spleen and the kidney is higher, the brain uptake is extremely low, and the tissues are paired68The physiological uptake of Ga-NOTA-AEEP-HAIYPRH was substantially consistent with the in vivo biodistribution profile of normal KM mice at the same time.The ratio of the percent ID/g of the tumor to the muscle is 1.90 +/-1.70, which indicates that the tumor targeting agent has certain targeting property on tumor tissues.
In example 168PET/CT imaging of Ga-NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice
1 nude mouse with U87MG tumor was injected via tail vein with the drug of example 168Ga-NOTA-Pip-HAIYPRH 11.1MBq, post injection PET/CT image dynamic acquisition (300s × 14). The scanned image is reconstructed by using the format of the OSEM3D, an image Region of interest (ROI) is outlined for calculation, and a metabolic curve is drawn. Statistical methods statistical analysis was performed using SPSS10.0 statistical software, with variance analysis, P < 0.05 being statistically significant for differences. The results are shown in fig. 1 and fig. 2, respectively.
The results show that the drug uptake of the tumor tissue becomes more and more obvious with the time, as shown in figure 1, the tumor tissue (indicated by an arrow) starts to be weakly imaged at 5min which is visible to the naked eye, and the radioactive uptake of the tumor tissue tends to be stable at 45-70 min. According to the metabolism curve in fig. 2, the drug is metabolized and distributed by the body after injection, and is rapidly taken by the tumor, and the ratio of the percent ID/g of the tumor/muscle is 2.17 +/-0.22 times from 5min to 70 min. Comparison with other tissues, e.g., liver uptake decreased slowly over time, but tumor uptake tended to increase slowly, indicating that example 1 is68Ga-NOTA-Pip-HAIYPRH is gradually metabolized and cleared by liver, kidney and the like in the circulation process in vivo, but is slowly concentrated at the tumor site.
To summarize the above, 11.1MBq was administered to tumor-bearing nude mice via the tail vein68Ga-NOTA-Pip-HAIYPRH can realize targeted tumor imaging.
In example 268PET/CT imaging of Ga-NOTA-AEEP-HAIYPRH in U87MG tumor-bearing nude mice
1 mouse with the U87MG tumor is taken and injected through tail vein68Ga-NOTA-AEEP-HAIYPRH 11.1MBq, post injection PET/CT image dynamic acquisition (300s × 14). The scanned image is reconstructed by using the format of the OSEM3D, an image Region of interest (ROI) is outlined for calculation, and a metabolic curve is drawn. Statistical methods statistical analysis was performed using SPSS10.0 statistical softwareBy variance analysis, the difference is more than 0.05 which has statistical significance. The results of the experiment are shown in FIGS. 3 and 4.
The results show that the drug uptake of the tumor tissue becomes more and more obvious with the time, as shown in fig. 3, the tumor tissue (indicated by an arrow) starts to be imaged when being visible to the naked eye for 5min, and the radioactive uptake of the tumor tissue tends to be stable when being 40min-70 min. According to the metabolism curve in fig. 4, the drug is metabolically distributed after entering the body, and the ratio of the% ID/g of tumor/muscle is 2.05 +/-0.10 times within 5min to 70 min. In contrast to other tissues, for example, liver uptake decreased slowly over time, but tumor uptake tended to increase slowly, indicating that68Ga-NOTA-AEEP-HAIYPRH is gradually metabolized and cleared by organs such as liver and kidney during the circulation process in vivo, but is slowly concentrated at the tumor site.
To summarize the above, 11.1MBq was administered to tumor-bearing nude mice via the tail vein68Ga-NOTA-AEEP-HAIYPRH, can realize targeted tumor imaging.
Imaging experiments prove that the two imaging agents have the tumor PET imaging function, but the advantages and the disadvantages of the two imaging agents also need to integrate the specificity and the sensitivity of the imaging agents and the radiation dose safety under different metabolic rates, and can be applied in comparison and selection in further preclinical or clinical phase I tests.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, 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 all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims (10)

1. A polypeptide precursor, wherein the structure of the polypeptide precursor is a targeting polypeptide with a bifunctional metal chelator NOTA-R-amino acid sequence of HAIYPRH, and the structural formula of the polypeptide precursor is as shown in formula I:
Figure FDA0002644744390000011
wherein A is CO;
b is NH;
and R is selected from a group shown in formula II or formula III:
Figure FDA0002644744390000012
the expression of the formula II or the expression of the formula III indicates the connection with the A in the formula I, and the expression of the formula II or the expression of the formula III indicates the connection with the B in the formula I.
2. The polypeptide precursor of claim 1, wherein the structural formula of the polypeptide precursor is formula IV or formula V:
Figure FDA0002644744390000013
Figure FDA0002644744390000021
3. a radioisotope-labeled polypeptide, wherein said radioisotope-labeled polypeptide is produced by labeling a precursor of the polypeptide of claim 1 or 2 with a radioisotope.
4. The radioisotope-labeled polypeptide of claim 3, wherein said radioisotope-labeled polypeptide is targeted to bind transferrin receptor.
5. The radioisotope-labeled polypeptide of claim 4, wherein said radioisotope comprises68Ga、64Cu、86Y and Al18F.
6. A method for preparing a radioisotope-labeled polypeptide as claimed in any one of claims 3 to 5, comprising the steps of:
(1) mixing the polypeptide precursor of claim 1 or 2 with a weak acid salt buffer solution to obtain a polypeptide precursor weak acid salt mixed solution;
(2) leaching the radioactive isotope with HCl to obtain radioactive isotope leacheate;
(3) mixing the polypeptide precursor weak acid salt mixed solution in the step (1) and the radioisotope eluent in the step (2);
(4) adjusting pH to 3.8-4.2, and heating to obtain the radioisotope labeled polypeptide.
7. A PET imaging agent comprising the radioisotope-labeled polypeptide of any one of claims 3 to 5.
8. Use of the PET imaging agent of claim 7 for the assessment of transferrin receptor expression levels.
9. Use of the PET imaging agent of claim 7 for the preparation of a diagnostic agent for imaging malignant tumors in which transferrin receptor expression is aberrant.
10. The use according to claim 9, wherein the malignant tumor with abnormal transferrin receptor expression comprises glioma mediated by c-Myc oncogene, prostate cancer, hepatocellular carcinoma, lymphoma, triple negative breast cancer, lung cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, bladder cancer, colorectal cancer.
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CN114796535A (en) * 2022-04-18 2022-07-29 南方医科大学南方医院 Targeting G-quadruplex polypeptide PET (polyethylene terephthalate) imaging agent as well as preparation method and application thereof
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