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

Abstract

The present 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. The structural formula of the PET imaging agent precursor of the present invention is: wherein M is a chelating group. The PET imaging agent precursor of the present invention has two cyclic peptide units, and the chelating group is linked by a glutamic acid. The distance between the two binding sequences in the dimer molecule is long enough so that it can simultaneously bind to the integrin receptor αvβ6 expressed on the cell surface, which can enhance the binding affinity and the tumor's uptake of the drug. The prepared PET imaging agent has a good ratio of target area to non-target area, lower normal tissue uptake and high tumor uptake, can be targeted and specifically taken up at the tumor site, and is more conducive to PET imaging of αvβ6-positive diseases.

Description

Technical Field

[0001] 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 Art

[0002] 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.

[0003] Currently, radiopharmaceuticals targeting integrin αvβ6 [ 68 Ga]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. 68 Ga-labeled radiopharmaceuticals have a short half-life. 68 Ge- 68 The Ga isotope generator produces a small amount of radionuclides, which cannot meet the needs of large-scale production or clinical diagnosis. 18 Among the F-labeled αvβ6-targeted radiopharmaceuticals, a peptide designed based on the hand, foot and mouth disease virus FMDV was used as the main component, and an αvβ6 peptide inhibitor A20FMDV2 with a designed sequence of NAVPNLRGDLQVLAQKVART was obtained, and then 18 F-FBA-A20FMDV2, on this basis, polyethylene glycol (PEG) was used to improve its tumor retention strength to prepare a probe. Its preliminary clinical application showed good tumor lesion imaging, but there was obvious gastrointestinal physiological uptake that affected tumor display. In addition, a molecular probe of cystine knot peptides (Knottins) was designed based on the amino acid motif RTDXL in FMDV that binds to αvβ6 18 F-FP-R01-MG-F2, in its initial clinical application, has good imaging of pancreatic cancer lesions, but it also has obvious gastrointestinal physiological uptake that affects the display of tumors. 18 The preparation process of F-labeled αvβ6-targeted radiopharmaceuticals is complex and time-consuming.

[0004] 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

[0005] 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 prepared PET imaging agent targeting integrin αvβ6 can be targeted and specifically taken up in the tumor site, and has good stability and ratio of target area to non-target area.

[0006] 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.

[0007] Therefore, a first aspect of the present invention provides a PET imaging agent precursor.

[0008] Specifically, the structural formula of the PET imaging agent precursor is:

[0009]

[0010] Wherein, M is a chelating group.

[0011] Preferably, the chelating group is selected from any one of the following structural formulas:

[0012]

[0013]

[0014] The second aspect of the present invention provides a method for preparing the PET imaging agent precursor according to the first aspect of the present invention.

[0015] Specifically, the method for preparing the PET imaging agent precursor comprises the following steps:

[0016] The dimeric polypeptide and the chelating agent are mixed and reacted to obtain the PET imaging agent precursor.

[0017] 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.

[0018] 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.

[0019] 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.

[0020] Preferably, the preparation process of the dimeric polypeptide is:

[0021] (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;

[0022] (2) Mixing the monomeric cyclic peptide obtained in step (1) with glutamic acid and lysine, and reacting them to obtain the dimeric polypeptide.

[0023] 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.

[0024] 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) to obtain the dimeric polypeptide.

[0025] Specifically, the PET imaging agent precursor is prepared by using the Fomc solid phase peptide synthesis method.

[0026] 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.

[0027] A third aspect of the present invention provides a PET imaging agent.

[0028] Specifically, the PET imaging agent contains the structure of the PET imaging agent precursor described in the first aspect of the present invention.

[0029] Preferably, the structural formula of the PET imaging agent is:

[0030]

[0031] Among them, R n+ represents a radioactive metal nuclide, and n is selected from a positive integer.

[0032] Preferably, the radioactive metal nuclide is selected from [Al- 18 F] 2+ , 68 Ga 3+ , 64 Cu 2+ Any one of .

