CN118307640A - PET (polyethylene terephthalate) developer precursor, PET developer, and preparation method and application of PET developer precursor and PET developer - Google Patents

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to a PET (polyethylene terephthalate) developer precursor, a PET developer, a preparation method and application thereof. The structural formula of the PET imaging agent precursor is as follows: Wherein M is a chelating group. The PET imaging agent precursor has two cyclic peptide units, and is linked with chelating group through one glutamic acid, and the two binding sequences in the dimer molecule are long enough to combine with integrin receptor alpha v beta 6 expressed on the cell surface, so that the binding affinity and the uptake of tumor to medicine can be enhanced, and the prepared PET imaging agent has good target area to non-target area ratio, lower normal tissue uptake and tumor high uptake, can be specifically absorbed at tumor sites in a targeting way, and is more beneficial to PET imaging of alpha v beta 6 positive diseases.

Description

A PET imaging agent precursor, a PET imaging agent, and a preparation method and application thereof

Technical Field

The invention belongs to the field of pharmaceutical technology, and particularly relates to a PET imaging agent precursor, a PET imaging agent, and a preparation method and application thereof.

Background technique

Integrins are dimeric transmembrane receptors that mediate the interaction between cells and the extracellular matrix (ECM), and play a key role in the signaling pathways that regulate cell differentiation, migration, proliferation, and apoptosis. Integrins are composed of α and β subunits, with a total of 24 αβ dimers. Among them, αvβ3 is mainly expressed on tumor neovascular endothelial cells and is closely related to tumor angiogenesis, while αvβ6 is only expressed in epithelial cells and is most closely related to the formation of cancer, and is called "cancer" integrin. As an epithelial-specific cell surface receptor, αvβ6 is undetectable in healthy adult epithelial cells, but is significantly upregulated in a variety of epithelial cell-derived cancers, including pancreatic cancer, making it a very attractive target for clinical detection and targeted therapy.

At present, the radiopharmaceutical targeting integrin αvβ6 [ ^68Ga ]Ga-DOTA-5G has a good uptake value, and the background radioactivity distribution in the gastrointestinal tract and other normal tissues is low, but the retention in the target area is still insufficient. At the same time, the ^68Ga -labeled radiopharmaceutical has a short half-life, and the ^68Ge - ^68Ga isotope generator produces a small amount of nuclides, which cannot meet the needs of large-scale production and clinical diagnosis. Among the ^18F -labeled radiopharmaceuticals targeting αvβ6, the peptide designed based on the hand, foot and mouth disease virus FMDV is the main one, and the αvβ6 peptide inhibitor A20FMDV2 with the designed sequence of NAVPNLRGDLQVLAQKVART is obtained, and then ^18F -FBA-A20FMDV2 is prepared. On this basis, polyethylene glycol (PEG) is used to improve its tumor retention strength to prepare a probe. Its preliminary clinical application shows that the tumor lesions are well imaged, but there is obvious physiological uptake in the gastrointestinal tract, which affects the tumor display. In addition, based on the amino acid motif RTDXL in FMDV that binds to αvβ6, a molecular probe of cystine knot peptides (Knottins) ¹⁸ F-FP-R01-MG-F2 was designed. It has good imaging of pancreatic cancer lesions in preliminary clinical applications, but there is also obvious gastrointestinal physiological uptake that affects the display of tumors. In addition, the preparation process of the above-mentioned ¹⁸ F-labeled αvβ6-targeted radiopharmaceuticals is complicated and time-consuming.

Therefore, there is an urgent need to provide a PET imaging agent precursor, wherein the prepared PET imaging agent targeting integrin αvβ6 can be targeted and specifically taken up in the tumor site and has good stability and a ratio of target area to non-target area.

Summary of the invention

The present invention aims to solve one or more technical problems existing in the above-mentioned prior art, and at least provide a beneficial choice or create conditions. Specifically, the present invention provides a PET imaging agent precursor, and the PET imaging agent targeting integrin αvβ6 prepared by the precursor can be targeted and specifically taken up in the tumor site, and has good stability and ratio of target area to non-target area.

