Targeted NPY polypeptide nuclide ligand as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a targeted NPY polypeptide nuclide ligand as well as a preparation method and application thereof. The structural formula of the targeted NPY polypeptide nuclide ligand is as shown in formula (1), wherein L is a metal chelating agent. The preparation method comprises the following steps: (1) adding tyrosine, an activating agent, a condensing agent and a solvent into Rink Amide MBHA Resin, reacting at room temperature for 1-5 hours, and grafting tyrosine on the Rink Amide MBHA Resin; (2) adding arginine, an activating agent, a condensing agent and a solvent, reacting at room temperature for 1-5 hours, grafting arginine, and sequentially grafting glutamine, arginine, tryptophan, isoleucine, proline, asparagine, asparagine, tyrosine, histidine, arginine and a metal chelating agent according to the steps, so as to obtain polypeptide resin L-Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin; and (3) adding the obtained polypeptide resin into a lysis solution, and reacting at room temperature for 1-5 hours to obtain the targeted NPY polypeptide nuclide ligand with good in-vivo and in-vitro stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a targeted NPY polypeptide nuclide ligand, its preparation method, and its application. Background Technology

[0002] Utilizing nuclear medicine imaging techniques to detect lesions throughout the body early and accurately provides clinicians with better guidance for treatment. Currently, molecular imaging modalities mainly include: molecular magnetic resonance imaging, optical imaging, targeted ultrasound, single-photon emission computed tomography (SPECT), and positron emission tomography (PET). Among these, nuclear medicine imaging (PET, SPECT) plays an increasingly important role in clinical practice due to its advantages of high sensitivity, strong tissue penetration, in vivo quantitative analysis, and the availability of multiple radionuclides.

[0003] Neuropeptide Y (NPY) is a 36-amino acid neuropeptide that exerts a variety of functions in vivo by activating specific receptors in the G protein-coupled receptor family, such as Y1, Y2, Y4, and Y5 receptors. It plays a crucial role in regulating appetite, energy metabolism, stress response, and mood processing. In specific brain regions, such as the hypothalamus, elevated NPY levels have been shown to be directly associated with increased food intake, thus affecting body weight and energy balance. NPY ligands, through binding to their receptors, participate in the regulation of cellular behavior, including cell adhesion, growth, differentiation, migration, and apoptosis, thereby influencing intracellular signaling pathways. Under pathological conditions, NPY expression patterns change significantly. In cancers such as breast cancer, glioma, ovarian cancer, and pancreatic cancer, NPY expression levels are often upregulated, and this upregulation is closely related to tumor invasiveness, metastatic potential, and poor patient prognosis. Besides its role in cancer, abnormal NPY expression is also associated with obesity, type II diabetes, cardiovascular disease, and certain mental illnesses. Therefore, the NPY receptor has become a potential therapeutic target for these diseases. Developing intervention strategies targeting the NPY receptor, such as using NPY antagonists or reducing NPY expression through RNA interference, may help control obesity, improve metabolic syndrome, and inhibit tumor progression. Therefore, real-time monitoring of changes in NPY expression levels is crucial for early cancer detection. Furthermore, NPY-targeting probes have potential applications in cancer diagnosis and treatment, especially in tumors with upregulated NPY expression. Combined with traditional chemotherapy, radiotherapy, or immunotherapy, NPY-targeted therapy may provide a more effective treatment option for patients with advanced cancer. Further exploration of the specific mechanisms of action of NPY in different diseases is needed to achieve precision medicine and personalized treatment.

[0004] To date, research on targeted drugs against the neuropeptide Y (NPY) receptor is still in its early stages, and no widely approved drugs have been marketed. The NPY receptor family includes multiple subtypes such as Y1, Y2, Y4, and Y5, each playing a unique role in physiological and pathological processes. Due to the specific roles of these receptors in different diseases, developing drugs targeting specific NPY receptors is highly complex and specific. Although the U.S. Food and Drug Administration (FDA) has not yet approved any drugs specifically targeting the NPY receptor, several candidate drugs have shown therapeutic potential in preclinical and clinical trials. In particular, drugs targeting the NPY Y1 receptor antagonist, the Y2 receptor agonist, and the Y5 receptor antagonist are being actively explored for applications in the treatment of obesity, diabetes, cardiovascular disease, and certain cancers. Furthermore, research on these drugs extends beyond therapeutic areas to early cancer screening. Future research will continue to explore the safety, efficacy, and applicability of these drugs, with the aim of bringing breakthrough treatments for related diseases. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned technical problems by providing a targeted NPY polypeptide nuclide ligand specifically for the imaging diagnosis of NPY Y1-positive tumor patients, which can provide medication guidance and real-time efficacy monitoring for patients receiving anticancer drug treatment.