[0033] Preferably, the PET imaging agent is selected from any one of the compounds of the following structures:

[0034]

[0035]

[0036] The fourth aspect of the present invention provides a method for preparing the PET imaging agent described in the third aspect of the present invention. Specifically, the method for preparing the PET imaging agent comprises the following steps:

[0037] The PET imaging agent precursor and the radioactive metal nuclide are reacted in a solution to prepare the PET imaging agent.

[0038] Preferably, when the radioactive metal nuclide is [Al- 18 F] 2+ When the anion exchange column is used to capture 18 F ions and blow dry with nitrogen gas for later use.

[0039] Preferably, when the radioactive metal nuclide is [Al- 18 F] 2+ When it is necessary to add aluminum salt to react.

[0040] Preferably, the aluminum salt comprises AlCl3.

[0041] Specifically, the main function of the aluminum salt is to complex with fluoride ions to obtain [Al- 18 F] 2+ The complex is chelated with the chelating group to obtain a PET imaging agent.

[0042] Preferably, the solution comprises at least one of sodium acetate and sodium chloride.

[0043] Preferably, the pH of the solution is 3-5; further preferably, the pH of the solution is 3.5-4.5.

[0044] Specifically, the solution is used to transfer the 18 F ions are eluted.

[0045] Preferably, the PET imaging agent precursor is first dissolved in a solvent.

[0046] Preferably, the solvent is selected from at least one of acetonitrile and DMSO.

[0047] 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.

[0048] 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.

[0049] Preferably, the AlCl3 is an AlCl3 solution, and the pH of the AlCl3 solution is 3-5; further preferably, the pH of the AlCl3 solution is 3.5-4.5.

[0050] Preferably, the concentration of the AlCl3 solution is 1.5-2.5 mM; further preferably, the concentration of the AlCl3 solution is 1.8-2.2 mM.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Preferably, ethanol and physiological saline are used to elute the C18 or HLB solid phase extraction column in sequence.

[0055] 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.

[0056] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0057] (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.

[0058] (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.

[0059] (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

[0060] Figure 1 It is a high-resolution mass spectrum of NOTA-E-[cyclo(RGD-Chg-E)K]2 of the present invention;

[0061] Figure 2 is the HPLC UV spectrum of NOTA-E-[cyclo(RGD-Chg-E)K]2 of the present invention;

[0062] Figure 3 For the present invention 18 Radio-HPLC spectrum of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2;

[0063] Figure 4 For the present invention18 Radioactive HPLC spectra of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 at 1h and 2h in vitro in PBS system, 1h and 2h in vitro in human serum (NHS) system, and 1h in vivo;

[0064] Figure 5 for[ 18 F]AlF-NOTA-SDM17 and the present invention [ 18 Micro-PET / CT images of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 at different time points within 60 minutes in H2009 tumor-bearing mice (αvβ6 positive);

[0065] Figure 6 for[ 18 F]AlF-NOTA-SDM17 and the present invention [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 changes in %ID / g values ​​of organs at different time points on 60-minute Micro-PET / CT;

[0066] Figure 7 for[ 18 F]AlF-NOTA-SDM17 and the present invention [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 uptake and inhibition imaging of H2009 tumor-bearing mice (αvβ6 positive) at different time points on Micro-PET / CT over 60 minutes.

[0067] Figure 8 For the present invention 18 Micro-PET / CT images of αvβ6-positive H2009 and MDA-MB-231 tumors (αvβ6-negative) in 60-minute F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2.

[0068] Fig. 9 For the present invention 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 and [ 18 F]AlF-NOTA-FAPI-42, 18 F-FDG was used for 60 minutes for Micro-PET / CT contrast imaging;

[0069] Fig.10 For the present invention 18F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 and [ 18 F]AlF-NOTA-FAPI-42, 18 F-FDG in H2009 tumor-bearing mice (αvβ6 positive) 60 minutes Micro-PET / CT contrast imaging at different time points of %ID / g value over time;

[0070] Fig.11 For the present invention 18 Biodistribution of [F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in H2009 tumor-bearing mice (αvβ6 positive) and MDA-MB-231 tumor-bearing mice (αvβ6 negative) over 60 minutes. DETAILED DESCRIPTION

[0071] 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 noted that the following embodiments do not limit the protection scope of the present invention.