The inventive concept of the present invention is as follows: the PET imaging agent precursor of the present invention has two cyclic peptide units and is linked to a chelating group via a glutamic acid. The distance between the two binding sequences in the dimer molecule is long enough to enable it to simultaneously bind to the integrin receptor αvβ6 expressed on the cell surface, thereby enhancing the binding affinity and the uptake of the drug by the tumor, thereby enabling the PET probe prepared from the PET imaging agent precursor to be targeted and specifically taken up in the tumor site, and having good stability and a ratio of the target area to the non-target area.

Therefore, a first aspect of the present invention provides a PET imaging agent precursor.

Specifically, the structural formula of the PET imaging agent precursor is:

Wherein, M is a chelating group.

Preferably, the chelating group is selected from any one of the following structural formulas:

The second aspect of the present invention provides a method for preparing the PET imaging agent precursor described in the first aspect of the present invention.

Specifically, the method for preparing the PET imaging agent precursor comprises the following steps:

The dimeric polypeptide and the chelating agent are mixed and reacted to obtain the PET imaging agent precursor.

Preferably, the reaction is carried out at a pH of 7-10; and/or the reaction temperature is 3-5° C., and/or the reaction time is 10-14 h.

Further preferably, the reaction is carried out at a pH of 8-9; the reaction temperature is 3.5-4.5°C; and the reaction time is 11-13h.

More preferably, the reaction is carried out at a pH of 8.5; the reaction temperature is 4° C., and the reaction time is 12 h.

Preferably, the preparation process of the dimeric polypeptide is:

(1) Glutamic acid is used as the first amino acid, and glycine, aspartic acid, glycine, and arginine are sequentially connected to obtain a cyclic peptide, and then the carboxyl end of the glutamic acid is connected to lysine to obtain a monomeric cyclic peptide;

(2) mixing the monomeric cyclic peptide obtained in step (1) with glutamic acid and lysine, and reacting them to obtain the dimeric polypeptide.

Preferably, step (1) is specifically as follows: using Fmoc-L-glutamic acid (fluorenylmethoxycarbonyl-L-glutamic acid 1-tert-butyl ester) (E) as the first amino acid, sequentially connecting L-cyclohexylglycine (L-Chg), aspartic acid (D), glycine (G), and arginine (R), using the N-terminal amino group and the glutamic acid side chain carboxyl group to cyclize to obtain the cyclic peptide c (RGD-Chg-E), and then the carboxyl end of glutamic acid (E) is connected to a lysine (K) to form a monomeric cyclic peptide c (RGD-Chg-E) K.

Preferably, step (2) is specifically to mix the monomeric cyclic peptide obtained in step (1) with glutamic acid (E) and lysine (K), and to react the amino groups of the side chains of glutamic acid (E) and lysine (K) in the monomeric cyclic peptide c(RGD-Chg-E)K to obtain the dimeric polypeptide.

Specifically, the PET imaging agent precursor is prepared by using the Fomc solid phase peptide synthesis method.

Preferably, in step (2), the reaction temperature is room temperature; the reaction time is 10-14 h; further preferably, the reaction time is 11-13 h; further preferably, the reaction time is 12 h.

A third aspect of the present invention provides a PET imaging agent.

Specifically, the PET imaging agent contains the structure of the PET imaging agent precursor described in the first aspect of the present invention.

Preferably, the structural formula of the PET imaging agent is:

Wherein, R ⁿ⁺ represents a radioactive metal nuclide, and n is selected from a positive integer.

Preferably, the radioactive metal nuclide is selected from any one of [Al- ¹⁸ F] ²⁺ , ⁶⁸ Ga ³⁺ , and ⁶⁴ Cu ²⁺ .

Preferably, the PET imaging agent is selected from any one of the compounds of the following structures:

The fourth aspect of the present invention provides a method for preparing the PET imaging agent according to the third aspect of the present invention. Specifically, the method for preparing the PET imaging agent comprises the following steps:

The PET imaging agent precursor and the radioactive metal nuclide are reacted in a solution to prepare the PET imaging agent.

Preferably, when the radioactive metal nuclide is [Al- ¹⁸ F] ²⁺ , the ¹⁸ F ions are captured by an anion exchange column and blown dry by nitrogen for later use.

Preferably, when the radioactive metal nuclide is [Al- ¹⁸ F] ²⁺ , an aluminum salt needs to be added to carry out the reaction.

Preferably, the aluminum salt comprises AlCl ₃ .

