Radiolabeled Exendin (9-39) polypeptide probe precursor and application thereof

Abstract

The invention relates to a radiolabeled Exendin (9-39) polypeptide probe precursor and application thereof, and belongs to the technical field of radiopharmaceutical chemistry and nuclear medicine diagnosis and treatment. And the precursors of the radioactive labeled Exendin (9-39) polypeptide probe are respectively as follows: Lys12 <-> NOTA <-> Exendin (9-39), Lys27 <-> NOTA <-> Exendin (9-39) and Asp09 <-> NOTA <-> Exendin (9-39). According to the radiolabeled Exendin (9-39) polypeptide probe precursor, a difunctional chelating agent 1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid (1, 4, 7-triazacyclononane-1, 4, 7-triacetic acid, NOTA) is modified at different amino acid sites (Asp09, Lys12 and Lys27) of polypeptide Exendin (9-39) serving as a basis, a radioactive polypeptide probe is constructed by labeling radionuclides, and by developing an insulinoma mouse model, the insulinoma mouse model is developed, so that the activity of the insulinoma mouse model is improved, and the activity of the insulinoma mouse model is improved. And screening the radioactive polypeptide probe with strong affinity and selectivity.

Description

Technical Field

[0001] The present invention relates to a radiolabeled Exendin(9-39) polypeptide probe precursor and its application, belonging to the technical fields of radiopharmaceutical chemistry and nuclear medicine diagnosis and treatment. Background Art

[0002] Insulinoma is a functional pancreatic neuroendocrine tumor and the most common cause of hyperinsulinemic hypoglycemia in adults. Surgical operation is the only way to cure insulinoma. Since most insulinoma lesions are small, pancreatic surgery is difficult, with many complications and difficult intraoperative exploration, accurate preoperative localization of insulinoma is the key to diagnosis and treatment.

[0003] Common imaging localization diagnostic methods for insulinoma include enhanced CT, magnetic resonance, endoscopic ultrasound, etc. The diagnostic accuracy of enhanced CT and magnetic resonance is about 60%-70%. The sensitivity of endoscopic ultrasound for insulinoma in the pancreatic head and neck can reach 80%-90%. However, endoscopic ultrasound has difficulty in diagnosing insulinoma in the pancreatic tail and ectopic insulinoma. Somatostatin receptor imaging is the standard imaging diagnostic method for gastroenteropancreatic neuroendocrine tumors. However, in insulinoma, its diagnostic sensitivity is only 30%-50%. Even today, about a century after insulinoma was recognized, its localization diagnosis still often becomes a clinical problem.

[0004] Insulinoma originates from pancreatic β cells. In recent years, a nuclear medicine imaging technique targeting pancreatic β cells, glucagon-like peptide-1 receptor (GLP-1R), has been established for the localization diagnosis of insulinoma. GLP-1R is the receptor with the highest expression level found in insulinoma so far, and more than 90% of insulinomas have the molecular feature of expressing GLP-1R. Nuclear medicine molecular imaging targeting GLP-1R has considerable clinical research and application value for insulinoma.

[0005] Currently, the tracers for GLP-1R imaging that have been studied more are exendin-4 labeled with radionuclides (such as 111 In, 99 mTc, 68 Ga, 18 F). Exendin-4 is a GLP-1R agonist containing 39 amino acids, has a high affinity for GLP-1R, and has good in vivo stability. In 2008, Wild et al. reported in the New England Journal of Medicine 111 In-DTPA-exendin-4 for diagnosing 2 cases of occult insulinoma, which was the first report of GLP-1R imaging used in clinical research. Since then, Christ et al. have reported a number of 111Study on using In-labeled exendin-4 for the localization diagnosis of insulinoma, with a diagnostic sensitivity of 95% (95% CI 74 - 100) and a positive predictive value of 83% (95% CI 62 - 94). Since 2015, multiple studies have successively reported 68 the use of 68Ga-labeled exendin-4 GLP-1R PET imaging for the localization diagnosis of insulinoma. Compared with previous GLP-1R imaging techniques, its obvious improvement is the use of a positron radionuclide to label exendin-4. The use of PET significantly improves the image resolution and signal-to-noise ratio, and thus also improves the detection sensitivity of insulinoma.

[0006] Currently, clinical studies on GLP-1R imaging at home and abroad all use radioisotope-labeled exendin-4. Exendin-4 is a GLP-1R agonist and is itself a drug used to treat type 2 diabetes, with biological activity. After injecting 68 the 68Ga-exendin-4 tracer, blood glucose will decrease (the average decrease in adults is 1.3 mmol / L, up to 2.6 mmol / L), increasing the risk of hypoglycemia in patients with insulinoma. Approximately 50% of patients need continuous intravenous infusion of glucose during the examination to maintain normal blood glucose. In the exploratory study of infantile congenital hyperinsulinism, it was found that in half of the children, after injecting 68 68Ga-exendin-4, blood glucose dropped below 1.5 mmol / L, increasing the risk of applying this technology.

