CN112043839A - Radioisotope-labeled peptide imaging agent targeting transferrin receptor and its application - Google Patents

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 peptide imaging agent targeting transferrin receptor and its application

technical field

The invention belongs to the field of preparation and application of biomedical imaging agents, in particular to a radioisotope-labeled polypeptide imaging agent targeting transferrin receptors and applications thereof.

Background technique

Positron Emission Tomography (PET) is the most cutting-edge medical molecular imaging technology. PET has significant advantages: (1) 3D functional imaging of radioactive probes can be obtained, showing the spatial distribution of biomolecular activities in human models; (2) non-invasive in vivo imaging modality enables real-time visualization of cancer-related processes and facilitates understanding of tumor evolution; ③ By combining with other imaging modalities such as magnetic resonance imaging (MRI) and computed tomography (CT) to form multimodality, structural and functional imaging can be obtained in the same examination. Based on these advantages, PET/MR, PET/CT, and whole-body PET dynamic parametric imaging allow the evaluation of multiple dimensions of biological signaling in a single examination.

The key to the development of PET molecular imaging technology is to develop effective PET radioactive imaging agents for disease-specific biomarkers. PET molecular imaging achieves functional imaging, and its clinical significance is much higher than that of CT and MRI structural imaging, and it is also superior to single-photon emission computed tomography (SPECT). The imaging agent used in SPECT is a single-photon type imaging agent. Radioisotopes ^99m Tc, ¹³¹ I, etc. The imaging principles of the two are different. PET imaging relies on electronic collimation and does not require a collimator. Therefore, the sensitivity of PET is more than 10 times higher than that of SPECT. The resolution of the SPECT system is 8-16 mm, while the resolution of the PET system is 2-8 mm. Clear and high diagnostic accuracy.

Transferrin receptor (TFRC) is a type II transmembrane glycoprotein whose main function is to mediate the internalization of iron-containing serum transferrin (Tf) into cells and provide iron for cell proliferation. Existing studies have shown that TFRC has been identified as one of the clinically relevant tumor imaging markers.

Existing studies are mostly based on the TFRC-Tf mechanism, using TF as a targeting ligand to design imaging agents. However, this mechanism has three disadvantages: (1) Endogenous Tf can compete with Tf ligand imaging agents for TFRC. ②Tf protein has a large molecular weight (76-81kDa), and it takes a long time to distribute in vivo. It requires radioisotopes with a particularly long half-life (such as ⁸⁹ Zr t _1/2 = 78.4h) to be labeled for PET imaging, which increases the extra radiation dose of the body. ; ③ The change of the spatial conformation of Tf protein can easily lead to its inactivation, which is not conducive to the targeting effect.

Peptide drugs have many advantages such as good tissue distribution pharmacokinetics, good permeability, low toxicity, no immunogenicity, chemical modification and radiolabeling easiness, so they are increasingly developed for imaging agent. However, peptide drugs also have shortcomings such as rapid metabolism of prototype drugs in vivo, short elimination half-life, and low bioavailability.

Therefore, how to obtain an imaging agent that can not only solve the above-mentioned defects of the prior art, but also has tumor PET imaging function, strong specificity, high sensitivity, and can ensure the safety of radiation dose under different metabolic rates. Tracking and imaging techniques have extremely high research value.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a polypeptide precursor;

Another object of the present invention is to provide a radioisotope-labeled polypeptide targeting transferrin receptor;

Another object of the present invention is to provide a method for preparing the above radioisotope-labeled polypeptide;

Another object of the present invention is to provide a PET imaging agent;

Another object of the present invention is to provide the application of the above-mentioned imaging agent in evaluating the expression level of transferrin receptor.

The technical scheme adopted by the present invention is:

A first aspect of the present invention provides:

A polypeptide precursor, characterized in that the structure of the polypeptide precursor is a bifunctional metal chelator NOTA-R-targeting polypeptide whose amino acid sequence is HAIYPRH, and the structural formula of the polypeptide precursor is as formula I:

Among them, A is CO;

B is NH;

R is selected from the group represented by formula II or formula III:

* in Formula II or Formula III represents connection to A in Formula I, and # in Formula II or Formula III represents connection to B in Formula I.

The polypeptide sequence HAIYPRH (ie, His-Ala-Ile-Tyr-Pro-Arg-His) can specifically bind to TFRC with a binding constant of 10 nM and a very high receptor affinity. And endogenous Tf protein will not inhibit but promote the specific binding of HAIYPRH polypeptide-modified carrier to TFRC on the surface of tumor cells. At present, there is no positron radioactive imaging agent prepared by using the sequence HAIYPRH polypeptide as a targeting ligand in China, and it is used in the research of malignant tumor receptor imaging.