Specifically, the main function of the aluminum salt is to complex with fluoride ions, and the obtained [Al- ¹⁸ F] ²⁺ complex is chelated with the chelating group to obtain the PET imaging agent.

Preferably, the solution comprises at least one of sodium acetate and sodium chloride.

Preferably, the pH of the solution is 3-5; more preferably, the pH of the solution is 3.5-4.5.

Specifically, the solution is used to elute the ¹⁸ F ions on the anion exchange column QMA.

Preferably, the PET imaging agent precursor is first dissolved in a solvent.

Preferably, the solvent is selected from at least one of acetonitrile and DMSO.

Preferably, the usage ratio of the PET imaging agent to the solvent is (2.7-22) μg: (9-55) μL; further preferably, the usage ratio of the PET imaging agent to the solvent is (3-20) μg: (10-50) μL.

Preferably, when aluminum salt is added for reaction, the usage ratio of the PET imaging agent precursor and the aluminum salt is (2.7-22) μg: (0.35-22) μL; further preferably, the usage ratio of the PET imaging agent precursor and the aluminum salt is (3-20) μg: (0.4-2) μL.

Preferably, the AlCl ₃ is an AlCl ₃ solution, and the pH of the AlCl ₃ solution is 3-5; further preferably, the pH of the AlCl ₃ solution is 3.5-4.5.

Preferably, the concentration of the AlCl ₃ solution is 1.5-2.5 mM; further preferably, the concentration of the AlCl ₃ solution is 1.8-2.2 mM.

Preferably, the reaction temperature is 80-120° C., and the reaction time is 8-25 min; further preferably, the reaction temperature is 90-110° C., and the reaction time is 10-20 min.

Preferably, after the reaction is completed, the process further includes adding water to dilute and enriching the product through a C18 solid phase extraction column or an HLB solid phase extraction column, and then cleaning the C18 solid phase extraction column or the HLB solid phase extraction column and drying it.

Preferably, after the C18 solid phase extraction column or the HLB solid phase extraction column is cleaned and dried, the C18 or HLB solid phase extraction column is rinsed, and the elution is collected after passing through a sterile filter membrane to obtain a PET imaging agent that can target αvβ6.

Preferably, ethanol and physiological saline are used to elute the C18 or HLB solid phase extraction column in sequence.

The fifth aspect of the present invention provides a use of the PET imaging agent precursor described in the first aspect of the present invention and/or the PET imaging agent described in the third aspect of the present invention in the preparation of a biological function diagnostic reagent.

Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

(1) The present invention discloses a PET imaging agent precursor targeting integrin αvβ6 dimer cyclic peptides. The PET imaging agent precursor has two cyclic peptide units and is linked to a chelating group via a glutamic acid. The distance between the two binding sequences in the dimer molecule is long enough to allow it to simultaneously bind to the integrin receptor αvβ6 expressed on the cell surface, thereby enhancing the binding affinity and the drug uptake by the tumor. The PET imaging agent prepared by the PET imaging agent precursor is an excellent new pharmacokinetic probe and is widely used in biological function imaging. The structure of the PET imaging agent includes a polypeptide, a chelating group and a radioactive metal nuclide, and has a good target area to non-target area ratio, stability, water solubility, low normal tissue uptake and high tumor uptake. It can be targeted and specifically taken up at the tumor site, which is more conducive to PET imaging of αvβ6-positive diseases and has high diagnostic sensitivity.

(2) The present invention selects a cyclic pentapeptide (RGD-Chg-E)K with small molecular weight, high selectivity and hydrophilicity as the core sequence, and obtains a PET imaging agent precursor targeting integrin αvβ6 by adopting the Fomc solid phase peptide synthesis method, wherein a dimeric polypeptide is first obtained, and then the amino group of glutamic acid is amidated and connected with a chelating group (NOTA) to obtain a PET imaging agent precursor (NOTA-E-[cyclo(RGD-Chg-E)K] ₂ ). This makes the distance between the two binding sequences in the dimer molecule long enough to enable it to simultaneously bind to the integrin receptor αvβ6 expressed on the cell surface, further enhancing the binding affinity and the tumor's uptake of the drug, and achieving a better diagnostic or therapeutic effect.