[0007] As a GLP-1R antagonist, Exendin-(9 - 39) has been explored in clinical trials in recent years for its effect in treating children with congenital hyperinsulinism. Clinical trial data show that Exendin-(9 - 39) significantly increases the fasting and postprandial blood glucose of children, reduces the fasting insulin level, and significantly reduces the incidence of hypoglycemia in children.

[0008] However, how to apply Exendin-(9 - 39) to the development of radioactive labeling probes, while achieving insulinoma imaging and avoiding causing a decrease in the patient's blood glucose and ensuring the safety of the examination, has become a technical problem urgently to be solved in this technical field. Summary of the Invention

[0009] The purpose of the present invention is to provide a radioactive-labeled Exendin(9 - 39) polypeptide probe precursor and its application, which can achieve insulinoma imaging while avoiding causing a decrease in the patient's blood glucose and ensuring the safety of the examination.

[0010] The above object of the present invention is achieved by the following technical solutions:

[0011] A radiolabeled Exendin(9-39) polypeptide probe precursor, namely: Lys 12 -NOTA-Exendin(9-39), Lys 27 -NOTA-Exendin(9-39), and Asp 09 -NOTA-Exendin(9-39).

[0012] Lys 12 Preparation of Lys

[0013] (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, using tert-butoxycarbonyl (BOC) to protect the amino acids with free amino groups in the polypeptide except Lys 12 outside.

[0014] (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography;

[0015] (3) Preparation of Lys 12 -NOTA-Exendin(9-39) radiolabeled probe precursor

[0016] Based on Exendin(9-39), select the site Lys 12 , label the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and prepare Lys 12 -NOTA-Exendin(9-39) radiolabeled polypeptide probe precursor.

[0017] Preferably, step (1) is specifically as follows:

[0018] First, wash the solid support Rink Amide AM Resin with dichloromethane; then wash the resin with N,N-dimethylformamide (DMF) to remove solvent residues; finally, wash with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids; add the amino acids of Exendin(9-39) in sequence, and use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) as coupling agents, dissolve them in N,N-dimethylformamide (DMF), and react with the resin; repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a mixed solution of TFA / TIS / H2O to cleave the peptide chain, release the peptide from the solid support, filter to remove the resin, and collect the solution.

[0019] Preferably, step (2) is specifically as follows:

[0020] The chromatographic column is a reverse-phase C18 column, mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30 - 60% mobile phase B to obtain Exendin(9-39).

[0021] Preferably, step (3) is specifically as follows:

[0022] Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and purify the product using a C18 SPE solid-phase extraction column after the reaction ends.

[0023] Lys 27 Preparation of the Lys

[0024] (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, and use tert-butoxycarbonyl BOC to protect the amino acids with free amino groups in the polypeptide except Lys 27 outside.

[0025] (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography.

[0026] (3) Preparation of the Lys 27 -NOTA-Exendin(9-39) radiolabeled probe precursor

[0027] Based on Exendin(9-39), select the site Lys 27, label the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and prepare Lys 27 -NOTA-Exendin(9-39) radiolabeled polypeptide probe precursor.

[0028] Preferably, the specific steps of step (1) are as follows:

[0029] First, wash the solid support Rink Amide AM Resin with dichloromethane, then wash the resin with N,N-dimethylformamide to remove solvent residues. Finally, wash with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids; add the amino acids of Exendin(9-39) in sequence, use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole as coupling agents, dissolve in N,N-dimethylformamide, and react with the resin. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a TFA / TIS / H2O mixed solution to cleave the peptide chain, release the peptide from the solid support, filter to remove the resin, and collect the solution.

[0030] Preferably, the specific steps of step (2) are as follows:

[0031] The chromatographic column is a reverse-phase C18 column, mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39).

[0032] Preferably, the specific steps of step (3) are as follows:

[0033] Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and after the reaction is completed, purify the product using a C18 SPE solid-phase extraction column.

[0034] Asp 09 The preparation of the Asp

[0035] (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis, and use tert-butoxycarbonyl BOC to protect the amino acids with free amino groups in the polypeptide except Asp 09 outside.

[0036] (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography;

[0037] (3) Asp 09 Preparation of radioactively labeled probe precursor of Asp

[0038] Based on Exendin(9-39), select the site Asp 09 , label the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and prepare the radioactively labeled polypeptide probe precursor of Asp 09 -NOTA-Exendin(9-39).