[0006] The targeted NPY polypeptide nuclide ligand in the technical solution of this invention has the structural formula shown in formula (1):

[0007] Where L is a metal chelating agent.

[0008] Further, the metal chelating agent is one or more of the following: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA), 1,4,7-triazacyclononane-N,N',N'-triacetic acid (NOTA), N-hydroxysuccinimide carbazide (HYNIC), (2S)-(1-tetrahydropyrimidin-2-one)-3-methylbutyric acid (TPA), diethyltriaminepentaacetic acid (DTPA), 2-[1,4,7-triazacyclononane-1-yl-4,7-bis(t-Bu ester)]-1,5-pentanoic acid (NODAGA), triethylenetetramine (TETA), cyclohexanediol and terephthalic acid copolymer (PCTA), and 1,4,7,10-tetra(aminocarbonylmethyl)-1,4,7,10-tetraazacyclododecane (DOTAM).

[0009] This invention also provides a method for preparing the above-mentioned targeted NPY polypeptide nuclide ligand, comprising the following steps:

[0010] (1) Add tyrosine, activator, condensing agent and solvent to Rink Amide MBHA Resin and react at room temperature for 1-5 h to graft tyrosine onto Rink Amide MBHA Resin.

[0011] (2) Add arginine, activator, condensing agent and solvent, react at room temperature for 1-5 h, graft arginine, and follow the above steps to graft glutamine, arginine, tryptophan, isoleucine, proline, asparagine, asparagine, tyrosine, histidine, arginine and metal chelating agent in sequence to obtain polypeptide resin L-Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin;

[0012] (3) Add the obtained polypeptide resin to the lysis buffer and react at room temperature for 1 to 5 hours.

[0013] Further, in step (1), the mass ratio of Rink Amide MBHA Resin, tyrosine, activator, and condensing agent is 3.0–8.0:2.0–5.0:2.0–5.0:2.2–5.5.

[0014] Preferably, the amino acids used in the above-mentioned methods for preparing NPY-targeting polypeptide nuclide ligands are all fluorene-methyloxycarbonyl-protected amino acids, including fluorene-methyloxycarbonyl-protected tyrosine (Fmoc-Tyr-OH), fluorene-methyloxycarbonyl-protected arginine (Fmoc-Arg-OH), fluorene-methyloxycarbonyl-protected glutamine (Fmoc-Gln-OH), fluorene-methyloxycarbonyl-protected tryptophan (Fmoc-Try-OH), fluorene-methyloxycarbonyl-protected isoleucine (Fmoc-Iso-OH), fluorene-methyloxycarbonyl-protected proline (Fmoc-Pro-OH), fluorene-methyloxycarbonyl-protected asparagine (Fmoc-Asp-OH), and fluorene-methyloxycarbonyl-protected histidine (Fmoc-His-OH).

[0015] Preferably, the amino acids used in the above-mentioned method for preparing the targeted NPY polypeptide nuclide ligand are all D-type amino acids or all L-type amino acids.

[0016] Furthermore, Rink Amide MBHA Resin is deprotected both before and after amino acid grafting to prepare for grafting the next amino acid or metal chelator.

[0017] As a preferred method, deprotection involves stirring Rink Amide MBHA Resin (with or without grafted amino acids) in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 20–30 min, and then washing the resulting solid fraction with DMF 3–5 times after filtration.

[0018] As a preferred option, the mass ratio of the amino acid grafted onto Rink Amide MBHA Resin to the amino acid to be grafted, the activator, and the condensing agent is 3.0–8.0:2.0–5.0:2.0–5.0:2.2–5.5.

[0019] Further, the activator is one or more of N,N'-diisopropylcarbodiimide (DIC), dicyclohexylcarbodiimide (DCC), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC).

[0020] Furthermore, the condensing agent is one or both of 1-hydroxybenzotriazole (HOBT) and N-methylmorpholine (NMM).