(3) The PET imaging agent of the present invention is an excellent new type of pharmacokinetic probe, and its preparation process is simple, efficient and high in yield.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a high-resolution mass spectrum of NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention;

Fig. 2 is the HPLC UV spectrum of NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention;

FIG3 is a radioactive HPLC spectrum of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention;

FIG4 is a radioactive HPLC spectrum of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in an in vitro PBS system at 1h and 2h, in an in vitro human serum (NHS) system at 1h and 2h, and in vivo at 1h;

FIG5 is a Micro-PET/CT image of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumor-bearing mice (αvβ6 positive) at different time points within 60 minutes;

FIG6 is a graph showing the change of %ID/g values of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention at different time points in organs on 60-minute Micro-PET/CT;

FIG7 is a graph showing the changes in %ID/g values at different time points of uptake and inhibition imaging of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumor-bearing mice (αvβ6 positive) on 60-minute Micro-PET/CT;

FIG8 is a 60-minute Micro-PET/CT image of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumors (αvβ6 positive) and MDA-MB-231 tumors (αvβ6 negative);

FIG9 is a comparative image of Micro-PET/CT of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention and [ ¹⁸ F]AlF-NOTA-FAPI-42 and ¹⁸ F-FDG for 60 minutes;

FIG10 is a graph showing the change in %ID/g values at different time points of 60-minute Micro-PET/CT contrast imaging of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention, [ ¹⁸ F]AlF-NOTA-FAPI-42, and ¹⁸ F-FDG in H2009 tumor-bearing mice (αvβ6 positive);

FIG. 11 is a graph showing the biodistribution of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumor-bearing mice (αvβ6 positive) and MDA-MB-231 tumor-bearing mice (αvβ6 negative) over a period of 60 minutes.

Detailed ways

In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

Example 1

A PET imaging agent targeting integrin receptor αvβ6 dimeric cyclic peptide, the structural formula of which is:

A method for preparing a PET imaging agent targeting integrin receptor αvβ6 dimeric cyclic peptides comprises the following steps:

(1) Preparation of cyclo(RGD-Chg-E)K: 0.03 mmol of polyacrylamide resin (PAM resin) was used as a solid phase carrier, Fmoc-L-glutamic acid (0.12 mmol) was selected as the first amino acid, and after being connected to the PAM resin, 20% by volume piperidine/dimethylformamide (DMF) was used to remove the N-terminal Fmoc protecting group, and O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU) (0.12 mmol) and 1-hydroxybenzotriazole (HOBt) (0.12 mmol) were used as condensation reagents. The agent is sequentially connected with L-cyclohexylglycine (L-Chg), aspartic acid (D), glycine (G), and arginine (R) with a molar weight of 0.12 mmol each to construct a peptide chain, and then the N-terminal amino group is cyclized with the carboxyl group of the glutamic acid side chain to obtain the cyclo(RGD-Chg-E) cyclic peptide; the carboxyl end of glutamic acid (E) is further connected with a lysine (K) to form a crude monomer cyclopeptide cyclo(RGD-Chg-E)K; the crude product is separated and purified by high performance liquid chromatography to obtain a cyclic polypeptide monomer cyclo(RGD-Chg-E)K (15 mg);

(2) Preparation of E-[cyclo(RGD-Chg-E)K] ₂ : In anhydrous DMF (2 mL), tert-butyloxycarbonyl (Boc)-protected activated glutamic acid ester (Boc-E(OSu) 2) (0.1 mmol) was mixed with the cyclic polypeptide monomer Cyclo(RGD-Chg-E)-K (10 mg) obtained in step (1) to obtain a mixture; the pH value of the mixture was adjusted to 8.5 using diisopropylethylamine (DIPEA), and the mixture was stirred at room temperature for one night to obtain a crude product Boc-E-[Cyclo(RGD-Chg-E)-K] 2; the mixture was then treated with anhydrous TFA (trifluoroacetic acid) for 5 min to remove the Boc protection, and then separated and purified by high performance liquid chromatography, and the fractions were collected and freeze-dried to obtain white powder E-[cyclo(RGD-Chg-E)K] ₂ (8 mg);