[0039] Preferably, the specific steps of step (1) are as follows:

[0040] First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane, then wash the resin with N,N-dimethylformamide to remove solvent residues. Finally, wash with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids. Add the amino acids of Exendin(9-39) in sequence. Use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole as coupling agents, dissolve them in N,N-dimethylformamide, and react with the resin. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a TFA / TIS / H2O mixed solution to cleave the peptide chain, release the peptide from the solid-phase carrier, filter to remove the resin, and collect the solution.

[0041] Preferably, the specific steps of step (2) are as follows:

[0042] The chromatographic column is a reversed-phase C18 column. Mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39).

[0043] Preferably, the specific steps of step (3) are as follows:

[0044] Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and purify the product using a C18 SPE solid-phase extraction column after the reaction.

[0045] A radioactively labeled Exendin(9-39) polypeptide probe, M-Lys12 -NOTA-Exendin(9-39), M-Lys 27 -NOTA-Exendin(9-39) and M-Asp 09 -NOTA-Exendin(9-39); M is 68 Ga, 67 Ga, 111 In, 99m Tc, 18 F[AlF], 64 Cu, 188 Re, 177 Lu, 90 Y.

[0046] Application of radioactively labeled Exendin(9-39) polypeptide probe in insulinoma imaging.

[0047] 68 Application of Ga radioactively labeled Exendin(9-39) polypeptide probe in insulinoma imaging.

[0048] 67 Ga, 111 In, 99m Application of Tc radioactively labeled Exendin(9-39) polypeptide probe in nuclear medicine SPECT imaging.

[0049] 18 F[AlF], 64 Application of Cu radioactively labeled Exendin(9-39) polypeptide probe in nuclear medicine PET imaging.

[0050] 188 Re, 177 Lu, 90 Application of Y radioactively labeled Exendin(9-39) polypeptide probe in the preparation of drugs for nuclear medicine radionuclide therapy.

[0051] The radioactively labeled Exendin(9-39) polypeptide probe precursor of the present invention is based on the polypeptide Exendin(9-39), and modifies the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (1,4,7-triazacyclononane-1,4,7-triacetic acid, NOTA) at its different amino acid sites (Asp 09 , Lys 12 and Lys 27 ), constructs a radioactively labeled polypeptide probe by labeling a radionuclide, and screens a radioactively labeled polypeptide probe with strong affinity and selectivity through the imaging of an insulinoma mouse model.

[0052] Beneficial effects:

[0053] The radiolabeled Exendin(9-39) polypeptide probe precursor of the present invention exhibits high affinity and high specificity for GLP-1R, and at the same time can avoid causing a decrease in the blood glucose of patients. It is a potential GLP-1R PET imaging agent and can be used for insulinoma imaging.

[0054] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this does not mean any limitation to the protection scope of the present invention. Brief description of the drawings

[0055] Figure 1 It is the structural formula of the raw material Exendin(9-39) used in Example 1 of the present invention;

[0056] Figure 2 It is the structural schematic diagram of NOTA-exendin(9-39) after labeling NOTA in Example 1 of the present invention;

[0057] Figure 3 It is the structural formula of three kinds of probes in the embodiments of the present invention;

[0058] Figure 4-1 It is Lys prepared in Example 1 of the present invention 12 -High performance liquid chromatography of NOTA-Exendin(9-39) molecule;

[0059] Figure 4-2 It is Lys prepared in Example 1 of the present invention 12 -Mass spectrometry characterization of NOTA-Exendin(9-39) molecule;

[0060] Figure 4-3 It is Lys prepared in Example 2 of the present invention 27 -High performance liquid chromatography of NOTA-Exendin(9-39) molecule;

[0061] Figure 4-4 It is Lys prepared in Example 2 of the present invention 27 -Mass spectrometry characterization of NOTA-Exendin(9-39) molecule;

[0062] Figure 4-5 It is Asp prepared in Example 3 of the present invention 09 -High performance liquid chromatography of NOTA-Exendin(9-39) molecule;

[0063] Figure 4-6 It is Asp prepared in Example 3 of the present invention 09- NOTA - Characterization of the mass spectrometry of Exendin(9 - 39) molecule;

[0064] Figure 5 In Example 4 of the present invention 68 Ga - Lys 12 Radio - HPLC chromatogram of Ga - Lys - NOTA - Exendin(9 - 39);

[0065] Figure 6 In Example 5 of the present invention 68 Ga - Lys 27 Radio - HPLC chromatogram of Ga - Lys - NOTA - Exendin(9 - 39);

[0066] Figure 7 In Example 6 of the present invention 68 Ga - Asp 09 Radio - HPLC chromatogram of Ga - Asp - NOTA - Exendin(9 - 39);

[0067] Figure 8 PET image of the mouse with insulinoma model in Application Example 1 of the present invention. Detailed implementation manners

[0068] Unless otherwise specified, the reagents and raw materials used in the preparation methods and detection methods described in the following examples and comparative examples are all commercially available products on the market, and the equipment used is all common equipment; the units are all weight units.