The invention utilizes the linking group to change the charge and hydrophilicity of the prototype polypeptide, thereby changing the pharmacokinetics and biodistribution of the prototype drug to a certain extent, so as to adapt to different indications and various clinical needs.

The bifunctional metal chelator NOTA is an emerging bifunctional chelating agent after DOTA, desferrioxamine (DFO), etc. It is the smallest ring ligand. It is a Ga ³⁺ ion radius of 0.76 angstroms, which is very suitable for triazide ring ligand holes, and the formed chelates have high thermodynamic and kinetic stability, while traditional bifunctional chelators, such as DOTA chelates are thermodynamically stable. low sex.

Wherein, formula II is also known as 4-amino-(1-carboxymethyl) piperidine (4-amino-(1-carboxymethyl) piperidine, Pip); formula III is also known as polyethylene glycol (polyethylene glycol, AEEP).

Pip can increase the lipid solubility of the compound, and AEEP can increase the water solubility of the compound. The increase in lipid solubility can facilitate the penetration of drugs through the blood-brain barrier and be developed for the imaging of malignant brain tumors.

Further, the structural formula of the above-mentioned polypeptide precursor is formula IV or formula V:

In the present invention, the tumor imaging marker TFRC is used as the target, HAIYPRH is used as the targeting polypeptide ligand, the nitrogen end of the targeting peptide HAIYPRH is modified by two linking groups (Pip and AEEP), and then combined with a bifunctional metal chelator 1,4 ,7-triazacyclononane-N,N',N" ^-triacetic acid ( ^NOTA ) is connected to form a polypeptide precursor ¹ (formula IV) with Polypeptide Precursor 2 (Formula V). In practice, the positron isotope metal coordinating moiety may be used to complex (also referred to as "coordinating") one or more metal positrons under chemical or physiological conditions Any part of the electron isotope. The positron isotope may include metal complexes of metal species such as ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁶ Y, ⁸⁹ Zr, and non-metal positron isotopes such as Al ¹⁸ F. The positron isotope metal coordination part and the positive Electron isotopes form complexes that are thermodynamically and kinetically stable to keep the complexes 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, coordinating moieties include polycarboxylic acids such as NOTA, DOTA, DTPA, DFO, or analogs or homologs thereof. For greater stability under physiological conditions, macrocyclic moieties (eg, triaza and tetraazamacrocycles) are generally preferred. In this embodiment, the macrocyclic metal coordinating moiety is NOTA.

A second aspect of the present invention provides:

A radioisotope-labeled polypeptide targeting transferrin receptors, the radioisotope-labeled polypeptide is composed of the above-mentioned polypeptide precursor and a radioisotope.

The radioisotope-labeled polypeptide targeting the transferrin receptor in the present invention was found through the in vivo distribution of normal KM mice and nude mouse human malignant glioma models, and small animal PET/CT in vivo imaging studies. The metabolism of liver and kidney is eliminated. After entering the body, it can be quickly concentrated in the tumor site, and the residence time in the tumor site is long. The tumor/muscle ratio increases with time and stabilizes in about 30 minutes. The image can effectively identify tumors. The radioactive uptake value between 30min-70min is ideal, and it is expected to be used as an imaging agent for malignant tumors with abnormal TFRC expression, and has market development potential.

Further, the above-mentioned radioisotope-labeled polypeptide targets and binds to the transferrin receptor.

Further, the above radioactive isotopes include at least one of ⁶⁸ Ga, ⁶⁴ Cu, ⁸⁶ Y and Al ¹⁸ F. Preferably, the above radioisotope is ⁶⁸ Ga.

Among current PET imaging agents, the positron radioisotope ⁶⁸ Ga is one of the most attractive isotopes for PET diagnostics for three reasons:

① The half-life of ⁶⁸ Ga is 67.7min, which is close to the physical half-life of ¹⁸ F, and the positron abundance is 89%. It is an ideal substitute for ¹⁸ F. Under the same diagnostic image standard, the radiation dose to the body is lower;

②Due to the rapid diffusion of peptides and small molecule drugs in the body, the half-life of ⁶⁸ Ga matches the pharmacokinetics of these molecules very well, so that the imaging agent can better localize the tumor and clear the blood faster. Imaging can be performed quickly, or continuous dynamic imaging can be started from 0s, while long half-life ⁸⁹ Zr-labeled macromolecules or antibody imaging agents need to circulate for many hours or even 2-3 days to reach a steady state;

③ ⁶⁸ Ga is mainly prepared by commercial ⁶⁸ Ge/ ⁶⁸ Ga generators, and can also be produced by cyclotron, so it can effectively control the cost and continuously produce, and the feasibility of the production market is higher than that of ¹⁸ F, ⁸⁹ Zr, etc., which are completely dependent on cyclotron produced isotopes.