[0072] 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.

[0073] Example 1

[0074] A PET imaging agent targeting integrin receptor αvβ6 dimeric cyclic peptide, the structural formula of which is:

[0075]

[0076] A method for preparing a PET imaging agent targeting integrin receptor αvβ6 dimeric cyclic peptides comprises the following steps:

[0077] (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 cyclic peptide 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);

[0078] (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, the Boc protection was removed, and the mixture was separated and purified by high performance liquid chromatography, and the components were collected and freeze-dried to obtain white powder E-[cyclo(RGD-Chg-E)K]2 (8 mg);

[0079] (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);

[0080] (4) PET imaging agents 18 Preparation of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2: The preparation process of PET imaging agent includes the following steps:

[0081] S1: H2 bombarded by medical cyclotron 18 OGet 18 F ions are captured using an anion exchange column (QMA column) 18 F ions, and then blow dry with nitrogen for later use;

[0082] 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 AlCl3 (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 a temperature of 100°C for 15 minutes. 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.

[0083] Figure 1 This is a high-resolution mass spectrum of the PET imaging agent precursor NOTA-E-[cyclo(RGD-Chg-E)K]2, where the abscissa m / z represents the mass-to-charge ratio of the ion. Figure 2 This is the 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.

[0084] Performance Testing

[0085] 1. Radiochemical Purity Determination

[0086] 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).

[0087] Take 10MBq of the reaction solution and 10MBq of the radioactive product PET imaging agent [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2, radiochemical purity was determined by radio-HPLC, and the radio-HPLC spectrum was as follows Figure 3 As shown, Figure 3 (a) is the radio-HPLC graph of the reaction solution; Figure 3 (b) is the radio-HPLC graph of the purified solution.

[0088] Depend on Figure 3 It can be seen that the yield of the developer in Example 1 of the present invention without attenuation correction is 67.5% ( Figure 3 (a)); [ 18 The radiochemical purity of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was >99%, and the retention time was 9.0-9.9 min ( Figure 3 (a)).

[0089] 2. Determination of lipid-water distribution coefficient

[0090] 3.7 MBq of the [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was added as a radioactive drug into a centrifuge tube (15 mL) containing 3 mL of n-octanol and 3 mL of PBS, and then sealed in a dry thermostat and shaken at room temperature for 5 min. The mixture 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. This was 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.

[0091] LogP = Log (n-octanol γ count / PBS γ count) (1);

[0092] 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.

[0093] The test results of the lipid-water distribution coefficient are shown in Table 1.

[0094] Table 1: Lipid-water distribution coefficient

[0095] Radiopharmaceuticals <![CDATA[[ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2]]> LogP -5.9±0.02

[0096] From Table 1 [ 18 The lipid-water distribution coefficient of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 shows that the PET imaging agent prepared in Example 1 of the present invention [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2, as a radioactive drug, is a water-soluble substance with strong hydrophilicity. Its excretion pathway in the body can be mainly rapidly metabolized through the kidneys. It is predicted that the uptake of radioactive drugs by other normal tissues may be low, and images with high overall contrast can be obtained.

[0097] 3. Stability test

[0098] HPLC determination 18 The stability of [F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in vivo and in vitro was analyzed by HPLC under the same conditions as those used for radiochemical purity testing.

[0099] (1) In vitro stability evaluation

[0100] Radio-HPLC was used to test the stability in the in vitro PBS system and human serum system, specifically:

[0101] The radioactive dose was 3.7 MBq. 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 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, which was repeated three times.

[0102] (2) In vivo stability assessment

[0103] A volume of 200 μL and a radioactive dose of 14.8 MBq [ 18F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was injected into 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 with a γ counting tube (30s / tube), and the radioactivity of each tube was measured with a γ counter for analysis.

[0104] PET imaging agents[ 18 The radioactive HPLC spectra of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in vitro PBS system at 1h and 2h, in vitro human serum (NHS) system at 1h and 2h, and in vivo in non-tumor-bearing mice at 1h are shown in the figure. Figure 4 shown.

[0105] Depend on Figure 4 It can be seen that [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 showed a single and consistent radiation peak in both in vivo and in vitro stability experiments, proving that it is stable in vivo and in vitro.