[0021] Preferably, grafting tyrosine onto Rink Amide MBHA Resin involves adding an activator to deprotected Rink Amide MBHA Resin and activating it for 3–10 min, then adding a solution containing tyrosine and a condensing agent dissolved in a solvent, reacting at room temperature for 1–5 h, and then washing the solid portion 3–5 times with a solvent after filtration.

[0022] Preferably, the solvents used in steps (1) and (2) include, but are not limited to, one or more of N,N-dimethylformamide (DMF), N,N-dimethylacetamide (DMAC), N-methylpyrrolidone (NMP), and dimethyl sulfoxide (DMSO).

[0023] Preferably, in step (2) when grafting the metal chelating agent, the molar ratio of the metal chelating agent to the condensing agent is 1.0:1.0~2.0.

[0024] Furthermore, the lysis solution in step (3) is a mixture of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane and water.

[0025] Furthermore, the volume percentages of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water in the lysis solution are 90–95%, 3–10%, 1–5%, and 1–5%, respectively.

[0026] As a preferred option, after reacting at room temperature in step (3), the mixture is filtered, the filtrate is precipitated, and the resulting precipitate is dissolved in acetonitrile and water, then filtered, purified, and freeze-dried.

[0027] Preferably, in step (3), purification is performed using a high-performance liquid chromatograph; and lyophilization is performed by pre-freezing with liquid nitrogen and then freezing with a lyophilizer for 40-60 hours.

[0028] The present invention also provides a probe for targeting NPY polypeptide nuclides, comprising the above-mentioned NPY polypeptide nuclide ligand and a radionuclide.

[0029] Furthermore, the radionuclide is 99m Tc, 68 Ga、 64 Cu、 111 In、 90 Y、 225 Ac、 137 Cs、 90 Sr、 125 I, 131 I, 153 Sm and 177 Any one of Lu.

[0030] Furthermore, the activity of the radionuclide is 0.8–2.5 mCi.

[0031] The preparation method of the above-mentioned NPY-targeting polypeptide nuclide probe includes the following steps: adding the NPY-targeting polypeptide nuclide ligand to an ammonium acetate solution, then adding a radioactive nuclide solution, heating the reaction and then cooling to room temperature.

[0032] Furthermore, the concentration of the ammonium acetate solution is 1.0–2.0 mol / L.

[0033] Furthermore, the volume ratio of ammonium acetate solution to the targeted NPY polypeptide nuclide ligand solution is 2–3:1.

[0034] Furthermore, the volume of the radionuclide solution is 20,000 to 40,000 times the volume of the NPY polypeptide nuclide ligand solution.

[0035] Furthermore, the heating reaction is carried out at a temperature of 30–80°C for a time of 5–20 minutes.

[0036] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0037] (1) Using a bifunctional metal chelator to couple with amino acids to obtain a targeted NPY polypeptide nuclide ligand, which has better in vitro and in vivo stability, and binding with a radionuclide to obtain a targeted NPY polypeptide nuclide probe, which can be used for early diagnosis and real-time effect monitoring of malignant tumors.

[0038] (2) The targeted NPY polypeptide nuclide probe has good biocompatibility and optimizes the pharmacokinetic properties of the probe, especially the clearance kinetics of non-tumor tissues, which can achieve better diagnostic and treatment effects.

[0039] (3) The obtained NPY-targeting polypeptide nuclide probe is suitable for guiding drug treatment and real-time efficacy monitoring in patients with cancers such as breast cancer, glioma, ovarian cancer and pancreatic cancer, as well as other tumors that can affect NPY expression changes;

[0040] (4) The obtained NPY-targeting polypeptide nuclide probes not only enhance the accuracy of tumor diagnosis, but also provide new tools for early cancer screening and treatment efficacy evaluation, which have important clinical significance and application prospects. Attached Figure Description

[0041] Figure 1 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L HPLC chromatogram of NPY;

[0042] Figure 2 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L MS spectrum of NPY;

[0043] Figure 3 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L Radio HPLC chromatogram of NPY;

[0044] Figure 4 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L Graph of in vitro stability analysis of NPY;

[0045] Figure 5 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L Graph of experimental results for NPY's lipid-water partition coefficient;

[0046] Figure 6 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L NPY uptake assay in MCF-7 cells;