(3) Preparation of PET imaging agent precursor NOTA-E-[cyclo(RGD-Chg-E)K] ₂ : Add 2 mg of chelating agent 2-[4,7-bis[2-(tert-butoxy)-2-oxoethyl]-1,4,7-triazacyclononane-1-yl]acetic acid (NOTA-bis(t-Buester)) to E-[Cyclo(RGd-Chg-E)-K] ₂ (8 mg) obtained in step (2), adjust the pH value to 8.5 with 0.1 M sodium hydroxide, and then incubate at 4°C overnight. After semi-preparative high performance liquid separation, freeze-drying is performed to obtain white powder PET imaging agent precursor NOTA-E-[Cyclo(RGD-Chg-E)-K] ₂ (9 mg);

(4) Preparation of PET imaging agent [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ : The preparation process of the PET imaging agent includes the following steps:

S1: ¹⁸ F ions are obtained by bombarding H ₂ ¹⁸ O with a medical cyclotron, and ^{the 18} F ions are captured by an anion exchange column (QMA column), and then dried by nitrogen gas for later use;

S2: 300 μL of acetonitrile and 120 μg of the imaging agent precursor obtained in step (3) were mixed in a reaction bottle, and 10 μL of AlCl ₃ (pH=4, 2 mM) was added. Then, the QMA column was eluted with sodium acetate solution (pH=3.9, 0.3 mL) and added to the reaction bottle. After shaking and mixing, the mixture was sealed and heated. The reaction was carried out at 100° C. for 15 min. After the reaction, a reaction solution was obtained. Water was added to the reaction solution for dilution and the product was enriched by a C18 solid phase extraction column. The C18 solid phase extraction column was then washed with 30 mL of water and dried. Finally, the C18 solid phase extraction column was rinsed with 1 mL of injection ethanol and 12 mL of physiological saline in turn, and the eluted solution was filtered through a sterile filter membrane and collected in a sterile vacuum bottle to obtain a PET imaging agent targeting integrin αvβ6 dimer cyclic peptide [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ .

Figure 1 is a high-resolution mass spectrum of the PET imaging agent precursor NOTA-E-[cyclo(RGD-Chg-E)K] ₂ , wherein the abscissa m/z represents the mass-to-charge ratio of the ion. Figure 2 is an HPLC UV spectrum of the PET imaging agent precursor NOTA-E-[cyclo(RGD-Chg-E)K] _2. The structure of the NOTA-E-[cyclo(RGD-Chg-E)K] ₂ precursor was determined by HPLC UV spectrum and high-resolution mass spectrometry.

Performance Testing

1. Radiochemical Purity Determination

The radiochemical purity of the radiopharmaceutical was determined by high performance liquid chromatography (HPLC), wherein the analysis conditions of HPLC were as follows: the analytical column was a ZORBAX Eclipse XDB-C18 column; the mobile phase was a 0.1% trifluoroacetic acid (TFA) aqueous solution in phase A and a 0.1% TFA acetonitrile solution in phase B; the elution gradient was as follows: 0-2 min, 90% phase A, 10% phase B, flow rate 1 mL/min; 2 min-14 min, phase A decreased to 20%, phase B increased to 80%, flow rate 1 mL/min; the ultraviolet detection wavelength was 220 nm, and the radioactivity detector was a radio-HPLC (BIOSCAN, USA).

10 MBq of the reaction solution after the S2 reaction in step (4) of Example 1 and 10 MBq of the radioactive product PET imaging agent [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ were taken, and the radiochemical purity was determined by radio-HPLC. The radio-HPLC spectrum is shown in FIG3 , wherein FIG3( a ) is the radio-HPLC spectrum of the reaction solution; and FIG3( b ) is the radio-HPLC spectrum of the purified solution.

As can be seen from FIG3 , the yield of the developer of Example 1 of the present invention without attenuation correction is 67.5% ( FIG3( a )); the radiochemical purity of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ is >99%, and the retention time is 9.0-9.9 min ( FIG3( a )).

2. Determination of lipid-water distribution coefficient

3.7 MBq of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ prepared in Example 1 was added as a radioactive drug into a centrifuge tube (15 mL) containing 3 mL of n-octanol and 3 mL of PBS, and sealed in a dry thermostat and shaken at room temperature for 5 min. The tube was allowed to stand until the n-octanol and PBS phases were completely separated. 500 μL of each phase was taken from each phase with a pipette and placed in a γ counting tube. The count was measured with a γ counter. The results were repeated three times and the average value was calculated. The LogP value was calculated according to formula (1), which was the lipid-water distribution coefficient.