[0106] 4. Micro PET / CT imaging test

[0107] According to the set scanning program, 60 minutes of PET 3D acquisition was performed, 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. On the radioactivity distribution image, the region of interest (ROI) was outlined along the edge of the tumor and each tissue, and the radioactivity count in each ROI was measured to calculate the percentage of injected dose per gram of tissue (%ID / g).

[0108] (1)[ 18 F]AlF-NOTA-SDM17([ 18F]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 Observation on the MicroPET / CT imaging effect of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in animal models of malignant tumors.

[0109] H2009 tumor-bearing mice (αvβ6 positive) were fixed on the scanning bed and injected via the tail vein [ 18 F]AlF-NOTA-SDM17 and [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 (7MBq / mouse), dynamic micro PET / CT imaging was performed for 60 minutes after injection to explore the imaging and dynamic organ distribution in vivo, 18 F]AlF-NOTA-SDM17 and the present invention [ 18 Micro-PET / CT images of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 imaging agent at different time points within 60 minutes in H2009 tumor-bearing mice (αvβ6 positive) are shown in the figure below. Figure 5 shown.

[0110] Depend on Figure 5 It can be seen that 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, and showed extremely low gastrointestinal uptake, which made the overall image background value lower. However, due to the rapid metabolism in the body, the unit price [ 18 F]AlF-NOTA-SDM17 had poor uptake and retention in the target tumor; compared with the monovalent [ 18 F]AlF-NOTA-SDM17 is not retained on the tumor. In contrast, the divalent ligand prepared in Example 1 of the present invention [ 18F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 showed stronger retention in the tumor in the imaging, and an image of drug uptake distribution with tumor uptake and renal metabolism as the main components was obtained 60 minutes after injection.

[0111] (2)[ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 specific imaging in Micro PET / CT.

[0112] [ 18 F]AlF-NOTA-SDM17 and [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was co-injected with an overdose (200 μg / mouse) of NOTA-SDM17 or NOTA-E-[cyclo(RGD-Chg-E)K]2 unlabeled prodrug for inhibition imaging in H2009 tumor-bearing mice (αvβ6 positive); then the bivalent [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 radiopharmaceutical was further used for dynamic 60-min micro PET / CT imaging of MDA-MB-231 tumor-bearing mice (αvβ6 negative).

[0113] [ 18 F]AlF-NOTA-SDM17 and the present invention [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 changes with time at different time points in organs on Micro-PET / CT for 60 minutes. Figure 6 As shown, Figure 6 (A) is 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 changes in %ID / g values ​​of organs at different time points on 60-minute Micro-PET / CT; Figure 6 (B) is 18 F]AlF-NOTA-SDM17 %ID / g value of organs at different time points on 60-minute Micro-PET / CT.

[0114] [ 18 F]AlF-NOTA-SDM17 and the present invention [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 uptake and inhibition imaging of H2009 tumor-bearing mice (αvβ6 positive) at different time points on Micro-PET / CT for 60 minutes. Figure 7shown.

[0115] The present invention 18 Micro-PET / CT images of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in H2009 tumors (αvβ6 positive) and MDA-MB-231 tumors (αvβ6 negative) at 60 minutes are shown in Figure 2. Figure 8 As shown, Figure 8 (a) is 18 60-minute Micro-PET / CT images of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2; Figure 8 (b) for 18 Figure 5. Changes of %ID / g values ​​of H2009 tumors (αvβ6 positive) and MDA-MB-231 tumors (αvβ6 negative) at different time points on 60-minute Micro-PET / CT.

[0116] Depend on Figure 6 and 7 As can be seen, inhibition imaging by co-injection of excess NOTA-SDM17 or NOTA-E-[cyclo(RGD-Chg-E)K]2 unlabeled prodrug made αvβ6-positive tumors 18 F]AlF-NOTA-SDM17 or [ 18 The uptake of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was significantly reduced.