Ga-68标记模块。In addition, the ^68Ga labeling method in the present invention is simple, short in time, and high in labeling rate, and the chemical synthesis conditions and basic process in manual labeling can be directly transformed into existing industrialized radiopharmaceutical production modules to realize industrialized production, such as

Ga-68 marker module.

In the examples of the present invention, two examples of radioisotope-labeled polypeptides are listed, namely ⁶⁸ Ga-NOTA-Pip-HAIYPRH and ⁶⁸ Ga-NOTA-AEEP-HAIYPRH.

The structural formula of above-mentioned ^68Ga -NOTA-Pip-HAIYPRH is shown in formula VI:

The structural formula of above-mentioned ^68Ga -NOTA-AEEP-HAIYPRH is shown in formula VII:

A third aspect of the present invention provides:

The preparation method of the above-mentioned radioisotope-labeled polypeptide comprises the following steps:

(1) mixing the above-mentioned polypeptide precursor with the weak acid salt buffer to obtain the weak acid salt mixture of the polypeptide precursor;

(2) eluting the above-mentioned radioisotope with HCl to obtain a radioisotope eluent;

(3) mixing the polypeptide precursor weak acid salt mixture in step (1) and the radioisotope eluent in step (2);

(4) Adjust the pH to 3.8-4.2 and heat to obtain a radioisotope-labeled polypeptide.

Wherein, the above-mentioned weak acid salt buffer can be selected from weak acid salt solutions, including one or more of sodium acetate, gentisic acid, and sodium phosphate, and buffered salt solutions familiar to those skilled in the art can also be used.

A fourth aspect of the present invention provides:

A PET imaging agent, the imaging agent component includes the above-mentioned radioisotope-labeled polypeptide.

Peptide PET imaging agents have great market value. With the promotion of large-scale PET/CT medical equipment, domestic related research is becoming more and more active. Compared with traditional nuclear medicine using ^99mTc imaging agents for SPECT imaging, this The imaging accuracy and sensitivity of the PET imaging agent in the invention are higher, which is one of the important ways for accurate diagnosis of tumors in clinical practice, and is also an important development direction of tumor molecular imaging drugs and medical technology.

After intravenous injection of the imaging agent of the present invention (containing ⁶⁸ Ga-NOTA-Pip-HAIYPRH or ⁶⁸ Ga-NOTA-AEEP-HAIYPRH), its in vivo biological distribution characteristics and PET/CT imaging effect. The pharmacokinetics and biodistribution of the two imaging agents share some common features. Both imaging agents are mainly metabolized by the liver and excreted by the kidneys of mice. Secondly, the spleen also has some physiological uptake, but in general, the physiological uptake and metabolic pathways of PET imaging agents do not affect the diagnosis of tumor tissue. This problem can be solved by delayed scanning. The results of in vivo PET/CT imaging and in vivo biodistribution of tumor-bearing nude mice showed that in addition to physiological uptake in the liver, kidney, and spleen, the tumor site also had significant radioactivity uptake, and showed radioactive concentration over time, which could effectively identify the tumor site.

A fifth aspect of the present invention provides:

Use of the above imaging agent in evaluating the expression level of transferrin receptor.

A sixth aspect of the present invention provides:

The application of the above imaging agent in the preparation of an imaging diagnostic reagent for malignant tumors with abnormal transferrin receptor expression.

Further, the above-mentioned malignant tumors with abnormal expression of transferrin receptor include glioma, prostate cancer, hepatocellular carcinoma, lymphoma, triple negative breast cancer, lung cancer, pancreatic cancer, renal cell mediated by c-Myc oncogene. cancer, ovarian cancer, bladder cancer, colorectal cancer.

In the tumor cell signaling pathway, activation of c-Myc oncogene can stimulate the transcription of TFRC in the system, so in glioma, prostate cancer, hepatocellular carcinoma, lymphoma, triple-negative breast cancer, TFRC is generally highly expressed in various malignant tumors such as lung cancer, pancreatic cancer, renal cell carcinoma, ovarian cancer, bladder cancer, and colorectal cancer, and the imaging agent in the present invention is mainly aimed at the expression level of transferrin receptor. Therefore, Glioma, prostate cancer, hepatocellular carcinoma, lymphoma, triple-negative breast cancer, lung cancer, pancreatic cancer, renal cell cancer, ovarian cancer, bladder cancer, colorectal cancer, etc. Early clinical diagnosis, staging and efficacy evaluation of malignant tumors.