LogP = Log (n-octanol γ count/PBS γ count) (1);

In formula (1), PBSγ counts represent the radioactive counts in the PBS phase; n-octanolγ counts represent the radioactive counts in the n-octanol phase; and Log is the logarithm with base 10.

The test results of the lipid-water distribution coefficient are shown in Table 1.

Table 1: Lipid-water distribution coefficient

Radiopharmaceuticals [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ LogP -5.9±0.02

It can be seen from the lipid-water distribution coefficient of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ in Table 1 that the PET imaging agent [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ prepared in Example 1 of the present invention is a water-soluble substance as a radioactive drug, has a strong hydrophilic property, and can be excreted mainly through the kidneys through rapid metabolism. It is predicted that the uptake of radioactive drugs by other normal tissues may be low, and an image with high overall contrast can be obtained.

3. Stability test

The in vivo and in vitro stability of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ was determined by HPLC. The HPLC analysis conditions were the same as those in the radiochemical purity test.

(1) In vitro stability evaluation

Radio-HPLC was used to test the stability in the in vitro PBS system and human serum system, specifically:

[ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ with a radioactive dose of 3.7 MBq was mixed with 300 μL of PBS and human serum, respectively, and then allowed to stand at room temperature for 1 h and 2 h before HPLC analysis. This was repeated three times.

(2) In vivo stability assessment

[ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ with a volume of 200 μL and a radioactive dose of 14.8 MBq was injected into the non-tumor-bearing mice through the tail vein. One hour after the injection, the nude mice were killed, and the blood of the nude mice was collected and mixed with 300 μL of acetonitrile to obtain a blood sample. The blood sample was then immediately centrifuged at a speed of 8000 rpm/min for 5 minutes. The supernatant was first passed through a filter membrane and then separated by HPLC. The eluate was collected, concentrated and injected into an analytical HPLC system. The eluate was collected using a γ counter tube (30 s/tube), and the radioactivity of each tube was measured using a γ counter for analysis.

The radioactive HPLC spectra of the PET imaging agent [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ in vitro PBS system at 1 h and 2 h, in vitro human serum (NHS) system at 1 h and 2 h, and in vivo in non-tumor-bearing mice at 1 h are shown in FIG4 .

As can be seen from FIG4 , [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ exhibited a single and consistent emission peak in both in vivo and in vitro stability experiments, proving that it is stable in vivo and in vitro.

4.Micro PET/CT imaging test

According to the set scanning program, PET 3D acquisition was performed for 60 minutes, and then low-dose CT scanning was performed for 10 minutes per bed. The image was reconstructed using the 3D iteration method (ordered subset maximum expectation method). The Inven Research Workplace (IRW2.2) workstation was used for data processing, and the coronal and sagittal planes were reconstructed after PET attenuation correction using CT data. The region of interest (ROI) was outlined along the edge of the tumor and each tissue on the radioactivity distribution image, and the radioactivity count in each ROI was measured to calculate the percentage of injected dose per gram of tissue (%ID/g).

(1) Observation of the MicroPET/CT imaging effects of [ ¹⁸ F]AlF-NOTA-SDM17 ([ ¹⁸ F]AlF-NOTA-SDM17 is a PET imaging agent targeting αvβ6 disclosed by Quigley NG, Tomassi S, DiLeva FS, et al. Click-Chemistry (CuAAC) Trimerization of an αvβ6 Integrin Targeting Ga-68-Peptide: Enhanced Contrast for in-Vivo PET Imaging of Human Lung Adenocarcinoma Xenografts. Chembiochem. 2020 Oct 1; 21(19): 2836-2843) and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ in animal models of malignant tumors.

H2009 tumor-bearing mice (αvβ6 positive) were fixed on the scanning bed and injected with [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ (7 MBq/mouse) through the tail vein. Dynamic micro PET/CT imaging was performed for 60 minutes after injection to explore the imaging and dynamic organ distribution in vivo. Micro-PET/CT images of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ imaging agents of the present invention at different time points within 60 minutes in H2009 tumor-bearing mice (αvβ6 positive) are shown in Figure 5.