[0047] Figure 7 For injection 68 Ga-NOTA- L PET / CT images of BALB / c Nude mouse MCF-7 breast cancer model after NPY for 30 min, 60 min and 120 min;

[0048] Figure 8 For injection 68 Ga-NOTA- D PET / CT images of BALB / c Nude mouse MCF-7 breast cancer model after NPY for 30 min, 60 min and 120 min;

[0049] Figure 9 For injection 68 Ga-NOTA- D PET / CT images of the BALB / c Nude mouse MDA-MB-468 breast cancer model after NPY for 30 min, 60 min and 120 min;

[0050] Figure 10 The results obtained from the examples 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L Pharmacokinetic results of NPY. Detailed Implementation

[0051] The technical solution of the present invention will be further described and illustrated below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are only for the purpose of helping to understand the present invention and are not intended to limit the specific scope of the present invention. Furthermore, the accompanying drawings used herein are merely for better illustrating the content disclosed in the present invention and do not limit the scope of protection. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commonly used materials in the art, and the methods used in the embodiments are all conventional methods in the art.

[0052] Example 1

[0053] The preparation method of the targeted NPY polypeptide nuclide ligand in this embodiment includes the following steps:

[0054] (1) Deprotection of Rink Amide MBHA Resin: Rink Amide MBHA Resin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF was 2:8) for 30 min, the reaction solution was filtered off, and the resulting solid part was washed with DMF 5 times.

[0055] (2) Dissolve 80g Fmoc-Tyr-OH and 40g HOBT in DMF, add 38g DIC to the deprotected Rink Amide MBHAResin and activate for 5min, then add DMF solution containing Fmoc-Tyr-OH and HOBT, react at room temperature for 3h to obtain Tyr-Rink Amide MBHAResin, and wash the solid part with DMF 3 times after filtration.

[0056] (3) Stir Tyr-Rink Amide MBHA Resin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80g Fmoc-Arg-OH and 40g HOBT in DMF, add 38g DIC to the deprotected Tyr-Rink Amide MBHAResin to activate for 5 min, then add DMF solution containing dissolved Fmoc-Arg-OH and HOBT, react at room temperature for 3 h to obtain Arg-Tyr-Rink Amide MBHAResin, filter and wash the solid part with DMF 3 times;

[0057] (4) Arg-Tyr-Rink Amide MBHAResin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF was 2:8) for 30 min. The reaction solution was filtered off. The resulting solid part was washed 5 times with DMF. 80 g of Fmoc-Gln-OH and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected Arg-Tyr-Rink Amide MBHAResin and activated for 5 min. Then, DMF solution containing Fmoc-Gln-OH and HOBT was added. The reaction was carried out at room temperature for 3 h to obtain Gln-Arg-Tyr-Rink Amide MBHAResin. The solid part was washed 3 times with DMF after filtration.

[0058] (5) Stir Gln-Arg-Tyr-Rink Amide MBHA Resin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80g Fmoc-Arg-OH and 40g HOBT in DMF, add 38g DIC to the deprotected Gln-Arg-Tyr-Rink Amide MBHA Resin and activate for 5 min, then add DMF solution containing dissolved Fmoc-Arg-OH and HOBT, react at room temperature for 3 h to obtain Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin, filter and wash the solid part with DMF 3 times;

[0059] (6) Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF was 2:8) for 30 min. The reaction solution was filtered off, and the resulting solid part was washed 5 times with DMF. 80 g of Fmoc-Try-OH and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin and activated for 5 min. Then, DMF solution containing Fmoc-Try-OH and HOBT was added and reacted at room temperature for 3 h to obtain Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin. The solid part was washed 3 times with DMF after filtration.

[0060] (7) Stir Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80 g Fmoc-Iso-OH and 40 g HOBT in DMF, add 38 g DIC to the deprotected Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin and activate for 5 min, then add DMF solution containing dissolved Fmoc-Try-OH and HOBT, react at room temperature for 3 h to obtain Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin, filter and wash the solid part with DMF 3 times;

[0061] (8) Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF was 2:8) for 30 min. The reaction solution was filtered off. The resulting solid part was washed 5 times with DMF. 80 g of Fmoc-Pro-OH and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin and activated for 5 min. Then, DMF solution containing Fmoc-Pro-OH and HOBT was added. The reaction was carried out at room temperature for 3 h to obtain Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin. The solid part was washed 3 times with DMF after filtration.