[0069] Based on Exendin(9 - 39) (as Figure 1 shown), three sites were selected: Lys 14 , Lys 27 and Asp 09 ; and the bifunctional chelating agent 1,4,7 - triazacyclononane - 1,4,7 - triacetic acid (NOTA) was respectively labeled to form three kinds of polypeptide probe precursors: Lys 12 - NOTA - Exendin(9 - 39), Lys 27 - NOTA - Exendin(9 - 39), Asp 09 - NOTA - Exendin(9 - 39), as shown in Table 1;

[0070] Table 1. Primary amino acid sequence of Exendin(9 - 39) modified with NOTA at different sites

[0071]

[0072] As shown Figure 1 in the figure, it is the structural formula of the raw material Exendin(9-39) used in Example 1 of the present invention; as shown Figure 2 in the figure, it is the primary amino acid sequence of NOTA-exendin(9-39) after labeling with NOTA in Example 1 of the present invention; as shown Figure 3 in the figure, it is the structural formula of three kinds of probes in the embodiments of the present invention; wherein, A is Asp 09 -NOTA-Exendin(9-39); B is Lys 12 -NOTA-Exendin(9-39); C is Lys 27 -NOTA-Exendin(9-39).

[0073] Example 1: Preparation of the radioactive labeled probe precursor of Lys 12 -NOTA-Exendin(9-39), the steps are as follows:

[0074] (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, and use tert-butoxycarbonyl BOC to protect the amino acids with free amino groups in the polypeptide except Lys 12 The specific steps are as follows:

[0075] First, wash the solid-phase carrier Rink Amide AM Resin resin with dichloromethane for 5 minutes, and wash it 3 times; then wash the resin with N,N-dimethylformamide (DMF) to remove solvent residues; finally, wash it with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids; Add 0.15 mmol of amino acids of Exendin(9-39) in sequence each time, and use 0.1 mmol of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) each as a coupling agent, dissolve them in N,N-dimethylformamide (DMF), and react with the resin for 30 minutes; repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a mixed solution of TFA (trifluoroacetic acid) / TIS (triisopropylsilane) / H2O (volume ratio: 95:2.5:2.5) to cleave the peptide chain, release the peptide from the solid-phase carrier, filter, remove the resin, and collect the solution;

[0076] (2) Remove TFA and other solvents by rotary evaporator, and purify using liquid chromatography, specifically including: the chromatographic column is a reversed-phase C18 column, mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30 - 60% mobile phase B to obtain Exendin(9 - 39);

[0077] (3) Lys 12 Preparation of Lys

[0078] -NOTA-Exendin(9 - 39) radioactive labeling probe precursor. Based on Exendin(9 - 39), select the site Lys 12 , and label the bifunctional chelating agent 1,4,7 - triazacyclononane - 1,4,7 - triacetic acid (NOTA) to prepare Lys 12 -NOTA-Exendin(9 - 39) radioactive labeling polypeptide probe precursor;

[0079] Specifically including: Dissolve 0.3 mmol of NOTA - NHS ester and 0.1 mmol of Exendin(9 - 39) in N,N - dimethylformamide (DMF), adjust the reaction system to pH 8, react at room temperature for 5 h. After the reaction, purify the product using a C18 SPE solid-phase extraction column, and determine that the final purity is greater than 95% by high-resolution mass spectrometry ESI - MS (Orbitrap Fusion Lumos, ThermoFisher, USA).

[0080] Example 2: Preparation of Lys 27 -NOTA-Exendin(9 - 39) radioactive labeling probe precursor, the steps are as follows:

[0081] (1) Synthesize Exendin(9 - 39) by solid-phase peptide synthesis method, and use tert-butyloxycarbonyl BOC to protect the amino acids with free amino groups in the polypeptide except Lys 27 The specific steps are as follows:

[0082] First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane for 5 minutes, wash it 3 times, then wash the resin with N,N-dimethylformamide (DMF) to remove solvent residues. Finally, wash it with N,N-diisopropylethylamine to activate the resin and prepare for amino acid coupling; Add 0.15 mmol of amino acids of Exendin(9-39) in sequence each time. Use 0.1 mmol each of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) as coupling agents, dissolve them in N,N-dimethylformamide (DMF), and react with the resin for 30 minutes. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a TFA / TIS / H2O mixed solution (volume ratio: 95:2.5:2.5) to cleave the peptide chain, release the peptide from the solid-phase carrier, filter to remove the resin, and collect the solution;

[0083] (2) Remove TFA and other solvents by vacuum rotary evaporation (Rotary Evaporator), and purify using liquid chromatography. Specifically, the chromatographic column is a reversed-phase C18 column, mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39);

[0084] (3) Preparation of Lys 27 -NOTA-Exendin(9-39) radioactive labeling probe precursor

[0085] Based on Exendin(9-39), select the site Lys 27 , label the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) to prepare Lys 27 -NOTA-Exendin(9-39) radioactive labeling polypeptide probe precursor;

[0086] Specifically, dissolve 0.3 mmol of NOTA-NHS ester and 0.1 mmol of Exendin(9-39) in N,N-dimethylformamide (DMF), adjust the reaction system to pH 8, react at room temperature for 5 hours. After the reaction is completed, purify the product using a C18 SPE solid-phase extraction column, and determine the final purity to be greater than 95% by high-resolution mass spectrometry ESI-MS (Orbitrap Fusion Lumos, ThermoFisher, USA).