The beneficial effects of the present invention are:

1. The present invention first utilizes the sequence HAIYPRH polypeptide as a targeting ligand to prepare a positron radioactive imaging agent, and uses it for the study of tumor receptor imaging. The polypeptide sequence HAIYPRH has a small molecular weight, low immunogenicity, and is compatible with Endogenous transferrin in vivo does not have a competitive effect and can specifically bind to transferrin receptors;

2. The imaging agent in the present invention is mainly eliminated by liver and kidney metabolism. After entering the body, it can be rapidly concentrated in the tumor site, and the retention time in the tumor site is long. It can be stable from left to right, and the image can effectively identify the tumor site. The radioactive uptake value between 30min and 70min is ideal. It is expected to be used as an imaging agent for malignant tumors with abnormal TFRC expression, and has market development potential;

3. The present invention also discloses a method for improving the structure of the HAIYPRH sequence polypeptide. The connecting group Pip can be used to increase its fat solubility, the connecting group AEEP can be used to increase its water solubility, and the improvement of the fat solubility can promote the drug to penetrate the blood-brain barrier, Developed for imaging of malignant brain tumors.

Description of drawings

Fig. 1 is the PET imaging of ^68Ga -NOTA-Pip-HAIYPRH of Example 1 in U87MG tumor-bearing nude mice;

Fig. 2 is the radioactive uptake and metabolism curve of tumor, liver and muscle tissue after administration of ^68Ga -NOTA-Pip-HAIYPRH of Example 1;

Fig. 3 is the PET imaging of ^68Ga -NOTA-AEEP-HAIYPRH of Example 2 in tumor-bearing nude mice;

Figure 4 is the radioactive uptake and metabolism curve of tumor, liver and muscle tissue after administration of ⁶⁸ Ga-NOTA-AEEP-HAIYPRH of Example 2.

Detailed ways

In order to make the invention purpose, technical solutions and technical effects of the present invention clearer, the present invention will be further described in detail below with reference to the specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, rather than for limiting the present invention.

The experimental reagents and instruments used, unless otherwise specified, are conventional consumables and reagents that can be obtained from commercial sources.

Reagents and Instruments

Reagents:

Ultrapure water, ultrapure hydrochloric acid (Merck, Germany), pharmaceutical grade absolute ethanol, sodium acetate and sodium citrate, Chelex-100 resin (100-200 mesh, Sigma, USA), 0.9% saline, MEM medium and Fetal bovine serum (Gibco, USA), 0.25% trypsin-EDTA digestion solution and 0.01M PBS buffer, isoflurane, 6 kinds of Fmoc-protected amino acids, fluorene methoxyl chloride (Fmoc-Cl), tert-butanol, 1 ,4,7-Triazacyclononane-N,N',N"-triacetic acid (NOTA), 4-amino-(1-carboxymethyl)piperidine, 3-[2-(2-aminoethyl) Oxy)ethoxy]-propionic acid, Fmoc-Leu-Wang resin, dimethylformamide (DMF), piperidine, 4-amino-(1-carboxymethyl)piperidine, N,N-diiso Propylethylamine (DIEA), N-methylpyrrolidone (NMP), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), trifluoroacetic acid (TFA) ), triisopropylsilane (TIS);

Among them, the structural formulas of the 6 kinds of Fmoc-protected amino acids are:

1.Fmoc-His-OH, the structural formula is:

2. Fmoc-Arg(Pbf)-OH, the structural formula is:

3. Fmoc-Pro-OH, the structural formula is:

4.Fmoc-Tyr(tBu)-OH, the structural formula is:

5.Fmoc-Ile-OH, the structural formula is:

6.Fmoc-Ala-OH, the structural formula is:

instrument:

⁶⁸ Ge/ ⁶⁸ Ga generator (Germany ITG company), radionuclide activity meter CRC-15PET, thin-layer radioactivity scanner (Radio-TLC), SN-695γ radioimmunoassay counter, analytical balance, multi-purpose incubator MH2800D (Tianjin Aotes Instruments Co., Ltd.), carbon dioxide incubator, animal anesthesia machine, water bath insulation system (Shanghai Yuyan Scientific Instrument Co., Ltd.), small animal PET/CT (Germany SiemenseInveonPET/CT), peptide solid-phase synthesizer (Symphony , PTI, USA), high performance liquid chromatography, electrospray ionization mass spectrometer (ESI-MS, Bruker Optics, USA).

Preparation of polypeptide precursors

1. Preparation of Fmoc-Pip-OH

Fmoc-Pip-OH can be obtained by protecting 4-amino-(1-carboxymethyl) piperidine with tert-butanol and Fmoc-Cl. Of course, it can also be prepared by other methods familiar to those skilled in the art.