[0062] (9) Stir Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80 g Fmoc-Asp-OH and 40 g HOBT in DMF, add 38 g DIC to the deprotected Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin and activate for 5 min, then add DMF solution containing dissolved Fmoc-Asp-OH and HOBT, react at room temperature for 3 h to obtain Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin, filter and wash the solid part with DMF 3 times;

[0063] (10) Stir Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80 g Fmoc-Asp-OH and 40 g HOBT in DMF, add 38 g DIC to the deprotected Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin and activate for 5 min, then add DMF solution containing dissolved Fmoc-Asp-OH and HOBT, react at room temperature for 3 h to obtain Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin, filter and wash the solid part with DMF 3 times;

[0064] (11) Stir Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin in a mixture of piperidine and DMF (volume ratio of piperidine to DMF is 2:8) for 30 min, filter off the reaction solution, wash the obtained solid part with DMF 5 times, dissolve 80g Fmoc-Tyr-OH and 40g HOBT in DMF, add 38g DIC to the deprotected Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin and activate for 5 min, then add DMF solution containing dissolved Fmoc-Tyr-OH and HOBT, react at room temperature for 3 h to obtain Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin, filter and wash the solid part with DMF 3 times;

[0065] (12) The Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF: 2:8) for 30 min. The reaction solution was filtered off, and the resulting solid fraction was washed 5 times with DMF. 80 g of Fmoc-His-OH and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin and activated for 5 min. Then, a DMF solution containing dissolved Fmoc-His-OH and HOBT was added, and the mixture was reacted at room temperature for 3 h to obtain His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA. Resin, after filtration, the solid fraction was washed three times with DMF;

[0066] (13) His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF: 2:8) for 30 min. The reaction solution was filtered off, and the resulting solid fraction was washed 5 times with DMF. 80 g of Fmoc-Arg-OH and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin and activated for 5 min. Then, a DMF solution containing dissolved Fmoc-Arg-OH and HOBT was added, and the mixture was reacted at room temperature for 3 h to obtain Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA. Resin, after filtration, the solid fraction was washed three times with DMF;

[0067] (14) Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin was stirred in a mixture of piperidine and DMF (volume ratio of piperidine to DMF: 2:8) for 30 min. The reaction solution was filtered off, and the resulting solid fraction was washed 5 times with DMF. 80 g of NOTA and 40 g of HOBT were dissolved in DMF. 38 g of DIC was added to the deprotected Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin for 5 min of activation. Then, a DMF solution containing dissolved NOTA and HOBT was added, and the mixture was reacted at room temperature for 3 h to obtain the polypeptide resin N-Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA. Resin, after filtration, the solid fraction was washed three times with DMF;

[0068] (15) The obtained peptide resin N-Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHAResin was added to a mixture of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water and reacted at room temperature for 3 h. The volume percentages of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water were 90%, 5.0%, 2.5%, and 2.5%, respectively. After filtration, the filtrate was precipitated in ice-cold ether. The precipitate was dissolved in acetonitrile and water, filtered, and purified by high-performance liquid chromatography. After pre-freezing in liquid nitrogen, it was lyophilized. The obtained NPY-targeting peptide nuclide ligand was designated as NOTA- D NPY (All amino acids used in this embodiment are D-type amino acids).

[0069] Example 2

[0070] The preparation method of the NPY-targeting polypeptide nuclide ligand in this example is the same as in Example 1, except that all the amino acids used are L-type amino acids. The final NPY-targeting polypeptide nuclide ligand is denoted as NOTA-. L NPY.

[0071] Application Examples

[0072] Take 30 μL of 1.5 M ammonium acetate solution and slowly add it to 10 μL of the above-mentioned 1 μg / μL targeted NPY polypeptide nuclide ligand (NOTA-). D NPY or Nota- L Add 300 μL of freshly rinsed radionuclide solution to the NPY solution.68 GaCl3, with a radioactive nuclide activity of 2 mCi, was heated at 40 °C for 10 min and then cooled to room temperature to obtain NPY peptide nuclide probes. 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L NPY.