[0087] Example 3: Asp 09 -Preparation of the radiolabeled probe precursor of NOTA-Exendin(9-39) is as follows:

[0088] (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, using tert-butoxycarbonyl (BOC) to protect the amino acids with free amino groups in the peptide except Asp 09 The specific steps are as follows:

[0089] First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane for 5 minutes, wash it 3 times, then wash the resin with N,N-dimethylformamide (DMF) to remove solvent residues. Finally, wash it with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids. Add 0.15 mmol of amino acids of Exendin(9-39) in sequence each time. Use 0.1 mmol of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) as coupling agents, dissolve them in N,N-dimethylformamide (DMF), and react with the resin for 30 minutes. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a mixed solution of TFA / TIS / H2O (volume ratio: 95:2.5:2.5) to cleave the peptide chain, release the peptide from the solid-phase carrier, filter, remove the resin, and collect the solution;

[0090] (2) Remove TFA and other solvents by rotary evaporation under vacuum, and purify it by liquid chromatography. Specifically, the chromatographic column is a reversed-phase C18 column, mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39);

[0091] (3) Asp 09 -Preparation of the radiolabeled probe precursor of Asp-NOTA-Exendin(9-39)

[0092] Based on Exendin(9-39), select the site Asp 09 , label the bifunctional chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) to prepare the radiolabeled polypeptide probe precursor of Asp-NOTA-Exendin(9-39). The specific steps are as follows: 09 The specific steps are as follows:

[0093] Dissolve 0.3 mmol of NOTA-NHS ester and 0.1 mmol of Exendin(9-39) in N,N-dimethylformamide (DMF), adjust the reaction system to pH 8, and react at room temperature for 5 h. After the reaction, purify the product using a C18 SPE solid-phase extraction column, and determine that the final purity is greater than 95% by high-resolution mass spectrometry ESI-MS (Orbitrap Fusion Lumos, ThermoFisher, USA).

[0094] Characterization of the radiolabeled probe precursor NOTA-Exendin(9-39) prepared in Examples 1-3 of the present invention:

[0095] Characterize the radiolabeled probe precursor using liquid chromatography (Agilent 1260 Infinity, Agilent, USA)-high-resolution mass spectrometry (Orbitrap Fusion Lumos, ThermoFisher, USA). Confirm the successful synthesis through accurate molecular weight comparison (error value ≤ 10 ppm) and secondary mass spectrometry fragment verification. The specific analysis conditions for liquid chromatography are as follows: the chromatographic column is a Phenomenex Luna 5u C18 column; mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B for a total of 20 minutes.

[0096] As Figure 4-1 shown, the high-performance liquid chromatography of the Lys 12 -NOTA-Exendin(9-39) molecule prepared in Example 1 of the present invention; as Figure 4-2 shown, the mass spectrometry characterization of the Lys 12 -NOTA-Exendin(9-39) molecule prepared in Example 1 of the present invention; as Figure 4-3 shown, the high-performance liquid chromatography of the Lys 27 -NOTA-Exendin(9-39) molecule prepared in Example 2 of the present invention; as Figure 4-4 shown, the mass spectrometry characterization of the Lys 27 -NOTA-Exendin(9-39) molecule prepared in Example 2 of the present invention; as Figure 4-5 shown, the high-performance liquid chromatography of the Asp 09 -NOTA-Exendin(9-39) molecule prepared in Example 3 of the present invention; as Figure 4-6 shown, the mass spectrometry characterization of the Asp 09 -NOTA-Exendin(9-39) molecule prepared in Example 3 of the present invention; the chemical purity is greater than 95%.

[0097] In the embodiments of the present invention, the chelating agent 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) can also be replaced with: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), HYNIC (6-hydrazinonicotinic acid), DTPA (diethylenetriaminepentaacetic acid), NODAGA (1,4,7-triazacyclononane-1-pentanedioic acid-4,7-diacetic acid).