The Fmoc-Pip-OH structural formula finally prepared in this example is as follows:

2. Preparation of Fmoc-AEEP-OH

Fmoc-AEEP-OH can be obtained by sequentially protecting 3-[2-(2-aminoethoxy)ethoxy]-propionic acid with tert-butanol and Fmoc-Cl. Of course, other methods familiar to those skilled in the art can also be used. method to prepare.

The Fmoc-AEEP-OH structural formula finally prepared in this example is as follows:

3. Preparation of NOTA-tBu(2)

NOTA-tBu(2) can be prepared by reacting NOTA with tert-butanol under the catalytic system of trifluoromethanesulfonic anhydride, or by reacting NOTA with potassium tert-butoxide. Of course, other methods familiar to those skilled in the art can also be used. method to prepare.

The structural formula of NOTA-tBu(2) finally prepared in this example is as follows:

4. Synthesis of NOTA-Pip-HAIYPRH polypeptide precursor

The NOTA-Pip- ^HAIYPRH polypeptide precursor in this example is synthesized using Fmoc-Leu-Wang resin through the Nα-Fmoc solid-phase peptide synthesis strategy, and the specific steps are as follows:

The resin was treated with 20% piperidine in DMF to remove the ^Nα -Fmoc protecting group. The following Fmoc-protected amino acids (3 equiv.) were then 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). In N-methylpyrrolidone (NMP) with 3 equiv of condensing agent O-(benzotriazol-1-yl)-N,N under the condition of 6 equiv N,N-diisopropylethylamine (DIEA) , N',N'-tetramethylurea quaternary ammonium hexafluorophosphate (HBTU) for coupling. After the extension was completed, the mixture was treated with a mixture of trifluoroacetic acid (TFA)/H ₂ O/triethylsilane (TIS) with a volume ratio of 95:2.5:2.5 for 4 h at room temperature for deprotection and cleavage 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 HPLC with a yield of 47%. The purified product was 96.8% pure by conventional HPLC analysis. The calculated molecular weight of NOTA-Pip-HAIYPRH peptide (C ₆₀ H ₉₁ N ₁₉ O ₁₅ ) was 1318.5, ESI-MS mass spectrometry analysis: result [M+H]+1319.8.

5. Synthesis of NOTA-AEEP-HAIYPRH polypeptide precursor

The NOTA- ^AEEP -HAIYPRH polypeptide precursor in this example is synthesized using Fmoc-Leu-Wang resin through the Nα-Fmoc solid-phase peptide synthesis strategy, and the specific steps are as follows:

The resin was treated with 20% piperidine in DMF to remove the ^Nα -Fmoc protecting group. The following Fmoc-protected amino acids (3 equiv.) were then 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 carried out with 3 equiv of the condensing agent HBTU in NMP under 6 equiv DIEA conditions. After the extension, the mixture was treated with TFA/H ₂ O/TIS in a volume ratio of 95:2.5:2.5 for 4 h at room temperature for deprotection and cleaved from the resin at the same time. 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 HPLC with a yield of 30%. The purified product was 97.4% pure by conventional HPLC analysis. The calculated molecular weight of NOTA-AEEP-HAIYPRH peptide (C ₆₀ H ₉₂ N ₁₈ O ₁₇ ) was 1337.5, ESI-MS mass spectrometry analysis: result [M+H]+1338.6.

The structure of the above-mentioned polypeptide precursor can be summarized as a bifunctional metal chelator NOTA-R-targeting polypeptide whose amino acid sequence is HAIYPRH, and the general structural formula of the above-mentioned polypeptide precursor is such as formula I:

Among them, A is CO;

B is NH;

The R is selected from the group represented by formula II or formula III:

* in the formula II or formula III represents a connection to A in the formula I, and # in the formula II or formula III represents a connection to B in the formula I.

R is 4-amino-(1-carboxymethyl)piperidine (Pip) (formula I) or polyethylene glycol (AEEP) (formula II).

Example 1 Preparation of ⁶⁸ Ga-NOTA-Pip-HAIYPRH

1. ^68Ga -labeled NOTA-Pip-HAIYPRH

Dissolve 20ug of the above-mentioned polypeptide precursor NOTA-Pip-HAIYPRH in 200ul of 1M sodium acetate solution, put it into a reaction flask, and extract 0.05mol/L HCl 5ml with a 5ml syringe to wash the ^68Ge / ^68Ga generator in stages. At a rate of 1 ml/min, collect 1.5 ml of high-activity ⁶⁸ Ga eluent in the middle, add it to the reaction flask, adjust the pH of the reaction solution to 3.8-4.2, seal it, and put it into a multi-function heater water bath at 90 °C for 10 min.

Measure the radiochemical purity of the product, if the radiochemical purity of the product is ≥80%, it indicates that the labeling is successful.