As can be seen from Figure 5, in the imaging of αvβ6-positive tumor-bearing mice, both small molecule highly hydrophilic radioactive drugs showed rapid metabolism in the body mainly in the kidneys, showing extremely low gastrointestinal uptake, which made the overall image background value lower. However, due to the excessively rapid metabolism in the body, the uptake and retention of the monovalent [ ^18F ]AlF-NOTA-SDM17 in the target tumor were poor; in contrast to the monovalent [ ^18F ]AlF-NOTA-SDM17 which was not retained in the tumor, the divalent ligand [ ^18F ]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ prepared in Example 1 of the present invention showed stronger retention in the tumor in the imaging, and an image with tumor uptake and kidney metabolism as the main drug uptake distribution was obtained 60 minutes after injection.

(2) Specific imaging of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ in Micro PET/CT.

[ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ were used for inhibition imaging in H2009 tumor-bearing mice (αvβ6 positive) by co-injection of an overdose (200 μg/mouse) of NOTA-SDM17 or NOTA-E-[cyclo(RGD-Chg-E)K] ₂ unlabeled prodrug; then the divalent [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ radiopharmaceutical was further used for dynamic 60 min micro PET/CT imaging in MDA-MB-231 tumor-bearing mice (αvβ6 negative).

The changes in %ID/g values of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention at different time points in organs on Micro-PET/CT for 60 minutes are shown in Figure 6, wherein Figure 6(A) is a graph showing the changes in %ID/g values of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ at different time points in organs on Micro-PET/CT for 60 minutes; Figure 6(B) is a graph showing the changes in %ID/g values of [ ¹⁸ F]AlF-NOTA-SDM17 at different time points in organs on Micro-PET/CT for 60 minutes.

FIG7 shows the changes in %ID/g values of [ ¹⁸ F]AlF-NOTA-SDM17 and [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention at different time points in uptake and inhibition imaging of H2009 tumor-bearing mice (αvβ6 positive) on 60-minute Micro-PET/CT.

The 60-minute Micro-PET/CT images of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumors (αvβ6 positive) and MDA-MB-231 tumors (αvβ6 negative) are shown in Figure 8 , wherein Figure 8(a) is a 60-minute Micro-PET/CT image of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ ; Figure 8(b) is a graph showing the change in %ID/g value of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ over time at different time points in H2009 tumors (αvβ6 positive) and MDA-MB-231 tumors (αvβ6 negative) on Micro-PET/CT for 60 minutes.

As can be seen from Figures 6 and 7, inhibition imaging by co-injection of an excess of NOTA-SDM17 or NOTA-E-[cyclo(RGD-Chg-E)K] ₂ unlabeled prodrug resulted in a significant reduction in the uptake of [ ¹⁸ F]AlF-NOTA-SDM17 or [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ by αvβ6-positive tumors.

As can be seen from Figure 8, compared with the continuous low uptake of the PET imaging agent [ ^18F ]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ prepared by the present invention by the MDA-MB-231 tumor (αvβ6 negative), the H2009 tumor (αvβ6 positive) showed a continuous and significant uptake of the imaging agent [ ^18F ]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ , which once again confirmed that the drug binding of [ ^18F ]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ through the tumor is specific, and the imaging agent can be specifically taken up in the tumor site, which has a good application prospect.

(3) Comparative imaging of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ , ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42.

After anesthetization of αvβ6-positive tumor-bearing mice, [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ was combined with ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42 ( ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42 are existing pan-tumor PET imaging agents) for static micro PET/CT imaging for 60 minutes. The same batch of models were imaged with different radioactive drugs at an interval of 48 hours.

The 60-minute Micro-PET/CT contrast imaging results of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention, ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42 are shown in FIG9 .

FIG10 shows the changes in %ID/g values at different time points of 60-minute Micro-PET/CT contrast imaging of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention with ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42 in H2009 tumor-bearing mice (αvβ6 positive).

As can be seen from Figures 9 and 10, compared with the existing pan-tumor PET imaging agents ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42, [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ showed lower normal tissue uptake and better image contrast, and had higher diagnostic sensitivity. αvβ6 divalent cyclic peptide radioactive drugs are more conducive to PET imaging of αvβ6-positive diseases than existing pan-tumor PET imaging agents.