[0098] Example 4: 68 Ga-Lys 12 Preparation of Ga-Lys-NOTA-Exendin(9-39)

[0099] (1) Use 5 mL of 0.1 M high-purity hydrochloric acid solution to wash the germanium-gallium generator (Eckert & Ziegler, 1850 MBq, 50 mCi) to obtain 68 GaCl3 hydrochloric acid solution;

[0100] (2) Take 1 mL and add 93 μL of 1.25 M sodium acetate solution, mix well, add 10 μL of Lys 12 -NOTA-Exendin(9-39) aqueous solution (1 mg / mL), react at 90 °C for 15 min, cool to room temperature, add 10 mL of ultrapure water and dilute;

[0101] (3) Pass through a plus C18 solid-phase extraction column for purification. First, wash with 5 mL of water, then elute with 2 mL of ethanol to obtain the product. Determine its radiochemical purity by radio-HPLC to obtain Ga-Lys 68 Ga-Lys 12 -NOTA-Exendin(9-39) with a radiochemical purity greater than 90%;

[0102] For the quality control of the radioactive molecular probe, the radiochemical purity is determined by radio-HPLC, which is higher than 90%. As Figure 5 shown, it is the Radio-HPLC spectrum of Ga-Lys 68 Ga-Lys 12 -NOTA-Exendin(9-39) in Example 4 of the present invention.

[0103] Example 5: 68 Ga-Lys 27 Preparation of Ga-Lys-NOTA-Exendin(9-39)

[0104] (1) Use 5 mL of 0.1 M high-purity hydrochloric acid solution to wash the germanium-gallium generator (Eckert & Ziegler, 1850 MBq, 50 mCi) to obtain 68 GaCl3 hydrochloric acid solution;

[0105] (2) Take 1 mL and add 93 μL of 1.25 M sodium acetate solution, mix well, add 10 μL of Lys 27 -NOTA-Exendin(9-39) aqueous solution (1 mg / mL), react at 90 °C for 15 min, cool to room temperature, add 10 mL of ultrapure water, and dilute;

[0106] (3) Purify through a plus C18 solid-phase extraction column. First, wash with 5 mL of water, then elute with 2 mL of ethanol to obtain the product. Determine its radiochemical purity by radio-HPLC to obtain 68 Ga-Lys 27 -NOTA-Exendin(9-39) with a radiochemical purity greater than 90%;

[0107] As Figure 6 shown, it is the Radio-HPLC spectrum of 68 Ga-Lys 27 -NOTA-Exendin(9-39) in Example 5 of the present invention.

[0108] Example 6: 68 Preparation of 09 Ga-Asp

[0109] (1) Use 5 mL of 0.1 M high-purity hydrochloric acid solution to wash the germanium-gallium generator (Eckert & Ziegler, 1850 MBq, 50 mCi) to obtain 68 GaCl3 hydrochloric acid solution;

[0110] (2) Take 1 mL and add 93 μL of 1.25 M sodium acetate solution, mix well, add 10 μL of Asp 09 -NOTA-Exendin(9-39) aqueous solution (1 mg / mL), react at 90 °C for 15 min, cool to room temperature, add 10 mL of ultrapure water, and dilute;

[0111] (3) Purify through a plus C18 solid-phase extraction column. First, wash with 5 mL of water, then elute with 2 mL of ethanol to obtain the product. Determine its radiochemical purity by radio-HPLC to obtain 68 Ga-Asp 09 -NOTA-Exendin(9-39) with a radiochemical purity greater than 90%;

[0112] As shown Figure 7 in the figure, it is the 68 Ga-Asp 09 -NOTA-Exendin(9-39) Radio-HPLC chromatogram in Example 6 of the present invention.

[0113] The radionuclide 68 Ga in the examples of the present invention can be replaced with 67 Ga, 18 F[AlF], 64 Cu, 177 Lu, 111 In, 188 Re, 90 Y, 99m Tc.

[0114] Application Example 1:

[0115] 68 Targeting study of Ga-labeled molecular probes ( 68 Ga-Lys 12 -NOTA-Exendin(9-39), 68 Ga-Lys 27 -NOTA-Exendin(9-39), 68 Ga-Asp 09 -NOTA-Exendin(9-39)) targeting study)

[0116] (1) Construct an insulinoma mouse model: Select the well-growing insulinoma cell line INS-1E, digest it with trypsin to make a single-cell suspension, adjust the cell density to 1×10 7 cells / mL, take 0.2 mL of the single-cell suspension, and subcutaneously inoculate it at the axilla of C57BL / 6 mice, and continue to raise for 14 days to complete the model construction;

[0117] (2) Inject 0.2 mL of 68 Ga-labeled probe ( 68 Ga-Lys 12 -NOTA-Exendin(9-39), 68 Ga-Lys 27 -NOTA-Exendin(9-39), 68 Ga-Asp 09 -NOTA-Exendin(9-39)) (0.2 mCi) into the tail vein of the model mice, and perform imaging at 30 min, 1 h, and 2 h through microPET respectively;

[0118] (3) Set up a blocking group, and use the excessive polypeptide probe precursor Asp09 -NOTA-Exendin(9-39) was injected into the insulinoma mice in the blocking group via the tail vein, and then 68 Ga-labeled probe was injected via the tail vein. 68 Ga-Asp 09 -NOTA-Exendin(9-39). The drug distribution differences between the blocking group and the experimental group were compared by microPET.