[0073] right 68 Ga-NOTA- D NPY and 68 Ga-NOTA- L NPY was analyzed by radioactive HPLC: An Agilent 1260 HPLC system equipped with a YMC-Pack ODS-A analytical column (250 × 4.6 mm L.DS-5 μm, 12 nm) was used. The gradient elution time was 20 min, and the flow rate was 1 mL / min. Mobile phase A was deionized water (containing 0.1% TFA), and mobile phase B was acetonitrile (containing 0.1% TFA). The elution gradient was set to 78% A and 22% B initially, and 58% A and 42% B after 20 min. The results are as follows: Figure 1 As shown, the MS spectrum is as follows Figure 2 As shown: From Figure 3 It can be seen that after purification by Sep-Pak C18 column, 68 Ga-NOTA- L NPY and 68 Ga-NOTA- D NPY radiochemical purity >98%.

[0074] Stability test: Take a certain amount 68 Ga-NOTA- L NPY or 68 Ga-NOTA- D NPY was added to physiological saline, fetal bovine serum, and human serum, and its radiochemical purity and stability were determined at different time points. The results are shown in [Figure number missing]. Figure 4 As shown, after 1 hour, 2 hours, and 3 hours 68 Ga-NOTA- L NPY and 68 Ga-NOTA- D NPY maintains a radiochemical purity of over 98% and good stability.

[0075] In vitro lipid-water partition coefficient experiment: Take equal volumes of 0.5 ml each of n-octanol and ultrapure water (or PBS) in an EP tube, and add approximately 1 uCi of... 68 Ga-NOTA- L NPY or 68Ga-NOTA- D The NPY polypeptide nuclide probe was sealed, mixed thoroughly, and shaken at room temperature for 5 min. The EP tube was then centrifuged at 15000g for 10 min. 100 μL of n-octanol and 100 μL of ultrapure water (PBS) were placed into gamma counter tubes, and the radioactivity count of each tube was measured using a gamma counter. LogP and LogD7.4 values ​​were calculated, and the results are shown below. Figure 5 As shown, it can be seen 68 Ga-NOTA- L NPY and 68 Ga-NOTA- D NPY has excellent water solubility.

[0076] In vitro cell uptake experiment: MCF-7 cells were evenly distributed into each well of a 24-well cell culture plate, with approximately 5 × 10⁶ cells per well. 4 Cells were incubated for 8-10 hours. Under aseptic conditions, 15 ml of culture medium without fetal bovine serum was added to a 15 ml centrifuge tube, and a certain activity level of purified cells was collected. 68 The Ga-NOTA-NPY radionuclide probe was added to the above solution to prepare a solution of approximately 1 uCi / ml, and shaken to mix. The old culture medium was discarded, and 0.5 ml of radioactive culture medium was added to each well (approximately 0.5 uCi / well). The cell culture plate was placed in a 37°C incubator for subsequent experiments. At 1 h, 2 h, and 3 h, the cell culture plate was removed (6 replicates per time point). The culture medium in the replicates was aspirated with a pipette and washed three times with 1800 μL PBS. The old culture medium and washing buffer were retained in a gamma counter tube. Cells were then digested with trypsin and collected again in a new gamma counter tube, washed three times with 1800 μL PBS. Finally, the gamma count in each tube was measured using a gamma counter, and the results were calculated. (See attached table). Figure 6 As shown, it can be seen 68 Ga-NOTA- D NPY probes have better targeting performance at the cellular level than 68 Ga-NOTA- L NPY probe.

[0077] PET imaging study: 6-week-old BALB / c Nude female mice, provided by Shanghai Yaokang Biotechnology Co., Ltd., were subcutaneously injected with MCF-7 human breast cancer cells into their right leg after two days of acclimatization in the animal facility. The injection volume was 0.1 mL (1×10⁻⁶). 7 Cells / mL dispersed in HBSS), and continued feeding for 2-3 weeks after injection until the solid tumor mass grows to 500-800 mm. 3 It is used for imaging experiments. 100-150 μCi / 0.2 mL is injected via the tail vein.68 Ga-NOTA- L NPY 68 Ga-NOTA- D NPY was injected for 30 min, 60 min, and 120 min, and small animal PET-CT imaging experiments were performed. The imaging results are as follows: Figure 7 and Figure 8 As shown: a 60-minute blocking experiment, illustrating... 68 Ga-NOTA- D NPY has good targeting properties; NPY-positive MCF-7 tumors are effective against... 68 Ga-NOTA- D NPY compounds exhibit high uptake, with clearly visible tumor uptake and no gallbladder uptake. The peptides remain at the tumor site for a longer period, which is more beneficial for tumor diagnosis. In the MDA-MB-468 breast cancer tumor model group with low NPY Y1 protein expression, tumor uptake... 68 Ga-NOTA- D The significant decrease in NPY indicates that the probe of this invention specifically binds to the NPY Y1 site. Figure 9 ).