2. Product purification

Use a syringe to draw the above-mentioned labeled product solution after the reaction, add it to the activated Sep-pak C-18 column, rinse free impurities ( ⁶⁸ Ga and ⁶⁸ Ge) with 5ml of normal saline to the waste liquid bottle, and finally use 0.5ml The Sep-pakC-18 column was rinsed with 60% ethanol to obtain a 60% ethanol solution of purified ^68Ga -NOTA-Pip-HAIYPRH.

Use paper chromatography to detect the radiochemical purity, when the radiochemical purity is ≥95%, proceed to the next step, otherwise continue to repeat the purification. The above radiochemical purity measurement method: the reaction solution is detected by Radio-TLC paper chromatography, the developing solvent is 0.1M sodium citrate buffer, the free ⁶⁸ Ga is close to the origin, and the ⁶⁸ Ga-labeled NOTA-Pip-HAIYPRH is distributed in the front of the solvent, The radiochemical purity of the product was calculated by the % area of the radioactive peak.

Add physiological saline to dilute about 10 times, filter with 0.22um sterile filter, and then use in animal experiments.

The structural formula of ^68Ga -NOTA-Pip-HAIYPRH prepared in this example is as follows:

Example 2 Preparation of ⁶⁸ Ga-NOTA-AEEP-HAIYPRH

1. ^68Ga -labeled NOTA-AEEP-HAIYPRH

Dissolve 20ug of the above-mentioned polypeptide precursor NOTA-AEEP-HAIYPRH in 200ul of 1M sodium acetate solution, put it into a reaction flask, and extract 0.05mol/L HCl 5ml with a 5ml syringe to wash the ^68Ge / ^68Ga generator in stages. At a rate of 1 ml/min, collect 1.5 ml of high-activity ⁶⁸ Ga eluent in the middle, add it to the reaction flask, adjust the pH of the reaction solution to 3.8-4.2, seal it, and put it into a multi-function heater water bath at 90 °C for 10 min.

Measure the radiochemical purity of the product, if the radiochemical purity of the product is ≥80%, it indicates that the labeling is successful.

2. Product purification

Use a syringe to draw the above-mentioned labeled product solution after the reaction, add it to the activated Sep-pak C-18 column, rinse free impurities ( ⁶⁸ Ga and ⁶⁸ Ge) with 5ml of normal saline to the waste liquid bottle, and finally use 0.5ml The Sep-pakC-18 column was rinsed with 60% ethanol to obtain a 60% ethanol solution of purified ^68Ga -NOTA-AEEP-HAIYPRH.

Use paper chromatography to detect the radiochemical purity, when the radiochemical purity is ≥95%, proceed to the next step, otherwise continue to repeat the purification.

Add physiological saline to dilute about 10 times, filter with 0.22um sterile filter, and then use in animal experiments.

Radiochemical purity measurement method: The reaction solution was detected by Radio-TLC paper chromatography, the developing solvent was 0.1M sodium citrate buffer, the free ⁶⁸ Ga was close to the origin, and the ⁶⁸ Ga-labeled NOTA-AEEP-HAIYPRH was distributed in the front of the solvent. The radiochemical purity of the product was calculated from the % area of the radioactive peak.

The ^68Ga -NOTA-AEEP-HAIYPRH structural formula prepared in this example is as follows:

Biodistribution test of the polypeptide imaging agent of the present invention

Biodistribution of ^68Ga -NOTA-Pip-HAIYPRH in Example 1 of the present invention in KM mice

Twelve KM mice, female, were randomly divided into groups, 3 mice in each group, fasted overnight for 12 hours, injected with 68Ga-NOTA-Pip-HAIYPRH 3.7MBq via tail vein, and blood was collected from the eyeballs 5, 30, 60, and 120 min after injection, respectively. The KM mice were killed by cervical dislocation, and the heart, lung, liver, spleen, kidney and other organs or tissues were harvested, weighed on an analytical balance, and the radioactive count was determined. After attenuation correction, the percent injection dose rate per gram of tissue (%ID/g) was calculated. . The experimental results are shown in Table 1.

Table 1. Biodistribution of 68Ga-NOTA-Pip-HAI in KM mice (%ID/g, mean±SD, n=3)

The results of in vivo distribution experiments show that ^68Ga -NOTA-Pip-HAIYPRH is rapidly distributed to various tissues and organs after entering the body circulation. The physiological uptake of 68Ga-NOTA-Pip-HAIYPRH is high in the liver and spleen, and the uptake in the brain is very low, indicating that it is not easy to pass through the blood-brain barrier. . The drug is water-soluble and is mainly eliminated by renal metabolism.