[0119] The results are as Figure 8 shown, which are the PET images of the insulinoma model mice in Application Example 1 of the present invention. Among them, A-C are the distribution of the radioactive molecular probe 68 Ga-Asp 09 -NOTA-Exendin(9-39) in the model mice at 30 min, 1 h, and 2 h. The molecular probe was significantly enriched in the tumor region within 1 h and was mainly metabolized through the kidneys and bladder. D is the 68 Ga-Lys 12 -NOTA-Exendin(9-39) distribution in the model mice at 1 h, with no enrichment in the tumor region. E is the 68 Ga-Lys 27 -NOTA-Exendin(9-39) distribution in the model mice at 1 h, with no enrichment in the tumor region. F is the blocking group. Before injecting 68 Ga-Asp 09 -NOTA-Exendin(9-39), an excessive amount of the precursor Asp was first injected to 09 -NOTA-Exendin(9-39) saturate the GLP-1R target, and then the radioactive probe 68 Ga-Asp 09 -NOTA-Exendin(9-39) was injected. After 1 hour, there was no obvious uptake in the tumor site on the imaging.

[0120] The above results indicate that only 68 Ga-Asp 09 -NOTA-Exendin(9-39) is effective.

[0121] The radioactive-labeled Exendin(9-39) polypeptide probe precursor and its application of the present invention are based on the GLP-1R antagonist exendin-(9-39) to develop a radioactive-labeled probe, which can avoid causing a decrease in the blood glucose of patients while realizing insulinoma imaging and ensure the safety of the examination.

[0122] In addition to 68 Ga, 67 Ga, 111 In,99m Tc can be used for SPECT imaging in nuclear medicine; 18 F[AlF], 64 Cu can be used for PET imaging in nuclear medicine; 188 Re, 177 Lu, 90 Y can be used for radionuclide therapy in nuclear medicine.

[0123] The radioactive labeled Exendin(9-39) polypeptide probe precursor and its application of the present invention have completed the chemical synthesis and mass spectrometry, high performance liquid chromatography characterization of the probe precursors Lys 12 -NOTA-Exendin(9-39), Lys 27 -NOTA-Exendin(9-39) and Asp 09 -NOTA-Exendin(9-39); through metal chelation reaction, the labeling of radionuclide 68 Ga is achieved, and the radiochemical purity of the products is all >90%; an insulinoma mouse model is constructed, and at the level of living animals, the pharmacokinetics and tumor targeting of the molecular probe are tested. It is found through experiments that among the three molecular probe precursors constructed by the present invention, Asp 09 -NOTA-Exendin(9-39) has good affinity and targeting for insulinoma.

[0124] The above is only the preferred embodiment of the present invention, and thus the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A radiolabeled Exendin(9-39) polypeptide probe precursor, namely: Lys 12 -NOTA-Exendin(9-39), Lys 27 -NOTA-Exendin(9-39) and Asp 09 -NOTA-Exendin(9-39). 2.Lys 12 -Preparation of the radiolabeled probe precursor of NOTA-Exendin(9-39) is as follows: (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, using tert-butoxycarbonyl (BOC) to protect the amino acids with free amino groups in the polypeptide except Lys 12 except; (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography; (3) Lys 12 -Preparation of radiolabeled probe precursor of NOTA-Exendin(9-39) Based on Exendin(9-39), the site Lys was selected 12 , and the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was labeled to prepare the Lys 12 -NOTA-Exendin(9-39) radiolabeled polypeptide probe precursor.

3. Lys as claimed in claim 2 12 -Preparation of a radiolabeled probe precursor of NOTA-Exendin(9-39), characterized in that: (1) The specific steps are as follows: First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane and then wash it with N,N-dimethylformamide (DMF) to remove residual solvents. Finally, wash it with N,N-diisopropylethylamine to activate the resin and prepare for amino acid coupling. Add the amino acids of Exendin(9-39) in sequence, use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole (HOBt) as coupling agents, dissolve them in N,N-dimethylformamide (DMF), and react with the resin. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a TFA / TIS / H2O mixed solution to cleave the peptide chain, release the peptide from the solid-phase carrier, filter to remove the resin, and collect the solution.

4. Lys as described in claim 3 12 -Preparation of a radiolabeled probe precursor of NOTA-Exendin(9-39), characterized in that: (2) The specific steps are as follows: The chromatographic column is a reversed-phase C18 column. Mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid. The mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39).