[0117] Figure 8 It can be seen that the PET imaging agent prepared by the present invention [ 18 Compared with the sustained low uptake of the imaging agent [F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2, H2009 tumors (αvβ6 positive) showed a significant 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 showed sustained and significant uptake, which once again confirmed that [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 binds specifically to the drug through the tumor, and the imaging agent can be specifically taken up in the tumor site, which has good application prospects.

[0118] (3)[ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 18 F-FDG,18 F]AlF-NOTA-FAPI-42 contrast imaging.

[0119] Will[ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 was respectively 18 F-FDG and [ 18 F]AlF-NOTA-FAPI-42( 18 F-FDG and [ 18 F]AlF-NOTA-FAPI-42 is an existing pan-tumor PET imaging agent) for micro PET / CT static imaging for 60 minutes, and different radioactive drugs were imaged in the same batch of models at an interval of 48 hours.

[0120] The present invention 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 18 F-FDG and [ 18 The 60-minute Micro-PET / CT contrast imaging results of F]AlF-NOTA-FAPI-42 are as follows Fig. 9 shown.

[0121] The present invention 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 18 F-FDG and [ 18 The %ID / g value of F]AlF-NOTA-FAPI-42 in H2009 tumor-bearing mice (αvβ6 positive) was changed over time at different time points in 60-minute Micro-PET / CT contrast imaging. Fig.10 shown.

[0122] Depend on Fig. 9 and 10 It can be seen that 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, with 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.

[0123] 5. Biodistribution test

[0124] Four H2009 tumor-bearing mice with high αvβ6 receptor expression and four MDA-MB-231 tumor-bearing mice with low αvβ6 receptor expression were selected and injected through the tail vein [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 (1.5 MBq / mouse), and then blood was collected at 60 min. The animals were killed by cervical dislocation, and the main organs and tumor lesions were separated, weighed, and the radioactivity count was measured to calculate the percentage injected dose per gram of tissue (%ID / g).

[0125] The present invention 18 The biodistribution of F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 in H2009 tumor-bearing mice (αvβ6 positive) and MDA-MB-231 tumor-bearing mice (αvβ6 negative) over 60 minutes is shown in Figure 2. Fig.11 shown.

[0126] Depend on Fig.11 It can be seen that the biodistribution experiment in 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.

[0127] 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 bind to the integrin receptor αvβ6 expressed on the cell surface at the same time, which can enhance the binding affinity and the tumor's uptake of the drug. It can be easily radiolabeled and a product with a radiochemical purity of 99% can be obtained [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2. [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 has good stability and hydrophilicity both in vivo and in vitro. 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 the gastrointestinal tract, 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 kidney metabolism, normal organ uptake is extremely low), but also can ensure the retention of drugs in the target area, showing stronger target retention, which is conducive to imaging and can be used for imaging of αvβ6-positive tumors (such as pancreatic cancer) and bone and joint diseases. 18 F-FDG and [ 18F]AlF-NOTA-FAPI-42, [ 18 F]AlF-NOTA-E-[cyclo(RGD-Chg-E)K]2 has better image contrast, which is beneficial for its clinical application.

[0128] 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

1. A PET imaging agent precursor, characterized in that: Its structural formula is:

2. The method for preparing the PET imaging agent precursor according to claim 1, characterized in that: The following steps are involved: The dimeric polypeptide and the chelating agent are mixed and reacted to obtain the PET imaging agent precursor.

3. The preparation method according to claim 2, characterized in that: The preparation process of the dimeric polypeptide is as follows: (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) The monomeric cyclic peptide obtained in step (1) is mixed with glutamic acid and lysine to react to obtain a dimeric polypeptide.

4. A PET imaging agent comprising the structure of the PET imaging agent precursor according to claim 1, characterized in that: Its structural formula is:

5. The method for preparing the PET imaging agent according to claim 4, characterized in that: The following steps are involved: PET imaging agent precursors, radioactive metal nuclides [Al- 18 F] 2+ The PET imaging agent is prepared by reacting in a solution.

6. The preparation method according to claim 5, characterized in that: The reaction temperature is 80-120° C., and the reaction time is 8-25 min.

7. Use of the PET imaging agent precursor according to claim 1 and / or the PET imaging agent according to claim 4 in the preparation of a biological function diagnostic reagent.

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