Biodistribution of ^68Ga -NOTA-AEEP-HAIYPRH in Example 2 of the present invention in KM mice

Twelve KM mice, female, were randomly divided into groups, 3 mice in each group, fasted overnight for 12 hours, injected with ^68Ga -NOTA-AEEP-HAIYPRH 3.7MBq via tail vein, and blood was collected from eyeballs 5, 30, 60, and 120 minutes after injection, respectively. , KM mice were killed by cervical dislocation, the heart, lung, liver, spleen, kidney and other organs or tissues were taken, weighed on an analytical balance, the radioactive count was determined, and the percentage injection dose rate per gram of tissue was calculated after attenuation correction (%ID/g ). The experimental results are shown in Table 2.

Table 2. Biodistribution of ^68Ga -NOTA-AEEP-HAIYPRH in KM mice (%ID/g, mean±SD, n=3)

The results of in vivo distribution experiments show that ^68Ga -NOTA-AEEP-HAIYPRH is rapidly distributed to various tissues and organs after entering the body circulation. The physiological uptake of 68Ga-NOTA-AEEP-HAIYPRH is high in the liver and spleen, and the uptake in the brain is extremely low, indicating that it is not easy to penetrate the blood and brain. barrier. The drug is water-soluble and is mainly eliminated by renal metabolism.

From Table 1 and Table 2, it can be seen that in normal KM mice, there are also some differences in the pharmacokinetics and biodistribution of the two imaging agents, the blood radioactivity uptake %ID of ^68Ga -NOTA-Pip-HAIYPRH /g higher than ^68Ga -NOTA-AEEP-HAIYPRH, the former has longer circulation time in vivo, slower metabolic clearance, and higher brain uptake, which has an important relationship with the chemical structure modification of the linker. The predicted LogD7.0 value of the linking group Pip is -2.57, and the predicted value of the LogD7.0 of the linking group AEEP is -3.66. Therefore, compared with the AEEP-modified imaging agent, the Pip-modified liposolubility is improved, and the imaging agent is similar to There may be increased serum albumin binding, decreased renal clearance, prolonged circulation in vivo, and increased binding to the target TFRC.

Targeting test of the polypeptide imaging agent of the present invention

Establishment of U87MG tumor-bearing nude mouse model

Balb/c nunu nude mice, SPF grade, female, 4-5 weeks old, 8 mice. The human glioblastoma cell line U87MG was selected for culture, culture conditions: MEM medium containing penicillin 100U/ml, streptomycin 0.1mg/ml, added 10% fetal bovine serum, carbon dioxide incubator temperature 37 ℃, CO ₂ 5%, 90% humidity, and cultured for 48 hours. Add 0.25% trypsin-EDTA digestion solution to digest and collect cells, centrifuge and wash 2 times with PBS buffer at 1000 rpm, adjust the cell concentration of cell suspension in MEM medium to 2.5×10 ⁷ cells/ml, and draw the cell suspension with a 1 ml syringe. Nude mice were inoculated with 5×10 ⁶ cells/cell in the right armpit, and the inoculation volume was 0.2 ml. 2 to 3 weeks after inoculation, when the tumor volume grows to 300-500 m ³ , it is used for in vivo biodistribution and imaging experiments.

Biodistribution of ^68Ga -NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice in Example 1

Three above-mentioned U87MG tumor-bearing nude mice, female, fasted overnight for 12 hours, were injected with 3.7MBq of ^68Ga -NOTA-Pip-HAIYPRH in Example 1 through the tail vein, blood was collected from the eyeball 60 minutes after the injection, and U87MG was sacrificed by cervical dislocation From tumor-bearing nude mice, organs or tissues such as heart, lung, liver, spleen, and kidney were taken, weighed on an analytical balance, and the radioactivity count was determined. After attenuation correction, the %ID/g was calculated. Statistical methods SPSS10.0 statistical software was used for statistical analysis, variance analysis was used, and P<0.05 was considered statistically significant. The experimental results are shown in Table 3.

Table 3. Biodistribution of ^68Ga -NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice after 60min injection (%ID/g, mean±SD, n=3)

The results showed that the physiological uptake of ^68Ga -NOTA-pip-HAIYPRH in the liver, spleen and kidney was relatively high, and the uptake in the brain was extremely low. The tumor/muscle %ID/g ratio was 3.08±0.45, indicating that tumor tissue also had significant uptake.