[0078] In addition, by comparing the pharmacokinetics of the probes (e.g. Figure 10 ),Discover 68 Ga-NOTA- D NPY probe ratio 68 Ga-NOTA- L The NPY probe had an increased circulation time in mice, consistent with PET-CT imaging results.

[0079] Finally, it should be noted that the specific embodiments described herein are merely illustrative of the spirit of the invention and are not intended to limit the implementation of the invention. Those skilled in the art can make various modifications or additions to the described embodiments or use similar methods to replace them; it is neither necessary nor possible to exemplify all embodiments here. However, these obvious variations or modifications derived from the essential spirit of the invention still fall within the scope of protection of the invention, and interpreting them as any additional limitation would contradict the spirit of the invention.

Claims

1. A ligand targeting NPY polypeptide nuclide, characterized in that, The structural formula is shown in equation (1): Where L is a metal chelating agent.

2. The targeted NPY polypeptide nuclide ligand according to claim 1, characterized in that, The metal chelating agent is one or more of the following: 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid, 1,4,7-triazacyclononane-N,N',N'-triacetic acid, N-hydroxysuccinimide carbazide, (2S)-(1-tetrahydropyrimidin-2-one)-3-methylbutyric acid, diethyltriaminepentaacetic acid, 2-[1,4,7-triazacyclononane-1-yl-4,7-bis(t-Bu ester)]-1,5-pentanoic acid, triethylenetetramine, cyclohexanediol and terephthalic acid copolymer, and 1,4,7,10-tetra(aminocarbonylmethyl)-1,4,7,10-tetraazacyclododecane.

3. A method for preparing the targeted NPY polypeptide nuclide ligand as described in claim 1, characterized in that, Includes the following steps: (1) Add tyrosine, activator, condensing agent and solvent to Rink Amide MBHA Resin and react at room temperature for 1-5 h to graft tyrosine onto Rink Amide MBHA Resin. (2) Add arginine, activator, condensing agent and solvent, react at room temperature for 1-5 h, graft arginine, and follow the above steps to graft glutamine, arginine, tryptophan, isoleucine, proline, asparagine, asparagine, tyrosine, histidine, arginine and metal chelating agent in sequence to obtain polypeptide resin L-Arg-His-Tyr-Asp-Asp-Pro-Iso-Try-Arg-Gln-Arg-Tyr-Rink Amide MBHA Resin; (3) Add the obtained polypeptide resin to the lysis buffer and react at room temperature for 1 to 5 hours.

4. The preparation method according to claim 3, characterized in that, Rink Amide MBHA Resin is deprotected before use and after amino acid grafting.

5. The preparation method according to claim 3, characterized in that, In step (1), the mass ratio of Rink Amide MBHAResin, tyrosine, activator, and condensing agent is 3.0–8.0: 2.0–5.0: 2.0–5.0: 2.2–5.

5.

6. The preparation method according to claim 3, characterized in that, The activator is one or more of N,N'-diisopropylcarbodiimide, dicyclohexylcarbodiimide, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; and / or the condensing agent is one or two of 1-hydroxybenzotriazole and N-methylmorpholine.

7. The preparation method according to claim 3, characterized in that, The lysis solution in step (3) is a mixture of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane and water.

8. The preparation method according to claim 7, characterized in that, The volume percentages of trifluoroacetic acid, 1,2-ethylenedithiol, triisopropylsilane, and water in the lysis buffer were 90–95%, 3–10%, 1–5%, and 1–5%, respectively.

9. A probe targeting an NPY polypeptide nuclide, characterized in that, It includes the targeted NPY polypeptide nuclide ligand and radionuclide as described in claim 1.

10. A method for preparing a targeted NPY polypeptide nuclide probe as described in claim 9, characterized in that, Includes the following steps: The targeted NPY polypeptide nuclide ligand was added to the ammonium acetate solution, followed by the addition of a radioactive nuclide solution. The mixture was heated to react and then cooled to room temperature.

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