5. Biodistribution test

Four mice each from the H2009 tumor-bearing mouse model with high expression of αvβ6 receptor and the MDA-MB-231 tumor-bearing mouse model with low expression of αvβ6 receptor were selected and injected with [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ (1.5 MBq/mouse) through the tail vein. Blood was then collected at 60 minutes. The animals were killed by cervical dislocation, and the main organs and tumor lesions were separated. The weights were measured and the radioactivity counts were measured, and the percentage injected dose per gram of tissue (%ID/g) was calculated.

The biodistribution of [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ of the present invention in H2009 tumor-bearing mice (αvβ6 positive) and MDA-MB-231 tumor-bearing mice (αvβ6 negative) for 60 minutes is shown in FIG11 .

As can be seen from Figure 11, the biodistribution experiment of tumor-bearing mice showed consistent results with Micro PET/CT, with kidney metabolism as the main factor, lower normal tissue uptake and high tumor uptake.

In summary, the imaging agent precursor NOTA-E-[cyclo(RGD-Chg-E)K] ₂ prepared by the present invention has two cyclic peptide units and a chelating group linked by a glutamic acid. The distance between the two binding sequences in the dimer molecule is long enough to enable it to simultaneously bind to the integrin receptor αvβ6 expressed on the cell surface, thereby enhancing the binding affinity and the drug uptake by the tumor. It can be easily radiolabeled, and a product [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ with a radiochemical purity of 99% can be obtained. [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ has good stability and hydrophilicity both in vivo and in vitro. In addition, [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ can be rapidly metabolized by the kidneys, resulting in very low drug uptake in normal tissues, especially in gastrointestinal images where no significant uptake was observed; [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ not only has better in vivo pharmacokinetics (mainly metabolized by the kidneys, with very low uptake by normal organs), but also ensures drug retention in the target area, showing stronger target retention, which is beneficial for imaging and can be used for imaging of αvβ6-positive tumors (such as pancreatic cancer) and bone and joint diseases. Compared with ¹⁸ F-FDG and [ ¹⁸ F]AlF-NOTA-FAPI-42, [ ¹⁸ F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K] ₂ has better image contrast, which is beneficial for clinical application.

The above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims (10)

1. A PET imaging agent precursor, characterized in that its structural formula is: Wherein, M is a chelating group. 2. The PET imaging agent precursor according to claim 1, characterized in that the chelating group is selected from any one of the following structural formulas: 3. The method for preparing the PET imaging agent precursor according to any one of claims 1 to 2, characterized in that it comprises the following steps: The dimeric polypeptide and the chelating agent are mixed and reacted to obtain the PET imaging agent precursor. 4. The preparation method according to claim 3, characterized in that the preparation process of the dimeric polypeptide is: (1) Glutamic acid is used as the first amino acid, and glycine, aspartic acid, glycine, and arginine are sequentially connected to obtain a cyclic peptide, and then the carboxyl end of the glutamic acid is connected to lysine to obtain a monomeric cyclic peptide; (2) Mixing the monomeric cyclic peptide obtained in step (1) with glutamic acid and lysine, and reacting them to obtain a dimeric polypeptide. 5. A PET imaging agent comprising the structure of the PET imaging agent precursor according to claim 1 or 2, characterized in that its structural formula is: Wherein, R ⁿ⁺ represents a radioactive metal nuclide, and n is selected from a positive integer. 6 . The PET imaging agent according to claim 5 , wherein the radioactive metal nuclide is selected from any one of [Al ¹⁸ -F] ²⁺ , ⁶⁸ Ga ³⁺ , and ⁶⁴ Cu ²⁺ . 7. The PET imaging agent according to claim 6, characterized in that the PET imaging agent is selected from the following structures: 8. The method for preparing the PET imaging agent according to any one of claims 5 to 7, characterized in that it comprises the following steps: The PET imaging agent precursor and the radioactive metal nuclide are reacted in a solution to prepare the PET imaging agent. 9 . The preparation method according to claim 8 , characterized in that the reaction temperature is 80-120° C., and the reaction time is 8-25 min. 10. Use of the PET imaging agent precursor according to any one of claims 1 to 2 and/or the PET imaging agent according to any one of claims 5 to 7 in the preparation of a biological function diagnostic reagent.