Biodistribution of ^68Ga -NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice in Example 2

Three above-mentioned U87MG tumor-bearing nude mice, female, fasted overnight for 12 hours, were injected with 68Ga-NOTA-AEEP- ^HAIYPRH 3.7MBq in Example 2 through the tail vein, blood was collected from the eyeballs 60 minutes after the injection, and the U87MG mice were sacrificed by cervical dislocation. From tumor-bearing nude mice, organs or tissues such as heart, lung, liver, spleen, and kidney were taken, weighed on an analytical balance, and the radioactivity count was determined. After attenuation correction, the %ID/g was calculated. Statistical methods SPSS10.0 statistical software was used for statistical analysis, variance analysis was used, and P<0.05 was considered statistically significant. The experimental results are shown in Table 4.

Table 4. Biodistribution of ^68Ga -NOTA-AEEP-HAIYPRH in U87MG tumor-bearing nude mice after 60min injection (%ID/g, mean±SD, n=3)

The results showed that the physiological uptake of ^68Ga -NOTA-AEEP-HAIYPRH by the liver, spleen and kidney was high, and the brain uptake was extremely low. The %ID/g ratio of tumor/muscle was 1.90±1.70, indicating that it has certain targeting to tumor tissue.

PET/CT imaging of ^68Ga -NOTA-Pip-HAIYPRH in U87MG tumor-bearing nude mice in Example 1

One of the above U87MG tumor-bearing nude mice was injected with 68Ga-NOTA-Pip- ^{HAIYPRH11.1MBq} in Example 1 via tail vein, and PET/CT images were dynamically acquired after injection (300s×14). The scanned image was reconstructed in OSEM3D format, the region of interest (ROI) was delineated for calculation, and the metabolic curve was drawn. Statistical methods SPSS10.0 statistical software was used for statistical analysis, variance analysis was used, and P<0.05 was considered statistically significant. The experimental results are shown in Figure 1 and Figure 2, respectively.

The results showed that the uptake of drugs by the tumor tissue became more and more obvious with the prolongation of time. As shown in Figure 1, the tumor tissue (indicated by the arrow) began to have weak imaging at 5 minutes, and the radioactive uptake of the tumor tissue tended to be higher at 45-70 minutes. Stablize. According to the metabolism curve in Figure 2, the drug was metabolized and distributed by the body after injection, and was rapidly taken up by the tumor. The %ID/g ratio of tumor/muscle from 5 min to 70 min was 2.17±0.22 times. Compared with other tissues, for example, the uptake in the liver decreases slowly over time, but the uptake in the tumor tends to increase slowly, indicating that the ^68Ga -NOTA-Pip-HAIYPRH in Example 1 is gradually metabolized by the liver, kidney, etc. Cleared, but showed slow accumulation at the tumor site.

In conclusion, administration of 11.1MBq ⁶⁸ Ga-NOTA-Pip-HAIYPRH to tumor-bearing nude mice through the tail vein can achieve targeted tumor imaging.

PET/CT imaging of ^68Ga -NOTA-AEEP-HAIYPRH in U87MG tumor-bearing nude mice in Example 2

One of the above-mentioned U87MG tumor-bearing nude mice was injected with ^11.1MBq of 68Ga-NOTA-AEEP-HAIYPRH via tail vein, and PET/CT images were dynamically acquired after injection (300s×14). The scanned image was reconstructed in OSEM3D format, the region of interest (ROI) was delineated for calculation, and the metabolic curve was drawn. Statistical methods SPSS10.0 statistical software was used for statistical analysis, variance analysis was used, and P<0.05 was considered statistically significant. The experimental results are shown in Figures 3 and 4.

The results showed that the uptake of drugs by the tumor tissue became more and more obvious as time went on. As shown in Figure 3, the tumor tissue (indicated by the arrow) began to be visualized at 5 minutes, and the radioactive uptake of the tumor tissue became stable at 40 minutes to 70 minutes. . According to the metabolism curve in Figure 4, the drug is metabolized and distributed after entering the body, and the %ID/g ratio of tumor/muscle within 5min to 70min is 2.05±0.10 times. Compared with other tissues, for example, the uptake in the liver decreased slowly with time, but the uptake in the tumor showed a slow increase trend, indicating that ^68Ga -NOTA-AEEP-HAIYPRH was gradually metabolized and eliminated by the liver, kidney and other organs during the circulation process in the body. The tumor site showed slow accumulation.

In conclusion, administration of 11.1MBq ⁶⁸ Ga-NOTA-AEEP-HAIYPRH to tumor-bearing nude mice through the tail vein can achieve targeted tumor imaging.

Imaging experiments have proved that both imaging agents have tumor PET imaging functions, but the advantages and disadvantages of the two should also be combined with the specificity, sensitivity and radiation dose safety of different metabolic rates, which can be used in further preclinical or Comparison and selection of applications in clinical phase I trials.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection 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:

wherein A is CO;

b is NH;

and R is selected from a group shown in formula II or formula III:

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:

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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