5. Lys as described in claim 4 12 -Preparation of a radiolabeled probe precursor of NOTA-Exendin(9-39), characterized in that: (3) The specific steps are as follows: Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and purify the product using a C18 SPE solid-phase extraction column after the reaction ends. 6.Lys 27 -Preparation of the radiolabeled probe precursor of NOTA-Exendin(9-39) is as follows: (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, using tert-butoxycarbonyl (BOC) to protect the amino acids with free amino groups in the peptide except Lys 27 except; (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography; (3) Lys 27 -Preparation of radiolabeled probe precursor of NOTA-Exendin(9-39) Based on Exendin(9-39), the site Lys was selected 27 , and the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was labeled to prepare the Lys 27 -NOTA-Exendin(9-39) radiolabeled polypeptide probe precursor.

7. Lys as claimed in claim 2 27 -Preparation of a radiolabeled probe precursor of NOTA-Exendin(9-39), characterized in that: (1) The specific steps are as follows: First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane, then wash the resin with N,N-dimethylformamide to remove residual solvents, and finally, wash it with N,N-diisopropylethylamine to activate the resin and prepare for amino acid coupling. Add the amino acids of Exendin(9-39) in sequence, use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole as coupling agents, dissolve them in N,N-dimethylformamide, and react with the resin. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis is completed, use a TFA / TIS / H2O mixed solution to cleave the peptide chain, release the peptide from the solid-phase carrier, filter to remove the resin, and collect the solution; (2) The specific steps are as follows: The chromatographic column is a reversed-phase C18 column. Mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid. The mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39); (3) The specific steps are as follows: Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and after the reaction, purify the product using a C18 SPE solid-phase extraction column. 8.Asp 09 -Preparation of the radiolabeled probe precursor of NOTA-Exendin(9-39) is as follows: (1) Synthesize Exendin(9-39) by solid-phase peptide synthesis method, using tert-butoxycarbonyl (BOC) to protect the amino acids with free amino groups in the peptide except Asp 09 except; (2) Remove TFA and other solvents by vacuum rotary evaporation and purify using liquid chromatography; (3)Asp 09 Preparation of radiolabeled probe precursor of NOTA-Exendin(9-39) Based on Exendin(9-39), the site Asp was selected 09 , and the bifunctional chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) was labeled to prepare the precursor of the radioactively labeled polypeptide probe Asp 09 -NOTA-Exendin(9-39); The specific steps of step (1) are as follows: First, wash the solid-phase carrier Rink Amide AM Resin with dichloromethane, then wash the resin with N,N-dimethylformamide to remove residual solvents. Finally, wash with N,N-diisopropylethylamine to activate the resin and prepare for coupling amino acids. Add the amino acids of Exendin(9-39) in sequence, use 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate and 1-hydroxybenzotriazole as coupling agents, dissolve them in N,N-dimethylformamide, and react with the resin. Repeat the above steps until the complete polypeptide Exendin(9-39) is synthesized. After the synthesis, use a mixed solution of TFA / TIS / H2O to cleave the peptide chain, release the peptide from the solid-phase carrier, filter to remove the resin, and collect the solution; The specific steps of step (2) are as follows: The chromatographic column is a reversed-phase C18 column. Mobile phase A is pure water containing 0.1% trifluoroacetic acid, and mobile phase B is acetonitrile containing 0.1% trifluoroacetic acid; the mobile phase gradient is 30-60% mobile phase B to obtain Exendin(9-39); The specific steps of step (3) are as follows: Dissolve NOTA-NHS ester and Exendin(9-39) in N,N-dimethylformamide, adjust the reaction system to pH 8, react at room temperature, and after the reaction, purify the product using a C18 SPE solid-phase extraction column.

9. A radioactively labeled Exendin(9-39) polypeptide probe, M-Lys 12 -NOTA-Exendin(9-39), M-Lys 27 -NOTA-Exendin(9-39) and M-Asp 09 -NOTA-Exendin(9-39); M is 68 Ga, 67 Ga, 111 In, 99m Tc, 18 F[AlF], 64 Cu, 188 Re, 177 Lu or 90 Y.

10. Application of radiolabeled Exendin(9-39) polypeptide probe in insulinoma imaging; specifically including: 68 Application of Ga radioactively labeled Exendin(9-39) polypeptide probe in insulinoma imaging; 67 Ga, 111 In, 99m Application of Tc radioactively labeled Exendin(9-39) polypeptide probe in nuclear medicine SPECT imaging; 18 F[AlF], 64 Application of Cu-radiolabeled Exendin(9-39) polypeptide probe in nuclear medicine PET imaging; 188 Re、 177 Lu、 90 Use of a Re, Lu or Y radioactively labeled Exendin(9-39) polypeptide probe in the preparation of a medicament for radionuclide therapy in nuclear medicine.

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